Studies on the Biology and Genetic Variation of Phomopsis on Grapevine
STUOTBS ON THE BTOIOGY AND GNNNTTC V¡'NTATION OF PHOMOPSIS ON GN¡.PBVINE
Reiny \ry.A. SchePer MSc, Wageningen Agricultural University
Thesis submitted for the degree of Doctor of Philosophy In The University of Adelaide
Department of Applied and Molecular Ecology Faculty of Agricultural and Natural Resource Sciences
August 2001.
Photograph on the Previous Page: Perithecia of Diaporthe viticola on a Chardonnay cane' +,+ .. i 'ñ Jt ; + 3 s*.
âa(,,\ f Ë' ,*J TABLE OF CONTENTS
Preface
Abstract I Declaration iii Acknowledgements iv Publications vi Abbreviations viii
Chapter I Introduction 1
Chapter 2 Literature review 2 2.1 Introduction 2 2.2 The genus PhomoPsis 2 2.2.I Taxonomy 2 2.2.2 Characteristics of the genus Phomopsis 4 2.2.3 cr-conidia and P-conidia 4 2.3 Distribution of the disease and taxonomy of Phomopsis on grapevine 2.3.1 Distribution of Phomopsis cane and leaf spot of grapevines 5 2.3.2 Taxonomy of Phomopsis on grapevine 6 2.3.3 Confusion in symptomatology and terminology 7 2.4 Symptoms of Phomopsis cane and leaf spot I 2.4.1 Symptoms in sPring 8 2.4.2 Symptoms in summer 9 2.4.3 Symptoms in autumn and winter 10 2.5 Ecology and epidemiology of P. viticola 11 2.5J Disease cycle l1 2.5.2 Ecology of P. viticola L2 2.5.3 Epidemiology of Phomopsis cane and leaf spot 13 2.6 Control of Phomopsis cane and leaf spot t6 2.6.L ManagementPractices t7 2.6.2 Resistance of grapevines to Phomopsis cane and leaf spot l7 2.6.3 The use of fungicides 18 2.7 Factors responsible for genetic diversity t9 2.7 .I Genetic variability of populations 19 2.7.2 Mutation 20 2.7.3 Migration 22 2.7.4 Random genetic drift 23 2.7.5 Natural selection 24 2.8 Genetic variation of Phomopsís on grapevine 25 2.8.I Sexualrecombination 26 2.8.2 Parasexualrecombination 27 2.9 Markers for genetic analysis of Phomopslï on grapevine 29 2.9.I PhenotYPicmarkers 29 2.9.2 Molecularmarkers 29 2.10 Applications of molecular markers 33 2.tl Summary 35 2.12 Aims of the project 35 2.L3 Addendum 36
Chapter 3 General materials and methods 37 3.1. Isolation of Phomopsis of grapevine 37 3.2 DNA extraction 43 3.2.I Extraction of DNA fromPhomopsis of grapevine 43 3.2.2 Extraction of DNA from micropropagated grapevine leaves 44 3.3 Construction of a DNA librarY 45 3.3.1 Ligation of Phomopsis DNA into pUC19 vector 45 3.3.2 Transformation of Escherichía coli 46 3.3.3 Screening for recombinant E colí colonies 47 3.3.4 Storage of DNA librarY 47 3.4 General molecular methods 48 48 3.4.I Plasmid DNA PreParation 3.4.2 Extraction of plasmid DNA inserts from agarose gels 49 3.4.3 Electrophoresis and Southern blot analysis 50 3.4.4 Radiolabellingandhybridisation 50
Chapter 4 Discovery and description of the teleomorph of Phomopsis taxon 1 53 4.1 Introduction 53 4.2 Materials and methods 53 4.2.I Isolation of the teleomorph of Phomopsis taxon 1 53 4.2.2 Microscopic examination of sexual structures 54 4.2.3 Identification of the teleomorph 54 4.2.4 Ascospore release 55 4.3 Results 55 4.3.1 Isolation of the teleomorph of Phomopsis taxon 1 55 4.3.2 Taxonomic description of the teleomorph 56 4.3.3 Identification of the teleomorph 56 4.3.4 Symptoms associated with Diaporthe viticola 56 4.3.5 Ascosporerelease 57 4.4 Discussion 57
Chøpter 5 Mating studies of Dìaporthe viticola 64 5.1 Introduction 64 5.2 Materials and methods 64 S.Z.l Induction of perithecia in vitro and the nature of the mating system 64 5.2.2 Conditions for perithecium development invitro 67 5.3 Results 68 5.3.1 Production of peritheciainvitro and the nature of the mating system 68 5.3.2 Conditions for perithecium development in vitro 76 5.4 Discussion 76 5.4.L Production of zone-lines and perithecia in vitro 76 5.4.2 The nature of the mating system 79 Chapter 6 Disease development in vineyards 83 6.1 Introduction 83 6.2 Materials and methods 83 6.2.I Monitoring of grapevines infected with Phomopsls taxon 1 83 6.2.2 Preliminary examination of budburst in bleached grapevine spurs 86 6.3 Results 87 6.3.I Monitoring of grapevines infected with Phomopsls taxon 1 87 6.3.2 Preliminary examination of budburst in bleached grapevine spurs 9T 6.4 Discussion 93
Chapter 7 Pathogenicity and seed transmission 102 7.1 Introduction 102 7.2 Materials and methods 103 7.2.L Preliminarypathogenicitystudy 103 7.2.2 Pathogenicity of D. viticola and Phomopsis on grapevine 105 7.2.3 Seedtransmission 106 7.3 Results 108 7.3.1 Preliminarypathogenicitytest 108 7.3.2 Pathogenicity of D. viticola and Phomopsis on grapevine 113 7.3.3 Seed transmission 113 7.4 Discussion trg
Chapter I Mycelial incompatibility in grapevine Phomopsís t25 8.1 Introduction 125 8.2 Materials and methods 127 8.2.L Mycelialincompatibilitytests 127 8.2.2 Data analysis 128 8.2.3 Mycelial incompatibility used to study mating behaviour r28 8.3 Results 129 8.3.1 Mycelialincompatibilitytests r29 8.3.1.1 Identification of morphological types of Phomopsis of grapevine 129 8.3.L2 Mycelial incompatibility in Phomopsis of grapevine 131 8.3.2 Estimate of variation within and between populations of Phomopsis taxon 1 t43 8.3.3 Mycelial incompatibility used to study mating behaviour r43 8.4 Discussion 143
Chapter 9 Development of taxon-specifÏc and RFLP markers, and a pilot study on genetic diversity in Phomopsís of grapevine t52 9.1 Introduction 152 9.2 Materials and methods 153 9.2.1 Screening genomic libraries of Phomopsis taxon I and 2 for taxon- specific and RFLP markers 153 9.2.2 Screening microsatellites for RFLP probes 155 9.2.3 RFLP data analYsis 155 9.3 Results L57 9.3.1 Screening genomic libraries of Phomopsls taxon 1 and 2 for taxon- specific and RFLP markers r57 9.3.2 Screening microsatellites t72 9.3.3 RFLP data analysis 173 9.4 Discussion 186
Chapter 10 General discussion 192 10.1 Introduction r92 10.2 Biology and epidemiology of Phomopsís of grapevine r92 10.3 New disease cycle for Phomopsis taxon 1 of grapevine t94 10.4 Genetic variation in Phomopsis of grapevine 196 10.5 Taxonomy of Phomopsis of grapevine 200 10.6 Implications for disease management 204
Appendix 206
Reþrences 212 ABSTRACT
phomopsis cane and leaf spot of grapevine, caused by the fungus Phomopsis viticola (Sacc.)
Sacc., may cause significant yield loss in viticultural regions with wet spring weather. Two main taxa of Phomopsls occur in Australian vineyards; they cause different symptoms and usually occur in different geographical areas, but co-exist in some areas. Taxon 2 causes the symptoms attributed to P. viticol¿ in other parts of the world. However, taxon 1 causes no symptoms during the growing season and only manifests itself in late winter as pycnidia on bleached canes. As very little is known about the biology, epidemiology and population structure of taxon 1, the progression of the disease was monitored and molecular and phenotypic markers were developed to study genetic diversity in this pathogen. To help distinguish the two taxa, molecular and phenotypic markers were also developed for taxon
2.
Studies on the biology of Phomopsis taxon I resulted in the discovery of a sexual phase. The teleomorph was described and was confirmed by the International Mycological
Institute as Diaporthe víticola (Nitschke). Perithecia of D. viticola wete found on 1- and 2- year-old canes of Chardonnay, Shiraz, Riesling, Cabernet and Pinot Noir in many vineyards
in southern Australia. The existence of the teleomorph is discussed in relation to genetic
variation in the pathogen and the management of the disease caused by Phomopsis taxon 1.
Disease caused by Phomopsis taxon I was monitored in five vineyards in the
Adelaide Hills, South Australia. Diseased spurs were tagged and observed for three
consecutive seasons; apparently healthy spurs were included in the study in the third season.
This study showed that taxon 1 may reduce and delay budburst. An attempt was made to
test the pathogenicity of isolates of both taxa of Phomopsis on grapevine in the glasshouse,
in order to confirm the data obtained from field studies. Seed transmission of both taxa was
demonstrated; all seedlings derived from berries infected with taxon 2 died, and967o of the
seedlings derived from berries infected with taxon 1 died, compared with 647o of the
controls.
The published disease cycles for Phomopsis cane and leaf spot may be applicable to
the disease caused by taxon 2 of Phomopsis found in Australia, however, they do not
describe the biology of taxon 1. A separate disease cycle, which focuses on the symptoms ll caused by phomopsis taxon 1, and includes the teleomorph and reduced budburst in spring, was devised.
Total DNA from both Phomopsis taxon I and taxon 2 was cloned into the plasmid
vector, pUC19. Taxon 1 DNA was cloned in the PsrI site and the SalI site was used for
taxon 2 DNA. Cloned sequences of both taxa were evaluated for taxon specificity. Three
putative taxon 1 specific probes with 3.5, 3.8 and 3.8 kb inserts, and two putative taxon 2
specific probes, 4.0 and 6.2 kb in size, were identified. The cloned sequences did not
hybridise to grapevine DNA nor to DNA from a range of higher fungi representing the
mycobiota of grapevine canes and stems.
Genetic variation in Phomopsis in Australia was investigated in a preliminary study
of 24 taxon l isolates, 13 taxon 2 isolates, two taxon 3 isolates and one taxon 4 isolate.
Microsatellite probes (CAT)5 and (GATA)a and cloned sequences of Phomopsis taxon 1
and taxon 2 identified genetic variation, within and between viticultural regions. However,
taxon 2 displayed more variation between regions than within regions and less variation
within regions than did taxon 1. All probes identified distinct differences between the DNA
of taxon 1 and taxon 2 isolates.
Mycelial incompatibility studies showed that32 taxon 1 isolates fell into 16 putative
mycelial compatibility groups (MCGs). A dark interaction zone or a growth-free zone was
present when incompatible isolates were paired on potato dextrose agar. All 16 taxon 2
isolates were compatible with each other, and incompatible with all taxon 1 isolates tested.
An in vitro mating system for Phomopsis taxon 1 was used in which paired isolates
produced perithecia on autoclaved Chardonnay canes derived from vines not treated with
fungicides. Several isolates appeared to be self-fertile, whereas others were self-sterile,
making interpretation of the mating type data difficult.
This study has helped to alleviate the confusion between the two main taxa of
phomopsis that infect grapevine in Australia. It provides information on the epidemiology
of the disease caused by taxon 1, and preliminary information on the genetic variation and
population biology of both taxa, including the characterisation of the sexual stage of
Phomopsis taxon 1. lll
DECLARATION
This work contains no material which has been accepted for the award of any other degree or diploma in any university or other tertiary institution and, to the best of my knowledge and belief, contains no material previously published or written by another person, except where due reference has been made in the text.
I give consent to this copy of my thesis, when deposited in the University Library, being available for loan or photocopying.
t/ / "t /2,,n t Sígned Døte lv
ACKNOWLEDGEMENTS
I would like to sincerely thank and acknowledge my supervisors, Dr Eileen Scott and Dr
Dara Melanson (formerly Whisson), for their advice, support, editing skills, occasional assistance with lab work and patience throughout this project, and for going out of their way to help me finish this thesis before leaving Australia by, for example, supplying me with references, reading and correcting chapters thoroughly and quickly, and staying in frequent contact via e-mail.
I would also like to thank professor Bob Symons, Dr Jim Hardie and the first Cooperative Research Centre for
Viticulture (CRCV) for supporting this work,
professor Otto Schmidt and the Department of Applied and Molecular Ecology for use of
facilities and resources,
Dr Belinda Stummer for her help with the molecular experiments and analysis, for teaching
me how to use GENSTAT 5 to generate dendrograms of genetic similarity for the RFLP
data, and for supplying the microsatellite primers and tissue cultured grapevine plantlets,
Ms Belinda Rawnsley (formerly Brant) and Ms Janine Lloyd for encouragement, support
and occasional assistance with lab and field work, and Ms Belinda Rawnsley for providing
photocopies of references after I left Adelaide'
Mr. Daniel Crane for his enthusiasm, assistance with lab and field work and for being a
dedicated CRCV summer student,
Dr Trevor V/icks and Dr Hugh Wallwork for advice and constructive criticism,
Dr Tan Nair, Dr Michael Priest and Ms Jill Merrin for supplyingPhomopsis cultures,
Mr. philip Iæppard @epartment of Statistics, The University of Adelaide) and Ms Helena
Oakey (Biometrics-SA) for their help in choosing the appropriate statistical analysis in
Chapter 6,
professor Margaret Sedgley for the use of her equipment and methodology for microscopic
examination of perithecia,
Dr Uli Theopold for assistance in the use of the cryocut microtome,
Dr P.F. Cannon (trVtr) for confîrmation of Diaporthe viticola, the vineyard owners for their cooperation, trial sites and materials,
Ms Jan Strawhorn, Ms Barbara Hall, Mr. Duncan Farquhar and Dr Darby Munro for driving me around the vineyards north of Melbourne, in the Coonawara and in Tasmania,
introducing me to vineyard owners and staff, and helping me collect samples.
Dr Greg Johnson, Dr Chris Green and Dr Andrew Highet for support and suggestions,
Dr Simon Walker and Ms Cathy Brand for assistance in using their micro-manipulators in
the attempt to isolate eight ascospores from single asci,
Carol Smith, Paul Ingram and Ernie Nagy for looking after my grapevines,
Ms Anke Johnson, Mr. Terry Feckner and M¡. Gary Taylor for help and support,
Ms Jennie Groom, Ms Emily Shepherd, Ms Sharon Clapham and Ms Sheila Cooper for
photographic assistance. vi PUBLICATIONS
Publications in Refereed Journals Scheper, R.W.A., Crane, D.C., Whisson, D.L. & Scott, E.S. (2000). The Diaporthe teleomorph of Phomopsis on grapevine. Mycological Research 104, 226-231. Melanson, D.L., Rawnsley, B. & Scheper, R.W.A. (2001). Molecular detection of Phomopsis taxa 1 and2 in grapevine canes and buds. Australasian Plant Pathology (submitted).
Publications in Industry Journals Scheper, R.W.A., Scott, E.S. & Whisson, D.L. (1995). The missing link in Phomopsis cane and leaf spot diseas e. The Australían Grapegrower & Winemaker 381, 23 - 25. Scheper, R.W.A., Scott, E.S. & Whisson, D.L. (1997). Phomopsis cane and leaf spot - Discovery of the sexual stage of P. viticola type 1. The Wine Industry Journal 12, 264 -265. Scheper, R.'W.4., Whisson, D.L. & Scott, E.S. (1997). Revised disease cycles for the two types of Phomopsis on grapevine. The Australian Grapegrower &Winemaker 405, 4L - 44. Whisson, D.L., Brant,8., Scheper, R.W.A. & Stummer, B.E. (1998). Detection of Phomopsis viticola in grapevine cane. The Australian Grapegrower & Winemaker 417,73 -75. Whisson, D.L., Brant, B., Stummer, B.E. & Scheper, R.W.A. (1998). Finding Phomopsis through molecular, classical techniques . Grap eGrow ers Aprllll||:l{ay, 44
Papers Presented at Professional Meetings Scheper, R.V/.A., Scott, E.S. & Whisson, D.L. (1995). Genetic diversity in Phomopsis viticolain Australian vineyards. Poster presented at the 10ù Biennial Australasian Plant Pathology Society Conference, Lincoln University, New Zealand,zS -30 August. Scheper, R.W.A., Scott, E.S. &'Whisson, D.L. (1996). Genetic variation in Phomopsis viticolain Australian vineyards. In: Proceedings of the 9ú Australian'Wine Industry Technical Conference (ed. C.R. Stockley et al.),p 190.'Winetitles, Adelaide, South Australia. Scheper, R.W.A., Scott, E.S. & Vy'hisson, D.L. (1996). Phomopsis viticola: pathogenicity, sexual stage and genetic variation. Poster presented at the First Meeting of the Australasian Mycological Society, Melbourne, Victoria, 29 Septembet - 2 October. Scheper, R.W.A., Scott, E.S. & Whisson, D.L. (1997). Field monitoring and a revised disease cycle of Phomopsis viticolataxon 1. Oral presentation at the 11ü Biennial Australasian Plant Pathology Society Conference, Perth, Western Australia, 29 September - 2 October. vll
(1997). Scheper, R.W.A., Crane, D.C., Scott, E.S., Whisson, D.L. & Stummer, B.E. Molecular and phenotypic characterisation of Phomopsis viticola in Australian vineyards. Poster presented at the 11th Biennial Australasian Plant Pathology Society Conference, Perth,'Western Australia, 29 September - 2 October. whisson, D.L., Brant,8., Stummer, B.E. & Scheper, R.w.A. (1998). Detection of phomopsis in grapevine canes using molecular and classical techniques. Poster presented at the 7th International Congress of Plant Pathology, Edinburgh, Scotland, 9-16 August 1998. Brant, B., Melanson, D.L. & Scheper, R.W.A. (1999). Phomopsis: molecular detection on grapevine cane. Oral presentation at the 12th Biennial Australasian Plant Pathology Society Conference, Canberra, ACT, 27-30 September 1999' vlll ABBREVIATIONS
ANOVA analysis of variance ATP adenosine 5'-triphosPhate CTAB cetyl trimethyl ammonium bromide CTP cytidine 5' -triphosphate EDTA ethylenediaminetetra-acetic acid DTT dithiothreitol
ôo gravitational force h hour(s) HSB high salt buffer IPTG isopropyl- p-D{hiogalactopyranoside kb kilobase pairs LB Luria-Bertani M molarity min minute NSW New South Wales OD optical density PDA potato dextrose agar pers. com. personal communication PDB potato dextrose broth PIPES piperazine-1,4-bis[2-ethanesulfonic acid] RO water reverse osmosis water SA South Australia
S second SDS sodium dodecyl sulPhate SSC saline-sodium citrate Tas Tasmania TE Tris-EDTA Tris tri s- (hydroxymethyl)- aminomethane uv ultra violet V Volts Vic Victoria WA water agar
X-gal 5 -bromo-4-chloro-3 -indolyl-p-D-galactoside I
1. INTRODUCTION
Throughout the viticultural world, Phomopsis viticola is well known as the pathogen which
causes phomopsis cane and leaf spot disease (Moller & Kasimatis, 1981; Moller et al',
l912;Hewitt & Pearson, 1988; Pscheidt & Pearson, 1989b; Bugaret, 1990; Emmett et aI',
lgg2). However, four taxa of Phomopsis on grapevines have recently been identified in
Australia (Merrin et a1.,1995). While these taxa were initially thought to be forms of P.
viticola, they may also be separate species of Phomopsis. Symptoms associated with these
different taxa have been confused. Furtherïnore, symptoms caused by Phomopsis have been
confused with those cause by Eutypa lata (Mroller and Kasimatis, 1981), other organisms
and abiotic factors (IPM Viticulture Research to Practice,1997). Little is known about the taxa of Phomopsis on grapevine in Australia. In order to
control phomopsis cane and leaf spot efficiently, with minimal fungicide input, it is
imperative that a better understanding of the biology, taxonomy and epidemiology of the
taxa is reached. The symptoms associated with the different taxa need to be verified, their
ability to cause disease established, and the genetic relationships between the taxa clarified.
In addition, the mode of reproduction of each taxon needs to be determined and taken into
account when developing improved disease management strategies, as sexual reproduction
enhances the likelihood of new genotypes appearing. Prior to the research presented in this
thesis, the perfect stage of P. viticola had been described only once, as Cryptosporella
viticola (Shear, 1911), but teleomorphs of the taxa of Phomopsis on grapevine in Australia
had not been described.
Nothing is known about the impact of sexual or asexual recombination on the
genetic diversity of the taxa of Phomopsis on grapevine. The use of molecular and
phenotypic markers in population genetic studies of Phomopsls on grapevine will provide
information on the level and structure of genetic diversity, thereby providing an indication
of the reproductive strategy.
The research reported in this study was aimed at clarifying aspects of the biology,
taxonomy and population structure of the taxa of Phomopsis on grapevines in Australia,
using both traditional and molecular techniques' )
2. LITERATURE REVIEW
2.1 Introduction
Very little is known about the four taxa of Phomopsís reported to infect grapevine in
Australia (Menin et a1.,1995). As most of the literature on Phomopsis cane and leaf spot concerns phomopsis viticola, this review focuses on this fungus and the disease it causes'
Merrin et al. (1995) suggested that Phomopsis taxon 1 fits the taxonomic description of P. viticola, although taxon 2 is associated with symptoms similar to those caused by P.
viticola.It was concluded that the taxa may be varieties of P. viticola or distinct species of
phomopsis. Because of this uncertainty, the fungus is referred to as "Phomopsis on
grapevine", unless specific research on P. viticolø is quoted'
This review discusses the characteristics of the genus Phomopsis, the taxonomy of
p. viticola, the disease with which it is associated, and the development and application of
markers that detect genetic diversity. As the experimental component of this project was
completed in 1997, this review concentrates on the information available up to 1997.
However, several papers published since then have reported research highly relevant to this
thesis; these papers are reviewed briefly in section 2.I3 and mentioned in discussion
sections, as appropriate.
2.2Thegenus PhomoPsis
2.2.1Taxonomy The genus Phomopsis belongs in the Mitosporic Fungi (formerly, the subdivision
Deuteromycotina, or the Fungi Imperfecti). The group encompasses the conidial states of
the Ascomycota and Basidiomycota, and the asexual fungi for which the perfect states have
not as yet been found (Sutton, 1973; Hawksworth et a1.,1995). Talbot (1971) stated that a
natural classification of Deuteromycotina is not possible. Species with conidial states that
are alike in morphology are placed in one form-genus of the Deuteromycotina, but they are
not necessarily phylogenetically related forms. A single genus of Ascomycotina or
Basidiomycotina may contain species with a number of conidial forms that are assigned to J different form-genera of the Deuteromycotina. Moreover, species of one form-genus do not necessarily have sexual states (when these are known) sufficiently alike to occupy the same genus of Ascomycotina or Basidiomycotina (Talbot, l97l)'
The genus Phomopsis is placed in the "class" Coelomycetes as the conidia are formed within a cavity lined by fungal and/or host tissue (Sutton, t973). The "class"
Coelomycetes contains many species that show very few distinctive morphological features.
Thus a large number of species has been distinguished almost solely on the basis of the different hosts or substrata on which they were first found and described. Many of those present species could, in fact, be synonyms, showing small morphological differences when
on different hosts or in different environments (Talbot,1971, Rehner & Uecker, 1994).
The Coelomycetes comprise two "orders", both consisting of microscopic parasites
or saprobes of plant material: the Melanconiales with acervuli, and the Sphaeropsidales
with pycnidia. The genus Phomopsis belongs to the latter "order" (Sutton, 1973). Originally,
species of Phomopsis were considered to be a section of the very large genus Phoma
(Sacc.), until Saccardo described the new genus, Phomopsis (Sacc.) (Saccardo, 1905). The
number of species in the genus is not known. Merrin et aI. (1995) referred to the existence
of g00 species or subspecies of Phomopsis, whereas Von Arx (1970) considered there to be
approximately 40 species of Phomopsis, although many more have been described as
herbarium specimens. Uecker (1988), however, lists approximately 180 Phomopsis-
Diaporthe connections in his world list of Phomopsis names'
Species of Phomopsis are considered to be anamorphs of Díaporthe (Von Am, l9¡O;Ainsworth,l97l; Sutton, Ig73), although teleomorphic states have been reported for
only 20To of the species (Uecker, 1983). Shear (1911) described the teleomorph of
phomopsis viticola as Cryptosporella viticola, on the basis of the l-celled ascospores of the 1973). As teleomorph ; Diaporth¿ ascospores are 2-celled (Shear, 1911; Müller & Von Am, not species of one form-genus of the Deuteromycotina can have sexual states that are
sufficiently alike to occupy the same genus of Ascomycotina (Talbot, l97l), it is possible
that most phomopsis species are anamorphs of Diaporthe while some are anamorphs of
Cryptosporel/ø. Consequently, since Diaporthe and Cryptosporellø belong to the same family, phomopsis species should be considered as having teleomorphs in the family
Diaporthaceae (Hawksworth et a\.,1983; Holliday, 1989)' 4
2.2.2 Characteristics of the genus Phomopsis
Phomopsis species have septate mycelium (von Arx, 1970), and reproduction occurs globose with a hard asexually by means of conidia produced in pycnidia. Pycnidia are a single textured, dark coloured wall which is well developed (Ainsworth, I97l)' have ostiole, and are eustromatic, consisting of fungal tissue only (Sutton, 1973). The conidia, conidiogenous cells are monophialidic and produce large numbers of enteroblastic (Sutton, Phomopsis in basipetal succession, through one opening in the cell wall 1973)' (a) and filiform 1- species produce two types of conidia: fusiform l-celled alpha conidia Both are exuded celled beta (p) conidia (scolecospores) (von Am, 1970; Holliday, 1989)' in cirrhi at maturity (Muntañola-Cvetkovic et aI',1985)'
2.2.3 a-conidia and P'conidia p-conidia fail to While phomopsis a-conidia can germinate and reproduce the species, the do so. Only occasionally have they been found to germinate to produce short mycelial to age threads (Gåirtel, 1972; Muntañola-cvetkovic et a1.,1985). In addition, they appear Thus p-conidia much more rapidly than the cr-conidia (Muntañola-Cvetkovic et at.,1985).
cannot be considered as disseminators of the pathogen'
Conidia of most Phomopsis species fall in a nÍurow size range. The vast majority the a- (937o) of the reported measurements of the cx,-conidia are 4-t2 ¡tm long, although of c-conidia vary conidia of a few species may be only 3 pm long, or reach 23 ¡t'm' V/idths 5 to 53 pm, but from 1 to 7 pm, but 76Vo are 2-3 pm. The B-conidia vary in length from 3'5 pm, but most 877o are within the 12-30 pm range. Their widths range from 0.35 to
(957o) areO.5-2 y,m wide (Uecker, 1988)'
Muntañola-Cvetkovic et aI. (1985) compared the ultrastructure of Phomopsis c,- and
p-conidia. Both are uninucleate; the g-conidia have an isodiametric nucleus in a central
position in the cytoplasm, the p-conidia have an elongated nucleus. Other differences are many large lipid that the cr-conidia contain mitochondria with numerous long cristae, and present in droplets situated at the poles. Polysaccharide (glycogen) and protein reserves are p-conidia, vacuoles, and in the form of granule rosettes in the cytoplasm. The in contrast,
have few mitochondria with a small number of cristae, and little storage material. droplet is Polysaccharide and protein reserves do not appeaf to be present. A large lipid 5 present in the p-conidia when they are inside the pycnidia, but in the cirrhi, only a few small lipid droplets are left. The early exhaustion of lipid reserves (starting before conidial germination) and the lack of other storage materials is probably one of the reasons for the apparent rapid ageing of p-conidia (Muntañola-cvetkovic et aL.,1985).
Previous research (Gåirtel, L972; Mihaljcevic et aL, 1985) indicates that different pycnidia within one species of Phomopsis may contain either one or both types of conidia'
The proportions of a- and p-conidia are reported to vary with seasonal changes, weather conditions (Hen et aI., 1983), the presence of other micro organisms such as Bacillus
subtilis, and the amount of available nutrients, including carbohydrates (Pezet, 1974).
Wehmeyer (cited by Muntañola-Cvetkovic et a\.,1985) stated in 1975 that the p-conidia are formed before the cr-conidia. Welch & Gilman (1948), however, reported that the production of the p-conidia in Diaporthe phaseolorum vaÍ. soiae is irregular and cannot be associated with specific cultures or with a specific growth phase.
Although p-conidia cannot be considered as disseminators of the fungus, they are corrmon in Phomopsls species. Moreover, pycnidia containing exclusively p-conidia are reported to survive from one year to the next (Mihaljcevic et a1.,1985). This indicates that these conidia must be important for the fungus. The significance of the p-conidia and their role in the etiology of the disease are poorly understood, and this requires further study.
2.3 Distribution of the disease and taxonomy of Phomopsis on grapevine
2.3.1 Distribution of Phomopsis cane and leaf spot of grapevines
Phomopsis cane and leaf spot, caused by Phomopsis viticola (Sacc.), has a cosmopolitan
distribution in temperate climates. The disease is widely distributed throughout the viticultural world and has been reported in Africa, Asia, Australia, Europe and North
America (Hewitt & Pearson, 19SS). In Australia, the disease is most corlmon in the middle
river Murray basin and higher rainfall districts of Victoria and New South Wales @mmett
et aI.,1992).In several vineyards of those states, the disease was of major concern during
the lgg2l93 and 1993194 grape seasons (Merrin et aI., 1995). The earliest record of 6
Phomopsis cane and leaf spot in Australian vineyards was in New South Wales (Noble ef a1.,1935).
2.3.2Taxonomy of Phomopsis on grapevine
The causal agent of phomopsis cane and leaf spot disease of grapevine was first recognised in 1880 by Saccardo, who named the fungus Phoma viticola. Later, after erecting the genus phomopsis (Saccardo, 1905; section 2.2.I), Saccardo changed the name to Phomopsis viticola (Saccardo, 1915). Reddick, unaware of Saccardo's earlier publication (Saccardo,
1880), found the same fungus in 1909 on diseased grape vines, and named it Fusicoccum viticolum (Reddick, 1909). Goidanich (1937) was the first to recognise the synonomy of
Fusicoccum viticolum and Phomopsis viticolai the older name Phomopsis viticola, therefore, took precedence. Pine (1958) confirmed this. However, apparently, neither
Goidanich (1937) nor pine (1953) examined the type material of F. viticolum or P. viticola.
In 1993, Merrin & Nair reported that at least two different phenotypes of Phomopsis had been observed on grapevines in Australia. Merrin et al. (1995) described two distinct taxa of Phomopsis (taxon I and taxon 2), plus two minor groups (taxon 3 and taxon 4). On the basis of morphology they suggested that taxon 1 fitted the current concept of P. viticola
(Sacc.), even though taxon 2 was associated with scarring on woody tissue and lesions on green tissues, similar to those caused by P. viticola (Moller et al., 1982; Emmett et al.,
lgg2) and F. viticolum (Reddick, 1909; Gregory, 1913). Taxon 1 was not associated with
these symptoms. Taxon 2 was considered to be a variety of P. viticola or a distinct species
(Merrin et a1.,1995). Compared with taxon l, isolates belonging to taxon 2had a slower
mycelial growth rate in vitro, red-brown instead of black pycnidia, larger ø-conidia (8.0-
11.8 ¡rm x2.0-3.2 ¡rm instead of 4.8-7.2 pm x I.4-2.2 Fm), a higher optimum temperature for a-conidium germination (35'C instead of 25oC), earlier sporulation in vitro (13 instead
of 20-23 days), and did not require light for sporulation. In addition, the a-conidia of taxon
1 were biguttulate and those of taxon 2 were not obviously biguttulate. Taxon 3 had some
characteristics of taxon 2 and others intermediate between taxon 1 and taxon 2. Mycelial
growth in vitro was slower than in taxon I but faster than in taxon 2, the cr-conidia were
larger (6.2-8.3 pm x 1.5-2 .2 pm) than in taxon 1 but smaller than in taxon 2, and the rate of
sporulation in vi¡o was the same as in taxon 2 (I3 days), as was the absence of a light 7 requirement for sporulation (Merrin et aI., 1995). Taxon 4, which lacks ct-conidia, was assumed to be a sterile form of P. vitícola.
The perfect stage of P. viticola has been described only once; Shear (1911) found perithecia of a Cryptosporella (Saccardo, 1877) species on a diseased vine. Single ascospores of this species grew into colonies that produced pycnidia identical to the pycnidia of Fusicoccum viticolum (Redd.). Shear (1911), therefore, concluded that
Cryptosporella viticola was the ascogenous form of F. viticolum, now known as P. viticola.
C. viticola could be the teleomorph of Phomopsis taxon 2 as this taxon is associated with symptoms similar to those caused by F. viticolum (Reddick, 1909; Gregory, 1913) andP. viticola (Moller et al., 1982: Emmett et a\.,1992), and the cx-conidia of the anamorph of C. viticola were, like those of taxon 2, not biguttulate. However, Shear was not able to grow the perfect stage from the imperfect, he attempted no inoculation experiments with ascospores, and the teleomorph has not been found again and has, therefore, not been confirmed.
2.3.3 Confusion in symptomatology and terminology
Phomopsis cane and leaf spot was formerly known as "dead-arm disease". Reddick (1909) listed the various names for this disease that were used by grapevine growers in the beginning of this century. The most commonly used name was "dead-arm" (Reddick, 1914), because descriptions of the disease mistakenly included dead arms or branches as a
symptom (Moller et a\.,1982). Until 1978 it was thought that the disease "dead-arm" was caused solely by P.
viticola, even though some symptoms, including dwarfed foliage, pruning wound cankers
and dead arms, were never found when healthy grapevines were inoculated with P. viticola
(Moller and Kasimatis, 1981). V/hen Moller and Kasimatis (1978) showed that Eutypa lata
(syn. Eutypa armeniacae) was pathogenic to grapevines and was also capable of inducing
dwarfed, chlorotic foliage and cankers on grapevines, it became clear that the two fungi had
been confused for many years: the symptoms typical of the disease "dead arm" had, in fact,
been caused by E latain combination with Phomopsis.
Much confusion derives from the fact that isolations from wood cankers caused by
E. lata, often yield Phomopsis sp. when both fungi occur in the vineyard. In addition, the 8 asexual state of E. lata on infected wood bears superficial resemblance to that of Phomopsis on grapevine. The pycnidia of both fungi can be found together on the dead bark of the cankers, where they exude a creamy-white mass of spores under high humidity, which dries to a yellow cirrhus (Moller & Kasimatis, 1978).
Since symptoms caused by E. Iata werc thought to be caused by P. viticola, literature on P. viticola before 1978 is not reliable. It is not certain whether research conducted on P. viticolabefore 1978, concems P. viticola, E. Iata, or a mixture of the two.
In addition, literature on P. viticola before 1995 may have confused symptoms caused by different taxa of Phomopsis on grapevines and referred to all taxa of Phomopsis found on grapevines as P. viticola.
2.4 Symptoms of Phomopsis cane and leaf spot
P. viticola can infect all green parts of grapevines @mmett et al., 1992): leaves, shoots, cluster stems (rachis), petioles and berries (Moller & Kasimatis, 1981; Moller et aI-, L982;
Hewitt & Pearson, 19SS). Symptoms are generally seen on portions of the basal three to six
internodes, although they can also be found on internodes along some shoots (Hewitt &
Pearson, 1988). Infection can lead to stunting of grapevines (Bugaret, 1990), diseased
mature berries (Lal & Arya, 1982) and reduced bunch set with yield losses of 20-387o
(Pscheidt & Pearson, 1989b).
2.4.1 Symptoms in sPring
The first symptoms of infection on the young leaves are tiny, dark brown to black spots with yellowish margins (Moller et al., 1982). The spots are rarely more than I mm in
diameter, irregular in shape, and the surrounding pale gleen or yellow haloes extend up to
2-3 mm beyond each spot. Merrin et al. (1995) described these symptoms for taxon 2 of
Phomopsis on grapevine only. I-eaf distortion occurs without tearing, although parts of
leaves may be killed when spots are numerous @mmett et a1.,1992). Even mildly infected
young leaves, particularly those with necrotic spots on the petioles, can turn yellow and
abscise (Bugaret, 1990; Emmett et aI., L992). 9
The first evidence of shoot infection is the occurence of small spots, similar to those found on the leaves. This infection usually occurs on the basal portion of the spring shoots (Moller et al., 1982; Emmett et aI., 1992), within 15 days of bud opening. The growth of these shoots is often inhibited (Bugaret, 1990). Of the four taxa of Phomopsis on grapevine found in Australia, only taxon 2 has been shown to cause these symptoms
(Merrin et a1.,1995).
Spots similar to those on the shoots and leaves appear on the flower cluster stems.
Occasionally the cluster stems are so badly infected that the flower clusters wither (Moller et a\.,1982; Emmett et a\.,1992), and the flowers may become necrotic, dry out and fall off
(Gåirtel, 1972).
2.4.2 Symptoms in surnmer
At the beginning of summer, the small black spots on the leaves expand and elongate into
5-10 mm long lesions (Moller & Kasimatis, l98l), reducing the photosynthetic area of the plant.
At the same time, the epidermal layers of the new shoots often crack at the infected parts, where the small, oblong shoot lesions have reached 3-6 mm in length. In areas where the elongated lesions are numerous, they coalesce and give a brownish-black, scabby appearance to parts of the shoots (Moller & Kasimatis, 1978; Moller et aI., 1982; Hewitt &
Pearson, 1988). These markings can become up to 5 cm long and 2 cm wide, and their cracked surfaces become roughened as canes swell and harden @mmett et a1.,1992).
Later in the summer, after shoots reach 30 to 60 cm in length, shoot breakage occurs in areas of heavy scabbing and lesion development. Severely sca:red shoots may break off at the base in strong winds (Moller at al., 1982; Bugaret, 1990; Emmett et aI., L992).
Shoots can also break just below the clusters (Moller et aI., 1982), reducing the cluster count and yield. Occasionally young grapes dry up and fall off if necrosis spreads as far as their point of attachment (Bugaret, 1990).
As the season progresses, the symptoms of Phomopsis cane and leaf spot become
obscured by vine growth and leaf cover. Normal leaves develop on subsequent nodes,
hiding the distorted basal leaves, and diseased vines may appear normal (Taylor & Mabbitt, 10
196I; Moller et a1.,1982; Hewitt & Pearson, 1988). Cracks in the epidermis and cortex of the shoots tend to heal and become rough as the tissues mature (Hewitt & Pearson, 1988).
2.4.3 Symptoms in autumn and winter
Late in the season, in cool weather conditions, fruit may become infected. Symptoms on berries are generally not extensive, with only isolated bunches affected on any one vine.
However, rain prior to harvest can be followed by the appearance of small light brown spots on clean berries, associated with early infection. The lesions enlarge quickly and become dark brown (Moller et a1.,1982;Lal & Aryu, 1982).
As autumn progresses, infected canes discolour and pycnidia develop (Bugaret,
1990; Emmett et a1.,1992). Cluster stems may become blighted and brittle from numerous infections, resulting in breakage of the cluster and loss of fruit (Hewitt & Pearson, 1988).
Infected berries become pulpy and rotted. Black fruiting bodies break through the skin of the grapes and yellow conidial masses exude (Moller et al., L982; Lal &' Arya, 1982).
Pycnidia occurring on berries are slightly larger than those which develop on the canes (Gregory, I9L3; Taylor & Mabbitt, l96L; Gåirtel, 1972). After the pycnidia mature, the berries shrivel and become mummified (Gåirtel, 1972; Moller et al., 1982; Hewitt &
Pearson, 1988).
In winter, pycnidia become prominent in the cortex of diseased l-year-old canes,
spurs, rachis stubs and old tendrils (Hewitt & Pearson, 1988). Severely infected canes
develop irregular dark brown to black patches intermingled with bleached areas. Infected
tissues, particularly around nodes, whiten as they dry out and become speckled with tiny
black pycnidia @mmett et al., 1992). Merrin et aI. (1995) found Phomopsis taxon 1 to be
associated with bleaching of l-year-old wood and flattish, dark lesions with a black border
and taxon 2 tobe associated with bleaching of l-year-old wood and heavy, obvious scars on
woody tissue.
During the late dormant season, the bark of infected canes and spurs bleaches white
in the areas between the lesions (Taylor & Mabbitt,196I; Moller et al., L982), especially on
basal portions of the canes. Severely affected canes or spurs exhibit an irregular, dark brown
to black discoloration intermixed with the whitish, bleached areas. Tissue at the nodes is
also whitish, and speckled with pycnidia. Severely affected canes and spurs are more l1 sensitive to low temperatures than healthy tissue; this can result in extensive killing of the spurs and weakening of the canes (Moller et al., 1982).
2.5 Ecology and epidemiology of P. viticola
2.5.1Disease cycle p. viticola is capable of long-term saprophytic survival (up to 4.5 years) on grapevines
(Moller & Kasimatis, 1981) and commonly occurs on dead grapevine wood (Moller &
Kasimatis, 1978). The fungus overwinters as mycelium and as pycnidia in infected tissues, particularly canes, spurs and bark @mmett et a1.,1992; Moller et aI., L982). Mycelium is also known to over-winter in dormant buds (Hewitt & Pearson, 1988; Bugaret, 1990). Infection generally occurs early in spring at the time of bud opening @ugaret,
1990). Water-borne conidia, released from pycnidia during rains, are apparently washed, splashed or spread by insects onto young vine foliage or flower-bunches @mmett et aI.,
Iggz).Infection occurs only when free moisture remains on the unprotected green tissue for many hours (section 2.5.3; Moller et a1.,1982). cr-conidia germinate in a temperature range of 1-37.C. At the optimal temperature of 23oC, infection may take place within a few hours in free water or in 98-100Vo relative humidity (Glirtel, L972; Hewitt & Pearson, 1988).
Infection usually occurs through the stomata (Gåirtel, 1972) and sometimes also through wounds (Emmett et aI., 1992). Symptoms appear 3-4 weeks after infection (Hewitt & pearson, 1988; Emmett et aI., 1992). During suÍtmer in warm, dry climates, the fungus is
relatively inactive in vine tissue, but growth resumes in autumn and the pycnidia develop
(Moller et al., 1982; Hewitt & Pearson, 1988; Emmett et al., L992). Infected canes and
spurs may continue to produce pycnidia and conidia for at least three seasons @mmett et
a1.,1992), and dead canes also may produce conidia for at least three more years (Pearson,
1eeo). t2
2.S.2Bcology of P. vifícolø
Known hosts of P. viticola are the Eurasian grapevine Vitis vinifuraL., the North American grapevine, V. rupestri.s Scheele (Hewitt & Pearson, 1988), and V. aestivales, V. lambrusca,
V. rotundiþIia and Ampelopsis quinqueþlia (IJecker, 1988). Experiments to determine whether the fungus can survive on other woody plant species, either as a saprophyte or a parasite, have not been described in the literature. Research in this area is important, because if other plants can be hosts, inoculum may originate from sources other than grapevines.
Inoculum of P. viticola consists of the water-borne g-conidia @mmett et a1.,1992).
The reservoir of inoculum depends on the parasitic and saprophytic activity of P. viticola (Moller & Kasimatis, 1978), which, in turn, depends on the weather (temperature, humidity), the climate, the season, and growth stage of the grapevine (Bugaret, 1990;
Emmett et aI., 1992). The inoculum density changes due to reproduction, death of the fungus and migration of a-conidia. Death can result from antagonism and autolysis.
Antagonism of P. viticola and autolysis have not been described in the literature, and merit investigation for their potential in management strategies.
Although the nature and function of the p-conidia are not known, they may have a role in the etiology of the disease. One possibility is that these conidia have a function similar to the microconidia of Botrytis cinerea. The sole function of the mononucleate microconidia is the spermatisation of B. cinerea sclerotia, leading to the formation of
apothecia (Hewitt & Pearson, 1988; Van der Vlugt-Bergmans et a1.,1993). Similarly, the p-
conidia of Phomopsis on grapevine might be involved in the sexual reproduction of the
fungus. In this case, their role would be the fertilisation of "female" structures in the
mycelium, leading to the formation of perithecia. However, C. viticola, has not been
described since 1911 (Shear, 1911) and, therefore, the role of the ascogenous stage is as
uncertain as the role of the p-conidia (Hewitt & Pearson, 1988). Future research in this area
might explain the role of the ascogenous stage as well as the p-conidia. l3
2.5.3 Epidemiology of Phomopsis cane and leaf spot
Plant disease results from the interaction of a host and a pathogen, influenced by environment. Important information concerning host populations is: cultivar, plant density, row width, plant height, canopy volume, geographical location, soil type, root length, and the host growth stage (Campbell & Madden, 1990).
Stages in shoot development of European grapevines were described by Eichhorn &
I-orenz (1977) and by Baggiolini (1952) (Table 2.I and Fig. 2.1). The stages are assumed to be similar in Australia. Young grapevine shoots are most susceptible to infection of P. viticola when bud opening occurs (stage D defined by Baggiolini, 1952). Young branches remain susceptible only at their herbaceous terminal ends, and rapid lengthening places them out of reach of the rain-splashed conidia. Therefore, infection generally takes place early in spring at the time of bud opening (section 2.5.L; Bugaret, 1990).
Another important factor in the development of Phomopsis cane and leaf spot disease is the physical environment; climate and weather. The macroclimate influences whether hosts can be found in a particular geographic area, and also which diseases can occur in that area. The microclimate within the plant canopy depends on variables such as temperature, humidity/ moisture, radiation and wind. Each of these quantities can have profound influences on the initiation and progress of epidemics (Campbell & Madden,
1990), including those of Phomopsis cane and leaf spot on grapevines. The environment
influences this disease through effects on various phases of the life cycle of P. viticola, as it
interacts with specific phases in the development of the gtapevine. As mentioned in section 2.5.I, rain is one of the most important environmental factors required for the disease
(Bugaret, 1986). At least 10 hours of rain (in spring) are required for conidium production
from pycnidia, and after conidium dispersal, a further 8 to 10 hours or more of very high relative humidity or surface wetness are required for infection @mmett et al., 1992).
Consequently evening rainfalls are most likely to favour infection (Bugaret, 1990). The
optimum temperature for conidium germination and fungal growth is 23oC, consequently P.
viticola appears to be inactive during hot summer weather @mmett et aI.,1992).
Epidemics of Phomopsis cane and leaf spot can occur when the growth stage of the
host plant is optimal for fungal infection (stage D; section 2.5.3), combined with the right
environmental conditions (prolonged rain). In addition, a low mean daily temperature of 5 t4
Table 2.1 Stages in shoot development of the grapevine
Eichhorn-Lorenz B
Winter dormancy: winter bud scales Winter bud: almost comPletelY 01 A more or less closed covered by two brownish scales 02 Bud swelling: buds exPand inside the bud scales 03 Brownish "wool" clearly visible B Bud swell 05 Bud burst: green shoot hrst clearly C Green shoot visible 07 First leaf unfolded and spread away D Leaves emerge: from shoot tips of leaves visible, bases still protected by "wool" 09 Two to three leaves unfolded E Leaves unfolded; first leaves spread away from shoot; internodes visible Four to six leaves unfolded; T2 Five to six leaves unfolded; F inflorescences clearly visible all inflorescences visible Inflorescences seParated and 15 Infl orescence elongating; G flowers closely pressed together spaced along shoot l7 Inflorescence fully develoPed; H Flowers seParated flowers separating 19 Beginning of flowering; first caps falling 2l Early flowering:257o of caPs fallen 23 Full flowering:50Vo of caPs fallen I Flowering 25 Late flowerin g: 807o of caPs fallen 27 Fruit set: young fruits beginning to J Fruit set swell, remains of flowers lost 29 Berries small; bunches begin to hang Berries pea-sized 31 Berries pea-sized; bunches hang K 33 Beginning of berrY touch L Berry touch ripening (Véraison); 35 Beginning of berry riPening; M Berry beginning of loss of green colour berries change colour mature 38 Berries ripe for harvest N Berries Main tendrils become woodY and 4T After harvest, O end of wood maturation brown (Aoûtement) 43 Beginning of leaf fall P Leaf fall 47 End of leaf fall Summarised from Eichhorn & Lorenz (1977) and Baggiolini (1952). l5
Figure 2.1 Stages in grapevine shoot development from dormant bud to leaf fall (Baillod &
Baggiolini,1993). See also Table 2.1. Stades repères de la vigne Dessrns de M. Baggiolini
A B c D Sortie des feuilles Bourgeon d'h¡ver Bourgeon dans le coton Pointe verte
E F G H séparés Feuilles étalées Grappes visibles Grappes séparées Boutons floraux
Y E Y
J K L Grappe fermée Floraison Nouaison Petit pois
M N o P feuilles Véraison Maturité Aoútement Chute des l6 to 7oC can increase the likelihood of an epidemic, because shoots grow slowly and thus remain short and very susceptible to P. viticola for a longer period of time (Hewitt &
Pearson, 19SS). Consequently, prolonged periods of rain and cold weather during early spring, are the prime factors in the development of an epidemic (Hewitt & Pearson, 1988). The inoculum density of the pathogen is the third important factor in the development of the disease. The rate of development, geographic extent, and duration of an epidemic are dependent on the dispersibility, availability and viability of infective pathogen propagules (a-conidia) (Campbell & Madden, 1990). Since P. viticola q,-conidia are mainly splash dispersed, the fungus spreads mostly within the vine rather than from vine to vine.
This keeps the spread of the disease within the vineyard localised, in close proximity to the inoculum source (Hewitt & Pearson, 1988). Long distance spread of P. viticola to new viticultural areas occurs primarily through the transfer of infected or contaminated propagation materials such as budwood, cane cuttings and young plants (Hewitt & Pearson,
1988; Merrin et al., 1995). The availability of P. viticola cr-conidia can increase significantly when periods of rain and cold weather continue for several days during early spring. Because of this build-up of inoculum, the disease becomes increasingly severe with each successive cool, wet spring (Hewitt & Pearson, 1988).
Information on the crop, the weather, the pathogen and their interactions can be used in conceptual and/or mathematical models, which can provide information to vineyard managers for use in making decisions about control of the disease (Kable et a1.,1990). It is, therefore, important to verify the epidemiology of the diseases caused by the major taxa of
Phomopsis on grapevine, so such models can be developed.
2.6 Control of Phomopsis cane and leaf spot
Although economic loss from Phomopsis on grapevine is minor in most years, in some years severe infection and losses can occur (Moller et a1.,1982). It is, therefore, important to know which methods can be used in order to control Phomopsis cane and leaf spot on grapevines. t7
2.6.1 Management Practices To avoid introducing phomopsis into vineyards, care should be taken to use disease-free propagating materials (budwood, cuttings, rootlings) when planting or replanting (Hewitt &
Pearson, 1988; Emmett et aL, Igg2). once the disease has appeared, diseased and dead wood should be removed during pruning, and the material ploughed into the soil, or destroyed by burning (Hewitt & Pearson, 1988; Emmett et aI., 1992). Canes or spurs damaged or infected by Phomopsis should not be used in the framework of vines @mmett et aI., 1992).
pruning methods may have a significant effect on the amount of disease present in a vineyard. For example, in top-wire cordon hedged vineyards the incidence of Phomopsis (head cane and leaf spot is significantly greater than in Umbrella Kniffin trained trained with long canes), hand-pruned vineyards, because in hedged vineyards dead canes are left behind after pruning. Dead canes continue to produce inoculum for at least 3 years, increasing the amount of inoculum from year to year (Pscheidt & Pearson, 19894; Pearson,
19e0).
Research conducted by Pearson (1990) on Phomopsis cane and leaf spot determined that hedged vines, sprayed with fungicides (section 2.6.3), develop about the same amount of disease as non-sprayed, hand-pruned, top-wire cordon trained vines. Consequently, growers who hedge their vineyards to reduce costs, may need to invest in a more intensified
spray program to control the disease.
2.6.2 Resistance of grapevines to Phomopsis cane and leaf spot
Although grapevine cultivars differ widely in relative susceptibility to Phomopsis cane and
leaf spot, no known cultivars of grapes are resistant to this disease (Hewitt & Pearson,
lggg). The only grapevine cultivar that has been found to be "almost resistant" to P. viticola
is Pinot Meunier (Bugaret, 1990). Two important factors in determining resistance or susceptibility of plants to
diseases are the mineral nutrition of the plant and the ability of the plant to produce
phytoalexins. For example, Kast (1991) found that both the severity of Phomopsis cane and
leaf spot, and the amount of o-conidia produced by the fungus, increased significantly when
nitrogen supply to grapevines in the field was increased. Moreover, V. vinifera produces a l8 number of stilbenes or "stress metabolites", which play a significant role in the resistance to fungi (Langcake, 1981; Kuc & Rush, 1985; Hoos & Blaich, 1988; Hoos & Blaich, 1990).
One of these, resveratrol, has been shown to inhibit mycelial growth of P. viticola in vitro
completely at concentrations above 50 ppm (Hoos & Blaich, 1990). More research on the
environment required to increase resistance of vines to Phomopsis may lead to control
measures which rely less on the use of fungicides.
2.6.3 The use of fungicides
Since resistant grapevine cultivars are not available, fungicides have been used for control
of Phomopsis cane and leaf spot disease. Propagation material can be disinfected with
quinolone (8-hydroxy-quinoline sulphate, sold as Chinosol W@), if permitted (Hewitt &
Pearson, 1988), to avoid introducing Phomopsis to new vineyards.
During late dormancy (2to 3 weeks before bud swell), eradicant chemical sprays,
such as sodium arsenite or dinoseb (2-sec-butyl-4,6-dinitrophenol, sold as Premerge@), may
be applied (if permitted) to kill the over-wintering pycnidia and conidia on the surface of
the vines, and lessen the risk of new shoot infection (Moller et a1.,1982; Hewitt & Pearson,
19SS). Alternatively, lime sulphur may be applied "over-the-trellis" to reduce the
production of cinhi from pycnidia (Gadoury et a1.,1994). Lime sulphur is used extensively
in Australia, as it also provides control for other fungal diseases, including early outbreaks
of powdery mildew (Bourbos & Skoudridakis, 1991), however, the effects of lime sulphur
on Phomopsis cane and leaf spot have not been assessed in Australian conditions. Two applications of a pre-infection (protectant) chemical, such as mancozeb
(dithiocarbamate, sold as Dithane@) or dithianon (quinone, sold as Delan@), are generally
recoÍrmended for Australian vineyards, the first at 50Vo budburst and again 2 weeks later (Magarey et al., 1994). When conditions favour disease development, one or more
additional applications may be necessary (Hewitt & Pearson, 1988; Emmett et aI., 1992),
while in severely infected vineyards, both dormant and spring treatments may be advisable
(Moller et a\.,1982).
Resistance of Phomopsis species to fungicides is not reported in the literature, but
there is some evidence for resistance in vineyards in eastern Australia (N.G. Nair, pers'
com.). The incidence of fungicide resistance in P. viticolø requires further study. l9
2.7 Factors responsible for genetic diversity
2.7.1 Genetic variability of populations
Isolates of many species of plant pathogenic fungi show diversity in characteristics such as morphology and pathogenicity. Fusarium orysporum, for example, is known to exist in many pathogenic forms (Bosland, 1988; Appel & Gordon, t994), and isolates of
Rhizoctonia solani show tremendous variation in both morphological and pathogenic characteristics (Otsrien, 1994). Substantial amounts of variation have been found among isolates of some Phomopsis species and their teleomorphs. For example, differences in the growth rate and pathogenicity were found among isolates of P. subordinaria @e Nooij &
Van Damme, 1988), and vegetative compatibility tests and "RAPD" (random amplified polymorphic DNA) markers revealed a considerable amount of variation among isolates of this fungus (Meijer et al., 1994). Similarly, vegetative compatibility tests revealed substantial variation rrmong isolates of Diaporthe ambigua, both within cankers and between rootstocks (Smit et aI., 1997). However, five isolates of a Phomopsis species infecting peach showed no signifîcant difference in virulence, nor multi-locus DNA fingerprints (Uddin & Stevenson, 1997).
There are two main sources of information about genetic variability in a natural population: a) studies on protein or DNA variability using electrophoresis techniques and b) the response of the population to selection pressure. A fungal population is likely to respond differently to selection imposed by a changed environment, e.g. through viticultural practices, when genetic variation is already present in the population, as compared with a genetically homogeneous population. For example, when the conditions first arise, the frequency of the required allele, e.g. fungicide resistance, may increase immediately in a heterogeneous population, while a delay in the response of the population may occur when this allele is absent or very rare (Maynard Smith, 1989)'
The four major factors that are responsible for genetic variability in phytopathogen populations; mutation, migration, genetic drift and selection (Ayala, 1983; Burdon, 1992) are discussed in sections 2.7 .2 to 2.7 .5. 20
2.7.2Mutation
A mutation may involve base substitutions in the DNA sequence, the inversion of a section of DNA, the insertion or deletion of single bases resulting in a "frame shift", or the duplication or deletion of a section of the DNA (Maynard Smith, 1989). Mutations are the ultimate source of all genetic variability (Ayala, 1983). Fungi with haploid mycelium will express non-lethal mutations immediately in the phenotype of the organism. As mutations in diploid or dikaryotic fungi are usually recessive, they can remain unexpressed (Agrios,
1938). Since the ploidy level of Phomopsis on grapevine is unknown, the direct influence of mutations on the fungus is also unknown. Anamorphs of many Ascomycetes ¿ìre haploid throughout the vegetative phase of their life cycle, but diploid and triploid isolates of
BjlÐ4Þ cinerea (anamorph of the Ascomycete Botryotinia fuckeliana) have also been found
(Van der Vlugt-Bergmans, 1993).
Mutations in extranuclear DNA, found in the mitochondria and in plasmid-like
DNAs of fungi, are also possible (Giese et al., 1990; Debets et al., 1994; Gessner-Ulrich &
Tudzynski , lgg4). Through mutations in extrachromosomal DNA, fungi may acquire the ability to tolerate previously toxic substances (e.g. fungicides), utilise new substances for
growth, and change their virulence toward host plants (Fincham g! 31., 1979; Agrios, 1988).
Cytoplasmic inheritance does not follow the Mendelian laws of genetics. Instead,
mitochondria are maternally inherited, without crossing over or recombination in their
DNA, and all copies in one individual are usually identical. Therefore mitochondrial DNA
can provide information on geographical structuring of the fungus (Maynard Smith, 1989).
However, mutations in mitochondrial DNA are more difficult to detect or characterise,
because they are transmitted by non-Mendelian mechanisms (Agrios, 1988).
Most mutations are assumed to be strongly deleterious and are, therefore, rapidly
eliminated from the population by natural selection, without increasing the genetic diversity (Aquadro, lgg2). NonJethal mutations that occur in sequences that do not involve the
phenotypic fitness of the fungus in the present conditions, are called "selectively neutral".
These mutations may contribute to the genetic diversity of a population (Kimura, 1983;
Maynard Smith, 1989; Aquadro, 1992).
Repetitive DNA sequences, which are often situated in the heterochromatin in
chromosomes, are usually not transcribed @oolittle & Sapienza, 1980; Orgel & Crick, 2l
1980). They do not seem to be essential, but can, nevertheless, comprise more than 307o of. the genome of a eukaryotic cell @oolittle & Sapienza, 1980; Maynard Smith, 1989).
Continual changes in composition, copy number and chromosomal location of repetitive
sequences have been observed for fungi (Flavell, 1986), with little effect on the phenotypic fitness @oolittle & Sapienza, 1980). However, mutations in repetitive DNA sequences can increase the overall levels of genetic diversity significantly (Flavell, 1986), because
genomic variation can accumulate @oolittle & Sapienza, 1980).
Highly repetitive DNA sequences, on the other hand, are short and are present in
very large numbers, often in tandemly arranged blocks (Maynard Smith, 1989). DNA
sequences that are repeated in "tandem" are known as "satellite" DNA (Thomas et aI.,
1993b). Other types of repetitive DNA present in eukaryotic genomes may be present on all
chromosomes, but concentrated in particular regions, for example near the centromere or at telomeric sites (Maynard Smith, 1989; Thomas et dl., 1993b). The so-called
"microsatellites", or "simple sequences", usually have an overall length of less than 100
base pairs (bp) and are made up of small repeat units that generally consist of fewer than
four nucleotides (Tautz, L9ï9;Thomas et a\.,1993b). Poly(G) and poly(A) are the simplest
of the microsatellites, while poly(GT) and poly(GA) are the most frequent, and poly(TC),
poly(CAC) and poly(GATA) have also been reported (Beckmann & Soller, 1990; Thomas
et a1.,1993a). Microsatellites occur in all eukaryotic genomes and can be expected once in
every 10 kb of DNA sequence (Tautz & Renz, 1984: Tautz, 1989). "Minisatellites" can
have an overall length of up to 20 kb, and are made up of larger repeating units (Jeffreys er
aI., 1985; Thomas et aI., 1993b). Tandemly repeated sequences, such as micro- and
minisatellites, are highly variable in size (Beckmann & Soller, 1990) due to mutations such
as transposition and unequal crossing over (Jeffreys et a\.,1985; Nakamura et a\.,1987).
Repeated DNA sequences can also include gene clusters and tandemly repeated
genes (Maynard Smith, 1989). Genetic variation in gene clusters may arise through
mutations in the introns. Similarly, variation in tandemly repeated genes, such as the
ribosomal RNA genes, may arise through accumulation of mutations in the intergenic
spacer (IGS) regions (Flavell, 1986; King & Schaal, 1989). Variation can also occur
through gene conversion and transposition. These mechanisms are the cause of molecular
drive; i.e. the fixation of mutations within populations, which causes greater within-species ,,. than between-species homogeneity in the repeats @over, 1982; Maynard Smith, 1989). In addition, variability of the copy number of tandemly repeated genes can be caused by unequal crossing over @over,1982: Maynard Smith, 1989)'
Mechanisms such as reciprocal translocation, deletion and transposition, are thought to be the cause of chromosomal length polymorphisms, commonly occurring in filamentous fungi in nature. These mutations may, therefore, be selectively neutral and occur at a high rate (Kistler & Miao, lgg2). Since the sexual stage of P. viticola is, apparently, rare (section
2.3.2), chromosome polymorphisms are likely to persist in this fungus. Transposable elements may provide a mechanism for increasing the mutation rate of fungal pathogens.
Retroviral-like transposable elements have been identified in Cladosporium fuIvum (- FulviafuIva) (McHale et a1.,1989) and also in some strains of MagnaporThe griseø. The presence of retrotransposable elements in M. grisea is correlated with the host range of the strains @obinson et aI., 1993; Skinner et a1.,1993). In Rhynchosporium secalis mutation appears to be one of the most important mechanisms of generating genetic variability, especially for pathogenicity (Goodwin et aL.,1994).
2.7.3Migration
The second process of evolutionary change is gene flow, or migration, which occurs when individuals migrate from one to another population. Migration does not change the gene frequencies for the species as a whole and has, therefore, fewer lasting consequences than the other processes of evolutionary change. Nevertheless, migration appears to be an
important source of genetic variation in populations of asexually reproducing fungi, such as
Rhynchosporium secalis (Goodwin et al., 1994). As a consequence of the migration
process, the allelic frequencies of the populations involved change, resulting in less
variation between populations, and more variation within populations (Hartl & Clark, 1989;
Ayala, 1983). Migration of Phomopsls within and between vineyards can occur via human
transport of infected or contaminated grapevine canes (section 2.5.3). This may lead to
migration of genetically diverse strains between populations. The subsequent gene flow
may be one cause of variation within populations of Phomopsis on grapevine. 23
Ascospores or cx,-conidia may allow migration of individuals between populations, but the frequency of successful establishment of new Phomopsis genotypes in other locations is unknown. Moreover, ascospores of P. viticola are evidently very rare and the splash-dispersed a-conidia are apparently rarely spread outside the vine (section 2.5.3). In contrast, splash-dispersed conidia of R. secalis appear to migrate quite easily (Goodwin er at.,1994). P. viticola seems to be a pathogen with high vertical (within-site cycle+o-cycle) and low horizontal (subpopulation to subpopulation) transmission efficiencies. The genetic divergence between local populations of such pathogens increases with increased host population subdivision, since the effects of localised selection and drift (sections 2.7.4 &'
2.7.5) go unchecked by geneflow (Burdon,1992).
Long-distance migration of Phomopsis on grapevine from other countries into
Australia has occurred at least once and more introductions may have followed. The epidemiological consequences of these introductions are unknown. In Australian populations of the wheat stem rust pathogen (Puccinia graminis vat. tritici), overseas introductions appear to have eliminated the pre-existing forms (Burdon et al., 1983).
However, possible introductions of P. viticola would not necessarily have the same impact on the population, since the latter disease is seasonal and episodic, whereas Phomopsis cane
and leaf spot is a more persistent disease (section 2.5). Therefore, the arrival of new
genotypes of Phomopsis on grapevine probably just provides variability, additional to that
occurring through mutation.
2.7.4 Random genetic drift
Because populations are finite in numbers, gene frequencies may change due to a chance
process known as random genetic drift, or simply, drift (Ayala, 1983; Maynard Smith,
1989). This chance process causes different populations of a species to drift apart
genetically, resulting in more variation between populations. The effect of random genetic
drift is dependent on the size of the population. The smaller the population, the more the
allelic frequency changes are due to genetic drift (Ayala, 1983; Maynard Smith, 1989). The
larger the population, the more similar the allelic frequency of the progeny will be to the
parental generation and the more polymorphic the population stays (Ayala, 1983; Aquadro,
1992). 24
For many pathogenic fungi, founder effects caused by migration from ancestral populations to new areas may reduce genetic variation within populations (Goodwin et al.,
1993). population s of Phytophthora infestans have been shown to contain very low levels of genetic variability in areas to which the pathogen was introduced most recently, while populations from its centre of origin in Mexico were highly variable (Tooley et al.' 1985;
Goodwin et aI.,lgg2c).R. secalis also appears to exhibit founder effects, since less genetic diversity occurs in Australia than in other continents (Goodwin et a1.,I993).In addition, the uniformity of isozyme phenotypes (section 2.9.2) in the Australian populations of wheat rust fungi may result from founder effects due to migration, establishment and asexual reproduction of a small number of pathogen genotypes (Burdon et a1.,1982; Burdon et aI.,
1983). In the same way, it is possible that populations of Phomopsis on grapevine in
Australia exhibit a founder effect, since it is very likely that only a small number of genotypes has been introduced in Australia. However, no research has been conducted into this area.
2.7.5 Natural selection
Of the four major factors that are responsible for genetic variability in populations, natural selection is the only one to promote the adaptation of organisms to their environment.
Selection induces the differential reproduction of alternative genetic variants, due to the fact that some variants increase the fitness of their carriers relative to the carriers of other
variants (Ayala, 1983).
Natural selection can occur as a result of competition for limited resources, or (Ayala, because of inclement weather and other aspects of the physical environment 1983)'
such as viticultural practices. Selection affects levels of within-population variation at both
selected and linked neutral sites (Aquadto,1992)-
Heterogeneous environments favour different genotypes in different sub-
environments, allowing the presence of genetically differentiated populations of a species
within short distances. This diversifying selection, therefore, prevents fixation and keeps
alternative alleles in the population for longer than expected under genetic drift alone
(Ayala, 1983). 25
Selection can become directional when environments change, due to parasites, competitors, fungicides, or viticultural practices, when organisms colonise a new territory with different conditions, or when (through mutation or gene flow) a new, favourable allele or genetic combination appears in the population. When this happens, different alleles will be favoured, the fitness of variant genotypes will shift, and the genetic constitution of the population will change (Ayala, 1983). Directional selection tends to sweep favoured alleles through a population to fixation, eliminating much of the variation at linked neutral sites, and causing less variation around the selected site than expected under mutation and drift alone (Aquadro, 1992).
The influence of natural selection on populations of Phomopsis on grapevine is unknown and is an area of study that has implications for the development of improved control methods. It is, for example, useful to know how likely it is that this pathogen will develop fungicide resistance or differences in pathogenicity due to selection pressure.
2.8 Genetic variation in Phomopsis on grapevine
The degree of variability in a population is also dependent on the reproductive biology of the fungus. For example, in sexually reproducing fungi, genetic variation in the progeny is introduced primarily through segregation and recombination of DNA during meiosis.
Asexually reproducing fungi often exhibit significantly less variation (Michelmore &
Hulbert, 1987; Agrios, 1938). However, asexually reproducing fungi with a rare or absent sexual phase, such as Rhynchosporium secalis (Goodwin et al., Igg}a) and Cladosporium fulvum (Curtis et aL,1994), show very high degrees of variation. New gene combinations can be generated by sexual and parasexual recombination (sections 2.8.1 &. 2.8.2), both of which are important mechanisms of variability.
Consequently, the reproductive biology of Phomopsis on grapevine has to be taken into
consideration when examining which factors are responsible for the generation and
maintenance of genetic variation within populations of each taxon of Phomopsis. 26
2.8.1 Sexual recombination
During sexual recombination in fungi, both inter- and intrachromosomal recombination occurs. It is, therefore, possible that 507o recombination occurs between markers th4t are situated on the same chromosome. For example, the nicB locus (nicotinic acid requiring) and phenB locus (phenylalanine requiring) of Aspergillus nidulans are located so far apart on chromosome VII, that they are inherited independently during meiosis (Clutterbuck, leeo).
Sexual recombination of phytopathogenic fungi has often been demonstrated in laboratories, but the importance of the sexual cycle in nature is often difficult to demonstrate (Michelmore & Hulbert, 1987).
Many "Pyrenomycetes" are homothallic and their perithecia are, therefore, usually selfed. Perithecia of crossed origin can be selected with the aid of auxotrophic markers in the laboratory @ncham et aI., 1979). Under non-selective conditions, however, the numbers of hybrid cleistothecia of the homothallic Aspergillus nidulans are low, compared to selfed cleistothecia (Scheper, 1992). Teleomorphs of Phomopsis species may be homothallic, as proposed for the teleomorphs of Phomopsis soiae (southern Diaponhe phaseolorum,Ploetz & Shokes, 1986), Phomopsis group 2 from elm (Brayford, 1990') and phomopsis ambigua (Diaporthe ambigua, Smit ¿r al., 1997), or heterothallic, as proposed for the teleomorph s of Phomopsis group I from elm (Brayford, 1990u) and P. subordinaria
(Diaporthe adunca, Linders & Van der Aa, 1995).
Sexual recombination is more likely to induce genetic variation in heterothallic than in homothallic fungi. In heterothallic fungi, all perithecia are of crossed origin, because haploid parent strains with identical alleles for the "mating type" locus are sexually incompatible. Mating can only occur between strains of opposite mating type (Michelmore
& Hulbert, 1987; Glass & Kuldau, 1992). New gene combinations within each taxon of. Phomopsis on grapevine could be
produced by sexual recombination between genetically diverse isolates, although sexual
reproduction appears to be rare. The frequency of outcrossing can have significant effects
on the rates of change in gene frequencies and, accordingly, on the rate of genetic response
to selection pressures. Therefore, it is important to determine whether the taxa of phomopsis on grapevine are homothallic or heterothallic, and how often sexual 2',1 recombination actually occurs. There are three options with respect to the sexual nature of P. viticola: i) P. viticola is heterothallic, but the probability that two thalli of opposite mating types meet, is very small and perithecia are rarely produced. The reason for this could be either that the two mating types are not present in the same geographical area, or that the splash-dispersed c- and p-conidia are not able to reach thalli of the complementary mating type; ii) P. viticola is homothallic, and can produce perithecia, but reproduction is mainly via conidia and the perithecia are rarely seen; iii) P. viticola has no sexual stage; the one reported by Shear (1911) was eroneous.
If sexual recombination in P. viticola does not occur frequently enough to generate high levels of recombination and genotypic diversity within a limited number of generations, mutation or parasexual recombination may be the major means of generating genetic variation.
2.8.2 P arasexual recombination
In many phytopathogenic fungi, the sexual cycle may be rare or non-existent in nature.
Genetic variation and recombination in such fungal populations is dependent on asexual recombination or mutation (Michelmore & Hulbert, 1987). Laboratory studies indicate that several fungi have the potential for asexual variation through parasexuality (Tinline &
MacNeill, 1969; Crawford et al., 1986; Agrios, 1988). The parasexual cycle i.e. the alternation from the haploid phase to the diploid phase and vice versa without the occu1¡ence of meiosis (Pontecorvo, 1956), involves hyphal fusion, heterokaryosis, karyogamy, recombination through mitotic crossover, and haploidisation (Tinline &
MacNeill, 1969; Michelmore & Hulbert, 1987; Agrios, 1988).
Although heterokaryosis and the parasexual cycle have been demonstrated in various fungi under laboratory conditions, their importance to the survival and evolution of plant pathogens in nature has been difficult to characterise. Forced heterokaryons are often unstable when selective pressure is removed (Michelmore & Hulbert, 1987). However, spontaneous formation of Aspergillus nidulans heterokaryons under non-selective circumstances has been found @ales et a1.,1983), and dual inoculation of barley seedlings with different isolates of Rhynchosporium secalis resulted in novel phenotypes that may be due to asexual recombination (Newman & Owen' 1985). 28
Vegetative incompatibility, or heterokaryon incompatibility, between strains can occur in the asexual stage of the fungal lifecycle. It prevents either the occurrence of
somatic anastomosis, or the formation of normal heterokaryons after anastomosis.
Heterokaryon incompatibility has been demonstrated in homothallic and heterothallic fungi'
and is described for several Ascomycete genera, such as Neurospora (Petkins, 1975; Mylyk, Ig75), teleomorphs of Aspergillus (Croft, 1985) and Podosporø @sser, 1956).
Recently, vegetative incompatibility has been described for several Phomopsis species, e.g.
p. oblonga (Brayford, 1990b) and P. subordinarlø (Meijer et al., 1994), and teleomorphs,
such as Diaporthe phaseolorum (Ploetz & Shokes, 1986; 1989) and D. ambigua (Smit er aI., IggT). Identical alleles at several loci are required for vegetative heterokaryon
formation, so that only closely related isolates are likely to anastomose (Michelmore &
Hulbert, 1987). The existence of vegetative incompatibility may break up populations into
reproductively isolated, genetically diverse sub-populations, called VCGs (vegetative
compatibility groups), in P. subordinariø (Meijer et a1.,1994) and D. ambigua (Smit et aI.,
IggT), and they are called AGs (anastomosis groups) in Rhízoctonia solani (Cubeta et aI.,
1ee3).
When heterokaryon incompatibility and sexual incompatibility occur in the same
species, they are usually independent of one another. However, mating-type associated
vegetative incompatibility occurs in Neurospora cra,ssa, where hyphal fusion and
heterokaryosis occur only between mycelia of the same mating type @eadle & Coonradt,
I944;Fincham et a1.,1979; Glass & Kuldau, 1992).
To date, it is unknown whether Phomopsis on grapevine generates variation by
asexual recombination. No information relating to anastomosis, somatic hybrids, or
incompatibility is available. If the fungus is able to form heterokaryons in nature, then mitotic recombination between different nuclei might occur, resulting in genetically different isolates. However, the frequencies of karyogamy in heterokaryons, mitotic
recombination, and non-disjunction in A. nidulans ¿ue very low (Pontecorvo & Käfer, 1958;
Käfer, 1977;Bos ef ø/., 1988). If these frequencies are similar in Phomopsis on grapevine, it
would be unlikely that much genotypic variation within populations would be generated by
parasexual recombination over a short period of time. 29
The use of genetic markers to estimate variation within and between populations of the four taxa of Phomopsis on grapevine (Merrin et a1.,1995), should provide information on the genetic structure of the populations. This may be useful in determining whether the four taxa can exchange DNA, whether the ascogenous stage of the fungus occurs in
Australia, whether phomopsis on grapevine is able to generate genetic variation asexually, and whether selection pressure due to viticultural practices can result in fungicide resistance or differences in pathogenicitY.
2.9 Markers for genetic analysis of Phomopsis on grapevine
2.9.1 Phenotypic markers In order to study the genetic s of Phomopsis on grapevine, easily scored characters or markers are required to provide information about the genotypes that are present in a population. Some naturally occurring markers are morphology of isolates in vitro, optimum temperature for mycelial growth, pycnidium colour, light requirement for sporulation, cirrhus colour, conidium size, the presence or absence of cr- and p-conidia and optimum temperature for conidium germination (section 2.3.2). Fungicide resistance, mating type and vegetative incompatibility are additional useful markers. V/hile none of the latter markers have been described for Phomopsis on grapevine, vegetative incompatibility was found to be a useful marker in P. subordinariafor identifying genetic variation (Meijer et a1.,1994).
2.9.2 Molecular markers
A range of molecular markers is available, including isozymes, RFLP and PCR markers and
molecular karyotypes. Molecular markers generated by RFLP and PCR have proved useful
in the genetic analysis of fungi and provide a large number of polymorphic, selectively neutral markers, which makes them suitable for analysis of diversity and population
genetics (Michelmore & Hulbert, 1987; Milgroom, 1995; McDonald, 1997; Stummer et al-'
2000). 30
Isozymes
Electrophoretic techniques for assaying isozymes make examination and quantification of genetic variation in natural populations possible. The characterisation of isozyme variation is one of the techniques used to examine both the genetic structure of phytopathogenic fungi, and the origin and nature of variation in populations (Burdon & Roelfs, 1985; I-eung & V/illiams, 1986; Goodwin et al., 1993). However, the technique failed to show any variation in P. subordinaria, as no polymorphisms were found with seven enzymes among
69 isolates from 23locations, making it unsuitable for identifying individual isolates of this fungus (Meijer et al.,Ig94).In most studies, only a few enzyme systems have been found to be useful in any one fungal species (Newton, 1937). Optimal conditions for extraction and activation need to be determined for each enzyme, and some isozymes are developmentally regulated and show tissue specificity (Michelmore & Hulbert, 1987). Therefore, when large numbers of markers are needed for the genetic analysis of a fungus, other molecular markers may be more apProPriate.
RFLP markers In recent years, the application of molecular biological techniques, such as DNA fingerprinting and detection of restriction fragment length polymorphisms (RFLP), has allowed advances in clarifying the taxonomic relationships of numerous species of fungi (Manicom et aI., 1987; Braithwaite et al., 1990; Levy et aI., I99I). RFLPs have gteat potential for genetic studies, as they are able to generate large numbers of genetic markers that are often multi-allelic. A DNA fingerprint is the pattern of DNA fragments produced with restriction analysis of cefain highly variable repeated DNA sequences within a
genome. They may be produced by hybridisation of oligonucleotide probes to restriction
fragments of genomic DNA (Day, 1987; Coddington & Gould, 1992), and show
polymorphisms between DNA sequences that are homologous to the probe, including
variation in non-transcribed regions, such as repeated DNA sequences (Michelmore & Hulbert, 1987). Repeated or repetitive nuclear DNA probes have been used to detect
variation within fungal phytopathogen populations that, in some cases, were nearly uniform
at isozyme loci'(Iævy et al., l99I; Goodwin et aI., L992c; Milgroom et al., 1992).
Ribosomal DNA probes (Vilgalys & Gonzalez, 1990; OBrien, 1994), as well as random 3l clones from genomic libraries (Christiansen & Giese, 1990; McDonald & Martinez, 1990;
Ueng & Chen, 1994; Evans et aI., 1997), can also be used to detect intraspecific variation between closely related isolates. A variety of DNA markers may, therefore, be able to detect genetic variation within and between populations of Phomopsis on grapevine.
Variability in mitochondrial DNA (mtDNA) RFLP patterns has been used extensively in the investigation of phylogenetic population biology of many fungi (Smith &
Anderson, 1989; Förster & Coffey, 1993; Appel & Gordon, L994; Fukuda et aI., 1994).
Fungal mtDNA is suitable for evolutionary studies because of its convenient size, relatively high copy number, the lack of methylation, ease of isolating purified mtDNA, and abundant polymorphisms (Bruns et aI., 1991). Moreover, mtDNA can be used as a cytoplasmic marker, to detect anastomosis (Michelmore & Hulbert, 1987), and as a maternally inherited marker, to track the dispersal of ascospores (Milgroom & Lipari, 1993). However, in some fungi, mtDNA RFLPs are not the best markers for population studies. In Aspergíllus nidulans, for example, no mtDNA variability in restriction fragments has been found (Croft,
1987), whereas the mtDNA in Cryphonectria parasitica is so polymorphic that, in some populations, almost every individual has a unique haplotype (Milgroom & Lipari, 1993).
PCR markers
The polymerase chain reaction (PCR) method (Saiki et aL.,1985; Mullis & Faloona, 1987) provides DNA markers that are easier to produce and analyse than are RFLP markers
(Thomas & Scott, 1993).In this case, polymorphisms are sought in the distance between two short target sequences, rather than the presence or absence of restriction endonuclease sites, as is the case for RFLPs (Skolnick & V/allace, 1988). The sequence of the amplified
DNA depends on the type of primer and on the reaction conditions (Saiki, 1989). PCR markers fall into two groups based on primer design (Thomas & Scott, 1993); those based on arbitrary primers (Welsh & McClelland, 1990; Williams et a1.,1990), and those known as sequence-tagged sites (STSs) (Olson et a\.,1989).
The first type of DNA polymorphism assay, which is based on the enzymatic amplification of random DNA segments with single primers of arbitrary nucleotide sequence, produces dominant RAPD (random amplified polymorphic DNA) markers
(Williams et aL.,1990), that are inherited in the Mendelian way. Prior sequence information 32 is not required for RAPD markers. Thus, the RAPD technique is a rapid and useful means of distinguishing closely related isolates by comparing polymorphisms in the genomic fingerprints (Welsh & McClelland, 1990). RAPD markers were used successfully by Meijer et aL (L994) to find variation in P. subordinaria and to calculate phenotypic distances.
Durand et aI. (1993) used RAPD markers to assess recombination following the parasexual cycle of Penicillium roqueþrti. ln Botrytis cinerea, RAPD markers showed genetic polymorphisms between strains and, indirectly, revealed information about the epidemiology and population genetics of the fungus (Van der Vlugt-Bergmans ¿t aI.,1993).
Also, relationships between Botrytis species can be determined by comparison of RAPD pattems (Gillings & Luck, 1993). Similarly, the RAPD technique may be a useful method of distinguishing and identifying isolates of Phomopsis on grapevine.
Sequence tagged sites (STSs) (Olson et aI., 1989), are amplified using primers designed from a known sequence. For example, ribosomal sequences, such as internal transcribed spacer regions (ITS), have been used to design primers for PCR analysis of
Cladosporium fulvum (Curtis et aI., 1994). Primers derived from the consensus sequences for the intron splice junctions (ISJ) can also be used for PCR analysis, as the junctions to exons are highly conserved sequences (Weining & Langridge, 1991), whereas introns are highly variable in sequence and length because little selection pressure occurs (section
2.7.2). The ISJ primer Rl was found to detect genetic variation within Uncinula necator on grapevines @vans et al., 1996). It is, therefore, possible that ISJ primers can detect polymorphisms between isolates of Phomopsis on grapevine.
The most informative, or polymorphic, STS markers appear to be those that amplify
DNA regions containing microsatellite repeat sequences (section 2.7.2) (Tautz, 1989;
Beckmann & Soller, 1990; Weber, 1990). Microsatellite sequences are found within unique
flanking sequences; they exhibit highly polymorphic length variation and represent
polyallelic loci when analysed as sequence-tagged sites (STSs) (Beckmann & Soller, 1990;
Thomas et al., 1993a). Microsatellite STSs have been used successfully to identify
grapevine cultivars, map the grapevine genome, determine relatedness between cultivars,
and study genetic variation (Thomas & Scott, 1993; Thomas et al., 1993\.Consequently,
this type of marker appears to be extremely useful for studies of relationship of individuals 33 within and between populations (Tautz, 1989), and may, therefore, also be of use for the genetic analysis of populations of Phomopsis on grapevine.
Molecular karyotypes
Pulsed-field gel electrophoresis technology allows the separation of large DNA molecules.
For lower eukaryotes this can be in the size range of complete chromosomes (Carle & Olson, 1984; Orbach et al., 1988). This technique, refined as contour-clamped
homogeneous electric field (CFIEF) gel electrophoresis, has been applied to the study of
fungal genomes (Chu er aI., 1986). Molecular karyotypes have been described for species
representing at least 22 generc of fungi, including nine genera of phytopathogenic fungi
(Mills & McCluskey, 1990). In some fungi, electrophoretic karyotyping has revealed the
existence of mini-chromosomes (Masel et a1.,1990; Poplawski et aI.,1992). This technique
may reveal polymorphisms between homologous chromosomes in different Phomopsis
isolates, as well as the existence of taxon or strain-specific mini-chromosomes. Differences
in karyotypes within or between populations of Phomopsis on grapevine may give more
information about the population genetics of the fungus.
2.10 Applications of molecular markers
P. viticola is placed in the form-genus Phomopsis because the morphology of the conidial
state is simila¡ to that of other Phomopsis species. Moreover, P. viticola has been
distinguished on the basis of the host plant (V. vinifera) on which it was first found and
described, and not on a genetic basis (section 2.2.L). P. viticola may, therefore, comprise
two or more species that have not been distinguished morphologically, or it may be part of a
larger species, together with Phomopsis "species" (synonyms, sectio¡ 2.2.I) that have been
found on different hosts. In order to classify Phomopsis species with confidence, and to
distinguish strains of Phomopsis on grapevine, molecular markers that detect variation are
essential. The taxonomic level at which a marker will be useful depends on how conserved
the marker is, compared with the exhibited diversity. Repetitive DNA sequences are known
to accumulate polymorphisms (section 2.7.2) and may, therefore, be good ma¡kers for
determining phylogenetic relationships within Phomopsis on grapevine, especially since 34 repetitive DNA sequence variants can become fixed in genetically isolated populations (section 2.7.4), resulting in differentiation between populations of the fungus.
Consequently, repetitive DNA sequences may provide a source of markers for discriminating between strains of Phomopsis on grapevine. Both RFLP and PCR techniques have been shown to be useful in the taxonomic identification of species and subspecies
(section 2.9.2).
Taxon-specific and species-specific molecular markers have the potential to be used for the detection of Phomopsis in infected grapevine tissue. These markers may be used to distinguish this fungus from other organisms and abiotic factors that cause similar symptoms on grapevine canes (IPM Viticulture Research to Practice, 1997).
Molecular markers may also be used to evaluate the genetic structure of populations of Phomopsis on grapevine, for use in epidemiological studies. The estimation of the extent and distribution of genetic variation within and between populations of the four taxa of
Phomopsis on grapevine in Australia may be useful in determining whether sexual reproduction and inter-population gene flow occur in the field. Population genetic models make it possible to determine the extent of gene flow and the primary mode of reproduction, by examining the distribution of genetic variation within and between populations (Tibayrenc et a1.,1991). If little variation is observed within a population and much variation between populations, then the fungus is likely to reproduce asexually; if gene flow occurs, genetic divergence among populations will be limited (section 2.7.3).
Techniques that can be used to estimate the frequencies of genotypes in populations are: isozyme variation; RFLPs; PCR analyses; and DNA sequencing (Tibayrenc et al., 1991).
RFLP and PCR markers, especially, may be useful in population studies of P. viticolc, since populations of this fungus are expected to be genetically homogeneous due to the absence or rarity of a sexual phase. If genetic variation in P. viticola and taxa of Phomopsis on grapevine in Australia is generated mainly by mutation, then genetically heterogeneous populations may consist mainly of clonally propagated strains, which are maintained by selection (section 2.7 .5).
Molecular markers could be used to detect and identify species and strains of
Phomopsis on grapevine, distinguish genotypes within the species, detect and quantify
genetic variation among field isolates, and as a tool for use in epidemiological and 35 population genetics studies of Phomopsis on grapevine. Fundamental knowledge about the epidemiology of the disease, and about the mechanisms by which the taxa of Phomopsis on grapevine can generate genetic variation, is necessary if we are to understand the etiology of phomopsis cane and leaf spot disease. This understanding may, eventually, lead to the development of improved strategies for disease management.
2.11 Summary
This review shows that, although many authors have described the symptoms and ecology of p. viticola, they may have described a mixture of several taxa of Phomopsis on grapevines rather than one species. Moreover, Koch's postulates have not been fulfilled for p. viticola nor for any of the taxa of Phomopsis found on grapevines in Australia. Little is known about Phomopsis on grapevine in Australia and, in order to improve disease management strategies, aspects of the biology, ecology, taxonomy and mechanisms of generating genetic variation of this fungus need to be understood. Increased knowledge of the epidemiology of the disease caused by the different taxa of Phomopsis on grapevine
may lead to the development of more efficient chemical, cultural and biological control
methods which could become part of an integrated disease management strategy.
The development of appropriate molecular and phenotypic markers for the taxa of
the pathogen, and possible applications of these markers for Phomopsis on grapevine, were
discussed.
2.12 Aims of the project
The aims of this project were:
i) to study the biology of the taxa of Phomopsís on grapevine in Australia, by conducting
field, glasshouse and laboratory studies; ii) to fulfil Koch's postulates for the four known taxa of Phomopsis on grapevine in
Australia; 36 iii) to develop phenotypic and molecular markers for distinguishing genetically different isolates of Phomopsls on grapevine;
iv) to investigate the genetic variation within and between populations and taxa of
phomopsis on grapevine, using molecular and phenotypic markers; and
v) to develop taxon-specific DNA-based markers to facilitate the detection of the fungus in
grapevine tissue.
2.13 Addendum
After the experimental work for this project had been completed, Phillips (1999) described
Diapor-the perjuncta Niessl. on grapevines in Portugal; a fungus with an anamorph similar
to phomopsjs taxon 1 in Australia. Using molecula¡ analysis of the nuclear ITS regions and
the mitochondrial small subunit rDNA, Mostert et al. (2001) confirmed that an Australian
taxon 1 isolate, a Portuges e D. perjuncta isolate and a South African D. periuncfa isolate
were the same species. In the same paper, Mostert et al. (2001) suggested that the
Australian Phomopsis taxon 2 was, in fact, P. viticola, that Phomopsis taxon 1 was D.
perjuncta, that taxon 3 was a separate species of Phomopsis and taxon 4 was a species of
Libertella. 37
3. GENERAL MATERIALS AND METHODS
3.1 Isolation of Phomopsis of grapevine
Procedures for isolation of Phomopsis of grapevines followed the guidelines in the Plant
Quarantine Standard of South Australia, January 1993, provided by the Department of
Primary Industries of South Australia.
Bleached or scarred canes were collected from dormant vines or from the ground of vineyards in Phylloxera-free regions in South Australia and Tasmania. The canes were cut into 10 to 13 cm sections and placed on glass rods, laid over moist paper towel in plastic containers (15 x 15 cm) and incubated at 15eC in the dark for 1 to 30 weeks. Cirrhi were removed with a sterile needle and suspended in 50 pl sterile reverse osmosis (RO) water containing 0.0057o Tween 20. For every cirrhus, the morphology of the conidia was checked by placing 10 pl of the suspension on a slide and examining at 400 x magnification. The remaining 4O pl were plated onto Difco potato dextrose agar (PDA) in a
9 cm Petri dish (ca 25 ml per dish) and incubated at 23eC. After 3 to 5 days a single colony was subcultured to a fresh PDA plate and to l.2To (wlv) water agar (Oxoid@ agar in RO water, WA). Both plates were incubated in a cycle of 12 h at 23eC in light (Philips TLD
18W33 3F, 380-780 nm and near ultraviolet, 380-400 nm) and 12 h at 14eC in darkness.
obtained from areas of Victoria. Pieces of Isolates were also ¡hylloxera-free bleached grapevine bark (5 mm2¡ were surface sterilised by submerging them sequentially in 70Vo ethanol for 30 s, sterile RO water, bleach (IVo available chlorine) for 2 min, sterile
RO water, 707o ethanol for 30 s and four rinses in sterile RO water, before placing them on WA in Petri plates. The plates were sealed with strips of Glad@wrap before they were transported to the laboratory where they were incubated at 23eC in darkness for 1 to 2 weeks. Cinhi were treated as described above. If no cirrhi appeared, small sections of white mycelial growth on the agar were transferred to fresh PDA and V/A plates and incubated in an alternating light / dark regime as described previously, until pycnidia and cirrhi formed.
For every cirrhus the morphology of the conidia was checked using a microscope.
Single spore-derived cultures were prepared by streaking a suspension of a-conidia
(104-10s ml-r) across a line marked on a thin (2 mm) layer of Phytagel (5 g l-t RO water + 38
Table 3.1 Origin of Phomopsis isolates used in this study.
Isolate Taxon Host Cultivar and Location Collection Date
1 A-C I Chardonnay Sutherland, Adelaide Hills, SA July 1994 1994 3 A-D 1 Chardonnay Sutherland, Adelaide Hills, SA July 1994 4 A-E 1 Chardonnay Sutherland, Adelaide Hills, SA July 5 1 Sauvignon Blanc Morialta, Adelaide Hills, SA July 1994 7 A-D I Chardonnay Pfitzner, Adelaide Hills, SA July 1994 1994 8 A-D 1 Chardonnay Pfitzner, Adelaide Hills, SA July 9 A-D I Chardonnay Pfitzner, Adelaide Hills, SA July 1994 10 1 Chardonnay Pfittzner, Adelaide Hills, SA July 1994 1994 1I A-C 1 Chardonnay Barratt, Adelaide Hills, SA July 12 A-T I Chardonnay Barratt, Adelaide Hills, SA July 1994 14 A-G I Chardonnay Ashton Hills, Adelaide Hills, SA July 1994 15 A-B 1 Chardonnay Ashton Hills, Adelaide Hills, SA July 1994 16 1 Chardonnay Ashton Hills, Adelaide Hills, SA July 1994 17 A-B I Chardonnay Ashton Hills, Adelaide Hills, SA July 1994 18 A-B I Chardonnay Ashton Hills, Adelaide Hills, SA July 1994 19 1 Chardonnay Ashton Hills, Adelaide Hills, SA July 1994 20 A-E I Chardonnay Hillstowe, Adelaide Hills, SA July 1994 2I A-C I Chardonnay Hillstowe, Adelaide Hills, SA July 1994 1994 22 B-E 1 Chardonnay Hillstowe, Adelaide Hills, SA July 23 B-E I Chardonnay Hillstowe, Adelaide Hills, SA July 1994 24 A-T 1 Chardonnay Hillstowe, Adelaide Hills, SA July 1994 25 I Pinot Noir Pfitzner, Adelaide Hills, SA July 1994 27 I Pinot Noir Pfitzner, Adelaide Hills, SA July 1994 31 A-D I Chardonnay Morialta, Adelaide Hills, SA July 1994 33 A-C I Chardonnay Hargrave, Adelaide Hills, SA July 1994 34 A-B I Chardonnay Hargrave, Adelaide Hills, SA July 1994 35 A-B 1 Chardonnay Hargrave, Adelaide Hills, SA July 1994 36 1 Chardonnay Hargrave, Adelaide Hills, SA July 1994 37 A-B I Chardonnay Hargrave, Adelaide Hills, SA July 1994 38 A-B I Chardonnay Hargrave, Adelaide Hills, SA July 1994 39 A-B I Chardonnay Hargrave., Adelaide Hills, SA July 1994 4l I Chardonnay Hargrave, Adelaide Hills, SA July 1994 42 A-B I Chardonnay Hargrave, Adelaide Hills, SA July 1994 43 B-C I Chardonnay Hargrave, Adelaide Hills, SA July 1994 44 A-B 1 Chardonnay Hargrave, Adelaide Hills, SA July 1994 45 A-B I Chardonnay Hargrave, Adelaide Hills, SA July 1994 46 2 Shiraz Penfolds, Padthaway, SA Mar. 1994 47 2 Shiraz Penfolds, Padthaway, SA Mar. 1994 48 1 Chardonnay Barratt, Adelaide Hills, SA Nov. 1994 50 A-E T Chardonnay Gartness Block, Coonawarra, SA Aug. 1994 51 A-C T Chardonnay Gartness Block, Coonawarra, SA Aug. 1994 52 A-D T Chardonnay Gartness Block, Coonawarra, SA Aug. 1994 53 A-C T Shiraz Mildara, Coonawarra, SA Aug. 1994 54 A-D T Shiraz Mildara, Coonawarra, SA Aug. 1994 55 T Shiraz Mildara, Coonawarra, SA Aug. 1994 56 T Shiraz Coona SA . 1994 Table 3.1 continued on the next page 39
Table 3.1 (continued)
Isolate Taxon Host Cultivar Location Collection Date
57 A-D T Riesling Ashton Hills, Adelaide Hills, SA June 1995 58 A-C T Chardonnay Ashton Hills, Adelaide Hills, SA June 1995 60 1 Riesling Ashton Hills, Adelaide Hills, SA June 1995 6l A-D 1 Riesling Ashton Hills, Adelaide Hills, SA June 1995 62 I Riesling Ashton Hills, Adelaide Hills, SA June 1995 63 A-B I Riesling Ashton Hills, Adelaide Hills, SA June 1995 64 A-B I Riesling Ashton Hills, Adelaide Hills, SA June 1995 65 A-C I Riesling Ashton Hills, Adelaide Hills, SA June 1995 66 1 Riesling Ashton Hills, Adelaide Hills, SA June 1995 67 I Chardonnay Ashton Hills, Adelaide Hills, SA June 1995 68 1 Chardonnay Ashton Hills, Adelaide Hills, SA June 1995 69 I Chardonnay Ashton Hills, Adelaide Hills, SA June 1995 70 I Chardonnay Ashton Hills, Adelaide Hills, SA June 1995 7t 1 Chardonnay Ashton Hills, Adelaide Hills, SA June 1995 72 I Chardonnay Hargrave, Adelaide Hills, SA June 1995 73 I Chardonnay Hargrave, Adelaide Hills, SA June 1995 74 I Chardonnay Hargrave, Adelaide Hills, SA June 1995 75 I Chardonnay Hargrave, Adelaide Hills, SA June 1995 76 2 Shiraz Southcorp, Padthaway, SA July 1995 77 2 Shiraz Southcorp, Padthaway, SA July 1995 80 T Chardonnay Gartness Block, Coonawarra, SA July 1995 81 T Chardonnay Gartness Block, Coonawarra, SA July 1995 82 T Chardonnay Gartness Block, Coonawarra, SA July 1995 83 T Riesling Ashton Hills, Adelaide Hills, SA June 1995 84 T Riesling Ashton Hills, Adelaide Hills, SA June 1995 85 T Shiraz Mildara, Coonawarra, SA July 1995 86 T Shiraz Mildara, Coonawarra, SA July 1995 "97 T Chardonnay Hargrave, Adelaide Hills, SA June 1995 u88 T Chardonnay Hargrave, Adelaide Hills, SA June 1995 89 T Riesling Ashton Hills, Adelaide Hills, SA Nov. 1995 90 I Riesling Ashton Hills, Adelaide Hills, SA Nov. 1995 9T 2 Chardonnay Clover Hill Vineyard, Tas July 1996 92 2 Chardonnay Clover Hill Vineyard, Tas July 1996 93 2 Chardonnay Clover Hill Vineyard, Tas July 1996 94 1 Chardonnay Clover Hill Vineyard, Tas July 1996 95 I Chardonnay Clover Hill Vineyard, Tas July 1996 96 1 Pinot Noir Freycinet, Hill Vineyard, Tas July 1996 97 1 Riesling Freycinet, Hill Vineyard, Tas July 1996 99 A-B I Chardonnay Freycinet, Hill Vineyard, Tas July 1996 100 I Chardonnay Freycinet, Hill Vineyard, Tas July 1996 101 1 Chardonnay Freycinet, Hill Vineyard, Tas July 1996 r02 A-C 1 Chardonnay Spring Vale Vineyard, Tas July 1996 103 I Cabernet Bream Creek, Tas July 1996 104 A-B 1 Cabernet Bream Creek, Tas July 1996 105 I Cabernet Bream Creek, Tas July 1996 106 1 Cabernet Bream Tas Jul 1996 Table 3.1 continued on the next page 40
Table 3.1 (continued)
Isolate Taxon Host Cultivar Location Collection Date
r07 1 Cabernet Bream Creek, Tas July 1996 108 A-B 1 Cabernet Bream Creek, Tas July 1996 109 A-B I Pinot Noir Marion's Vineyard, Tas July 1996 110 2 Chardonnay Marion's Vineyard, Tas July 1996 111 1 Chardonnay Marion's Vineyard, Tas July 1996 IT2 1 Chardonnay Marion's Vineyard, Tas July 1996 113 I Chardonnay Marion's Vineyard, Tas July 1996 o1 14 A-D 1 Grenache Two V/ells, Adelaide Plains, SA Aug. 1996 br r5 A-B 1 Cabernet Fern Hill, Vic Mar. 1996 116 I Cabernet Fem Hill, Vic Mar. 1996 II7 T Riesling Lillydale, Vic Aug. 1995 118 T Riesling Lillydale, Vic Aug. 1995 119 I Riesling Lillydale, Vic Mar. 1996 r20 1 Riesling Lillydale, Vic Mar. 1996 L2T 1 Riesling Lillydale, Vic Mar. 1996 r22 1 Riesling Lillydale, Vic Mar. 1996 r25 A-B I Chardonnay Hargrave, Adelaide Hills, SA Aug. 1996 126 1 Chardonnay Hargrave, Adelaide Hills, SA Aug. 1996 r27 1 Chardonnay Hargrave, Adelaide Hills, SA Aug. 1996 t28 1 Riesling Ashton Hills, Adelaide Hills, SA Aug. 1996 r29 1 Riesling Ashton Hills, Adelaide Hills, SA Aug. 1996 131 T Chardonnay Freycinet, Hill Vineyard, Tas July 1996 r32 T Cabernet Bream Creek, Tas July 1996 t33 T Cabernet Bream Creek, Tas July 1996 t34 T Riesling Freycinet, Hill Vineyard, Tas July 1996 135 T Chardonnay Clover Hill, Tas July 1996 136 T Pinot Noir Spring Vale Vineya¡d, Tas July 1996 t37 T Pinot Noir Marion's Vineyard, Tas July 1996 138 T Pinot Noir Marion's Vineyard, Tas July 1996 r39 A-D T Chardonnay Marion's Vineyard, Tas July 1996 140 1 Chardonnay Hillstowe, Adelaide Hills, SA Aug. 1996 r4t A-B I Chardonnay Hillstowe, Adelaide Hills, SA Aug. 1996 142 1 Chardonnay Barratt, Adelaide Hills, SA Aug. 1996 r43 I Chardonnay Hillstowe, Adelaide Hills, SA Aug. 1996 144 I Chardonnay Hargrave, Adelaide Hills, SA Aug. 1996 r45 A-D I Chardonnay Hargrave, Adelaide Hills, SA Aug. 1996 r46 A-C T Riesling Ashton Hills, Adelaide Hills, SA Aug. 1996 150 A-B 1 Riesling Ashton Hills, Adelaide Hills, SA Nov. 1996 151 A-F 1 Riesling Ashton Hills, Adelaide Hills, SA Nov. 1996 152 A-B I Chardonnay Hargrave, Adelaide Hills, SA Nov. 1996 153 A-B I Chardonnay Hargrave, Adelaide Hills, SA Nov. 1996 DAR 6946r 1 Cabernet Drumbourg, Vic July 1992 DAR 69467 1 Chardonnay Yarra Valley, Vic July 1992 DAR 69488 I Chardonnay Adelaide Hills, SA t99t DAR 69489 1 Chardonnay Adelaide Hills, SA l99t DAR 57591 2 Shiraz Muswellbrook, NS'W Dec. 1986 Table 3.1 continued on the next page 4l
Table 3.L (continued)
Isolate Taxon Host Cultivar Location Collection Date
DAR 69457 2 Nyora Griffith, NSw Sep. 1991 DAR 69460 2 Waltham Cross Swan Hill, Vic July 1992 DAR 69477 2 Shiraz Rutherglen, Vic Iuly 1992 DAR 69476 2 Shiraz Barossa Valley, SA Nov. 1992 DAR 69486 2 Sultana Mildura, Vic July 1992 DAR 69458 3 Chardonnay Yarra Valley, Vic Oct. 1991 JM 139 J nla Nuriootpa, SA nla DAR 69484 4 nla Hunter Valley, NSV/ Feb.1992 VRU OO31 2 Chardonnay Griffith, NSIW nla VRU OO35 2 Sultana Mildura, Vic nla VRU 0036 2 Cab. Sauvignon Crawford River, Condah, Vic nla VRU OO43 2 nla nla nla VRU OO48 2 Sultana Mum¡mbateman, NSW nla VRU OO5O 2 Sultana þmple, Vic nla VRU OO74 2 nla Cape Jaffa, SA nla VRU OO84 2 Shiraz Padthaway, SA nla u Single ascospore isolates were made of these isolates, with a micro-manipulator b The o-conidia of these isolates we¡e 7-8.5 pm long. T Teleomorph, these isolates were collected from perithecia of Diaporthe viticola (Chapter 4) A-G When isolates were derived from the anamorph, up to seven cirrhi (A-G) were taken from different areas of one grapevine cane. When isolates were derived from the teleomorph, uP to seven cinhi (A-G) were taken from agar that was inoculated with ascospores from one perithecium. SA = South Australia, Vic = Victoria, Tas = Tasmania, NSW = New South Wales. n/a = not available DAR isolates and the JM isolate were gratefully received from Mr M. Priest (Curator, DAR, NSW Agriculture). The VRU isolates \ryere gratefully received from Dr N.G. Nair (NSW Agriculture)' 42
Table 3.2 Single conidium- and single ascospore-derived cultures of Phomopsis of grapevrne.
Isolate Taxon Culture derived from:
50 D.l T Conidium 51 C.1 T Conidium 54 8.1 T Conidium 54 D.l T Conidium 87 .1 T Ascospore 88.1 T Ascospore 9l .1 2 Conidium 95 .1 I Conidium 99 4.1 I Conidium 100 .1 1 Conidium 101 .1 I Conidium 103 .1 1 Conidium 104 4.1 I Conidium 110 .1 2 Conidium DAR 69467.1 I Conidium DAR 69489.1 1 Conidium DAR 69457.1 2 Conidium DAR 69486.7 2 Conidium DAR 69458.1 3 Conidium T Teleomorph, these isolates were collected from perithecia of Diaporthe viticola (Chapter 4) DAR isolates were gratefully received from Mr M. Priest (Curator, DAR, NSW Agriculture). 43
200 mg CaClzl-r¡ in each 9 cm Petri dishes. The plates were incubated at 23"C fot 16 h and then scanned along the line using a dissecting microscope (x 220) in a laminar-flow cabinet.
Isolated germinating a-conidia were selected and a 2 mm square of agar was cut around each conidium with a sterile scalpel. The square was transferred to a PDA plate with a
sterile needle (Johnson & Booth, 1983).
Hyphal tip-derived cultures were prepared by streaking a suspension of cr,-conidia
incubated at23"C for 3 days. 1104-t05 ml-r¡ with a wire loop on WA plates. The plates were Using a dissecting microscope (x 220) in a laminar-flow cabinet, hyphal tips (cø 25 ¡tm
long) from single colonies were transferred to PDA plates with a sterile scalpel.
Isolates derived from the asexual stage of taxa of Phomopsis of grapevine were
numbered using the code #X.* where # = a number representing a grapevine cane, X = a letter representing a pycnidium on the cane and * = a number representing a single
conidium-derived isolate. Isolates derived from perithecia were numbered using the code pycnidium #X. * where # = a number representing a perithecium, X = a letter representing a grown on agar inoculated with ascospores derived from the perithecium and * = a number
representing a single conidium-derived isolate, or using the code #.* where # = a number
representing a perithecium and * = â nurrber representing a single ascospore-derived isolate. Single conidium- and hyphal tip-derived cultures as well as bulk isolates, which
were derived from single cirrhi, (Tables 3.1 and 3.2) were maintained on PDA and WA
plates in an alternating light / dark regime as described above.
3.2 DNA extraction
3.2.1 Extraction of DNA from Phomopsís of grapevine
Sterile Difco potato dextrose broth (PDB) (100 ml) with ampicillin (50 pg ml-r,Boehringer
Mannheim), penicillin (100 units ml-r, Boehringer Mannheim) and streptomycin (100 pg
ml-I, Boehringer Mannheim) in a 250 ml Erlenmeyer flask, was inoculated with two cirrhi
or 10 to 15 small pieces of aerial mycelium of a given Phomopsis isolate, and incubated for
5 to 8 days at 23"C in darkness on an orbital shaker, set at 25 rpm. The mycelium was
harvested by vacuum on sterile miracloth (Calbiochetnt¡ in a Buchner funnel and washed 44 three times with sterile nanopure water. The dried mycelium (0.5 g) was folded in aluminium foil, frozen in liquid nitrogen and stored at -80oC'
Total genomic DNA was extracted using a method modified from Raeder & Broda
(1935). Frozen mycelium (0.5 g) was ground to a fine powder in liquid nitrogen. The ground mycelium was brushed into a 16 ml Oak Ridge centrifuge tube, suspended in 4 ml extraction buffer (200 mM Tris-HCl pH 8.5, 500 mM NaCl, 25 mM EDTA, 0.5 % SDS) (1:1) and vortexed. The slurry was extracted with an equal volume of phenol/ chloroform and the phases were separated by centrifugation at 13,000 g for t hour. The upper aqueous (20 phase was transferred to a clean Oak Ridge tube containing 140 ¡rl of RNase A solution mg ml-l), incubated at 37"C for 7 min, extracted with an equal volume of chloroform and centrifuged for 10 min at 13,000 g. The upper phase was transferred to another Oak Ridge tube and 5 M NaCl was added to make the volume 4 ml (final concentration of 0.5 to 1 M
NaCl). DNA was precipitated with two volumes of cold absolute ethanol and centrifuged for 10 min at 13,000 g. The pellet was washed twice in cold 70 7o ethanol, vacuum dried and dissolved in 50-100 ¡rl TE buffer (10 mM Tris-HCl, lmM EDTA, pH 8)' The DNA concentration was estimated by running 2 pl on a I Vo agarose gel (50 V, in TAE buffer, appendix) and comparing the intensity of the band with a Lambda DNA standard (appendix) after ethidium bromide staining. DNA yields ranged from 10 to 16 pg g-t mycelium. The DNA was stored at -20"C.
3.2.2Bxtraction of DNA from micropropagated grapevine leaves
Grapevine DNA was extracted from micropropagated grapevine cv Cabernet Sauvignon
tissue (received from Dr B.E. Stummer) using a method modified from Doyle & Doyle
(1983). Fresh leaf tissue (1 g) was ground to a powder in liquid nitrogen, suspended in 7.5
ml of CTAB buffer (27o CTAB, 20 mM EDTA, 100 mM Tris-HCl, pH 8, 1'4 M NaCl, 0'2 30 min. Genomic DNA was Vo þ-mercaptoethanol) at 60oC and incubated at 60oC for
extracted by adding an equal amount of chloroform/ isoamyl alcohol (24:I) to the slurry and
placing it on a rotating disc for 20 min. The aqueous and organic phases were separated by
centrifugation at 1,600 g for 5 min. The nucleic acids were precipitated from the aqueous
phase by adding 0.67 volumes of cold isopropanol. After 10 min to 2 hours on ice, the
precipitate was hooked out of solution with a sterile glass rod, transferred to a new tube and 45 washed with cold 76 7o ethanol containing 10 mM ammonium acetate overnight at 4"C.
The ethanol was poured off and the pellet was vacuum-dried for 2 min and dissolved in 400
pl TE buffer. RNA was removed either during restriction erzyme digestion or as a separate
step by adding RNase A to a final concentration of 10 pg ml-r and incubating at37"C for 30 min. The DNA was re-precipitated by adding ammonium aceta;te (pH 7.7) to a final
concentration of 2.SMrin 3 ml followed by 2.5 volumes of cold absolute ethanol. The DNA
was hooked out of the solution, transferred to a new tube, air dried, dissolved in 300 pl TE
buffer and stored at -2}"C.DNA yields ranged from 20 to 24 ¡tgg-r leaf tissue.
3.3 Construction of a DNA librarY
3.3.1 Ligationof Phomopsís DNA into pUC19 vector phomopsis DNA was ligated into the plasmid pUC19 using methods modified from
Sambrook et aI. (1989). Approximately 2 pg pUC19 DNA was digested with the restriction
enzymes SaI[ or Psfl, according to the manufacturer's instructions @oehringer Mannheim).
The enzyme was removed by phenol/chloroform (1:1) and chloroform/isoamyl alcohol ea:D extraction, after which the DNA was precipitated with two volumes of cold absolute ethanol. The digested vector DNA was dephosphorylated with 0.5 units of calf intestinal
alkaline phosphatase (CIP) (Promega) in 50 pl CIP reaction buffer (5 mM Tris-HCl, pH 9,
0.1 mM MgCl2, 0.01 mM ZnClz,0.l mM spermidine) for 30 min at 37"C. The volume was
increased to 200 pl with TE buffer, extracted twice with phenol/chloroform and once with
chloroform/isoamyl alcohol (24:l). The DNA was precipitated with two volumes of cold
absolute ethanol for 30 min at -20"C, washed withTOTo ethanol, dried and dissolved in 10
pl TE buffer. The DNA concentration was estimated by running 1 pl on a I7o agarose gel as
described in section 3.2.L. Approximately 2 pg of total Phomopsis DNA was digested with the restriction
enzymes SøII or PsrI in a volume of 40 pl. The enzyme was removed by phenoVchloroform
(1:1) and chloroform/isoamyl alcohol (24:I) extraction followed by precipitation of the
DNA with two volumes of cold absolute ethanol. Following centrifugation and drying, the 46
DNA was dissolved in 15 pl TE buffer and the concentration was estimated as described above.
Insert (Phomopsis) to vector (pUC19) DNA molar ratios, measured in available picomole ends (Sambrook et aI., 1989), were calculated prior to ligation. Six ligation reactions were performed: in four reactions the insert:vector ratio was 1:4, I:2, l:1 or 2:1, in
one control reaction no insert was added and in the second control reaction no insert or
DNA ligase was added. The ligation reactions were performed in 30 pl containing 50 ng
linearised dephosphorylated pUCl9 DNA, 50 to 350 ng digested total Phomopsis DNA, 5
units of T4 DNA ligase (Promega), ligation buffer and 1 mM ATP. The reaction took place in a 20oC waterbath placed in a refrigerator overnight, with the temperature slowly
dropping to 4"C.
3.3.2 Transformation of Escherìchia coli
Competent cells of E. coli DH5a were prepared by growing a single bacterial colony
overnight in a 10 ml standing culture of Luria-Bertani (LB) broth at 37oC. A 1 ml aliquot
this culture was added to 100 ml LB broth and incubated at37"C in an orbital shaker at200
rpm until the ODoooom was 0.4 - 0.5. The cells were pelleted by centrifugation at 5,000 g for
5 min at4"C,resuspended in 40 ml cold 0.1 M MgCl2, centrifuged again, resuspended in 40
ml cold 0.1 M CaClz and placed on ice for 15 min. The cells were centrifuged once more
and resuspended in 6 ml cold 0.1 M CaClz containing l57o glycerol. The competent cells
were dispensed into 30 Eppendorf tubes, frozen in a dry ice-ethanol bath and stored at - g0'c.
The competent cells (100 pl) were transformed with 30 pl ligated DNA (100 to 400
ng DNA, section 3.3.1) in a sterile 15 ml glass Core*@ tube on ice. Three control
transformations were caried out in the same way: the first contained linearised pUC19 and
no phomopsls DNA; the second contained linearised pUC19 DNA, no Phomopsis DNA
and no T4 DNA ligase; the third contained circular pUC19 DNA only. After 30 min on ice,
the cells were heat shocked at 42"C for 2 min and placed back on ice for I min. A volume
of 4 ml LB broth (37"C) and 40 ¡tl of 2 M MgCl2 were added and the cells were incubated
for 45 min at 37"C. After centrifugation for 2 min at 5,000 g, the cells were resuspended in
600 pl LB broth and six 100 pl aliquots were spread out on LB agar containing 75 pg ml-t 47 ampicillin, 1.25 mM IPTG (isopropylthio-p-D-galactoside) and 50 pg ml-r X-gal (5-bromo-
4-chloro-3 -indolyl- p-D-galactoside) and incubated overni ght at 37 " C.
3.3.3 Screening for recombinant E. colí colonies
White colonies containing recombinant DNA were transferred in duplicate, and in a grid pattern, to LB agar containing 75 pg d-l ampicillin (LBamp agar) and to positively charged nylon membranes (Boehringer Mannheim) placed on LBamp agar (Grunstein & Hogness,
1975; Sambrook et a1.,1939). A blue, non-recombinant colony was included on each agar plate. The plates were incubated overnight at 37"C. The bacteria on the nylon membranes were lysed by placing the membranes colony side up for 20 min on Whatman 3MM paper saturated with 10mM glucose, 25 mM Tris-HCl, pH 8, 10 mM EDTA and 4 mg ml-r lysozyme. The released DNA was denatured by placing the membranes on V/hatman 3MM paper saturated with 1.5 M NaCl, 0.5 M NaOH and l%o SDS for 20 min, and neutralised by placing the membranes on three changes of IVhatman 3MM paper saturated with 1 M Tris- HCl, pH 7.5, 10 min each change. After partially air drying, the membranes were submerged in 2 x SSC and the dead bacterial cells were wiped off with non-absorbent cotton wool. DNA was cross-linked to the darnp membranes using the Stratalinker@ UV crosslinker (Bio Rad) set at 150 mJ. The membranes were stored dry at room temperature.
Clones containing Phomopsis DNA were identifîed by hybridising radio-labelled total Phomopsis DNA to the DNA on the nylon membranes. Radiolabelled probes were prepared as described in section 3.4.4. After the membranes were hybridised and washed
(section 3.4.4),they were exposed to X-ray film (Kodak X-Omat) at -80oC, inside a cassette containing intensifier screens. The strength of the hybridisation signal was recorded for each clone.
3.3.4 Storage of DNA librarY
Single E. coli colonies containing Phomopsis DNA inserts were grown overnight at 37oC in
5 ml LB broth containing 100 pg ml-r ampicillin (LBamp broth). A 1 ml sample was added to 5 ml fresh LBamp broth and incubated for 2.5 hours in an orbital shaker set at 200 rpm
and 37oC. A 350 ¡rl volume of the bacterial suspension was transferred to an Eppendorf 48 tube conraining 150 pl sterile glycerol/LB broth (1:1) and 1 pl ampicillin (50 mg ml-r¡. The bacterial stock cultures were stored at -80oC.
3.4 General molecular methods
3.4.1 Plasmid DNA preparation
In order to screen a large number of cloned inserts as potential Southern hybridisation probes, a crude plasmid preparation, as described by He et aI. (1990), was used. Single bacterial colonies containing putative clones were grown overnight in 5 ml LBamp broth at
37"C. A I ml aliquot was added to 5 ml fresh LBamp broth and incubated for 2.5 hours in an orbital shaker (200 rpm) at37"C. The cells were pelleted (2 min at 12,000 g), washed in sterile nanopure water and resuspended in 600 pl LiCl solution (2.5 M LiCl, 50 mM Tris-
HCI pH8, 4Vo (wlv) Triton X-100, 62.5 mM Na2EDTA, filter sterilised). After cell lysis, the
DNA was extracted in an equal volume of phenoUchloroform (1:1) and the phases were separated by centrifugation at 15,000 g for 10 min. An equal volume of chlorofoÍn was added to the aqueous phase, mixed well and centrifuged. DNA was precipitated from the aqueous phase with 2 volumes of cold absolute ethanol and 0.1 volume of 5 M ammonium acetate for 30 min at -20"C and pelleted for 15 min at 15,000 g. The pellet was washed twice in cold 70Vo ethanol, vacuum dried and dissolved in 20 pl TE buffer.
Some recombinant plasmids replicated slowly, and very little plasmid was recovered using the method described above. These plasmids were grown as follows. Single colonies containing these plasmids were grown overnight in 5 ml LBamp broth at 37"C to late log phase. One ml of this culture was added to 20 ml Tenific Broth (1.27o tryptone, 2.47o yeast extract,0.47o glycerol (v/v), 17 mMKHzPO¿,72rnNI çIIPO+) (Tartof &Hobbs, 1987;
Sambrook et al.,l9S9) containing 100 pg ml-r ampicillin in 125 ml flasks and incubated for
2.5 hours in an orbital shaker set at 200 rpm and 37oC, until the ODoooo- reached 0.4. At this time, 100 pl chloramphenicol (34 mg ml-r) was added (final concentration 170 pg ml-t) and the flasks were incubated overnight at 37oC in an orbital shaker set at 200 rpm, to enhance replication of plasmids while inhibiting replication of the bacterial chromosome
(Clewell, L972). The cells were pelleted (5,000 g for 5 min at 4"C), washed in 4 ml ice cold 49
STE (0.1 M NaCl, 10 mM Tris-HCl pH 8, I mM EDTA), resuspendedin2 ml LiCl solution
(see above) and the plasmids were extracted as described above.
pure plasmid DNA was isolated with the Promega Wizard@ PIus SV Minipreps
DNA Purification System kit. Cloned inserts from these plasmids were used as Southern
hybridisation probes (section 3.4.4).
3.A.2Ðxtraction of plasmid DNA inserts from agarose gels
A piece of agarose gel containing the relevant DNA fragment was excised from the gel and
the DNA was extracted by "fÍeeze squeezing" or "gene cleaning" (Thuring et al., 1975;
Vogelstein & Gillespie, 1979). For the "freeze squeeze" method, the piece of agarose
containing the DNA band was put in a plastic bag (3 x 4 cm) and the bag was sealed by
heating. The agarose was froze¡ for I h and then squeezed between two fingers in the top
comer of the bag, so the liquid ran to the opposite corner. This was repeated until all the
liquid was in the bottom corner. The top corner of the bag (with the gel) was subsequently
cut off and the liquid transferred to an Eppendorf tube. Sterile nanopure water was added
until the volume was 200 pl and 200 pl phenol/ chloroform was added and mixed well.
After centrifugation at 15,000 g for 5 min the aqueous phase was transferred to a new
Eppendorf tube and 0.1 volume of 5 M ammonium acetate and 2 volumes cold 1007o
ethanol were added. DNA was precipitated for 5 h at -20"C. The pellet was washed twice
withTyVo ethanol, dissolved in 15 pl TE buffer and stored at -20"C;1 pl was run on a gel to
determine the DNA concentration.
For the "gene clean" method, the GENECLEAN tP rc¡t BIO 101 was used. The
piece of agarose gel containing the relevant DNA fragment was transferred to an Eppendorf
tube and 3 volumes of 6 M NaI were added. After incubation for 5 min at 55oC, 10 pl
"home made" silica milk (Boyle & Iæw, 1995) was added and the tube was placed on ice
for 5 min. The silica milk-DNA complex was pelleted at 15,000 g for 5 s and washed three
times with 450 ¡rl ice cold new wash (Geneclean@). The DNA was eluted by resuspending
the pellet in 15 pl TE buffer, incubating for 2 min at 55oC and pelleting the silica milk' The
supernatant was transferred to a new Eppendorf tube and stored at -20oC:'1 pl was run on a
gel to determine the DNA concentration as described previously. 50
3.4.3 Electrophoresis and Southern blot analysis
Agarose gel electrophoresis was carried out according to Sambrook et aL (1989). DNA
from Phomopsis, grapevine or recombinant plasmids was digested with a restriction enzyme (SøII or PsrI for the plasmids) overnight at 37oC, according to the manufäcturer's
(Boehringer Mannheim) instructions. The DNA fragments were fractionated in a l7o ot 2Vo
agarose gel (depending on the size of the fragments) in TAE buffer (appendix) at 25 to 65
V for 2 to 18 hours (depending on the size of the gel). Hindfr-digested Lambda DNA was
used as a size marker. Gels were stained with ethidium bromide to visualise the DNA using IIV-light.
DNA fragments in an agarose gel stained with ethidium bromide were denatured by
soaking the gel in denaturing solution (1.5 M NaCl, 0.5 M NaOH, a minimum of two times
the volume of the gel) for 30 min followed by an equal volume of neutralising solution (1.5 M NaCl, 0.5 M Tris-HCl pH 7.4, 1 mM EDTA) for 30 min. The denatured DNA was
transferred to a positively charged nylon membrane (Boehringer Mannheim) overnight,
using the capillary transfer method of Southern (1975). The position of the gel slots was
marked on the membrane with a soft (2B) pencil. The membrane was rinsed for 2 min in 5
x SSC and blotted dry on Whatman 3MM filter paper. DNA was cross-linked to the
membrane using a Stratalinket@ UV crosslinker (Bio Rad) set at 150 mJ. The membranes
were stored dry at room temperature.
3.4.4 Radiolabelling and hybridisation
Total Phomopsis DNA was used as a probe for the colony blots (section 3.3.3) and to screen
Southern blots of the plasmid preparations. Approximately 40 ng of total Phomopsis DNA
was radiolabelled by the random primer method with 30 pCi a-32P-dCTP (Feinberg &
Vogelstein, 1983) using the Megaprime DNA labelling system (Amersham). The mixture
was incubated at37oC for 30 to 60 min.
Cloned Phomopsis DNA inserts (50 ng) that had been extracted from the gel
(section 3.4.2) were radiolabelled using the random primer method with 30 pci cr-32P-
dCTP. When the DNA inserts were not separated by gel electrophoresis, the clones (50-100
ng) were digested with Pvøtr and mixed with 1 ¡ll (0.1 pg) each of the pUCl9 Pvun. specific
primers P19S1 and P19S2 (appendix). Sterile nanopure water was added to give a total 5t volume of 35 pl, the mixture was denatured at 100'C for 5 min and placed on ice' Ten ¡rl labelling buffer (Amersham), 3 pl (30 pci) of cx,-32P-dcTP and 2 ¡tl (2 u) Klenow enzyme were added and the mixture was incubated at 370c for 30 to 60 min.
Unincorporated nucleotides were separated from the labelled DNA in a Sephadex
G-100 mini-column (medium grade, suspended in TE buffer) in a Pasteur pipette with a small plug of glass wool in the neck. The pipette with the glass wool plug had been autoclaved for 20 min at I2I'C and placed in an oven set at 240"C for 6 h. The labelled
DNA solution (50p1) was loaded onto the top of the column, allowed to run in and TE buffer was then added to fìll the column. The first radioactive fraction contained the labelled probe, the unincorporated nucleotides remained in the column. Radiolabelled probes were denatured at 100"C for 5 min, cooled on ice and added to the hybridisation solution.
Microsatellite probes ((cAT)s, (GATA)4, (GAA)0, (GACA)4 and (GGAT)a) were 3', terminal end-labelled with terminal deoxy-nucleotidyl transferase (Boehringer Mannheim) according to manufacturer's instructions. Microsatellite DNA (50 ng or 10 pmoles in a 1 pl volume) was mixed with 10 pl of 5 x terminal transferase buffer (Boehringer Mannheim), 5 pl 25 mM CoClz, 28 pl sterile nanopure water, a pl (a0 pCi¡ cr-32P-dCTP and 2 pl (50 U) terminal transferase, and incubated at 37"C for t h. The labelled DNA was separated from the unincorporated nucleotides by running it through a Biogel P-30 column in a Pasteur pipette with glass wool in the neck that had been autoclaved fot 20 min at IZIoC and placed in an oven set at 240"C for 6 h. Microsatellite probes were added directly to the hybridisation solution
Hybridisation was carried out in a rolling-bottle hybridisation oven. The positively charged nylon membranes were separated by nylon mesh inside a 30 cm bottle. The membranes were pre-hybridised overnight and hybridised for 18-20 h in 10 ml hybridisation
solution (appendix) containing 200 ¡rl single stranded sonicated salmon sperTn DNA (10 mg ml-r). pre-hybridisation and hybridisation usually took place at 65oC, except when
microsatellite probes were used, in which case pre-hybridisation and hybridisation were
ca:ried out at 5oC below the Tm of the microsatellite. After hybridisation with the phomopsis DNA probes, the membranes were washed twice in the bottle with 2 x SSC,
0.12o SDS at 65"C for 20 min and twice in a container with 1 x SSC, 0.1% SDS, shaking at 52
65oC for 15 min. Following hybridisation with microsatellite DNA, the membranes were
washed once in the bottle for 20 min with 6 x SSC, 0.17o SDS at 5oC below the Tm of the
microsatellite, twice in the bottle for 15 min with 3 x SSC, 0.17o SDS, and once, with
shaking for 20 min in a container with 3 x SSC, 0.17o SDS all at 5oC below the Tm of the
microsatellite. The membranes were exposed to X-ray film (Kodak X-Omat) at -80oC,
inside a cassette containing intensifier screens. 32p-labelled DNA was removed from the membranes before re-hybridising by
immersing the membranes in 0.1 x SSC, 0.57o SDS at 100'C for 15 min and washing them
i¡2 x SSC. The filters were re-used up to 15 times. 53
4. DISCOVERY AND DESCRIPTION OF THE TELEOMORPH OF
PHOMOPSIS TAXON I.
4.1 Introduction
Sexual reproduction is an important mechanism for generating and maintaining genetic
variation within populations (section 2.8.1). Variation in populations of a phytopathogen
may be beneficial for survival in heterogeneous environments (Maynard Smith, 1978;
Bell,1982) and for adaptation to fungicides and other management practices. The sexual
stage of P. viticola has been described only once (Shear, 1911), as Cryptosporella viticola,
and has not been found since (section 2.3.2). Teleomorphs of other Phomopsis species
belong to the genus Diaporthe (Uecker, 1988; section 2.2.I).
Asci or ascospores of many Ascomycetes are actively released by the apothecia (e.g.
Tapesiayallundae,H. Wallwork, pers. com.), cleistothecia (e.g. Uncinulanecator, Gadoury
& Pearson, 1990) or perithecia (e.g. members of the Sordariaceae, Ingold & Hudson, 1993).
Perithecia of D. aduncc, however, do not eject the ascospores forcibly; the ascospores are
exuded in mucilage (Linders, L994).
In this chapter, the teleomorph of Phomopsis taxon 1 is described in detail, and the
mechanism for ascospore release is discussed. Perithecia of Diaporthe viticolø (Nitschke,
1867), but not of Cryptosporella viticola, were discovered on grapevine canes infected with
Phomopsis taxon l. Diaporthe vitícola was first described by Nitschke (1867) but has never
before been linked to Phomopsis cane and leaf spot disease.
4.2 Materials and methods
4.2.1 Isolation of the teleomorph of På omopsís taxon I
Grapevine canes that displayed symptoms associated with taxon 1 were collected from
vineyards and placed in moist conditions at 15oC (section 3.1). The canes were incubated for3to8months. 54
4.2.2 Microscopic examination of sexual structures
Mature and immature perithecia were excised with a scalpel from Chardonnay and Shiraz canes. Some perithecia were frozen in Tissue-Tek@ O.C.T. 4583 Compound (Miles Inc.,
Elkhart, USA) and sectioned at 7-10 pm at -27"C using a Reichert-Jung cryocut 1800 microtome. Sections were placed on Sigma silane-prepru slides and allowed to dry for 5 min at 45oC. Sections were mounted in water or 80Vo glycerol, and photographed at various magnifications using a Zeiss phase contrast microscope.
Alternatively perithecia were fixed in 3Vo glutaraldehyde (Probing & Structure,
Kirwan, Qld, Australia) in 0.025 M phosphate buffer, p}J7 at4oC for 70 h. Fixed perithecia were dehydrated with methoxy ethanol, ethanol, propanol and butanol sequentially, as described by Feder & O'Brien (1968), and embedded in glycol methacrylate (Sigma) monomer mix (GMA) for 4 days with two changes of GMA. The perithecia were then transferred to gelatin capsules containing GMA and polymenzed at 60oC for 2 to 4 days.
Perithecia were sectioned using a Sorval JB4 microtome with glass knives. Sections (2-4 pm) were placed on a drop of RO water on a microscope slide, dried ovemight at 60oC, stained with periodic acid-Schiff's reagent (PAS) and toluidine blue O (TBO) (Feder &
O'Brien, 1968; Sedgley, lg82), and mounted in Surgipath@ micromountru mounting medium. Mounted sections were viewed and photographed at various magnifications using a Zeiss Axiophot microscope.
Asci and ascospores were obtained from perithecia of Chardonnay and Shiraz canes by pricking the perithecium with a sterile scalpel. The exudate was suspended in 507o lactic acid and transferred to a microscope slide. Photographs were taken at various magnifications using a Vanox interference microscope. Melzer's reagent was used to establish whether the asci were operculate (Hawksworth et a1.,1983).
4.2.3 ldentification of the teleomorph
Material was sent to the International Mycological Institute, Surrey, UK, for confirmation of identification. Perithecia were lodged in the dried reference collection, as IMI
368581 and 368584. Perithecia were also lodged in the Waite Fungal Herbarium (ADW
17036 and L7O37). A bulk isolate (54) and three single conidium-derived cultures (50D.1,
5lC.1 and 54B.1), gtown from ascospores, were submitted for examination. One of these, 55 either 50D.1 or 5lC.1, was placed in the dried reference collection as IMI 368582. An other isolate, either 548.1 or 54,was placed in the culture collection as IMI368583 (appendix).
4.2.4 Ãscospore release
Chardonnay and Riesling canes, collected from two vineyards in the Adelaide Hills and one
Tasmanian vineyard, were incubated as described in section 3.1. Forty-five mature
perithecia were excised from the canes and mounted on small pieces of Blu-Tackru which
had been placed inside sterile 9 cm Petri dishes, beside two pieces of sterile damp Wettexru
cloth and incubated at 23"C. The Petri dishes had double sided adhesive tape stuck to the
inside of the lid, and were sealed with strips of Glad@wrap. The distance between the tip of
the perithecium neck and the Petri dish lid was approximately 1 mm, 3 Írm or 5 mm; five
perithecia within the same Petri dish were placed at the same distance from the lid. After 36
days, the strips of Glad@wrap and the Wettexru cloth were removed from three plates and 1
g of silica gel was added to these plates. The pieces of double sided adhesive tape were
examined fortnightly at 400 x magnification after staining with ammoniacal congo red, and
replaced with fresh pieces of tape.
4.3 Results
4.3.1 Isolation of the teleomorph of Plr omopsis taxon 1
Many grapevine canes that produced cirrhi with conidia shown to belong to taxon 1,
produced perithecia after the pycnidia ceased to exude conidia. Perithecia were produced
only on canes with narrow black lines (Fig. 4.1). Formation of such lines, often termed 'zone-lines' (Webber, 1981; Brayford, 1990b), is a characteristic feature of the genus
Diap orthe Nitschke (V/ehmeyer, 1993).
Perithecia were found on 1- and 2-year-old canes of Chardonnay, Riesling, Shiraz,
Cabernet and Pinot Noir collected from many vineyards in South Australia, Victoria and
Tasmania. Grapevine canes collected from June to August required incubation in moist
conditions at 15oC before perithecia matured. Asci and ascospores from these perithecia
grew into colonies typical of Phomopsis taxon 1 when placed on WA or PDA (Table 3.1). 56
Pruned canes that were collected from the floor of the vineyard in November did not need to be incubated for the perithecia to mature: dried ascospores, but no asci, were present inside perithecia with the necks broken off. When placed on WA or PDA, the ascospores germinated to form characteristic colonies, pycnidia and conidia of Phomopsis taxon 1
(isolate 89, Table 3.1).
A.3.ZTaxonomic description of the teleomorph
Ascomata perithecioid, on canes, scattered or in groups, numerous, black, ampulliform to globose, up to 400 ¡rm in diameter, with prominent long necks that are often twisted or bent
and sometimes branched (Fig. 4.1). The necks can be up to 3 mm long, are 90-110 pm in diameter and have an apical ostiole. Ascomatal wall 13-35 pm thick, two-layered, the outer
layer 2-5 cells thick and composed of rather thick-walled, brown, strongly flattened cells, 7-
17 pm in size (textura angularis), the inner wall with some layers of hyaline, more strongly flattened cells with thinner walls, partly dissolving at maturity @ig. a.2 A, B and C). The
long neck has the same two layers.
Asci numerous, subclavate to cylindrical, ascus wall thin, unitunicate, sessile,40-58
x 7-9 pm, usually 8-spored, evanescent; apically with a refractive, non-amyloid ring and
with an indisrincr stalk (Fig. 4.3).
Ascospores biseriate, hyaline, ellipsoid or fusoid, medianly uniseptate, slightly or
not constricted at septum, often with 2-4larye guttulae, slightly acute at the ends,9.5-15 x
2.5-4.0 pm (Fig.4.3).
4.3.3 ldentification of the teleomorph Dr P.F. Cannon (IM, pers. com.) conf,rrmed the teleomorph as Diaporthe viticola
(Nitschke, 1867; appendix). However, Dr E. Punithalingam (IMI, pers. com.) identified the
bulk isolate 54 and the single conidium-derived cultures, 50D.1, 5lC.1 and 54B.1, grown
from ascospores as Phomopsís sp. and not as P. viticol¿ (Sacc.) Sacc.
4.3.4 Symptoms associated with Diaporthe vìtícola
The symptoms associated with the teleomorph were most obvious in winter, when the area
within the zone-lines on bleached spurs and canes became dark brown to black. Nitschke 57
(1867) noticed this also: he wrote that the stroma has a black edge and that the wood surface is a brownish /blackish colour because of the stroma, but he did not mention the bleaching of the canes. In the vineyard, the canes look as if someone drew on them with a black marker (Fig. a.a).
4.3.5 Ascospore release
No asci or ascospores were found on the double sided adhesive tape, however, ascospores were occasionally found in a yellowish drop of mucilage at the tip of the perithecial neck. In dry conditions, the ascospores sometimes dried out into a powder.
4.4 Discussion
The occurrence of the sexual cycle may be of great importance to the host-pathogen interaction as it may lead to greater variation within the population due to recombination and segregation, allowing Phomopsis taxon 1 to respond to new selection pressures.
The name Diaporthe viticola has not been considered recently (P.F. Cannon, IN{I, pers. com.). In the only comprehensive monograph of the genus, Wehmeyer (1933) treated it as part of the D. medusaea complex. which is characterised largely by the long ascomatal necks. D. viticola has never before been linked to Phomopsis cane and leaf spot disease.
Shear (1911) has provided the only description of a sexual stage of P. viticola.He found that single ascospores of Cryptosporella viticola grew into colonies that produced
pycnidia identical to those of Fusicoccum vitícolun (Redd.). He concluded that C. viticola
was the teleomorph of F. viticolum (section2.3.2), which caused lesions on green grapevine
shoots (Reddick, 1909; Gregory, 1913), similar to those associated with Phomopsis taxon2
(Menin et a1.,1995). This suggests that C. viticola could be the sexual stage of Phomopsis
taxon 2. Also, Shear (1911) described cx,-conidia as being 7.5-ß pm x 2-5 ¡tm and not
biguttulate, suggesting that they may have been cr-conidia of Phomopsis taxon 2, which are hypothesis that g-11.8 ¡tmx2-3.2 pm (Menin et a1.,1995, section 2.3.2). This supports the phomopsis taxon 2 may be synonymous with F. viticolum, and that this fungus and P.
viticola(Sacc.) Sacc. may differ. The o-conidia of P. viticola (Sacc.) Sacc. are 7 pm x 4 ¡rm 58
Figure 4.1 Perithecia (p) and zone-lines (z) of D. viticola on a 1-year-old Shiraz cane, scale bar = 0.5 mm (A) and on a l-year-old Chardonnay cane, scale bar = 1 mm (B). FIF, # ||b!..-,:. *: r,J r#r
tr¡ -'i-.*- Fì rF ü tt
I
i I I -C. I {b.*. t TI s-Ft1a\. l.* - *ù\ v 59 Figure 4.2 Sections through perithecia of D. viticola. A. Section through a mature perithecium (cryocut at -27"C,8 !rm), photographed using a Zeiss phase contrast microscope. Scale bar = 15 pm. B. Section (3 t¡m) through a perithecium, fixed in glutaraldehyde, embedded in GMA, stained with periodic acid-Schiff's reagent and toluidine blue, and photographed using a Zeiss Axiophot microscope. Scale bar = 65 pm. C. Section (3 pm) through a perithecium on a Chardonnay cane, fixed in glutaralde-hyde, embedded in GMA, stained with periodic acid-Schiff's reagent and toluidine blue, and photographed using a Zeiss Axiophot microscope. Scale bar = 200 pm. A B # ^ C 0 .4 .'ir ¡ 60 Figure 4.3 Asci of D. viticola in 50Vo lactic acid, photographed using a Vanox interference microscope. Each ascus contains eight ascospores. Scale bar = 10 pm. 61 Figure 4.4 Bleached Chardonnay cane with characteristic black marks (see arrows), a symptom associated with D. viticola, as seen in the vineyard in spring. 62 (Saccardo, 1880), of similar size to the cr-conidia of Phomopsis taxon I which are 4.8-7.1 pers. ¡tm x 1.4-2.2 ttm (Merrin et a1.,1995, section 2.3.2). Dr E. Punithalingam (IMI, com.) identified isolates of taxon 1,3 and 4,that \'/ere sent to him by Ms S.J. Merrin in 1992, as P. viticola (Sacc.) Sacc. Three years later, and immediately prior to his retirement, he identified bulk isolate 54 and the single conidium-derived cultures, 50D.1, 51C.1 and 54B.1, grown from ascospores as Phomopsls sp. and not as P. viticola (Sacc.) Sacc., because "the conidial width is much n¿urower than quoted in the original account". However, the cr-conidia of these isolates were of similar size as those of the taxon 1 isolates sent in 1992. Goidanich (L937) transferred F. viticolum to the genus Phomopsis as P. viticola (Redd.) Goid., apparently without knowing that Saccardo (1915) had already described P. viticola (Sacc.) Sacc., and apparently without examining Reddick's type material. Apparently, neither did Pine (1958), when he agreed with Goidanich and considered P. viticola (Sacc.) Sacc. to be the correct name for F. viticolum. Pine (1958), however, did not compare the two holotypes. Taxon 1 may, in fact, be P. viticola (Sacc.) Sacc., with teleomorph D. viticola, and taxon 2 may be F. viticolum (Redd.), with teleomorph C. viticola. It is obvious, and has been acknowledged by IMI, that there is a need for a comprehensive revision of the genus (appendix). The characteristic symptoms on the cane, associated with the teleomorph, have been confused with sclerotia of Botrytis cinerea in Australia. B. cinerea can also cause bleaching of canes (Marois et aL, 1992), and the black sclerotia can appear similar to the flat, black marks that are associated with D. viticola. The observation of ascospores in a drop of muscilage suggests that they may be exuded rather then actively expelled (section 4.3.4). However, additional experiments, looking at the effect of temperature, humidity and light regime on ascospore release should be conducted. Ingold & Hudson (1993) stated that ascospores in Pyrenomycetes are usually actively discharged. However, there are some exceptions, for example in Chaetomium sp. and in D. adunca the asci break down within the perithecium and a mass of spores mixed with mucilage oozes out through the ostiole (Ingold & Hudson, L993; Linders, 1994). Similarly, asci of D. viticola broke down inside the perithecium (section 4.3.2) and 63 ascospores appeared to ooze out through the ostiole. The ascospores of D. viticola sometimes dried out into a powder at the tip of the long neck (section 4.3.4). Dried ascospores may be wind dispersed, as the long necks of the perithecia may lift the spores out of the boundary layer of still air surrounding the cane. Ascospores may also be splash dispersed. If ascospores do infect grapevines and cause disease, they may provide a source of airborne inoculum for long range dispersal of the fungus. If the ascospores are wind-blown, control strategies targeted at splash dispersed conidia may be of little use. Consequently, the reproductive biology of the two taxa of Phomopsis of grapevines has to be taken into consideration when decisions about control and management of the disease are made. 64 5. MATING STI]DIES OF DIAPORTHE VITICOLA 5.1 Introduction The discovery of the teleomorph of Phomopsis taxon 1 implies that sexual recombination may be an important mechanism of generating variability in this fungus. The ability to generate new genotypes may make Phomopsis taxon 1 more adaptable to changes in the environment. However, the impact on the population structure depends on the genetic nature of the mating system, since sexual recombination is more likely to induce genetic variation in heterothallic than in homothallic fungi (section 2.8.1). If mating types exist, they could be used as markers for population genetic studies. Diaporthe species may be heterothallic, as proposed for D. adunca (Linders & Van der Aa, 1995) or homothallic, as proposed for southern D. phaseolorum (Ploetz & Shokes, 1980) and D. ambigua (Smit et aL, 1997). Nothing is known about the genetics of D. viticola, and perithecia have never been produced in culture. In this study, a technique was developed to induce the sexual cycle in vitro, and a preliminary study of the nature of the mating system of D. viticol¿ was conducted. In addition, isolates of Phomopsis taxon 2 and 3 were tested for sexual compatibility with isolates of taxon 1,2 and3. 5.2 Materials and methods 5.2.1 Induction of peritheciaín vítro and the nature of the mating system Attempts were made to induce the sexual stage of Phomopsis taxon I in vitro on micropropagated grapevines, autoclaved Chardonnay cane pieces and nutrient media. Experiment 1 Ten micropropagated grapevines, cv Cabernet Sauvignon, with three leaves each (received from Dr B.E. Stummer), were inoculated in vitro with conidia from the taxon 2 isolates DAR 69471 and DAR 69476, or with ascospores or conidia collected in November 1996 from canes infected with Phomopsis taxon 1 (Table 5.1). The conidia were collected from diseased canes with or without zonelines. Pycnidia and perithecia were induced to form by 65 placing diseased canes in moist conditions at 15oC (sections 3.1 and 4.2.1). Asci and ascospores were obtained from perithecia by pricking them with a sterile scalpel. Suspensions of ascospores (mixtures of 3x10s-lx10u ascospo."s ml-l and 2x10a-2x105 asci ml-r) and conidia (mixtures of several cirrhi, 5x10s-1x106 c-conidia ml-l) belonging to taxon 1, as well as conidium suspensions (9x10s-1x10ó c-conidia ml-l) from the taxon 2 isolates DAR 69471 and DAR 69476 were made in sterile RO water containing 0.0057o Tween 20. Small droplets (3 pl) were pipetted aseptically onto the three leaves until the entire surface was covered. One control plantlet was inoculated with sterile RO water containing 0.005Vo Tween 20. The micropropagated grapevines were kept at room temperature on the laboratory bench (approx. 23"C) for I month and then put at 4oC or 15oC in darkness, or kept at room temperature, for up to 8 months. Experiment 2 Perithecia were produced by a technique modified from Brayford (1990u). Young (5 months old) and l-year-old Chardonnay canes, derived from vines not treated with fungicides, were autoclaved twice (I2I"C for 20 min), cut into 3 cm pieces, split in two lengthways and immediately autoclaved twice again. A young and a l-year-old piece of cane were placed, end to end, on two pieces of moist, sterile filter paper inside each 9 cm Petri dish. Ten taxon I isolates from the Adelaide Hills and the Coonawana (Table 5.2), as well as ten taxon I isolates from two vineyards in the Adelaide Hills (Table 5.3) were paired in all combinations, and one isolate from the first group was paired with an isolate from the second group. Two groups of ten isolates, rather then one group of 20 isolates, were used, to reduce the number of Petri plates required for the experiment (448 instead of 844 plates). If Phomopsis of grapevines is a heterothallic fungus, it is likely that both mating types will be present among 20 isolates. One isolate from the first group was crossed with an isolate from the second group, a strategy which was expected to allow the determination of the mating type of all20 isolates. Most isolates in the first group were gro,wn from hyphal tips, except for isolate 50D.1 which was a single conidium-derived isolate (Table 3.2), and isolates 50E and 524, which were derived from a single cirrhus, "bulk" isolates (Table 3.1). Isolates 174 and 17B 66 were derived from two pycnidia2 mm apart on the same cane and separated by a zone-line; isolates 184 and 18B were derived from the same piece of bark (25 mm2), and isolates 504-E were derived from a single perithecium (Table 3.1). All isolates in the second group were "bulk" isolates. Isolates 1514-F were derived from pycnidia on the same cane. This cane had many zone-lines, and cirrhi were taken from areas on either side of the zone-lines. Isolates 1524-B were also derived from one cane, and isolates 1534-B from a different cane, both with zone-lines (Table 3.1). Two mycelial plugs of one isolate were placed on one side of the canes, and one mycelial plug of an other isolate.on the other side of the canes (Fig. 5.1). In this way, the mycelium of the two isolates always met on both canes. Four replicates were made of each cross and of selfed isolates and the uninoculated control. The Petri plates were sealed with strips of Glad@wrap and placed in the dark at 23"C for 1 week, by which time the canes were colonised. One ml of sterile RO water was added to moisten the filter paper, and two sets of replicates were transferred to 15"C in darkness and the remaining two were placed at 4oC in darkness. The plates were examined 3, 6 and 1l weeks after inoculation. In addition, one single ascospore-derived, one hyphal tip-derived and six single conidium-derived taxon 1 isolates, four single conidium-derived taxon 2 isolates, two taxon 3 isolates and one taxon 4 isolate were paired with themselves and with taxon I isolate 154, which did not produce perithecia when paired with itself, As a positive control, two taxon I isolates that did produce perithecia when selfed, were paired with themselves and with isolate 154. As a negative control, isolate 154 was paired with itself. Attempts were also made to cross the four single conidium-derived taxon 2 isolates in all combinations, and the taxon 3 and 4 isolates with a single conidium-derived taxon 2 isolate (Table 5.4). The additional crosses were carried out in duplicate using the method described in experiment 24, but were incubated at 15oC in darkness only. Experiment 3 The isolates used in experiment 2 were paired in duplicate on PDA, V/A and Difco lima bean agar (LBA), ca 25 ml in 9 cm Petri dishes, by applying four or six mycelial plugs of different isolates to the medium. All isolates were also paired with themselves. The plates 67 were incubated in the conditions described in section 3.1, and examined 4, 8, 12 and 24 weeks after inoculation. Experiment 4 A large number (>50) of plates of ca 25 ml of PDA, Vy'A, carnation leaf agar (CLA, comprising WA with autoclaved carnation leaf pieces, approx. 25 mm2, on the surface, Merrin et al., 1995), LBA and Difco corn meal agar (CMA) were inoculated with ascospores (mixtures of 10s-106 ascospores ml-l and 104-10s asci ml-l) or conidia (mixtures of several cirrhi, 105 cr-conidia ml-t) collected from diseased canes and prepared as described previously (experiment l). Spore suspensions were streaked over the agar with a wire loop. The agar plates were incubated in the conditions described in section 3.1. 5.2.2 Conditions for perithecium developmentin vítro Two environmental factors which may influence the development of perithecia were examined: light regime and temperature. Fourteen Petri dishes containing sterile Chardonnay canes were inoculated with "bulk" isolate I25A which produced perithecia by itself at 15oC in the conditions described in experiment 2 (section 5.2.1; Table 5.3). Two Petri dishes were placed at 15oC in darkness, two were placed at approximately 23oC on the laboratory bench, and two were incubated in a cycle of 12 h at 23"C in white and near ultraviolet light, and 12 h at 14oC in darkness (section 3.1). The remaining eight Petri dishes were placed first in the dark at approximately 23"C for I week and, when the canes were colonised, two plates were transferred to the laboratory bench at approximately 23oC, two were kept at approximately 23"C in darkness, two were transferred to 15"C in darkness, and two were transferred to an incubator set at L2 h at 23"C in white and near ultraviolet light, and 12 h at 14oC in darkness (section 3.1). An uninoculated Petri dish containing sterile Chardonnay canes was placed next to each pair of inoculated plates. The plates were examined 3 weeks after inoculation and weekly thereafter. 68 5.3 Results 5.3.1 Production of peritheciaín vítro and the nature of the mating system Experiment 1 Perithecia containing ascospores were formed on micropropagated grapevines that were inoculated with ascospores or a mixture of taxon 1 conidia from canes with zone-lines (Table 5.1). Plantlets that had been placed in the dark at 15oC, I month after inoculation, bore mature perithecia 5 months after inoculation. Two of these plantlets produced pycnidia and cirrhi, typical of Phomopsis taxon l, prior to perithecium development. The plantlet that was placed in the dark at 4"C, I month after inoculation, bore immature perithecia 8 months after inoculation. The plantlet that was kept at approx. 23"C on the laboratory bench, did not bear perithecia. The uninoculated plantlet and plantlets that were inoculated with conidia of taxon I from canes with no zonelines or with conidia of taxon 2 isolates, did not bear perithecia. Experiment 2 Production of perithecia and formation of zone-lines Three weeks after inoculation, the autoclaved Chardonnay canes and the filter paper inside some Petri dishes showed vague lines where the mycelium of the isolates met (Fig. 5.1). Six weeks after inoculation, 7Vo of the young Chardonnay canes had zone-lines @ig. 5.2 A). The l-year-old canes did not have zone-lines. Eleven weeks after inoculation, small, immature perithecia were present on some canes that were incubated at 4"C, whereas perithecia with viable ascospores 'were present on most young and l-year-old canes that were incubated at 15oC (Fig.5.2 B). The perithecia, asci and ascospores matched the descriptions given in section 4.2.3 and, on WA or PDA, the ascospores germinated to produce colonies typical of Phomopsis taxon 1. Of the 111 crosses (222 Petri plates) that were placed at 15oC in darkness, 15 (30 plates) did not produce perithecia or zone-lines. Two crosses produced perithecia or zone- lines in only one of the replicate plates, therefore, in total 32 plates did not contain perithecia or zone-lines (Table 5.5). Eight crosses produced zone-lines and perithecia in one replicate but produced an irregular clumped growth in the other replicate plate. Three 69 crosses produced zone-lines and perithecia in one replicate and only zone-lines on the young canes in the other replicate plate. Of the 178 plates that contained perithecia, the young canes bore perithecia in 175 plates, of which 160 also had zone-lines. The remaining young canes with perithecia (15 plates) were covered in thick mycelium which may have obscured the zone-lines. In three plates that contained perithecia, the young canes did not bear perithecia and two of these canes had zone-lines (Table 5.5). Moreover, three of the four plates without perithecia but with zone-lines on the young canes, had a duplicate plate that contained canes with both perithecia and zone-lines. The l-year-old canes bore perithecia in 160 plates, and 31 of these canes also had zone-lines (Table 5.5). Zone-lines and perithecia were not produced in the uninoculated controls. The nature of the mating sYstem Of the first group of ten taxon I isolates, five did not form perithecia when selfed (Table 5.2). These isolates also did not form perithecia when paired with other isolates that did not self. Four of these five isolates were derived from hyphal tips (154, 184, 188 and 508) and one \ryas a "bulk" isolate (52A). The remaining isolates produced perithecia when selfed and when paired with other isolates, except for the single conidium-derived isolate 50D.1, which was capable of selfing, but produced only zone-lines and no perithecia when paired with isolate 184. Three of the five isolates that produced perithecia when selfed, were derived from hyphal tips (174, 17B and 504), and one was a "bulk" isolate (50E). Isolates 504-E were derived from a single perithecium, yet isolate 50B did not produce perithecia when selfed, whereas isolates 504, 50D.1 and 50E did (Table 5.2). The ten bulk isolates of the second group were all capable of selfing, and perithecia were produced in every combination (Table 5.3). Two of the seven single conidium-derived taxon 1 isolates did not form perithecia when selfed or paired with isolate 154 (Table 5.4). The remaining five isolates and the single ascospore-derived isolate produced perithecia when selfed and when paired with isolate 154, after 14 weeks. The positive controls produced perithecia after l0 weeks and the negative control (isolate 15A) did not produce perithecia. The taxon 2,3 and 4 isolates did not produce perithecia when selfed or when paired with isolate 15A, and perithecia 70 Table 5.1 Production of perithecia and pycnidia of D. víticola on micropropagated grapevines in vitro incubated in various conditions. uinoculum room temperature l5oC / dark 4"C I dark (applied until leaves covered) 1 month 8 months 2 months 5 months 5 months 8 months omixture of asci and ascospores mycelium mycelium immature obtained from Pinot Noir from perithecia Marion's Vineyard in Tasmania "mixture of asci and ascospores mycelium mycelium mature obtained from Chardonnay from perithecia Hargrave in South Australia "mixture of asci and ascospores mycelium mycelium mature obtained from Chardonnay from pycnidia pycnidia perithecia Hillstowe in South Australia cirrhi cinhi dmixture of asci and ascospores mycelium mycelium obtained from Chardonnay from pycnidia pycnidia Clover Hill in Tasmania otaxon I cinhi from cane without mycelium mycelium mycelium zone-lines, obtained from Riesling pycnidia pycnidia pycnidia fromAshton Hills in SA "taxon I cirrhi from cane without mycelium mycelium mycelium zone-lines, obtained from Riesling pycnidia pycnidia pycnidia from Ashton Hills in SA cirrhi cirrhi cirrhi "taxon I cinhi from 3 canes with mycelium mycelium mature zone-lines, obtained from Char- pycnidia pycnidia perithecia donnay from Hargrave in SA cirrhi cinhi otaxon 2 (DAR 6947L) cinhi mycelium mycelium mycelium pycnidia pycnidia pycnidia cinhi cinhi cinhi "taxon 2 (DAR 69476) cinhi mycelium mycelium mycelium pycnidia pycnidia pycnidia cirrhi cirrhi cinhi dstcrile RO water, no fungal no fungal containing 0.0O5Vo Tween 20 gowth growth ' Taxon I inoculum (asci, ascospores and conidia) was derived from naturally infected gtapevine canes, collected from vineyards in Tasmania and the Adelaide Hills in South Australia. b Plantlets were transferred to 4oC after I month at room temperature (approx. 23"C). " Plantlets were transferred to 15oC after 1 month at room temperature (approx. 23"C). d Plantlets were maintained at room temperature (approx. 23"C). 7I Figure 5.1 Experimental design for production of perithecia in vitro, 3 weeks after inoculation. Two mycelial plugs of one Phomopsis isolate were placed on one side of the sterile Chardonnay canes, and one mycelial plug of an other isolate was placed on the other side of the canes. Cane A is a l-year-old cane, cane B is a 5 months old cane. The diameter of the Petri dishes was 9 cm. 1534 A - tuÌ ,-z B :- - -E -'*-i ¿ 151F 72 Figure 5.2 A 5-month-old Chardonnay cane inoculated in vitro with isolates 174 and 504 of Phomopsrs taxon 1 and incubated at lsoc. A. Six weeks after inoculation, zoneJines are present (see arrow). Scale bar = 1 mm. B. Eleven weeks after inoculation, zoneJines (z) and fertile D. viticola perithecia (p) are present. Scale bar = 1.2 mm. A B 73 Table 5.2 Results of pairwise crosses, in duplicate at 15oC, made between ten isolates of Phomopsis taxon I from the Adelaide Hills and the Coonawarra, on autoclaved grapevine canes in vitro. 15A I7 t7B 184 18B 504 508 50D.1 50E 52A 52A +^ + + + + 508 + + + + + + + + + 50D.1 u+ + + b + + + + 508 + + + 504 + + + + + + 188 + + 184 + + 17B ; + + 17A + + 15A Isolates 154, 174, l7B, 184, l8B, 50,A' & 508 were derived from hyphal tips, 50D.1 is a single conidiumderived isolate (Table 3.2) and 50E and 52A are bulk isolates (Table 3.1). Isolates 504 - 50E were derived from a single perithecium, isolate 524 from a different perithecium. + Indicates formation of fertile perithecia after incubation at 15"C for 11 weeks. - Indicates no perithecium formation. ' One replicate plate did not contain perithecia, zone-lines or irregular clumped growth. b Zone-lines were formed on the young cane in one of the replicate plates. Table 5.3 Results of pairwise crosses, in duplicate at 15oC, made between teno isolates of Phomopsis taxon 1 from two vineyards in the Adelaide Hills, on autoclaved grapevine canes in vitro. r25A 151A 151C 151D l5lE 151F L52A t52B 153A 1538 b15A + 1538 + + + + + + + + + + 1534 + + + + + + + + + I52B + + + + + + + + I52A + + + + + + + 15lF + + + + + + 15lE + + + + + 15lD + + + + 15lC + + + 1514 + + r25A + u Isolates I25A - l53B (bulk isolates) are listed in Table 3.1. b Isolate 154 from the first group was paired only with isolate 1524 of this group + indicates formation of fertile perithecia after incubation at 15oC for 11 weeks. 74 Table 5.4 Eight isolates of Phomopsls taxon 1, four taxon 2 isolates, two taxon 3 isolates and one taxon 4 isolate were paired with themselves and with Phomopsis taxon I isolate 154. The four taxon 2 isolates were paired in all combinations and the taxon 3 and 4 isolates were paired with Phomopsis taxon 2 isolate 110.1. selfed 154 110.1 91.1 DAR 69486.t u51A u51C.1 088.1 + + u95.1 + + u994.1 + + "1oo.l + + "101.1 + + ÐAR 69467.r + + "91.1 '110.1 'DAR 69457.1 'DAR 69486.r dDAR 694s9 orM 139 DAR 69484 rr7A + + rrz5A + + cl5A + Indicates formation of fertile perithecia after incubation at 15oC for 14 weeks. - Indicates no perithecium formation. " Isolates of Phomopsis taxon 1; all isolates twere derived from single conidia, except isolate 514 which was derived from a hyphal tip. b Single ascospore-derived isolate of D. viticola. " Single conidium-derived isolates of Phomopsis taxon 2. o Taxon 3 "bulk" isolates. " Taxon 4 "bulK'isolate. r Positive controls (taxon 1 isolates). s Negative control (taxon 1 isolate). 75 Table 5.5 Production of zone-lines and perithecia of D. viticola on autoclaved young and 1- year-old Chardonnay canes in 222 Petri plates placed at 15oC in darkness. number of plates young canes 1-year-old canes 31 zone-lines & perithecia zone-lines & perithecia Lt7 zone-lines & perithecia perithecia L2 zone-lines & perithecia 9 perithecia & thick mycelium perithecia 6 perithecia & thick mycelium 2 zone-lines perithecia 1 thick mycelium perithecia 4 zone-lines 8 irregular clumped growth 32 No perithecia or zone-lines were present on the canes. '76 were not produced when the four taxon 2 isolates were crossed in all combinations or when the taxon 3 and 4 isolates were paired with taxon 2 isolate 110.1 (Table 5.4). Experiments 3 and 4 Perithecia failed to develop on PDA, WA, CLA, LBA and CMA inoculated with ascospores, mixtures of conidia or mycelial plugs. 5.3.2 Conditions for perithecium developmentín vítro Perithecia developed on canes inoculated with isolate 125A, first in the Petri dishes that were placed at 15"C in darkness. After 9 weeks immature perithecia were present in both replicate plates and after 13 weeks mature perithecia were present (Table 5.6). Perithecia also developed in both Petri dishes that were placed at approximately 23"C in darkness for I week and then transferred to 15oC in darkness. In these plates, perithecia were not yet visible after 9 weeks, but mature perithecia were present after 13 weeks. One of the plates that was placed at23"C in darkness for I week and then transferred to an incubator set at 12 h at 23"C in white and near ultraviolet light, and L2 h at 14oC in darkness contained immature perithecia after 13 weeks. The other plate contained only mycelium. 5.4 Discussion 5.4.1. Production of zone-lines and perithecia ín vítro The teleomorph of Phomopsis taxon 1 was produced successfully in vitro on micropropagated grapevines and on sterile Chardonnay canes. Perithecia did not form on agar plates inoculated with ascospores, mixtures of conidia or mycelial plugs. Isolates of Phomopsis taxon 2,3 and 4 did not produce perithecia on micropropagated grapevines nor on sterile Chardonnay canes. Moreover, these isolates did not produce perithecia when paired with a taxon 1 isolate that did not produce perithecia, when selfed, or when paired with other taxon 2 isolates. The vague lines which were observed where mycelia met on autoclaved Chardonnay canes and filter paper, 3 weeks after inoculation, were considered to be caused by mycelial 77 Table 5.6 Conditions for the development of perithecia of D. viticola on sterilised Chardonnay canes inoculated with isolate I25A in vitro, in duplicate. incubation conditions number of weeks after inoculation 3-6 7-8 9 13 uimmature 15oC in darkness mycelium immature perithecia fruiting bodies perithecia aimmature approx. 23"C in darkness mycelium pycnidia pycnidia fruiting bodies uimmature aimmature approx. 23"C in lightb mycelium mycelium fruiting bodies fruiting bodies uimmature cycle of 12hat23"Cin light' mycelium "immature mycelium and 12 h at l4oC in darkness fruiting bodies fruiting bodies aimmature nimmature approx. 23"C in darkness for I mycelium perithecia week, then l5"C in darkness fruiting bodies fruiting bodies aimmature oimmature approx. 23"Cin darkness for 1 mycelium mycelium week, then approx.23"C in fruiting bodies fruiting bodies lightb uimmature uimmature approx. 23"C in darkness for I mycelium immature week, then cycle of 12 h at23oC fruiting bodies fruiting bodies perithecia in light' and 12 h at 14oC in in one plate darkness only u Immature fruiting bodies are either immature pycnidia or immature perithecia. b Petri dishes were placed on the laboratory bench. " Petri dishes were placed in an incubator set at 12h at23"C in white and near ultraviolet light, and L2h at 14oC in darkness. 78 incompatibility (chapter 8), were morphologically different from zone-lines and were not associated with the sexual stage. This study suggests that the formation of zone-lines on grapevine canes is associated with the formation of perithecia. It had earlier been observed that perithecia were produced only on naturally infected canes with zone-lines when incubated at 15oC; canes without zone-lines produced only pycnidia (section 4.3.1). Micropropagated grapevine plantlets (section 5.3.1, experiment 1) that were inoculated with a mixture of conidia from canes with no zone-lines, did not produce perithecia. Plantlets that were inoculated with ascospores or conidia from canes with zone-lines, did produce perithecia, although the latter treatment was unreplicated, so requires further experimentation. Moreover, formation of perithecia on sterile, young grapevine canes in vitro was associated with the presence of zone-lines, except in a few cases, when the zone-lines may have been concealed by of thick mycelium, or when zone-lines were present and perithecia did not form on the canes (section 5.3.1, experiment 2). However, when zone-lines or perithecia were present on their own on a young cane, the duplicate plate contained canes with both zone-lines and perithecia. The only exception was the cross of isolates 50D.1 and 184: no perithecia were formed, but zonelines were present on the young cane in one plate. Formation of perithecia was expected in this cross, since isolate 50D.1 was capable of selfing. Webber (1981) reported zone-line formation in the inner bark of dead elm colonised by Phomopsis oblonga. Brayford (1990b) discussed the significance of zone-line formation by the two Phomopsis groups from elm. However, the lines Brayford (1990b) discussed were apparently formed on agar when the mycelia of two incompatible isolates met. It is, therefore, possible that the antagonistic reaction that Brayford (1990b) described may not be related to the zone-lines that are present on the elm tissue, but instead was a vegetative incompatibility reaction (chapter 8). The relationship between the presence of perithecia and the presence of zone-lines on elm was not discussed. Perithecia developed fastest on grapevine canes that were placed directly at 15oC: immature perithecia ,were present after 9 weeks. In an incubator set at 12h at23"C in white and near ultraviolet light and 12 h at 14oC in darkness, perithecia developed slowly, and perithecia did not develop on grapevine canes placed at 23"C (section 5.3.2). At 4"C perithecia developed very slowly (section 5.3.1, experiment I and 2). Light was not 79 required for perithecium formation. Since 23oC appears to be unsuitable for perithecium development, it is possible that perithecia develop in winter and spring, and mature in spring, when temperatures of approximately 15oC would be expected. Linders & Van der Aa (1995) found that the number of sporulating pycnidia of D. adunca was negatively related to the number of perithecia formed later on the same Plantago stems. This was not investigated in D. viticola, but pycnidia or cirrhi were not found on sterile grapevine canes ín vitro prior to perithecium formation, although pycnidia were found on canes when perithecia did not form. However, pycnidia and cirrhi were found on micropropagated grapevines prior to perithecium formation (Table 5.1). In Phomopsis of grapevines, environmental conditions may influence the balance between sexual and asexual reproduction. 5.4.2The nature of the mating system Studies on the nature of sexual reproduction were inconclusive, and failed to demonstrate that Phomopsis taxon I exhibits heterothallism or homothallism. Some isolates, including six single conidium-derived isolates and a single ascospore-derived isolate, produced perithecia when grown singly on sterilised grapevine canes. This is similar to the homothallic fungi, Phomopsis group two from elm and southern D. phaseolorum, which produced perithecia when grown singly on sterilised elm twigs and soybean stems, respectively (Ploetz & Shokes, 1986; Brayford, 1990). Other isolates, including two single conidium-derived isolates, did not produce perithecia when selfed, nor did they do so when paired with other self-sterile isolates. If there are two mating types present in Phomopsis taxon 1, the self-sterile isolates may have the same allele for mating type. Isolates that were self-fertile usually produced perithecia when paired with isolates that were self-sterile (Table 5.2), however, these isolates can not be assumed to be of the opposite mating type, since it is possible that the perithecia are the product of selfing. The existence of self-fertile and self-sterile isolates of Phomopsis tâxon 1, that were derived from the same perithecium (isolates 504-E, Table 5.2), and appear to be similar in all other aspects, can be explained in several ways. One possibility is that D. víticola is a homothallic species in which a mutation to self-sterility, unrelated to mating type, is present. Another possibility is that the self-sterile, hyphal tip-derived isolate 508 did not 80 produce perithecia because of an inhibition caused by the presence of two or more different nuclei in its mycelium. However, self-fertility may be interpreted by a mechanism other than homothallism. There are two models in the literature that may explain the situation in Phomopsis taxon 1: the fungus may be pseudo-homothallic, or mating type switching may occur. In pseudo-homothallic fungi, such as Neurospora tetrasperma and Podospora anserina, four binucleate ascospores are formed per ascus @odge, 1927; Franke, 1957). The two nuclei in each ascospore contain different mating types, so the dikaryotic mycelium is self-fertile (Nauta, 1994). However, single ascospore-derived isolates of Neurospora tetrasperma can be obtained that are heterothallic. These ascospores are smaller and contain only one nucleus with only one allele for mating type @odge,1927). Conversion of mating type is thought to occur in several fungi. In Sclerotinia trifoliorum all mature asci display ascospore dimorphism, with 4:4 segregation of small and large ascospores (Uhm & Fujii, 1983o). The size of the ascospores does not influence the morphology, growth rate or pathogenicity of the resulting single ascospore-derived isolates. However, single ascospore isolates derived from small spores are always self-sterile, whereas those derived from large spores are self-fertile and produce apothecia with asci that exhibit the 4:4 segregation for spore size (Uhm & Fujii, 1983"). Uhm & Fujii (1983b) suggested that S. trifoliorum has bipolar heterothallism: isolates derived from small ascospores have mating type S and isolates derived from large ascospores have mating type L. S-isolates are always self-sterile but can be fertilised by microconidia of L-isolates. This was confirmed by Rehnstrohm &. Free (1993) who mated L- and S-isolates and demonstrated the segregation of genetic markers involved in mycelial incompatibility into the progeny. Uhm & Fujii (1983b) suggested that the self-fertile L-isolates are capable of switching their mating type from L to S with subsequent pairing of L and S nuclei in the same thallus. Fertilisation of sclerotia with microconidia, therefore, does not occur, but the resulting asci do show segregation for S and L spores (Fujii & Uhm, 1988). Another filamentous ascomycete in which regular unidirectional reversal of mating type has been described is Chromocrea spinulosa (Mathieson, L952; Perkins, 1987). As in S. triþIiorum, each ascus contains four self-sterile small ascospores and four self-fertile large ascospores (Mathieson, 1952). A similar pattern of self-sterile and self-fertile 8l segregation occurs in Glomerella cingulafa, although ascospores are all the same size. In this fungus, mating type B mutates readily to b, and reverse mutations from b to B can occasionally occur (Wheeler, 1954; Perkins, 1987). Other species in which both self- fertility and self-sterility have been reported are Ceratostomella fimbriata, which is similar to S. triþIiorum and C. spinulosa in that ascospore progeny of self-fertile strains include both self-fertile and self-sterile strains (Olson, 1949), and Nectria haematococca (Matuo & Snyder, 1973). The genetics of the mating systems of these species is unclear. Asci of D. viticola contain eight ascospores and are, therefore, more similar to S. triþIiorum and C. spinulosa than to N. tetrasperma and P. anserina. However, the ascospores have two cells each with one nucleus @.L. Melanson, pers. com.) that may be the product of mitotic division. It could be that Phomopsis taxon I has a mating system similar to that of S. trifoliorum and C. spinulosa. That would explain why some isolates appear to be homothallic and others heterothallic, even when they are derived from a single perithecium (isolates 504-E, Table 5.2). However, the possibility that D. viticola is a homothallic species in which a self-sterility mutation, unrelated to mating type, is present cannot be discounted. Therefore, more research is needed. Ascosporogenesis should be examined cytologically, to determine whether the nuclei in the two cells of the ascospores are the product of mitosis or meiosis; single ascospore-derived isolates, derived from single asci and perithecia, should be paired with themselves to determine the ratio of self-sterile to self- fertile isolates among the progeny of perithecia; self-fertile, single ascospore-derived isolates should be examined (e.g. isolate 88.1) to determine whether asci containing four self-fertile and four self-sterile progeny are regularly produced; and attempts should be made to cross self-fertile isolates with self-sterile isolates. Progenies of such crosses should be assessed to determine whether they differ genetically from each other and from the parent isolates. Linders & Van der Aa (1995) studied the role of the cr- and p-conidia in relation to the formation of perithecia in D. adunca. Their conclusion was that the q-conidia function as spermatia in this heterothallic fungus, although they did not try to co-inoculate Plantago stems with mycelium of opposite mating types. In our study, conidia were not used to fertilise mycelium, and pycnidia or cirrhi were not found on the sterilised grapevine canes 82 prior to perithecium formation. It is, therefore, unlikely that o- or p- conidia function as spermatia in the self-fertile isolates of Phomopsis taxon 1. However, cl-conidia from the self-fertile isolates may be able to fertilise the mycelium of the self-sterile isolates of Phomopsís taxon l, as is the case in S. tífoliorum (Rehnstrohm & Free, 1993). p-conidia may also have a role in fertilising self-sterile isolates. 83 6. DISEASE DEVELOPMENT IN VINEYARDS 6.L Introduction Little is known about the disease caused by Phomopsis taxon I on grapevines. Most research on symptoms, ecology, epidemiology and control of Phomopsis cane and leaf spot, as described in sections 2.4, 2.5 and 2.6, was carried out before the different taxa of Phomopsis on grapevines (Merrin et al., 1995) were identified. It appears, however, that most of the literature on this disease concerns Phomopsis taxon 2, as this taxon is associated with scarring and bleaching of l-year-old wood, dark spots with yellow haloes on leaves and dark, elongated lesions on young shoots and petioles (Merrin et aI., 1995). Therefore, the pathogenicity of Phomopsis taxon 2 in Australian vineyards is recognised (N.G. Nair and T. Wicks, pers. com.). The disease associated with Phomopsis taxon 1, which became prevalent in the late 1980s in Australia (D.L. Melanson, pers. com.), appeared to result in milder symptoms. This taxon was not found to cause symptoms on green gtapevine tissues (Merrin et aI., 1995) and in the vineyards, l-year-old canes were bleached but apparently not scarred. The development of the disease associated with Phomopsls taxon t has not been studied in Australian vineyards, and the importance of this taxon has yet to be defined. In this chapter, the progression of the disease associated with Phomopsrs taxon I in the field is described. Disease incidence, measured as percentage of bleached spurs, was monitored in five grapevine plots in four successive seasons. In addition, a preliminary study is presented in which the incidence of budburst on infected grapevine spurs and canes was compared with the incidence of budburst on apparently healthy spurs and canes. 6.2 Materials and methods 6.2.1 Monitoring of grapevines infected with Phornopsís taxon 1 As the symptoms caused by Phomopsis taxon 1 on grapevines were uncertain, grapevines were assessed for the presence of bleached spurs and canes, the presence of pycnidia on these spurs and canes, stunted growth of new shoots, and incidence of budburst. In addition, 84 the presence of scars on woody tissues, brittleness of infected canes and the presence of necrotic lesions and/or pycnidia on green tissues were noted. The disease incidence in each of the vineyards was measured as percentage of l- year-old spurs that were bleached and speckled with pycnidia. In Ashton Hills (Chardonnay and Riesling), all spurs in every other row in the grapevine plots were scored "bleached" or "not-bleached"; in Barratt's and Hargrave's vineyards, all spurs in every third row in the Chardonnay plots wcre scored; in Hillstowe and Sutherland's vineyard, all spurs in each of the rows in which spurs had been tagged (nine and 11, respectively), were scored. In 1994, each grapevine plot was examined twice before budburst, in August, to check whether the spurs had started to bleach, and in September, to determine the percentage of spurs bleached. In 1995 and 1996, the disease incidence was determined in September just prior to budburst.In 1997 the disease incidence was determined in August, due to lack of time. In August L994, when the buds were dormant, approximately 100 l-year-old bleached Chardonnay spurs or canes were randomly tagged in each of five vineyards in the Adelaide Hills (Ashton Hills, Ba:ratt's vineyard, Hargrave's vineyard, Hillstowe and Sutherland's vineyard), as were approximately 100 l-year-old bleached Riesling spurs or canes in the Ashton Hills vineyard. Spurs or canes were tagged only if there was no visual sign of infection by organisms other than Phomopsis taxon 1. From each plot of 100 tagged spurs or canes, a 5 mm2 piece of bleached grapevine bark from ten of the spurs was surface sterilised, placed on WA in Petri plates at 23eC (section 3.1) and incubated for 2 to 4 weeks, to verify that Phomopsls taxon I was present on the spurs. Also, bleached canes collected from the ground were placed in moist conditions at 15eC for I to 10 weeks (section 3.1) and examined for the presence of Phomopsis taxon 1. No apparently healthy spurs were tagged as controls, since there were too few in each plot to allow a comparison between bleached and apparently healthy spurs. In Barratt's and Hargrave's vineyards, no spurs or canes were found that were free of both bleaching and infection by organisms other than Phomopsis taxon 1; in Ashton Hills, 2Vo of the Riesling spurs and 57o of the Chardonnay spurs were apparently healthy, and in Hillstowe and Sutherland's vineyards, 47o of the spurs in the grapevine plots was apparently healthy. Only spurs were tagged in Barratt's and Sutherland's vineyards. In Ashton Hills and Hillstowe, L07o of the material tagged consisted of canes, and in Hargrave's vineyard,25Vo 85 of the material tagged consisted of canes. For convenience, however, tagged spurs and canes will be referred to as 'tagged spurs' in the rest of this chapter, except where bleached spurs were compared with bleached canes. In the season of 199411995, each grapevine plot was examined twice before budburst (August and September) to note scarring of tagged bleached spurs and to check if the buds had started to swell. After the buds started to burst in September, all tagged spurs wcrc cxamined once every fortnight, until fruit set in December, for the presence of necrotic lesions and/or pycnidia on green tissues, stunted growth of new shoots, and incidence of budburst. The same spurs were examined again in June 1995, after leaf fall, but before pruning, in order to assess more accurately whether buds and new shoots/canes had died, broken off, or were stunted. Stages in grapevine shoot development described by Baggiolini (1952, Table 2.2 andFig.2.I) were used to describe the developmental stage of the vines. Tagging of spurs and canes was repeated in the two following growing seasons (199511996 a¡d 199611997), as above, except that in Hargrave's vineyard I07o of the sample consisted of canes, and Sutherland's vineyard was no longer included in the trial. The presence of Phomopsis taxon 1 on the spurs and in the vineyard in both 1995196 and 1996197 was verified as above, except that pieces of bleached bark from five tagged spurs from each vineyard, were placed on WA. In the third season (1996197), 99 apparently healthy Chardonnay spurs in Barratt's vineyard and 38 apparently healthy Chardonnay spurs in Hillstowe were included as controls. Tagged spurs were examined as above, except that they were examined only once in spring (October/1.{ovember), when the vines were in stages G, H or I @aggiolini, t952), and in 1997 the grapevine plots were examined only once prior to budburst (in August), because of lack of time. To prevent damage to the vines from the tags being too tight, each year all tags were removed in winter after pruning, and new l-year-old spurs and canes were tagged prior to budburst. As much as possible, spurs derived from spurs that were tagged in the previous season were tagged again. However, sometimes the spur had been pruned off and sometimes both buds had died or become stunted, so there was no spur to tag. In these cases, different spurs, if possible from the same vine, were tagged. Approximately 70-857o of the tagged spurs in growing seasons 1995196 and 1996197 were derived from spurs that had been tagged in the previous season. 86 6.2.2Preliminary examination of budburst in bleached grapevine spurs The tagged bleached Chardonnay spurs in each of the four vineyards (Ashton Hills, Barratt's vineyard, Hargrave's vineyard and Hillstowe) and Riesling spurs in Ashton Hills (section 6.2.1) were also assessed for budburst and stunted growth of new shoots in the three successive growing seasons. Apparently healthy Chardonnay spurs in Barratt's vineyard and Hillstowe were included in the 1996197 season as controls (section 6.2.I). The buds/new shoots on the tagged spurs were examined in spring (October/l{ovember) when the vines were in stages G, H or I (Baggiolini, 1952) and in early winter (June) after leaf fall but prior to pruning. In spring, shoots that were longer than 5 cm were classified as normal size shoots (designated 'n'), shoots that were shorter than 5 cm were classified as stunted (designated 's') and buds that did not burst were classified as dead (designated 'd'). In June, shoots that were longer than25 cm and shoots that had borne bunches of grapes were classified as normal size shoots ('n'). Shoots that were shorter than 25 cm and had borne no berries were classified as stunted ('s'). Buds that did not burst and shoots that were broken off or had died were classified as dead ('d'). As each tagged spur had two buds, each spur was classified into one of six categories (nn, ns, nd, ss, sd or dd), depending on the health of the two buds. For example, a spur on which one bud grew into a shoot of normal size and the other produced a stunted shoot, was designated 'ns'. The numbers of spurs in each of the six categories were used to compare different treatments, using 12 analysis (Pagano, 1986), as follows: budburst on the apparently healthy spurs in Barratt's vineyard and Hillstowe in 1996197, was compared with budburst on bleached spurs in the same vineyards and season; budburst on bleached Riesling spurs in Ashton Hills in June 1996 was compared with that of bleached Riesling canes in the same vineyard and date; and budburst on bleached Riesling spurs was compared with budburst on bleached Chardonnay spurs in Ashton Hills in the three consecutive seasons. 87 6.3 Results 6.3.1 Monitoring of grapevines infected with Phornopsís taxon L The disease incidence was determined in September of most years (Table 6.1), as many new spurs in Barratt's vineyard and in Hillstowe were not bleached until September. In September 1994 and 1995, the disease incidence was high (90Vo-I007o) in all grapevine plots, except for Barratt's vineyard, where 707o and 657o of the l-year-old spurs were bleached and bore pycnidia, i¡ 1994 and 1995, respectively (Table 6.1). However, the remaining 3OVo and357o of the spurs did not look healthy and appeared to be infected with organisms other than Phomopsis or had been damaged by hail or wind. In September 1996, the disease incidence was high (907o-98%o) in Ashton Hills and Hargrave's vineyard, but lower in Barratt's vineyard (73Vo) and Hillstowe (86Vo, Table 6.1). This, combined with fewer symptoms caused by organisms other than Phomopsis in Barratt's vineyard and in Hillstowe, allowed for tagging of 99 and 38 apparently healthy spurs, respectively. In August 1997, the disease incidence was high in Ashton lltlls (997o and L00Vo), lower in Hargrave's vineyard (85Vo) and low in Barratt's vineyard (4OVo). As the new shoots in Hillstowe had not bleached by August, no data were available for this vineyard in 1997 (Table 6.1). In Barratt's vineyard,2To of the new shoots that were formed on the apparently healthy control canes were bleached, compared with 55Vo of the new shoots from the bleached canes. Phomopsis taxon 1 was isolated from 907o of the bark samples taken from tagged bleached spurs from all grapevine plots in September 1994, from 84Vo of the samples collected in September 1995 and from 887o of the samples collected in September 1996. Pycnidia of Phomopsis taxon I and/or perithecia of D. viticola (Chapter 4) were found on 957o of the bleached canes collected from the vineyard floors. Zone-lines and black marks on some bleached l-year-old and2-year-old spurs were also observed in all grapevine plots. Pycnidia were observed on l-,2- and 3-year-old bleached spurs. Observations of new shoots developing from tagged spurs revealed necrotic lesions on petioles, young leaves and stems in Barratt's (7Vo of the new shoots), Hargave's (lIVo) and Sutherland's (2Vo) vineyards in spring and summer of 1994. Five young shoots with 88 necrotic lesions from each of these vineyards were taken for further examination and culture, but Phomopsis was not isolated from any of the lesions. In the spring and summer of 1995, necrotic lesions were found on petioles, young leaves and stems in Ashton Hills Chardonnay (5Vo of the new shoots), Ashton Hills Riesling (5Vo) and Hargave's vineyard (20Vo). Again, Phomopsis was not isolated from any of the five shoot samples taken from each of the vineyards. Bleached l-year-old wood that had some scarring at the base of the spurs was found on 5Vo of new spurs in Ashton Hills Riesling, on 4Vo of new spurs in Ba:ratt's and on 167o of new spurs in Hargrave's vineyards in winter 1995, and on 6Vo of new spurs in Ashton Hills Chardonnay and on 4Vo of new spurs in Barratt's vineyard in winter 1996. No samples of scarred wood were taken, since the sca:red spurs were bleached and bore pycnidia and were, therefore, likely to be infected with Phomopsis, making it impossible to tell whether the scarring was caused by Phomopsis or by other biotic or abiotic factors. In spring (October/1.{ovember) 1994, when most shoots were in stages E, F or G, some of the buds had not burst (Fig. 6.1): 13.57o of the buds from the tagged spurs in the Ashton Hills Chardonnay plot 15.57o in the Ashton Hills Riesling plot; 7.57o in Barratt's vineyard; 227o in Hargrave's vineyard; 2I7o in Hillstowe and l07o in Sutherland's vineyard had not burst (Table 6.2). A few weeks later @ecember), when the shoots were in stages I and J, approximately half of the delayed buds had burst, but the shoots were very small (< 5cm) and many looked weak and sickly. Buds that had not burst by December 1994 were dead in June 1995 and some of the very small shoots were also dead in June 1995. Bleaching of new shoots was not yet apparent in June. In September, however, many spurs were bleached and speckled with pycnidia in all grapevine plots. In Sutherland's vineyard less than l%o of new shoots were bleached in June 1995, but in September of the same year, 90Vo of the l-year-old spurs were bleached and bore pycnidia (Table 6.1). In the two following seasons, death of buds and stunting of young shoots was recorded in both spring and early winter. Many stunted shoots, shorter than 25 cm, that had borne no berries were observed in early winter in all locations. More dead or stunted buds and shoots were found in early winter than in spring (Table 6.2).In both spring 1996 and winter 1997, in both Barratt's vineyard and Hilstowe, the bleached spurs bore many more dead and stunted buds and shoots than did the control spurs. For example, in spring L996, 89 Figure 6.1 The buds of this bleached grapevine spur, typical of infection with Phomopsis taxon 1, did not burst in November. 90 Table 6.1 Disease incidence, measured as percentage of bleached spurs, in five vineyards in the Adelaide Hills, in three successive growing seasons after pruning and just prior to budburst (September L994,1995 and 1996 and August 1997). vineyard r994 1995 r996 t997 Ashton Hills Chardonnay 90 90 90 99 Ashton Hills Riesling 97 100 98 100 Barratt 70u 65u 73b 40" Hargrave 98 100 93 85 Hillstowe 92 95 g6b d Sutherland 90 90 ' The remaining 30Vo and 357o of the spurs were damaged by organisms other than Phomopsis and./or hail and wind. b Apparently healthy spurs were tagged as controls in these vineyards in this season. " The low score is probably due to the early scoring of bleaching in 1997 (August instead of September). d August was too early in the season to score bleaching in this vineyard. - No data available. Table 6.2 Percentage of buds and shoots on tagged bleached or apparently healthy spurs that were dead or stunted, in five vineyards in the Adelaide Hills, in spring and early winter of three grape growing seasons. Vineyard bleached spurs bleached spurs bleached spurs apparently healthy spurs spring'94 spring'95 winter'96 spring'96 winter'97 spring'96 winter'97 Ashton Hills C. 13.5 10.9 14.3 13.6 L7.6 Ashton Hills R. 15.5 10.0 23.3 8.3 16.2 Barratt 7.5 12.4 25.0 16.7 29.5 3.2 9.6 Hargrave 21.9 18.1 38.2 9.5 18.9 Hillstowe 20.9 16.7 27.3 12.3 33.7 3.9 15.8 Sutherland 10.0 Ashton Hills C. Ashton Hills Chardonnay. Ashton Hills R. Ashton Hills Riesling. No data available. 9l 16J7o of the buds/shoots from tagged bleached spurs in Barratt's vineyard were dead or stunted, compared with 3.2Vo of the buds/shoots from the control spurs. Similarly, in Hillstowe, l2.3Vo of the buds/shoots from tagged bleached spurs were dead or stunted, compared with 3 .9Vo of the buds/shoots from the control spurs (Table 6.2) . Only a few shoots from tagged spurs broke off: two young shoots had broken off in Hillstowe in spring 1994 as had one new cane in Ashton Hills Riesling in June 1997 and two shoots in Ashton Hills Chardonnay in June 1997. 6.3.2 Preliminary examination of budburst in bleached grapevine spurs For each tagged spur, the health of the two buds/shoots was recorded in spring and in early winter (Table 6.3). In spring, the tags were often difficult to find amongst the foliage and, occasionally, pruning had already started when the spurs were examined in June (e.9. Barratt's, winter 1996). This explains the difference in the number of tagged spurs found in spring and in early winter (Table 6.3). In all grapevine plots, most tagged bleached spurs bore two normal sized shoots (nn), in both spring and winter of the three growing seasons, except in Hargrave's vineyard in June 1996 when 46 out of 110 spurs were classified nn, and in Hillstowe in June 1997 when 44 out of 104 spurs were classified nn (Table 6.3). In all grapevine plots, in both spring and winter of the three growing seasons, the majority of the spurs that did not bear two normal sized shoots, bore one normal sized shoot (ns or nd). Only a small percentage of the tagged bleached spurs bore two buds or shoots that were dead or stunted (ss, sd or dd) in spring (OVo-7.6Vo) and winter (3.6Vo-18.27o) of the three seasons. Bleached Riesling canes and spurs were scored separately for budburst in Ashton Hills, in June 1996 (Tables 6.3 and 6.4). 77.37o (17 of 22) of the buds/shoots from the tagged canes were assessed as dead or stunted, compared with I6.7Vo (30 of 180) of the buds/shoots from the tagged spurs. 12 analysis (Pagano, 1936) was used to compare budburst on apparently healthy spurs with budburst on bleached spurs in Barratt's vineyard and in Hillstowe, in spring 1996 and early winter 1997. This analysis was also used to compare budburst on bleached Riesling spurs with that of bleached Riesling canes in Ashton Hills in June 1996. The data used for the comparison were extracted from Table 6.3 and are presented in Table 6.4. 92 To meet the requirements of the 12 tests, contingency tables were produced in which the outcome categories that had expected frequencies (f,) < 5 were pooled (Table 6.5). It was not possible to do this for the data for Hillstowe in spring 1996 (Table 6.5C) nor for canes and spurs in Ashton Hills (Table 6.5E), as the sample size was not large enough to create a table with two columns and three rows which would give an expected frequency in each cell of at least 5. If the outcome categories of Table 6.5C and E are pooled in order to create tables of two rows and two columns, then each expected frequency should be at least 10 (Pagano, 1986), but the sample sizes were too small to meet these requirements. Therefore, 12 could not be calculated for the data in Table 6.5C and E. The 12 analysis was, therefore, not used to compare budburst of bleached Riesling spurs with that of bleached Riesling canes, nor was it applied to compare bleached and control Chardonnay spurs in Hillstowe in spring 1996. V/hen budburst on bleached spurs was compared with that on apparently healthy spurs in Barratt's vineyard in spring 1996 and in winter Lgg7,12 values of 20.6 with df = 2 and 23.0 with df = 3, respectively, were obtained (Table 6.5A and B). With a critical probability level (aJevel) of 0.01, the critical 742 value is 9.2 if df = 2, and the critical 12 value is 11.3 if df = 3 (Pagano, 1986). Since the obtained X2 values (20.6 and 23.0, respectively) are larger than the critical 12 values (9.2 and 11.3, respectively), the frequency distributions in Table 6.54 and B are significantly different. The difference between the control and the bleached spurs was highly significant in Barratt's vineyard, in both spring 1996 and early winter 1997. In Hillstowe, in June lgg7, a742 value of 7.6 with df = 2 (Table 6.5D) was obtained. The critical y2 value is 9.2 if cr = 0.01 and df = 2, and the critical 12 value is 6.0 if cL = 0.05 and df = 2 (Pagano, 1986). Since the obtained 12 value (7.6) is less than the critical 12 value for cr = 0.01, but larger than the criticaì 742 value for cx = 0.05, the difference between the contiol and the bleached spurs was significant. In the same way, budburst on bleached Riesling spurs in Ashton Hills was compared with that on bleached Chardonnay spurs in the same vineyard, in three consecutive seasons. In spring I9g4,1995 and 1996, the obtained 12 values were less than the critical 742 value for q, = 0.05; budburst on bleached Riesling and Chardonnay spurs was the same. However, in winter Lgg6, a 12 value of 8.4 with df = 3 was obtained. The critical 12 value is 11.3 if s 93 = 0.01 and df = 3, and the critical y2 value is 7.8 if c¡¿ = 0.05 and df = 3 (Pagano, 1986). Since the obtained 12 value (8.4) is less than the critical 72 value for cr = 0.01, but larger and than the critical 742 value for cr = 0.05, the difference between the bleached Riesling Chardonnay spurs was significant; significantly more buds from bleached Riesling spurs had died or become stunted, than did those from bleached Chardonnay spurs in Ashton Hills in June 1996. In June 1997, there was a highly significant difference between the frequency distribution of bleached Chardonnay and Riesling spurs (262o5t= 1I.43 and12";r= 9.2 for cr = 0.01). However, rryhen the data in categories ns and nd were pooled, no statistically significant difference was found between budburst on bleached Riesling and Chardonnay spurs (X2oar=0.23 and X2"n = 3.8 for cr = 0.05). 6.4 Discussion The primary aim of the observations made in the vineyards in the first season (1994-1995), was to verify the symptoms associated with infection of grapevines by Phomopsis taxon 1 and to distinguish them from symptoms associated with other factors. Bleaching of 1- and 2-year-old canes and spurs, black spots indicating the presence of fruiting-bodies, zone- lines and/or black marks on bleached spurs, stunted growth of new shoots and reduced budburst were the symptoms that were observed most commonly on spurs infected with taxon 1. Pycnidia were not observed on green tissues or berries, and neither the occasional necrotic lesions found on green tissues, nor scarring of woody tissues could be linked to infection by Phomopsis taxon 1. Infection with taxon I appears to be symptomless in spring and summer and, like the disease caused by Phomopsis leptostromiþrmis on lupins, appears to have features of a latent disease (Cowling et dI., 1984; Wood &. Sivasithamparam, 1989). The necrotic lesions on green tissues and scarring of woody tissues may have been caused by hail, wind, mite or insect damage. Only a few infected canes broke off, but the observations did not involve actively bending canes to check for brittleness. Moreover, the most severely infected canes, which may have been the most brittle, were usually pruned out in winter. The symptoms listed above support those described by Merrin et al. (L995) for Phomopsis taxon 1. They associated this taxon with 94 Table 6.3 Number of bleached or control Chardonnay or Riesling spurs from four vineyards and of bleached Riesling canes and spurs from one of the vineyards, that were placed in each of six outcome categories in spring and winter of three growing seasons. Vineyard: Ashton Hills, Chardonnay outcome bleached spurs bleached spurs bleached spurs spring'94 spring'95 winter'96 spring'96 winter'97 nn 79 91 84 87 76 NS 1l 614 152 nd il 810 829 SS 2 22 31 sd I 2I 0t dd 0 l1 T2 total 104 110 L12 Lt4 111 Vineyard: Ashton Hills, Riesling outcome bleached spurs bleached spurs bleached spurs early winter'96 spring'94 spring'95 winter'96 spring'96 winter'97 spurs canes nn 75 86 68 86 75 662 NS 5 28 l0 12 80 nd t4 6 11 7t4 10 I SS 0 o2 01 20 sd 5 26 o2 06 dd I 46 01 42 total 100 100 101 103 105 90 l1 Vineyard: Barratt, Chardonnay outcome bleached spurs bleached spurs bleached spurs control spurs spring'94 spring'95 winter'96 spring'96 winter'97 spring'96 winter'97 nn 85 82 3L 73 58 87 8r NS 7 159 18 14 66 nd 8 54 1l 28 0 11 SS 0 33 T2 00 sd 0 02 29 00 dd 0 01 01 0l total 100 105 50 105 L12 93 99 Vineyard: Hargrave, Chardonnay outcome bleached spurs bleached spurs bleached spurs spring'94 spring'95 winter'96 spring '96 winter'97 nn 7l 74 46 92 80 NS ll l8 22 713 nd 22 ll 22 l0 L2 SS 3 25 1l sd 4 110 06 dd I 25 t2 total t12 108 110 lll tt4 Vineyard: Hillstowe, Chardonnay outcome bleached spurs bleached spurs bleached spurs control spurs spring'94 spring'95 winter'96 spring'96 winter'97 spring'96 winter'97 nn 69 72 61 82 44 35 26 ns 20 15 23 13 30 L6 nd 8 16 15 920 26 ss 6 13 03 00 sd 2 o4 03 00 dd 0 l4 24 00 total 105 105 1r0 r06 104 38 38 nn: both shoots normal size; ns: one shoot normal size, one shoot stunted; nd: one shoot normal size, one shoot dead; ss: both shoots stunted; sd: one shoot stunted, one shoot dead; dd: both shoots dead. 95 Table 6.4Data displayed in Table 6.3 used for A2 analysis to compare bleached spurs with apparently healthy spurs (4, B, C, D) and bleached spurs with bleached canes (E). Vineyard: Barratt, Chardonnay A outcome spring 1996 B outcome early winter 1997 bleached control bleached control nn 73 87 nn 58 81 ns 186 NS 146 nd 11 0 nd 28 11 ss 10 ss 20 sd 20 sd 90 dd 00 dd l1 total 105 93 total T12 99 Vineyard: Hillstowe, Chardonnay c outcome spring 1996 D outcome early winter 1997 bleached control bleached control nn 82 35 nn 44 26 ns 13 I ns 306 nd 92 nd 206 SS 00 ss 30 sd 00 sd 30 dd 20 dd 40 total 106 38 total 104 38 Vineyard: Ashton Hills, Riesling E outcome early winter 1996 spurs canes nn 662 ns 80 nd 10 1 ss 20 sd 06 dd 42 total 90 11 nn: both shoots normal size; ns: one shoot normal size and one shoot stunted; nd: one shoot normal size and one shoot dead; ss: both shoots stunted; sd: one shoot stunted and one shoot dead; dd: both shoots dead. 96 Table 6.5 Contingency tables of the data displayed in Table 6.4, with the expected frequencies (fi) for each of the categories in brackets. The degrees of freedom (dfl) and the obtained 742 for each of the data sets are written underneath each table. Vineyard: Barratt, Chardonnay A outcome spring 1996 B outcome early winter 1997 bleached control bleached control nn 73 (84.8) 87 (7s.2) 160 nn s8 (73.8) 8l (6s.2) 139 ns t8 (12.7) 6 (11.3) 24 ns 14 (10.6) 6 (9.4) 20 nd+ t4 (7.4) 0 (6.6) I4 nd 28 (20.7) 11 (18.3) 39 105 93 198 ss+ 12 (6.e) 1 (6.1) 13 T12 99 2tL df =2, X2 =20.6 df = 3, X2 =23.0 Vineyard: Hillstowe, Chardonnay C outcome spring 1996 D outcome early winter 1997 bleached control bleached control nn 82 (86.1) 35 (30.e) tL7 nn 44 (sr.3) 26 (r8.7) 70 ns 13 (10.3) l (3.7) T4 ns 30 (26.4) 6 (e.6) 36 nd+ 11 (e.6) 2 (3.4) 13 nd+ 30 (26.4) 6 (e.6) 36 106 38 r44 104 38 r42 df = 2, yJ = notpossible to calculate df =2, T,l=7.6 Vineyard: Ashton Hills, Riesling E outcome early winter 1996 canes nn 66 (60.6) 2 (7.4) 68 NS 8 (7.1) 0 (0.e) 8 nd+ 16 (22.3) 9 7) 25 90 l1 101 df=2, = tlot to calculate nd+ outcomes nd, ss, sd and dd combined, to meet the requirement of the test that the expected frequency (f,) in each cell is at least 5, for tables where the number of rows or columns is greater than 2. ss+ outcomes ss, sd and dd combined. 97 bleached l-year-old wood with "flattish, dark lesions with a dark border", now known to be zone-lines and stromata associated with perithecia of D. víticola (Chapter 4, Scheper et aL, 2000). In addition, Merrin et al. (1995) determined that Phomopsis taxon I did not cause scarring of woody tissue nor symptoms on young growth, described previously (Moller et a1.,1982; Emmett et aL,1992; section 2.4), and conidial morphology differed from that of the Phomopsis associated with lesions and scaring, which was designated taxon 2. In studies conducted overseas, on grapevines displaying lesions and scarring symptoms, which are associated with taxon 2 infection in Australia, Bugaret (1990) mentioned stunting of grapevines caused by P. viticola andPscheidt and Pearson (1989b) described reduced bunch set. However, stunted growth of new shoots and reduced budburst has not been mentioned by these authors in relation to infection of grapevines by Phomopsis (section 2.4). Itt older literature, however, when symptoms on grapevines caused by Phomopsis and Eutypa lata were still confused (section 2.3.3), Punithalingam (1979) writes P. viticola causes drying of buds, and Pine (1959) found that 797o of buds on canes showing symptoms of dead-arm disease, did not burst when those canes were planted in soil; the dead-arm fungus was subsequently isolated from747o of the unburst buds. This may mean that Phomopsis taxon 2 on grapevines may also cause bud-death and stunted growth, but more research in this area is needed. Phomopsis vaccinii, however, is known to cause death of buds on blueberry twigs (Milhollan d, 1982). Monitoring of tagged, bleached spurs in the two following seasons, and of tagged control spurs in the last season, confirmed the findings of the first season. However, in order to establish that Phomopsis taxon I causes the symptoms described above, Koch's postulates must be fulfilled. In all five vineyards, a larger percentage of buds and shoots was observed to be dead or stunted in early winter than in spring (Table 6.2). Possible reasons for this are: a) some shoots that were stunted in spring were dead in early winter (June); b) some shoots broke off in summer or autumn and were counted as dead in June; c) the measurement of "stunted shoots" was more conservative in spring than in early winter, so a number of shoots that were, in fact, stunted, were counted as normal length shoots in spring. The data collected in early winter are probably more reliable than those collected in spring, as the outcome of 98 each bud is known in June, whereas in spring it is sometimes too early to predict whether a bud will burst and whether a short shoot will produce berries. Significantly fewer buds from apparently healthy spurs died or were stunted, than did those from bleached spurs in Barratt's vineyard in spring 1996 and winter 1997 and in Hillstowe in winter 1997. This suggests that bud-death and stunted growth of new shoots may be caused by infection of the vines with Phomopsis taxon 1. Moreover, a few buds on the control spurs may have been infected with Phomopsis, causing some of the bud-death and stunted growth found among the controls. Preliminary testing, using a molecular probe (clone pT1P180, Chapter 9), has detected Phomopsis taxon I in diseased buds as well as in a small number of buds from apparently healthy spurs that had failed to burst (Melanson el al.,2O0I; B. Rawnsley, pers. com.), suggesting that bleaching symptoms may be unreliable indicators for disease associated with taxon t. Phomopsis sojae has also been isolated from apparently healthy plants (Kulik, 1984). The possibility that bud-death and stunted gowth on the control spurs may be caused by Phomopsis taxon 1, without causing symptoms, implies that there may be a more significant difference between healthy and bleached spurs than was detected in this study. The occurrence of bud-death and stunted shoots on bleached Riesling and Chardonnay spurs was compared in Ashton Hills. In spring of all three seasons, the distribution of the outcomes (nn, ns and the combined outcomes nd, ss, sd and dd) for budburst on bleached Riesling spurs and Chardonnay spurs was similar. Likewise, in June 1997, similar percentages of buds grew into normal size shoots ¿rmong the bleached Riesling (83.87o) and Chardonnay (82.47o, Table 6.2) spurs, although there were significantly more dead buds on the Chardonnay spurs. However, in June of the previous year (1996), the percentage of dead buds and dead and stunted shoots, was significantly higher for the Riesling (23.3Vo) than for the Chardonnay (I4.4Vo,Table 6.2).In addition, the disease incidence in the Riesling plot was higher than in the Chardonnay plot in the winters of 1994,1995 and 1996 (Table 6.1). While these observations may indicate that Riesling is marginally more susceptible to infection by Phomopsis taxon 1 than is Chardonnay, the disease would need to be monitored for several more years to confirm this. In Hargrave's vineyard, in the first season (1994195), many of the vines were cane- pruned or pruned using the Scott-Henry method, while others were spur-pruned. However, 99 many buds failed to burst in rows where vines were cane-or Scott-Henry-pruned and the owner decided, therefore, to spur-prune most vines in winter 1995. In the following two seasons, the percentage of buds/shoots that died or was stunted, declined to approximately half (Table 6.2), and the disease incidence decreased by approximately IlVo (Table 6.1). Similarly, a preliminary study in Ashton Hills, comparing budburst on bleached Riesling spurs with that on bleached Riesling canes (Tables 6.4E and 6.5E), showed that a larger percentage of buds/shoots on canes were dead or stunted compared with those on spurs. However, this result was not statistically significant, due to the small number of canes observed. Nevertheless, the observation that bud-death decreased in Hargrave's vineyard when it was converted from cane- or Scott-Henry-pruned to mainly spur-pruned, may indicate that spur pruning is a better management strategy to control disease associated with Phomopsis taxon 1 in cool and wet climates than is cane pruning, although a large number of bleached canes and spurs would need to be monitored over time to confirm this. A possible explanation may be that there is less inoculum present in spur-pruned vineyards, providing that the pruned, diseased canes are removed from the vineyard floor and destroyed. Pscheidt & Pearson (1989o), who examined vines that displayed lesion and scarring symptoms, also found that pruning methods may have a significant effect on the amount of disease in a vineyard (section 2.6.I). However, they compared vines which were hand-pruned using Umbrella Kniffin, Top-Wire Cordon and hedged Top-Wire Cordon systems. Reduction of inoculum by selectively pruning diseased shoots resulted in a significant reduction of Phomopsis shoot blight of peach trees (Uddin and Stevenson, 1998). A similar study on grapevines, where plots of vines with all bleached spurs removed, are compared with plots of unpruned vines, would show whether the amount of Phomopsis taxon I inoculum influences the percentage of dead buds and stunted shoots, or the disease incidence in the following season. However, such a study will need to be carried out in vineyards with disease incidences lower than 50Vo, otherwise in the pruned plot, few spurs will be left to give rise to new shoots. Whether or not death of buds and stunted growth of young shoots, which may be due to infection by Phomopsis taxon 1, translates into yield loss is uncertain. More research is needed to assess this. For instance, the weight of bunches from control spurs should be compared with the weight of bunches from bleached spurs in the same vineyard. In the 100 USA, Pscheidt & Pearson (19S9b) detected reduced bunch set with yield losses of 20-30Vo in seasons conducive to the disease. However, they were investigating grapevines that displayed the lesion and scarring symptoms associated with taxon 2 in Australia. The disease incidence and percentage of buds and shoots that died or were stunted, shown in Tables 6.1, 6.2 and 6.3, varied from season to season and from vineyard to vineyard. This may be caused by different weather conditions which influence the microclimate within the vine canopy and, therefore, the inoculum density and the progression of the disease. However, weather data from the Bureau of Meteorology are not likely to be very useful, since each vineyard in the Adelaide Hills is likely to have a different microclimate due to differences in the terrain as well as vine canopy management, spacing, pruning methods, and other characteristics of the vineyards and vines @.L. Melanson, pers. com.). Therefore, in order to assess the effect of the weather on the disease associated with Phomopsis taxon 1, weather data (rainfall, wind speed, humidity, temperature) should be collected from each of the vineyards, preferably using weather stations located within each grapevine plot. The analyses in this chapter were done using the nonparametnc y2 test. This test was chosen, because a) the analyses involved nominal data, i.e. the six discrete and mutually exclusive outcome categories, and b) population characteristics such as the distribution of the populations and the homogeneity of variance were unknown. The 12 test is unaffected by such variations in population characteristics. Analysis of Variance to compare vineyards and seasons was not deemed appropriate, given that no hypothesis had been formulated that could be tested with this analysis. Within this context, it would be difficult to identify statistical difference with statistical meaningfulness. Any possible differences between vineyards and/or seasons could be due to any number of unknown interacting variables including weather, vineyard and shoot debris management, age of the vines, size of the vineyards, and the vineyard characteristics that contribute to the unique microclimate of each vineyard, as mentioned above. Without an initial hypothesis, no matter what the result of an ANOVA, the analysis would not contribute information relevant to Phomopsis taxon 1. Uddin & Stevenson (1993) used ANOVA to compare four treatments of peach trees infected with a Phomopsis 101 sp. in three seasons and two counties in Georgia, USA, but they too did not compare seasons or counties. In conclusion, the field studies described in this chapter confirm that Phomopsis taxon I is associated with bleaching of woody tissues, without scarring of those tissues. In addition, the results indicate that stunted growth of new shoots and reduced budburst occur more frequently on bleached spurs than on apparently healthy spurs. However, the field results need to be verified by glasshouse experiments, in which grapevines are inoculated with Phomopsis taxon 1 conidia and D. viticola asci and ascospores (Chapter 7). r02 7. PATHOGENICITY AND SEED TRANSMISSION 7.1 Introduction The two main taxa of Phomopsis on grapevine in Australia are associated with different symptoms. Taxon 2 is associated with scarring and bleaching of l-year-old wood, dark spots with yellow haloes on leaves and dark elongated lesions on young shoots and petioles (Merrin et aI., 1995) and is considered to be P. viticola elsewhere in the world (Mostert e/ al., 2001). Taxon 1 is only associated with bleaching of 1- and 2-year-old wood and reduced budburst. In South Australia, a large percentage of grapevine spurs showed bleaching symptoms associated with Phomopsis taxon 1, with disease incidence in individual vineyards ranging from 65Vo to t007o (Chapter 6). Moreover, significantly fewer buds on bleached spurs burst, compared with apparently healthy spurs. Koch's postulates have not been fulfilled for any of the taxa of Phomopsis on grapevines and none of the taxa have been shown to cause symptoms on grapevines in a glasshouse. Infection of plants by other Phomopsis spp. has been achieved with or without prior wounding of the host (Milholland, 1982;Herr et aI., L983; De Nooij & Van Damme, 1988; Williamson et al., I99I; Linders, 1994; Shivas, 1994: Crump et aI., 1996; Uddin & Stevenson, L997). Blueberry, lupin and Emex australis plants were inoculated by spraying to "run-off' with conidial suspensions (106 cr-conidia ml-l) of P. vaccinü, P. leptostromiþrmis and P. emicis, respectively (Milholland, 1982; Williamson et al., l99I; Shivas, 1994), while wounded Plantago lanceolata L. plants and peach trees were inoculated with a single drop of conidial suspension of P. subordinaria and Phomopsis sp., respectively (De Nooij & Van Damme, 1988; Uddin & Stevenson, 1997). Therefore, an attempt was made to test the pathogenicity and mode of infection of isolates of Phomopsis on grapevines in the glasshouse in order to fulfil Koch's postulates and confirm observations made in the field. In addition, possible seed transmission of representative isolates of Phomopsis was examined, to check whether symptoms caused by this fungus would appear on grapevines grown from infected seeds. 103 7 .2 Materials and methods 7 .2.1 Preliminary pathogenicity study A preliminary pathogenicity test was conducted to determine whether wounding of grapevine plants was required for infection by Phomopsis. A. Plant material Hardwood cuttings of V. viniþra cy. Chardonnay, clone Il0B1, disinfected with quinolone (8-hydroxy-quinoline sulphate, sold as Chinosol W@), were obtained from Registered Source Areas (Riverland Vine Improvement Committee, Barmera, South Australia). The cuttings were hydrated by submerging them in RO water for 24 h, RO water containing Milton bleach (0.0O047o available chlorine) for 24 h and RO water for 24 h (Neldner et al., lgg3),before dipping in lime sulphur (50 ml l-t¡ to prevent bud mite infection. The cuttings were propagated using a technique adapted from Mullins & Rajasekaran (1981), which ensures that formation of adventitious roots precedes budburst. Cuttings were cut directly below the lowest bud, and placed in sterile sand in a thermostatically controlled heated container (25"C at the bases of the cuttings) in a cold room (4"C) in darkness for 5-6 weeks. The sand was kept moist by spraying with water on alternate days. Twenty-six rootlings were then planted into 20 cm diameter pots containing U.C. potting mixture (Baker, 1957) and placed in a glasshouse with a day temperature of 23oC (8.00 am - 7.00 pm) and night temperature of 14oC - I7"C. After 6 weeks, the rootlings, now with shoots, were fertilized fortnighrly with a complete water soluble fertilizer (1.6 g l-r Aquasot@ (¡fpf 23:4:18), Hortico, Laverton North, Victoria), until leaf fall in late autumn. Predatory mites (Phytoseíulus persimilis, BioProtection Pty Ltd, Warwick, Queensland) were released in the glasshouse to control two-spotted mites (Tetranychus urticae). B. Inoculation and assessment of symptoms Inoculum was produced by subculturing nine taxon 1 isolates, four taxon 2 isolates and three taxon 3 isolates on PDA and CLA as described in section 3.1, until pycnidia with cirrhi had formed on the majority of plates. Of these isolates, four taxon I isolates, three taxon 2 isolates and two taxon 3 isolates, listed in Table 7.1, produced pycnidia with cirrhi and were used to inoculate the grapevines. The cirrhi were removed with a sterile needle 104 and suspended in 1 ml sterile RO water containing 0.0057o Tween 20 as a surfactant. Concentrations were adjusted with the aid of a haemocytometer to 106 o-conidia ml-r. The viability of the conidia was assessed by streaking aliquots on PDA, incubating for 8 h at 23"C anddetermining the percentage germination, which ranged between 80Vo and lOO7o. The grapevines were inoculated in early summer 1994 (December), 10 days after planting the cuttings, when the buds were in stages D and E (Baggiolini, 1952). Two methods were used. The first was modified from that used by Milholland (1982), Williamson et aI. (1991) and Shivas (L994), except that 4 ¡rl droplets were placed on the breaking buds until "run-off'. The second method was modified from that of De Nooij & Van Damme (1988) and Uddin & Stevenson (1997), except that a 10 ¡¿l drop of conidial suspension (106 a-conidia ml-l) was placed on breaking buds and the wound was made through the drop with a sterile 0.41 mm needle. Each Phomopsis isolate was inoculated onto the breaking buds of two grapevine plants. One plant was inoculated with the non- wound method, and the other plant with the wound method. Control plants were "inoculated" using both methods, with sterilised RO water containing 0.005Vo Tween 20. The inoculated grapevines were arranged randomly on the glasshouse bench, interspersed with uninoculated plants. The inoculated buds were covered immediately with clear polyethylene bags, to simulate a dew period, which were removed 70 h later. Six weeks after inoculation (January 1995), disease severity was evaluated by assessing the percentage of necrotic area per leaf according to Emmett's Phomopsis leaf spot assessment key @mmett, R.W.,1989, unpublished). After leaf fall, in winter (August 1995), the plants were examined for bleaching and scarring of the woody tissues and pruned to two buds per spur. In spring 1995, the vines were examined twice (October and November) for symptoms associated with infection with Phomopsis, and they were examined and pruned in August 1996 and July 1997. In July 1997, excised canes were placed in moist conditions at 15oC (section 3.1) and incubated for 1 to 4 months. to assess the formation of pycnidia or perithecia as an indicator of infection. Three of the uninoculated grapevines that did not show any symptoms of infection with phomopsis, were inoculated with ascospore suspensions 110s-10ó ascosPores ml-l¡ of D. viticola. in spring 1995 (October), when the buds were in stages D-F (Baggiolini, 1952). The ascospores were obtained from perithecia on Chardonnay, Riesling and Shiraz canes 105 collected in vineyards in South Australia (isolates 82, 83, 84 and 85) and were suspended in 200 pl sterile RO water containing 0.O05Vo Tween 20. The plants were inoculated by placing 4 pl droplets on the breaking buds until "run-off', and covering with clear polyethylene bags, as described above. The viability of the ascospores, assessed by percent germination on PDA at23"C after 8 h, was 80Vo - L007o. One month after inoculation, the vines were assessed for symptoms, then re-examined and pruned in August 1996 and July IggT.In July lgg7, excised canes were surface sterilised, placed in moist conditions at 15'C (section 3.1) and incubated for I to 4 months to assess infection. T.2.2Pathogenicity of D. vi.ticola and Phornopsís on grapevine A. Plant material Chardonnay rootlings, clone I10V1, were obtained from Ken Carypidis, PO Box 258, Virginia, SA. The rootlings were soake d in 0.5Vo (w/v) Chinosol W@ for 4 h in at 4oC, drained, and stored in a sealed black plastic bag at 4oC. The rootlings were rehydrated and dipped in lime sulphur as described in section7.2.I. Forty-five rootlings with healthy root systems were planted in 25.5 cm diameter pots containing recycled soil and placed in a glasshouse with a day temperature of zO"C - 25oC and night temperature of 13oC - 18oC. The rootlings were fertilized as described in section7.2.I. Miticide sprays (a mixture of propargite, sold as Omite@ and closentizine, sold as Apollo@, in rotation with a mixture of kelthane (dicofol) and tetradifon, sold as Mastalvlite@) were applied by the glasshouse staff approximately I month after inoculation in October and Novembet 1996 and again in January 1997. Predatory mites were released in the glasshouse when the rootlings were planted and after the sprays were applied. B. Inoculation and assessment of symptoms Ascospores of D. viticola were obtained from perithecia on Cabernet, Chardonnay, Pinot Noir and Riesling canes collected in vineyards in South Australia and Tasmania. Inoculum was produced by suspending asci and ascospores in sterile RO water containing 0.0057o Tween 20 (10s-10u ascospo."s ml-r). Similarly, cirrhi were suspended in sterile RO water containing O.005Vo Tween 20 (10s-106 conidia d-t). The cirrhi were obtained from pycnidia on Chardonnay and Riesling canes collected in vineyards in South Australia and 106 Tasmania, because isolates of Phomopsis subcultured on various fungal media did not produce enough cinhi for inoculation purposes. Before use, the conidia were examined with a light microscope to check whether they belonged to taxon I or taxon 2. The viability of the ascospores and conidia was assessed by placing 5 p¿l of each inoculum on sterile cellophane on WA, incubating the plates as described in section 3.1 for 2 days and examining the pieces of cellophane for germination of spores under a light microscope. The grapevines were inoculated in spring 1996 when the shoots were in stages E and F (Baggiolini, 1952) and were 4 to 10 cm tall. As the buds on the rootlings did not burst synchronously, 12 plants were inoculated on the 30th of September, nine on the 11ü of October and 19 on the lSth of October (Table 7.2). One shoot per vine was inoculated by placing 75 x 4 ¡rl droplets on the shoots. Control plants were inoculated with sterilised RO water containing 0.0057o Tween 20. Five vines remained uninoculated. The inoculated grapevines were aranged randomly on the glasshouse bench, interspersed with the uninoculated plants, and covered with clear polyethylene bags as described in section 7.2.I. The vines were assessed for symptoms 4 to 6 weeks after inoculation (15th of November), 3 months after inoculation (January 1997) and in winter after leaf fall (August 1997). 7.2.3 Seed transmission Healthy Chardonnay berries, clone I10V1, were collected from the Coombe vineyard at the Waite Campus of the University of Adelaide in March 1995. Total soluble solids of 35 individual berries, measured with a hand-held refractometer (Nippon Optical Works Co., Ltd) at 20"C, varied between 22.4o and 27.6"8nx, with an average of 24.9"8nx. The bunches were washed thoroughly to remove residues of wettable sulphur and copper oxychloride. The bunches were surface sterilised by spraying with 95Vo ethanol, leaving them for 5 min and, subsequently, submerging them in bleach (l7o avallable chlorine) for I min followed by three rinses in sterile RO water. The bunches were stored for 3 days, at 2"C, in beakers filled with sterile RO water covered with Parafilm@. Three days later, the bunches were dried on paper towels and all berries that were shriveled, burst, scarred or otherwise damaged were removed with sterile tweezers. As berries are prone to fungal and bacterial contamination because of their high sugar content, the bunches were surface sterilised again using a technique modified from Petrini (1986), by sequential immersion in 107 757o ethanol for 60 s, sterile RO water, bleach (2Vo available chlorine) fot 2 min, sterile RO water, 757o ethanol for 30 s and three rinses in sterile RO water, before drying on sterile filter paper in a laminar flow cabinet. Inoculum was produced by subculturing a total of II2 taxon 1 isolates, six taxon 2 isolates and three taxon 3 isolates on PDA and CLA as described in Chapter 3, until pycnidia with cinhi were present. One day before inoculation, 17 taxon I isolates, four taxon 2 isolates and two taxon 3 isolates (Tables 7.3 &.7.4, except taxon 1 isolate 248), which had produced pycnidia with cirrhi, were selected to inoculate the berries. Cirrhi were removed with a sterile needle and suspended in sterile RO water containing 0.0057o Tween 20. Concentrations were adjusted with a haemocytometer to 5 x 10s cr-conidia ml-I. In addition, a suspension of conidia from taxon 1 isolate 24E, which had been stored in sterile RO water containing 0.0057o Tween 2O at 4"C for 3 months, was also used to inoculate berries. The viability of the conidia was assessed by placing 5 pl of each inoculum on sterile cellophane on WA and PDA, spreading the drop with a sterile loop and incubating the plates at23"C for 8 h. The pieces of cellophane were examined as described above. Eight berries, from eight different bunches, were placed in each of 54 sterile 250 ml polycarbonate tubs (Disposable Products Ltd, South Australia). Each of the 24 conidial suspensions \ryas used to inoculate two tubs of berries, by placing a 10 pl droplet onto each pedicel. As controls, berries in three tubs were inoculated with sterile RO water containing 0.O05Vo Tween 20 and the remaining three tubs were not inoculated. The tubs were arranged randomly in an incubator at 23"C for 3 months in a cycle of 16 h light (Philips TLD l8W 133 3F,380-780 nm) and 8 h dark. The berries were scored for berry colour and presence of mycelium, 3 days after inoculation, and for berry colour, presence and colour of mycelium, and presence of pycnidia, 1, 3 and 4 weeks after inoculation. Two months after inoculation, Phomopsis was re-isolated from the benies by excising pieces of the skin (2 mm2¡ and the pulp (2 mm3¡, surface sterilising by immersion jn707o ethanol for 30 s followed by three rinses in sterile RO water, before placing them on WA. The plates were incubated as described in section 3.1. Beny sections were observed for the presence of Phomopsls after 1 and 4 months. Seeds were separated from the pulp and skins, rinsed in sterile RO water, surface sterilised ín70Vo ethanol for 30 s and rinsed three times in sterile RO water. They were then 108 placed in 54 steril e 250 ml tubs containing sterile, moist perlite, and incubated at 2"C for 12 weeks. Seeds were surface sterilised again in 707o ethanol for 30 s, rinsed in sterile RO water, and germinated on sterile moist filter paper in 9 cm Petri dishes at 20oC (Gadoury & Pearson, 1991). In early suÍuner (6 December 1995), the seedlings were planted into l0 cm diameter pots containing U.C. potting mixture (Baker, 1957) and placed in a glasshouse with a day temperature of 23'C (8.00 am - 7.00 pm) and night temperature of l4oC - L7"C. The seedlings were fertilised fortnightly as described in section 7.2.I. The plantlets were examined for symptoms in autumn and winter (May and August L996), and were re- examined and pruned in July 1997. The excised twigs were surface sterilised, placed in moist conditions at 15oC (section 3.1) and incubated for 1 to 4 months to assess if infection had occurred by the appearance of pycnidia or perithecia' 7.3 Results 7.3.1 Preliminary pathogenicity test Six weeks after inoculation, one of the vines that had been wounded and treated with water and Tween had a small amount of necrosis (0.8Vo) on the first leaf, but the remaining controls were healthy (Table 7.1). The four vines treated with taxon 1 isolates using the non-wound technique, showed some necrosis (0.47o - 57o) of the inoculated leaves. Iæaves that had formed after inoculation were app¿uently healthy. The vines inoculated with the same isolates using the wound-inoculation technique, showed a similar level (IVo - 3Vo) of necrosis of the inoculated leaves and the vine inoculated with isolate 24E also had one dead 1¡¿af Q00Vo necrosis, Table 7.1). However, no necrotic spots with yellow haloes, typically associated with taxon 2 (section 2.4.I), were detected on any vines inoculated with taxon I isolates. In contrast, leaves of all three vines inoculated with taxon 2 isolates using the non- wound technique, showed the characteristic lesions (Table 7.1). The vine inoculated with isolate DAR 69457 had black spots with yellow haloes on the first (37o necrosis, Fig. 7.1) and the second (0.4Vo necrosis) leaf. Similarly, two leaves on the vine inoculated with isolate 46 developed the characteristic spots, while the first leaf on the vine inoculated with isolate DAR 69486 was dead and the subsequent three leaves also showed the spots (Table 109 7.I).Lr,aves that had formed after inoculation, were apparently healthy. All breaking buds inoculated with taxon 2 isolates using the wound-inoculation technique died, and a second and, occasionally, a third shoot was formed, which was apparently healthy. The four vines inoculated with the taxon 3 isolates using the non-wound and the wound techniques were generally healthy, although they had a small amount of necrosis (07o - L67o, Table 7'1). In winter, after leaf fall, no bleaching or scarring of woody tissues was observed on any of the vines, except slight bleaching of one cane on the vine inoculated with taxon 2 isolate DAR 69486 using the wound-inoculation technique. No pycnidia were observed on any of the vines. Two vines died in winter; one was a control plant and the other had been wound-inoculated with taxon 2 isolate 46. In October 1995, the seven control plants showed no symptoms of infection with Phomopsis. Three of the control plants had one or more shoots in stages D-F (Baggiolini, 1952, Table 7.1), the remaining control plants had 20 - 40 cm long shoots. The vines inoculated with the four taxon 1 isolates showed no symptoms of disease. All shoots were 20 - 40 cm long, except for those inoculated with isolate 42A and one shoot inoculated with isolate 248, which were in stages B-E (Table 7.I). Of those inoculated with taxon 2 isolates, only the vine non-wound-inoculated with isolate 46 had a spur with scarring; the scarring extended the entire 2 cm base of the spur. Vines that were inoculated with isolates DAR 69457 and DAR 69486 did not show symptoms typical of taxon 2.The shoots on the vines inoculated with taxon 2 isolates were 20 - 40 cm long, except for those non-wound- inoculated with isolates DAR 69457 and DAR 69486, which were in stages F and E, respectively (Table 7.1). The shoots on the vines inoculated with isolates belonging to taxon 3 were 20 - 40 cm long, except for the vine non-wound-inoculated with isolate DAR 69458 which had buds in stages D and E (Table 7.1). One month later, the basal 2 cm of the spur with scarring symptoms was still scarred but not bleached. Four of the inoculated vines that had both shoots shorter than 20 cm in October, now had one shoot that was growing strongly and one that remained weak. In August 1996, neither bleaching of woody tissue nor pycnidia were observed and the scarring of the spur inoculated with taxon 2 isolate 46 had become less evident. The weak shoots on the vines wound-inoculated with isolates 248 and 42A and non-wound- 110 Figure 7.1 Chardonnay leaf showing necrotic spots with yellow haloes typical of infection with Phomopsis taxon 2, 6 weeks after inoculation with taxon 2 isolate DAR 69457, without wounding, in the glasshouse. 111 Table 7.1 Symptoms observed on Chardonnay grapevines over a period of 3 years, after inoculation with Phomopsis isolates in the preliminary pathogenicity test. Symptoms were observed 6 weeks after inoculation (January 1995), 1 year after inoculation in spring (October 1995), in August 1996 and in July 1997. uleaf bstage Phomop sis Isolate, Taxon Symptoms of Shoots Health of Canes Health of Canes & Method of Inoculation Jan. 1995 Oct. 1995 Aue. 1996 July 1997 238 Tl non-wound necrosis (3Vo &5Vo) both > 20 cm both healthy 1007o healthy 248 T1 non-wound necrosis (0.8Vo) both > 20 cm both healthy 25Vo dead 388 Tl non-wound necrosis (0.4Vo & 0.8Vo) both > 20 cm both healthy 257o stunted 42A Tl non-wound necrosis (0.87o & 0.8Vo) B&B I dead, I healthy 507o stunted 238 Tl wound necrosis (0.87o) both > 20 cm both healthy 50Vo dead 248 Tl wound 1 dead, I necrosis (37o) E&>20cm I dead, I healthy 50Vo dead, 388 Tl wound necrosis (O.8Vo) both > 20 cm both healthy 507o dead 42A Tl wound no symptoms C&D I dead, t healthy 1007o healthy DAR 69457 T2 non-wound spots (37o &O.4Vo) F&F both healthy '508o d &5OVo s DAR 69486 T2 non-wound 1 dead, 3 x spots (I.6Vo) E&E I dead, t healthy 1007o healthy 46 T2 non-wound spots (1.57o &.37o) both > 20 cm* both healthy 5OVo dead rs\Eo DAR 69457 T2 wound bud death both > 20 cm both healthy d &25Vo s DAR 69486 T2 wound bud death both > 20 cm both healthy 507o dead 46 T2 wound bud death DAR69458 T3 non-wound no symptoms D&E 1 dead, I healthy 50Vo dead JM 139 T3 non-wound necrosis (0.8Vo) both > 20 cm both healthy 75Vo dead s2OVo D4R69458 T3 wound necrosis (O.8Vo) both > 20 cm both healthy d &60Vo s r25Eo JM 139 T3 wound necrosis (I.6Vo) both > 20 cm both healthy d & 507o s water & Tween non-wound no symptoms both > 20 cm both healthy 25Vo dead water & Tween wound necrosis (O.8Vo) both > 20 cm both healthy 207o dead uninoculated no symptoms both > 20 cm both healthy 25Vo stunted uninoculated no symptoms both > 20 cm both healthy 507o dead, uninoculated no symptoms F&F c uninoculated no symptoms F&F d d uninoculated no symptoms D&>20cm e uninoculated no svmDtoms " For each treatment, one bud was inoculated; 0 to 4 leaves grown from each inoculated bud showed symptoms. o Shoots were scored according to Baggiolini's (1952) stages, except when the shoots were more than 2O cmlong, as the young vines did not produce inflorescences and stages G-N 'were not relevant. Shoots that were not stunted were designated "> 20 cm". " The young shoots were inoculated with asci and ascospores of isolate 82 in October 1995. o The young shoots were inoculated with asci and ascospores of isolates 83 and 84 in October 1995. " The breaking bud was inoculated with asci and ascospores of isolate 85 in October 1995. t Of the four buds, 507o or 257o died before or after budburst and 50Vo or 25Vo of the shoots became stunted. s Of the five buds,207o died before budburst and 607o became stunted. * Scaring visible on the spur, but no stunted shoots present. - The vine died in winter 1995. tt2 inoculated with isolates 42A, DAR 69486 and DAR 69458 had died (Table 7.1). All other inoculated vines, as well as the control vines, had healthy new canes. In July 1997 , no bleaching or scarring of woody tissues or pycnidia were observed on any of the vines. Dead buds and stunted shoots occurred on both the control and inoculated vines: 20Vo to 50Vo of the buds and shoots on the control vines had died or were stunted compared with 07o to 507o of the buds on vines inoculated with taxon I isolates; \Vo to 75Vo of the buds on taxon 2 inoculated vines and 50Vo to 80Vo of the buds on taxon 3 inoculated vines (Table 7.1). In August 1997, 1 month after prunings from inoculated and control vines were placed in moist conditions at 15oC, one cane from the vine wound-inoculated with'isolate 238, showed zone-lines, pycnidia and cirrhi containing cr-conidia (6-7 pm) and p-conidia. The remaining canes did not show signs of infection with Phomopsis. By November, immature perithecia had also developed on the cane from the vine inoculated with isolate 23B.In addition, pycnidia and cirrhi containing cr-conidia (10-11 pm) and p-conidia were present on canes from the vine inoculated with isolate 46. In November 1995, I month after inoculation with ascospores of D. viticola, the shoots inoculated with isolates 83 and 84 showed no symptoms of infection. The shoot which was inoculated with isolate 85 had died. One of the shoots inoculated with isolate 82 had a leaf with extensive necrosis around the edges (25Vo necrosis), the other shoot was apparently healthy. In August 1996, no bleaching or scarring of woody tissues or pycnidia were observed on any of the vines. All infected buds and shoots had grown into healthy canes, except for the bud inoculated with isolate 85, however, a new healthy cane had grown from a bud situated next it. In July 1997, there was still no bleaching or scarring of woody tissues and pycnidia were not present. As above, dead buds and shoots were observed: jVo to 67Vo of the buds on vines inoculated with D. viticola isolates died, compared with 25 7o to 50Vo of the buds and shoots on the control vines. The prunings from the vines inoculated with ascospores showed no signs of infection with Phomopsis, I,2 or 4 months after they were placed in moist conditions at 15"C. 113 T.3.2Pathogenicity of D. vìtícola and' Phomopsis on grapevine The viability of the conidia and ascospores assessed by germination on cellophane over WA, ranged from 307o to 907o (Table 7 .2). Four to 6 weeks after inoculation, most vines had mite damage, obscuring any symptoms which may have been causedby Phomopsis or D. viticola. Fourteen vines had leaves with necrotic spots surrounded by yellow haloes, with the area affected ranging from 27o to 507o. However, the vines with leaf spots included uninoculated vines, vines treated with water and Tween, vines inoculated with ascospores and vines inoculated with conidia from taxon I and taxon 2 isolates (Table7.2). Three months after inoculation, all vines were covered in mites and their webs. The control plants showed the same damage as the inoculated vines. In winter, neither scarring of woody tissues nor pycnidia were observed on any of the vines. Some canes were slightly bleached (42Vo), but this occurred for all treatments. The length of the inoculated canes was 15 to 150 cm and the length of the uninoculated canes and those treated with water and Tween was 30 to I20 cm (Table 7 .2). Both control and inoculated vines were among the 27Vo of the vines that bore berries. There was no relationship between the presence of berries and the presence of symptoms. 7.3.3 Seed transmission Germination of conidia was greater on cellophane over PDA than on cellophane over WA. On PDA, most isolates had a germination rate of 95 - l00Vo, except isolates 20C,238 and 38B, which had a rate of 707o (Table 7 .3). Three days after inoculation, the inoculated berries had become slightly red and white mycelium was growing on the pedicels. The uninoculated berries and those treated with water and Tween were still green and no mycelium was observed. One week after inoculation, all inoculated berries were covered with white mycelium and most were red./brown in colour, except the 16 berries inoculated with taxon I isolate 248, one berry inoculated with taxon I isolate 23C and two berries inoculated with taxon 2 isolate 46, which were still green. The uninoculated berries and those treated with water and Tween were also green and no mycelium was visible' Ll4 Table 7.2 Date of inoculation, origin and viability of Phomopsis and D. viticola isolates used to inoculate Chardonnay rootlings, and the symptoms observed on the vines 4 to 6 weeks after inoculation (15 November) and in winter (August). uSymptoms, Inoculation Inoculum Spore Cane Length Date Germination 15 Nov. 1996 Aue. 1997 30 Sept. 96 T1, Hargrave's, SA, Chardonnay 907o none (mite damage) 60 cm, B 30 Sept. 96 Tl, Hargrave's, SA, Chardonnay 907o none (mite damage) 40 cm,50 cm 30 Sept. 96 Tl, Hargrave's, SA, Chardonnay 907o none (mite damage) 80 cm 30 Sept. 96 T1, Hargrave's, SA, Chardonnay 907o leaf spots (57o) 40 cm,60 cm 30 Sept. 96 Tl, Ashton Hills, SA, Riesling 807o leaf spots (IOVo &20Vo) 30 cm, B 30 Sept. 96 T2, Clover Hill, Tas, Chardonnay 307o none (mite damage) ll0 cm, B, G 30 Sept. 96 D, Bream Creek, Tas, Cabernet 507o leafspots (307o &50Vo) 60 cm 30 Sept. 96 D, Bream Creek. Tas, Cabernet 507o none (mite damage) 15 cm,70 cm 30 Sept. 96 D, Freycinet, Tas, Chardonnay 70Vo none (mite damage) 40 cm,50 cm 30 Sept. 96 D, Marion's, Tas, Chardonnay 4OVo none (mite damage) 30 cm, 100 cm, B, G 30 Sept. 96 water and Tween none (mite damage) 60 cm, B, G 30 Sept. 96 water and Tween none (mite damage) 80 cm 11 Oct. 96 Tl, Hillstowe, SA, Chardonnay 80Vo none 70 cm, B 1l Oct.96 Tl, Hillstowe, SA, Chardonnay 907o none (mite damage) 15 cm, 30 cm,50 cm 11 Oct. 96 D, Bream Creek, Tas, Cabernet 9ÙVo none 70 cm 11 Oct. 96 D, Freycinet, Tas, Riesling 75Vo none 70 cm l1 Oct. 96 T2, Clover Hill, Tas, Chardonnay 9O7o leaf spots (mite damage) 40 cm, G 11 Oct. 96 D, Clover Hill, Tas, Chardonnay 9OVo leaf spots (mite damage) 100 cm, G 1l Oct.96 D, Spring Vale, Tas, Pinot Noir 9ÙVo leaf spots (27o) 150 cm 1l Oct.96 water and Tween none 120 cm, B, G 11 Oct. 96 water and Tween none (mite damage) 90 cm 18 Oct.96 D, Marion's, Tas, Pinot Noir 9O7o scarred leaf stalks (mites) 60 cm, B 18 Oct. 96 D, Marion's, Tas, Pinot Noir 9OVo leaf spots (27o) 80 cm l8 Oct.96 D, Freycinet, Tas, Pinot Noir 9ÙVo leaf spots (27o) (mtes) 50 cm, B 18 Oct. 96 D, Ashton Hills, SA, Riesling 757o leaf spots (2Vo & 3Vo) 40 cm, 50 cm l8 Oct. 96 T1, Hargrave's, SA, Chardonnay 60Vo leafspots (2Vo &57o) 60 cm l8 Oct. 96 Tl, Hargrave's, SA, Chardonnay 85Vo leaves dead due to mites 25 cm, 30 cm 18 Oct. 96 D, Freycinet, Tas, Chardonnay 80Vo none (mite damage) 40 cm,70 cm, B, G 18 Oct.96 D, Freycinet, Tas, Chardonnay TOVo leaf spots (157o) 80 cm, B 18 Oct. 96 D, Marion's, Tas, Chardonnay 75Vo none (mite damage) 90 cm l8 Oct. 96 D, Marion's, Tas, Chardonnay 757o leaf spots (27o, 37o & 5Vo) 80 cm, G 18 Oct. 96 D, Marion's, Tas, Chardonnay 80Vo leaf spots (157o) 90 cm, B 18 Oct. 96 D, Marion's, Tas, Chardonnay 757o leaf spots (ZVo,2Vo &4Vo) 70 cm, G 18 Oct. 96 D, Bream Creek, Tas, Cabernet SOVo none (mite damage) 100 cm, B 18 Oct.96 D, Bream Creek, Tas, Cabernet 80Vo none (mite damage) 20 cm, B l8 Oct. 96 D, Marion's, Tas, Pinot Noir 9O7o none (mite damage) 90 cm, B, G 18 Oct. 96 water and Tween none 30 cm,40 cm, B 18 Oct. 96 water and Tween leaf spots (27o) 40 cm,50 cm 18 Oct. 96 water and Tween none (mite damage) 70 cm, B 18 Oct. 96 water and Tween none (mite damage) 100 cm, G control uninoculated none (mite damage) 30 cm control uninoculated none 60 cm control uninoculated none (mite damage) 70 cm,70 cm, B control uninoculated none 40 cm control uninoculated leaf spots (57o) 80 cm, B, G TI Phomops¡s taxon I conidia, T2 Phomopsis taxon 2 conidia, D D. viticola asci and ascospores. B the cane was slightly bleached, G grapes were present. ' Symptoms that may be caused by Phomopsis infection' 115 Three and 4 weeks after inoculation, most infected berries bore pycnidia (Table 7.3). Four weeks after inoculation, all inoculated berries had white or pale yellow/green mycelium and most berries were black or red in colour, except those inoculated with isolate 24E which were green. The uninoculated berries and those treated with water and Tween remained green without mycelium. Phomopsis mycelium grew from all pieces of skin and pulp of berries placed on WA, 2 months after inoculation, except those inoculated with isolate 24F, the uninoculated berries nor those treated with water and Tween. One month after the pieces were placed on agar, pycnidia with cirrhi were present on those inoculated with taxon 1 isolates 84, 18, 2OC,238,4I,45A, DAR 69467.1, DAR 69489.I, taxon 2 isolates 46,47, DAR 69486.1' DAR 69458 and taxon 3 isolate JM 139. Since isolate 248 fuled to produce mycelia on the tissue or seeds and did not affect the seedlings over the course of the experiments, it will not be referred to again. It is possible that long-term storage at 4"C may have affected the ability of the conidia to infect grape tissue. Seeds derived from both infected and control berries germinated, except those from berries inoculated with isolate JM 139 (Table 7.4).The percentage of seeds that germinated was similar among those derived from the inoculated berries, uninoculated berries and berries treated with water and Tween. Between l5%o and 597o of the seeds obtained from berries infected with taxon 1 isolates, and between 3I7o and 577o of those from taxon 2 infected berries germinated, compared with 367o and 46Vo for the controls. The seeds and germinated seeds from berries inoculated with Phomopsis were covered in mycelium. The majority of the seeds also bore pycnidia with cirrhi, while those from the control berries had no mycelium or pycnidia. Seedlings obtained from berries inoculated with isolates belonging to taxon 1 and the raxon 3 isolate DAR 69458 had roots of 2 - 5 mm long with brown or black tips. Often the roots were yellow and had started to rot. Most seedlings from berries inoculated with taxon 1 isolates 238,23C,318 and 454 died within a month @ecember 1995), while most of those inoculated with the remaining taxon 1 isolates were still alive (Table 7.4). All seedlings from berries inoculated with isolates belonging to taxon 2 had black or brown roots and most were covered in mycelium and were dead within a month. The remaining 116 seedlings had green cotyledons and were alive in December 1995 (Table 7.4). The seedlings from the control berries were apparently healthy with roots of I - 5 cm in length. In autumn (May) 1996,6 months after planting, five of the 14 seedlings derived from uninoculated berries and berries treated with water and Tween were alive. All seedlings from berries inoculated with isolates of taxa 2 and 3 had died. Only 14 of the 59 planted seedlings, derived from berries inoculated with taxon 1 isolates, had survived, which corresponded to seven of the 18 isolates (Table 7.4). In winter (August) 1996, seedlings derived from control berries and berries inoculated with isolates 18, 388, 41 and DAR 69489 were still alive. No bleaching or scarring of woody tissues or pycnidia were observed on any of the seedlings. In July 1997, only a small number of seedlings from uninoculated and inoculated berries were alive and had gro,wn into apparently healthy vines (Table 7.4). Five of the 14 control seedlings were still alive, compared with two out of four seedlings derived from berries inoculated with isolate 41 and one out of four seedlings from those inoculated with isolate DAR 69489. Pruned twigs, 15 cm long and 2 mm thick, from the living and dead plantlets derived from berries inoculated with taxon 1 isolates 18,248,388,41 and DAR 69489 and from plantlets derived from the controls, were placed in moist conditions at 15'C. After I month (August 1997), perithecia containing asci and viable ascospores were present on the prunings from the vine derived from a berry inoculated with isolate DAR 69489 and in September zone-lines appeared. Also in September, pycnidia with cinhi were present on the prunings from the dead vines derived from berries inoculated with isolates 18 and 38B. The cr-conidia were 6-7 pm long and grew into cultures typical of taxon 1, when placed on WA. In addition, prunings from the vines derived from berries inoculated with isolate 4l had zone-lines and immature perithecia, which matured in November. tt7 Table 7.3 Viability of Phomopsis taxa I,2 and 3 isolates used to inoculate Chardonnay berries and the presence of pycnidia on the berries, 3 and 4 weeks after inoculation. Isolate & Taxon Viability on 3 weeks after inoculation 4 weeks after inoculation WA PDA Vial 1 Yial2 Vial I YialZ 8A T1 75Vo 987o 0 + I + 18 T1 757o 98Vo I 0 + + zOC T1 7O7o 0 0 0 I 23B- T1 07o 7ÙVo + + + ++ 23C T1 7j%o 907o ++ ++ ++ ++ 23D T1 9ÙVo I 0 + + 238 T1 95Vo + + ++ + 248 T1 I00Vo I007o 0 0 0 0 318 T1 75Vo 1007o 0 0 0 0 38A T1 I007o ++ + +++ ++ 388 T1 30Vo 707o + + + + 4T T1 IO07o 1007o 0 0 I 0 42A T1 90Vo L007o ++ +++ ++ +++ 428 T1 1007o + ++ +++ +++ 45A T1 l00Vo I007o +++ +++ +++ +++ DAR 69467.1 T1 40Vo 1007o 0 0 I I DAR 69489 T1 l00%o 0 0 I I DAR 69489.1 T1 887o l00Vo 0 0 I I 46 T2 65Vo 1007o +++ +++ +++ +++ 47 T2 807o 987o ++ +++ +++ +++ DAR 69486 T2 60Vo 957o ++ +++ +++ +++ DAR 69486.1 T2 307o 95Vo +++ +++ +++ +++ DAR 69458 T3 75Vo 1007o +++ + +++ + JM 139 T3 207o IO0Vo + + ++ + uwater & Tween 0Vo 0 0 0 0 0 0 uuninoculated 0 0 0 0 0 0 Tl isolates belonging to Phomopsis taxon L, T2 Phomopsis taxon 2, T3 Phomopsis taxon J t uninoculated berries and those treated with water and Tween were distributed in three vials' - the viability of the inoculum was not tested on this agar medium' 0 no pycnidia present, I small immature pycnidia present, +, ++, +++ increasing number of mature pycnidia present on the berries. 118 Table 7.4 Number of seeds obtained from berries inoculated with isolates of Phomopsis taxa 1, 2 and 3 and from the controls, the percentage seed germination after I month on moist filter paper, the number of seedlings that survived, and the number of vines that were alive 6 and 18 months after planting in U.C. potting mixture (Baker, 1957). uBerries I Month after Placing Seeds on Moist Filter Number of Living Vines Inoculated Paper aT20"C (6 Dec. 1995) with Isolate Number 7o Seeds Number of Seedlings May 1996 July 1997 I Alive & Taxon of Seeds Germinated I Dead 8A T1 13 38 0 5 1 0 18 Tl t4 29 0 4 1 {< zOC TI 19 37 3 4 0 0 238 TI 15 40 5 1 0 0 23C TI 20 40 7 I 0 0 * 23D T1 15 33 1 4 0 238 T1 I6 t9 0 J 0 0 248 T1 t7 59 0 10 4 4 318 TI 13 15 2 0 0 0 38A T1 t7 18 I 2 0 0 ,1. 388 T1 I4 57 3 5 1 4I T1 12 42 1 4 4 2 42A T1 15 20 0 3 0 0 428 TI L4 2T 0 3 I 0 45A T1 12 25 3 0 0 0 DAR 69467.1 T1 13 38 1 4 0 0 DAR 69489 T1 L4 43 2 4 2 I DAR 69489.1 T1 I4 29 2 2 0 0 46 T2 2L 57 10 2 0 0 47 T2 16 38 5 1 0 0 DAR 69486 T2 I7 4L 7 0 0 0 DAR 69486.1 T2 16 31 4 I 0 0 DAR69458 T3 r6 38 I 5 0 0 JM 139 T3 9 0 0 0 0 0 water & Tween I3 46 0 6 2 2 uninoculated 22 36 0 8 J J there were 16 berries per isolate, 24 berries treated with water and Tween, and24 uninoculated berries. t the last vine had died a few weeks prior to the date of scoring. il9 7.4 Discussion The aim of this study was to fulfil Koch's postulates by inoculating healthy grapevines with isolates of Phomopsis in a glasshouse and re-isolating the fungi from plants that display symptoms typical of the disease. Two inoculation methods that simulated possible natural modes of infection were tested: i) splashing of spores onto breaking buds to allow infection through natural openings such as stomata, as proposed by Gärtel (1972, section 2.5.1) and ii) wound-inoculation, to test for infection through wounds @mmett et al., 1992) or transmission of the fungus by insect vectors, as studied by Linders (1994).In addition, a seed transmission experiment was conducted, with the aim of i) demonstrating that the disease can be carried from the seed of an infected berry into the resultant seedling, and ii) to examine the effect of the different taxa on seedling development. The non-wound-inoculation method, in the preliminary pathogenicity test, resulted in the development of the characteristic necrotic spots with yellow haloes on leaves of all three vines non-wound-inoculated with isolates belonging to taxon 2. Moreover, the only vine that developed a scarred spur, typical of taxon 2 infection, had been inoculated without wounding, with a taxon 2 isolate, the previous year. This implies, as has been demonstrated for P. vaccinii and Phomopsis on peach trees (Milholland, 1982; Uddin & Stevenson, 1997), that wounding is not a prerequisite for the experimental infection of grapevine by Phomopsis taxon 2. In the experiment conducted here, wound-inoculation with taxon 2 resulted in death of the wounded buds. For isolates belonging to taxa 1 and 3, little difference was observed between the two inoculation methods. I-eaves on vines inoculated using both methods showed some necrosis, 6 weeks after inoculation. One of the vines, wounded and treated with water and Tween, also had a leaf with some necrosis, indicating that some of the necrotic lesions may have been caused by mites, white flies or needle damage. Based on these findings, the non-wound-inoculation technique was then used to test the pathogenicity of Phomopsis taxa 1 and 2 and of D. viticola on grapevines (section 7 .2.2). Unfortunately, any possible symptoms due to infection by the fungi were obscured by severe mite infestations in this experiment, and the results are not discussed further. The grapevines used in the preliminary pathogenicity test of Phomopsis taxon I and 2 isolates r20 (section 7 .2.I), were in a different glasshouse and did not suffer severe mite infestation. These vines were monitored over a period of 3 years for the development of symptoms attributed to infection by these two taxa (section 2.4; Chaptet 6). Vines inoculated with Phomopsls taxon 1 did not show symptoms of disease due to Phomopsis in the year of inoculation. This agrees with the field studies (Chapter 6), where no symptoms were found in spring and summer. Vines infected with taxon 1, like soybean and lupin plants infected with P. sojae and P. leptostromiþrmis, respectively, appear to remain symptomless while the fungus is growing within the plant (Kulik, 1984; Williamson et a1.,1991). One year after inoculation, in spring, three out of eight vines inoculated with taxon 1 had stunted shoots and/ or dead buds, compared with one out of seven control vines that had a single stunted shoot. This concurs with the field study (Chapter 6), which indicated that bud-death and stunted growth of new shoots may be associated with infection of vines with Phomopsis taxon 1. Similarly, P. vaccinii has been reported to cause bud-death on blueberry twigs (Milholland, 1982). Because of the small number of grapevines that displayed symptoms of infection, no attempt was made to re-isolate taxon 1 from the affected shoots and buds, as their removal may have affected subsequent growth of the fungus in the vines. As in the field studies, stunted growth and bud-death in this study was scored most reliably in winter, almost 2 yeas after inoculation, when the fate of the buds was known. Other symptoms observed in the field studies (Chapter 6), such as bleaching of l- and 2-year-oldcanes, fruiting-bodies and zone-lines, were not observed in the glasshouse at any time during the 3-year study. However, 3 years after inoculation, a cane removed from a vine inoculated with taxon 1 isolate 23B and incubated, developed zone-lines, pycnidia with cirrhi containing conidia typical of Phomopsís .taxon I and immature perithecia. This implies that mycelium of taxon I grew within the vine for 3 years, without bleaching the canes. It is possible that this occurs in the vineyard; grapevines may not express symptoms of infection for a number of years after initial infection. Research on the duration of the latent period is required. Vines inoculated with Phomopsls taxon 2 using the non-wound technique, had leaves with necrotic spots surrounded by yellow haloes, while the buds that were inoculated using the wound-inoculation technique died. Leaves and shoots that formed after T2I inoculations were apparently healthy. This indicates that the lesions were caused by localised infection of unwounded green tissues. However, no attempt was made to re-isolate Phomopsis from the leaf spots. One year after inoculation, only one vine displayed scarring and cracking typical of taxon 2 infection (Merrin et a1.,1995). Stunted growth and death of young shoots was less prevalent among vines inoculated with taxon 2 than those inoculated with taxon 1. Only one of five living vines inoculated with taxon 2 had stunted shoots, compared with one of seven remaining control vines that had a single stunted shoot. No pycnidia were observed on the canes, up to 3 years after inoculation with taxon 2. However, as for taxon I isolate 238, a cane pruned from a vine inoculated with isolate 46, 3 years previously, developed pycnidia with cirrhi containing conidia typical of Phomopsis taxon 2, following incubation. This cane had grown from the spur that displayed scarring symptoms 1 year after inoculation, but the sample used here did not display symptoms. This implies that mycelium of this taxon 2 isolate grew within the vine for 3 years and may have been responsible for the scarring at the base of the cane, but without causing bleaching and without forming pycnidia. Vines inoculated with taxon 3 did not show symptoms typical of infection with Phomopsis in the year of inoculation, as was the case for vines inoculated with taxon 1. One year after inoculation, one of four vines inoculated with taxon 3 had two stunted shoots, one of which died later in the season. No bleaching or scarring was observed during the 3 years and the fungus was not recovered from canes following incubation. It is suggested that taxon 3 may have failed to infect the vines. Hewitt & Pearson (1983) suggested that bleaching of scared canes, infected with P. viticola, resulted from the production of numerous pycnidia which lift the epidermal layer, admitting air beneath it and giving the surface a white to silvery sheen. The absence of bleached canes on inoculated vines in the glasshouse may be due to the lack of pycnidia. Emmett et aI. (1992), however, suggested that the woody tissues whiten as they dry out. The absence of pycnidia, perithecia, zone-lines or bleaching symptoms on vines in the glasshouse may be due to the inoculation methods used. The use of other methods, that do not simulate natural modes of infection, such as inserting colonised toothpicks or agar inoculum plugs in wounds (Hen et aI., 1983; Crump et aI., L996; Uddin & Stevenson, t22 L997), may be more successful in producing these symptoms, as more inoculum enters the plants. However, two vines were shown to be successfully inoculated, as pycnidia developed after shoot excision and incubation. The absence of pycnidia in the glasshouse, but their development during incubation, ffiây be due to environmental effects of a glasshouse setting, such as low humidity and the absence of rain. Moreover, yearly re- infection by either of the taxa may also be required for disease expression. It may also be possible, however, that either of the taxa of Phomopsis of grapevine needs to grow inside the canes for a number of years before disease is expressed. Additional research is required to determine the growth habit of Phomopsis within grapevine canes and events controlling pycnidium production. The results obtained for Phomopsis taxon 1 and 2 werc similar to those of Shivas ¿l aI. (I99I), who tested the pathogenicity of biotypes A and B of P. leptostromiþrmis on Lupinus angustþIius L. plants in a glasshouse. These authors observed that isolates of biotype B produced bleached lesions and "pinprick" stromata on the plants, while isolates of biotype A failed to produce symptoms. Only biotype B was re-isolated from the inoculated plants. Likewise, in this study, vines inoculated with taxon 2 developed lesions and vines inoculated with taxon 1 failed to produce symptoms. However, both taxa were re- isolated from inoculated grapevines. In the seed transmission experiment, most of the Phomopsis isolates tested, colonised the berries and produced pycnidia. Infection with Phomopsis did not appear to affect the subsequent seed germination rate, but all of the seedlings derived from infected berries showed signs of infection and rotting of the roots. Within taxon l, four isolates (238, 23C, 3LB and 454) appeared to be more pathogenic than the other isolates. Most seedlings derived from berries inoculated with these isolates died within I month of germination. In contrast, seedlings derived from berries inoculated with four less pathogenic isolates (18, 38B, 41 and DAR 69489) were still alive 1 year after planting. Similar percentages of seedlings infected with isolates 41 and DAR 69489 and control seedlings were alive, 18 months after planting, indicating that these isolates are not pathogenic to grapevine seedlings. Isolates belonging to taxon 2 were considered more pathogenic than the taxon I isolates, as all seedlings derived from the infected berries had rotting roots and died approximately 1 month later. The pathogenicity r23 of taxon 3 isolate JM 139 is unknown, as none of the seeds derived from berries inoculated with this isolate germinated. The pathogenicity of taxon 3 isolate DAR 69458 appeared to be similar to that of taxon I isolates of intermediate pathogenicity. Most prunings from seedlings derived from taxon 1 infected berries developed pycnidia or perithecia following incubation. This implies that mycelium of taxon 1 grew within these vines for 18 months and that taxon I can be transmitted via seeds. However, nonc of the grapevine twigs were bleached in the glasshouse, possibly because the plants were too young or because pycnidia were not present on the twigs prior incubation. In conclusion, the experiments described in this chapter indicate that stunted shoots and dead buds occur more frequently on vines inoculated with Phomopsis taxon 1 than on those inoculated with taxon 2 or the controls. This agrees with the findings in Chapter 6. Both a scarred cane and leaves with necrotic spots and yellow haloes developed on vines inoculated with Phomopsis taxon 2, which agrees with the findings by Merrin et aI. (1995). Moreover, both taxon I and taxon 2 werc re-isolated from inoculated grapevines. While this suggests that these two taxa cause the symptoms with which they are associated in the field, it cannot yet be claimed that Koch's postulates have been fulfilled. First, the fungi were isolated from canes that did not display symptoms of the diseases, and second, not all symptoms associated with the diseases appeared on vines in the glasshouse; in vitro incubation of the canes was required to induce formation of spore forming bodies. In addition, stunting and bud-death symptoms occurred on less than half of the vines inoculated with taxon 1 isolates (1 year after inoculation), taxa 1 and 2 were isolated from only one vine each, one control vine developed a small amount of necrosis (6 weeks after inoculation), one control vine had a stunted shoot (1 year after inoculation), and there was a lack of repetition. The seed transmission experiment was apparently unsuccessful in producing symptoms of disease caused by Phomopsis on young vines, although D. viticola was re-isolated from l8-month-old plants grown from seeds infected with taxon 1. The absence of symptoms associated with taxon I infection may have been due to problems with the seed or experimental conditions. Therefore, optimal conditions need to be defined before repeating this type of experiment. However, it is also possible that symptoms may appear when the vines are mature. In order to fulfil Koch's postulates conclusively, a more extensive, well-replicated pathogenicity test needs to be conducted, in which vines are t24 grown in conditions conducive to the development of pycnidia, perithecia and zone-lines, and with isolation of Phomopsis from affected tissues, such as stunted shoots and dead buds in the case of taxon I and leaf lesions and scarred canes in the case of taxon 2. 125 8. MYCELIAL INCOMPATIBILITY IN GRAPEVINE PHOMOPilS 8.1 Introduction Vegetative incompatibility is a valuable tool for the analysis of the population structure of fungi. It is a phenotypic marker that can be used to study the reproductive strategy, population structure and genetics of plant pathogenic fungi (Kistler, 1997; section 2.9.1). Using this type of marker, the relationship of individuals within a population at multiple loci can be examined simultaneously because, in ascomycetes, vegetative incompatibility is known to be the result of the action of alleles at several distinct loci (section 2.8.2). Thus, the more closely related two isolates are, the more likely they are to be vegetatively compatible and, therefore, within the same VCG (section 2.8.2). However, this marker is not useful in determining the degree of relatedness of isolates (I-eslie, 1993; Kistler, 1997). Knowledge of vegetative incompatibility may provide information on the importance of sexual reproduction in the population. A sexually reproducing population is expected to have a large number of VCGs represented in the population (I-eslie, 1993). In an asexual population, however, each VCG will form a genetically isolated sub-population with specific phenotypes and VCGs may be lost to genetic drift so the population will become less diverse (Croft & Jinks, 1977; Correll, I99L; Iæslie, 1993). Moreover, fungi that rely primarily on asexual reproduction often demonstrate more phenotypic variation between VCGs than within VCGs (Correll, 1991; Kistler, 1997). A consequence of vegetative incompatibility is the reduction of the spread of infectious agents (Caten, 1972; I-eslie, 1993), as the absence of anastomosis between different fungal individuals prevents the transmission of hypovirulence factors such as double-stranded RNAs (dsRNA, I-eslie, 1993) and deleterious cytoplasmically transmitted mycoviruses (Brasier, L996). Some strains of Diaporthe ambigua have been found to contain an endogenous virus-like dsRNA segment and to be hypovirulent; the large number of VCGs in this fungus (Smit et a1.,1996) may limit the spread of this dsRNA segment. The main basis for scoring vegetative incompatibility and assigning isolates to VCGs is the barrage zones that form when incompatible isolates are co-cultivated on agar. Vegetatively compatible isolates do not form a barrage; they grow into each other without t26 altering their morphology (I-eslie, 1993). Incompatible isolates of several Phomopsis species form barrages when their mycelia meet, including Diaporthe phaseolorum (Ploetz & Shokes, 1986), Phomopsis subordinan¿ (Meijer et aI., 1994) and Diaporthe ambigua (Smit et aI., 1997). Hyphae fuse in the barrage zone, but the resulting heterokaryotic cells are rapidly destroyed through a degenerative and lytic reaction (I-eslie, 1993). This lysis of hyphae was observed in the barrage zones of D. phaseolorum (Ploetz & Shokes, 1986) and an unknown species designated as Phomopsis group one from elm (Brayford, 1990b). Macroscopically, the barrages of D. phaseolorum weÍe brown on PDA, and those of D. ambigua weÍe black on freshly prepared oatmeal agar (Ploetz & Shokes, 1986; Smit ¿l aI., L997). Phomopsis group one from elm formed dark brown or black barrages or cleared zones with weak pigmentation on malt agar (Brayford, 1990b), and P. subordinaria sometimes formed aerial mycelium at the barrage zone, or a growth-free zone (Meijer et aI., tee4). A characteristic feature of the genus Diaporthe is the formation of nalrow, black zone-lines in the substratum (Wehmeyer, 1933). Webber (1981) reported zone-line formation in the inner bark of dead elm colonised by Phomopsis oblonga, and Brayford (1990b) suggested that these zone-lines are formed when vegetatively incompatible isolates meet. Zone-lines found on grapevine canes colonised by Phomopsis taxon I (chapter 4) may also be associated with vegetative incompatibility within this fungus. Because vegetative incompatibility may play a major role in regulating population structure of the pathogen, in territorial integrity of individual genets and in limiting the spread of mycoviruses and dsRNA, information obtained from vegetative incompatibility studies may have wider ecological significance than those provided by most other genetic markers (Brasier, 1996). This chapter addresses incompatibility in Phomopsis taxon 1. As microscopic studies of anastomosis, heterokaryon formation and cell lysis in the barage zone were not performed, the incompatibility reactions will be referred to as mycelial incompatibility, rather then vegetative incompatibility, and compatible isolates will be grouped into mycelial compatibility groups (MCGs). r27 8.2 Materials and methods 8.2.1 Mycelial incompatibility tests Mycelial incompatibility in Phomopsis of grapevine was investigated by inoculating PDA plates (ca 25 ml in a 9 cm Petri dish) in duplicate, by applying seven mycelial plugs to the medium. One plug was applied in the middle of the plate and six plugs were placed in a ring around the isolate in the middle. Pairings were repeated, as necessary, to obtain a clear response. PDA was used because D. phaseolorum and P. subordinariahave been reported to produce clearly visible barrages on this medium (Ploetz & Shokes, 1986; Meijer et aL, Igg4). The plates were incubated as described in section 3.1, and examined 1,3,5 and 7 weeks after inoculation. Different colony morphologies among Phomopsis isolates are listed in Table 8.1. Isolates were termed "incompatible" if they formed a dark balrage zone at the boundary, or if a growth-free zone was formed between the isolates (Meijer et aI., 1994). Compatible isolates grew together, forming a seamless junction. In experiment 1, 12 taxon 1 isolates were co-cultivated with each other and with themselves. The morphology of each isolate was recorded I week after inoculation and the appearance of each incompatibility reaction was recorded 5 or 7 weeks after inoculation. On the basis of the compatibility tests each isolate was assigned to an MCG (Table 8.2). In experiments 2 and 3, 11 taxon 1 isolates and one taxon 2 isolate (Table 8.3), and eight taxon 1, one taxon 2 and two taxon 3 isolates (Table 8.4) were tested for mycelial incompatibility. The morphology of these isolates was recorded I week after inoculation. Subsequently, a series of experiments was established to group all of these isolates into MCGs. The details of these experiments are provided in section 8.3.1.2. In experiment 4, 16 isolates of Phomopsis taxon 2 were tested for mycelial incompatibility. Morphology was recorded 1 week after inoculation (Table 8.5). In experiment 5, Phomopsis taxon 1 isolate DAR 69488 and taxon 4 isolate DAR 69484 were co-cultivated with isolates representative of the MCGs established previously (Tables 8.2, 8.3 and 8.4), with taxon 3 isolates JM 139 and DAR 69458.1, and with themselves. Taxon 4 isolate DAR 69484 was also co-cultivated with taxon 2 isolates DAR 69486.I,110.1, VRU 0048 and VRU 0084. Isolates VRU 0048 and VRU 0084, which had been tested against 14 other taxon 2 isolates (Table 8.5), were co-cultivated with isolates r28 representative of the MCGs shown in Tables 8.2 and 8.3, and with taxon 3 isolates JM 139 and DAR 69458.I, taxon 4 isolate DAR 69484, and with themselves (Table 8.6). The origins of isolates used in this study are recorded in Table 8.7, along with MCG. 8.2.2Data analysis Variation in colony morphology and mycelial incompatibility within and between three populations in South Australia (Table 8.8) were analysed using Shannon-V/eaver indices (I1). The frequencies of the colony morphology groups and MCGs in these populations were calculated, and phenotypic diversity was estimated using Shannon and Weaver's information statistic (Shannon & Weaver, 1949; Bowman et a1.,1971) as: g = -},'p,lnp, i=l where p¡ is the fraction of the whole sample represented by the iú phenotype (colony morphology group or MCG) from a population with s phenotypes. When all isolates have the same phenotype, H = 0, and when all have different phenotypes, l/ has the greatest value (ln n, for a sample of size n) (Hutcheson, 1970; Peet, L974; Anagnostakis et aI., 1986). Using this statistic, the total diversity can be divided into components (Zhang et al., L987), enabling the calculation of the proportions of the total diversity due to variation within and between populations (Goodwin et aI., 1992u). In order to compare the phenotypic diversity in the different locations, the estimated diversity values were normalised, such that // = Hlln n, and .I/ ranges from 0 (all isolates have the same phenotype) to I (each isolate is unique). This was necessary because the sample sizes varied among locations (Goodwin et a1.,1993). 8.2.3 Mycelial incompatibility used to study mating behaviour Mycelial incompatibility was used as a genetic marker to investigate compatibility amongst progeny from single perithecia. Isolates that originated from the same perithecium were tested for mycelial incompatibility with each other and with themselves (Table 8.9), as described in section 8.2.1. r29 8.3 Results 8.3.1 Mycelial incompatibility tests 8.3.1.1 ldentification of morphological types of Phomopsis of grapevine Five different morphological types were identified among the isolates of Phomopsis of grapevine (Table 8.1, Figs. 8.1 and 8.2). Four of the morphological types were found among isolates of Phomopsis taxon 1. Most taxon 1 isolates had either uniform and dense mycelium (uf-type, 13 of 31 isolates) or sparse mycelium (s-type, 12 of 31 isolates), while five taxon 1 isolates had fluffy mycelium (fe-type), and one had mycelium with concentric rings (cf-type, Tables 8.2,8.3 and 8.4). Some isolates of Phomopsis taxon I derived from pycnidia on the same cane, differed in morphology, for example, isolates 174 and 178, 184 and 18B, and l25A and 1258, whereas others had the sÍìme morphology, for example, isolates 1504 and 1508, 1514 to 151F, I52A and 1528, and 1534 and 153B (Tables 8.2, 8.3 and 8.4). Certain isolates derived from single perithecia also differed in morphology, for example, isolates 50A to 50E, and 514 and 51C.1 (Table 8.2). Isolates belonging to all four morphological types of taxon 1 were isolated from Ashton Hills (12 isolates). In another vineyard in the Adelaide Hills (Hargrave, six isolates) and in the Coonawarra district (seven isolates), three of the four types were found (uf, s and fe). In Tasmania (four isolates) and the Yarra Valley (one isolate), only type s was found. The two taxon 3 isolates were of type cf and fe (Table 8.4). Mycelium of the Phomopsis taxon 2 isolates was generally darker in colour and denser than that of isolates of taxa 1 and 3. Three of the morphological types were found among taxon 2 isolates (Table 8.1 and Fig. 8.2). Most taxon 2 isolates (10 of 16) had thick mycelium in the colony centre with sparse mycelium at the colony edge and pycnidia throughout the colony (fsp{ype); this morphological type was not present among taxon I and 3 isolates. Three taxon 2 isolates were of type cf with or without pycnidia, and three of type uf with or without pycnidia (Table 8.5). Phomopsis taxon 2 isolates from South Australia had morphological types fsp or uf. In Victoria, all three morphological types of taxon 2 werc found; isolates from Mildura and Irymple were types cf or uf, with or without pycnidia. In New South Wales, the fsp-type and 130 Table 8.1 Colony morphologies of isolates of Phomopsis of grapevine on PDA. Morphological type Abbreviation Taxon sparse mycelial growth s-type taxon 1 fluffy mycelium at the colony edge fe-type taxal&3 concentric rings of dense and fluffy mycelial growth cf-type taxa 1, 2 &.3 (with pycnidia) (cfp-type) (taxon 2) uniform, dense and fluffy mycelial growth uf+ype taxal &2 (with pycnidia) (ufp{ype) (taxon 2) thick and fluffy in the centre, but sparse mycelial growth fsp-type taxon 2 at the colony edge, with pycnidia Some taxon 2 isolates with morphological types cf and uf produced pycnidia in some Petri plates, the morphological types of these isolates were scored as cfp and ufp. 131 the cfp-type were found. Both isolates from Griffith, New South Wales, were type fsp, as were the two isolates from Tasmania. There was no evidence of mycelial incompatibility after I week in culture. 8.3.1.2 Mycelial incompatibitity in Phomopsís of grapevine Interactions between isolates were termed compatible when no visible incompatibility reaction occurred. However, because of the different morphologies, these isolates did not always form a seamless junction where their mycelia met (Ftg. 8.1). Self-pairings, used as controls, were always compatible and the mycelia merged to form a seamless junction. Three weeks after inoculation, faint pigmentation was visible where the mycelium of some isolates met. After 5 weeks, barrages were visible at the boundaries of most incompatible isolates, whereas in some pairings, reactions remained unclear at 7 weeks. These latter isolates were tested again, this time with four replicates, against each other and themselves on PDA. The incompatibility reactions often differed in appearance (Figs 8.1 and 8.2). Both the width of the barrage zone and the intensity and colour of the pigmentation varied for the different isolate combinations. In several cases, barrages of aerial mycelium or zones without hyphae were formed between incompatible isolates. In a few cases, pycnidia were formed at the boundary. The types of incompatibility reactions were recorded in experiment I (Table 8.2), but in subsequent experiments no differentiation between the types of reactions was made. The first 12 taxon 1 isolates tested (experiment 1) were grouped into six MCGs, five of which had only one member, and one which had seven members (MCG 5). Two mycelial incompatibility reactions occurred within MCG 5: isolate 51C.1 was incompatible with isolates 154 and 508, while apparently compatible with the remaining five members of MCG 5 (Table 8.2). The mycelium of isolates 174 and 17B was incompatible. These isolates were derived from pycnidia of a single cane, separated by a zone-line. Isolates l8A and 188, derived from a single cane without zone-lines, were also incompatible. Mycelium of isolate 504 was incompatible with mycelium of isolates 508 - 50E, all of which were derived from a single perithecium (Table 8.2). r32 The morphology of the mycelial incompatibility reaction between MCG 1 (isolate 524) and the seven isolates that belong to MCG 5, was always a pink barrage zone (Table 8.2). The reactions between MCG I and MCGs 2,3,4 and 6 were different to the reaction between MCG 1 and MCG 5. The morphology of the mycelial incompatibility reaction between MCG 2 (isolate 184) and the seven isolates that belong to MCG 5, was always a growth-free zone. Likewise, the incompatibility reactions between MCG 3 (isolate 188) and the seven isolates of MCG 5 were mostly a growth-free zone, with or without pink pigmentation, but brown pigmentation occurred once. The incompatibility reactions between MCG 4 (isolate 174) and the seven isolates of MCG 5 consisted mostly of a grey barage zone, but brown pigmentation and a growth-free zone with pink pigmentation occurred in two combinations of isolates. The incompatibility reactions between MCG 6 (isolate 504) and the seven isolates of MCG 5 were mostly a grey barrage zone, but a growth-free zone and a ridge of aerial mycelium occurred in both replicates of two combinations of isolates (Table 8.2). The 1l Phomopsis taxon I isolates used in experiment 2 (Table 8.3) were grouped into five MCGs, two of which had only one member, two had two members each, and one had five members. The mycelium of isolates 1504 and 1508 was compatible; these isolates were derived from pycnidia from a single cane, separated by a zone-line. Isolates l5lA to 15lF were also derived from pycnidia on a single cane, each separated from the others by zone-lines and were compatible with each other. Isolates I52A and 152B were derived from pycnidia from a single cane, separated by a zone-line, and were shown to be incompatible in one of the two replicate plates, and in one of the four repeated replicate plates. The remaining plates did not show an incompatibility reaction. Isolates 1534 and 1538 were also derived from pycnidia from a single cane, separated by a zone-line, and were incompatible. However, mycelia of isolates l52B and 1538 were compatible while isolates L52A and 1534 were incompatible (Table 8.3). The 1l Phomopsis taxon 1 isolates and the taxon 2 isolate (Table 8.3) were tested against MCGs 1 to 6. MCG 5 was represented by isolates 51C.1 and 508. Five taxon 1 isolates (151A to 151F) that were compatible with each other, reacted differently when paired with the two isolates belonging to MCG 5: isolates 1514 and 15lF had compatible reactions with isolate 51C.1 but were incompatible with isolate 508 in one of the two 133 replicates; isolate 15lC was incompatible with isolates 50B and 51C.1 in one replicate plate and compatible in the other; and isolates 15lD and 15lE were compatible with isolates 508 and 51C.1. V/hen the inconclusive reactions were repeated, isolates 1514 and 151F were incompatible with isolate 50B in one of the four replicates. Isolate 15lC was incompatible with isolate 50B in two of four replicates and incompatible with isolate 5lC.1 in one of four replicates. The five isolates were placed into MCG 5. The remaining six taxon I isolates were incompatible with isolates belonging to MCGs 1 to 6, and were grouped into MCGs 7 to 10. MCGs 7 and 9 each had two members. MCGs 8 and 10 had one member each. Phomopsis taxon 2 isolate DAR 69486.1was incompatible with MCGs 1 to 10 (Table 8.3). In experiment 3, eight taxon I isolates (Table 8.4) were grouped into six MCGs, four of which had only one member and two of which had two members each. The mycelia of two isolates that were derived from pycnidia from a single cane, separated by a zone-line (isolates l25A and 1258), were compatible, and the mycelia of two isolates from different vineyards in Tasmania (isolates 95.1 and 100.1) were compatible (Table 8.4). The eight taxon 1 isolates, one taxon 2 isolate and two taxon 3 isolates (Table 8.4) were tested against MCGs 1 to 10 and taxon 2 isolate DAR 69486.L. Taxon 1 isolates 1254 and 1258 were placed into MCG 5 and the remaining taxon I isolates were grouped into MCGs 11 to 15. Taxon 2 isolates 110.1 and DAR 69486.I were incompatible with MCGs 1 to 15, but compatible with each other. The taxon 3 isolates JM 139 and DAR 69458.1 were compatible with each other, but incompatible with MCGs 1 to 15 and incompatible with taxon 2 isolates 110.1 and DAR 69486.I (Table 8.4). In experiment 4, the 16 Phomopsis taxon 2 isolates, listed in Table 8.5, were all compatible with each other. In experiment 5, Phomopsis taxon 1 isolate DAR 69488 was found to be incompatible with MCGs I to 15, although a visible incompatibility reaction with isolates 152B and 1538 (MCG 9) occurred in one of the replicatè plates only. When repeated, the incompatibility reaction was visible in one of four replicate plates. Isolate DAR 69488 was also incompatible with isolates belonging to taxon 3, and was placed into MCG 16. Taxon 4 isolate DAR 69484 was incompatible with taxon 1 isolates belonging to the MCGs I to 15, incompatible with three taxon 2 isolates (DAR 69486.I, 110.1, VRU 0048), incompatible with taxon 2 isolate VRU 0084 in one of the two replicate plates and in one of four repeated 134 replicate plates, and incompatible with the taxon 3 isolates JM 139 and DAR 69458.1 in one of the two replicate plates. V/hen repeated, a visible incompatibility reaction was present between isolates DAR 69484 and DAR 69458.1 in two of four replicate plates, and between isolates DAR 69484 and JM 139 in none of the four replicate plates. Taxon 2 isolates VRU 0048 and VRU 0084 were found to be incompatible with the isolates representative of MCGs I to 10, and incompatible with the taxon 3 and 4 isolates in at least onc of the two replicate plates. V/hen the inconclusive reactions were repeated, one or two of the four replicate plates showed a visible incompatibility reaction (Table 8.6). The isolates in each MCG, their origin and the ability of each isolate to produce perithecia (Chapter 5) are summarised in Table 8.7. Twelve of the 16 MCGs of Phomopsis taxon I contained only one isolate. MCG 7 contained two isolates that were derived from a single cane, and had the same morphological type. MCG 9 contained two self-fertile isolates with the same morphology that were derived from different canes collected in the same vineyard. MCG 13 contained two self-fertile isolates, from different vineyards in Tasmania, that had the same morphological type. MCG 5 contained 14 of the 3l taxon 1 isolates. All isolates that were classified as MCG 5 were obtained from two vineyards in the Adelaide Hills or from the Coonawarra. Nine of the isolates had morphological type uf, four had type s, and one had type fe. Both self-sterile and self-fertile isolates were found within MCG 5. In 1994, isolates belonging to MCG 5 were obtained from both the Adelaide Hills (Ashton Hills) and the Coonawarra. In 1996, isolates belonging to MCG 5 were isolated again from Ashton Hills, and MCG 5 was also isolated from a different vineyard (Hargrave) in the Adelaide Hills (Table 8.7). In 1994, isolates belonging to four MCGs (2,3, 4 & 5), were obtained from Ashton Hills in South Australia. In 1996, isolates belonging to two MCGs (5 e.7) were isolated from Ashton Hills, and isolates belonging to four MCGs (5, 8, 9 A 10) were obtained from Hargrave in South Australia (Table 8.7). In total, 12 MCGs of Phomopsis taxon 1 were isolated from South Australia, three from the Coonawarra and ten from the Adelaide Hills. In the Adelaide Hills, five MCGs were isolated from Ashton Hills and four from Hargrave. In addition, three different MCGs were isolated from Tasmania. 135 Figure S.L Morphological types and the first signs of mycelial incompatibility among Phomopsis taxon 1 isolates on PDA, 2 weeks after inoculation. Morphological types uf (uniform, dense and fluffy mycelium), fe (fluffy mycelium at the colony edge) and s (sparse mycelium) are visible. Mycelial incompatibility between isolates is not yet visible, except for the barrage of aerial mycelium between isolate 52A, and isolates 504 and 174. Self- pairings of isolates 50A, 50D.1 and 52A are compatible, the mycelia merge to form a seamless junction. Compatible isolates with different morphologies, such as isolates 17B, 508, 50D.1, 5lA and 51C.1 do not form a seamless junction where their mycelia meet. The diameter of the Petri dishes was 9 cm. Errc*I4 '. þ, ,f re".L {e l"r rS r¿a-"l S. 136 Figure 8.2 Mycelial incompatibility among Phomopsis isolates on PDA, 3 weeks after inoculation. All isolates have morphological types uf (uniform, dense and fluffy mycelium), except for taxon 2 isolate DAR 69486.I, which has type cfp (concentric rings of fluffy mycelium, with pycnidia). Seamless junctions between mycelia of compatible isolates are visible, and grey barrage zones a¡e visible where incompatible isolates meet (see arows). A, B. Upper and lower surfaces, respectively. Isolate l50A did not grow. C, D. Upper and lower surfaces, respectively. The number 2 represents taxon 2 isolate DAR 69486.t. .A B C Ð 137 Table 8.2 Results of mycelial incompatibility tests between 12 isolates of Phomopsis taxon I from the Adelaide Hills and the Coonawarra, on PDA. 15A L7A t7B 184 18B 504 508 50D.1 50E 514 51C.1 52A 52A P A P P,F B,F P,A P P P P P UG 5lC.1 UG G F P,F G 51A P,F F F F 50E G F F G 50D.1 G F P,F G 508 G F P,F G 50A ; F ¿ F F 188 B G P,F B 184 F G F t7B G T7A B 15A uffeufcffefeufs sufs fe 545236555551 Isolates 154, 174, 17B, 184, 188, 504, 50B & 5lA were derived from hyphal tips, 50D.1 and 51C.1 were single conidium-derived isolates (Table 3.2) and 50E and 524 were bulk isolates (Table 3.1). Isolates 174 and 178 were derived from separate pycnidia on a single cane, separated by a zone- line. Isolates 184 and 18B were derived from two different pycnidia on a single cane without zonelines. Isolates 504 - 50E were derived from a single perithecium, isolates 514 and 51C.1 were derived from a different single perithecium, and isolate 524 from a third perithecium. A, B, F, G & P indicate an incompatibility reaction between two isolates: A = ridge of aerial mycelium; B = brown or yellow pigmentation; F = growth-free zone; G = grey pigmentation and P - pink pigmentation. - the isolates were compatible. " incompatibility reaction within MCG 5. Morphological types: uf = uniform, dense and fluffy mycelium; cf = concentric rings of dense and fluffy mycelium; fe - fluffy mycelium at the colony edge and s = sparse mycelium. 138 Table 8.3 Results of mycelial incompatibility tests between 11 isolates of Phomopsís taxon 1 from two vineyards in the Adelaide Hills and one isolate of Phomopsís taxon 2 from Victoria, and seven isolates representative of MCGs I to 6 (Table 8.2) on PDA' DAR 150 150 15 1 t5r 151 15 1 15r t52 152 153 153 69486.r A B A C D E F A B A B 1538 I I I I I I I I I I 1534 I I I I I I I I I I I52B I I I I I I I I t52A I I I I I I I I l 15lF I I I 15lE I I I 15lD I I I 15lC I I I 1514 I 150B I l50A I DAR 69486.1 524 (1) I I I I I I I I I I I I 184 (2) I I I I I I I I I I I I 188 (3) I I I I I I I I I I I I 17 A (4) I I I + I I I I I I I sOB (5) I I I + + + + I I I 5lC.1 (5) I I I + I I I I soA (6) I I I I I ; I I I I I I morph. cfp s S uf uf uf uf uf uf uf uf uf MCG taxon 2 7 7 5555589109 Single conidium-derived isolate DAR 69486.1 (taxon 2) is listed in Table 3.2, and isolates 1504 - 1538 (bulk isolates) are listed in Table 3.1. Isolates 1504 and 1508 were derived from pycnidia on a single cane, separated by a zone-line. Isolates 1514-F were derived from pycnidia on a single cane, each separated from the other pycnidia by zone-lines. Isolates 1524 and 1528 were derived from pycnidia on a single cane, separated by a zoneline, and isolates 1534 and 1538 were derived from pycnidia on a single cane, separated by a zone-line. Isolates 524, 184, 188, 174, 508, 5lC.1 and 504 represent MCGs I to 6; MCGs are in brackets. I indicates an incompatibility reaction between two isolates. - the isolates were compatible. + an incompatibility reaction between two isolates was visible in less than 5IVo of the replicates. Morphological types: cfp = ç6¡çentric rings of dense and fluffy mycelium, with pycnidia; s = sparse mycelium and uf = uniform, dense and fluffy mycelium. 139 Table 8.4 Results of mycelial incompatibility tests between eight isolates of Phomopsis taxon 1 from Tasmania, South Australia and Victoria, one isolate of taxon 2 from Tasmania, and two isolates of taxon 3 from South Australia and Victoria, and 14 isolates representative of MCGs I to 10 and taxon 2 (Tables 8.2 and 8.3) on PDA. DAR DAR 95.1 100.1 103.1 1044 r25A r25B 110.1 JM DAR 69467.1 ó9489. l 139 69458. l DAR 69458, I I I I II I I I I JM 139 I I I II I I I I 110.1 I I I II I I I I25B I I I II I I25A I I I II I 1044 I I I II 103.1 I I 100.1 I I I I 95.1 I DAR 69489.1 I l DAR 69467.1 524 (l) I I + II I I I I I I 184 (2) I I I II I I I I I I r88 (3) I I I II I I I I I I r7 A(4) I I I ++ I + + I I I 5OB,5IC.1 (5) I I I II I I I I sOA (6) I I I II I I I I I I 1504, 1s0B (7) I I I II I I I I I I 1s2A (8) I I I II I I I I I I 1528, l53B (9) I I I II I I I I I I 1s3A (10) + I I II I I I I I I DAR 69486.1 I I I II I I I I + morph. s s s ss S fe s fsp cf fe laxon? taxon 3 taxon 3 MCG 11 t2 T3 13 t4 155 5 Isolates DAR 69467.1, DAR 69489.L,95.1, 100.1 and 103.1 were single conidium-derived taxon I isolates (Table 3.2). Taxon I isolate 1044 was derived from a hyphal tip and isolates 1254 and 1258 were bulk isolates (Table 3.1). Taxon 2 isolate 110.1 and taxon 3 isolate DAR 69458.1 were single conidium-derived isolates (Table 3.2). Bulk isolate JM 139 (taxon 3) is listed in Table 3.1. Isolates 125A and l25B were derived frompycnidia on a single cane, separated by a zoneline. Isolates 524 (1) to 1534 (10) represent MCGs 1 to 10, the MCGs are in brackets. Isolate DAR 69486.L is a single conidium-derived taxon 2 isolate, used in a previous experiment (Table 8.3). I indicates an incompatibility reaction between two isolates. - the isolates were compatible. + an incompatibility reaction between two isolates was visible in less than 5l%o of the replicates. Morphological types: s = sparse mycelium; fe = fluffy mycelium at the edge; fsp = ¡u¡¡t mycelium in the centre, sparse at the colony edge, with pycnidia and cf = concentric rings of fluffy mycelium. 140 Table 8.5 Morphology of the 16 isolates of Phomops¡s taxon 2 from New South Wales, Victoria, South Australia and Tasmania, used in mycelial incompatibility tests on PDA. Isolate Morphological Origin of the isolate Date of collection type VRU OO31 fsp Griffith, NSW nJa VRU OO35 cfp Mildura, Vic nla VRU 0036 uf Crawford River, Condah, Vic nla VRU OO43 fsp nla nla VRU OO48 cfp Mum¡mbateman, NSW nla VRU OO5O ufp Irymple, Vic nJa VRU OO74 fsp Cape Jaffa, SA nla VRU OO84 uf Padthaway, SA nla DAR 57591 fsp Muswellbrook, NSW 1986 DAR 69457.r fsp Griffith, NSW r99t DAR 69460 fsp Swan Hill, Vic t992 DAR 69471 fsp Rutherglen, Vic 1992 DAR 69476 fsp Barossa Valley, SA t992 ucf DAR 69486.t Mildura, Vic 1992 91.1 fsp Clover Hill, Tas r996 110.1 fsp Marion's Vineyard, Tas 1996 Isolates VRU 0031 to VRU 0084, and isolates DAR 69460, DAR 69471, DAR 69476 and DAR 5759I are listed in Table 3.1. Isolates DAR 69457.1, DAR 69486.1,91.1 and 110.1 were single conidium-derived isolates (Table 3.2). All isolates were compatible with each other. Morphological types: fsp = ¡u¡¡t mycelium in the centre and sparse at the colony edge, with pycnidia; cfp = çsn..ntric rings of dense and fluffy mycelium, with pycnidia; uf = uniform, dense and fluffy mycelium; ufp = u¡¡¡.rm, dense and fluffy mycelium, with pycnidia and cf = concentric rings of dense and fluffy mycelium. ' Isolate DAR 69486.1 produced pycnidia in a previous experiment and was type cfp (Table 8.3). n/a = information not available. I4L Table 8.6 Results of mycelial incompatibility tests of Phomopsís taxon I isolate DAR 69488, taxon 2 isolates VRU 0048 and VRU 0084, and taxon 4 isolate DAR 69484 with isolates representative of MCGs I to 15, two taxon 2 isolates, two taxon 3 isolates, and themselves. DAR 69488 VRU OO48 VRU OO84 DAR 69484 52A (1) I I I I 18A (2) I I I I 188 (3) I I I I r7 A (4) I I I I soB (s) I I I I s1c.1 (s) I I I I sOA (6) I I I I lsoA (7) I I I I lsoB (7) I I I I ls2A (8) I I I I 1s2B (e) + I I I 1s3B (e) + I I I 1s3A (10) I I I I DAR 69467.1 (11) I nla nla I DAR 69489.t (t2) I nla nla I 9s.1 (13) I nla nla I 100.1 (13) I nla nla I 103.1 (14) I nla nla I 1044 (ls) I nla nla I VRU 0048 (taxon 2) nla I VRU 0084 (taxon 2) nla + 110.1 (taxon 2) nla I DAR 69486.1(taxon 2) nla I DAR 69458.1 (taxon 3) I + + + JM 139 (taxon 3) I + I + selfed ' MCG I6 taxon 2 taxon2 taxon 4 Isolates DAR 69488, VRU 0048, VRU 0084 and DAR 69484 are listed in Table 3.1. Isolates 524 (1) to l04A (15) represent MCGs 1 to 15, the MCGs are in brackets. Taxon 2 isolates VRU 0048 and VRU 0084 are listed in Table 3.1. Taxon 2 isolates 110.1 and DAR 69486.1 and taxon 3 isolate DAR 69458.1 were single conidium-derived isolates (Table 3.2). Taxon 3 isolate JM 139 is listed in Table 3.1. I indicates an incompatibility reaction between two isolates. - the isolates were compatible. + an incompatibility reaction between two isolates was visible in less than 5LVo of the replicates. n/a not available, these isolates were not co-cultivated. r42 Table 8.7 MCGs in Phomopsls of grapevine, the ability of the isolates to produce perithecia and their origin. "MCG Isolates 'Self-fertile Origin or sterile I 52/' self-sterile Coonawarra, SA, Chardonnay perithecium r994 2 184 self-sterile Ashton Hills, SA, Chardonnay pycnidium t994 3 188 self-sterile Ashton Hills, SA, Chardonnay pycnidium t994 4 T7A self-fertile Ashton Hills, SA, Chardonnay pycnidium t994 5 154 self-sterile Ashton Hills, SA, Chardonnay pycnidium 1994 5 l7B self-fertile Ashton Hills, SA, Chardonnay pycnidium r994 5 508 self-sterile Coonawarra, SA, Chardonnay perithecium r994 5 50D.1 self-fertile Coonawarra, SA, Chardonnay perithecium t994 5 50E self-fertile Coonawarra, SA, Chardonnay perithecium t994 5 514 self-sterile Coonawarra, SA, Chardonnay perithecium t994 5b 5lC.1 self-sterile Coonawarra, SA, Chardonnay perithecium r994 5 r25A self-fertile Hargrave, SA, Chardonnay pycnidia r996 5 I25B Hargrave, SA, Chardonnay pycnidia t996 5b 15lA self-fertile Ashton Hills, SA, Riesling pycnidia t996 5b l5lC self-fertile Ashton Hills, SA, Riesling pycnidia r996 5b 15lD self-fertile Ashton Hills, SA, Riesling pycnidia r996 5b 15lE self-fertile Ashton Hills, SA, Riesling pycnidia t996 5b 15lF self-fertile Ashton Hills, SA, Riesling pycnidia r996 6 504 self-fertile Coonawa¡ra, SA, Chardonnay perithecium t994 7 l50A Ashton Hills, SA, Riesling pycnidia r996 7 1508 Ashton Hills, SA, Riesling pycnidia 1996 8 t52A self-fertile Hargrave, SA, Chardonnay pycnidium t996 9 T52B self-fertile Hargrave, SA, Chardonnay pycnidium t996 9 1538 self-fetile Hargrave, SA, Chardonnay pycnidium t996 10 1534 self-fertile Hargrave, SA, Chardonnay pycnidium r996 11 DAR 69467.1 self-fertile Ya:ra Valley, Vic, Chardonnay pycnidium t992 t2 DAR 69489.1 Adelaide Hills, SA, Chardonnay pycnidium t99r 13 95.1 self-fertile Clover Hill, Tas, Chardonnay pycnidium 1996 13 100.1 self-fertile Freycinet, Tas, Chardonnay pycnidium r996 I4 103.1 Bream Creek, Tas, Cabemet pycnidium t996 l5 1044 Bream Creek, Tas, Cabernet pycnidium r996 t6 DAR 69488 Adelaide Hills, SA, Chardonnay pycnidium 1991 taxon 2 taxon 2 isolates self-sterile sixteen isolates, see Table 8.5 taxon 3 JM 139 self-sterile Nuriootpa, SA pycnidium nla taxon 3 DAR 69458.1 self-sterile Yarra Valley, Vic, Chardonnay pycnidium 1991 taxon 4 DAR 69484 self-sterile Hunter Valley, NSW pycnidium r992 All isolates are listed in Tables 3.1 or 3.2. n/a not available. " the Phomopsis taxon 1 isolates were grouped into MCGs I to 16; taxon2formed a 17ù MCG, taxon 3 an 18ú, and taxon 4 a 19ü' o incompatibility reactions occurred between these isolates and a small number of other isolates that were placed in MCG 5. " data from Chapter 5. - these isolates were not tested for their ability to produce perithecia. 143 The two taxon 3 isolates that were obtained in South Australia and Victoria belonged to the same MCG (Table 8.7). 8.3.2 Estimate of variation within and between populations of Pftomopsís taxon 1 Population s of Phomopsis taxon 1 in South Australia showed high levels of variation with respect to colony morphology and mycelial incompatibility, with I/'varying between 0.41 and0.74 (Table 8.8). Shannon-Weaver indices (Il) were used to divide diversity into within and between population components. More of the diversity occurred within the three populations (79.57o) than between them (20.5Vo). Using colony morphology as a marker, 90Vo of the variation occurred within the populations; analysis of mycelial incompatibility showed that 69Vo of the variation occurred within the populations (Table 8.8). Variation in colony morphology within MCG 5 and within the total collection of Phomopsis taxon I isolates was also determined. The Shannon-Weaver index within MCG 5 was I/ = 0.83 (Ë/'= 0.31), within the group of remaining MCGs (all MCGs with known colony morphology combined, except MCG 5) H - L.20 (H' = 0.42), and within the total collection of taxon 1 isolates with known colony morphology H = I.L4 (H' = 0.33). Therefore, more of the diversity occurred within MCG 5 (89Vo) than between MCG 5 and the other MCGs (LIVo). 8.3.3 Mycelial incompatibility used to study mating behaviour Mycelial incompatibility occurred among the progeny of two perithecia from the Coonawarra (Table 8.9). Two progeny of another perithecium from the Coonawarra were compatible with each other, as were three progeny of a perithecium from the Adelaide Hills (Table 8.9). 8.4 Discussion This pilot study involved 32 isolates of Phomopsis taxon 1, 16 isolates of Phomopsls taxon 2, andonly two taxon 3 isolates and one taxon 4 isolate, due to the lack of availability of the t44 Table 8.8 Shannon-'Weaver indices (/1) calculated using data for colony morphology and mycelial incompatibility, used to estimate phenotypic diversity within and between populations of Phomopsis taxon I in South Australia. Normalised Shannon-Weaver indices (H) are shown in brackets. Colony morphology Mycelial incompatibility Mean Il Ashton Hills (n = 12) r.t2 (0.4s) r.23 (0.s0) l.l8 (0.47) HHargrave (n = 6) 0.87 (0.48) 1.33 (0.74) 1.10 (0.6r) ll Coonawara (n = 7) 1.08 (0.ss) 0.80 (0.41) 0.e4 (0.48) Fl mean r.02 t.L2 t.07 .Flcombined(n=25) 1. l3 t.63 1.38 a Fl mean 907o 697o 79.5Vo Il combined bll combined - Il mean l0Vo 3lVo 20.57o Ël combined a percentage of variation that occurred within populations. b percentage of variation that occurred between populations 145 Table 8.9 Results of mycelial incompatibility tests between isolates of Phomopsis taxon I derived from four single perithecia, on PDA. 50A 508 50D.1 50E 51A 51C.1 50E I C C C 5lC.1 C C 50D.1 I C C 514 C 508 I c 504 C 548 1 54C 54D.1 146A 146B I46C 54D.1 I C c L46C C C C 54C I C I46B C C 548.1 C 146A C Isolates 50D.1,5IC.1,548.1 & 54D.1 were single conidium-derived isolates (Table 3.2), isolates 504, 508, 514 & 54C were derived from hyphal tips, and isolates 508, 1464, 146B &' 146C were bulk isolates (Table 3.1). Isolates 504 - 50E were derived from a single perithecium, isolates 5lA and 51C.1 were derived from a second perithecium, and isolates 548.1 - 54D.1 were derived from a third perithecium, all from the Coonawarra, South Australia. Isolates 146A - 146C were derived from a single perithecium from the Adelaide Hills, South Australia. I indicates an incompatibility reaction between two isolates' C the isolates are compatible. t46 latter two taxa. In terms of colony morphology, taxon I and 2 displayed a similar amount of variation. Taxon I isolates displayed more variation, in terms of mycelial incompatibility types, than did taxon 2. Among the taxon 1 isolates examined, four different morphological types and 16 MCGs were identified. Three different morphological types were identified among the taxon 2 isolates examined, and mycelium of all taxon 2 isolates was compatible, but incompatible with the 16 MCGs of taxon I and the isolates of taxa 3 and 4. The taxon 2 isolates were then grouped into a 17th MCG, the isolates belonging to taxon 3 became the 18th MCG and the taxon 4 isolate the 19th MCG. Two dominant morphological types were identified within Phomopsis taxon 1, uniform and dense mycelium (uf-type, 42Vo) and sparse mycelium (s-type,39Vo). However, the number of isolates with morphological type uf may be over-represented because isolates 1514-151F were derived from thesamepieceofcane andmay, therefore, representone strain. Similarly, isolates 1504 and 1508 were derived from the same cane. If these isolates are also identical, 427o of the isolates would have morphological type s and35Vo type uf. Variation in colony morphology in taxon 1 within vineyards in South Australia was higher than the variation between vineyards or regions in South Australia. This may be due to the occurrence of sexual reproduction in the population, latent infections on nursery rootlings caused by several different strains, or instability of the colony morphology marker in vitro. In Tasmania only one morphological type (s-type) was isolated from three vineyards in different regions, but the sample size (four isolates) was too small to draw conclusions about the variability. One dominant morphological type was identified within Phomopsis taxon 2: thick mycelium in the colony centre with sparse mycelium at the colony edge with pycnidia (fsp- type, 63Vo\ The distribution of the three morphological types found within taxon 2 was similar in Victoria, South Australia and New South Wales. In Tasmania, however, only type fsp was found within taxon 2 isolates. Sixteen MCGs were identified among the Phomopsls taxon 1 isolates examined. Assuming that mycelial incompatibility has a genetic basis, one locus with 16 alleles, or four loci with two alleles would be necessary to form 16 MCGs, because Phomopsis is haploid. However, if the latter is true, to explain the incompatibility with isolates belonging to Phomopsís taxon 2, the 17th MCG, a fifth gene would be necessary. Five loci with two r47 alleles would still be needed if the isolates belonging to taxa 3 and 4 were grouped into two additional MCGs (18th and tgth IvtCG¡. Other Phomopsis species with polygenic inheritance of mycelial incompatibility include Phomopsis group one from elm (Brayford, 1990b) and P. subordinaria (Meijer et aI., 1994). Further work is required to determine the actual number of loci involved and whether or not there are multiple alleles' The morphology of the mycelial incompatibility reactions often differed in Phomopsis taxon 1. This phenomenon was also described in P, subordinaria (Meijer et al., 1994) and in O. novo-uhzri (Brasier, 1984; Brasier, L996). Meijer et aI. (1994) did not differentiate between types of reactions tbrmed tbr the classification of the isolates into VCGs. Brasier (1984, 1996), however, described five morphological reaction categories for O. novo-ulmi, ranging from the fully vegetatively incompatible (w-reaction) through partially vegetatively incompatible (n-reaction) to the weakly incompatible (l- and lg- reactions) and the compatible (c-) reaction. In this fungus, isolates of the same w-VCG can be partially vegetatively incompatible if they have a different allele governing the n- reaction. Vegetative incompatibility in O. novo-ulmi is, therefore, multigenic, heterogenic and epistatic (Brasier, 1996). The present study showed that mycelial incompatibility in Phomopsis taxon 1 is most likely multigenic, and possibly heterogenic. Where recorded, the morphology of the mycelial incompatibility reaction between the MCG 1 isolate and isolates of MCG 5 was always a pink barrage zone, and the reaction between MCG 2 and isolates of MCG 5 was always a growth-free zone. This difference in reaction may be the result of different genes affecting the different reactions. However, more research is needed to examine whether different genes cause different mycelial incompatibility reactions in Phomopsis taxon 1. Incompatibility in Phomopsis may be epistatic, if incompatibility between isolates of taxon 1 and taxon 2 is caused by a gene other than those which cause incompatibility within taxon 1. This gene could then be seen as dominant, since all taxon I MCGs would have the same allele of this gene. In general, less variation is expected within VCGs than between VCGs (sections 2.8.2 and 8.1). Isolates of D. phaseolorun f.sp. meridionalis, Phomopsis group one from elm and P. subordinaria showed less variation in a spectrum of characteristics (virulence, growth rates, fungicide sensitivity, mating type, morphology and RAPD polymorphisms) within VCGs than between VCGs (Ploetz & Shokes, 1989; Brayford, 1990b; Meijer et aI., 148 1994).In the current study on Phomopsis of grapevine, however, there appears to be more variation in morphology within MCG 5 than between MCG 5 and the remaining MCGs. A large amount of phenotypic variation within VCGs is usually associated with fungi that do not rely primarily on asexual reproduction (section 8.1). However, morphology is a very variable character with many subtle differences, which makes it difficult to use as a marker for the identification of isolates (Meijer et al., L994). Morphological differences may also be a result of genetic changes in the isolates induced during storage and culture. The number of VCGs in a population is influenced by the number of loci that affect vegetative incompatibility and by the frequency with which sexual outcrossing occurs. Populations will tend to have fewer VCGs when outcrossing is rare or non-existent (I-eslie, 1993; Viljoen et al., L997).In this study, a relatively large number of MCGs was found in South Australia, Tasmania and the different vineyards and regions in South Australia. Moreover, variation in mycelial incompatibility in South Australia was higher within populations than between populations of Phomopsis taxon 1. The high degree of MCG diversity within vineyards and regions in South Australia may be due to sexual reproduction in the population. It is also possible that rootlings possessed latent infections caused by several MCGs which then dispersed asexually. Changes from year to year in the composition of MCGs of Phomopsis taxon I appeared to occur in Ashton Hills in South Australia. Only one MCG found in 1994 (VCG 5) was also present in 1996. Similarly, replacement of the VCGs was found in P. subordinariø infecting Plantago lanceolata in a location where the teleomorph was frequently found. In a location where one VCG dominated, the teleomorph was seldom present, which suggests a major role of the teleomorph in the occurrence of VCGs (Meijer et al., 1994). However, some asexually reproducing fungi have a high incidence of VCG diversity which is thought to be caused by the influx of conidia from other sites (Glass & Kuldau, L992). This is probably not the case for Phomopsis of grapevine, as the conidia are splash-dispersed over relatively short distances. A more detailed study, involving a number of locations over time, could provide information on the role of the teleomorph in Phomopsis taxon l. The spatial distribution of VCGs in Phomopsis species has been reported to vary. Ploetz & Shokes (1989) found that some VCGs of D. phaseolorum f.sp. meridionalis were r49 widespread over large areas. In Phomopsls group one from elm, P. subordinaria and D. ambigua populations, large numbers of VCGs were found, with each location generally having its own unique set of VCGs (Brayford, 1990b; Meijer et a|.,1994; Smit ¿r at., L997). Populations of O. novo-ulmi, however, are initially clonal or near clonal in terms of vegetative compatibility, but rapidly become highly heterogeneous (Brasier, 1996). Among the isolates of Phomopsrs taxon 1, one predominant MCG (MCG 5) was found, comprising ca 447o of the isolates. The remainder of the isolates formed a heterogeneous group, in which 15 different MCGs were present among 18 isolates. However, the number of isolates belonging to MCG 5 may be exaggerated because isolates 1514-15lF may represent one strain (see above). If that were the case, and isolates l50A and 1508 were also identical, only 377o of the strains would be of MCG 5,7.57o of the isolates would be of MCG 9, and 7.57o of the isolates would be of MCG 13, while 48Vo of the isolates formed a heterogeneous group, in which 13 different MCGs were present ¿ìmong 13 isolates. There are a number of possible reasons why MCG 5 was more common in South Australia than the other ten MCGs that occurred in that state. MCG 5 may have a competitive advantage over isolates of other MCGs, or its prevalence may be due to a founder effect. For example, in D. phaseolorun f.sp. meridionalis, VCG I comprisedT9Vo of all isolates and was more widespread than other VCGs (Ploetz & Shokes, 1986). However, this VCG did not possess superior virulence, rapid growth or insensitivity to fungicides. Thus, these characteristics did not account for the prevalence of this VCG, which was, therefore, considered to be due to a founder effect (Ploetz & Shokes, 1989). Likewise, epidemic populations of Ophiostoma ulmi in Europe are also largely composed of one VCG (Brasier, 1988). Similarly, it is possible that MCG 5 isolates became established in South Australia before isolates from other MCGs spread to this region, but further study is needed to test this hypothesis. Mycelial incompatibility occurred among the progeny of two perithecia of Phomopsis taxon 1, which is indicative that outcrossing had occurred. Apparently D. viticola has the capacity to outcross, despite previous findings that D. viticola can produce perithecia when selfed (Chapter 5). The homothallic fungus D. ambigu¿ also has the capacity to outcross (Smit et a1.,1997). 150 There did not appear to be a relationship between mycelial incompatibility and mating system. This study suggested that zone-lines on grapevine canes were not formed because vegetatively incompatible isolates met. Isolates derived from either side of zone-lines on canes were more often compatible than incompatible. Moreover, incompatible isolates have been obtained from single canes without zone-lines. V/hen Brayford (19905 assumed that the zone-lines in the inner bark of dead elm colonised by P. oblonga were formed when vegetatively incompatible isolates met, he apparently did not examine whether isolates obtained from either side of a zone-line in elm tissue were incompatible. Observations on Phomopsis taxon 1, reported in chapter 5, suggest that the formation of zone-lines on grapevine canes is associated with the formation of perithecia. Representative isolates of Phomopsis taxon 2 werc incompatible with isolates representative of taxon 1 MCGs I to 16 and with isolates belonging to taxa 3 and 4. As in the two groups of Phomopsis isolated from elm, isolates from one group (taxon 1) contained a relatively large number of different MCGs, indicating considerable genetic heterogeneity in the natural populations of this fungus, while very little variation was found in the second group (taxon 2). The heterogeneous group one of Phomopsis from elm was heterothallic and the homogeneous group two was homothallic (Brayford, 1990" & 1990b). Similarly, in Phomopsis of grapevine the difference in mycelial incompatibility within the two taxa corresponds with what is known about their mating systems. The teleomorph of the heterogeneous taxon I is abundant (chapters 4 and 5), while the teleomorph of the apparently homogeneous taxon 2 is unknown. The differences in mycelial incompatibility within the two taxa support the view that they are distinct taxa or species, with no evidence of genetic exchange. The intensity of the incompatibility reactions varied among isolates. Sometimes isolate combinations formed barrages only weakly, making it difficult to see whether isolates were compatible or incompatible. This has also been observed in southern D. phaseolorum (Ploetz & Shokes, 1986) and in P. subordinaria (Meijer et aI., 1994). Anagnostakis (1982) attributed this to single gene differences among loci determining vegetative incompatibility. When incompatibility reactions are difficult to see macro- scopically, additional measures may be useful. The anastomosis reactions between the l5l isolates can be studied microscopically (section 8.1), or complementary auxotrophic nitrate non-utilising (nit) mutants of the isolates can be paired on medium without nitrate (Masel er a1.,1996: Viljoen et a1.,1997). However, the use of a different medium may also increase the strength of the incompatibility reaction. Smit et al. (1997), for example, apparently did not come across weakly "barraging" isolate combinations of D. ambigua on freshly prepared oatmeal agar. These methods should be used to examine whether isolate 5lC.1 and isolates 15lA to l51F belong to MCG 5. If isolate 51C.1 can form a heterokaryon with isolates 154 and 508, it belongs to MCG 5, and if it cannot form a heterokaryon with isolates of MCG 5, it is of a different MCG. Similarly, isolates 1514 to 151F should be tested against isolates 50B and 51C.1 for heterokaryon formation. In theory, however, vegetative incompatibility within a VCG can be explained by the existence of cumulative vegetative compatibility (VC) loci on separate linkage groups (A.J.M. Debets, pers. com.). For example, A is a VC locus on linkage group I and B a VC locus on linkage group II. Isolates with allele combinations A1B¡ and AzBz, and AtBz and AzBt are incompatible, while all other combinations (eg. A1B¡ and Aßù are compatible. In the case of cumulative VC loci, horizontal transfer of genetic material between incompatible isolates is possible since an isolate ArBr is compatible with AzBt and the latter isolate is compatible with isolate AzBz. Molecular studies (Chapter 9), however, show that isolate 15lC is very different from the other MCG 5 isolates tested (isolates 50D.1 and 51C.1), while mating type studies (Chapter 5) showed mycelia of isolates l25A and 15lC to be incompatible (Ftg. 5.1). This indicates that isolates 151A-151F may not belong to MCG 5. Isolates within a VCG are potentially capable of exchanging genetic information via a parasexual process. The importance of this ability depends on the structure of the population, the number of partners with which an isolate could exchange DNA (Iæslie, 1993), and on the importance of the sexual stage. In taxon 1 a large number of potential VCGs have been found in natural populations. This suggests a very low occurrence of heterokaryon formation and, therefore, the frequency with which genetic diversity is generated via the parasexual cycle would be very low. In taxon 2, however, all isolates appeared to be compatible and the sexual stage has not been found. t52 9. DEVELOPMENT OF TAXON-SPECIFIC AND RFLP MARKERS, AND A PILOT STUDY ON GENETIC DIVERSITY TN PHOMOPSß OF GRAPEVINE 9.L Introduction RFLP markers have been used to study genetic diversity within and between populations of fungi, and for the taxonomic identification of species and subspecies (section 2.9.2). Generally, probes used to detect RFLPs have been cloned from DNA of the species being studied, or synthesised as oligonucleotide sequences consisting of tandem repeats of a core sequence @eScenzo & Harrington, 1994). The advantage of using DNA cloned from both taxon 1 and 2 of Phomopsis of grapevine, is that taxon-specific and species-specific markers can be developed. Such markers have the potential to be used for the detection of the pathogen in asymptomatic, infected grapevine tissue, and may be used to distinguish Phomopsis from the many other fungi that cause similar symptoms on grapevine canes (section 2.10). Some advantages of using synthetic microsatellite probes are that (i) they are relatively inexpensive to prepare, (ii) the core sequence and number of repeats can easily be changed (Ali et a1.,1986), (iii) the DNA fingerprints generated are highly reproducible, and (iv) they are effective in detecting RFLPs in a variety of fungi and other organisms (DeScenzo & Harrington, 1994). However, they are not specific and must be used with pure cultures. Molecular markers were developed for the study of genetic diversity in natural population s of Phomopsis of grapevine and the relatedness of the four taxa identified thus far in Australia. DNA from one isolate each of taxon 1 and taxon 2 was cloned and both taxon-specific markers and sequences that detected genetic diversity within and between the taxa were selected. The amount of genetic variation among 24 isolates of taxon 1, 13 of taxon 2, two of taxon 3 and one of taxon 4 was investigated, using the clones and synthetic microsatellite probes. Genetic similarities were calculated between all pairs of isolates in order to determine the relatedness of the different taxa. 153 9.2 Materials and methods 9.2.1 Screening genomic libraries of Phomopsis taxon 1 and 2 for taxon-specifTc and RFLP markers Total genomic DNA of DAR 69467.1, a single conidium-derived isolate of Phomopsis taxon 1, was digested to completion with restriction endonuclease PsrI and cloned as described in section 3.3. Clones were numbered using the code pTlP# where p = plasmid, T1 = taxon 1, P = PsfI and # = a number. The second DNA library was constructed using DNA from DAR 69457.1, a single conidium-derived isolate of taxon 2, digested to completion with S¿lI (section 3.3). Clones were numbered using the code pT2S# where p = plasmid, T2 = taxon 2, S = Søl[ and# = a number. Plasmids containing Phomopsis DNA inserts were identified using colony blots that were hybridised, in three separate reactions, with radiolabelled total DNA of taxon 1 isolate DAR 69467.I, totalDNA of D.viticolaisolate 50D.l,andtotalDNAof taxon2isolate DAR 69457.I (section 3.3.3). Taxon 1 and taxon 2 clones that showed medium to strong hybridisation signals on colony blots, or hybridised only with DNA from the taxon 1 and D. viticola isolates, or only with DNA from the taxon 2 isolate, were isolated using the crude plasmid DNA preparation method described in section 3.4.I. Phomopsis DNA inserts were separated from vector DNA by digestion with the appropriate restriction enzyme (Psfl or Sa[I), fractionated in 10 cm long IVo agarcse gels, transferred to positively charged nylon nitrocellulose membranes, and hybridised, as above, with radiolabelled total DNA from the three isolates, in three separate reactions (sections 3.4.3 and 3.4.4). Using this method, the size of the inserts was determined, and pUC19 DNA was shown to hybridise to Phomopsis DNA. When multiple inserts were found, the inserts were numbered with a capital letter, insert A representing the largest insert in a recombinant plasmid, insert B the second largest insert, and so on. The hybridisation signal for each insert was compared with the intensity of the corresponding band in the agarose gel after ethidium bromide staining, to obtain an estimate of the copy number. Recombinant plasmids containing putative taxon-specific inserts or those giving a medium to high signal, were isolated using the Promega Wizard@ P/¿¿s SV Minipreps DNA Purification System kit, according to the manufacturer's instructions. Inserts were separated 154 from vector DNA by restriction enzyme digestion, followed by electrophoresis in l7o agarose gels, and recovered using the "gene clean" or the "fÍeeze squeeze" method (section 3.4.2). They were then radiolabelled and screened for their ability to distinguish different isolates, by hybridisation to Southern blots of restriction enzyme digested total DNA from different Phomopsis isolates (Table 9.1) and grapevine. A 3 ttg amount of Phomopsis DNA and grapevine DNA were digested with PsrI and DraI, respectively, in 100 pl, fractionated in 20 cm long lVo agarose gels, transferred to positively charged nylon nitrocellulose membranes, and hybridised with 50 ng radiolabelled insert DNA (sections 3.4.3 and3.4.4). Each probe was hybridised to total DNA from: 24 taxon 1 isolates including D. viticola; 13 taxon 2 isolates; two taxon 3 isolates; and one taxon 4 isolate (Table 9.1). For the purposes of this analysis, the atypical isolates 1148 and 115B (section 3.1) were included in taxon l. Fourteen taxon 1 isolates, 13 taxon 2 isolates and one taxon 4 isolate were present on two separate Southern blots, enabling banding patterns on different blots to be compared after hybridisation with the different probes (Table 9.1). The taxon 2 clones were hybridised to a Southern blot containing 60 ng SølI- digested total DNA from six taxon 2 isolates (DAR 69457.I, DAR 69486.L, 46,77,91.1, 110.1) to determine the specificity and the degree of genetic variation. A putative taxon 2-specific clone was screened by hybridisation to another Southern blot with 60 ng Sa/I-digested total DNA from grapevine, seven taxon I isolates (DAR 69467.1, DAR 69489.1,42A,95.1, 994.1, 103.1, l14B), four D. viticola isolates (50D.1, 5lC.1, 85, 88.1), six taxon 2 isolates (DAR 69457.1, DAR 69486.I, 46,77,9I.1, 110.1), and taxon 3 isolate DAR 69458 (section 3.4.3). The clone was radiolabelled using the pUC19 Pvall-specific primers Sl and 52 (section 3.4.4). A control hybridisation using pUClg DNA as the probe, demonstrated that the vector hybridised to digested and undigested total DNA of Phomopsis taxon 1 and taxon 2. In addition, cloned Phomopsis taxa 1 and 2 inserts were hybridised to a Southern blot, prepared by Mrs. B. Rawnsley, containing 100ng PsrI restricted DNA from Aspergillus nidulans, Botrytis cinerea, an unknown pycnidial fungus isolated from grapevine cane, and apparently healthy grapevine canes and berries. 155 9.2.2 Screening microsatellites for RFLP probes Synthetic microsatellite probes (GATA)4, (CAT)5, (GAA)6, (GACA)4 and (GGAT)4 (received from Dr B.E. Stummer) were screened by hybridisation to Southern blots of I pg of digested total DNA from taxon I isolate DAR 69467.1, D. viticola isolate 51C.1 and taxon 2 isolate DAR 69486.LTotal Phomopsis DNA was digested with PstI, Sa[I and DraI in 40 pl and fractionated in a 20 cm long lVo agarose gel, and with HaeU., S¿a3AI and Hpan in 40 ¡rl and fractionated in a 20 cm long 27o agarose gel. The DNA was transferred to positively charged nylon nitrocellulose membranes, and hybridised with 50 ng of each of the microsatellite probes (sections 3.4.3 and3.4.4). Endonuclease/microsatellite probe combinations which produced DNA fingerprints with the most distinct bands and detected differences between the isolates, were used to study genetic variation among the Phomopsis isolates. The selected microsatellite probes were hybridised to Southern blots of 3 pg of Psrl-digested total DNA from the isolates listed in Table 9.1 and 3 pg of Dral-digested grapevine DNA. Twenty-eight of the 40 Phomopsis isolates were present on two Southern blots and were hybridised twice with each probe, enabling the comparison of banding patterns on different Southern blots. 9.2.3 RFLP data analysis RFLP fingerprints, generated by selected microsatellite sequences and cloned DNA fragments used as probes on Southern blots of Psrl-digested total Phomopsis DNA, were analysed to estimate genetic variation of isolates within and between geographical regions and to determine the genetic relationship of the isolates. For each of the 40 Phomopsis isolates listed in Table 9.1, restriction fragments were scored as being present (1) or absent (0). Only distinct bands were scored. Variation within and between two populations of taxon I in South Australia and within and between taxon I and taxon 2 of Phomopsis were analysed using Shannon- Weaver indices (I/¡. The frequencies of the RFLP phenotypes generated by each of 14 probes in these populations were calculated, and phenotypic diversity was estimated using Shannon and Weaver's information statistic (Shannon & Weaver, 1949) as in section 8.2.2, except that p¡is the fraction of the sample represented by the ln nfÏ-p banding pattern. To 156 Table 9.I Phomopsis isolates used for screening of RFLP probes. ulsolate Taxon Origin Date of Number of Collection Southern Blots T7A 1 Ashton Hills, Adelaide Hills, SA, 1994 I 50D.1 T Gartness Block, Coonawarra, SA, 1994 2 51C.1 T Gartness Block, Coonawarra, SA, r994 1 85 T Mildara, Coonawarra, SA, 1995 I 87.r T Hargrave, Adelaide Hills, SA, r995 2 88.1 T Hargrave, Adelaide Hills, SA, r995 2 95.1 1 Clover Hill, Tamar Valley, Tas, r996 2 99A.1 1 Freycinet, Hill Vineyard, east Tas, r996 2 o1l4B 1 Two Wells, Adelaide Plains, SA, r996 I olr5B I Fern Hill, Vic, r996 2 r33 T Bream Creek, east Tas, r996 2 135 T Clover Hill, Tamar Valley, Tas, r996 2 r36 T Spring Vale Vineyard, east Tas, r996 2 1394 T Marion's Vineyard, Tamar V., Tas, 1996 1 139B T Marion's Vineyard, Tamar V., Tas, r996 1 139C T Marion's Vineyard, Tamar V., Tas, t996 I 140 1 Hillstowe, Adelaide Hills, SA, t996 2 1414 I Hillstowe, Adelaide Hills, SA, r996 2 1418 I Hillstowe, Adelaide Hills, SA, 1996 2 15lC 1 Ashton Hills, Adelaide Hills, SA, r996 I t538 1 Hargrave, Adelaide Hills, SA, r996 I DAR 69461 1 Drumbourg, Vic, 1992 I DAR 69467.1 1 Yarra Valley, Vic, r992 2 DAR 69488 1 Adelaide Hills, SA, 1991 2 77 2 Southcorp, Padthaway, SA, 1995 2 91.1 2 Clover Hill, Tamar Valley, Tas, r996 2 I 10.1 2 Marion's Vineyard, Tamar V., Tas, r996 2 DAR 57591 2 Muswellbrook, NS{, 1986 2 DAR 69457.1 2 Griffith, NSW, 1991 2 DAR 69460 2 Swan Hill, Vic, 1992 2 DAR 69486.1 2 Mildura, Vic, 1992 2 VRU OO31 2 Griffith, NSW, nla 2 VRU 0036 2 Crawford River, Condah, Vic, nla 2 VRU OO48 2 Mum¡mbateman, NS\ry, nla 2 VRU OO5O 2 þmple, Vic, nla 2 VRU OO74 2 Cape Jaffa, SA, nla 2 VRU OO84 2 Padthaway, SA, nla 2 DAR 69458 3 Yarra Valley, Vic, I99L I JM 139 J Nuriootpa, SA, nla I DAR 69484 4 Hunter Valley, NSW, r992 2 T teleomorph of taxon 1, collected from perithecia of D. viticola (Chapter 4). n/a = collection date not available n Isolates 50D.1, 5lC.1, 91.1, 95.1,994.1, 110.1, DAR 69457.1, DAR 69467.I & DAR 69486.I are single conidium-derived isolates, and 87.1 & 88.1 are single ascospore-derived isolates obtained from two perithecia on the same cane (Table 3.2). Isolates l7A, 1394, 1398 & 139C were derived from hyphal tips, and the remaining isolates are bulk isolates (Table 3.1). Isolates 1394, 1398 & l39C were derived from a single perithecium. b The a-conidia of these isolates did not match the description of taxon I (section 3.1). r57 compare the diversity of the two taxa, the Shannon-Weaver indices were nonnalised (Goodwin et aL,1993). Genetic similarities among all pairs of the 40 Phomopsis isolates were calculated using Nei & Li's (1979) index of genetic similarity for RFLP comparisons (S*y), with the formula: S*y = 2N"y/(N*+Ny), where N*, is the number of restriction fragments shared by the two isolates x and y, and N* and N, are the numbers of fragments in the fingerprints of isolates x and y, respectively (Nei &L|1979; Lynch, 1990). The Unweighted Pair Group Method with Arithmetic Mean (UPGMA) was used for cluster analysis of the S*, values (Rohlf, 1985), and dendrograms of genetic similarity for the RFLP data were generated. Calculations were carried out using the GENSTAT 5 Package (Numerical Algorithms Group, Oxford). 9.3 Results 9.3.1 Screening genomic libraries of Phomopsis taxon 1 and 2 for taxon-specific and RFLP markers A. Plasmid DNA preparations of selected clones Fifty taxon 1 clones that showed medium to strong hybridisation signals on colony blots, and./or hybridised with DNA from taxon I isolate DAR 69467.1 and D. viticola isolate 50D.1 only, and not with DNA from taxon 2 isolate DAR 69457.1, were selected (section 3.4.I, Fig.9.1). Fourteen clones contained multiple inserts, and eight plasmids apparently did not contain an insert. A total of 18 Phomopsis taxon 1 inserts hybridised with DNA from the taxon 1 and D. viticola isolates only. Using hybridisation signal as a guide, nine of these putative taxon l-specific DNA inserts were classified as low copy, four as medium copy and five as high copy clones (Table 9.2). A total of 46 Phomopsis taxon 1 inserts showed weak hybridisation to taxon 2 DNA. Thirty-four taxon 2 clones that showed medium to strong hybridisation signals on colony blots, and/or hybridised with DNA from taxon 2 isolate DAR 69457.I only, and not with DNA from the taxon 1 and D. viticola isolates, were isolated. Eleven clones contained multiple inserts, and six plasmids apparently did not contain an insert. Two taxon 2 inserts, r58 Figure 9.1 Example of crude plasmid preparations of Phomopsls taxon I clones, and the corresponding autoradiographs after hybridisation with total DNA from Phomopsís taxon I or taxon 2. The cloned sequences originated from single conidium-derived isolate DAR 69467.r A. Plasmid preparation of Phomopsis taxon I clones, digested with Psfl, fractionated in a l%o aflarose gel and stained with ethidium bromide. Lanes 1-7: pTlPlO, pT1P84, pTlP95, pT1P99, pTlP180, pTlP2l6,pT1P238. Hindil-digested l. DNA size markers are shown to the left. B. Corresponding autoradiograph after hybridisation with total Phomopsis taxon 1 DNA. C. Corresponding autoradiograph after hybridisation with total Phomopsls taxon 2 DNA. Total DNA from both Phomopsis taxon I and taxon 2 hybridised to the plasmid vector, pUC19. A B C Àt234561 Àt234561 ì"r234567 23.1 tn 9.r !- 6.6 - ¡ 4.4 O a o O a - l-.'5 2.3 - 2.0 a a ¡ o r59 Table 9.2 Insert size and estimated copy number of the putative taxon-specific Phomopsis clones identified using the crude plasmid DNA preparation method. Inserts originated from single conidium-derived isolates DAR 69467.1 (taxon 1) and DAR 69457.1(taxon 2). Cloneu Taxon Insert Size (kb) Copy NumberÞ pTlP10 1 3.5 high pTlP45, insert A I 3.7 low pT1P87 1 2.5 low pT1P88, insert B I 1.9 low pTlP95 1 3.5 high pT1P180 1 3.8 high pTlP182 1 5.2 low pTlPl88, insert A 1 5.2 low pTlPl99 I 5.0 medium pTLP2I6 I 3.8 high pTIP256 1 7.0 medium pT1P260 1 1.9 low pTLP267 1 3.8 high pTlP27I, insert A I 4.r medium pT1P284, insert B I 2.2 low pT1P286 I 5.8 low pT1P311, insefi D I 1.8 low pTIP322 1 12.0 medium pT2522 2 4.0 high pT2S34, inseft A 2 6.2 low u taxon-specifrc Phomopsis DNA inserts of recombinant plasmids containing multiple inserts are named insert A (largest of the inserts), insert B (second largest of the inserts) or insert D (fourth largest of the inserts). o number was estimated using the hybridisation signal of the crude plasmid preparations, hybridised"opy with total Phomopsis DNA. 160 one low copy and one high copy, hybridised with DNA from the taxon 2 isolate only (Table 9.2). A total of 29 taxon 2 inserts hybridised with taxon l, D. viticola and taxon 2 DNA. B. Putative taxon-speciflrc clones Inserts of the high copy putative taxon 1- specific clones pT1P10, pT1P95, pT1P180 and pTIP2l6, the insert of the putative taxon 2-specific clone pT2S22 and the largest insert (insert A) of taxon 2 clone pT2S34 (Table 9.2), were hybridised to Southern blots of PsrI- digested total DNA from the Phomopsis isolates listed in Table 9.1 and DrøI-digested grapevine DNA. The original data of these hybridisations are presented in the Appendix. Each of the four putative taxon l-specific clones generated RFLP fingerprints containing 15 to 30 bands in 20 of the 24 taxon I isolates tested and each of the clones identified 17 different banding patterns among the 20 taxon 1 isolates (Table 9.4A).Isolates 139A, 1398 and 139C had identical banding patterns, as did isolates 87.1 and 88.1. Clone pT1P180 did not hybridise with DNA from taxon 2 (Fig. 9.2),3 or 4, nor from taxon I isolates 114B, 1158, 15lC and 1538. Clones pTlPlO and pT1P95 gave identical banding patterns with all isolates tested. Both clones hybridised weakly to a 4.8 kb DNA fragment present in ten of 13 taxon 2 isolates (absent in isolates VRU 0084, VRU 0036 and DAR 69486.1), and to a 15 kb fragment in isolate VRU 0036. The clones did not hybridise to DNA from taxon 3 or 4, nor from taxon I isolates 1148, 1158, 151C and 1538. Clone pTlP2L6 showed weak hybridisation to a4.7 kb DNA fragment present in ten of 13 taxon 2 isolates (absent in isolates VRU 0084, VRU 0036 and DAR 69486.1), and to a 13.8 kb DNA fragment in isolate VRU 0036, and to three DNA fragments (2.9 kb, 4.2 kb and 5.8 kb) present in isolates 15lC and 1538. It did not hybridise with DNA from taxon 3 and 4 isolates nor taxon 1 isolates IL4B, 1158. None of the inserts hybridised to DNA from grapevine, A. nidulans, B. cinerea or the unknown fungus from grapevine canes. The two putative taxon 2-specific clones did not hybridise to DNA from taxon 1, 3 or 4, nor to DNA from grapevine, A. nidulans, B. cinerea or the unknown fungus from grapevine canes. Insert A of clone pT2S34 hybridised with all Phomopsis taxon 2 isolates tested (Fig.9.2, Table 9.4A). Clone pT2S22 hybridised with nine of 14 taxon 2 isolates and did not hybridise with PsrI- or Søll-digested DNA from taxon 2 isolates DAR 69486.1, VRU 0036, VRU 0084,46 and 77 (F|g 9.3, Table 9.44). l6r Both putative taxon 2-specific clones hybridised to only one fragment of .S¿l[- digested total DNA from six taxon 2 isolates. Insert A of clone pT2S34 hybridised to a 6.2 kb fragment of Søll-digested total DNA from the source isolate (DAR 69457.1), which was the size of the insert. When the insert was hybridised to Pstl-digested total DNA from 13 taxon 2 isolates, three bands appeared for each isolate. In 12 isolates, including the source isolate, DNA fragments of 1.8 kb, 1.9 kb and 3.4 kb showed homology with the clone (Fig. 9.2); the inconsistent band is probably the result of partial digestion by the restriction endonuclease PsrI. Isolate VRU 0036 also had three homologous fragments, but they were 1.78 kb, 1.9 kb and 3.4 kb. Clone pT2S22 hybridised to a 4 kb fragment of Sall-digested total DNA from the source isolate (Fig 9.3), which is the size of the insert. However, the clone hybridised to several DNA fragments greater than 20 kb, as well as a number of smaller fragments (4.6-20 kb) of Psrl-digested total DNA from the source isolate and the other taxon 2 isolates with homology to the insert (Fig. 9.3). C. Non-specific RFLP fingerprinting clones Two taxon I clones (pT1P84, insert B and pT1P99) and three taxon 2 clones (pT2S12, insert B, pT2S21, insert A and pT2S2I, insert B) that hybridised with both taxon 1 and taxon 2 DNA (Table 9.3), were hybridised to Southern blots of Psrl-digested total DNA from the isolates listed in Table 9.1 and DraI-digested grapevine DNA. Taxon I clone pT1P84 (insert B) recognised 10 to 19 DNA fragments among 20 taxon I isolates, while only one to three bands appeared for taxon 1 isolates 1148, 1158, 15lC and 1538, 13 taxon 2 isolates and two taxon 3 isolates (Table 9.48).Isolate 1148 and the taxon 3 isolates had identical banding patterns, and one of the two bands was present in isolate 1158. The three bands found for isolates 15lC and 1538 were unique and the probe did not hybridise to total DNA from the taxon 4 isolate. In total, 20 different banding patterns were identified among24 taxon I isolates (Tables 9.4B and 9.5, Fig. 9.4). Taxon I clone pT1P99 hybridised with one to 11 DNA fragments among the taxon 1 isolates (Fig. 9.4) and only one unique fragment (3.8 kb) for the taxon 2 isolates, the taxon 3 isolates and isolates 114B and 115B (3.1 kb) andisolates 151C and 1538 (3.6 kb). In total, 16 different RFLP banding patterns were identified among 24 taxon I isolates (Tables 9.48 and 9.5). t62 Figure 9.2 Southern blot of total DraI-digested DNA (approx. 3 pg lane-r) from grapevine (lane 1), and total Psfl-digested DNA (approx. 3 pg lane-r) from five Phomopsis taxon 2 isolates (lanes 2-6), four D. víticola isolates (lanes 7-10) and four taxon 1 isolates (lanes 11-14), hybridised with a taxon l-specific probe (A) and a taxon 2-specific probe (B). Hindm-digested l, DNA size markers, in kb, are shown to the left. Isolates from left to right: vRU 0048, vRU 0050, '77, 91.I, 110.1, 85, 1394, 1398, 139C, DAR 6946I, 95.1, 1414, 1418. A. Taxon l-specific insert of clone pT1P180 used as probe. B. Taxon 2-specific insert A of clone pT2S34 used as probe. A B I 2 3 4 5 6 7 8 9 1011121314 r 2 3 4 5 6 7 8 9 10 11 t2 13 74 23.t - 9.4 - 6.6 - 4.4 - ** I* 2.3 2.O FU. 3 t63 Figure 9.3 Southern blots of Phomopsis DNA hybridised with the radiolabelled putative Phomopsis taxon 2-specific clone pT2S22. A. Autoradiograph of total ,SøIl-digested DNA (approx. 60 ng lane-r) from grapevine (lane l), six Phomopsis taxon 2 isolates (lanes 2-7), one taxon 3 isolate (lane 8), four D. viticola isolates (lanes 9-I2) and seven taxon I isolates (lanes 13-19). Hindfr-digested À DNA size markers, in kb, are shown to the left. Phomopsis isolates from left to right: DAR 69457.I, DAR 69486.r,77, 46,91.1, 110.1, DAR 69458,50D.1, 5lC.1, 85, 88.1, DAR 69489.1, DAR 69467 .r, 95.1, 99A.1, 103. 1, rr4B, 42A. B. Autoradiograph of total Psrl-digested DNA (approx. 3 pg lane-r¡ from 13 taxon 2 isolates (lanes l-13) and one taxon 4 isolate (lane 14). Hindfr-digested l, DNA size markers, in kb, are shown to the left. Phomopsis isolates from left to right: DAR 69457.1, DAR 57591, VRU 0031, VRU 0048, VRU 0074, VRU 0084, 77,91.1,110.1, VRU 0036, vRU 0050, DAR 69460, DAR 69486.r, DAR 69484. A B t234 6 7 I 9 10111213141516171819 r234567891011121314 . fltl 23.t - 23.t - 9.4 - 9.4 - 6.6 6.6 - - 4,4 - 4.4 - 2.3 23- 2.0 2.t - rt t't 164 Taxon 2 clone pT2S2l (insert A) identified three different RFLP banding patterns among the 13 taxon 2 isolates (Tables 9.4B and 9.5). The clone hybridised to six or seven fragments among the taxon 2 isolates, while only one to three faint bands appeared for the taxon 1,3 and 4 isolates (Table 9.48). The band for isolates 1148 and 1158 was also present in the taxon 3 isolates but not in any of the other isolates, and the band for isolates 151C and 1538 was also present in the taxon 4 isolate and the taxon 1 isolates except isolates 1l4B and 1158. Taxon 2 clones pT2SI2 (insert B) and pT2S2l (insert B) each identified only one banding pattern among the 13 taxon 2 isolates (Tables 9.4B and 9.5, Fig. 9.4); the weaker inconsistent bands (Fig. 9.4) are probably the result of partial digestion by the restriction endonuclease Psfl. Insert B of pT2S21 hybridised to three fragments of the taxon 2 isolates, while only one or two bands appeared for the taxon 1 and taxon 3 isolates. Isolates l14B and 1158 had the same banding pattern as the taxon 3 isolates (Tables 9.4B and 9.5). Insert B of pT2S12 hybridised to six fragments of the taxon 2 isolates, compared toonlytwofragments amongthetaxon l andtaxon 3 isolates. Isolates 1148 and 1158 had the same bands as the taxon 3 isolates and these bands were not present in taxon I and taxon 2 (Table 9.4B). The clone hybridised to a 4.7 kb fragment of SøIl-digested total DNA from the source isolate and other taxon 2 isolates, which is the size of the insert. Three probes (pT1P99, insert B of pT2SI2 and insert B of pT2S21) revealed identical banding patterns for isolates 1l4B and 1158 and the taxon 3 isolates. Clone pT1P84 (insert B) revealed identical banding patterns for isolate 1148 and the taxon 3 isolates, while isolate 115B was identical apart from one missing band (Tables 9.4B and 9.5, Fig. 9.5). The four probes did not hybridise to total DNA from the taxon 4 isolate. In addition, insert B of pT1P84, clone pT1P99 (Frg. 9.5) and insert B of pT2S21 identified isolates 15lC and 153B as being different from the other taxon 1 isolates. None of the putative RFLP fingerprinting clones hybridised to DNA from A. nidulans, B. cinerea or the unknown fungus of grapevine canes, and the insert of pTlP99 was the only clone that hybridised to grapevine DNA. 165 Table 9.3 Putative RFLP Phomopsis clones that were used to probe total DNA from isolates listed in Table 9.1. Insert size, estimated copy number and hybridisation signal of the clones were identified using the crude plasmid DNA preparation method and hybridisation to radiolabelled total Phomopsis taxon I and taxon 2 DNA as probes. Inserts originated from single conidium-derived isolates DAR 69467.I (taxon 1) and DAR 69457.1 (taxon 2). Cloneu Insert size (kb) Hybridisation signal to taxon 1 DNA taxon 2 DNA pT1P84, insert B 6.3 strong weak pTlP99 4.4 strong weak pT2SI2, insert B 4.7 medium strong pT2SZI, insert A 7.5 weak strong pT2S2I, insert B 3.8 weak medium ^ Phomopsis DNA inserts of recombinant plasmids containing multiple inserts are named insert A (largest ofthe inserts) or insert B (second largest ofthe inserts). r66 Figure 9.4 Southern blot of total Psrl-digested DNA from Phomopsls isolates (approx. 3 pg lane-r), hybridised with taxon 1 (A., B) and taxon 2 clones (C, D). ArB. Phomopsis taxon I isolates, lanes 1-13: DAR69488, 140, 1414, 1418,50D.1,87.1, 88.1, 95.1, 99A.1, 133, 135, 136, DAR 69467.I. A. Hybridised with the multi-copy clone pT1P84 (insert B). B.Hybridised with the medium copy clone pT1P99. C, D. Phomopsis taxon 2 isolates, lanes 1-13: DAR 69457.I, DAR 5759L, VRU 0031, vRU 0048, VRU 0074, VRU 0084, ',77,9L.1, 110.1, VRU 0036, VRU 0050, DAR 69460 and DAR 69486.I. C. Hybridised with the medium copy clone pT2Sl2 (insert B). D. Hybridised with the low copy number clone pT2S2L (insert B). A B r 2 3 4 5 6 1 8 9 l0ll1213 I 2 3 4 5 ó 7 8 9 t0lt1213 - 23.t - FT 94 t - IT) - 66 t -l- lt 44 ü IO. l t F t 23 ¡t ll Tt í* 20 -¡ C D | 2 3 4 5 6 7 8 9 10 11 t2l3 1 2 3 4 5 6 7 8 9 10111213 23 I 94 66 44 lrlrb -I)- - - 23 -2.0- l¡--(¡ =+ ¡tt rE I ü r{¡t------_¡l _ r- rrlüIEtrr c 4* 's {Ë î¡ ,* {þ t67 Figure 9.5 Southern blot of total Psrl-digested DNA (approx. 3 ¡rg lane-r¡ from taxon 3 isolates (lanes I-2),a taxon 4 isolate (lane 3), D. viticola isolates (lanes 4-6) and taxon I isolates (lanes 7-ll), hybridised with taxon I (4, B) and taxon 2 clones (C). Hindfr- digested À DNA size markers, in kb, are shown to the left. Phomopsis isolates from left to righr: DAR 69458, JM 139, DAR 69484,50D.1, 88.1, 135, I7A,1148, 1158, l5lc, 1538. A. Taxon 1 clone pT1P84 (insert B) used as probe. A faint 5 kb fragment was visible in lane 11 of the original autoradiograph. B. Taxon I clone pT1P99 used as probe. C. Taxon 2 clone pT2SI2 (insert B) used as probe. A r2345678910 11 23.1 - 9.4 - It 6.6 - 4.4 - t 2.3 - B 1.2345678910 11 23.r - ; 9.4 - 6.6 - 4.4 - Ir; 2.3 - c I 2 3 4 5 6 7 8 9 10 1l t 23.r - 9.4 - 6.6 - 4.4 - rr r; rl 2.3 - ?.-ts Table 9.44 The presence or absence of each restriction fragment in Psrl-digested DNA from 24 Phomopsis taxon 1 isolates, 13 taxon 2 isolates, two taxon 3 isolates and one taxon 4 isolate, using putative taxon-speciflrc Phomopsls clones. Isolate Probeu oTlPlO & oTlP95 pTlPlS0 pTlP2l6 oT2S22 oT2S34 insert A Taxon I l7A 00100001m01000000000m01 0010000000ün00 010001001010001000101 I I 0l r 100000 0000010ü)000r0000üxxn0000100010000010010000101000000 0m00000 0000 50D.1 00 l 0000 r 000 I 0001 00100 I 00 l 00r l 000 l 0000000 l0l0l 1010010010000101 l l0l l 100000 100001m0000100000010000001000 l r m0l 10010010101000000 000000ü) 0000 5lc.l 00l0l0l 1000001001m010101001 l l 10r0000000 1010010000010100m101 l00l 1010000 100001 0000001 0001000001000000101m01 010100101010000ff) 000000,00 0000 85 0ü)r l0l00l r00010000010100m101 l r 00ff)0010 l 0 l0l 00 lm000l m0l m0l l0l l 0l 0000 10100100000000000001000000001m01000001 1001010001 0100 00000000 0000 87.1 & 88.1 ün0101000r001 001m000001ün010100100000 001 r0l0l 000m1000000010100000000 0ün0100010000000010ün000lm0l 010001fino1000000m000 00000000 0000 95.1 0100t001 1m0m01001000000001momr000000 l00l r 001 01001000100101 r0l000lm0 1010000100000000001m00000100r 000001 10010010000ff)1000 00000000 0000 99A.1 0r001 001 r0l 10000101000üxn0l I I I l0l 01001 0 100100010010100100101 l l0l l l l0r l I l 0000000m l 000 l 0000 l 00000 I 000 I 0 I 000 I 0 I 0 I 00 I 0 I 0 I I 00 I I I 00000000 0000 I l4B M MfiXXXXXNOOOOOüXXXXNO 00000m0 m00 I l58 W 00000000 fino 133 001 001 0 I 00t 00 I 00r 0l 000000001 0l I 00m00000 001001000001m0100101 1000t 000000 10000üxxn0000000100000m0100101000001 100000100000000 00000000 0000 135 001 0r 001 r 01001001010000101001 l0lm0l0l0l l0l0l00l00l 1001000101 l l0l l 10001 l 10010ü)0ü)10001m0100m000000101000101 I lün010100001 I 00m0000 0000 136 00r 0001 0000001 001 0 l 00000ü)0 I I I I ün0000ü) l 0 l 00 l 000001 00001 mol r 001 I 0 10000 l 0000010000000ün010100000100101000101 1m010100000000 o0000000 0000 l39A,B,C 001m01m010000100000100m0100m00000000 l 0l 00001 üxnOl 00 10001 l l0r 0000000 100m1000000ün0000010000t m00l0000ml0l00l0lün000ü) 00000000 0000 140 00r 00010001 1m00100000m0000001000000m0 000101 0l I m0ml 00000r l 0l 00000m0 fixn0r000001ün0000000100001000001001000010010100m00 00000000 0000 l4tA 0001001 1m01000100000ü)0100001 m000000fi) l0 l 001 m000001 00 I 00001 I 0l I 000(n0 l0ü)0100000fixxn0001000100ün00l0ml0l0l00l0l ün00000 00000000 0000 l4lB 0001001010r ün010001 0000100000010000ün0 l 0l 0l mol m00l l 000001 I 0000000m0 10m000000m01000m10010000m01000001010010m00001000 00000000 0000 t5lc M w r fin00000000 I 00000000 I 000m00 mom000 0000 l53B M M r üxn000ün0 I 00000000 I 000m00 000uxn0 00ü) DAR 69461 0lün0100001000100100100101001 I 10001finO l 00r 00000000000001001 l l 0l 00üm00 lm0l000üxxn0000m0r0000001001 l0ml0l00r0l 0100000000 00000000 0000 DAR 69467.1 0l finOl0l0l000l0l0l00ml I 00001010101 0001 l00l 00 I m000mt 000101 l 00101 001 l 0 100000m100000000010010m0000101m01 0100001m01001010 00000000 0000 DAR 69488 mol 001 0 l 0r l 0000 l 01000m I 00001 fin001 l m0 l0l 0r l0l00l0lfixnOl0l 1m0lofixno 1000010ün000001000100000010001 lün0l00lm0l 100000000 00000000 0000 Taxon 2 77 00000000000000000r0fin0000000000000fin00 W M1000000000000000000 00000000 I ll0 I l0.l 0000000000000000010üxm00000000000000000 0000M 00000000000000m000000000000000000 l 00000000000m00000 llllllll 1ilo NSW phen m00000000000000010m00000000m00üx)0000 00M0000 0000000000000000000000000000000000 l 000m0000000000000 lllllill I110 VRU 0036 I W 0 l 00000m00000ün0000000000000000000000000000m000000 00000000 I l0l vRU 0084 M00000 000000000000000000000ün00000000000000000000000000000 00000000 ln0 DAR 69486.r M 0m000000000000000000000000000000000000000m000000000 00000000 lll0 Taxa3&4 DAR 69458 M 00m0000 flno JM I39 M 000fixm000m0000 00000m0 fino DÂR ÁO¿R¿ m M000000üxn0üxn000000 00000000 0000 " presence and absence ofrestriction fragments are denoted by I and 0, respectively. NSW phen: New South Wales phenotype includes isolates 91.1, DAR 5759I, DAR 69457.1, DAR 69460, VRU003I, VRU 0048, VRU 0050 and VRU 0074. Table 9.48 The presence or absence of each restriction fragment in Psrl-digested DNA from 24 Phomopsis taxon 1 isolates, 13 taxon 2 isolates, two taxon 3 isolates and one taxon 4 isolate, using putative RFLP fingerprinting Phomopsis clones. Isolate Probe" pTlP84 insert B pTlP99 pT2Sl2 pT2S2l insert A pT2S2l insert B insert B Taxon I l7A 00 l 0 l 0000 l 00000 l 00000 l 0 r 0000000 I 00000 I 00 I 0 I 000000 I 00000 I 00 00110000000001w 001 1000000 010000000000010000 10010000 50D.1 I 0 I 0 l 00 I 0000000 I 0000000 r 0000000 I 00000 I I 0 I 0 I I 00 I 0000 I 00 I 000 001 001 1000000 010000000000010000 10010000 5lc.l I 0 I 0 I 00 I 0 I 00000 I 00000000 r 000000 I 00000 I 0 I 00 I 000 I 0000 I 0 I 0000 001 001 1000000 000100000000010000 r00r0000 85 I I I 00 I 000 I 000 I 000000 l 000 I 00 I 000 I 0000 I 0 I 0 I 0 I 00 I 000 I 0 I 000 I 00 001 10001 I 001 1000000 000100000000010000 10010000 87.1 & 88.1 I l 0000 I 00 l 000 l 000000 l 000 l 000000 I 0000 I 0 I 000 I I 0 I 000 I 00000000 0000 r 00000 l 000000000 I 00000000000000 l 001 1000000 000r00000000010000 10010000 95. l I 0 I 0000 I 0 I 0000 l 00 I 000000 l 00 I 0000 I 00 I 0000 I 0 I 0000 I 000 I 000000 I 0 1000 l 00 l 000010 I 0 I 00 I 000000 I 000 l 100 001 1000000 000100000000010000 10010000 994.1 I 0 I 0000 I 0 I 00000 I 00000 I 0000 I 00 I 0 I 000000 I 0 I 0 I 0 I 000 I I 00 I 000 I 0 00r000101000000100000000000100010000 001 1000000 000000000000010000 10010000 ll4B 0000000000000cÐ I 00000000000000000000 I m wr000000 1000100000 0000000001 00000000 01000100 1l58 IM 1000000 1000100000 000000000100000000 01000100 133 r000r000r 0 r 00000 l l 000 l 000000001 000000 00rm1000000000 00r 1000000 000000000000010000 10010000 r35 I 0 I 000000 I 0000 I 0 I 000 l 000 l 0000 I 0 I 000 I 00 I 00 I I I 0 I 000 I 0 I 0 I 000 I 0 I I 00 I I 00000000 I 000000001 000 r 00 l 0000 001 1000000 010000000000010000 10010000 t36 l0 10000 1000000010100000010000 l0 100000001001 100000001000000 00 001 1000000 010000000000010000 r0010000 l39A,B,C I r001001000100000000000010000001000000001 00000000101000000 00 I I 00000000000000000000000000000000 001 1000000 000r00000000010000 10010000 140 000100r00r010000000001000000000100001 000000000000000000001 0000000000000 I I 001 1000000 000100000000010000 10010000 l4lA l l 00 r 00 l 00 l 0000 l 00000 l 0000 l 0000 I 00000 I 00 I 0 I I 00000 I I 0000 r 00 001 I 000000000 1000000001 00000000 I 0000 001 1000000 000100000000010000 10010000 t4lB l r 00 r 00 l 0000 r 00 l 00000 l 0000 I 0000 I 0000 I 00000 I I 0 I 0 I 0000000000 00000000000000000000 r 0000000000000 l l 001 1000000 000100000000010000 10010000 15lc r 00000100001000000000000000 M1000000000000 001 1000000 000000000000010000 00010000 l53B M I 00000 I 0000 I 000000000000000 m1000000000000 001 1000000 000000000000010000 00010000 DAR 6946r l 0 l 000 l 0000 I 000000 I 00000 l 000000 I 00000 I I 000 I 000000 I 0 I 000 I 00 001 1000000 000000000000010000 10010000 DAR 69467.1 l I 00000 I 0 I 00000 I 000 I 00000 l 000 r 0 I 00 I 00 I 0 I 00 I I 0 I 00 I I 000 I 000 I 00000000000r000001000000010000010000 001 1000000 000100000000010000 10010000 DAR 69488 I l 000 I I 00 I 0 I 00000000 r l 0000 l 0000 I 0000 I I 0000 I 000000 I 0 r 000000 001 1000000 000100000000010000 10010000 Taxon 2 77 l00l 010001 1 1 il 10000100100100001 I 0010001 I I l0.l w I 0000000 l 00 l m 0000000000000000000 r 0000000000000000 0 I 000 I I I I I 00001001 10010001 I l 0010001 l NSW phen M l 0000000 r 00 I w 0000000000000000000 I 0000000000000000 0 I 000 I I I I I 00001001 r0010001 l l 0010001 I vRU 0036 l 00 l 000000000000000000000000 0l000lllu 10100010100100001 I 001000r r VRU OO84 wr0000000r001M 0l000lllll 10100010100100001 I 0010001 l DAR 69486.1 Mr00000001001 01000lllll 10000100100100001 I 0010001 I Taxa3 &.4 DAR69458 000000000000000r M W1000000 1000100000 000000000100101000 01000100 JM 139 0000000000000001 M M1000000 1000100000 000000000100101000 01000100 DAR 69484 0000000000 010000000010010000 00000000 " presence and absence ofrestriction fragments are denoted by 1 and 0, respectively. NSW phen: New South Wales phenotype includes isolates 91.1, DAR 57591, DAR 69457.1, DAR 69460, VRUOO3I, VRU 0048, VRU 0050 and VRU 0074 Table 9.4C The presence or absence of each restriction fragment in Psrl-digested DNA from24 Phomopsis taxon 1 isolates, 13 taxon 2 isolates, two taxon 3 isolates and one taxon 4 isolate, using three microsatellite probes. Isolate Probe" ____ _ (GATA)4 (CATX (cAA)6 t7 A. 000000000101000000000000001010000000001010100010001 000001001001 l0l t0l0r l00l l0l t00l0l00l 10100100101000010 001000010000100000001000000 50D.1 000000000101000000000000001010000000001010100010001 000001001001r0110101100110il0010100010100100101000010 0010000r0010100000001000000 5lc.l 000000000101000000000000001010000000001010100010001 00000100100110110101100u0110010100110100100101000010 001000010010100000001000000 85 000000000101000000000000001010000000001010100010001 000001001001 l0l l0t0l l00l l0l l00l0l00l 10100100101000010 001000010010100000001000000 87.1 & 88.r 00001 010000000001010000010000 000001001001 l0l l0l00l0l0l0l0r0l0l00l 10r00000101000010 00000001 1010100000001000000 95. l 00000000010000000000100000101 0010000001010000010000 000000r01 l0l l0l l0l I r000001 10001 l00l r 10100100101000010 0000000000 l 0 l 0000000 I 000000 99A.1 0000000001 1000000000000000 l0 10000000001010 100100001 00000 l 00 r 00 I I 0 I I 0 I 0 I I 0 I I 00 I I 00 I 0 I 000 I 0 I 00000 I 0 I 0000 I 0 0000000 l 0 r 00 10000000 I 000000 lt4B w I 0 I 0000 I 00 I 000 I 00000000000 000101000001001 t0l0l 10001 I I I 10010001 100100000001 10100 00000000000 r 000000000000000 l158 M I 0000 I 00 I 000 I 0000000000 I 00010100000100r l0l 1010000r I I 1001000t 100100100001 10t00 00000000000 l 000000000000000 133 0000000001010000000000000010100000000010101000r000r 00000 r 00 I 00 I 00 I l 0 r 0 l r 0 I I 00 I I 00 I 0 r 000 I 0 I 00 I 00 I 0 I 0000 I 0 00 I 0000 l 00 l 0 I 0000000 l 000000 135 000000000 l 0000 I 0 I 000000 I 00 I 0 I 000000000 I 0 I 0 I 000 I 000 I 00000 l 00 l 00 l I 0 I I 0 I 0 r I 0 I I 00 I I 00 I 0 I 000 I 0 r 00000 I 0 I 0000 I 0 0000000 r 0 100 I 0000000 l 000000 136 000000000 10r0000000000000010t 0000000001010100010001 00000 I 00 I 00 r 00 l l 0 I 0 I I 0 I I 00 I I 00 I 0 I 000 I 0 I 00 I 00 I 0 I 0000 I 0 00 r 0000 r00 l 0 I 0000000 I 000000 l39A,B,C 00000000010 1000000000000001010000000001010100010001 00000 l 00 r 00 I I 0 I I 0 l 0 l r 00 I l 0 I I 00 I 0 I 000 I 0 I 00 I 00 I 0 I 0000 I 0 0000000 I 00 I 0 I 0000000 I 000000 140 1010000000001010000010000 000001001001 l0l l0l00l0l0l0l0l0l0l00l 10100000t01000010 000000010010100000001000000 l4lA 00000000010100r00000000000101000000000 1010100010001 000001001001 l0l r0l0r l00l l0l l00t0l00l 1010010010r000010 00 I 0000 r 00 l 0 l 0000000 I 000000 t4lB 000000000000000r000000000010100000000010 10000010000 00000 r 00 r 00 l l 0 r l 0 r 00 I 0 I 0 I 0 I 0 I 0 I 0 I 00 I I 0 I 00000 I 0 I 0000 I 0 0000000 l 00 l 0 r 0000000 I 000000 l5lc w I 0 I 000000000 I 0 I 00000 I 0000 000000000000000001001010100010001001 l0l 00000101000000 0000000000 l 0 I 0000000 l 000000 l53B w I 0 I 000000000 I 0 I 00000 I 0000 00000000000000000100 l0l0l000l0m 100 I 10100000 101000000 0000000000 I 0 I 0000000 l 000000 DAR 69461 00000000010100000000000000101000000000 I 01010001000 I 00000 I 00 I 00 I I 0 l r 0 r 0 l I 00 I I 0 I I 00 I 0 I 000 I 0 I 00 I 00 I 0 10000 I 0 00 10000 l 00 l 0 l 0000000 I 000000 DAR 69467.1 0000000 I 000 l 00 l 0 l 000000000 I 0 I 000000000 r 0 I 0 I 00 I 0000 I 00000 l 00 l 00 I l 0 I l 0 r 0 l l 0 I I 00 I I 00 I 0 I 000 I 0 I 00000 I 0 I 0000 I 0 00000000 r 0 l 0 r 0000000 I 000000 DAR 69488 000000000000000100000000001010000000001010000010000 00000 r 00 l 00 l l 0 I I 0 I 00 I 0 I 0 I 0 I 000 I 0 I 00 I I 0 I 00000 I 0 I 0000 I 0 0000000 r l 0 l 0 I 0000000 l 000000 Taxon 2 77 I 0000000000000000 I I 000000 I 0 I 00 I 00 I 00 I 00 I 0000000 I 0 I 0 00000 I 0 I I 0 I 0 I l 00 l I 0 I 0 I 0000 I 0 I 0 I I 0 I 00 I 000 I 00 I 00 I 00 I 00 I 100010000000001 0 I l0 l 001 I I I I I l0.l I 0 I 00 r 00 l 0000000000 I 00 I 00 I 0 I 00 I 00 I 00 I 00 I 0000000 I 0 I 0 l r l0l00l l0l r r l00l r0101000010101001000000100101 l00l00t 10001000000000101 l0l00l r I I I NSW phen I 0 I 00 I 00 I 0000000000 I 00 I 00 I 0 I 00 I 00 I 00 I 00 I 0000000 I 0 I 0 r l l0l00r l0l I I t00l l0l0l0000l0l0l 101000100100101 l00l00l 10001000000000101 l0l00r r l I I VRU 0036 r 0 r 0000000000 I 00000 l 00000 I 0 I 0 r I 00 I 00 I 00 I 0 I 00000 I 0 I 0 l l0l0l0r r0l r l l00l l0l0l0000l0l0r l0l00l l00l00l0l l00l00l l00l I 100000000101 l0l00l I I I I VRU OO84 r 0000 l 000000000000 l 000000 I 0 I 00000 I 00 I 00 I 0000 I 00 I 0 I 0 000 r 0 l 0 r 00 l 0 I I 00 I r 0 I 0 I 0000 I 0 I 00 I 0 I 00 I 000 I 00 I 0 I 000 I 00 I 100000000000001 r0r0l00l l I I I DAR 69486.r l 0 l 00000000000000 I 0000000 I 0 I 00 I 00 I 00 I 00 I 0000000 I 0 I 0 0000000 l r 00 r 0 l 00 I I 0 I 0 I 0000 I 0 I 00 I 0 I 00 I 000 I 00 I 00 I 00 I 00 I 10000010000000001 l0l00l l I I I Taxa3 & 4 DAR 69458 000000000000000001000000010100001000000100000000100 000101000001 l0l t0l t0l000l l0l I 1010010100100100001 10100 00000000000 I 0000 I 0 I 00 I 00000 JM I39 000000000000000001000000010100000000000r00000000100 00010101010110il0110100001011t01001010010010000110100 00000000000 I 0000 l 0 I 00 I 00000 DAR 69484 0 l0 100 I 00000 10000 1000 I 00 I 00 I 00 I 000 I 0 I I 00000 I 0000000 I I 00 I I 0 I l00l r l0l0l l0l0l00t l0l l00l00l l0t00l00l000l0loo 0 l 00000 r 0000 I I 00 l 0 I 00000000 " presence and absence ofrestriction fragments are denoted by I and 0, respectively NSW phen: New South Wales phenotype includes isolates 91.1, DAR 57591, DAR 69457.1, DAR 69460, VRU003t, VRU 004g, VRU 0050 and VRU 0074. t7t Table 9.5 Summary of RFLP banding patterns identified among 24 Phomopsis taxon I isolates, 13 taxon 2 isolates, two taxon 3 isolates and one taxon 4 isolate, digested with PsrI, after hybridisation with six Phomopsis taxon 1 clones, five taxon 2 clones and three microsatellite probes. Isolate Probe" IO & 95 848 99 180 216 I2B 2TA 2TB 22 344 GATA CAT GAA Taxon I ¡ß t< * * * t7A M P XDD HH 'ß 50D.1 * tß F rlc * M P XDD trNN 51C.1 tß {< F ,ß * M a XDD HH NN 85 rß * {< * * M a XDD IIH NN 87.1 & 88.1 rß rt * ¡F tß M a x* JJ OO 95. l t * t * rß M a X't *{< 99A.1 * * * ,ß ,1. M R x* KK PP 1l4B B G N S Y* *QQ 1l5B * G N S Yt' *QQ 133 * rtc ; M R XDD LL NN 135 * * * M P x* KK PP 136 {< ,1. * M P XDD LL NN l39A,B,C * * * M a XDD trr t40 t< H {< M a XEE JJ RR I4TA * * * M a x* HH NN t41B * H ,1. M a XEE JJ RR l5lC c I K M R ZFF MM SS 1538 C I K M R ZFF MM SS DAR 6946r * rt F {. rl. M R XDD trNN ,F t ,1. {< * DAR 69467.1 M a x* KK 'l' DAR 69488 ,* t l. * * M a XEE *oo Taxon 2 77 A D J L o T AA-CC,K TT 110.1 A D J L o U AA BB CC GG TT NSW phen A D J L o U AA BB CC GG TT VRU 0036 rl. D J t o V AA-T* ,F VRU OO84 D J o V AA-CC* {< DAR 6948ó.1 D J o T AA-CC* rl. Taxon 3 DAR 69458 B G N w Y rf t UU JM 139 B G N w Y * * UU Taxon 4 DAR 69484 rF ¡1. ,t * ' probes: l0 & 95 = pTlPlO & pTlP95; 848 = pT1P84 insert B; p! = pTlP99; 180 = pTlPlSO; 216 = pTlP216; 1/l = pT2Sl2 insert B;2LA = pT2S2I insert A; 2lB = pT2S2L insert B; 22 = pT2S22i 34A = pT2S34 insert A; GATA = (GATA)¿; CAT = (CAT)5; GA'r{ = (GAA)o. NSW phen: New South Wales phenotype includes isolates 91.1, DAR 57591, DAR 69457.1, DAR 69460, VRU 0031, VRU 0048, VRU 0050 and VRU 0074, which had identical fingerprints with all probes used. A to UU: isolates with the same letter(s) had identical RFLP f,rngerprinting pattems '* isolates with unique RFLP fingerprints. - no DNA fragments hybridised to the probe. r72 9.3.2 Screening microsatellites The hybridisation of five microsatellite probes to total DNA from a Phomopsis taxon I isolate, a D. viticolø isolate and a taxon 2 isolate, digested with six different endonucleases, revealed similar RFLP fingerprints for the taxon I and the D. viticola isolates, and a different fingerprint for the taxon 2 isolate, in every endonuclease/microsatellite probe combination tested (Fig. 9.6). Microsatellite probes (GATA)¿, (CAT)5, (GAA)6, and (GGAT)a produced RFLP fingerprints with distinct bands when hybridised to Phomopsis DNA digested with PsrI or SaïL, but not to DNA digested with DraI, HaefÍÍ, Sau3AI and Hpan' (Fig. 9.6). Microsatellite (GACA)4 did not produce distinct bands. Psrl-digested DNA resulted in more distinct bands and the endonuclease/micro- satellite probe combinations PsIV(GATA)¿, PsrV(CAT)s and PstA (GAA)ó detected variation within and between the two main taxa (Fig. 9.7). Microsatellite (GATA)¿ hybridised to five to 11 fragments amongthe24 taxon I isolates, 11 to 14 fragments among the taxon 2 isolates, fîve fragments of taxon 3 isolate JM 139, and to six fragments of taxon 3 isolate DAR 69458, five of which were also present in isolate JM 139. The probe hybridised to 13 fragments of the taxon 4 isolate. In total, 11 different RFLP fingerprint patterns were identified among24 taxon 1 isolates and five different fingerprint patterns were identified among 13 taxon 2 isolates (Tables 9.4C and 9.5). Microsatellite (CAT)5 hybridised to 19 to 22 fragments among 22 taxon 1 isolates, 11 fragments of taxon I isolates 151C and 1538, 18 to 27 fragments among the taxon 2 isolates, 23 fragments of taxon 3 isolate JM 139, 22 fragments of taxon 3 isolate DAR 69458,2I of which were also present in isolate JM 139, and 26 fragments of the taxon 4 isolate. One band was present in all taxon L,2,3 and 4 isolates. Taxon I isolates 114B and l15B had 20 bands with 18 bands in common, 16 of the 18 bands were also present in the taxon 3 isolates. In total, 10 different RFLP fingerprint patterns were identified among 24 taxon 1 isolates and six different fingerprint patterns were identified among 13 taxon 2 isolates (Tables 9.4C and 9.5). The hybridisation of microsatellite (GAA)6 resulted in three to six bands among22 taxon 1 isolates, one band of taxon 1 isolates 114B and 1158, l0 to 13 bands among the 13 173 taxon 2 isolates, four bands of the taxon 3 isolates and six bands of the taxon 4 isolate. The band in isolates 1l4B and 115B was also present in the taxon 3 isolates. In total, l0 different RFLP fingerprint patterns were identified among 24 taxon 1 isolates and four different fingerprint patterns were identified among 13 taxon 2 isolates (Tables 9.4C and e.s). 9.3.3 RFLP data analysis A. Estimate of variation within and between populations and taxa Shannon-Weaver indices (.I/) were calculated using Psrl-digested Phomopsis DNA and RFLP banding patterns generated by 11 of the 14 probes (Table 9.6). Clones pT2S22 and pT2S34 insert A were excluded from the analysis of the two taxa because they did not hybridise with any of the taxon 1 isolates and clone pT1P180 was excluded because it did not hybridise with any of the taxon 2 isolates (Table 9.5). Since clones pT1P10 and pT1P95 gave identical banding patterns for all isolates tested, only one was included. Isolates 87.1 and 88.1, which were obtained from the same cane and had identical RFLP fingerprints with all probes, were considered to be clonal and were counted as one isolate. Similarly, isolates 1394, B and C, which were derived from a single perithecium, had identical RFLP fingerprints with all probes. Isolates 114B,1158, 15lC and 1538 were not included in the analysis of taxon 1, because they had RFLP banding patterns very different from the other taxon I isolates. After these modifications, the sample sizes of the Adelaide Hills and the Tasmanian populations of taxon 1 were the same, making it possible to compare the diversity of the populations without normalising the indices (Goodwin et a|.,1993). Both the Adelaide Hills and the Tasmanian population of taxon I showed high levels of variation with the five taxon I probes and the microsatellite probes, with more of the variation occurring within the two populations (63Vo - 72Vo) than between (28Vo - 37Vo). Two of the three taxon 2 probes did not reveal any variation within or between the populations, and probe pT2S2I insert A showed that SIVo of the variation occurred within the populations. Overall, a high level (average TIVo) of diversity existed within the taxon 1 populations (Table 9.64). r74 On average, more variation was found within taxa 1 and 2 (607o) than between the taxa. However, probes pT2S12 insert B and pT2S21 insert B identified lÙOVo variation between the two taxa (Table 9.68). Taxon 2 showed much lower levels of variation than taxon I with the four taxon I probes and the microsatellites, but the two taxa exhibited similar levels with the taxon 2 probes (Table 9.6C). Microsatellites (GATA)¿ and (CAT)5 identified more variation within taxon 2 than did the clones, but the opposite was true for the taxon I isolates using the taxon 1 clones (Table 9.6C). B. Estimate of genetic similarities among Phomopsís isolates The genetic similarities among all pairs of the 40 Phomopsis isolates (Table 9.1) were calculated using Pstl-digested DNA and RFLP fingerprints generated by i) five taxon I clones (Table 9.7), ä) five taxon 2 clones (Table 9.8) and iii) three microsatellite probes Írmong the taxon 1 isolates (Table 9.9) and taxon 2, 3 and 4 isolates (Table 9.10). The percentage similarity, S"r, values were used to construct three dendrograms @igs 9.8, 9.9 and 9.10) which clearly separated taxon 1 and taxon 2. The five taxon 1 clones did not detect any similarity (S*v) in the banding patterns between isolates of taxon 1,3 and 4 and those of taxon 2 (Fi9.9.8). The taxon l probes revealed that the taxon 3 isolates were very closely related to isolates 1148 (S*y = I007o) and 1158 (S*y - 80Vo), and that the similarity between isolates 15lC and 1538 and the remaining 20 taxon 1 isolates was low (4.77o - 17.ïVo, Table 9.7). The similarity between the2O taxon I isolates (excluding 114B, 1158, l51C and 1538) and the taxon 3 isolates (including 1l4B and 1158) varied between 07o and8.97o (data not presented). The taxon I probes resolved the 20 taxon 1 isolates into two groups with, on average, 33.9Vo similarity (Frg. 9.S); one consisted of five isolates, the other of 15. There was no similarity between: the taxon I isolates and the taxon 4 isolate (Fig. 9.8); the taxon 3 isolates (including 1148 and 1158) and taxon 4 (Frg. 9.8); and the taxon 3 isolates (including 1148 and 1158) and isolates 15lC and 1538 (data not presented). The five taxon 2 clones did not detect any similarity in banding patterns between the taxon 2 isolates and the isolates of taxon 1, 3 and 4 (Table 9.8). The probes detected two major groups within taxon 2, one with four isolates and the other with nine isolates (Fig. t75 Figure 9.6 Southern blots of restriction enzyme digested total DNA from Phomopsis taxon 1 isolate DAR 69467.I, D. viticola isolate 51C.1 and taxon 2 isolate DAR 69486.I (approx. 1 pg lane-r) hybridised with microsatellite probe (CAT)5. Ã. Phomopsis DNA fractionated in a IVo agarose gel, Lane 1, DAR 69467.IlPstI;Lane 2, 1ICJ|PstI; Lane 3, DAR 69486.11 PstI; Lane 4, DAR 69467.L|9a[I;Lane 5, 51C.1/ Sa[I;Lane 6, DAR 69486.11 Sail; Lane7, DAR 69467.LlDraI; Lane 8, 51C.1/ DraI; Lane 9, DAR 69486.1/ Dral; Hindfr-digested À DNA size markers, in kb, are shown to the left. B. Phomopsls DNA fractionated in a 2Vo agarose Sel. Lane 1, DAR 69467 .LlHaeW;Lane 2,slc.Ll Haefr; Lane 3, DAR 69486.11 Haefr; Lane 4, DAR 69467.IlSau3AI; Lane 5, slc.ll Sau3AI;Lane 6, DAR 69486.11 Sau3AI; Lane7, DAR 69467.IlHpan; Lane 8, slc.ll Hpan; Lane 9, DAR 69486.11 Hpan. Híndfr-digested À DNA size markers, in kb, are shown to the left. A B r23456189 123456789 23.t - ,èr. . I 6.6 - ';È i|b 9.4 - _ffi 4.4 - 6.6 - o,r * t *= t t:, ##ffi 2.3 - 4.4 - # *#t 2.0 - :ä' 4F, # '.'-.: ": -Ë r"' ffi!1t;'í r.,,¡' i.& 2.3 - .Þ¡ 2.0 - * Ç s' 0.6 - # # r76 Figure 9.7 Southem blot of total DraL-digested DNA (approx. 3 pg lane-r) from grapevine (lane 1), and total Psrl-digested DNA (approx. 3 ¡rg lane-r) from five Phomopsis taxon 2 isolates (lanes 2-6), four D. viticola isolates (lanes 7-10) and four taxon 1 isolates (lanes ll-14), hybridised with microsatellite probes (CAT)5 (A) and (GATA)4 (B). ¡/¿ndltr- digested À DNA size markers, in kb, are shown to the left. Phomopsis isolates from left to right: vRU 0048, vRU 0050, 77,9I.1, 110.1, 85, 1394, 1398, 139C, DAR 6946I,95.r, l4IA,1418. A B I 2 3 4 5 6 7 8 9 10111213t4 r234567891011121314 23.L *rg ,,r,a 23.t - 'll1 ., ð,ff 9.4 9.4 - :¡f4 ü* * 6.6 - :t rrË Ç.- rltr; 6.6 - |o* *-ã-,ï-h *¡f 4.4 - ,1,.!r 4.4 - {* lq f * {4ñÞ ; +dÈi **il * *"* *t*, l4 Iffi [; 2.3 - 2.3 - 2.0 - I 2.0 - * t ili f,* *s *fl¡*'I*e -ì lr id::, ¡ !*, r77 Table 9.6 Shannon-Weaver indices (I1) calculated using data on RFLP banding patterns produced by 1l probes, used to estimate diversity within and between two geographical regions in Australia for Phomopsis taxon I (A), and to estimate diversity within and between taxon 1 and taxon 2 of Phomopsis (B). Normalised Shannon-Weaver indices (É1) were calculated for taxa 1 and2 of Phomopsis (C). Probes' taxon 1 taxon2 microsatellites l0 & 95 848 99 180 216 l2B 21A 2lB GATA CAT GAA Mean A Il Adelaide Hills 1.79 r.79 1.56 1.79 t.79 0.00 0.45 0.00 r.24 l.0l 1.33 l.16 (n=6) fI Tasmania r;19 1;t9 t.79 t;Ì9 t;19 0.00 r.l0 0.00 r.24 1.33 1.33 1.2'l (n=6) 11 mean 1.79 r;t9 1.68 l;19 1.79 0.00 0.78 0.00 1.24 r.t1 1.33 1.21 11 combined 2.48 2.48 2.3't 2.48 2.48 0.00 0.96 0.00 t.75 1.86 1.8ó 1.70 (n = 12) b ¡1 -"un 72% 72Vo 7l7o 727o 72Vo 817o TlVo 63Vo 717o 717o Il combined B 11 Taxon I 2.83 2.83 2.56 2.83 0.00 0.96 0.00 I .66 I .84 r.79 | .73 (n = L7) H Taxon2 0.69 0.00 0.00 0.69 0.00 0.83 0.00 1.04 t.29 0;t9 0.53 (n = 13) H mean t;76 r.42 1.28 1.76 0.00 0.90 0.00 1.35 1.56 r.29 l.l3 11 combined 2.59 2.29 2.r3 2.59 0.68 1.59 0.68 2.08 2.29 2.04 1.90 (n = 30) Il mean 68Vo 62% 60Vo 687o ÙVo 57Vo OVo 65Vo 68Vo 63% 60Vo Il combined c f/ Taxon I l 0.90 l 0.00 0.34 0.00 0.59 0.65 0.63 0.61 II Taxon2 0.27 0.00 0.00 0.27 0.00 0.32 0.00 0.41 0.50 0.31 0.2r " probes: 1Q = pTlPlO; 84B = pTlP84 insert B; ÇJ = pT1P95; 99 = pT1P99; 1$Q = pTlP180; 216 = pTIP2L6; l2B = pT2Sl2 insert B; 2IA = pTZSZI insert A; 2IB = pT2S2I insert B; GATA = (GATA)¿; CAT = (CAT)s; GAA - (GAA)6. b percentage of variation that occurred within populations. 'percentage of variation that occurred within the taxa. n number of isolates. 178 9.9). The similarity between the two groups varied between 65.27o andTLITo (Table 9.8). There was no similarity between the taxon 1 and the taxon 3 isolates (including I l4B and 1158). In contrast to the results for the taxon 1 clones, the similarity between the taxon I isolates (excluding 114B and 1158) and the taxon 4 isolate averaged 27 .2Vo (Fig 9.9). However, similar to the taxon I clones, there was no similarity between the taxon 3 (including 1l4B and 1158) and the taxon 4 isolates (Table 9.8 and Fig. 9.9). The three microsatellite probes detected similarities between 20 taxon I isolates (excluding 1148, 1158, 151C and 1538) and the taxon 2 isolates of, on average, L9.LVo (data not presented). The 20 taxon 1 isolates displayed a slightly higher average level (31.77o) of similarity with the taxon 3 isolates, including 1148 and 1158, and with the taxon 4 isolate (39.6Vo, data not presented). The similarity between the 20 taxon I isolates and isolates 151C and 1538 varied between 53.9Vo and 77.67o (Table 9.9). The microsatellite probes resolved the 20 taxon I isolates into three groups; isolate 95.1 had, on average, 69.2Vo similarity with the remaining taxon 1 isolates, which were divided into a group of five and a group of 14 isolates with, on average ,78.6Vo similarity @g. 9.10). The group of five isolates was the same group that was resolved by the taxon I probes. Taxon 2 isolates had a low level of similarity with: the taxon 3 isolates together with 114B and 1l58 (I6Vo to 327o); the taxon 4 isolate (23.37o to 32.3Eo); and isolates 15lC and 1538 (8.6Vo fo 13.87o, Table 9.10). The similarity between the taxon 3 isolates, including 114B and 1158, and the taxon 4 isolate was on average 4l.7Vo (Fig. 9.10). Similarities among the 20 taxon I isolates (excluding 114B, 1158, 15lC and 1538) detected with the taxon 1 clones were lower, on average (4I.5Vo), than those detected with the microsatellite probes (85.1Vo, Tables 9.7 and 9.9). However, similarities among the 13 taxon 2 isolates detected with the taxon 2 clones resembled those detected with the microsatellite probes (averaging 85.27o and89.9Vo, respectively; Tables 9.8 and 9.10). t79 Table 9.7 The percentage similarity, S"r, detected with the five clones (pTlP95, pTlP180, pTIP2I6, pT1P84 insert B and pT1P99) derived from taxon I isolate DAR 69467.I, among the taxon 1 isolates. t7A 100 50D.1 66.7 r00 51C.1 49.5 66.0 100 85 32.3 42.9 48.2 100 87. l 26.8 3r.6 33.7 47.5 100 88.1 26.8 3r.6 33.7 47.5 100 100 22.5 )') \ 100 95.1 27.r ^')') 34.9 36.5 99A.1 40.7 50.8 49.2 43.9 24.4 24.4 4t.9 100 114B 4.7 3.6 3.6 3.2 4.4 4.4 0 2.6 100 I 158 0 0 0 J,J 4.5 4.5 0 0 80.0 r00 133 38.5 48.4 59.3 35. l 32.5 32.5 25.5 41.4 0 0 100 135 35.1 47.2 50.4 42.r 34.5 34.5 40.0 62.6 0 0 48.2 100 r36 32.9 53. I 69.4 40.4 32.2 32.2 39.6 45.8 4.2 0 65. I 50.4 139A 42.1 56.2 49.4 48.4 33.3 33.3 37.0 36.7 0 0 40.5 34.6 1398 42.1 56.2 49.4 48.4 33.3 33.3 37.0 36.7 0 0 40.5 34.6 l39C 42.t 56.2 49.4 48.4 33.3 JJ.J 37.0 36.7 0 0 40.5 34.6 140 39.5 31.5 33.7 31.6 48.7 48.7 15.2 27.5 5.1 5.3 32.4 25.5 1414 52.9 60.0 54.0 45.3 31.5 31.5 3l.t 45.0 4.0 0 37.7 34.7 l4lB 29.3 40.0 31.6 39.6 45.2 45.2 24.5 26.r 8.9 4.6 25.0 27.6 l5lc t7.o 13.3 10.0 6.t 12.2 12.2 6.4 5.0 0 0 13.3 4.9 l538 t7.o 13.3 10.0 6.1 12.2 12.2 6.4 5.0 0 0 13.3 4.9 DAR 35.7 49.5 45.4 42.7 32.6 32.6 34.0 44.4 0 0 41.5 39.0 6946r DAR 28.O 35.4 44.3 28.6 33.3 33.3 31.0 54.r 3.2 0 32.7 53.7 69467.r DAR 44.9 52.9 43.r 46.3 50.6 50.6 28.6 44.3 3.9 3.9 43.7 42.3 69488 r7A s0D.l 51C.1 85 87.1 88.1 95.1 994.1 l14B 1158 133 135 136 100 l39A 49.4 100 l39B 49.4 100 100 l39C 49.4 100 100 100 t40 27.2 30.6 30.6 30.6 100 l41A 47.8 57.8 57.8 s7.8 2t.7 r00 t4lB 29.9 38.5 38.5 38.5 4t.o 44.9 100 15lc 7.7 4.7 4.7 4.7 r4.0 1 1.1 12.2 100 153B 7.7 4.7 4.7 4.7 14.0 l l.1 t2.2 100 100 DAR 49.4 52.5 52.5 52.5 35.0 48.4 27.9 11.8 11.8 100 69461 DAR 43.8 )o) 29.2 29.2 229 39.3 37.3 9.0 9.0 42.3 100 69467.1 DAR 38.3 42.4 42.4 42.4 47.l 50.0 48.4 14.3 14.3 45.2 38.5 100 69488 DAR DAR DAR 136 139A l39B 139C 140 141A l41B 151C 1538 69461 69467.1 69488 180 Table 9.8 The percentage similarity, S*r, detected with the five clones (pT2S3a insert A, pT2S22, pT2Sl2 insert B, pT2S21 insert A and pT2SZl insert B) derived from taxon 2 isolate DAR 69457.I, among the isolates belonging to taxon 2,3 and 4 and isolates 114B, 115B, 151C and 1538. ^77 100 u 9l.l 7 t.r 100 u I10.1 7 L.t 100 100 " DAR 57591 7 r.l 100 100 100 u DAR 69457.r 7 t.t 100 100 100 100 " DAR 69460 7 t.t 100 100 100 100 100 u DAR 69486.1 100 7t.l 7l.r 7Lt 7 T.T 7I.I 100 , VRU OO3I 7 t.t 100 100 100 100 100 7 T.I 100 " vRU 0036 86.5 65.2 65.2 65.2 65.2 65.2 86.5 65.2 100 'vRU 0048 7 L.I 100 100 100 100 100 7I.I 100 65.2 100 " vRU 0050 7I.I r00 100 100 100 100 7 t.l 100 65.2 100 " vRU 0074 7 r.l 100 100 100 r00 r00 7 r.l 100 65.2 100 " vRU 0084 91.9 69.6 69.6 69.6 69.6 69.6 91.9 69.6 94.7 69.6 o o¡,R og¿sg 0 0 0 0 0 0 0 0 0 0 b JIr¿ t39 0 0 0 0 0 0 0 0 0 0 " DAR 69484 0 0 0 0 0 0 0 0 0 0 o r14B 0 0 0 0 0 0 0 0 0 0 d tt5B 0 0 0 0 0 0 0 0 0 0 d t5tc 0 0 0 0 0 0 0 0 0 0 o 1538 0 0 0 0 0 0 0 0 0 0 DAR DAR DAR DAR VRU VRU VRU 77 91.1 il0.1 57591 69457.t 69460 69486.1 003 l 0036 0048 " vRU 0050 100 " vRU 0074 100 100 " vRU 0084 69.6 69.6 100 o oRn og+58 0 0 0 100 b JM r39 0 0 0 100 100 " DAR 69484 0 0 0 0 0 100 d u4B 0 0 0 83.3 83.3 0 100 o ll5B 0 0 0 83.3 83.3 0 100 100 d 15lc 0 0 0 0 0 28.6 0 0 100 d t53g 0 0 0 0 0 28.6 0 0 100 100 VRU VRU VRU DAR DAR 0050 oo74 0084 69458 JM 139 69484 l14B 1158 15lC 1538 u otaxon otaxon taxon 2 isolates; 3 isolates; " taxon4 isolate; I isolates. l8l Table 9.9 The percentage similarity, S*y, detected with three microsatellite probes ((GATA)4, (CAT)5 and (GAA)6), among the taxon 1 isolates. t7A 100 50D.1 97.t 100 5lC.1 98.6 98.6 100 85 98.6 98.6 100 r00 87.r 77.6 77.6 79.4 79.4 100 88.1 77.6 77.6 79.4 79.4 100 100 95.1 70.6 70.6 72.5 72.5 67.7 67.7 100 994.r 85.3 85.3 84. I 84. I 73.9 73.9 63.6 100 lt4B 39.3 36.1 38.7 38.7 37.9 37.9 33.9 33.9 100 ll58 36. I 32.8 35.5 35.5 34.5 34.5 33.9 33.9 88.5 100 133 92.8 95.7 94.3 94.3 75.8 75.8 68.7 86.6 33.3 33.3 100 135 85.7 85.7 84.5 84.5 74.6 74.6 64.7 9t.2 32.8 32.8 87.0 100 136 92.8 95.7 94.3 94.3 75.8 75.8 68.7 86.6 33.3 33.3 100 87.0 l39A 95.7 98.6 97.t 97.1 78.8 78.8 71.6 86.6 36.7 JJ.J 94.1 87.0 1398 95.7 98.6 97.t 97.1 78.8 78.8 7 r.6 86.6 36.7 33.3 94.r 87.0 139C 95.7 98.6 97.1 97.1 78.8 78.8 71.6 86.6 36.7 33.3 94.r 87.0 140 80.0 80.0 81.8 81.8 96.8 96.8 69.8 76.2 39.3 35.7 78.1 76.9 1414 97.2 97.2 98.6 98.6 78.3 78.3 71.4 82.9 38.1 34.9 93.0 86.1 l418 80.0 80.0 81.8 81.8 96.8 96.8 69.8 76.2 39.3 35.7 78. l 76.9 151C 59.3 59.3 6r.8 6l.8 74.5 74.5 53.9 53.9 26.7 22.2 60.4 55.6 l53B 59.3 59.3 61.8 61.8 74.5 74.5 53.9 53.9 26.7 22.2 60.4 55.6 DAR 97.r 100 98.6 98.6 77.6 77.6 70.6 85.3 36.1 32.8 95.6 85.7 69461 DAR 80.0 829 81.7 81.7 74.6 74.6 61.8 85.3 32.8 32.8 84.1 85.7 69467.t DAR 80.0 80.0 81.8 81.8 96.8 96.8 69.8 't6.2 35.7 32.r 78.3 76.9 69488 r7A 50D.1 51C.1 85 87.1 88.1 95.1 99A.1 1l4B l15B r33 135 136 100 l39A 94.t 100 l39B 94.1 100 100 r39C 94.t 100 100 100 140 78. l 81.3 81.3 81.3 100 l4lA 93.0 95.8 95.8 95.8 80.6 100 l4lB 78.1 81.3 81.3 81.3 100 80.6 100 l5lc 60.4 60.4 60.4 60.4 77.6 60.7 77.6 100 l53B 60.4 60.4 60.4 60.4 77.6 60.7 77.6 100 100 DAR 95.7 98.6 98.6 98.6 80.0 97.2 80.0 59.3 59.3 100 6946t DAR 84.1 84.1 84.1 84.1 73.9 83.3 73.9 55.6 ss.6 82.9 100 69467.r DAR 78.1 81.3 81.3 81.3 96.7 80.6 96.7 73.5 73.5 80.0 76.9 100 69488 DAR DAR DAR 136 139A l39B 139C 140 141A l41B 15lC l53B 6946t 69467.1 69488 t82 Table 9.10 The percentage similarity, S*r, detected with three microsatellite probes ((GATA)4, (CAT)5 and (GAA)6), among the isolates belonging to taxon 2, 3 and 4 and isolates 1148,1158, 15lC and 1538. u77 100 u gl.1 83.0 r00 " 110.1 82.6 98.0 100 'DAR 57591 83.0 r00 98.0 100 " DAR 69457.l 83.0 100 98.0 100 100 u DAR 69460 83.0 100 98.0 100 100 100 u DAR 69486.1 87.8 80.0 79.6 80.0 80.0 80.0 100 " vRU 0031 83.0 100 98.0 100 100 100 80.0 100 'vRU 0036 84.5 87.6 85.4 87.6 87.6 87.6 79.6 87.6 r00 'vRU 0048 83.0 100 98.0 100 100 100 80.0 100 87.6 100 'vRU 0050 83.0 100 98.0 100 100 100 80.0 100 87.6 100 " vRU 0074 83.0 100 98.0 100 100 100 80.0 r00 87.6 100 " vRU 0084 85.7 76.r 75.6 76.r 76.1 76.1 77.5 76.r 77.9 76.1 b oAn 6g¿58 29.3 21.7 19.8 2r.7 2t.7 21.7 28.2 2r.7 27.9 2t.7 b ¡tvt t39 32.0 24.1 ))') 24.r 24.t 24.r 3l.0 24.1 30.2 24.1 c DAR 69484 31.8 3 r.3 31.9 3 1.3 31.3 3 1.3 31.0 3l.3 32.3 3r.3 d tt4B 29.0 23.4 21.3 23.4 23.4 23.4 30.8 23.4 30.0 23.4 d l15B 23.2 18.2 16.0 18.2 18.2 18.2 24.6 18.2 25.0 18.2 d t5rc t2.9 8.6 8.8 8.6 8.6 8.6 13.8 8.6 11.0 8.6 d r53B t2.9 8.6 8.8 8.6 8.6 8.6 13.8 8.6 I1.0 8.6 DAR DAR DAR DAR VRU VRU VRU 77 91.1 110.1 57591 69457.1 69460 69486.1 0031 0036 0048 'vRU 0050 r00 'vRU 0074 100 100 " vRU 0084 76.1 76.r 100 o oAR 69¿s8 2r.7 2t.7 27.4 100 o Jl¿ 139 24.1 24.1 30.1 93.8 100 c DAR 69484 31.3 31.3 23.3 44.2 46.8 100 d ll4B 23.4 23.4 32.8 75.9 69.0 36.6 100 o lt5B 18.2 18.2 26.9 75.9 72.4 39.4 88.5 r00 d 15lc 8.6 8.6 10.0 t9.6 t5.7 31.3 26.7 22.2 100 o t53g 8.6 8.6 10.0 19.6 15.7 31.3 26.7 ))') 100 100 VRU VRU VRU DAR DAR 0050 0074 0084 69458 JM 139 69484 l14B l15B 15lC 1538 'taxon 2 isolates; taxon 3 isolates; "taxon4 isolate; taxon 1 isolates. 183 Taxon Origin Isolate AH DAR 69488 AH 87. l AH 88. r AH r40 AH l4lB AH t4lA Tas I 39A Tas I 398 Tas l 39C Vic DAR 69461 SA 50D.1 AH t7A Tas ¡33 Tas l3ó SA 5lc.l SA 85 Tas 99A.1 Tas 135 Vic DAR ó94ó7.I Tas 95. I atypicâl AH t5tc atypical AH l538 atypical SA I t48 3 Vic DAR 69458 3 SA JM ¡39 atypical Vic I l58 4 NSrr¡r' DAR ó9¡184 ) SA 17 2 Tas 9t.l , Tas I t0.l ) NSW DAR 5759r a NSW DAR 69457.r 1 Vic DAR 694ó0 a NSW vRU 0031 a NSW VRU (xX8 a Vic vRU 0050 1 SA vRU 0074 a Vic DAR 69486.t a SA vRU 0084 I Vic vRU 0036 100 80 60 40 20 0 7o Similarity (S,r) Figure 9.8 Computer-generated dendrogram of 40 Phomopsis isolates, showing genetic similarity, S'r, calculated from RFLP data, generated by five clones derived from taxon 1 isolate DAR 69467.1. The origin of the isolates is described in more detail in Table 9.1. AH = Adelaide Hills in South Australia, SA = South Australia other then the Adelaide Hills. 184 Taxon Origin Isolate SA 5lc.r SA 85 AH 87. I AH 88. I Tas 95. l Tas l39A Tas l39B Tas I 39C AH 140 AH l4lA AH l4lB Vic DAR ó9/tó7.1 AH DAR 69488 AH I7A SA 50D.r Tas r35 Tas r3ó Tas 99A.1 Tas r33 Vic DAR 694ól alypical I AH l5rc atypical I AH t53B 4 NSW DAR 69484 atypical I SA t t48 at¡pical I Vic l l58 3 Vic DAR 69458 3 SA IM I39 a Tas 91. I Tas I t0.t 1 NSW DAR 5759t a NSW DAR 69457.1 ) Vic DAR 69¡160 1 NSW vRU 003¡ ) NSW vRU 0048 a Vic vRU 0050 ) SA vRU 00?4 ) Vic VRU æ36 a SA VRU æt4 a SA 77 a Vic DAR 694E6.I lm 80 60 40 20 0 7o Similarity (S,r) Figure 9.9 Computer-generated dendrogram of 40 Phomopsis isolates, showing genetic similarity, Srr, calculated from RFLP data, generated by five clones derived from taxon 2 isolare DAR 69457.1. The origin of the isolates is described in more detail in Table 9.1. AH = Adelaide Hills in South Australia, SA = South Australia other then the Adelaide Hills. 185 Taxon Origin Isolate AH DAR 69488 AH 87. l AH 88. I AH l¡10 AH l4lB AH l4lA SA 5l c.l SA 85 AH t7A SA 50D.1 Vic DAR ó94óI Tas l39A Tas r398 Tas r39C Tas t33 Tas t36 Tas 99A,1 Tas 135 Vic DAR 69467.t Tas 95. I atypical AH l5lc atypical AH l53B atypical SA I l4B at)?ical Vic ¡ t58 3 Vic DAR 69458 3 SA JM I39 4 NSrril DAR 69484 a Tas 9t.t a NSW DAR 5759r ) NSW DAR 69457.r ) Vic DAR 69460 t NSV/ vRU 0031 a NSV/ VRUüX8 Vic vRU 0050 a SA vRU 0074 ) Tas Ir0.l a Vic VRU OO3ó a SA VRU OO84 a SA 77 1 Vic DAR 69¡18ó.1 100 80 60 40 20 7o Similarity (S.r) Figure 9.10 Computer-generated dendrogram of 40 Phomopsis isolates, showing genetic similarity, S*r, calculated from RFLP data, generated by microsatellite probes (GATA)¿, (CAT)s and (GAA)o. The origin of the isolates is described in more detail in Table 9.1. AH = Adelaide Hills in South Australia, SA = South Australia other then the Adelaide Hills. 186 9.4 Discussion Genomic clones and microsatellite sequences were used as probes to assess genetic variation within and between taxa of Phomopsis. All probes used in this study detected variation between isolates and differentiated between isolates of taxon 1 and taxon 2. The l0 clones, prepared from isolates of taxon 1 and taxon 2, differed in their ability to detect variation. Some may be suitable for detection and taxonomic determination of isolates, whereas others may be used to detect genetic variation between closely related isolates. Putative taxon-specific clones, that showed no homology to DNA from several other fungi or grapevine tissue, were developed for taxa I and 2 of Phomopsis. Taxon 1 clone pTlPl80 hybridised with DNA from all 20 typical taxon 1 isolates but not with DNA from the four atypical taxon I isolates nor with DNA from other taxa. This clone detected a large amount of variation within taxon 1 and was a highly repetitive sequence. Similarly, taxon 2 clone pT2S34 (insert A) hybridised only with taxon 2 isolates. However, it revealed little variation within this taxon. The putative taxon 2-specific clone pT2S22 hybridised only with DNA from taxon 2, but failed to hybridise with five of the 14 isolates tested. This clone hybridised to a 4 kb fragment of Sall-digested DNA and to several large DNA fragments of Pstl-digested DNA from the source isolate. While this indicates that the clone may be a tandem repeat sequence, it is not present in all isolates and could be of mitochondrial origin, or the result of infection by a DNA plasmid. This hypothesis should be tested by analysis of mitochondrial DNA and by determining whether extra- chromosomal DNA is present in taxon 2 isolates. In addition, mitochondrial DNA can be used as a cytoplasmic marker to detect anastomosis (Michelmore & Hulbert, 1987), and to study the phylogenetic population biology of the fungus (section 2.9.2). RFLP markers specific to taxon I and taxon 2 could be used to detect and distinguish the two taxa of Phomopsis on grapevine canes. The high copy clone pT1Pl80 may be useful in a DNA-based diagnostic test for the detection of taxon 1 in grapevine tissue. However, since pT2S34 (insert A) appears to be low copy, it may be less useful for detection purposes, although the signal could be amplified by PCR. Clone pT1P99 was a useful fingerprinting probe for genetic studies of taxon I because the banding pattern was easy to score and variation between isolates was detected. 187 Similarly, the five taxon 2 clones resulted in banding patterns that were easy to score for all the taxa. None of the taxon 1 clones hybridised with the taxon 4 isolate, and only one of five taxon 2 clones (clone pT2S2I, insert A) hybridised with taxon 4 DNA. Four isolates (1148, 1158, 15lC and 1538), initially identified as taxon 1 based on morphology, were shown by DNA analysis to be different from this taxon. All clones, except the taxon 2-specific pT2S22 and pT2S34 (insert A), identified isolates 1148 and 1158 as similar to taxon 3. The size of the cr-conidia of isolates 1148 and 1158 further supports the allocation of these isolates to taxon 3 (sections 2.3.2 and 3.1, Merrin et aI., 1995). All five taxon 1 clones identified isolates 15lC and 1538 as different from taxon 1; pT1P10 (= pTlP95) and pTlP180 did not detect any homology. The five taxon 1 clones resolved multiple bands in all 20 taxon 1 isolates and were likely to be repeat-sequence probes. Likewise, taxon 2 clone pT2S2I (insert A) resolved multiple bands in the taxon 2 isolates as did clone pT2S22 for nine of 14 taxon 2 isolates. These probes were useful for detecting variation within populations. The taxon I clones detected most variation among isolates belonging to taxon 1, whereas the microsatellite probes detected similar amounts of variation within taxon I and within taxon 2. The use of highly discriminatory probes, such as the taxon 1 clones and the microsatellite probes, provides a means of studying the population structure and the predominant mode of reproduction in the vineyard (I-evy et a1.,1991). In Phomopsis taxon 1, variation in RFLP banding patterns was higher within than between geographical regions, as shown by the Shannon-V/eaver indices. This variation may be due to the occurrence of sexual reproduction in the regions, or to multiple introductions via latent infections on rootlings. Similarly, Goodwin et al. (1992^) found more variation in pathogenicity within than between populations of Rhynchosporium secalis and speculated that a high rate of spontaneous mutations, a parasexual cycle or an unknown sexual stage may maintain intrapopulation variability. Another possibility is that multiple migration events from different source populations have occurred (Goodwin et a1.,1993). Little variation was detected among taxon 2 isolates with the taxon 1 and 2 probes and the microsatellite probes. Either the markers are not sufficiently polymorphic or the level of genetic diversity of the 13 taxon 2 isolates collected from South Australia, 188 Tasmania, New South Wales and Victoria is low. This suggests that sexual recombination in Phomopsis taxon 2 is either absent or rare. The sexual stage of Phomopsis taxon 2 has not been found in Australia. Additional isolates need to be analysed to determine whether a sexual stage exists and to determine the extent and basis for the observed variation. Transportation of canes infected with taxon 2 may have caused the appearance of the New South'Wales phenotype in Victoria (isolates DAR 69460 and VRU 0050), South Australia (isolate VRU 0074) and Tasmania (isolate 91.1). The Tasmanian isolate 110.1 had banding patterns identical to those of the New South Wales phenotype with all probes except microsatellite probe (CAT)5. This isolate may be a descendant of the New South Wales phenotype that has undergone one or more mutations. Additional probes are required to test this. The taxon 2 probes detected significantly less variation among the taxon 2 isolates than did the taxon 1 probes among the taxon 1 isolates. This may be because most taxon 1 probes were high copy number clones, whereas the taxon 2 probes, except pT2S22, were medium and low copy clones. Additional markers need to be developed to determine the extent of variation within taxon 2 in Australia. Most probes used in this study detected more variation within rather than between taxa and may not be useful for identifying the taxa. However, the two taxon-specific finge¡printing probes, pTlPl80 and pT2S34 insert A, may be used to identify the respective taxa. Another two probes, pT2Sl2 insert B and pT2S21 insert B, detected diversity between the two taxa and not within; these probes may be useful for the taxonomic identification of isolates. Cluster analyses of the genetic similarities among the 40 Phomopsis isolates clearly separated the four taxa. In all three cases, isolates 1l4B and 1l5B clustered with the taxon 3 isolates. The dendrogram generated using the taxon 1 probes showed that isolates 15lC and 1538 differed from the taxon I isolates, forming a putative fifth group of Phomopsis. The taxon 2 and microsatellite probes showed these isolates to be marginally different from the taxon I isolates, but these probes detected fewer polymorphic bands than did the taxon I probes. Isolates 151C and 1538 are self-fertile and capable of producing perithecia of D. viticola (Chapter 5) and it is, therefore, likely they were derived from taxon I through mutation and/or sexual recombination, but this requires further investigation. The taxon I 189 probes resolved the 20 typical taxon 1 isolates into two distinct groups, whereas the microsatellite probes identified a third category, consisting of isolate 95.1. Again, this might be because the microsatellite probes detected fewer polymorphic bands than did the taxon I probes. There was no similarity in banding patterns between isolates of taxon 1 and taxon 2 using the clones as probes. However, the two taxa were shown to have homologous sequences. The 27.27o similarity between the taxon 1 isolates and the taxon 4 isolate, detected by the taxon 2 probes, may have been exaggerated because none of the taxon 1 probes hybridised with taxon 4, and only one of the five taxon 2 probes hybridised with the taxon 4 isolate. Additional probes are required to resolve this. The observation that the taxon I clones are more polymorphic than the three microsatellite sequences indicates that the former are probably non-coding and that they target a larger number of loci than do the microsatellite probes (McDonald, 1997). Similarly, Stummer et al. (2000) found that Uncinula necator specific DNA probes detected higher levels of diversity than did microsatellite PCR primers. McDonald and Martinez (1991) found that different DNA fingerprinting probes, which hybridised to unrelated repetitive DNA sequences present on several different chromosomes, may provide different measurements of genetic diversity among individuals in the same Septoria tritici population. This may be caused by different recombination and/or mutation frequencies at different RFLP loci. In this study, it is possible that the microsatellite probes targeted coding regions in the DNA of taxon 1, whereas the taxon I clones targeted regions of the DNA that undergo mutation and recombination at a higher frequency. Sequencing of the taxon 1 clones may reveal why they are more polymorphic than the microsatellites. The dendrograms generated with the taxon 1 probes and the microsatellite probes, showed taxon I to consist of a heterogeneous group of isolates. The only isolates with identical fingerprinting patterns with every probe, were isolated from the same cane. The sexual stage (Chapter 4) may, therefore, play a significant role in the population biology of taxon 1. The dendrograms generated with the taxon 2 probes and the microsatellite probes, divided taxon 2 into two groups; one homogeneous group of nine isolates, eight of which had identical fingerprints with every probe, and one heterogeneous group of four isolates. The homogeneous group may have resulted from the introduction of one isolate into 190 Australia, which then reproduced clonally. Isolates of the heterogeneous group may have been introduced separately into Australia, or they may have evolved from the homogeneous group by mutation, or may be the result of sexual or asexual recombination. More research is needed to clarify this. However, clonal reproduction and gene flow may be important in the population biology of Phomopsrs taxon 2. The microsatellite and Phomopsis probes were used to calculate genetic similarities among the Phomopsis isolates. However, the calculation of Nei's genetic diversity statistic using RFLP markers, is correct only if: i) each DNA band represents only one genetic locus; ii) each locus is observed only as a single DNA fragment length; iii) none of the loci are genetically linked; iv) no loóus is subject to natural selection and v) no locus is linked to a gene which is affected by selection (Nei, 1973; Brown & Simpson, 1994). The characteristics of the probes used in this study, therefore, should be investigated to validate the findings. As linkage between some loci is likely to occur (Goodwin et al., I992b; Milgroom et aI., L992), some of the RFLP markers used in this study may be unsuitable for calculating diversity statistics and should be removed from the analyses. If the markers are found to be inappropriate for calculating diversity statistics, then new markers would need to be developed, such as single locus or low-copy RFLP probes (Nei er a1.,1983). McDonald & Martinez (1991) used several single-site RFLP loci to assign a multilocus haplotype to isolates of Septoria tritici by combining allelic data from different probelenzyme combinations. Using this analysis, the genetic similarity between two isolates is the percentage of shared alleles at the single-site RFLP loci. The taxon I and the microsatellite probes were not suitable for this type of analysis, since they hybridised to highly repeated sequences. In such cases, individual bands of the hybridisation profiles should only be scored as loci or alleles if crossing experiments and segregation analysis have been done. In the absence of crossing experiments and segregation analysis, the different banding patterns should be defined as RFLP phenotypes. McDonald & Martinez (1991) also calculated genetic similarity of the same S. tritici isolates based on the fraction of shared bands in DNA fingerprints, as was done in the present study. The researchers compared the two different methods and found that the two measures of genetic similarity were not correlated for any of the three DNA fingerprinting 191 combinations tested. They suggested, therefore, that DNA fingerprints may not provide an accurate measure of genetic similarity among clones in fungal populations. The probes that were used in this study to generate RFLP fingerprints of 40 Phomopsis isolates should be used with additional restriction enzymes in order to verify the grouping. These fingerprints could then be compared with the fingerprints generated by the probe/Psrl combinations and used in more extensive analysis of the population genetics. Also, the stability of the fingerprinting patterns for each of the clones needs to be investigated over successive asexual generations of the isolates, since only probes that are stable over time and generations can be used in population studies. t92 10. GENERAL DISCUSSION 10.1 Introduction The two main taxa of Phomopsis of grapevine in Australia, previously identified by Merrin et aL (1995), were examined. The biology and pathogenicity of Phomopsis taxon 1 was studied and compared with that of taxon 2, which is associated with symptoms attributed to P. viticola in other regions of the world. This resulted in more information on the epidemiology of the disease associated with Phomopsis taxon 1. Molecular and phenotypic markers were developed to study genetic variation within and between populations of Phomopsis taxon 1. Molecular markers specific to taxa I and 2 were developed to enable detection of the fungus in symptomless vine material. A third, less common, taxon of grapevine was also examined. Phomopsis taxon 2 corresponded with P. viticola described in phylogenetic analyses and morphological studies reported by Mostert et al. (2O01), after the experimental work for this thesis was completed. Topics discussed in this chapter are: the pathogenicity of taxon 1; the role of the teleomorph; and aspects of the taxonomy of Phomopsis of grapevine. As two complementary approaches were used, the discussion is divided into sections which address the different studies. The biology and epidemiology of the two main taxa of Phomopsis of grapevine in Australia are considered first (section 10.2) and visualised in two revised disease cycles (section 10.3). The genetic diversity of Phomopsis of grapevine, studied using molecular and phenotypic markers is then discussed (section 10.4). Both approaches contributed information on the taxonomy of Phomopsis of grapevine (section 10.5). Both studies provided information which may be used in the development of disease management strategies (section 10.6). L0.2 Biology and epidemiology of Phomopsis of grapevine Morphological and field studies on Phomopsis taxon I resulted in the discovery of the teleomorph, identified as Diaporthe viticola by Dr P.F. Cannon (IMI, pers. com.), supported the symptoms attributed to taxon 1 by Menin et aI. (1995) and suggested that this taxon 193 may be associated with bud-loss and stunted growth of grapevine shoots. In addition, preliminary pathogenicity testing supported the assumption that taxon 2 can cause necrotic spots with yellow haloes and scarring of canes. This study provided new information on the epidemiology of the disease caused by Phomopsis taxon 1. The discovery of the teleomorph suggests the potential for infection by ascospores. These spores may provide a source of airborne inoculum for long range dispersal of the fungus. Because the optimum temperature for the development of perithecia was l5oC, it is likely that they develop in winter and spring, and mature in spring, similar to the pycnidia. Ascospores were capable of infecting micropropagated grapevines (section 5.3.1), however, it is unknown if ascospores infect grapevines in the field. This needs to be determined and spore trapping experiments should be conducted to investigate when ascospores and conidia are dispersed, and the proportions of each, during the grape growing season. This would give information about their dissemination and relative importance. Prior to this project, relatively little research had been carried out on taxon 1 and, because of its asymptomatic growth, the pathogenicity of taxon I was in question. This study, however, suggests that taxon 1 is associated with bud-death and stunted growth on grapevines, although further studies are required to confirm this and to fulfil Koch's postulates. Moreover, long-term asymptomatic growth of taxon I within canes could result in a gradual decline in productivity by limiting the transportation of nutrients (Melanson er a1.,2001). Both long-term saprophytic survival and over-wintering in grapevine buds have been described for P. viticola (Pine, 1959; Hewitt & Pearson, 1988). In this study, both major taxa of Phomopsis of grapevine in Australia were found to survive within canes for 3 years. In the field, buds may become infected through subcuticular growth of the fungus or through direct infection (Melanson et a1.,2001). In the glasshouse study, buds appear to become infected through subcuticular growth, although dead buds were not tested for the presence of Phomopsis. Young shoots infected with taxon 2, possibly, through subcuticular growth, did not develop necrotic spots with yellow haloes and remained symptomless like those infected with taxon 1. Scarring of a cane occurred 10 months after inoculation with taxon 2, but more research is required to find out how long either of the taxa needs to grow inside the canes before disease is expressed and pycnidia develop. Long-term survival 194 within grapevine canes and over-wintering in buds prolong the opportunity for dissemination of the fungus. Asymptomatic growth of Phomopsis taxon 1 makes disease development, yield loss and long term health and productivity of an infected grapevine difficult to monitor. It is, for example, unknown whether taxon 1 is an endoph¡e which may become pathogenic when vines are stressed, or a pathogen with a latent period. Similarly, relatively little is known about the disease caused by taxon 2 in Australia. While reduced bunch set and yield loss in the USA were described by Pscheidt & Pearson (1989b), it is uncertain whether or not taxon 2 causes yield loss in Australia, and the long term health and productivity of grapevines infected with taxon 2 has not been monitored. 10.3 New disease cycle for Phomopsis taxon L of grapevine Published disease cycles of Phomopsis cane and leaf spot, as summarised in section 2.5J, do not take into account the existence of two, or possibly more, taxa of Phomopsis infecting grapevine. This makes it difficult for grape growers to distinguish between the taxa and to determine which is present in the vineyard. A disease cycle showing the course of symptoms associated with Phomopsis taxon 1 is proposed, which includes the teleomorph and the symptoms described in Chapters 6 and 7 (Fig. 10.1A). As bleaching of canes and spurs was found to be associated with infection of grapevines with Phomopsís taxon 1, but scarring and necrotic lesions were not, the name "Phomopsis cane blight" is proposed for the disease caused by taxon 1. In late winter, diseased canes and spurs are bleached and speckled with pycnidia, and perithecia may also be present. Narrow black zone-lines or black marks (Fig. 4.4) are usually visible on the bleached spurs when perithecia are present. Perithecia usually mature after the pycnidia, and ascospores may be released when the humidity is high for several hours. a-conidia germinate in a temperature range of 5-30'C (Merrin et a1.,1995). Infection by ascospores or cr-conidia in the vineyard has not been demonstrated. In spring, budburst on bleached spurs appears to be reduced and shoots may be stunted (Chapters 6 and 7). During summer in Australia, symptoms of the disease are hard to see. The fungus may grow 195 Figure. 10.1 Revised disease cycles for the two major taxa of Phomopsis infecting grapevine in Australia. A. Disease cycle of Phomopsis cane blight, associated with Phomopsls taxon 1. B. Disease cycle of Phomopsis cane and leaf spot, associated with Phomopsis taxon 2. A I l4,nnuR 1 SEXUAL R!PRODUCTION^sÐruAL RTPRODUCTION bleuched c¡no wltl¡ frultlng bodlcs .¡St* pycnkllum wltlt lporcs lsPffil I rwutttN I (" I wcukcttctl cnnc Þ oscxutl sPotts scxuol sporcs / INFITC'TION / fungus rchtivolY lnactlvc I fsffiER rcduccd l¡udl¡utsl nnd ¡tur¡lcd gfowth tnfly occllf B ASEXUAL f wbtrpRl RI!NRODUCTION scnÍcd nnd spur wlth frultlng botllcn (PYcnldln) pycnidlunr wlth sporcs rffil l¡rowrt cntckn tlltl sct¡rs on yotlng cnlls nscxuol sporcs lNffDcTroN fungus rulnlivclY inuclivc fffiRl sp0l¡ smnll bluck spots with- ycllow ltnlocs on lcof on younB sltoot t96 inside the green tissues without causing symptoms until winter, when the fruiting bodies develop, or, as for P. viticola, it may be relatively inactive in vine tissue @mmett et al., L992). Weakened canes have been associated with Phomopsis infection (IPM Viticulture Research to Practice, L997), and have been observed, in autumn, in vines infected with taxon 1 in one vineyard (D.L. Melanson, pers. com.). As for P. viticola, infected canes and spurs may continue to produce pycnidia and conidia for at least three seasons @mmett e/ aI., 1992; Pearson, 1990). A modified disease cycle for Phomopsis cane and leaf spot, caused by taxon 2, is also presented (Fig. 10.18) for comparison (Scheper et a1.,1997). This disease cycle differs from published disease cycles in that neither infected grapes nor bleached spurs without scarring are shown, because the latter are associated with taxon 1 infection, and bunch rot caused by taxon 2 is very rare in Australia (R.W.Emmett, pers. com.). 10.4 Genetic variation in Phomopsis of grapevine Both phenotypic and molecular markers discriminated between isolates of taxon 1 and taxon 2 infecting grapevine in Australia. Colony morphology and self-fertility were the only markers examined that were not capable of distinguishing between the taxa. Mycelial compatibility grouped taxa2,3 and 4 into three separate MCGs and the taxon 1 isolates into 16 additional MCGs. Pathogenicity testing on seedlings showed that taxon 2 was generally more pathogenic than either taxon I or 3. The molecular markers differentiated between the four taxa. This study indicated that the teleomorph may play a significant role in the population biology of taxon 1. First, the variation in colony morphology, mycelial incompatibility and RFLP banding patterns within populations or geographical regions was higher than the variation between populations or regions. In addition, cluster analyses of genetic similarities among the isolates generated with RFLP probes showed this taxon to be heterogeneous. Furtheûnore, a large number of MCGs was found, mycelial incompatibility occurred among the progeny of two perithecia and there appeared to be more variation in morphology within MCG 5 than between MCG 5 and the remaining MCGs. Although this r97 high degree of diversity within taxon 1 is likely to be due to sexual reproduction in the population, the results need to be supported by spore-trapping experiments and studies of the infectivity of ascospores, followed by mating studies and molecular analysis of cultures derived from single ascospores. The possibility of multiple introductions of taxon I via latent infections on rootlings should also be investigated. Sexual recombination is more likely to result in genetic variation in heterothallic than in homothallic fungi (Michelmore & Hulbert, !987; Brayford, 1990u & 1990b; Glass & Kuldau, L992).It is, therefore, important to determine whether taxon 1 is homothallic or heterothallic. Studies on the nature of sexual reproduction, however, \ryere inconclusive. Some isolates were self-fertile, while other isolates did not produce perithecia when selfed, nor did they do so when paired with other self-sterile isolates. Taxon 1 may be a homothallic species in which a mutation to self-sterility is present, or a heterothallic species in which mating type switching occurs. The finding that D. viticola is apparently capable of outcrossing, also supports the hypothesis that sexual recombination may be involved in the generation and maintenance of genetic variation within populations of this fungus. However, research using molecular markers is required to determine the frequency of sexual outcrossing and whether recombination occurs. Isolates of taxon 2 exhibited less variation than taxon I in terms of mycelial compatibility and RFLP markers. As the sexual stage of Phomopsis taxon 2 has not been found in Australia and the sexual stage of P. viticola has not been observed since 1911 (Shear, 1911), it is likely that sexual recombination is infrequent or does not occur in taxon 2. Moreover, mycelium of all taxon 2 isolates examined appeared to be compatible, suggesting that heterokaryon formation may occur in the vineyard and that parasexual recombination, together with mutation, may be the major means of generating genetic variation in this taxon. A parasexual cycle, which results in mitotic recombination, has been demonstrated for a number of imperfect fungi and ascomycetes. For instance, the high levels of variation found in Rhynchosportum secalis were attributed to a possible high rate of spontaneous mutations, a possible parasexual cycle, an unknown sexual stage or multiple migration events from different source populations (Goodwin et aI., L992u & 1993). Rare sexual recombination events may also occur in Phomopsis taxon 2 in the field, but are as yet r98 undetected. Research on the occurrence of a sexual or parasexual cycle, the mutation rate and the occuffence of migration events is required. The low level of genetic variation detected for Phomopsis taxon 2 in Australia may result from founder effects due to migration, establishment and asexual reproduction of a small number of genotypes. This hypothesis is supported by a recent investigation of Phomopsis on grapevine by Mostert et aI. (2001), in which they showed that taxon 2 has also been found in the U.S.A., South Africa, Turkey and several countries in Europe, and taxa I and 3 in Portugal and South Africa. It is, therefore, likely that taxa I,2 and 3 were introduced separately into Australia, and they may all exhibit lower levels of variation in Australia than in other continents. Detailed comparison of numerous isolates from each of these countries, using molecular markers, is required to identify the origin of the taxa of Phomopsis on grapevine and to establish whether variation in other continents is greater than in Australia. The markers developed in this study were useful for identifying phenotypes or genotypes. However, in order for a marker to be useful for distinguishing genetically different isolates or identifying an isolate, the marker locus must be somatically stable. The stability of the markers used in this study should, therefore, be examined. The stability of the colony morphology marker was questioned in section 8.4 and analyses of genetic diversity using this marker may, therefore, be unreliable. The sampling method employed is very important for the study of genetic variation. To obtain preliminary estimates of variability, isolates from a wide geographical area should be sampled and examined. For example, Christiansen and Giese (1990) studied 28 isolates of Erysiphe graminis f . sp. hordeí from several countries in Europe and the USA as a preliminary study. Similarly, but on a smaller scale, the preliminary investigation of genetic variation in Phomopsrs taxon 2 included 13 isolates from ten different locations in four states in Australia. The study of taxon 1 included 24 isolates from eight locations in three states. To determine within-population variation, large numbers of isolates need to be sampled from a small area. An example of such a study is that by McDonald and Martinez (1990), in which the genetic diversity among 93 isolates of Septoria tritici from a single wheat field was investigated. A preliminary investigation of within-population variation was conducted for taxon 1 among six isolates from the Adelaide Hills and among six t99 isolates from Tasmania, using RFLP markers, and among l2 isolates from Ashton Hills, six from Hargrave's and seven from the Coonawarra, using MCG and colony morphology markers. High levels of variation within each of the populations were observed. However, further research with a larger number of isolates from a single vineyard is required. To obtain information regarding migration of the fungus and genetic relationships among populations, many isolates from several populations need to be examined. The survey by Goodwin et al. (1993) of genetic variation in R. secalis populations in Australia, Europe, Japan and the USA in which they suggested that R. secalis migrated from a common centre of origin, is a good example of such research. The results of the preliminary investigation of Phomopsis on grapevine in Australia reported in this thesis, indicated that genetic variation within taxon I occurred within and between populations and that genetic variation was also present in taxon 2,but at a lower level. In future research, many taxon I isolates should be sampled from several vineyards in the Adelaide Hills to study genetic variation within and between vineyards, and the data compared with a similar number of isolates from other grape growing regions in Australia. In addition, many taxon I isolates should be sampled over several years from a single vineyard to study genetic variation within and between years, to investigate the relative importance of the sexual stage versus the clonal long-term survival of the fungus within canes. Similar studies should be conducted for taxon 2. Both the molecular markers and the mycelial incompatibility tests suggest that taxon I and 2 are genetically isolated and do not undergo sexual or parasexual genetic recombination with each other. This is similar to the two biotypes of Colletotrichum gloeosporioides, which cause different diseases, form separate VCGs (Viljoen et a1.,1997) and are genetically distinct (Masel et al., 1996). However, in the field, environmental conditions favouring a rare sexual or parasexual event between the taxa could occur. For example, Cisar et aI. (1994) have shown that sexual recombination can occur between some widely divergent, normally asexual, biotypes of C. gloeosporioides under particular environmental conditions. Experiments by Masel et aI. (1996) on the same fungus suggested that a horizontal transfer of a 2 Mb chromosome from one biotype to the other has taken place in nature, even though these biotypes were vegetatively incompatible. Such rare recombination events may also occur between the taxa of Phomopsis of grapevine and may play a role in the generation of genetic variation. 200 Putative taxon-specific clones, that showed no homology to DNA from several other fungi or grapevine tissue, were developed for taxa I and 2 of Phomopsis. The taxon I clone pTlPl80 recognises a repeated sequence specific to taxon I and identifies genetic variation within this taxon, whereas the taxon 2-specific clone pT2S34 (insert A) revealed little variation within this taxon. The taxon l-specific clone provides a useful tool for the detection of Phomopsis taxon 1 in grapevine tissue, particularly since this taxon is asymptomatic during the growing season. A similar high copy taxon 2-specific probe has been developed (Melanson ¿t a1.,200I).Identification of Phomopsís in grapevine canes or buds may provide an early indication of infection before symptoms are visible. Other uses of taxon-specific markers include i) the potential to monitor the spread of Phomopsis within and between vineyards and geographical areas, ii) the ability to test grapevine cuttings for the presence of Phomopsis before planting or for use in pathogenicity tests and iii) the potential to determine origin, evolution and genetic structure of Phomopsis populations. 10.5 Taxonomy of Phomopsis of grapevine The confusion about the taxonomy of Phomopsis of grapevine is being resolved gradually (Phillips, 1999; Mosteft et a1.,2001). Merrin et al. (1995) described two distinct taxa of Phomopsis (taxon 1 and taxon 2), plus two minor groups (taxon 3 and taxon 4) on grapevines in Australia. Based on a repoft by Dr E. Punithalingam (IMI, pers. com., 1992), which identified isolates of taxon 1, 3 and 4 (DAR 69459, 69464 and 69484) as P. viticola (Sacc.) Sacc. (IMI 352478, 352479 and 352481, see appendix), Merrin et aI. (1995) reported that taxon 1 fitted the current concept of P. viticola (Sacc.), while taxon 2 might be a variety of P. viticola or a distinct species. Three years later, the same facility identified the single conidium-derived cultures, 50D.1, 51C.1 and 54B.1, grown from ascospores (IMI 368582 and 368583, see appendix) as Phomopsis sp. and not as P. viticola (Sacc.) Sacc., because "the conidial width is much narower than quoted in the original account", although the cr-conidia were, apparently, similar in size to those of the taxon 1 isolates sent 1n L992.Indeed, the cr-conidia of taxon I are much smaller (4.8-7.2 x L4-2.2 pm) than those reported for P. viticola by Saccardo (1880), Reddick (1909) and Pine (1958), viz. J x 20r 4 ltm, 6.3-II.2 x L.7-2.8 pm and 8-10 x 3 pm, respectively. Moreover, taxon I does not produce lesions on vines typical of the disease caused by P. viticola, and its culture morphology differs from that reported for P. vitícola (Pine, 1958). The morphological data suggest that Phomopsls taxon 1 may belong to a species distinct from P. viticola. To add to the confusion, the teleomorph of taxon I was first confirmed as Diaporthe viticola (Nitschke, 1867) by Dr P.F. Cannon (IMI, pers. com.) in 1995 (section 4.3.3; appendix; Scheper et a|.2000). Subsequently, Phillips (1999) compared two samples of D. viticola on grapevines sent from Australia with a species isolated from grapevine in Portugal, identified as Diaporthe perjuncta Niessl., and concluded they represented the same species. This was subsequently verified by molecular analysis of the nuclear ITS regions and the mitochondrial small subunit rDNA (Mostert et a1.,200I). Phillips (1999) suggested the name D. perjuncta was appropriate for the teleomorph and cited previous research on the morphology of several species (Traverso, 1906; Wehmeyer, 1933; Kobayashi, L97O; Ananthapadmanaban, 1989). In addition, a Phomopsis species on grapevines in Portugal was indistinguishable from the anamorph of the Australian D. viticola isolates and bore little resemblance to P. viticola, although a name for the anamorph was not proposed (Phillips, 1999). The designation Phomopsls taxon I remains, therefere, appropriate. Apparently, the teleomorph of taxon t has characters, such as the shape and size of the ascospores, that could place it in D. viticolalD. meduseae, D. eres or D. perjuncta (V/ehmeyer, 1933; Phillips, L999). Although the long ascomatal necks of the teleomorph would place it in D. viticolalD. meduseae, these could be a result of the long period of incubation in humid conditions used in this study, implying that it could also be D. eres or D. perjuncta. Molecular markers, such as the taxon l-specific and other fingerprinting markers developed in this study, may be used to determine the species and the degree of similarity with other Phomop sisl Diaporthe species. At the start of this project, taxon 2 was thought to be either a variety of P. viticola or a different species (Merrin et aL, 1995). However, taxon 2 produces a-conidia similar in size (8-1L.8 x 2-3.2 pm) to those produced by P. viticola (Pine 1958; Hewitt and Pearson 1988), lesions produced on vines are typical of the disease caused by P. víticola, and its morphology in culture resembles that reported for P. viticola (Pine, 1958). This suggests 202 that Phomopsis taxon 2 may be P. viticola, although taxon 2 cr-conidia do not contain guttules, which is a characteristic feature of P. viticolø. Molecular and phenotypic markers used in this study clearly distinguished between taxon I and taxon 2. Similarly, Mostert ¿t aI. (2001) reported that molecular analysis of the nuclear ITS regions and the mitochondrial small subunit rDNA clearly distinguished taxon 1 from taxon 2. Moreover, the Australian taxon 2 isolate 91 clustered with P. viticola isolates originating from other regions of the world (Mostert et a1.,2001). The taxon 2-specific clones developed in this study and by Melanson et al. (2001) may be used to verify the findings by Mostert et aI. (200I) by examining more isolates from Australia and other continents. The perfect stage of P. viticolø was described only once as Cryptosporella viticola (Shear, 1911), and has not been found again. It is possible that a teleomorph exists, but is rare and the development of the ascomata may require environmental conditions very different to those required by the teleomorph of taxon 1. This is the case with the two groups of Phomopsis from elm (Brayford, 1990'). Perithecial production by the heterothallic group 1 was repressed by cold incubation and fruiting occurring after 2-3 months at 20-25oC in a 12 h light/dark cycle. In contrast, perithecial production by the homothallic group 2 was enhanced by incubating at low temperatures (5-10'C) for 2-3 months in darkness, followed by a period at 22"C to stimulate ascospore differentiation. Similarly, a teleomorph of Phomopsis taxon 2 on grapevines may require specific environmental conditions. If the sexual stage is found and differs from that of taxon 1, it would provide conclusive evidence that the two taxa are separate species. The four Phomopsis taxon 3 isolates were clearly different from taxa I and 2, when analysed using the phenotypic and molecular markers. Similarly, Mostert et aI. (2001) found that taxon 3 isolates from grapevines in Australia, South Africa and Portugal and two Plnmopsis isolates from Pyrus and Protea species in South Africa clustered together using molecular analysis, and were distinct from Phomopsis taxa I and 2. It was suggested that taxon 3 is a separate species, designated Phomopsls sp. I (Mostert et a1.,2001). Phomopsis taxon 4 was assumed by Merrin et aI. (L995) to be a sterile form of taxon 1. However, none of the taxon 1 clones used in this study hybridised to taxon 4 DNA. Moreover, only one of five taxon 2 clones hybridised to taxon 4 DNA; this clone also hybridised to DNA from taxa L,2 and 3, but not to DNA from grapevine or other fungi that 203 were tested, and did not detect any similarity in the banding patterns between the taxon 2 isolates and the taxon 4 isolate. Taxon 4 was clearly different from the other taxa and can not be considered a sterile form of taxon 1. Mostert et aI. (200I) considered an Australian taxon 4 isolate to be a species of Libertella, the anamorph of Eutypa. Cluster analyses of genetic similarities identified a fifth group of Phomopsis on grapevines in Australia. This group, consisting of two isolates from the Adelaide Hills (151C and 1538), frây have been derived from taxon 1 through mutation and/or sexual recombination, but this requires further investigation. It is likely that a number of species of Phomopsis infect grapevine. In addition to the four taxa identified by Merrin et aI. (1995) and the putative fifth group identified in this study, Kuo & I-eu (1998) identified a new pathogen, Phomopsis vitimegaspora, on grapevines.in Taiwan. This species has large cr-conidia similar in size to those of the anamorph of C. viticola (Shear, 1911). Moreover, Mostert et al. (2001) recently isolated Phomopsis amygdalí from grapevines in South Africa, and suggested that an Italian isolate from grapevines be designated as a new species of Phomopsis. Recent molecular research has suggested that at least some Phomopsis species have a much wider host range than previously suspected (P.F. Cannon, IMI, pers. com.). P. amygdali and Phomopsis taxon 3, for example, also occur on other unrelated hosts (Mostert et a1.,2001). In response to repoÍs that some species of Phomopsis were not restricted to a particular host genus, Rehner & Uecker (1994) compared the ITS region of 5l isolates from different plants and found similar nucleotide sequences, suggesting that Phomopsís species infect more than one host, or that host switching occurs frequently. Therefore, it is possible that Phomopsís species occurring on plants such as fruit trees and eucalypts, which commonly surround vineyards in Australia, may also infect grapevines. Indeed, specific parts of the lifecycle could occur on such hosts, such as a possible teleomorph of taxa 2 or 3. Recently, Yuan et aI. (1995) found P. eucalypticola on eucalypts in Australia, the cr- conidia were similar in size (5.0-7.5 pm x 2.O-3.O pm) to those of Phomopsis taxa I and 3 on grapevines. If this species is non-host specific, it may be related to taxon 3 or perhaps isolates l51C and 1538 on grapevine. It would be prudent to conduct reciprocal pathogenicity studies using Phomopsis isolates found on grapevine and other hosts. In 204 addition, the application of molecular markers would assist in resolving questions on host specificity of Phomopsis species. 1.0.6 Implications for disease management In order to develop timely and effective control strategies with reduced chemical input, it is imperative to have a sound understanding of the biology of the taxa of Phomopsis and the effect of the diseases they cause on the productivity of the grapevine. This study provided knowledge about taxon 1 that could be utilised in the field. The reproductive biology of the two main taxa of Phomopsis infecting grapevines in Australia has to be taken into consideration in making decisions about control and management of the disease. For instance, the discovery of the sexual phase of taxon 1 may affect management of Phomopsis cane blight. If ascospores infect grapevines, they may provide a source of airborne inoculum for long range dispersal of the fungus and the disease. Consequently, control strategies targeted at splash dispersed conidia may be of little use. Moreover, current fungicide spray regimes for control of Phomopsis are primarily based on taxon 2 infection (Rawnsley & Wicks, 2000) and may not be suitable for control of taxon 1, because of differences in biology and epidemiology of the two taxa. More research is needed to establish which spray regime, if any, is appropriate for control of taxon 1 and whether it is required in all areas. The occurrence of the sexual cycle of Phomopsis taxon I may allow the pathogen to respond to new selection pressures, and increase the likelihood of fungicide resistance developing. It is, therefore, important to adopt strategic methods of applying fungicides and to improve hygiene by removing diseased cane material. As with Phomopsis of peach trees (Uddin & Stevenson, 1998), selective pruning of bleached grapevine canes appeared to reduce the disease associated with Phomopsis taxon I (Chapter 6), perhaps by reducing the inoculum. The field study indicated that spur pruning may be a better strategy for management of disease caused by taxon I in cool and wet climates than is cane pruning. Another way to improve hygiene is to ensure that planting material is disease-free. Taxon- specific molecular markers would be a useful, but not infallible, tool to achieve this. 205 In order to make disease management decisions, it is important to know which disease is present in the vineyard and whether Phomopsis taxon 1 reduces yield. In this project, the symptoms associated with infection of grapevines by taxon 1 were distinguished from symptoms associated with other factors. The new disease cycle, which illustrates these symptoms, will assist growers and viticulturists to distinguish between the two major taxa of Phomopsis of grapevine in Australia. Moreover, when more conclusive evidence is available about the effect of both taxa on yield in Australian conditions, the disease cycles may assist in making more informed decisions about the type and timing of control measures. Taxon-specific molecular markers have considerable potential in research on the biology of Phomopsis, the effect of the disease on the productivity of the grapevine and may ultimately contribute to more timely and effective control strategies with reduced chemical imput. The markers could be used to identify the fungus in canes and buds, assist in the supply of disease-free planting material, monitor the efficacy of control procedures, and could facilitate decisions on cultural practices to control the disease. In conclusion, this study resulted in the identification of the sexual stage of Phomopsis taxon 1, which may be responsible for the high level of genetic variation observed among isolates of this taxon. Phomopsis taxon I was also found to be associated with stunted growth of shoots and bud-death in vineyards. In view of the distinct differences between taxon 1 and taxon 2, the disease associated with taxon t has been named Phomopsis cane blight and a new disease cycle was devised. The information obtained about taxon I will enable researchers to monitor the disease in the field as an aid towards more informed management strategies. However, pathogenicity testing and yield loss data are required before recommendations on control can be made. 206 APPENDIX Tris-acetate (TAE) buffer 40 mM Tris 20 mM sodium acetate 1 mM EDTA pH adjusted to 7.8 with acetic acid Prepared as a 50 x solution, stored at 4"C. Tris-EDTA buffer (TE) buffer 10 mM Tris-HCl I mM EDTA pH adjusted to 8.0. Gel loading buffer 0.4 M urea 57o sucrose 5 mM EDTA, pH 7 0.OL7o bromophenol blue Prepared as a 10 x solution, stored at -20oC. Calf intestinal alkaline phosphatase (CIP) reaction buffer (Promega) 50 mM Tris-HCl, pH 9 1 mM MgCl2 0.1 mM ZnCl2 I mM spermidine T4 DNA ligation buffer (Promega) 30 mM Tris-HCl, pH 7.8 l0 mM MgCl2 10 mM DTT 0.5 mM ATP pUCl9 Pvull specific primers primer Sl = 5'ACAGCTATGACCATG 3' primer 52 = 5'TCCCAGTCACGACGT 3' t Prepared as a 0.1 pg pf solution of each primer and stored at -20"C. Hybridisation solution in 10 ml: 2 ml Denhardt's solution 3 nìl5 x HSB 5 lú, 6Vo Dextran sulphate (filter sterilised, stored at 4"C) Denhardt's solution 2Vo gelatin (Difco, Bacto) 2Vo fic oll@ 400 (Pharmac i a) 2Vo polyvinyl pynolidone (PVP 360,000, Sigma) 107o sodium dodecyl sulphate (SDS) 57o tetrasodium pyrophosphate Dissolved in sterile nanopure water at 65'C while stirring, and filtered through sterile filter paper in sterile funnel into sterile bottle at 65oC. 5xHSB 3M sodium chloride 100 mM PIPES 25 mM NazEDTA pH adjusted to 6.8 with sodium hydroxide, and autoclaved. 207 Communications with the International Mycological Institute I-etter to IMI, dated 2211211995, requesting information on the Phomopsis cultures sent to IMI by Ms S.J. Merrin in 1992. Report H273192/YA3 Communication to Ms S.J. Merrin by Dr E. Punithalingam, reproduced with the permission of Dr M. Priest, 15 August 2001. Report D426l95lYA3 Communication about Phomopsis isolates 50D.1, 51C.1 and 54B.1 by Dr E. Punithalingam. Report D426l95lYA3 Communication about Diaporthe viticola by Dr P.F. Cannon DEPARTMBNT OF CROP PROTECTION Head of Dcpartment: Tel: (08) 3031269 Professor Otto Schmidt Fax: (08) 3794095 Wednesday ,22 December 1995 The biosystematics Services International Mycological Institute Surey, TW20 9TY, UK Report: D426195/Y A3 Our ref: Diaporthe viticola,RS Dear Dr Saddler, Thank you for your letter of 2l Novenrber 1995. The inforrlation on Phomopsis tti.ticr¡la and Dia.porthe vitic:ola has been very, helpful. Would it be possibìe to send nre information on the Phomopsis cnltures that Jill Merrin and Dr Tan Nair sent to you? The numbers of their cultures are as follows: DAR 69457 lMl 352477 representative of Taxon 2 DAR 69459 IMI .35247tì taxon 1 DAR 69464 IMI 352479 taxon 3 DAR 69484 IMI 3524tì I representative of Taxon 4 DAR 69488 IMI 3-524tì3 taxon 1 Dr Nair has given nre pernrission to request this infomration as the original report cannot be located. Inclucled is their publication in Australasian Plant Pathology. I look fonv¿rrcl to hearirrg fron-r vou and thank you very nruch for your assistance. Yours sincerely, Reiny Scheper I NTERNATIONAL MYCOLOGICAL I NSTITUTE IDENTIFICATION SERVICES lort: Yr273lezlvþ3 tr ref: grapevine lr IMI nber number Identification and comments 14 lL']-.(11 L 3524n Phomopsis sp. A dried example of rhis isolate has been placed in the IMI herbarium. Report from Dr E. Punithalingam. þ r-rr I 352./-78 Phomopsis viticola (Sacc.) Sacc. This produced only alpha conidia and their measurements are at the lower end of 1þs ¡¡nge for P. viticola. A d¡ied example of this isolate has been placed in the IMI herbarium. Report from Dr E. Punithalingam. L¡ )-¿'n -l 352479 Phomopsis viticola (Sacc.) Sacc. A dried example of this isolate has been placed in the IMI herbarium. Repof from Dr E. Punithalingan. 352480 Not identilied. A report on this will follow in due course. i r.L)'r (t 352481 Phomopsis viticola (Sacc.) Sacc, This produced only beta conidia which a¡e ideutical to those of P. viticola. A dried example of this isolate has been placed in the IMI herbarium. Report from Dr E. Punithalingam. ù2L 352482 Phomopsis viticola (Sacc.) Sacc. A dried example of this isolate has been placcd in the IMI herbarium. Report from Dr E. Punithalingam. L42-+.. . t 352¿183 Not identilied. My comment for IMI 35?M also applies to IMI 35283. This material has been discarded. Report from Dr E. Punithalingam. tn955 3524€/. Phomopsis sp. This isolate is similar to la (IMI 3524m in colonial and conidial characters. Both isolates produced only ellipsoid conidia measurins*+ 95-12 x 2-3 to thosé of Goid., which is a later homonym of P Sacc. has been placed in the IMI herbarium. Report from Dr E. Punithalingam rilc ¡ll ¡tason¡blc carc is t¡kcn to cnsure thc acq¡racy lnd reliability of an idcntification rcPort Prcparcd by lhc Institulc, no liability cen bc rcccpted by thc lnst¡tutc, its mcmbcrs, staf[ or açnts in respcc of læ, damagc or injury (*ficthcr fatal or othcrwisc), horsoaar causcd, wtrich may bc suffcrcd as ¡ r6utt of lhc idcntif¡cat¡on. I NTERNATIONAL MYCOLOGICAL INSTITUTE B¡OSYSTEMATIC S ERVICES rort: D426/es lv þ3 tr ref: Diaporthe viticola, RS tr IMI nber number Identification and comments Phomopsis sp. The following rePort has been made Dr B.C.S I¡ all houesty I am u¡able to confirm the isolate as P. acc) Sacc the conidial width ts much na¡¡ower tlan quoted in the original accotrnt. It migbt bc P. viticota var. øn'tpetop.rid¡s Grove which has Danow conidi4 or eveB P- irkola (Reddick) Goid. which has couidia of similar size to yor¡rs. Tbere a¡e also the narnes P. antpelina (Berk. & Mâ. Curris) Grove and P. cordifolia (Brur) Died. recorded from the genus Øru. To my kno*ledge no-one has ca¡ried out a.Ey ryPe- based revisioua¡y studies on tlese fungi and r.r¡ü rhat is done no specific epitheS c-' be r:sed *irh confidencc. A d¡ied presen'ed gxagp_l1o! this_lsolale ÞI bccn placed in t-he L\fI d¡ied reference colleclion. 'Report from Dr E. Pudt-batilgan. , o2(k\ t 3ó8583 Pbomopsis sp. Coomeots for 2 (I:\II 3ó3582) appl¡'to this isolate. This Lsol¿tc has been placed i¡ ttre L\fI Cultu¡e Collection, Report from Dr E. Punitlali-¡ea-8. íela"t ,,ît t (,', i"'J- þ\*- r.- (-).)-l \-- a,'^i --r' l .l ,-È S'" ',r:s-) r-j Lr-r.rr 'Jr. '--'-" r'F | /'' ir ii¡\ ái no liability cea bc titc ¡tt æ¡son¡bte care b t¡lcn ¡o cnsur€ rhc rcrr¡racy ¡nd rcliabiliry of en identilication tlPon P¡cPsrGd by thc Institutc, rcccptcd by ùc lrutirurg is mcmbc6 sotl or rgpns in rcspccr of 1o.., damags or injury (*'hcrhcr fatal or othersisc), horaocrær cruc4 *trich mry bc ¡uffcrcd ¡s ¡ ¡csult of ¡hc ¡dcnlificrliorr- I NTERNATIONAL MYCOLOGICAL I NSTITUTE B IOSYSTEMATIC SERVICES Report: D426/es /Y A3 Your ref: Diaporthe viticola, RS Your IMI number number Identifrcation and comments 1 368581 Diaporthe viticola Nitschke. The na-e Diapofthe viticola already exists; it was introduced by Nitschke n hts þrenomycetes Getmanici p.264 (186Ð. The na-e has not been considered recently, so far as I am aware, but in the only comprehensive monograph of the genus Wehmeyer (1933) treated it as part of the D. meùtsaeø complex This is characterized largely by the long ascomatal necks present on your specimen, and other features such as ascospore size also fit with Wehmeyer's studies. The neme ¡snains of uncertain application pending modern studies. Recent molecular research has suggested that at least some Diapofthe (and by inplication Phomopsis) species have a much wider host rânge than has hitherto been suspected, so introducing new nâmes should be done with especial caution. Nevertheless, a modern description and illustrations of the fungus would be of considerable value whatever nâme is eventually given to it. This material has been placed in the IMI dried reference collection. Report from Dr P.F. Cannon. ¿ 368582 Not identified. A report on this will follow in due course. l 368583 Not identified. 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