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FACTORS ASSOC~ATED WITH CONIOTHYRIUM
CANKER OF EUCAL YPTUS IN SOUTH AFRICA
By
leonel Merwe van Zyl
Submitted in fulfilment of the requirements for the degree
Doctor of Philosophy
In the Faculty of Science, Department of Microbiology and Biochemistry,
University of the Orange Free State, South Africa
May 1999
Promoter: Prof. M.J. Wingfield
Co-Promoters: Dr. T.A. Coutinho
Prof. B.D. Wingfield
DECLERA TION
I, the undersigned, hereby declare that the thesis submitted herewith for the
degree, Philisophiae doctoriae, to the University of the Orange Free State,
contains my own independent work. This work has hitherto not been submitted
for any degree at any other university of faculty.
LJ&
July 1999
Dedicated to my family
A PHILOSOPHER'S life is spent in discovering that, of the half-dozen truths he
knew when a child, such an one is a lie, as the world states it in set terms; and
then, after a weary lapse of years, and plenty of hard thinking, it becomes a truth
again after all, as he happens to newly consider it and view it in a different
relation with the others.
A Soul's Tragedy, Part II.
TABLE OF CONTENTS
Acknowledgements I
Preface TI
CHAPTER 1
The genus Coniothyrium in plant pathology, with special reference to
species that cause disease on Eucalyptus
1.0 Introduction 1
2.0 Coniothyrium Corda 2
3.0 Importance of the genus Coniothyrium in pathology 3
4.0 Coniothyrium canker of Eucalyptus 9
5.0 Conclusions 13
6.0 References 15
CHAPTER2
Morphological, cultural and pathogenic characteristics of Coniothyrium
zuluense isolates from different plantation regions in South Africa
Abstract 37
Introduction 38
Materials & Methods 39
Results 40
Discussion 42
References 44
CHAPTER 3
Genetic variation among field isolates of the Eucalyptus canker pathogen,
Coniothyrium zuluense
Abstract 51
Introduction 52
Materials & Methods 53
Results 57
Discussion 59
References 62
CHAPTER4
Morphological and molecular relatedness of geographically diverse
isolates of Coniothyrium zuluense from South Africa and Thailand
Abstract 72
Introduction 73
Materials & Methods 74
Results 80
Discussion 83
References 85
CHAPTER 5
A synergistic relationship between the Eucalyptus canker pathogen,
Coniothyrium zuluense, and two Pantoea species
Abstract 102
Introduction 103
Materials & Methods 104
Results 107
Discussion 111
References 113
CHAPTER 6
Polygalacturonase production by the Eucalyptus canker pathogen,
Coniothyrium zuluense, and two Pantoea species
Abstract 147
Introduction 148
Materials & Methods 150
Results 153
Discussion 155
References 157
CHAPTER 7
Partial cloning of a disease resistance gene from two Eucalyptus grandis
clones
Abstract 165
Introduction 166
Materials & Methods 168
Results 170
Discussion 171
References 172
Summary 188
Opsomming 191
I
ACKNOWLEDGEMENTS
I would like to thank Jesus, my best friend, for always being with me and
showing me that in good and in bad times I can rejoice in the love He has
for me.
It is my wish to express my sincere gratitude towards the following people and
institutions. Without your assistance the completion of this study would not have
been possible. I cannot list each person by name, there are too many of you
who helped me during the past couple of years, but I am sincerely grateful to you
all.
My loving parents for their love and support. You give meaning to unconditional
love. Therefore, I offer you all of my love. To the rest of my family I owe no less.
Thank you for everything on the long list of what you mean to me.
My FABI family in Pretoria and Bloemfontein. Mike for his guidance and support,
for teaching me forest pathology, for giving me all the opportunities to learn more
and become more. Also, for his never ending enthusiasm about the wonderful
world of fungi, science and life. Teresa, for her guidance, support, patience and
above all, friendship. So too, has Brenda added much more than just insight to
this project. Within FABI there are a number of people who have all contributed
to greater and smaller degree, to my learning experience and my life. You
cannot imagine how much I love being part of this family of friends and
colleagues.
Mariaane Wolfaardt for all her guidance and friendship.
The National Research Foundation (NRF), the South African Forestry Industry
and the Department of Microbiology and Biochemistry, University of the Orange
Free State, contributing funds and provided the facilities and opportunities
needed to complete this project. A very big word of gratitude for what you all
made possible.
II
PREFACE
The production of Eucalyptus is of considerable economic importance to the
South African forestry industry. More than 50 % of the annual timber produced is
derived from a range of Eucalyptus species, hybrids and clones. Exotic plants,
established in monoculture are often susceptible to infection by various
pathogens and Eucalyptus trees are no exception. A number of diseases have,
thus, been reported to affect Eucalyptus propagation in this country and
elsewhere.
In September 1988, a new and devastating Eucalyptus stem canker disease was
observed for the first time in the Zululand forestry region of KwaZulu-Natal. The
causal agent was identified as a species of Coniothyrium. Five Coniothyrium
species are known to be associated with leaf diseases of Eucalyptus. However,
the species associated with stem cankers in South Africa was considered to be
unique. The fungal pathogen was, therefore, described as Coniothyrium
zuluense due to its origin and occurrence in the Zululand forestry area.
Since the discovery of Coniothyrium canker in South Africa, it has become
important to have an effective management strategy against this disease.
Currently, the most reliable method of reducing losses due to this disease is
through the planting of disease resistant species and clones of Eucalyptus. In
order to effectively manage Coniothyrium canker, it is essential to gain
knowledge regarding the biology, as well as the population characteristics of the
pathogen. Information pertaining to these characteristics will make it possible to
predict the relative durability of selected disease resistant clones.
Very little is currently known about Coniothyrium zuluense or the disease that it
causes. This thesis represents the first in a series of studies involving various
III
aspects of the population biology and factors influencing development of
Coniothyrium canker. Each chapter has been written as an individual entity,
although a close relationship exists between research represented in each of
these units. A degree of repetition between chapters has been unavoidable.
As an introduction, the thesis commences with a literature review on important
aspects of the genus Coniothyrium. Firstly, the taxonomic uncertainties and
problems linked to the genus are considered. Furthermore, the role of
Coniothyrium in plant pathology is discussed. The main focus of this review
considers the occurrence of Coniothyrium species associated with Eucalyptus
trees, either as saprophytes or parasites. Specific attention is given to C.
zuluense and the likely impact that the disease might have in South Africa. A list
of Coniothyrium species causing disease, as well as their ecological importance
is also presented.
Since the discovery of Coniothyrium canker in South Africa, considerable effort
has been expended on obtaining knowledge of this pathogen. During surveys,
we collected a large number of C. zuluense isolates from severely infected
Eucalyptus species and clones. In chapter two, I consider variability in
morphology, cultural and virulence characteristics of C. zuluense. The primary
goal here was to consider possible variability in the population structure of the
pathogen. A high degree of variability in pathogenicity would be indicative of a
genetically diverse population and vice versa.
In chapter three, the population diversity of C. zuluense is investigated. The
diversity of a pathogen population is indicative of the durability in resistance of
selected disease resistant clones. Data pertaining to genetic diversity also
reflects on the mode of reproduction, as well as the possible origin of the
pathogen. More diverse populations are, thus, more likely to overcome disease
resistance in selected clones and it would be more likely that the pathogen
originated locally.
IV
Coniothyrium zuluense is known only in South Africa. In 1996, however, a
Coniothyrium sp. causing similar disease symptoms on an E. camaldulensis
clone in Thailand, was observed. In chapter four, the phylogenetic relationships
between the Thailand Coniothyrium species and C. zuluense is investigated
using molecular techniques. Molecular evidence was needed to determine the
identity of the Coniothyrium species from Thailand and to show its relatedness to
C. zuluense. Morphological and pathogenicity tests on the Coniothyrium sp. from
Thailand were also conducted to support molecular data.
No information is available regarding the biology and factors influencing disease
development in C. zuluense. During disease surveys, it was noted that bacteria
commonly exude from necrotic cankers on severely infected Eucalyptus clones.
lsolations from cankers have shown that two bacteria commonly occur, together
with C. zuluense. In chapter five, I consider the identity of these two bacteria
using a diagnostic nutrient utilisation method (Biolog's Microplate technique)
together with DNA sequences. Pathogenicity tests on Granny Smith apples, as
well as on a susceptible E. grandis clone were also conducted to investigate the
importance of both bacteria in disease development.
In chapter six, levels of polygalacturonase (PG) activity in C. zuluense isolates
varying in pathogenicity to a susceptible E. grandis clone, and in two bacterial
species, are determined. PG is considered to be the first cell wall-degrading
enzyme produced during plant-pathogen interactions and has been identified as
a determining factor in disease development for both fungal and bacterial plant
pathogens. The results obtained would give an indication of the possible
relationship between C. zuluense and the two bacteria that accompany it in
nature.
The activation of resistance genes, as well as pathogenesis-related proteins, has
been linked positively to disease resistance in various fungal and bacterial
v
pathogens. In a previous study, it was shown that significant differences in
disease resistance of two E. grandis clones (ZG 14 and TAG 5) to C. zuluense
infection, was evident. In chapter seven, the presence of such genes is
investigated. Possible differences between disease resistance in the two E.
grandis clones are also consistent. The justification for undertaking this study
was to determine whether molecular markers to screen clones for disease
susceptibility or resistance might emerge. Such markers would accelerate
breeding for improved disease resistant Eucalyptus clones.
This thesis expands our knowledge of C. zuluense and factors influencing its
pathogenicity. It is my sincere hope that the research encompassed in this
document will contribute towards an increased knowledge pertaining to C.
zuluense and also towards the improvement of Eucalyptus propagation in South
Africa.

CHAPTER 1
The genus Coniothyrium in plant pathology, with special reference
to species that cause disease on Eucalyptus
1.0 INTRODUCTION
Species of Eucalyptus L' Heritier are of considerable economic importance, both in
Australia where they are native, and in many other countries, where they have been
successfully introduced for plantation development. Not only do they represent a
major timber resource, but these trees are also used for distillates, tannins, essential
oils, nectar, pollen, the production of rayon and viscose, as well as for firewood
(Poynton, 1979; Turnbull, 1991). In South Africa, more than 50 % of timber
production annually is derived from various Eucalyptus species, of which the most
important is E. grandis Hill ex Maid. (Anonymous, 1995). Eucalyptus species in
South Africa are managed on a medium-length to short rotation for the production of
sawlogs, telephone and transmission poles, mining timber, rough building and
fencing materials (Poynton, 1979). The greatest production of industrial eucalypt
wood, however, is for the pulp and paper industry and mainly in the form of bleached
kraft pulp (Turn bull, 1991).
Where exotic trees are established in plantation monocultures, they are more
threatened by pathogens than in natural forests. In South Africa, a number of
diseases have been reported on various species and clones of Eucalyptus and these
cause serious economic losses. Cryphonectria canker, caused by Cryphonectria
cubensis (Bruner) Hodges, is one of the most serious Eucalyptus canker diseases in
South Africa (Wingfield et al., 1989). Other stem and root diseases include
Botryosphaeria canker caused by Botryosphaeria dothidea (Mong.:Fr.) Ces & De Not
(Smith et al., 1994) and Pythium and Phytophthora root rot (Linde et al., 1994).
2
A serious stem canker disease, apparently unknown elsewhere in the world, was first
observed in the Zululand forestry region of KwaZulu-Natal, in September of 1988 on
a single clone of E. grandis (Wingfield et al., 1997). It has subsequently become
widespread in the area and occurs, not only on a wide range of E. grandis clones, but
also on hybrids of this and other species (Goutinho et al., 1997; Wingfield et al.,
1997). The causal fungus was identified as Coniothyrium zuluense Wingfield, Crous
& Goutinho (Wingfield et al., 1997).
Coniothyrium zuluense is of considerable concern to the South African forestry
industry, as well as to other forestry groups elsewhere in the world. Its impact on
forestry has necessitated investigations on strategies to reduce losses. The aim of
this review is to summarise relevant knowledge pertaining to C. zuluense. Particular
attention is also given to taxonomic problems linked to the genus Coniothyrium
Corda, as well as the importance of other Coniothyrium species previously described
as Eucalyptus pathogens.
2.0 CONIOTHYRIUM CORDA
Coniothyrium is one of the oldest genera in the Goelomycetes and also one of the
largest (Reisinger et al., 1977). The genus includes 800 described species that vary
considerably in pycnidium structure, conidium and conidiophore morphology (Sutton,
1980). Coniothyrium was first described in 1821 as Clisosporium Fr. (Fries, 1823),
and was subsequently changed to Coniothyrium in 1840 (Corda, 1840). However, in
1859 the name was changed to Monoplodia Westd. and in 1917 to Asteropsis Frag ..
During 1923 it was renamed as Coniothyrinula Petrak (Petrak, 1923), but was later
transferred back to, Coniothyrium Corda.
Sutton (1971b) stated that various authors preferred the conservation of the gen.us
Coniothyrium Sacc. (type species C. fuckelii Sacc.), rather than Clisosporium Fr. or
Coniothyrium Corda. This was in contrast to the published proposal in the
International Code of 1935, which stated that Coniothyrium Corda emend. Sacc.
(type species, C. diplodiella (Speg.) Sacc.) should be conserved, rather than using
earlier homonyms. Subsequently, Coniothyrium Corda, lectotype species C.
3
palmarum Corda, was conserved against Clisosporium Fr. (type species C. lignorum
Fr.), and published in the International Code, 1956.
Sutton (1971 b) stated that the selection of C. palmarum as lectotype species for the
genus Coniothyrium, was unfortunate. Coniothyrium palmarum is characterised by
annellidic conidiogenous cells, thus, restricting the generic name to a limited number
of species. The majority of species described in Coniothyrium are, however, similar
to C. fuckelii in having phialidic conidiogenous cells. Therefore, they are incorrectly
placed in the genus Coniothyrium. Sutton (1971 b, 1980), therefore, proposed that
many species currently described in Coniothyrium, should be accommodated in
Microsphaeropsis Hëhn. Microsphaeropsis, type species M. olivacea Hóhn, is
congeneric with C. fuckelii and, thus, provides a more suitable generic place for many
Coniothyrium species.
Minter et al. (1982, 1983a, b) proposed a re-definition of the stages of
conidiogenesis. They concluded that all conidia previously described as "annello-
conidia", and most conidia described as "phialo-conidia" are all holoblastic and that it
is no longer appropriate to distinguish between phialides and anneIIides in most
instances (Minter et aI., 1982, 1983a, b). It is, thus, no longer necessary to separate
the genera Coniothyrium and Microsphaeropsis. Taxonomic mycologists, however,
have not changed their approach to identify fungal isolates and it is clear that revision
is needed to determine which of the 800 described taxa should be retained in
Coniothyrium and which of these should be accommodated in Microsphaeropsis.
Ideally mycologists should incorporate molecular techniques, such as sequence
analysis together with traditional morphological and ultra-structural studies in making
such a decision. This would be extremely difficult as cultures are not available for
most taxa in question.
3.0 IMPORTANCE OF THE GENUS CONIOTHYRIUM IN PATHOLOGY
Species of Coniothyrium are known to survive either as saprophytes, hyperparasites
of various plant pathogens, human pathogens, or as plant pathogens on a wide
range of plant hosts (Tables 1 and 2). Coniothyrium is best known for species such
4
as C. fuckelii and C. minitans Campbell, which are well known pathogens,
saprophytes, and hyperparasites of plants, animals including humans and insects
(Tables 1 and 2). The main focus of this review is, however, on the importance of
Coniothyrium spp. as pathogens of Eucalyptus.
3.1 Coniothyrium fuckelii: A plant and human pathogen
Coniothyrium fuckelii is considered to be the anamorph of the ascomycete
Leptosphaeria coniothyrium (Fuckel.) Sacc. (Sutton, 1971a). This fungus is known
as a serious plant pathogen of various plants (Table 1). Its primary hosts are Rosa
Thunb. and Rubus L. species, on which it causes graft canker (Sweets et al., 1982;
Muthaiyan et al., 1992) and cane blight (Williamson & Jennings, 1992), respectively.
The fungus has also been reported as a hyperparasite of nematodes (Clovis & Nolan,
1983), as well as a human pathogen (Kiehn et al., 1987).
Kiehn et al. (1987) diagnosed C. fuckelii as the causal agent of a liver infection in a
patient suffering from "acute myelogenous leukemia". In vitro antifungal testing
suggested susceptibility to both amphotericin Band ketoconazole. After several
weeks, however, the patient refused further treatment and later died. An autopsy
was refused. A second report of human infection with C. fuckelii was reported by
Scheil (unpublished data), where a "cutaneous phaeohyphomycosis" was described
in a 14 year old girl. The "erythematous plaque" was treated with ketoconazole with
no effect. The lesion was then surgically removed.
3.2 Coniothyrium minitans: A fungal biocontrol agent
Coniothyrium minitans is a sclerotial mycoparasite of Sclerotinia sclerotiorum (Lib.)
de Bary (Adams, 1990; Whipps et al., 1991; Wipps & Gerlagh, 1992; Tu, 1997).
Infection of S. sclerotiorum by the hyperparasite results in the destruction of hypha I
cells (Huang & Kokko, 1987, 1988; Huang & Kozub, 1991; Whipps & Gerlagh, 1992;
Tu, 1997) and sclerotial tissues (Huang & Kokko, 1987; Whipps et al., 1991; Gerlagh
et al., 1996; McLaren et al., 1996). Several studies have revealed that the mode of
hyperparasitism of C. minitans on hyphal cells, involves the direct penetration of the
5
host hyphae and the degradation of host cell walls (Adams, 1990; Whipps & Gerlagh,
1992; Tu, 1997).
Sclerotinia sclerotiorum is the causal agent of white mold, also known as sclerotinia
rot and sclerotinia wilt, on a wide range of hosts and has a world-wide distribution on
numerous field crops and vegetables (Huang & Kokko, 1987; McLaren et al., 1994;
McQuilken & Whipps, 1995; Tu, 1997). Most of the biocontrol studies involving C.
minitans have been concerned with its use as an inoculant applied either to foliage
(Harrison & Stewart, 1988; Gerlagh et al., 1996, 1999) or, more frequently, to soil for
the control of sclerotia forming pathogens (Whipps, 1987; Budge & Whipps, 1991;
Whipps et a/., 1992; Whipps et al., 1993; McLaren et al., 1996). Studies have,
however, also indicated that C. minitans is important in natural biological control of S.
sclerotiorum in the field (Adams, 1990; McLaren et al., 1994; Tu, 1997). It has been
shown that when C. minitans is applied to soil as a solid-substrate inoculum, it can
infect sclerotia of S. sclerotiorum year-round and effectively reduce their number and
viability (Budge et al., 1995; Gerlagh et al., 1996, 1999).
Biocontrol measures using C. minitans against the white mold fungus (S.
sclerotiorum) has been extensively studied (see Table 2). Many of the emerging
results from this study, however, have yet to be practically applied. This is mainly
due to the fact that biocontrol agents are subjected to strict registration guidelines.
Another major problem regarding the use of C. minitans, lies in the quantity of solid-
substrate preparations that are required for effective control (Whipps & Gerlagh,
1992).
3.3 Pathogens or saprophytes of Eucalyptus
To date, 11 Coniothyrium species have been described on Eucalyptus. Six of these
are referred to as "true" Coniothyrium species, characterised by anneIIidie
conidiogenous cells (Sutton, 1980). Four species, previously described in
Coniothyrium have since been re-described, and are now accommodated in the
genus Microsphaeropsis (Sutton, 1971b, 1980). This genus is currently used for
species similar to Coniothyrium, but with phialidic conidiogenous cells (Sutton, 1971b,
1980). The fifth Coniothyrium sp. was re-described and is currently accommodated
6
in the genus Fairmaniella Petrak & Syd. (Sutton, 1980). Morphological
characteristics, as well as disease symptoms of fungi formerly described in
Coniothyrium, are presented in Tables 3A and 3B.
3.3.1 Microsphaeropsis
Most of the Microsphaeropsis spp. formerly described in Coniothyrium, occur as
saprophytes on eucalypts (Sutton, 1974, 1980). The type species, Microsphaeropsis
olivacea (Bonord: Hëhn) Sutton (Basionym, C. olivaceum Bonord. apud. Fuckel.)
occurs as a saprophyte on E. tieltolle Fr. Muell. and has been reported from Australia,
India and the USA (Sutton, 1980; Sinclair et al., 1987). Similarly, Microsphaeropsis
eucalypti (Fragoso) Sutton (Basionym, C. olivaceum Bonord var. eucalypti Fragoso),
as well as M. globulosa (Camara) Sutton (Basionyms, C. globulosum Camara; C.
olivaceum Bonord var. eucalypti Fragoso; M. eucalypti (Fragoso) Sutton; C. eucalypti
Fragoso), are apparently of no significance to the Eucalyptus industry, in that they
occur as saprophytes on old leaves of E. globulus Labill in Portugal (Sutton, 1'"971b).
The only Microsphaeropsis sp. causing disease on Eucalyptus species, is.
Microsphaeropsis callista (H Syd.) (Basionym, C. callistum H Syd.) (Sutton, 1971 b).
This fungus was reported from Australia as a pathogen on living leaves of E.
haemastoma Sm. causing separate, circular to irregular shaped lesions up to 5 mm in
diam. (Sutton, 1971b). Leaf spots sometimes coalesce (Sutton, 1971b). Disease
symptoms are similar on both sides of the leaf with raised edges separated from
healthy tissue by brown to purplish brown lines surrounded by diffuse halos of brown
to purplish brown discolouration (Sutton, 1971 b; Cabral, 1985). This pathogen is,
however, not considered to be of great economic importance in Australia (Sutton,
1971b).
3.3.2 Fairmaniella
The genus Fairmaniella is monotypte with F. Ieprosa (Fairm.) Petrak & Syd.
(Synonyms, C. leprosum Fairman; Melanconium eucalypticola Hansford) as the only
species (Sutton 1971b, 1980). Fairmaniella leprosa causes disease symptoms that
vary for different Eucalyptus species (Sutton, 1971b, 1980). This fungal pathogen
7
causes lesions on leaves and shoots of E. fasciculosa in Australia (Sutton, 1971b,
1980; Swart, 1988), as well as E. globulusfrom Chile (Sutton, 1971b, 1980; Wingfield
et al., 1995). Lesion diam. varies between 3 - and 15-(20) mm and is typically
circular to elliptical or irregular in shape. The upper surfaces of lesions are mottled
pale to medium brown and surrounded by slightly raised ridges (Sutton, 1971b,
1980). The central region of the lower surface is characterised by grayish brown
discolouration (Sutton, 1971b). Lesions on leaves of E. clttiodore Hook., collected
from Zambia, have been shown to vary between 1 - and 7 mm in diam.. Lesions
ranged from minute circular flecks to larger irregular lesions that were medium brown,
paler in the centre with distinct dark brown raised edges (Sutton, 1971b, 1980).
Similar symptoms were also reported from E. robusta Sm. in Hawaii and from two
unknown Eucalyptus species in New Zealand (Sutton, 1971b, 1980).
Fairmaniella leprosa has also been reported from South Africa, where it was found to
cause distinct, round, cork-like lesions on leaves of E. globulus in the Franschhoek
and Stellenbosch areas of the Western Cape Province (Crous et al., 1989a, b, c).
Lesions occur 4 m above the ground on mature, older leaves (Crous et al., 1989a,. b,
c). This pathogen is, however, not considered to be of great economic importance in
South Africa, due to its limited host range and distribution (Crous et al., 1989a, b, c).
3.3.3 Coniothyrium senso stricto
Only six of the Coniothyrium spp. associated with Eucalyptus trees remains in the
genus Coniothyrium. This is due to Sutton's proposal (1980) that species producing
conidia from phialides should be accommodated in either Microsphaeropsis or
Fairmaniella. Differences in morphology, as well as in disease symptoms associated
with these Coniothyrium species are presented in Tables 4A and 48.
Coniothyrium ahmadii Sutton (synonym, Coniothyrium eucalypti Ahmad.) and C.
kallangurense Sutton & Alcorn, are not considered to be of any economic importance
to the Eucalyptus forestry industry. Coniothyrium ahmadii occurs on twigs and
branches of Eucalyptus species in Pakistan (Sutton, 1974, 1980). Its importance as
a pathogen is, however, not known. Coniothyrium kallangurense is a saprophyte on
leaves of E. microcorys F. Muell. in Australia (Sutton, 1975).
8
In 1971, a leaf disease on E. leptophylla in Australia was ascribed to C. eucalypticola
Sutton (Sutton, 1971b). This disease was characterised by circular to elliptical
lesions, ranging between 2 - 10 mm in diam .. Pale brown, slightly raised edges were
evident due to the pronounced exudation of conidial masses spreading over the leaf
surfaces (Sutton, 1971b). Subsequently, Swart (1986) distinguished two additional
Coniothyrium species as pathogens on Eucalyptus, C. parvum Swart and C. ovatum
Swart. Coniothyrium parvum causes necrotic leaf spots on E. melliodora A. Cunn. ex
Schau. and E. regnans F. Muell. in Australia. Lesions vary between 1 - 1.5 mm in
diam. (Swart, 1986). Coniothyrium ovatum causes necrotic leaf spots (1 mm in
diam.) on three Eucalyptus species, E. dives Schau., E. macrorhyncha F. Muell. Ex
Benth. and E. obliqua L'Herit. (Swart, 1986). These species are, however, of no
economic importance.
During 1988, Crous et al. reported that C. ovatum is the causal agent of leaf spots on
E. c/adocalyx F. Muell. and E. lehmannii (Preiss ex Schau.) in South Africa. Leaf
spots occur mainly on the lower branches of mature trees, and on young coppice
undergrowth, causing a prominent discolouration of the upper surface of juvenile
leaves (Crous et al., 1988). The leaf spots are irregular and dispersed randomly over
the leaves. They are dark purple to almost black in the middle, changing to purplish-
brown towards the edges (Crous et al., 1988). The pathogen is, however, not
considered to be of any significance to the local forestry industry due to the
insignificance of susceptible species.
Coniothyrium zuluense Wingfield, Crous & Coutinho has recently been described as
the causal agent of a devastating Eucalyptus stem canker disease in South Africa
(Wingfield et al., 1997). Comparison of this pathogen with previously described
Coniothyrium species from Eucalyptus, suggested that the species is new to Science
(Wingfield et al., 1997). The following section of this review will summarise all
relevant information known about C. zuluense.
9
4.0 CONIOTHYRIUM CANKER OF EUCAL YPTUS
Coniothyrium canker was first observed in the Zululand forestry region of KwaZulu-
Natal Province in South Africa in September 1988, where it occurred on a single
clone of E. grandis (Wingfield et al., 1997). Since its discovery, the pathogen has
become widespread and affects various Eucalyptus species, clones and hybrids.
This disease has rapidly become one of the most serious problems affecting the
Eucalyptus forestry in South Africa.
4.1 Morphological and diagnostic characteristics
According to Wingfield et al. (1997) mycelium of C. zuluense is situated internally
within the host tissue and is medium to dark brown in colour. The mycelium is
branched, septate, thick-walled and smooth to verruculose, ranging between 1.5 - 3
IJm in diam.. Pycnidia occur as single or aggregated structures. They are typically
intra- or sub-epidermal, globose to depressed ranging between 60 - 120 IJm in width
and 60 - 80 IJm in height. Pycnidial walls are composed of two to three layers of
dark brown textura angularis. Conidiogenous cells are characteristically annellidic,
pale brown, smooth, doliiform to reniform in shape, ranging between 4 - 8 x 2.5 - 3.5
IJm in size (Fig. 1A). Conidia are medium brown, thick-walled, smooth to verruculose
and broadly ellipsoidal (Fig. 1B). Apices of conidia are obtuse and bases sub-
truncate to bluntly rounded ranging between (4 -) 4.5 - 5 (- 6) x 2 - 2.5 (- 3.5) IJm in
size.
The fungus is extremely slow growing on artificial media (Wingfield et al., 1997).
Average colony diam. after 21 days on Potato Dextrose Agar (PDA) is 40.5 mm at 30
°C. The slow growing nature of C. zuluense, has been attributed to its biotrophic
nature (Coutinho et al., 1997; Wingfield et al., 1997). Optimal growth temperature is
found to be 30°C, although C. zuluense is able to grow at temperatures, ranging
from 15 to 30°C (Coutinho et al., 1997; Wingfield et al., 1997).
When grown on PDA at 30°C, it was observed that colonies are irregular, pale
olivaceous, with an outer olivaceous grey band of mycelium that is characterised by a
10
pale, mouse grey, margin (Fig. 2A). According to Wingfield et al. (1997) colony
margins tend to be smoother at lower temperatures. They also observed that when
colonies are viewed from below, four bands of colour are evident. The outer two
bands are olivaceous with the third band greenish-black and the forth band in the
centre of the colonies is rust coloured (Fig. 28).
4.2 Symptoms and Damage
Initial infections occur on young, green stem tissue during the growing season
(Coutinho et al., 1997; Wingfield et al., 1997). This gives rise to small (2 - 5 mm
diam.), discrete, necrotic lesions on the stem (Fig. 3). These small lesions coalesce
to form large necrotic patches (Fig. 4) (Coutinho et al., 1997; Wingfield et al., 1997).
These patches give rise to spindle-shaped swellings that are often cracked and
exude copious amounts of red / brown kino (Fig. 5 and 6). This is especially evident
in highly susceptible Eucalyptus species, clones and hybrids (Coutinho et al., 1997;
Wingfield et al., 1997).
Severely susceptible Eucalyptus clones are characterised by the development of a
series of stem cankers along the entire stem (Fig. 7). Cankers coalesce causing
large zones of dead cambium that causes the underlying xylem to dry out. The dried
wood cracks and checks (Fig. 8). Epicormic shoots or branches are often observed
in highly susceptible stands (Fig. 9). This is due to the partial gird ling of stems by the
cankers (Coutinho et al., 1997; Wingfield. et al., 1997). Epicormic branches
subsequently also become diseased and die at their apices. It was also reported that
in an extremely susceptible E. grandis clone (ZG 14), top die-back occurred due to
the girdling effect of cankers, resulting in a loss of height growth (Fig. 10) (Wingfield
et al., 1997).
4.3 Distribution and host range
Infection by C. zuluense is most severe in the Zululand forestry region of KwaZulu-
Natal (Coutinho et al., 1997; Wingfield et al., 1997). This region is typified by a sub-
tropical climate. Field reports show that the fungus also occurs in the Mpumalanga
Province of South Africa (Wingfield et al., 1997). All indications are, however, that
11
the disease is substantially less severe in those areas with temperate climates. The
distribution is, therefore, probably limited to sub-tropical climates that are apparently
required for growth and spread of the pathogen (Coutinho et al., 1997; Wingfield et
al., 1997).
Coniothyrium zuluense was first reported on a single E. grandis clone (Wingfield et
al., 1997). Since its discovery, the disease has become common and damaging in all
E. grandis stands derived from seed, as well as many other E. grandis clones
(Wingfield et al., 1997). In addition, hybrid clones of E. grandis with E. urophylla S.T.
Slake and E. camaldulensis Dehnh. previously believed to be disease resistant, have
started to show signs of infection (Coutinho et al., 1997; Wingfield et al., 1997).
Although Eucalyptus species are the only known hosts of C. zuluense, the disease is
not known in Australia, where most of the Eucalyptus species are indigenous.
According to Wingfield et al. (1997), this might suggest that C. zuluense is native to
South Africa. They proposed that the fungus might occur on native Myrtaceae in
South Africa and that it could have developed the capacity to infect Eucalyptus
species. This view was based on similar findings with Eucalyptus rust caused by
Puccinia psidii Winter (Ferreira, 1981; Coutinho et al., 1998). The latter fungus is not
known in Australia, but is common and damaging in South and Central America,
where it apparently originated from native Myrtaceae.
4.4 Dispersal and Infection
The distribution of Coniothyrium canker is probably determined by humid conditions
needed for the growth and spread of the pathogen (Coutinho et al., 1997; Wingfield
et al., 1997). The incidence of cankers in plantations varies greatly, depending upon
climatic conditions and Eucalyptus species, clones and hybrids planted (Coutinho et
al., 1997; Wingfield et al., 1997). Infection is strongly favoured by relatively high
rainfall and temperatures above 25°C (Wingfield et al., 1997). This lowers the
potential for serious damage to Eucalyptus species in other parts of South Africa with
low rainfall and temperatures.
12
Very little is known about the biology of C. zuluense (Coutinho et al., 1997; Wingfield
et al., 1997). It has, however, been shown that once conidia germinate, the germ
tubes infect the stems directly through the epidermis of the young tissue (Wingfield,
unpublished data). The means by which conidia are spread is, however, still
unknown. It has been proposed that conidia are dispersed during rain and by wind
which is typical of most pycnidial Coelomycetes (Wingfield et al., 1997). Conidia,
suspended in rainwater, flowing down stems might provide opportunities for
secondary infections lower down on stems (Coutinho et al., 1997, Wingfield et al.,
1997).
4.5 Host susceptibility
Variation in resistance to Coniothyrium canker exists within and among Eucalyptus
species (Coutinho et al., 1997; Wingfield et al., 1997). Various E. grandis clones
currently available for planting are highly susceptible (Coutinho et al., 1997; Wingfield
et al., 1997). However, certain E. grandis clones are moderately resistant to C.
zuluense infection (Coutinho et al., 1997; Wingfield et al., 1997). Some hybrid clones
of E. grandis with E. urophylla S. T. Slake, E. camaldulensis or E. nitens (Deane et
Maid.) Maid. are highly resistant to C. zuluense infection. These hybrid clones would,
therefore, be excellent choices for planting in high hazard areas.
There is considerable inter- and intraspecific variation in susceptibility to C. zuluense.
This may reflect differences in provenances of E. grandis that vary in their relative
susceptibility, or to the low virulence of the pathogen. However, the threat of C.
zuluense to South African forestry is dependent on the susceptibility of Eucalyptus
species, clones and hybrids planted. Eucalyptus gran dis , is extensively planted in
South Africa and is highly susceptible to this pathogen (Wingfield et al., 1997). It is,
therefore, important for the South African Forest Industry not to plant clones
susceptible to C. zuluense in areas where this pathogen is likely to be problematic.
For this reason, clones and hybrids should be screened for susceptibility to the
pathogen.
13
4.6 Management strategies
Currently, the most reliable management strategy to reduce the impact of
Coniothyrium canker, is by selecting clones and 'hybrids that show disease resistance
(Coutinho et al., 1997; Wingfield et al., 1997). However, there are indications that
clones previously believed to be resistant to infection are beginning to show signs of
infection (Wingfield et al., 1997). This is an indication that virulence in the pathogen
is changing (Coutinho et al., 1997; Wingfield et al., 1997).
Wingfield et al. (1997) were not able to find a sexual state for C. zuluense and
suggested that the fungus probably propagate asexually. If this is true, one should
expect that the fungus would have difficulties adapting to environmental changes,
such as the introduction of disease resistant clones. Knowledge regarding the
population structure of C. zuluense in South Africa is, therefore, of crucial importance
for programmes aimed at reducing the impact of this disease. The amount of genetic
diversity within the population of C. zuluense would also provide some insight into the
origin of the fungus.
5.0 CONCLUSIONS
Taxonomic problems with the genus Coniothyrium have resulted in considerable
confusion for many taxonomists. Sutton's studies (Sutton, 1971b, 1980) resulted in a
more precise concept for the genus, limiting species of Coniothyrium to only those
producing conidia from anneIIides. The position of the more than 800 species that
have been described in the genus remains uncertain and must await further study.
Only six of the previously described 11 Coniothyrium species known from Eucalyptus
species produce conidia from anneIIides. The rest of the species have been
accommodated in either Microsphaeropsis or Fairmaniella. It is currently fairly easy
to establish whether newly collected Eucalyptus fungi belonging in Coniothyrium,
differ from other species known on this host.
14
All Coniothyrium species on Eucalyptus, are either saprophytic or weak leaf-spotting
pathogens. This is in sharp contrast to the recently described Eucalyptus stem
canker pathogen, C. zuluense. This pathogen has caused extensive losses in
plantation forestry in the Zululand areas of the KwaZulu-Natal Province, South Africa.
Coniothyrium zuluense has already caused considerable damage and it has the
potential to cause serious losses in the future. Very little is known about this fungus,
and research is needed to reduce its economic impact. The only long-term control
strategy for this disease is by breeding and selection of disease resistant trees.
However, in order to capitalise on disease resistance, knowledge regarding the
population structure of C. zuluense in South Africa would be useful. Information
regarding the genetic composition of the pathogen, together with programmes aimed
at screening various Eucalyptus clones, species and hybrids for disease resistance
are needed to successfully manage the disease in future.
Very little is currently known about C. zuluense in South Africa. It is hoped that
studies contained in this thesis will contribute towards our understanding of the
pathogen as a whole. This will be relevant, not only to South Africa but also to other
countries where Eucalyptus is grown. If the pathogen is not of Australian origin, it
might also threaten native Myrtaceae in that country.
15
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\
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26
Table 1. List of Coniothyrium species occurring as pathogens on different plant
hosts.
Coniothyrium spp. Host Symptoms References
C. clematidis-rectae Clematis Jacq. spp. Withering Slavekoorde, 1974;
Petrak HowelIs, 1993
C. concentricum (Desm.) Yucca filamentosa L. Parasitic effect Crisan et ai., 1980
Sacc.
C. conorum Sacc. & Picea ajanensis (Lindl. De-foliage Man'ko & Azbukina,
Roum. et Gord.) Fiseh. ex Carr 1992
C. diplodiella Vitis Engelm. spp. White rot Tanaka et ai., 1977;
(Spegazzini) Sacc. Abelentsev, 1980
Vine-cluster drying-off Belli et ai., 1970;
Bisiach & Battino-
Viterbo, 1973; He et
ai., 1991
Italia grapevine rachis Conte et al., 1984
dieback
Grape stalk necrosis Shin et ai., 1984
Artabotrys White rot Locci & Quaroni,
hexapetalous L. 1972; Shreemali, 1973
C. ficicola Ornamental plants Leaf blight Dayakar-Yadav &
Rao, 1978
C. fragariae (Oudem.) Fragaria Ouch. spp. Blight Kovacs, 1969; Jarvis
Sutton & Hargreaves, 1972
C. fuckelii Sacc Malus Borkh. spp. Blister canker & bark Koganezawa &
necrosis Sakuma, 1980
Bambusa balcooa Dieback & blight Rahman & Khisha,
Roxb. 1981; Rahman et al.,
1983
Rubus L. spp. Cankers Ellis et al., 1984
Corchorus capsularis L. Leaf spot Ali & Saikia, 1991
Juniperus communis L. Leaf and shoot death Humphreys-Jones,
1977a
Prunus persica (L.) Dieback Zaharia & Rafaila,
Batsch 1977
Persimmon L. spp. Leaf spot Agarwala & Kondal,
1971
Populus L. spp. Sooty moulds Bao et ai., 1992
Prunus cornuta L. Leaf spot Ram, 1979
Quercus myrsinaefolia Greyish leaf blight Kaneko, 1982
Thunb.
Rosa rugosa Thunb. Blight Muthaiyan et ai., 1992
Rosa spp. Stem cankers Sweets et al., 1982
Blight Dishon et ai., 1978
Thuya orientalis L. Leaf & shoot death Humphreys-Jones,
1980
Rubus idaeus L. Cane blight Williamson &
Hargreaves, 1981;
Jennings, 1982;
Baudry et ai., 1993;
Williamson et ai., 1983
27
Coniothyrium spp. Host Symptoms References
C. fuckelii Rubus idaeus Cane blight Williamson et aI.,
1984; Williamson &
Jennings, 1992
C. hellebori Cooke & Helleborus L. spp. Leaf spot Fox, 1994
Massee
C. phyllachorae Maubl. Zea mays L. Tarspot Hock et aI., 1992a, b
C. olivaceum Bonorden Citrus L. spp. Soil sickness Reddy et aI., 1983
C. pini Pinus pine a L..; P. Leaf blight & dieback Bellar & Bayaa, 1993
halepensis Miller; P.
brutia Ten.
C. piricola Poteb., cf Plumeria L. spp. Leaf blight Rafaila & Dinulescu,
1977
C. pyrinum (Sacc.) J. Malus spp. Leaf spot Kondal & Agarwala,
Sheld 1974
C. prunicolum Plumeria spp. Spots on leaves, Glits, 1984
shoots & fruits
C. quereinum Quercus petraea Inhibit respiration (high Newsham et aI., 1992
(Bonorden) Sacc. (Mattuschka) Liebl. S02)
C. viburni Viburnum burkwoodii Leaf blotch Humphreys-Jones,
Thunb. 1977b
C. wernsdorffiae Laubert Rosa spp. Fire blight Protsenko &
Chelyshkina, 1973;
Semina et aI., 1982;
Semina et al., 1991
Coniothyrium sp. a Atriplex versiearia Hew. Dieback Cother & Gilbert, 1994
ex Benth.
Cupressus Miller spp. Lesions on stems Luisi & Triggiani, 1977
Cupressus spp. Lesions on stems Motta & Saponaro,
1982
Picea abies L. Powdery mildew Venn, 1983
Populus trichocarpa Branch cankers Shirnina, 1983
Protea repens (L.) L. Tip blight Serfontein & Knox-
Davies, 1990
Rosa spp. Stem cankers Vargas et aI., 1990
Simmondsia chinensis Leaf blight Young & Alcorn, 1981
(Link.) C. Schneider
a = Not specified
28
Table 2. Coniothyrium species used as biological control agents.
Coniothyrium spp. Host Hyperparasitism Reference
c. fuckelii Sacc. Globodera rostochiensis Mycoparasite Clovis & Nolan, 1983
(nematode)
C. minitans Campbell Sclerotinia sclerotiorum Mycoparasite Voras, 1969; Jones &
(Lib.) de Bary Johnson, 1970;
Ghaffar, 1972; Huang &
Hoes, 1976; Huang,
1977, 1979; Trutmann
et al., 1980; Huang,
1981; Trutmann et al.,
1982; Chaban &
Yakubova, 1983;
Fedulova, 1983;
Grisenko et al., 1983;
Huang, 1983; Phillips &
Price, 1983; Tribe,
1984; Tu, 1984;
Monaco, 1989; Phillips,
1989; Adams, 1990;
Whipps & Budge, 1990;
Budge & Whipps, 1991;
Huang & Kozub, 1991;
Whipps et al., 1991;
Lueth et al., 1992;
Sesan & Csep, 1992;
Whipps et al., 1992;
Whipps & Gerlagh,
1992; Verdam et al.,
1993; Sandys-Winsch
et al., 1993; Whipps,
1993; Whipps et al.,
1993; Whipps & Budge,
1993; McLaren et al.,
1994
Botrytis cinerea Pers.:Fr Mycoparasite Sesan & Tica, 1990
Sclerotium cepivorum Mycoparasite Ahmed & Tribe, 1977;
Berk Oliveira et al., 1984
C. olivaceum Sclerotinia sclerotiorum Mycoparasite Ivancia, 1992
Bonorden
C. sporulosum (W. Alternaria alternata Mycoparasite Turhan, 1993
Gams & Domsch) (Fr.:Fr.) Keissler
" Rhizoctonia solani KOhn Mycoparasite Turhan, 1990
Table 3A. Morphological characteristics of species of Coniothyrium, Microsphaeropsis and Fairmaniella on Euceiyptus?
c. palmarum C. fuckelii M. callista M. eucalypti M. globulosa M.olivacea F.leprosa
Conidiomata
Pycnid ia/Acervuli Pycnidia pycnidia pycnidia pycnidia pycnidia Pycnidia acervuli
Size (urn diam.) 150 115-370 150-200 100 100 200-300 100-200
Shape Globose sub-globose sphaerical - sphaerical - sphaerical - Globose
I globose .globose globoseOccurrence sub-stomatal sub-stomatal sub-epidermal sub-epidermal sub-epidermal Sub-epidermal sub-stomatal
Ostiole circular / circular / circular / not papillate / not papillate / not Circular
central central protruding protruding protruding
Pycnidial walls I Thick thick thick thick thick Thin thick
Conidiogenous cells
Annellidic / Annellidic phialidic phialidic phialidic phialidic Phialidic phialidic
Phialidic
Shape doliiform - ampulliform doliiform - doliiform - doliiform - Doliiform - ampulliform
cylindrical ampulliform ampulliform ampulliform ampulliform
Sizes (urn) 15-12 x 3-5 4-4.5 x 5-6 4-7 x 2.5-4 4-9 x 3-5 4-9 x 3-5 4-6 x 3-4 3-5 x 2.5-3.5
Colour hyaline to pale hyaline hyaline hyaline hyaline Hyaline hyaline
brown
Conidia
Colour Brown medium brown brown dark brown dark brown Pale brown pale brown
Thickness thick-walled thick-walled thick-walled thick-walled thick-walled Thin-walled thick-walled
Septate 0-1 euseptate Aseptate aseptate aseptate aseptate Aseptate aseptate
Size (urn diam.) 6-8.5 x 4.5 2-2.6 x 3.6-4.5 8.8 x 4.5-5.5 6-7 x 5-6 6-7 x 5-6 4.8 x 2.5-5 3-5 x 2.5-3.5
Shape Cylindrical; elliptical elliptical; globose; sub- globose; sub- Oval - ellipsoidal elliptical /obovate
spherical; obtuse apex; globose- globose-
eliptical; truncate base pyriform; pyriform;
clavate truncate base truncate base
"References Sutton, 1980 Sutton, 1971b Sutton, 1971b Sutton,1971b Sutton,1971b Sutton,1980 Sutton,1971b
tv
\0
Table 3B. Symptoms and hosts of species of Microsphaeropsis and Fairmaniella associated with Eucalyptus spp. a
M. cal/ista M. eucalypti M. globulosa M.olivacea F./eprosa
Hosts I E. haemastoma E. globulus E. globulus E. ficifolia E. fasciculosa
E. globulus
E. citriodora
E. robusta
Geographic Australia Portugal Portugal Australia, India & USA Australia, Chile,
distribution Hawaii, Zambia, New
Zealand, South Africa
Symptoms lesions on leaves saprophyte on leaves saprophyte on leaves Saprophyte on leaves lesions on leaves and
shoots
Size (mm diam.) 1-5 - - - 1-20
aReferences Sutton, 1971b; Sutton, 1971b; Sutton, 1971b Sutton, 1980 Sutton, 1971b;
Cabral, 1985 Sutton, 1974 Crous et al., 1989a,b
(jj
o
!

Table 4B. Comparison of symptoms and hosts of species of Coniothyrium ex Eucalyptus. a
c. ahmadii C. eucalypticola C. kallangurense C.ovatum C. parvum C. zuluense
Hosts I Eucalyptus spp. E. leptophylla E. microcorys E. dives E. melliodora E. grandis
E. macrorhyncha E. regnans and various E.
E. obliqua grandis hybrid
E. cladocalyx clones
E.lehmannii
Geographic Pakistan Australia Australia Australia Australia South Africa
distribution South Africa
Symptoms branches leaf spots saprophyte necrotic leaf necrotic leaf necrotic stem
spots spots cankers
Size (mm) I - 2-10 - 1 1-1.5 varies
aReferences Sutton, 1974 Sutton, 1971b Sutton, 1975 Swart, 1986 Swart, 1986 Wingfield et al.,
Crous et al., 1997
1988 Coutinho et al.,
1997
W
N
33
Fig. 1. Conidiogenous cells and conidia of' Coniothyrium zuluense. (A)
Characteristic annellidic conidiogenous cell (bar = 4 urn). (8) Single-celled ovoid
conidia (bar = 10 urn).
(from: Wingfield et al., 1997)
34
Fig. 2. Colour characteristics of single-conidial Coniothyrium zuluense cultures,
grown on Potato Dextrose Agar (PDA). (A) Colonies viewed from above are
irregular, pale olivaceous with an outer olivaceous grey band of mycelium. (8)
Colonies viewed from below possessed a rust colour centre with an olivaceous fringe.
I

36
Figures 7 - 10. Symptoms associated with Coniothyrium canker on Eucalyptus
grandis in South Africa.
Fig. 7. Severely infected trees ultimately develop a series of stem cankers along the
stems.
Fig. 8. In susceptible trees cankers coalesce and form large cracks along the stems.
Fig. 9. Epicormic shoots produced from cankered stem, indicate partial girdling due
to cankers.
Fig. 10. Top die-back due to the girdling effect of cankers.

35
Figures 3 - 6. Symptoms associated with Coniothyrium canker on Eucalyptus
grandis in South Africa.
Fig. 3. Initial infections are visible as discrete necrotic lesions on young green stem
tissue.
Fig. 4. Small necrotic lesions coalesce to form large necrotic patches.
Fig. 5. Spindle-shaped malformation on severely infected trees.
Fig. 6. Necrotic cankers are often cracked and exude copius amounts of red/brown
kino.

37
CHAPTER2
Morphological, cultural and pathogenic characteristics of
Coniothyrium zuluense isolates from different plantation regions in
South Africa
Coniothyrium canker, caused by Coniothyrium zuluense, is a serious stem canker
disease of Eucalyptus species in sub-tropical regions of South Africa. This disease
is typified by necrotic bark lesions that coalesce to form large kino-impregnated
cankers along the stems. The strategy currently used to manage Coniothyrium
canker in plantations is to deploy Eucalyptus species or clones that are resistant to
the disease. Considerable success has already been achieved in this regard, but
the long-term durability of resistance is of concern. Thus, forest managers are
interested in the genetic diversity of the pathogen and its potential to overcome
disease resistance in planting stock. In this study, 344 isolates of C. zuluense from
different plantation regions in South Africa were compared on the basis of colony
colour, conidial morphology, growth characteristics on agar and virulence to a
susceptible E. grandis clone. Conidia of all C. zuluense isolates measured were
similar in size and shape. The fungus is slow growing in culture, which is indicative
of its apparent biotrophic habit, with optimum growth observed at 30°C. Isolates of
C. zuluense displayed considerable variation in colony colour and pathogenicity in
inoculated trials. Variation in morphology and pathogenicity amongst isolates
suggests that C. zuluense has been present in South Africa for an extended period
of time, or that it is changing rapidly due to strong directional selection pressures.
38
The forestry industry in South Africa relies almost exclusively on the planting of
exotic species of Pinus and Eucalyptus. These genera are planted in approximately
equal proportions and about 1.5 million hectares of land is currently afforested
(Anonymous, 1995). Planting of Eucalyptus clones is a common practice and
results in large, genetically uniform stands. These plantations are at risk from
damage due to pests and diseases (Wingfield, 1990; Wingfield & Kemp, 1994). The
current means of reducing losses due to disease is by planting of disease resistant
species and clones of Eucalyptus (Wingfield et al., 1991). Strategies to ensure that
large numbers of disease resistant clones are planted and that a high degree of
genetic diversity is maintained in clonal plantations, have, therefore, been
implemented (Wingfield, 1990).
Coniothyrium zuluense Wingfield, Crous & Coutinho is a serious stem canker
pathogen of Eucalyptus trees in South Africa (Coutinho et al., 1997; Van Zyl et al.,
1997; Wingfield et al., 1997). Disease symptoms were first noted on a single clone
of Eucalyptus grandis Hill ex Maid. at Honey Farm plantation in the Zululand region
of the KwaZulu-Natal province. Since its discovery in 1988, various Eucalyptus
species, clones, and hybrids have displayed symptoms of infection. The earliest
symptoms of infection by C. zuluense on trees are small, discrete, necrotic lesions
on the young, green bark. These lesions coalesce to form large necrotic cankers on
the stems that exude copius amounts of kino. Epicormic shoots are commonly
produced from stems of cankered trees, indicative of partial girdling. In severely
infected clones, the tops of trees die, due to the girdling effect of the cankers
resulting in loss of growth in height (Coutinho et al., 1997; Van Zyl et al., 1997;
Wingfield et al., 1997).
Coniothyrium zuluense, and the canker disease associated with it, were first
observed nine years ago (Wingfield et al., 1997). Currently, no information is
available concerning the population characteristics of the pathogen. Knowledge
regarding fungal population structures is important to programmes aimed at
reducing the impact of disease, as this must affect the likely durability of disease
resistant clones. The aim of this study is, therefore, to consider variability in
39
morphological, cultural and virulence characteristics, amongst a large collection of
C. zuluense isolates.
MATERIALS AND METHODS
Isolates and cultures
A survey of C. zuluense in nine Eucalyptus growing regions of KwaZulu-Natal was
conducted during 1995 and 1996. Pieces of bark, showing characteristic disease
symptoms, were collected from each tree sampled. These bark samples were
incubated in Petri dishes containing moist filter paper at 30°C to induce production
of pycnidia. Conidial masses from 172 pycnidia were then washed onto the surface
of agar in Petri dishes containing 2 % w/v water agar (20 g agar (Biolab); 1 I distilled
H20) and spread across the medium surface. Plates were incubated for 19 - 24 hr
at 30°C. Single germinating conidia were then lifted from each plate with the aid of
a dissecting microscope and sterile syringe needle. The germinating conidia were
transferred to sterile 9 cm diam. Petri dishes containing 15 ml of 4 % w/v Potato
Dextrose Agar (PDA) (24 g Potato Dextrose extract (Merck); 1 g Yeast extract
(Merck); 1 g Glucose (Merck); 40 g agar (Merck); 1'1 distilled H20) and incubated at
30°C. Isolates produced in this manner were stored on PDA slants in screw-
capped tubes at 4°C.
Colony and conidia morphology
A total of 344 single conidial isolates of C. zuluense were transferred to 4 % w/v
PDA plates in order to observe colony colour and growth characteristics in culture.
Colony colour was rated using mycological colour charts of Rayner (1970). Spore
morphology was determined by measuring the length and width of thirty conidia per
pycnidium. Conidia from ten randomly selected pycnidia (each from different trees)
were measured for each of the nine plantation regions sampled.
40
Growth studies
Growth rates and temperature requirements were determined for each of the 344 C.
zuluense isolates collected. Isolates were transferred to PDA (five mm diam.
mycelial plugs) with three plates for each temperature and isolate to be tested.
Plates were incubated in the dark at temperatures ranging from 10 to 35 aC, at five-
degree intervals for 30 days. Growth of isolates was determined by measuring
colony diameter.
Pathogenicity tests
Pathogenicity tests were conducted on six-month-old trees of an E. grandis clone
(ZG 14) that is known to be highly susceptible to Coniothyrium canker under natural
conditions in KwaZulu-Natal. Twenty trees were inoculated with each of the 344
different single conidial isolates. Inoculations were done by removing a 10 mm
diameter disc of bark from the trees at breast height, and replacing this with a PDA
disc of agar bearing the fungus, or an uninoculated disc in the case of the 20
controls. Inoculation wounds were covered with masking tape to prevent
desiccation of the inoculum. Lesion length was measured six weeks after
inoculation. This experiment was repeated using all pathogenic isolates and 20
randomly selected non-pathogenic isolates.
RESULTS
Colony and conidial morphology
Isolates of C. zuluense varied considerably in colony colour (Figs 1, 2). Surface
colony colour of all 344 isolates screened in this study, varied from olive grey
(V23"lb), isabella (19"i), greenish glaucous (331I1f) to a grayish olive (21"") colour.
41
Colonies viewed from below were either black or rust coloured with white margins.
There was no predominant colony colour for isolates from any specific region.
Conidia of all isolates measured were similar in size and shape. Conidia, from
lesions on trees from all nine regions sampled, were pale brown in colour, thick
walled, smooth to verruculose and broadly ellipsoidal. The apices of these conidia
were obtuse and the bases sub-truncate to bluntly rounded (Table 1).
Growth studies
Coniothyrium zuluense is slow growing in culture, which is indicative of its apparent
biotrophic habit (Wingfield et al., 1997). The fungus failed to grow at 10°C (Fig.3).
At 15°C the mean colony diameter for all isolates was 12.6 mm. Best growth was
observed at 30 °C (61.4 mm diam.) followed by 25°C (39.3 mm diam.), 35°C (30.9
mm diam.) and 20°C (25.1 mm diam.), respectively.
Pathogenicity tests
Isolates of C. zuluense differed markedly in their relative pathogenicity (Fig. 4). Of
the 344 isolates tested, 269 (78 %) appeared to be non-pathogenic. Lesion lengths
for these isolates varied between 10 mm and 20 mm (Fig. 4). The remaining 75 (22
%) isolates produced lesions varying between 21 mm and 61 mm long. Lesion
lengths obtained from pathogenicity trials were divided into pathogenicity ranges
(Fig.4).
Control inoculations developed no symptoms and inoculation points were covered
with callus tissue (Fig. 5A). The most pathogenic isolates of C. zuluense gave rise
to a distinct swelling of the stem tissue around the inoculation site after six weeks
(Fig. 5B). Tissue surrounding the inoculation points was necrotic (Fig. 5B). These
symptoms were similar to those associated with natural infections.
42
DISCUSSION
Morphological and cultural characteristics of C. zuluense presented in this study
were similar to those published by Wingfield et al. (1997). Data regarding spore
morphology and growth characteristics were also consistent with those previously
published (Coutinho et al., 1997; Wingfield et al., 1997). Results of colony colour
and pathogenicity studies, however, showed evidence of considerable variation in
isolates of C. zuluense.
There was a wide diversity in colony colour of C. zuluense isolates. The majority of
isolates (66 %) were olive grey which is consistent with results of Wingfield et al.
(1997). The remaining (34 %) isolates had colony colours varying between isabella,
greenish glaucous and grayish olive. It is of interest that there were no consistent
patterns of colony colour in terms of origin of isolates.
A considerable degree of variation was observed in pathogenicity of the isolates
tested in this study. It was particularly interesting that 78 % of all C. zuluense
isolates were not able to cause any disease on the susceptible E. grandis clone. A
relatively small number (22 %) of isolates were able to cause necrotic canker
lesions. All isolates used had been collected and variability in pathogenicity is
unlikely be limited to age of isolates.
No sexual state has been found for C. zuluense, despite the fact that considerable
effort has been made to find such structures (Coutinho et al., 1997; Wingfield et al.,
1997). The assumption is, therefore, made that the fungus predominantly exists in
an asexual form and would thus have a limited capacity to change. Sexual
reproduction combines genes in a population continually into new combinations that
could subsequently lead to a rapid increase in virulence, whereas asexually
reproducing fungi possess a limited number of different gene combinations
(Anagnostakis & Kranz, 1987; McDonald & McDermott, 1993). The presence of
spermatogonia in some cultures, however, suggest that a sexual state may occur,
but has yet to be discovered (Coutinho et al., 1997; Wingfield et al., 1997). If it is
43
present, it seems unlikely to occur on Eucalyptus which have been very carefully
examined, but it could be present on a native South African plant species. The
sudden appearance of the disease in South Africa, as well as diversity of colony
types and pathogenicity, favours the hypothesis that C. zuluense originated from
native plants in this country.
Eucalyptus clones that are highly susceptible to C. zuluense fail to grow effectively,
which leads to significant losses to the South African forestry industry. A large
number of clones that are currently available for planting, show susceptibility to
infection (Coutinho et al., 1997; Van Zyl et al., 1997; Wingfield et al., 1997). There
is also evidence to suggest that clones previously known to be resistant, are
beginning to show signs of infection (Coutinho et a/.,1997; Wingfield et al., 1997).
This indicates that the virulence of the pathogen is changing. In plantation
programmes, regular deployment of new resistant clones and hybrids, impose
strong directional selection on pathogen populations. This is especially true for
asexual reproducing fungi because they must constantly adapt to changes in their
environment in order to survive (McDonald & McDermoU, 1993). Such pressure
might have lead to the pathogenic variation in C. zuluense.
Eucalyptus species are being propagated extensively outside Australia (where most
of these species are native) with about 8 million hectares currently grown in
plantations (Wingfield et al., 1997). Coniothyrium zuluense is a potential threat to
these plantations, particularly in areas with a tropical or sub-tropical climate and
dedicated efforts are, therefore, needed to avoid the spread of this fungus to other
countries. In this regard, it is important to note that control strategies can only be
successful if populations, instead of individuals are targeted (McDonald &
McDermoU, 1993). Future research will, therefore, focus on understanding the
population structure of C. zuluense. Such information will be valuable in
understanding the evolution of the population in response to the deployment of new
disease resistant clones.
44
REFERENCES
Anagnostakis, S.L. & Kranz, J. (1987). Population dynamics of Cryphonectria
parasitica in a mixed hardwood forest in Connecticut. Phytopathology 77, 751-
754.
Anonymous (1995). Extract of South African Forestry Facts for the year 1993 /
1994. Forestry Owners Association, South Africa.
Coutinho, T.A., Wingfield, M.J., Crous, P.W. & van Zyl, L.M. (1997) Coniothyrium
canker: A serious new disease in South Africa. In Proceedings of the IUFRO
Conference on Silvicultural and Improvement of Eucalyptus, pp 78-83,
Salvador, 24-29 August, Brazil.
McDonald, B.A. & McDermott, J.M. (1993). Population genetics of plant pathogenic
fungi. BioScience 43,311-319.
Rayner, R.W. (1970). A mycological colour charl. Commonwealth Agricultural
Bereaux: Kew, Surry.
Van Zyl, L.M., Wingfield, M.J. & Coutinho, T.A. (1997). Diversity among isolates of
Coniothyrium zuluense, a newly recorded Eucalyptus stem pathogen in South
Africa. In Proceedings of the IUFRO Conference on Silviculture and
Improvement of Eucalypts. Vo1.3. pp. 135-141. Salvador, Bahia, Brazil, 22-
27 August.
Wingfield, M.J. (1990). Current status and future prospects of forest pathology in
South Africa. South African Journal of Science 86,60-62.
Wingfield, M.J., Crous, P.W. & Coutinho, T.A. (1997). A serious canker disease of
Eucalyptus in South Africa caused by a new species of Coniothyrium.
Mycopathologia 136, 139-145.
Wingfield, M.J. & Kemp, G.H.J. (1994). Diseases of Pines, Eucalypts and Wattle.
In Forestry Handbook (ed. The South African Institute of Forestry), pp. 231-
249. South Africa.
Wingfield, M.J., Swart, W.J. & Kemp, G.H.J. (1991). Pathology considerations in
clonal propagation of Eucalyptus with special reference to the South African
situation. In Proceedings of the IUFRO International Symposium for Intensive
Forestry: The Role of Eucalypts, pp. 811-820. Durban, South Africa.
45
Table 1. Differences in conidial size of isolates of Coniothyrium zuluense, collected
from nine different plantation regions in KwaZulu-Natal.
Plantation a Number of isolates Width and length of conidia (urn) b
Aboyni 11 2 - 3.1 (2.6) x 3.5 - 5.1 (4.5)
Fairbreeze 16 2.1 - 3.5 (2.5) x 3.5 - 5 (4.1)
Futululu 70 2.5 - 3.6 (2.8) x 3.5 - 4.5 (4.1)
Honey Farm 50 2.5 - 3 (2.6) x 3 - 5.2 (4.3)
Palm Ridge 55 2 - 3 (2.8) x 4.3 - 5.6 (4.8)
Shire 10 2.6 - 2.8 (2.7) x 3.5 -6 (4.5)
Teranera 28 2.7 - 3.3 (2.8) x 4 - 4.3 (4.1)
Teza 81 2.5 - 3.6 (2.8) x 4.5 - 4.7 (4.5)
Trust 23 2.5 - 2.8 (2.6) x 4.2 - 4.5 (4.3)
"Plantations in Zululand, KwaZulu-Natal Province, South Africa
bEach size measurement represents the range of conidial lengths (averages in
parenthesis) and widths, computed from an average of 30 conidia derived from 10
randomly collected pycnidia.
46
Fig. 1. Differences in surface colony colour of 344 Coniothyrium zuluense isolates
grown on PDA in Petri dishes for 30 days at 30°C. Bars represent the number of C.
zuluense isolates in each colour class, and colours are those of Rayner (1970).
250 226
twi)
5 200 -
s0a 150 -
LI.
0 100 -ewt: 65
i 50 - .Ë"'" 29 r---I··. 24
;z:) 0 I
COLONY COLOUR
C10LlVE GREY DGREENISH GLAUCOUS
DISABELLA DGRAYlSH OLIVE
47
Fig. 2. Differences in surface colony colour of Coniothyrium zuluense isolates
grown on PDA in Petri dishes for 30 days at 30°C. Surface colony colour varied
from (A) olive grey (V23"lb), (C) isabella (19"i), (E) greenish glaucous (331I1f) to a
(G) grayish olive (21"") colour. Colonies viewed from below (B, 0, F, H) were either
black or rust coloured with white margins. Colours are those of Rayner (1970).

48
Fig. 3. Ranges of average colony diameters of 344 single conidial isolates of
Coniothyrium zuluense after incubation for 30 days at different temperatures
between 10 to 35°C. Bars represent the minimum and maximum colony diameters
for each temperature. Average colony diameters are represented by horizontal
bars.
100 ,----------------,
AVERAGE 86.4
80 ------------------------------------------------------------------------
I- 60 61.4--- -- -- --- --- --- -- --------- ----- -- ---- --- --- --- ---- - ----5.6.4- -- -- -- -- --ia:: 46.540
Cl
~ 20
o9 15.1o 0~ 12 11.4~5~.9 ~
10 15 20 25 30 35
TEPJPERATURE RANGES (OC)
49
Fig. 4. Ranges of lesion lengths associated with inoculation of 344 Coniothyrium
zuluense isolates. Bars represent the number of C. zuluense isolates within each of
five different lesion ranges produced after inoculation of a susceptible Eucalyptus
grandis clone (ZG 14). Each of the 344 isolates was inoculated onto the stems of
20 trees and means computed. Different letters differ significantly (P = 0.05)
according to Tukey's procedure for comparison of means (CV = 24.6 %).
t 300wi)
S 250
0
t-i) 200LL
0 150
w0:: 100
a: l
z::
e:» 50 1 b 1 c 2d
0
LESION LENGTH
50
Fig 5. Lesions associated with the inoculation of Eucalyptus grandis clone, ZG 14,
with isolates of Conithyrium zuluense. (A) Inoculation with an avirulent isolate of C.
zuluense, showing no lesion development. (8) Inoculation with a virulent isolate,
showing extensive lesion development.

51
CHAPTER 3
Genetic variation among 108 isolates of Eucalyptus stem canker
pathogen, Coniotbyrlum zuluense
A serious stem canker disease, caused by Coniothyrium zuluense, has become one
of the most important diseases on Eucalyptus species in South Africa. Knowledge
pertaining to the population structure of C. zuluense is crucial for selecting disease
resistant clones for future planting. The durability of disease resistance in these
clones will depend strongly on the genetic variability of the pathogen. The aim of this
study is thus to determine the genetic diversity of the C. zuluense population in South
Africa. In order to assess genetic variability, 108 C. zuluense isolates were selected
based on their pathogenicity to a susceptible Eucalyptus clone. Isolates originated
from nine different plantation regions in Zululand, KwaZulu-Natal. Amplified fragment
length polymorphism (AFLP) markers were used to determine the genetic diversity
and population structure of C. zuluense. Amplified fragments were scored as
discrete characters and analysed by cluster analysis. The level of genetic diversity
was relatively low, but higher than expected for an asexually reproducing pathogen.
Equally low variation was evident between the different plantation regions. Genetic
similarity values suggested a significant population differentiation between different
plantation regions (sub-populations). Interpretation of results, thus, indicates that
gene flow, together with selection, may be responsible for most of the gene diversity.
It is expected that new epidemics would not be as a result of the emergence of new
aggressive strains, but would rather be due to the introduction of susceptible
Eucalyptus spp., together with environmental conditions favouring disease
development.
The world-wide planting of Eucalyptus spp. has continued to increase with large new
industrial plantations and new pulp mills emerging regularly. In South Africa, for
\'J \
52
example, commercial Eucalyptus accounts for more than 50% of all newly afforested
areas in South Africa (Anonymous, 1995). A number of serious diseases have been
reported to occur on various species and clones of Eucalyptus for the first time in
South Africa during the course of the past decade (Linde et al., 1994; Smith et al.,
1994; Wingfield et al., 1989). Amongst these diseases, was the first report of a
serious stem canker disease caused by Coniothyrium zuluense Wingfield, Crous &
Coutinho in 1988 in KwaZulu-Natal (Wingfield et al., 1997).
Coniothyrium zuluense has been described only from South Africa and is suspected
to be endemic to the country (Wingfield et al., 1997). A number of Coniothyrium
species have previously been described as being pathogens of Eucalyptus (Sutton,
1975, 1980; Swart, 1986). These fungi are, however, only associated with leaf spots
on eucalypts. It, therefore, appears that the South African Coniothyrium sp.
associated with stem cankers, is unique. However, it is possible that the disease
occurs in areas of origin of Eucalyptus, but in an ecologically balanced situation, thus
making it inconspicuous.
Initial disease symptoms of Coniothyrium canker are visible as small necrotic lesions
on young stem tissue. On clones that are highly susceptible, these lesions merge to
give rise to large patches of dead, black bark that is often cracked and exudes copius
amounts of kino (Coutinho et al., 1997; Wingfield et al., 1997). In cases of severe
infection, epicormic shoots are produced on the stems around the spindle-shaped
swelling of the stems and the tops of trees begin to die (Coutinho et al., 1997;
Wingfield et al., 1997).
Currently, the most reliable management strategy to reduce the impact of
Coniothyrium canker is by selecting clones and hybrids that show disease resistance
(Wingfield et al., 1997). Field trials have, however, shown significant variation in the
susceptibility of different Eucalyptus clones, species and hybrids (Van Zyl et al.,
1997; Wingfield et al., 1997). In forestry ecosystems, environmental changes, such
as the deployment of new disease resistant clones and hybrids, impose strong
directional selection on pathogen populations that have to adapt to such changes in
the environment in order to survive (McDonald & McDermott, 1993). Control
53
strategies must, therefore, target a population instead of an individual if they are to be
effective (McDonald & McDermott, 1993).
There is little information available regarding the genetic make-up of C. zuluense in
South Africa. No sexual structures of C. zuluense have been found in South Africa,
indicating that the fungus reproduces only asexually (Wingfield et al., 1997). This
would lead to clonal lineages within a population (McDonald, 1997). Considerable
variation has, however, been observed in virulence levels of C. zuluense isolates
(Van Zyl et al., 1997). The genes that are involved in host-specificity represent a
small fraction of genes in the pathogen that may be subjected to host selection
(Leung et al., 1993). Analysis of C. zuluense field isolates with molecular markers
would, therefore, give a more precise measure of their genetic relatedness and origin.
The genetic diversity within a population will also give an indication of the level of
sexual or asexual reproduction in the population (McDonald, 1997).
Knowledge of the genetic structure of C. zuluense will yield valuable information
about the life-cycle of the fungus, as well as the durability of the resistance of
different Eucalyptus clones. The aim of this study is, thus, to determine the
population diversity of C. zuluense in South Africa. For this purpose, Amplified
Fragment Length Polymorphism (AFLP) technology was used to test the relation
between various isolates.
MATERIALS AND METHODS
Fungal isolates
Isolates of C. zuluense were obtained from cankers on the stems of severely infected
Eucalyptus clones, originating from nine different plantation regions in KwaZulu-Natal
(Fig. 1). Single conidial isolates were generated as previously described (Van Zyl et
al., 1997). A total of 108 single conidial isolates of C. zuluense were selected based
on their pathogenicity to a susceptible Eucalyptus grandis clone, ZG 14 (Van Zyl et
al., 1997) (Table 1).
54
Pathogenicity characteristics for the selected isolates ranged between avirulent (-),
intermediate (I) and virulent (+). Isolates were randomly selected from 344 isolates
previously collected, and were composed of 43 avirulent, 42 intermediate and 23
virulent isolates. Inoculum preparation and pathogenicity tests were performed as
previously described (Van Zyl et al., 1997).
Molecular comparisons
DNA extractions. Nucleic acid was extracted from all 108 Eucalyptus stem canker
isolates. Total genomic, high molecular weight DNA was extracted from all isolates
grown in 250 ml of enriched Potato dextrose broth (24 g Potato Dextrose extract
(Merck); 1 g Yeast extract (Merck); 1 g Glucose (Merck); 1 I distilled H20) in 500 ml
Erlenmeyer flasks. Cultures were incubated at 30°C on rotary shakers for seven
days. Mycelium was then harvested by filtration through Whatmann no. 1 filter paper
and dried using several layers of paper towel. Care was taken to remove all agar-
plugs. Two grams of dried mycelium was ground to a fine powder in liquid nitrogen
with a mortar and pestle. Ten ml of extraction buffer (100 mM Tris-HCI, pH 8; 50 mM
EDTA; 500 mM NaCI; 1.25 % SOS; 10 mM B-mercaptoethanol; 4 mM Spermidine; 1
mM Spermine; 1 mM Phenylmethylsulfonyl fluoride (PMSF)), maintained at 65°C,
was added to each isolate. The resultant slurry was transferred to a sterile centrifuge
tube and stored at - 20 °C until all samples were ready for further processing.
Samples were incubated in a water bath at 65°C for 60 min with frequent mixing.
Potassium acetate (0.4 volumes of SM stock) was added to each sample, transferred
to 30 ml centrifuge tubes, and incubated on ice for 20 min. The supernatant was
collected by centrifugation for 15 min at 17000 x g at 4°C. The aqueous phase was
transferred to clean 30 ml glass corex tubes and nucleic acid was precipitated by
adding 0.58 volumes of ice cold isopropanol. Tubes were placed at -20°C
overnight. The precipitate was collected by centrifugation for 10 min at 5000 x g at 4
°C. Nucleic acid pellets were washed with 5 ml of 70 % ethanol and incubated
overnight at 4°C.
55
Pellets were collected by centrifugation at 5000 x g for 10 min and air dried in a fume
hood at room temperature. Nucleic acid was re-suspended in 500 ~I sterile water for
60 min at 37°C. Nucleic acid suspensions were transferred to 1.5 ml Eppendorf
tubes and centrifuged for 10 min at 10000 x g. The supernatant, containing the DNA,
was collected in sterile 1.5 ml Eppendorf tubes and stored at - 20°C. All DNA
extracts were quantified by fluorometry and adjusted to a final concentration of 30
ng/~I.
Amplified Fragment Length Polymorphism's (AFLP) analysis
Restriction, ligation, and amplification were performed as described by Vos et al.
(1995). Genomic DNA (500 ng) from each sample was incubated for 16 hours at 25 °
C in a solution containing 10 U/IJl Cfo1 and 5 U/IJl Mse1 (Boehringer Mannheim,
USA), 1 x restriction-ligation buffer (10 mM Tris-HAc, pH7.5; 10 mM MgAc; 50 mM
KAc; 5 mM OTT), 1 U/IJl T4 DNA Ligase, 50 pmol/IJI Cfo1-adaptors, 50 pmol/ul Mse1-
adaptors (Table 2), 10 mg/ml RNase and 100 mM spermidine. The final sample
..,
volume was increased to 100 IJlwith sterile water. After ligation the reaction mixture
was diluted 10 - fold with T.1E buffer (10 mM TRIS-HCI, pH8.0; 10 mM EDTA) and
stored at -20°C. These ligated fragments served as templates in the amplification
reaction.
A pre-selective PCR (+ 1 reaction) amplification reaction was performed in 20 IJl PCR
reaction mix containing 5 IJl of the diluted DNA, 0.5 IJl of each +1 primer (10 prnol/ul)
(Table 2), 100 mM Tris-HCI, pH 8.0, 15 mM MgCI2, 500 mM KCI, 25 mM MgCb, SU/IJl
Taq DNA Polymerase (Boehringer Mannheim, USA) and 250 IJM of dNTP. Initial
denaturation was performed at 94°C for 1min, followed by 30 cycles of 30 sec at 94 °
C, 60 sec at 56°C (primer annealing) and 60 sec at 72 °C (final chain elongation).
The amplification PCR products were diluted 1a-fold in 1 x T.1 E and used as
templates in the second amplification.
The second amplification (+ 3 reaction) was performed using primers derived from
the first set of primers with additional nucleotides at the 3' end (Table 2). The Cfo1
primer was HEX fluorochrome-Iabeled and the Mse1 primer was FAM fluorochrome-
56
labeled (AmpFISTR ProfiIer PCR Kit, Perkin-Elmer, Norwalk, Conn.). Fluorescent
AFLPs were amplified under the following conditions: 0.2 IJl of the HEX
fluorochrome-Iabeled Cf01 + 3 primer (50 prnol/pl) was added to a 20-1J1PCR
reaction solution containing 5 IJl of the diluted + 1 pre-amplification mix, 0.41J1of the
FAM fluorochrome-Iabeled Mse1 + 3 primer, 5 U/IJl Taq DNA Polymerase
(Boehringer Mannheim, USA), 10 x Buffer (100 mM Tris-HCI, pH 8.0; 15 mM MgCI2;
500 mM KCI, pH8.3), 25 mM MgCI2 and 250 IJM of dNTP. The following temperature
profile was used: 12 cycles of 30 sec at 94°C, 30 sec at 65 °C, 60 sec at 72°C,
where the annealing temperature was subsequently reduced by 0.7 °C after each
cycle. The amplification was continued for 22 cycles of 30 sec at 94°C, 30 sec at 56
°C, and 60 sec at 72°C. All amplification reactions were performed using a Hybaid
Omnigene thermocycler (Hybaid, Middlesex, UK).
Electrophoresis and visualisation of AFLP peR products.
PCR products (1.51J1)were combined with 3 hnol TAMRA fluorescent-Iabeled
GeneScan 500 internal size standard (ABI), 1.6 IJl formamide, and 0.3 IJl 25 mM
EDTA (pH 8.0) containing 50 mg/ml blue dextran. This mixture was heat denatured
for 3 min at 95 oe, and immediately cooled on ice. Samples were loaded on a 5 %
denaturing polyacrylamide gel in 1 x TBE (Tris-borate EDTA, pH 8.0) and
electrophoresed for 2.5 hours at 1680 watts using the GS 36A-2400 run module.
Data was processed by GeneScan Analysis software (version 2.02) to produce a gel
image. PCR fragments for individual samples were automatically sized by the
GeneScan software using a comparison of the mobility of the internal lane size
standard to that of the sample fragment.
AFLP data analysis
Each polymorphic AFLP fragment was treated as a unit character and scored as
present (1) or absent (0) in all isolates. The experiments were repeated, and only
reproducible bands were scored. The index of genetic similarities (FST) of Nei & Li
(1979) was used to calculate pairwise genetic distances. Genetic diversity (HT) was
calculated as H = 1 - FST (Nei, 1973). Sub-population diversity, Hs, was estimated as
57
the mean diversity of AFLP markers among regions. The diversity between regions,
DST, was determined as the difference between HT and the mean diversity among all
regions (Hs). Differentiation among sub-populations is defined as the percentage of
diversity between regions out of the fatal diversity. Unweighted Pair-Group Mean
Arithmetic (UPGMA) was used for cluster analysis of the pairwise similarity matrix
that generated a dendrogram representing the genetic similarity among fungal
isolates. UPGMA analysis was carried out using NCSS97 (Visual Components, UK).
RESULTS
AFLP analysis. Averages of 44 fragments were obtained for each C. zuluense
isolate, ranging from 40 to 491 bp. Polymorphic fragments were distributed across
the entire size range with the major proportion (72 %) between 89 - 309 bp. The
total number of fragments scored across each of the 108 C. zuluense isolates, was
84 fragments for primer combination 1(HEX fluorochrome-Iabeled Cfo1 + 3 primer)
and 75 fragments for combination 2 (FAM fluorochrome-Iabeled Mse1 + 3 primer), of
which 13 (8.2 %) were monomorphic for all isolates, while 146 (91.8 %) displayed
informative polymorphism's. No differences were encountered on the AFLP profile of
the control check, based on replicate lanes of DNA, which were run on each gel.
DNA from a duplicate set of 20 C. zuluense isolates was extracted and amplified with
primer combinations to test for repeatability. Less than 1 % of the bands were
evaluated differently.
When NSCC97 (Visual Components, 1997) analysis was performed with each primer
combination individually, UPGMA groupings were almost identical to those when both
primer combinations were analysed together (data not shown). Spearman's Rank
Correlation Coefficient (rs) between genetic distances based on primer combination 1
and 2, was 34 % (rs = 0.34). This low level of correlation suggests that each primer
combination provided somewhat different and, therefore, complementary information.
However, correlation of genetic distances for both primer combinations (Primer
combination 1 and 2) with those of either Combination 1 or primer Combination 2,
were 92 % (rs = 0.92), respectively. Thus, each primer combination individually,
58
would have given an approximation of the entire data set, but sufficient differences
existed between combinations that both were necessary.
Genetic distance analysis. The total gene diversity, HT, was 19.5 %, with an
average within region diversity, Hs, of 18.3 %. Diversity between regions, OST, was
therefore 1.2 %, which is 6.2 % of the total diversity. Thus, approximately 6.2 % of
the overall gene diversity were accounted for by differences among regions. The
remaining 93.9 % was attributed to variation within regions. FSTvalues were also
used as a measure of population differentiation between and within C. zuluense
isolates collected from different plantation regions (FsT = 0.195; P <0.01).
Average FSTvalues are presented in Table 3. Results indicate that significant genetic
variation occurs among isolates of C. zuluense within most individual plantation
regions (FST = 0.195; P < 0.01). There was, however, no significant variation in
genetic similarity (GS) among fungal isolates collected from four of the nine plantation
regions used in this study. Isolates collected from Aboyni (FST = 0.098, 90.2 % GS),
Shire (FsT = 0.034, 96.6 % GS), Teranera (FST = 0.018; 98.2 % GS) and Trust (FST
= 0.089; 91.1 % GS) plantation regions showed no significant (P < 0.01) variation,
indicative of a highly clonal population. Average genetic similarity among fungal
isolates, within each individual plantation region, varied between 79.1 % (20.9 %
dissimilarity) (FST= 0.288; P <0.01) for Futululu and 98.2 % (1.8 % dissimilarity) (FST
= 0.018, NS) for Teranera plantation (Table 3).
Significant differentiation between the nine plantation regions was clearly evident.
Differentiation (FST) among the different plantations (sub-populations) is defined as
the percentage of diversity between regions out of the total diversity. The similarity
percentages among fungal isolates ranged from 71.2 % (FST = 0.288; P < 0.01) to
83.8 % (FST= 0.162; P <0.01), with an average similarity of 78.8 % (FST= 0.212; P <
0.01) (Table 3). It was evident that isolates from the Teranera plantation region, were
more genetically distant, ranging from 71.2 % (28.8 % dissimilarity) (FST= 0.288; P <
0.01) to 77.3 % (22.7 dissimilarity) (FST = 0.227; P < 0.01) similarity. There was no
correlation between geographic distance and FSTvalues (rs = 0.34, NS).
59
The similarity matrix was used to cluster the data using the unweighted pair-group
method, with an arithmetic average (UPGMA) algorithm. The dendrogram reflected
the average genetic distance, 19.5 %, between isolates of C. zuluense collected from
nine different plantation regions within KwaZulu-Natal (Fig. 2). What was also
evident, is that no specific cluster or grouping was visible among the 108 C. zuluense
isolates, irrespective of the fact that they varied in pathogenicity to Eucalyptus.
DISCUSSION
This study showed that the AFLP technique is useful for the characterisation of
intraspecific variation among the C. zuluense population. In this study, 40 % of
markers were polymorphic in a collection of 108 C. zuluense isolates from nine
plantation regions. The large number of polymorphism's detected with AFLP analysis
has the advantage that markers that appear unreliable can be discarded. This
approach also has a clear advantage in terms of the proportion of the genome being
analysed per reaction. Population diversity estimates are, therefore, more accurate
than estimates based on few loci in the genome. The data also showed that the
majority of AFLP markers segregate independently of one another and the risk of
overestimating variation is low.
A pathogenicity study conducted during 1997 showed a significant variation in
pathogenicity of C. zuluense isolates to a susceptible E. grandis clone (Van Zyl et al.,
1997). This is indicative of a diverse fungal population. Results from the present
study, however, revealed that C. zuluense in South Africa is represented by a
relatively low level of genetic diversity. AFLP analysis also indicated that no group
having similar pathogenicity characteristics formed a specific cluster. This is in
contrast to results obtained by Pongam et al. (1999) who showed with AFLP markers
that isolates of Leptosphaeria maculans (Desmaz.) Ces. & De Not. from North
Dakota, Western Canada, Georgia and the UK formed one tightly clustered AFLP
group and were mostly of the same pathogenicity group. Thus, pathogenicity data
alone may not reflect the true genetic variability and evolutionary history of the
isolates. Isolates that are genetically distinct may have similar or identical
60
pathogenicity patterns due to the fact that they were exposed to the same selection
pressures by a common set of hosts.
The relatively low but well established level of genetic diversity in C. zuluense
suggests that there may be a high level of asexual reproduction in the fungus.
Genetic diversity values would be expected to be much higher for mainly sexual
reproducing fungi (McDonald & McDermott, 1993). This conclusion is further
supported by the fact that a low genetic variation was observed within each of the
sampled regions. Fungi capable of only asexual reproduction have been found to
have a lower degree of genetic diversity than organisms that reproduce sexually
(McDonald & McDermott, 1993; Wolf & McDermott, 1994; Milgroom, 1996). The low
level of genetic diversity obtained in this study is in agreement with genetic diversity
values reported for the asexually reproducing fungus, Rhynchosporium secalis (Oud)
J.J. Davis (Goodwin et al., 1993). The authors estimated a genetic diversity value of
between 0.16 and 0.29 in Australia, Norway and the USA (Goodwin et al., 1993).
Results of the current study thus also suggest that C. zuluense reproduces asexually,
but that it has been present in South Africa for an extended period of time.
In the present study, a moderate, but significant level of geographic differentiation
was found between isolates of C. zuluense collected from individual plantation
regions. This is in contrast to other pathogens that are known to be predominantly
clonal, for example, Phytophthora infestans (Mont.) de Bary in the Netherlands (GsT=
0.06) (Fry et al., 1991) and Mycosphaerella graminicola (Fuckel.) Schroter
populations in California and Oregon (GST= 0.039) (Boeger et al., 1993). In these
studies, GsT, is a measure of genetic differentiation, which is similar to FST (Nei,
1973). Values of GSTand FSTnot significantly different from zero would indicate no
differentiation between populations. The geographic differentiation value obtained in
this study is, however, similar to levels of geographic differentiation reported for
Pyrenopeziza brassicae B. Sutton & Rawlinson (FsT = 0.16; Majer et al. 1998).
Therefore, the relatively high FSTvalue for C. zuluense also suggests that there are
distinct geographic sub-populations of the fungus within the Eucalyptus growing
areas of Zululand, KwaZulu-Natal.
61
Genetic variation within fungal populations is known to be influenced either by
ecologically important or selective neutral variation (McDermott & McDonald, 1993;
McDonald & McDermott, 1993; Anderson & Kohn, 1995; Milgroom, 1995, 1996;
Milgroom & Fry, 1997). Milgroom & Fry (1997) referred to ecologically important
variation as traits that affect fitness and are thought to be under selection. Asexually
reproducing pathogens, such as C. zuluense, are forced to constantly adapt to
changes in their environment in order to survive. The development of disease
resistant Eucalyptus clones or hybrids imposes strong directional selection on the
pathogen population, leading to increased numbers of individual genotypes. Thus,
genetic diversity is directly influenced by selection, meaning that genotypes with the
highest fitness will increase over a period of time (McDonald et al., 1989).
Selective neutral variation does not affect fitness and is also known to be affected by
evolutionary forces, such as mutations, mating systems, gene flow or migration and
population size (McDermott & McDonald, 1993; Anderson & Kohn, 1995; Milgroom,
1995, 1996). Cluster analysis of data from the current study showed no evidence for
one or two genotypes being widespread as would be expected of predominantly
asexual pathogens. This would suggest that epidemics of Coniothyrium canker are
the result of genetic flow of local populations, rather than the emergence of
aggressive strains of C. zuluense that have spread throughout the Zululand forestry
area. In the absence of any movement of gametes among populations, it is expected
that genetic drift will lead to random changes in allele frequencies for neutral loci in
different populations (Boeger & McDonald, 1991). Even if there were only limited
movement of genes among populations, a correlation between genetic and
geographic distance among populations should be observed (Boeger & McDonald,
1991). Results of this study showed no correlation between FST values and
geographic distance and, therefore, strongly support our hypothesis that genetic flow
can be considered to be the main factor contributing towards genetic variation within
the C. zuluense population.
This study provides valuable information regarding future management strategies of
Coniothyrium canker. In many situations, ecologically important variation (traits
effected by selection) is relevant for disease management (Leung et al., 1993;
Anderson & Kohn, 1995; Milgroom & Fry, 1997). However, an understanding of
62
ecological, as well as selective neutral variation, is necessary since it helps to predict
how a pathogen population will respond to the implementation of new disease
resistant clones. Population genetic data presented in this paper suggest that new
epidemics would not be due to the emergence of aggressive strains (increase in
virulence) spreading rapidly through the country. Outbreaks would rather be due to
the introduction of new susceptible Eucalyptus clones, species or hybrids, together
with environmental conditions that favour disease development.
Population genetic data presented in this paper indicated there is a low level of
genetic variation in C. zuluense, suggestive of asexual reproduction. Disease
outbreaks would thus be due to the introduction of susceptible Eucalyptus species
combined with environmental conditions conductive to infection. The low, but well-
established genetic variation is also suggestive that the fungus has been present in
this country for an extended period of time.
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65
Table 1. Coniothyrium zuluense isolates differing in colony morphology and
pathogenicity that were used in amplified fragment length polymorphism (AFLP)
analysis.
lsolate" PlantationD Pathogenicity Grouplnq"
1 CRY 949 Aboyni
2 CRY 950
3 CRY 951 "
4 CRY 952 "
5 CRY 953
6 CRY 954
7 CRY 955 Futululu
8 CRY 956 "
9 CRY 957 "
10 CRY 958 +
11 CRY 959 "
12 CRY 960 " +
13 CRY 961 "
14 CRY 962 "
15 CRY 963
16 CRY 964 "
17 CRY 965 "
18 CRY 966 "
19 CRY 967
20 CRY 968 "
21 CRY 969 "
22 CRY 970 "
23 CRY 971 "
24 CRY 972 "
25 CMW 1772 Honey Farm +
26 CMW 1778
27 CMW 2100 " +
28 CRY 973
29 CRY 974 +
30 CRY 975 +
31 CRY 976 "
32 CRY 977 "
33 CRY 978 "
34 CRY 979 " +
35 CRY 980
36 CRY 981
37 CRY 982
38 CRY 983 " +
39 CRY 984 "
40 CRY 985
41 CRY 986 "
42 CRY 987
66
Isolate" Plantationb Pathogenicity Grouplnq"
43 CRY 988 Palm Ridge
44 CRY 989 "
45 CRY 990
46 CRY 991
47 CRY 992
48 CRY 993 "
49 CRY 994
50 CRY 995
51 CRY 996
52 CRY 997 "
53 CRY 998 " +
54 CRY 999
55 CRY 1000
56 CRY 1001 +
57 CRY 1002
58 CRY 1003
59 CRY 1004 Shire
60 CRY 1005 "
61 CRY 1006 "
62 CRY 1007 +
63 CRY 1008 Teranera
64 CRY 1009 "
65 CRY 1010 "
66 CRY 1011 +
67 CRY 1012
68 CRY 1013
69 CRY 1014
70 CRY 1015
71 CRY 1016 Teza
72 CRY 1017
73 CRY 1018 +
74 CRY 1019 " +
75 CRY 1020 " +
76 CRY 1021 +
77 CRY 1022 +
78 CRY 1023 +
79 CRY 1024 +
80 CRY 1025 " +
81 CRY 1026 " +
82 CRY 1027
83 CRY 1028
84 CRY 1029
85 CRY 1030 "
86 CRY 1031
87 CRY 1032 " +
88 CRY 1033 "
89 CRY 1034
67
lsolate" PlantationD Pathogenicity Grouplnq"
90 CRY 1035 Teza
91 CRY 1036
92 CRY 1037 "
93 CRY 1038 "
94 CRY 1039
95 CRY 1040
96 CRY 1041 "
97 CRY 1042
98 CRY 1043
99 CRY 1044 Trust
100 CRY 1045
101 CRY 1046 "
102 CRY 1047 " +
103 CRY 1048 "
104 CRY 1049 "
105 CRY 1050 "
106 CRY 1051 Fairbreeze
107 CRY 1052
108 CRY 1053 "
a CRY and CMW numbers refer to Coniothyrium zuluense isolates used in
this study.
b Isolates were collected from nine different Eucalyptus growing plantation
regions in the Zululand forestry area of KwaZulu-Natal, South Africa.
C Pathogenicity grouping of different isolates based on the degree of virulence
towards susceptible Eucalyptus grandis clone, ZG 14 (+ = pathogenic, - = non-
pathogenic, I = intermediately pathogenic). Pathogenicity was determined in a
previous study (Van Zyl et al., 1997).
68
Table 2. Sequences of primers and adaptors used for AFLP analysis.
Name Enzyme Type Sequence
CA Cfo1 Adaptor 5'-GACGATGAGTCCTGAACG-3'
3'-TACTCAGGACTT-5'
MA Mse1 Adaptor 5'-GACGATGAGTCCTGAG-3'
3'-TACTCAGGACTCAT-5'
C-T Cfo1 Primer + 1 5'-GATGAGTCCTGAACGCT-3'
C-TCC Cfo1 Primer + 3 5'-GATGAGTCCTGAACGCTCC-3'
C-TGG Cfo1 Primer + 3 5'-GATGAGTCCTGAACGCTGG-3'
M-A Mse1 Primer + 1 5'-GATGAGTCCTGAGT AA-3'
M-AGT Mse1 Primer + 3 5'-GATGAGTCCTGAGTAAGT-3'
M-AGC Mse1 Primer + 3 5'-GATGAGTCCTGAGT AAGC-3'
Table 3. Average FST values of the Coniothyrium zuluense population". Bold numbers indicate FST is significantly different from
zero at P <0.01
Plantations 1 2 3 4 5 6 7 8 9
1 Aboyni 0.098
2 Futululu 0.191 0.209
3 Honey Farm 0.199 0.208 0.185
4 Palm Ridge 0.179 0.203 0.192 0.170
5 Shire 0.192 0.198 0.211 0.187 0.034
6 Teranera 0.236 0.260 0.227 0.237 0.263 0.018
7 Teza 0.162 0.200 0.185 0.156 0.208 0.215 0.151
8 Trust 0.211 0.212 0.210 0.222 0.258 0.274 0.216 0.089
9 Fairbreeze 0.175 0.221 0.214 0.194 0.222 0.288 0.185 0.227 0.186
a Average FST values for Coniothyrium zuluense isolates were calculated among and between plantations, based on the
values obtained from the F-statistic of Nei & Li (1979).
0\
\0
Mase~
LESOTHO
SOUTH AFRICA Zululand
"... .~r!p
ZULULAND
...........................
~
10km
71
Fig. 2. Dendrogram of 108 Coniothyrium zuluense isolates based on AFLP data
using UPGMA cluster analysis of pairwise distance data. The scale represents
genetic distance obtained using the equation of Nei & Li (1979).
CRY 1029
CRY 961
CRY 996
CRY 995
CRY 965
CRY 966
CRY 1028
CRY 1034
CRY 1010
CRY 1011
CRY 1015
CRY 1009
CRY 1014
CRY 1013
CRY 1012
CRY 1008
CRY9n
CRY 1050
CRY 1049
CRY 1048
CRY 1047
CRY 1045
CRY 1046
CRY 1044
CRY 974
CRY 975
CMW2100
CRY 963
CRY 964
CRY 959
CRY 960
CRY 956
CRY 955
CRY 1043
CRY 1042
CRY 1040
CRY 1041
CRY 1039
CRY 1001
CRY 1000
CRY 1002
CRY 976
CRY 973
CMW1772
CRY 1031
CRY 1033
CRY 1032
CRY 1024
CRY 991
CRY 990
CRY 989
CRY 992
CRY 988
CMW1n8
CRY 984
CRY 999
CRY 981
CRY 979
CRY 1022
CRY 1025
CRY 978
CRY 982
CRY972
CRY969
CRY 971
CRY 1053
CRY 1027
CRY 962
CRY 1021
CRY 1003
CRY 997
CRY 998
CRY 994
CRY 993
CRY 970
CRY 967
CRY 1006
CRY 1007
CRY 1005
CRY 1004
CRY 968
CRY 958
CRY 957
CRY 1052
CRY 1037
CRY 1038
CRY 1030
CRY 1026
CRY 983
CRY 1017
CRY986
CRY 980
CRY 987
CRY 985
CRY 1051
CRY 1035
CRY 1036
CRY 1023
CRY 1020
CRY 1019
CRY 1018
CRY 1016
CRY 950
CRY 949
CRY 951
CRY 954
CRY953
I I I I I I I I I
0.35 0.26 0.18 0.09 0.00
Nei's Genetic Similarity
72
CHAPTER4
Morphological and molecular relatedness of geographically diverse
isolates of Coniothyrium zuluense from South Africa and Thailand
Coniothyrium canker, caused by Coniothyrium zuluense, is a serious stem canker
disease of Eucalyptus species in sub-tropical parts of South Africa. A Coniothyrium
sp. associated with similar symptoms on E. camaldulensis was observed in 1996 in
Thailand. It was previously thought that C. zuluense was restricted to South Africa.
The aim of this study is, thus, to compare South African isolates of C. zuluense with
isolates of the Coniothyrium sp. from Thailand at the morphological and molecular
level. Results of morphological comparisons indicate that the South African and
Thailand isolates are the same. This was further confirmed when all Coniothyrium
isolates associated with stem cankers on Eucalyptus spp. grouped together in a
single major clade for both rDNA sequence data and AFLP analysis. This clade was
distant from isolates of other Coniothyrium spp. included for comparative purposes.
Although the Coniothyrium isolates from South Africa and Thailand resided in two
separate clades, these were closely related and, we believe that the isolates from
Thailand represent C. zuluense. This is, thus, the first record of the important
Eucalyptus stem canker pathogen, C. zuluense, outside South Africa.
73
Eucalyptus species are native to Australia, but approximately 8 million hectares of
Eucalyptus plantations have been established, mostly in tropical and sub-tropical
countries of the world (Anonymous, 1995). The success of Eucalyptus propagation,
however, is often hampered by their susceptibility to fungal diseases. These
diseases include both stem, root and leaf diseases, and have been shown to cause
considerable economic losses on various Eucalyptus species, clones and hybrids
(Park & Keane, 1984; Florence et al., 1986; Hodges et al., 1986; Ferreira, 1989;
Conradie et al., 1990; Linde et al., 1994; Smith et al., 1994; Crous & Wingfield, 1994,
1996).
Coniothyrium canker caused by the recently described Coniothyrium zuluense
Wingfield, Crous & Coutinho, is a serious Eucalyptus stem canker pathogen from
South Africa (Wingfield et al., 1997). This fungus was first reported in 1988 in an
isolated area in Zululand, KwaZulu-Natal, on a single clone of E. grandis Hill: Maid.
The earliest signs of infection by C. zuluense on trees are small, discrete, necrotic
lesions on the young, green bark (Coutinho et al., 1997; Wingfield et al., 1997).
These lesions coalesce to form large necrotic patches on the stems from which
copius amounts of red kino exude (Coutinho et al., 1997; Wingfield et al., 1997).
Epicormic shoots are commonly produced in the cankered areas, indicative of partial
girdling of the stems. In severely infected clones, the tops of trees die, due to the
girdling effect (Coutinho et al., 1997; Wingfield et al., 1997).
Since its discovery in 1988, C. zuluense has become widespread throughout
Eucalyptus growing areas of Zululand and occurs on a wide range of E. grandis
clones and hybrids, as well as other species of Eucalyptus (Coutinho et al., 1997;
Wingfield et al., 1997). An intensive disease survey was undertaken during 1995 and
1996, that lead to the discovery that C. zuluense isolates differ considerably in
surface colony colour and pathogenicity to a susceptible E. grandis clone (Van Zyl et
al., 1997). It was, therefore, hypothesised that more than one species of
Coniothyrium might be responsible for disease in South Africa.
A number of Coniothyrium spp. have been reported as pathogens of Eucalyptus
leaves in the past. In Australia, C. eucalypticola Sutton, C. ka/langurence Sutton &
Alkorn, C. ovatum Swart and C. parvum Swart are associated with leaf spot on
74
various Eucalyptus spp. (Sutton, 1975, 1980; Swart, 1986). Coniothyrium ahmadii
Sutton has been isolated from eucalypt leaf spots in Pakistan (Sutton, 1974). The
only report of a pathogenic Coniothyrium sp. from South Africa, prior to the discovery
of C. zuluense, was of leaf spot caused by C. ovatum (Crous et al., 1988). In this
case, C. ovatum was isolated from leaf spots occurring mainly on the lower branches
of mature E. cIadocalyx F. Muell. and E. lehmannii Preiss:Schauer trees in
Stellenbosch, Western Cape province.
During 1996, a survey of diseases of eucalypt plantations in the Sinai area of
Thailand was undertaken. This led to the discovery of a serious stem canker disease
of E. camaldulensis Dehnh. Disease symptoms were found to be very similar to
those caused by C. zuluense from South Africa. Coniothyrium zuluense is, however,
only known from South Africa and was hypothesised to be endemic to this country
(Coutinho et al., 1997; Wingfield et al., 1997). Based on superficial morphological
and cultural characteristics, the Thailand isolates were identified as representing a
species of Coniothyrium. The aim of this study is, therefore, to compare isolates of
C. zuluense with those of the Coniothyrium sp. from Thailand, based on
morphological comparisons, rDNA sequence data and Amplified Fragment Length
Polymorphism (AFLP) analysis.
MATERIALS AND METHODS
Isolates
Six single conidial isolates of a Coniothyrium sp. were collected from severely
infected E. camaldulensis trees from the Sinai region of Thailand (Table 1).
lsolations were made from segments of symptomatic material that were placed in
humidity chambers to induce the formation of fungal fruiting bodies. Single conidial
isolates were obtained as described by Van Zyl et al. (1997). Each isolate originated
from a different tree.
75
Nine single conidial isolates of C. zuluense were collected from nine Eucalyptus
plantation regions in Zululand, KwaZulu-Natal, South Africa (Table 1). These isolates
were chosen to be compared with those from Thailand, based on their differences in
surface colony colour and pathogenicity to a susceptible E. grandis clone (Van Zyl et
al., 1997). Colony colour characteristics of these isolates varied between olive grey
(V23""b), greenish glaucous (33'''\ isabella (19"1) and grayish olive (21"") (Van Zyl et
al., 1997). The selected isolates represented three non-pathogenic, four isolates of
intermediate pathogenicity and two isolates with high levels of pathogenicity.
Three Coniothyrium spp. other than C. zuluense were included for comparative
purposes (Table 1). These included C. ovatum Swart, a leaf-spotting pathogen
isolated from E. diversicolor in Stellenbosch, Western Cape, South Africa; C.
palmarum Corda (CBS 758.73), the lectotype species of Coniothyrium and C. fuckelii
Sacc. (CBS 132.26) that causes stem cankers on various Rosa Thunb. and Rubus L.
species. Massarina corni Sh. (CBS 496.64) was included as an outgroup (Table 1).
All isolates were grown on Petri dishes containing 15 ml of an enriched 4 % w/v
Potato Dextrose Agar (PDA) (24 g Potato Dextrose extract (Difco); 1 g Yeast extract
(Difco); 1 g Glucose (Difco); 40 g agar (Difco); 1 I distilled H20). Plates were then
incubated at 30°C for 10 days. Isolates are maintained in the culture collection of
the Forestry and Agricultural Biotechnology Institute (FABI), University of Pretoria,
and will be deposited in other recognised international culture collections.
Morphological comparisons
Colony and conidial morphology. Coniothyrium isolates from Thailand were grown
on PDA (39 g PDA (Difco); 1 I distilled H20) at 30°C. Measurements (length and
width) using a Zeiss Axioskop light microscope were made of mature conidia (thirty
from each structure). Colony colour was rated using mycological colour charts of
Rayner (1970). Colony colour and conidial measurements for the Thailand isolates
were compared with those of C. zuluense isolates associated with Eucalyptus stem
canker in South Africa (Van Zyl et al., 1997).
76
Growth studies. Growth rates and temperature requirements for the South African
and Thailand isolates were determined on PDA. The PDA plates were inoculated
with 5 mm mycelial plugs removed from the margins of actively growing colonies and
placed, face down, at the centre of the plates. Plates were maintained at
temperatures ranging from 10 to 35°C, at five degree intervals in the dark for 30
days. A total of 3 plates were incubated for each isolate at each temperature. Two
diameter measurements were obtained from each colony, perpendicular to each
other. A total of 6 measurements were thus taken at each temperature for every
isolate studied, and the mean growth computed.
Greenhouse pathogenicity trials
All six Thailand and nine South African isolates were used in greenhouse inoculation
trials. Isolates were grown on enriched 4 % PDA for two weeks prior to inoculation.
Pathogenicity tests were conducted on six-month-old E. grandis trees of the clone ZG
14, which is known to be highly susceptible to Coniothyrium canker under natural
conditions (Wingfield et al., 1997). Twenty trees were inoculated for each isolate
tested. A small wound (10 mm diameter) was made on the stem of each tree by
removing the bark and exposing the cambium. Mycelial plugs, of similar size,
overgrown with the test fungi, were placed into each wound with the mycelium facing
the cambium. Inoculation wounds were covered with masking tape to prevent
desiccation of the inoculum. For control inoculations, sterile PDA plugs were used
and inserted into wounds on the stems of five trees. Mean lesion lengths were
assessed after six weeks and statistical differences for each isolate were determined
using Tukey's studentised range test (P = 0.05). The experiment was repeated once.
Molecular comparisons
DNA extractions. Nucleic acid was extracted from all Eucalyptus stem canker
isolates, as well as, isolates included in the study for comparative purposes (Table 1).
Total genomic, high molecular weight DNA was extracted from all isolates by
culturing them in 250 ml of enriched Potato dextrose broth (24 g Potato Dextrose
extract (Merck); 1 g Yeast extract (Merck); 1 g Glucose (Merck); 1 I distilled H20) in
77
500 ml Erlenmeyer flasks. Cultures were incubated at 30 aC on rotary shakers for
seven days. Mycelium was then harvested by filtration through Whatmann no. 1 filter
paper and lyophilised.
DNA was isolated using the technique of Raeder & Broda (1985) with some
amendments. One gram of dried mycelium was ground to a fine powder in liquid
nitrogen with a mortar and pestle. Ten ml of extraction buffer (100 mM Tris-HCI, pH
8.0; 50 mM EDTA, pH 8.0; 500 mM NaCI; 1.25 % SOS; 10 mM B-mercaptoethanol; 4
mM Spermidine; 1 mM Spermine; 1 mM Phenylmethylsulfonyl fluoride (PMSF)),
maintained at 65 oe, was added to each isolate and incubated in a water bath at 65 °
C for 60 min with frequent mixing. The aqueous phase was collected after
centrifugation and phenol/chloroform (1:1 phenol to chloroform) extractions were
performed until the interface was completely clean. Precipitation of the nucleic acids
was done using 3 M NaAc (0.1 v/v) and isopropanol (0,6 v/v) followed by overnight
incubation at - 20 aC. After centrifugation, to harvest the nucleic acids, and washing
with 70 % EtOH, the pellet was re-suspended in 200 III sterile water. One III of
RNaseA (10 mg / ml) was added to the re-suspended sample and left at 37 aC
overnight to degrade all RNA in the sample. All DNA extracts were quantified by
fluorometry and adjusted to a final concentration of 30 ng/Ill.
DNA sequence comparisons
Polymerase Chain Reaction. The ITS1 and ITS2, as well as the 5.8S gene of the
ribosomal RNA operon were amplified using the polymerase chain reaction (PCR)
(Saiki et al., 1988). Amplifications were performed using ITS primers ITS 1 (5'-
TCCGTAGGTGAACCTGCGG-3') and ITS 4 (5'-GCTGCGTTCnCATCGATGC-3')
(White et al., 1990). The PCR reaction mixture (100 Ill) included 2 units of Taq DNA
Polymerase (Boehringer Mannheim, Germany), 10 x reaction buffer (Boehringer), 4.5
mM MgCI2 (Boehringer), 250 mM dNTPs and 0.5 III of each primer (100 pM).
Amplification reactions were done in a Hybaid Omnigene Temperature Cycler
(Hybaid, Middlesex, U.K.). Denaturation was performed at 95 aC for 30 sec, followed
by primer annealing at 55 aC for 45 sec. Chain elongation took place at 72 "C for 2
min. These steps were repeated for 35 cycles. Final chain elongation took place at
78
72°C for 5 min. PCR products were electrophoresed in 1.5 % agarose gels, stained
with ethidium bromide, and visualised using UV light. Amplification reactions were
done in duplicate.
DNA sequencing and data analysis. All PCR products were purified using a
QIAquick PCR Purification Kit (QIAGEN, Germany). PCR products were sequenced
in both directions using the Big Dye Cycle Sequencing kit with Amplitaq@ DNA
Polymerase, FS (Perkin-Elmer, Warrington, UK) on a ABI PRISM™ 377 DNA
Autosequencer (Perkin-Elmer). Primers ITS 1 and ITS 4 were used in the sequence
reaction. The nucleotide sequences were manually aligned. Phylogenetic
relationships among species were determined using the Heuristic search option in
PAUP (Phylogenetic Analysis Using Parsimony), with gaps treated as missing data
(Swofford, 1985). Confidence intervals were determined using DNA BOOTSTRAP
analysis (Bootstrap confidence intervals on DNA parsimony) (1000 replicates)
(Felsenstein, 1993).
Amplified Fragment Length Polymorphism (AFLP) analysis
Restriction, ligation, and amplification were performed as described by Vos et al.
(1995). Genomic DNA (500 ng) from each sample was incubated for 16 hours at 25 °
C in a solution containing 10 U/IJl Cfo1 and 5 U/IJl Mse1 (Boehringer Mannheim,
USA), 1 x restriction-ligation buffer (10 mM Tris-HAc, pH7.5; 10 mM MgAc; 50 mM
KAc; 5 mM OTT), 1 U/IJl T4 DNA Ligase, 50 prnol/ul Cfo1-adaptors, 50 prnol/ul Mse1-
adaptors (Table 2), 10 mg/ml RNaseA and 100 mM spermidine. The final sample
volume was increased to 100 !.IIwith sterile water. After ligation the reaction mixture
was diluted 10 - fold with T.1 E buffer (10 mM TRIS-HCI, pH8.0; 10 mM EDTA) and
stored at -20°C. These ligated fragments served as templates in the amplification
reaction.
A pre-selective PCR (+ 1 reaction) amplification reaction was performed in 20 !.IIPCR
reaction mix containing 5 !.IIof the diluted DNA, 0.5 !.IIof each +1 primer (10 prnol/ul)
(Table 2), 100 mM Tris-HCI, pH 8.3, 15 mM MgCI2, 500 mM KCI, 25 mM MgCI2, 5U/1J1
Taq DNA Polymerase (Boehringer Mannheim, USA) and 250 IJM of dNTP. Initial
79
denaturation was performed at 94°C for 1min, followed by 30 cycles of 30 sec at 94 °
C, 60 sec at 56°C (primer annealing) and 60 sec at 72 °c (final chain elongation).
The amplification PCR products were diluted 10-fold in 1 x T.1 E buffer and used as
templates in the second amplification.
The second amplification (+ 3 reaction) was performed using primers derived from
the first set of primers, with additional nucleotides at the 3' end (Table 2). The Cf01
primer was HEX fluorochrome-Iabeled and the Mse1 primer was FAM fluorochrome-
labeled (AmpFISTR Profiler PCR Kit, Perkin-Elmer, Norwalk, Conn.). Fluorescent
AFLPs were amplified under the following conditions: 0.2 IJl of the HEX
fluorochrome-Iabeled Cfo1 + 3 primer (50 prnol/ul) was added to a 20-1J1PCR
reaction solution containing 5 IJl of the diluted + 1 pre-amplification mix, 0.41J1of the
FAM fluorochrome-Iabeled Mse1 + 3 primer, 5 U/IJl Taq DNA Polymerase
(Boehringer Mannheim, USA), 10 x Buffer (100 mM Tris-HCI, pH 8.0; 15 mM MgCI2;
500 mM KCI, pH8.3), 25 mM MgCI2 and 250 IJM of dNTP. The following temperature
profile was used: 12 cycles of 30 sec at 94°C, 30 sec at 65 °C, 60 sec at 72°C,
where the annealing temperature was subsequently reduced by 0.7 °C after each
cycle. The amplification was continued for 22 cycles of 30 sec at 94°C, 30 sec at 56
°C, and 60 sec at 72°C. All amplification reactions were performed using a Hybaid
Omnigene thermocycler (Hybaid, Middlesex, UK).
Electrophoresis and visualisation of AFLP peR products. PCR products (1.51J1)
were combined with 3 fmol TAMRA fluorescent-Iabeled GeneScan 500 internal size
standard (ABI), 1.6 IJl formamide, and 0.3 IJl 25 mM EDTA (pH 8.0) containing 50
mg/ml blue dextran. This mixture was heat denatured for 3 min at 95°C, and
immediately cooled on ice. Samples were loaded on a 5 % denaturing
polyacrylamide gel in 1 x TBE (Tris-borate EDTA, pH 8.0) and electrophoresed for
2.5 hours at 1680 watts using the GS 36A-2400 run module. Data were processed
by GeneScan Analysis software (version 2.02) to produce a gel image. PCR
fragments for individual samples were automatically sized by the GeneScan software
using a comparison of the mobility of the internal lane size standard to that of the
sample fragment.
80
AFLP data analysis. Each polymorphic AFLP fragment was treated as a unit
character and scored as present (1) or absent (0) across all isolates. The
experiments were repeated, and only reproducible bands were scored. The index of
genetic similarities was calculated according following the Nei & Li (1979) definition of
similarity. Unweighted pair-group mean arithmetic (UPGMA) was used for cluster
analysis of the pairwise similarity matrix that generated a dendrogram representing
the genetic similarity among fungal isolates. UPGMA analysis was carried out using
NCSS97 (Visual Components, UK).
RESULTS
Morphological comparisons
Colony and conidial morphology. Conidia of the Thailand Coniothyrium isolates
investigated were 4.1 - 5.2 urn long and 2.5 - 3.5 urn wide (Table 3) whereas,
conidia of C. zuluense from South Africa were 3.5 - 5.6 urn long and 2.1 - 3.6 urn
wide (Van Zyl et al., 1997). All conidia used in this study were thick walled, smooth
and broadly ellipsoidal. The apices were obtuse and the bases sub-truncate too
bluntly rounded.
The colour of the Coniothyrium isolates from Thailand were all a grayish olive (21"")
colour (Table 3). South African isolates of C. zuluense vary from an olive grey
(V23"llb), isabella (19"i), greenish glaucous (331I1f) to a grayish olive (21"") colour
(Table 3) (Van Zyl et al., 1997). All South African and Thailand isolates viewed from
below were black or rust coloured with white margins.
Growth studies. In growth studies, isolates of the Coniothyrium spp. from Thailand
and C. zuluense had growth optima at 30°C (Table 4). Thailand isolates failed to
grow at 10°C and 15 °C. Coniothyrium zuluense isolates were, however, able to
grow at 15°C, although very slowly.
81
Pathogenicity tests. Results showed that none of the Thailand isolates, screened
for pathogenicity, were able to cause disease (Table 5). Results were consistent
between repetitions. Significant differences in lesion development were, however,
evident among C. zuluense isolates from South Africa. Those isolates previously
defined as having high levels of pathogenicity produced significantly larger lesions (P
= 0.05) than those isolates having intermediate and low levels of pathogenicity (Table
5). These isolates also differed significantly among each other in their capacity to
cause disease. There was no significant difference (P = 0.05) between lesion lengths
for the isolates of intermediate pathogenicity. Significant differences in lesion
development were, however, observed for isolates previously described as having
intermediate and low levels of pathogenicity. Isolates previously defined as being
non-pathogenic produced no lesions in this study. No symptoms developed on trees
inoculated as controls (Table 5). The inoculated pathogen was consistently re-
isolated from the lesions on inoculated trees and never from control trees.
Molecular comparisons
DNA sequence comparisons. Sequence data were manually aligned by inserting
gaps (Fig. 1). Alignment of the DNA sequence data within the ITS 1 and ITS 2
regions proved to be variable between all the different species studied. A Heuristic
search from the aligned DNA sequence data (573 characters) of the ITS 1, 5.8S, and
ITS 2 regions of the rRNA operon, produced one most parsimonious tree (Fig. 2) of
458 steps (Cl = 0.930, HI = 0.070, RI = 0.934). A thousand replicate bootstrap
analyses were done to ascertain the confidence intervals of the branch points of the
tree. Phylogenetic analysis was done using midpoint rooting. Three major clades
emerged. All the Coniothyrium isolates associated with stem cankers on Eucalyptus
grouped together in a single major clade. This clade appeared clearly distant from
isolates of other Coniothyrium species included for comparative purposes. Bootstrap
analysis showed that the branch point separating Eucalyptus stem canker isolates
from the other Coniothyrium species, had a confidence interval of 100 %.
82
Isolates representing C. zuluense and the Coniothyrium sp. from Thailand could be
sub-divided into two distinct sub-groups. The one group included C. zuluense
isolates from South Africa (91 % bootstrap value). The second sub-clade included
the Coniothyrium isolates from Thailand (99 % bootstrap value). Coniothyrium
palmarum formed a single, strongly supported (100 % bootstrap value) clade. The
three remaining Coniothyrium spp. grouped together in a third clade (99 % bootstrap
value). This clade was sub-divided into two distinct sub-groups. Massarina corni and
C. ovatum grouped together (100 % bootstrap value) with C. fuckelii, but in a
separate though strongly supported (99 % bootstrap value) sub-group.
AFLP analysis. A total of 177 scorabie AFLP markers were included in this study.
Of these, 9 (5.1 %) were monomorphic for all the species in the study while 168 (94.9
%) displayed informative polymorphism's. Fragment size ranged from 40 to 499 bp.
A similarity matrix based on the similarity coefficient of Nei & Li (1979) was produced
using all 177 (polymorphic and monomorphic) fragments (Table 6). Pairwise genetic
similarity revealed an average similarity of 82.3 % (17.7 % dissimilar) among isolates
of the Coniothyrium sp. from Thailand and 77.6 % similarity (22.4 % dissimilar)
among the C. zuluense isolates. The average percentage similarity shared between
isolates of C. zuluense from South Africa and those of the Coniothyrium sp. from
Thailand was 76.1 % (23.9 % dissimilar). Genetic similarity ranged from 61.7 % to
86.2 %. UPGMA analysis of the similarity matrix yielded a dendrogram (Fig. 3) which
grouped the South African and Thailand isolates together in a single clade.
UPGMA analysis of the similarity matrix grouped all of the Coniothyrium isolates
associated with stem cankers on Eucalyptus separate from the four species included
for comparative purposes. This is in agreement with results obtained for sequence
data. Average percentage similarity between the Eucalyptus stem canker pathogens
and the other Coniothyrium spp. was 11.2 % for C. fuekeIii, 18,2 % for C. palmarum,
37.3 % for M. corni and 43.4 % for C. ovatum.
83
DISCUSSION
In this study, morphological comparisons, pathogenicity tests and molecular
comparisons strongly support the view that the Coniothyrium sp. from Thailand is the
same as C. zuluense, which causes Eucalyptus stem canker in South Africa. This is
also the first report of C. zuluense outside South Africa.
Based on morphological comparisons, the Coniothyrium sp. from Thailand is virtually
indistinguishable to C. zuluense from South Africa. Conidial measurements of the
Thailand isolates were within size ranges published for C. zuluense (Van Zyl et al.,
1997; Wingfield et al., 1997). Thailand isolates, however, differed from C. zuluense
in that they were a grayish olive colour and failed to grow at 15°C. This was in
contrast to characteristics published for C. zuluense from South Africa (Van Zyl et al.,
1997). Isolates of the latter fungus varied between an olive grey, greenish glaucous,
isabella or grayish olive colour and were able to grow at temperatures ranging from
15 to 35°C (Van Zyl et al., 1997). Optimal growth temperature for both Coniothyrium
species was, 30°C. Although some differences' in colony colour and temperature
requirement for growth were observed, we do not believe that these are sufficient to
separate the isolates, from the two regions, into different taxa.
Pathogenicity tests on young trees in the greenhouse showed that the Thailand
Coniothyrium sp. was not pathogenic to E. grandis. lts role in tree disease in
Thailand is, therefore, uncertain. However, only a small number of isolates from
Thailand were available for study. Pathogenicity studies conducted in South Africa
during 1997 (Van Zyl et al., 1997), showed that only 22 % of 344 C. zuluense isolates
collected, were able to cause lesions on a susceptible E. grandis clone (Van Zyl et
al., 1997). Most of these isolates (78 %) collected from severely infected trees in the
field were, thus, not able to cause disease. In the future, we would, however, hope to
collect a sufficient number of isolates from Thailand and to conduct pathogenicity
tests on established trees in that country. Such tests will expand our understanding
of the role that C. zuluense has in Eucalyptus disease in Thailand.
84
Phylogenetic analysis of sequence data from the ribosomal RNA operon, confirmed
that the Thailand isolates and C. zuluense are the same. The ribosomal RNA operon
is well known to be an extremely useful source of genetic data for taxonomic
comparisons at species level (Blanz & Unseld, 1986; White et al., 1990; Kurtzman,
1992; Wingfield & Wingfield, 1993; Mitchell et al., 1995; Wingfield et al., 1996a, b;
Witthuhn et al., 1998). Data analysis of the present study showed that the South
African and Thailand isolates produced a single clade, separate from the other
related Coniothyrium spp. used for comparative purposes. However, Coniothyrium
isolates from the two regions formed distinct sub-groups within this major clade. This
might suggest that C. zuluense in Thailand is in the process of diverging away from
C. zuluense in South Africa, due to environmental influences.
Genetic similarity between Thailand isolates and C. zuluense, as determined by
AFLP analysis, confirmed that they are the same. AFLP analysis is a novel PCR
fingerprinting technique which selectively amplifies DNA fragments, corresponding to
unique positions on the genome (Zabeau & Vos, 1993; Vos et al., 1995). This
technique has previously been shown to be extremely useful in determining genetic
similarities between different fungal populations (Majer et al., 1996; Majer et al.,
1998; Pongam et al., 1999). Results of the current study showed that percentage
genetic similarity values between South African and Thailand genotypes, ranged from
61.7 % to 86.2 %, with an average similarity of 76.1 %. Data, thus, suggest that
genetic differences between the Thailand isolates and C. zuluense are evident, but
that they clearly share a common origin.
This report represents the first record of C. zuluense outside South Africa. It is,
however, intriguing to consider the possible origin of C. zuluense on Eucalyptus
species. Wingfield et al. (1997) suggested that the fungus is native to South Africa.
This hypothesis was based on the fact that the disease is not known elsewhere in the
world, especially in Australia where Eucalyptus is native. They suggested that the
fungus might have originated from native Myrtaceae, and had developed the capacity
to infect Eucalyptus. This is similar to the situation with Eucalyptus rust, caused by
Puccinia psidii Winter, which is not known in Australia, but is common and damaging
in South and Central America where it apparently originated from native Myrtaceae
(Coutinho et al., 1998). Results of the present study indicate that C. zuluense occurs
85
elsewhere in the world. Surveys to find possible alternative hosts, both native and
introduced must, therefore, be conducted in sub-tropical and tropical Eucalyptus
growing areas.
South African C. zuluense isolates display considerable variation in colony colour and
virulence (Van Zyl et al., 1997). Large variations in virulence are widely associated
with diverse pathogen populations that are influenced by a number of factors,
including the capacity for sexual reproduction in the fungus. Organisms capable of
sexual reproduction have higher genetic diversity than those reproducing only
asexually (McDonald & McDermott, 1993; Wolf & McDermott, 1994; Milgroom, 1996).
Coniothyrium zuluense is known to reproduce only asexually (Wingfield et al., 1997),
thus, the large variation in virulence was not expected. One hypothesis has been
that more than one species of Coniothyrium might be responsible for disease in
South Africa. Sequencing results in this study have, however, shown that only a
single, yet highly variable species of Coniothyrium, is responsible for cankers on
Eucalyptus species in South Africa.
The discovery of C. zuluense outside South Africa is important and shows that the
fungus is more widespread than previously believed (Wingfield et al., 1997). Surveys
for this disease in other subtropical and tropical Eucalyptus growing areas of the
world are required. to provide information regarding its origin. Furthermore,
pathogenicity tests with a wider range of isolates should be undertaken in Thailand.
Such tests will allow us to compare the susceptibility of different Eucalyptus spp.,
clones and hybrids. The ultimate aim will be to avoid the disease, which could be
achieved through selection of disease resistant planting stock.
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DNA sequence data. Mycologia 90,96-101.
89
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Table 1. List of fungal isolates used in DNA sequence comparisons.
Species Isolate no." Origin Collector
Coniothyrium zuluense CRY 957 Zululand, KZN, South AfricaD L.M. van Zyl
" CRY 964 " "
CRY 1000
CRY 1017 "
CRY 1023 " "
CRY 1047 " "
CRY 1049 " "
" CRY 1056 " "
CRY 1057 " "
C.ovatum CO 1 Western Cape, South Africa L.M. van Zyl & M.J. Wingfield
Coniothyrium sp. CMW 5231 Thailand "
CMW 5232
" CMW 5233 "
CMW 5234 "
CMW 5235
CMW 5236 " "
C. palmarum CBS 758.73 Israel Y. Pinkas
C. fuckelii CBS 132.26 Netherlands F. Laibach
Massarina corni CBS 496.64
"lsolates are maintained in the culture collection of the Tree Pathology Co-operative Programme, Forestry and
Agricultural Biotechnology Institute (FABI), University of Pretoria. CBS refers to the Centraal Bureau voor
Schimmelcultures, Baarn, Netherlands.
bKZN refers to the KwaZulu-Natal Province in South Africa. IDo
91
Table 2. Sequences of primers and adaptors used for AFLP analysis.
Name Enzyme Type Sequence
CA Cfo1 Adaptor 5'-GACGATGAGTCCTGAACG-3'
3'-TACTCAGGACTT-5'
MA Mse1 Adaptor 5'-GACGATGAGTCCTGAG-3'
3'-TACTCAGGACTCAT-5'
C-T Cfo1 Primer + 1 5'-GATGAGTCCTGAACGCT-3'
C-TCC Cfo1 Primer + 3 5'-GATGAGTCCTGAACGCTCC-3'
C-TGG Cfo1 Primer + 3 5'-GATGAGTCCTGAACGCTGG-3'
M-A Mse1 Primer + 1 5'-GATGAGTCCTGAGT AA-3'
M-AGT Mse1 Primer + 3 5'-GATGAGTCCTGAGT AAGT -3'
M-AGC Mse1 Primer + 3 5'-GATGAGTCCTGAGT AAGC-3'
Table 3. Comparison of important morphological characteristics of Coniothyrium zuluense and a Coniothyrium sp. from Thailand
Morphological characteristics
Fungus Isolate no. Colony colour" Conidium lengthb Conidium width Source of data
C. zuluense CRY 1017 21"11 4.6 - 4.7 (4.5) 2.6 - 3.1 (2.8) Van Zyl et al., 1997
CRY 1023 33"lf 4.5 - 4.7 (4.5) 2.6 - 3.6 (2.8)
" CRY 1056 V231111b 4.5 - 4.6 (4.5) 2.5 - 2.9 (2.7)
CRY 1047 " 4.2 - 4.5 (4.3) 2.5 - 2.8 (2.6)
CRY 1049 19"1 4.1 - 4.3 (4.2) 2.7 - 2.8 (2.5)
CRY 957 V23"lb 3.5-4.5 (4.1) 2.5 - 3.6 (2.8)
CRY 964 33111f 3.5 - 4.2 (4.0) 2.5 - 2.9 (2.6)
CRY 1000 21"11 4.3 - 5.6 (4.8) 2.1 - 3.2 (2.8)
CRY 1057 33"lf 4.3 - 5.2 (4.8) 2.4 - 3.5 (2.7)
Coniothyrium sp. CMW 5231 21"11 4.2 - 4.5 (4.3) 2.5 - 2.8 (2.5) This study
" CMW 5232 " 4.1 - 4.4 (4.3) 2.5 - 2.8 (2.5)
" CMW 5233 " 4.4 - 4.7 (4.5) 2.6 - 3.2 (2.8) "
CMW 5234 " 4.4 - 4.8 (4.5) 2.5 - 3.5 (2.8)
" CMW 5235 " 4.5 - 5.2 (4.8) 2.6 - 3.1 (2.8)
CMW 5236 " 4.4 - 4.8 (4.5) 2.5 - 2.8 (2.8)
"Colour classes are those published by Rayner (1970). Colour codes represent the following: olive grey (V23111Ibg),reenish
glaucous (33"lf), isabella (19"1) and grayish olive (21"11).
bAil measurements are listed in urn. Values are the mean of 30 measurements.
\0
IV
Table 4. Growth of Coniothyrium zuluense isolates compared with those of the Coniothyrium sp. from Thailand.
Growth Studies"
(average colony diameter (mm)
Temperature (OC)
Fungus Isolate number 10 15 20 25 30 35
Coniothyrium zuluense CRY 1017 o abc 7.5 b 16.5 a 25.4 a 39.4 a 16.2 a
" CRY 1023 Oa 14.2 b 26.4 be 42.3 be 66.8 cd 35.6 e
" CRY 1056 Oa 11.7 b 23.8 ab 36.5 b 59.6 be 27.9 be
" CRY 1047 Oa 11.8 b 24.1 ab 34.5 ab 53.8 b 26.5 b
" CRY 1049 Oa 15.2 be 30.9 e 50.5 d 75.4 e 40.1 d
" CRY 957 Oa 15.4 be 30.2 e 53.6 d 80.1 e 40.1 d
" CRY 964 Oa 8.9 b 17.5 a 27.8 a 43.6 a 23.5 b
" CRY 1000 Oa 9.8 b 20.1 a 32.1 ab 50.6 b 24.5 b
" CRY 1057 Oa 10.4 b 19.8 a 28.4 a 42.1 a 19.8 ab
Coniothyrium sp. CMW 5231 Oa Oa 27.4 be 38.7 be 62.4 e 15.8 a
" CMW 5232 Oa Oa 23.5 ab 35.8 b 58.4 be 14.4 a
" CMW 5233 Oa Oa 28.4 be 41.6 e 66.5 cd 16.9 a
" CMW 5234 Oa Oa 23.3 ab 40.2 e 58.9 be 14.8 a
" CMW 5235 Oa Oa 24.0 ab 41.5 e 62.4 e 15.7 a
" CMW 5236 Oa Oa 24.2 ab 38.2 be 59.9 be 14.8 a
"Growth was measured after incubating cultures for 30 days in the darkness. -\
bEach value represents an average of six measurements. \0w
'Each value with a different letter differs significantly at P = 0.05 from the others for that specific temperature range (CV = 13.45 %).
94
Table 5. Lesion lengths associated with inoculations using Coniothyrium isolates
from South Africa and Thailand.
Pathoqenicity"
Fungus Isolate number Lesion length (mm)
Coniothyrium zuluense CRY 1017 23.6 bC
II CRY 1023 56.4 e
CRY 1056 10 a
II CRY 1047 37.6 d
II CRY 1049 10 a
II CRY 957 17.8 b
II CRY 964 10 a
II CRY 1000 27.8 c
CRY 1057 23.1 b
Coniothyrium sp. CMW 5231 10 a
II CMW 5232 10 a
II CMW 5233 10 a
CMW 5234 10 a
II CMW 5235 10 a
CMW 5236 10 a
CONTROL 10 a
aSix-month-old trees of a susceptible Eucalyptus grandis clone (ZG 14) were
inoculated under glass-house conditions.
bEach value is an average of 20 measurements. CV = 11.3 %
'Values followed by different letters differ significantly at P = 0.05.
Table 6. Pairwise distance matrix based on the F-statistic of Nei & Li (1979) converted to a percentage, i.e. %Sij = 1-Sij
CMW5232 100
CMW5233 91.27 100
CMW5236 74.96 68.75 100
CMW5235 82.17 74.96 83.82 100
CMW5234 83.82 78.7 88.43 86.94 100
CMW 5231 91.96 86.18 71.95 77.79 81.33 100
CRY 1017 82.17 82.17 72.97 82.17 78.7 79.59 100
CRY 1023 80.47 76.86 70.91 78.7 76.86 75.92 85.41 100
CRY 1056 71.95 71.95 62.93 73.98 69.84 70.91 77.79 71.95 100
CRY 1049 79.59 69.84 60.4 69.84 71.95 76.86 73.98 69.84 61.68 100
CRY 1047 82.17 80.47 68.75 72.97 72.97 77.79 88.43 80.47 71.95 83 100
CRY 1000 79.59 75.92 60.4 81.33 65.34 76.86 89.16 81.33 78.7 66.5 83 100
CRY 1057 85.41 76.86 78.7 82.17 85.41 75.92 86.94 85.41 71.95 67.64 80.47 77.79 100
CRY 964 76.86 72.97 64.15 70.91 72.97 79.59 78.7 76.86 67.64. 79.59 82.17 69.84 74.96 100
CRY 957 77.79 69.84 71.95 79.59 79.59 78.7 81.33 83 68.75 68.75 73.98 78.7 79.59 75.92 100
M .comi 38.08 29.49 19.26 48.82 29.49 36.06 38.08 41.91 47.17 27.11 24.62 53.47 33.96 41.91 43.72 100
C.ovatum 48.82 38.08 24.62 51.96 38.08 47.17 45.47 41.91 47.17 40.03 33.96 56.35 38.08 45.47 43.72 95.23 100
C.fuckelii 24.62 -21.27 -1.15 19.26 6.57 10.04 13.29 6.57 10.04 10.04 29.49 16.36 13.29 -1.15 16.36 -506 -506 100
C.palmarum 2.85 -15.44 -5.48 2.85 10.04 6.57 -5.48 2.85 -56.2 -21.3 -27.9 -21.3 2.85 -27.9 -10.2 -506 -431 -506 100
\0
Vl
96
Fig. 1. Aligned sequences of the ITS 1 and ITS 2 regions, as well as the conserved
5.8S RNA gene. CMW numbers represent the Thailand Coniothyrium isolates while
CRY numbers refer to Coniothyrium zuluense isolates from South Africa. Sequence
data generated for C. palmarum, C. fuckelii and Massarina corni were included for
comparative purposes. N indicates unknown bases and a dash indicates a gap in the
sequence inserted to achieve the alignment.
CRY 1056 CTCCCAACCC CCCATCGAA- --TTTTCCAA ACCATGTTGC GCCTCGGGGG -CGACCCGGC CATCGCGC-C GGTGGCCCCC GGTGGACCCC TCCAACTCTG
CRY 1023 CTCCCAACCC CCCATCGAA- --TTTTCCAA ACCATGTTGC GCCTCGGGGG -CGACCCGGC CATCGCGC-C GGTGGCCCCC GGTGGACCCC TCCAACTCTG
CRY 1057 CTCCCAACCC CCCATCGAA- --TTTTCCAA ACCATGTTGC GCCTCGGGGG -CGACCCGGC CATCGCGC-C GGTGGCCCCC GGTGGACCCC TCCAACTCTG
CRY 1000 CTCCCAACCC CCCATCGAA- --TTTTCCAA ACCATGTTGC GCCTCGGGGG -CGACCCGGC CATCGCGC-C GGTGGCCCCC GGTGGACCCC TCCAACTCTG
CRY 1047 CTCCCAACCC CCCATCGAA- --TTTTCCAA ACCATGTTGC GCCTCGGGGG -CGACCCGGC CATCGCGC-C GGTGGCCCCC GGTGGACCCC TCCAACTCTG
CRY 1,049 CTCCCAACCC CCCATCGAA- --TTTTCCAA ACCATGTTGC GCCTCGGGGG -CGACCCGGC CATCGCGC-C GGTGGCCCCC GGTGGACCCC TCCAACTCTG
CRY 957 CTCCCAACCC CCCATCGAA- --TTTTCCAA ACCATGTTGC GCCTCGGGGG -CGACCCGGC CATCGCGC-C GGTGGCCCCC GGTGGACCCC TCCAACTCTG
CRY 964 CTCCCAACCC CCCATCGAA- --TTTTCCAA ACCATGTTGC GCCTCGGGGG -CGACCCGGC CATCGCGC-C GGTGGCCCCC GGTGGACCCC TCCAACTCTG
CMW 5231 CTCCCAACCC CCCAT-G--- --TTTTCC-A A-CCATGTTG -CCTCGGGGG -CGACCCGGC CATCGCGGGC CGGGGCCCCC GGTGGACCCC TCCAACTCTG
CMW 5234 CTCCCAACCC CCCAT-G--- --TTTTCC-A A-CCATGTTG -CCTCGGGGG -CGACCCGGC CATCGCGGGC CGGGGCCCCC GGTGGACCCC TCCAACTCTG
CMW 5235 CTCCCAACCC CCCAT-G--- --TTTTCC-A A-CCATGTTG -CCTCGGGGG -CGACCCGGC CATCGCGGGC CGGGGCCCCC GGTGGACCCC TCCAACTCTG
CMW 5236 CTCCCAACCC CCCAT-G--- --TTTTCC-A A-CCATGTTG -CCTCGGGGG -CGACCCGGC CATCGCGGGC CGGGGCCCCC GGTGGACCCC TCCAACTCTG
CMW 5232 CTCCCAACCC CCCAT-G--- --TTTTCC-A A-CCATGTTG -CCTCGGGGG -CGACCCGGC CATCGCGGGC CGGGGCCCCC GGTGGACCCC TCCAACTCTG
CMW 5233 CTCCCAACCC CCCAT-G--- --TTTTCC-A A-CCATGTTG -CCTCGGGGG -CGACCCGGC CATCGCGGGC CGGGGCCCCC GGTGGACCCC TCCAACTCTG
C. ovatum GCGGCGGCCT TAAATCGCGC CAAATGGC-A CCCTTTGTTT ACGGAGTACC G-TTCGTTTC C-T------- ---------C GGTGGG---- ----------
M. corni GCGGCGGCCT TAAATCGCGC CAAATGGC-A CCCTTTGTTT ACGGAGTACC G-TTCGTTTC C-T------- ---------C GGTGGG----
C. palmarum TGACCCGCCC TGTCT-G--- --AATATATA CCCC-TGTTT ATTGCGTACT ACTT-GTTTC CTT------- ---------- GGTGGG---- ----------
C. fuckelii TCACGCGCCG CAT-TCCTGC ---AT-CCT- ----TTTTTT ACGAAGCACC -TTTCGTTTC CTTCGGCGGG CAACCTGCC- GCTGGAA-CT T--AACAAAA
110 120 130 140 150 160 170 lBO 190 200
CRY 1017 CATCT-TTGC GTCT-GAG-- -T------CA ---CAAAAT- ---------- --------TA AA-T------ ------CAA- --TCAAAACT TTCAACAACG
CRY 1056 CATCT-TTGC GTCT-GAG-- -T------CA ---CAAAAT- ---------- --------TA AA-T------ ------CAA- --TCAAAACT TTCAACAACG
CRY 1023 CATCT-TTGC GTCT-GAG-- -T------CA ---CAAAAT- ---------- --------TA AA-T------ ------CAA- --TCAAAACT TTCAACAACG
CRY 1057 CATCT-TTGC GTCT-GAG-- -T------CA ---CAAAAT- ---------- --------TA AA-T------ ------CAA- --TCAAAACT TTCAACAACG
CRY 1000 CATCT-TTGC GTCT-GAG-- -T------CA ---CAAAAT- ---------- --------TA AA-T------ ------CAA- --TCAAAACT TTCAACAACG
CRY 1047 CATCT-TTGC GTCT-GAG-- -T------CA ---CAAAAT- ---------- --------TA AA-T------ ------CAA- --TCAAAACT TTCAACAACG
CRY 1049 CATCT-TTGC GTCT-GAG-- -T------CA ---CAAAAT- ---------- --------TA AA-T----~- ------CAA- --TCAAAACT TTCAACAACG
CRY 957 CATCT-TTGC GTCT-GAG-- -T------CA ---CAAAAT- ---------- --------TA AA-T------ ------CAA- --TCAAAACT TTCAACAACG
CRY 964 CATCT-TTGC GTCT-GAG-- -T------CA ---CAAAAT- ---------- --------TA AA-T------ ------CAA- --TCAAAACT TTCAACAACG
CMW 5231 CATCTCTTGC GTCT-GAG-- -T------CA ---CAAAA-- ---------- --------TA AAAT------ ------CAA- --TCAAAACT TTCAACAACG
CMW 5234 CATCTCTTGC GTCT-GAG-- -T------CA ---CAAAA-- ---------- --------TA AAAT------ ------CAA- --TCAAAACT TTCAACAACG
CMW 5235 CATCTCTTGC GTCT-GAG-- -T------CA ---CAAAA-- ---------- --------TA AAAT------ ------CAA- --TCAAAACT TTCAACAACG
CMW 5236 CATCTCTTGC GTCT-GAG-- -T------CA ---CAAAA-- ---------- --------TA AAAT------ ------CAA- --TCAAAACT TTCAACAACG
CMW 5232 CATCTCTTGC GTCT-GAG-- -T------CA ---CAAAA-- ---------- --------TA AAAT------ ------CAA- --TCAAAACT TTCAACAACG
CMW 5233 CATCTCTTGC GTCT-GAG-- -T------CA ---CAAAA-- ---------- --------TA AAAT------ ------CAA- --TCAAAACT TTCAACAACG
C. ovatum -----CTTGC CT-----GCC ATGACCCCCA ACCCAAAACC C-TTATGTAG -TAGCCAGTA ACCTTCAGTA AGTAAACAAA A-TCAAAACT TTCAACAACG
M. corni -----CTTGC CT-----GCC ATGAGGACCA ACCCAAAACC C-TT-TGTAG -TAGC-AGTA ACCTTCAGTA AGAAACAATA --TCAAAACT TTCAACAACG
C. palmarum -----CTTGC C-C----GCC AAAAGGACAC CTATAAAACC TCTTGTAATT ---GC-AGTC AGCGTCAGAA AAACTTAATA ATTCAAAACT TTCAACAACG
C. fuckelii CCTTTTTTGC ATCTAGCAT- -TACCTGTTC TGATAAAAAA CAATCGTTA- ---------- ---------- ---------- ---CAAAACT TTCAACAACG
I"Q
-...l
CRY 1056 GATCTCTTGG TTCTGGCATC GATGAAGAAC GCAGCGAAAT GCGATAAGTA ATGTGAATTG CAGAATTCAG TGAATCATCG AATCTTTGAA CGCACATTGC
CRY 1023 GATCTCTTGG TTCTGGCATC GATGAAGAAC GCAGCGAAAT GCGATAAGTA ATGTGAATTG CAGAATTCAG TGAATCATCG AATCTTTGAA CGCACATTGC
CRY 1057 GATCTCTTGG TTCTGGCATC GATGAAGAAC GCAGCGAAAT GCGATAAGTA ATGTGAATTG CAGAATTCAG TGAATCATCG AATCTTTGAA CGCACATTGC
CRY 1000 GATCTCTTGG TTCTGGCATC GATGAAGAAC GCAGCGAAAT GCGATAAGTA ATGTGAATTG CAGAATTCAG TGAATCATCG AATCTTTGAA CGCACATTGC
CRY 1047 GATCTCTTGG TTCTGGCATC GATGAAGAAC GCAGCGAAAT GCGATAAGTA ATGTGAATTG CAGAATTCAG TGAATCATCG AATCTTTGAA CGCACATTGC
CRY 1049 GATCTCTTGG TTCTGGCATC GATGAAGAAC GCAGCGAAAT GCGATAAGTA ATGTGAATTG CAGAATTCAG TGAATCATCG AATCTTTGAA CGCACATTGC
CRY 957 GATCTCTTGG TTCTGGCATC GATGAAGAAC GCAGCGAAAT GCGATAAGTA ATGTGAATTG CAGAATTCAG TGAATCATCG AATCTTTGAA CGCACATTGC
CRY 964 GATCTCTTGG TTCTGGCATC GATGAAGAAC GCAGCGAAAT GCGATAAGTA ATGTGAATTG CAGAATTCAG TGAATCATCG AATCTTTGAA CGCACATTGC
CMW 5231 GATCTCTTGG TTCTGGCATC GATGAAGAAC GCAGCGAAAT GCGATAAGTA ATGTGAATTG CAGAATTCAG TGAATCATCG AATCTTTGAA CGCACATTGC
CMW 5234 GATCTCTTGG TTCTGGCATC GATGAAGAAC GCAGCGAAAT GCGATAAGTA ATGTGAATTG CAGAATTCAG TGAATCATCG AATCTTTGAA CGCACATTGC
CMW 5235 GATCTCTTGG TTCTGGCATC GATGAAGAAC GCAGCGAAAT GCGATAAGTA ATGTGAATTG CAGAATTCAG TGAATCATCG AATCTTTGAA CGCACATTGC
CMW 5236 GATCTCTTGG TTCTGGCATC GATGAAGAAC GCAGCGAAAT GCGATAAGTA ATGTGAATTG CAGAATTCAG TGAATCATCG AATCTTTGAA CGCACATTGC
CMW 5232 GATCTCTTGG TTCTGGCATC GATGAAGAAC GCAGCGAAAT GCGATAAGTA ATGTGAATTG CAGAATTCAG TGAATCATCG AATCTTTGAA CGCACATTGC
CMW 5233 GATCTCTTGG TTCTGGCATC GATGAAGAAC GCAGCGAAAT GCGATAAGTA ATGTGAATTG CAGAATTCAG TGAATCATCG AATCTTTGAA CGCACATTGC
C. ovatum GATCTCTTGG TTCTGGCATC GATGAAGAAC GCAGCGAAAT GCGATAAGTA GTGTGAATTG CAGAATTCAG TGAATCATCG AATCTTTGAA CGCACATTGC
M. corni GATCTCTTGG TTCTGGCATC GATGAAGAAC GCAGCGAAAT GCGATAAGTA GTGTGAATTG CAGAATTCAG TGAATCATCG AATCTTTGAA CGCACATTGC
C. palmarum GATCTCTTGG TTCTGGCATC GATGAAGAAC GCAGCGAAAT GCGATAAGTA GTGTGAATTG CAGAATTCAG TGAATCATCG AATCTTTGAA CGCACATTGC
C. fuckelii GATCTCTTGG TTCTGGCATC GATGAAGAAC GCAGCGAAAT GCGATAAGTA GTGTGAATTG CAGAATTCAG TGAATCATCG AATCTTTGAA CGCACATTGC
310 320 330 340 350 360 370 380 390 400
CRY 1017 GCCCT-CTGG TATTCCGGAG GGCATGCCTG TTCGAGCGTC ATT-ACACCA --CTCCAGCC -TCGCTGGGT ATTGGGCGCC GCGGCCTCC- GCGCGCCTTA
CRY 1056 GCCCT-CTGG TATTCCGGAG GGCATGCCTG TTCGAGCGTC ATT-ACACCA --CTCCAGCC -TCGCTGGGT ATTGGGCGCC GCGGCCTCC- GCGCGCCTTA
CRY 1023 GCCCT-CTGG TATTCCGGAG GGCATGCCTG TTCGAGCGTC ATT-ACACCA --CTCCAGCC -TCGCTGGGT ATTGGGCGCC GCGGCCTCC- GCGCGCCTTA
CRY 1057 GCCCT-CTGG TATTCCGGAG GGCATGCCTG TTCGAGCGTC ATT-ACACCA --CTCCAGCC -TCGCTGGGT ATTGGGCGCC GCGGCCTCC- GCGCGCCTTA
CRY 1000 GCCCT-CTGG TATTCCGGAG GGCATGCCTG TTCGAGCGTC ATT-ACACCA --CTCCAGCC -TCGCTGGGT ATTGGGCGCC GCGGCCTCC- GCGCGCCTTA
CRY 1047 GCCCT-CTGG TATTCCGGAG GGCATGCCTG TTCGAGCGTC ATT-ACACCA --CTCCAGCC -TCGCTGGGT ATTGGGCGCC GCGGCCTCC- GCGCGCCTTA
CRY 1049 GCCCT-CTGG TATTCCGGAG GGCATGCCTG TTCGAGCGTC ATT-ACACCA --CTCCAGCC -TCGCTGGGT ATTGGGCGCC GCGGCCTCC- GCGCGCCTTA
CRY 957 GCCCT-CTGG TATTCCGGAG GGCATGCCTG TTCGAGCGTC ATT-ACACCA --CTCCAGCC -TCGCTGGGT ATTGGGCGCC GCGGCCTCC- GCGCGCCTTA
CRY 964 GCCCT-CTGG TATTCCGGAG GGCATGCCTG TTCGAGCGTC ATT-ACACCA --CTCCAGCC -TCGCTGGGT ATTGGGCGCC GCGGCCTCC- GCGCGCCTTA
CMW 5231 GCCCT-CTGG TATTCCGGAG GGCATGCCTG TTCGAGCGTC ATT-ACACCA --CTCCAGCC -TCGCTGGGT ATTGGGCGCC GCGGCCTCC- GCGCGCCTTA
CMW 5234 GCCCT-CTGG TATTCCGGAG GGCATGCCTG TTCGAGCGTC ATT-ACACCA --CTCCAGCC -TCGCTGGGT ATTGGGCGCC GCGGCCTCC- GCGCGCCTTA
CMW 5235 GCCCT-CTGG TATTCCGGAG GGCATGCCTG TTCGAGCGTC ATT-ACACCA --CTCCAGCC -TCGCTGGGT ATTGGGCGCC GCGGCCTCC- GCGCGCCTTA
CMW 5236 GCCCT-CTGG TATTCCGGAG GGCATGCCTG TTCGAGCGTC ATT-ACACCA --CTCCAGCC -TCGCTGGGT ATTGGGCGCC GCGGCCTCC- GCGCGCCTTA
CMW 5232 GCCCT-CTGG TATTCCGGAG GGCATGCCTG TTCGAGCGTC ATT-ACACCA --CTCCAGCC -TCGCTGGGT ATTGGGCGCC GCGGCCTCC- GCGCGCCTTA
CMW 5233 GCCCT-CTGG TATTCCGGAG GGCATGCCTG TTCGAGCGTC ATT-ACACCA --CTCCAGCC -TCGCTGGGT ATTGGGCGCC GCGGCCTCC- GCGCGCCTTA
C. ovatum GCCCTTC-GG TATTCCGTTG GGCATGCCTG TTCGAGCGTC ATTTAATCAA --CTCAAGCC CTGCTT-GGT GT-GGGTGTT T--G-TTCC- GCC-----TC
M. corni GCCCTTC-GG TATTCCGTTG GGCATGCCTG TTCGAGCGTC ATTTAATCAA ---TCAAGCC CTGCTT-GGT GT-GGGTGTT T--G-TTCC- GCC-----TC
C. palmarum GCCCTTC-GG TATTCCGTGG GGCATGCCTG TTCGAGCGTC ATTT-GTACC --CTCAAGCT TTGCTT-GGT GTTGGGTGTT T--G--TCCC GTG----TTA
C. fuckelii GCCCTTC-GG TATTCCATGG GGCATGCCTG TTCGAGCGTC ATT--CTACA CCCTCAAGCT CTGCTT-GGT GTTGGGCGTC T--G--TCCC GCC----TTC
\0
00
CRY 1056 AATGTCTCCG GCCGAGCCGA CCGTCTCCAA GCGTTGTGGC ACAA-CTGTT TCGCTTTCGG GA-CCGGTCC ---GGC-GAC GCGCCGTTAA ACCCTTTCAC
CRY 1023 AATGTCTCCG GCCGAGCCGA CCGTCTCCAA GCGTTGTGGC ACAA-CTGTT TCGCTTTCGG GA-CCGGTCC ---GGC-GAC GCGCCGTTAA ACCCTTTCAC
CRY 1057 AATGTCTCCG GCCGAGCCGA CCGTCTCCAA GCGTTGTGGC ACAA-CTGTT TCGCTTTCGG GA-CCGGTCC ---GGC-GAC GCGCCGTTAA ACCCTTTCAC
CRY 1000 AATGTCTCCG GCCGAGCCGA CCGTCTCCAA GCGTTGTGGC ACAA-CTGTT TCGCTTTCGG GA-CCGGTCC ---GGC-GAC GCGCCGTTAA ACCCTTTCAC
CRY 1047 AATGTCTCCG GCCGAGCCGA CCGTCTCCAA GCGTTGTGGC ACAA-CTGTT TCGCTTTCGG GA-CCGGTCC ---GGC-GAC GCGCCGTTAA ACCCTTTCAC
CRY 1049 AATGTCTCCG GCCGAGCCGA CCGTCTCCAA GCGTTGTGGC ACAA-CTGTT TCGCTTTCGG GA-CCGGTCC ---GGC-GAC GCGCCGTTAA ACCCTTTCAC
CRY 957 AATGTCTCCG GCCGAGCCGA CCGTCTCCAA GCGTTGTGGC ACAA-CTGTT TCGCTTTCGG GA-CCGGTCC ---GGC-GAC GCGCCGTTAA ACCCTTTCAC
CRY 964 AATGTCTCCG GCCGAGCCGA CCGTCTCCAA GCGTTGTGGC ACAA-CTGTT TCGCTTTCGG GA-CCGGTCC ---GGC-GAC GCGCCGTTAA ACCCTTTCAC
CMW 5231 A-TGTCTCCG GCCGAGCCGA CCGTCTCCAA GCGTTGTGGC ACAA-CTGTT TCGCTTTCGG GA-CCGGTCC ---GGCAGAC GCGCCGTTAA ACCCTTTCAC
CMW 5234 A-TGTCTCCG GCCGAGCCGA CCGTCTCCAA GCGTTGTGGC ACAA-CTGTT TCGCTTTCGG GA-CCGGTCC ---GGCAGAC GCGCCGTTAA ACCCTTTCAC
CMW 5235 A-TGTCTCCG GCCGAGCCGA CCGTCTCCAA GCGTTGTGGC ACAA-CTGTT TCGCTTTCGG GA-CCGGTCC ---GGCAGAC GCGCCGTTAA ACCCTTTCAC
CMW 5236 A-TGTCTCCG GCCGAGCCGA CCGTCTCCAA GCGTTGTGGC ACAA-CTGTT TCGCTTTCGG GA-CCGGTCC ---GGCAGAC GCGCCGTTAA ACCCTTTCAC
CMW 5232 A-TGTCTCCG GCCGAGCCGA CCGTCTCCAA GCGTTGTGGC ACAA-CTGTT TCGCTTTCGG GA-CCGGTCC ---GGCAGAC GCGCCGTTAA ACCCTTTCAC
CMW 5233 A-TGTCTCCG GCCGAGCCGA CCGTCTCCAA GCGTTGTGGC ACAA-CTGTT TCGCTTTCGG GA-CCGGTCC ---GGCAGAC GCGCCGTTAA ACCCTTTCAC
C. ovatum AGCGCGT--G GA-------- ----CTC--- -------GCC TCAAA-T-T- -CCATT--GG CAGCCGGTAT GTTGGC---- ---------- -----TTC--
M. corni AGCGCGT--G GA-------~ ----CTC--- -------GCC TCAAA-T-T- -CCATT--GG CAGCCGGTAT GTTGGC---- ---------- -----TTC--
C. palmarum --TGCGT--G GACTCGCC-- -----T---- ---------- TAAAGC---- --GATT--GG CAGCCGGCAT ATTGGCCGTG GAGCAGCAGT ACA--TTCAG
C. iuckelii -GCGCG-C-G GACTCGCCCC AAAT-TCATT GGCAGCGG-- TCC------- ----TT--GC C------TCC TCTCGCGCAG CACAA-TTGC GTCTGCGGGG
510 520 530 540 550 560 570
CRY 1017 CAAAGGTTGA CG-------- ----TCG-GA T-CAAGTAGG GATACCACGC TGAACTTAAG CATATCAATA AGC
CRY 1056 CAAAGGTTGA CG-------- ----TCG-GA T-CAAGTAGG GATACCACGC TGAACTTAAG CATATCAATA AGC
CRY 1023 CAAAGGTTGA CG-------- ----TCG-GA T-CAAGTAGG GATACCACGC TGAACTTAAG CATATCAATA AGC
CRY 1057 CAAAAGTTGA CC-------- ----TCG-GA T-CAAGTAGG GATACCACGC TGAACTTAAG CATATCAATA AGC
CRY 1000 CAAAAGTTGA CC-------- ----TCG-GA T-CAAGTAGG GATACCACGC TGAACTTAAG CATATCAATA AGC
CRY 1047 CAAAGGTTGA CC-------- ----TCG-GA T-CAAGTAGG GATACCACGC TGAACTTAAG CATATCAATA AGC
CRY 1049 CAAAGGTTGA CC-------- ----TCG-GA T-CAAGTAGG GATACCACGC TGAACTTAAG CATATCAATA AGC
CRY 957 CAAAGGTTGA CC-------- ----TCG-GA T-CAAGTAGG GATACCACGC TGAACTTAAG CATATCAATA AGC
CRY 964 CAAAGGTTGA CC-------- ----TCG-GA T-CAAGTAGG GATACCACGC TGAACTTAAG CATATCAATA AGC
CMW 5231 CAAAGGTTGA CC-------- ----TCG-GA T-CATGTAGG GATACCACGC TGAACTTAAG CATATCAATA AGC
CMW 5234 CAAAGGTTGA CC-------- ----TCG-GA T-CATGTAGG GATACCACGC TGAACTTAAG CATATCAATA AGC
CMW 5235 CAAAGGTTGA CC-------- ----TCG-GA T-CATGTAGG GATACCACGC TGAACTTAAG CATATCAATA AGC
CMW 5236 CAAAGGTTGA CC-------- ----TCG-GA T-CATGTAGG GATACCACGC TGAACTTAAG CATATCAATA AGC
CMW 5232 CAAAAGTTGA CC-------- ----TCG-GA T-CATGTAGG GATACCACGC TGAACTTAAG CATATCAATA AGC
CMW 5233 CAAAGGTTGA CC-------- ----TCG-GA T-CATGTAGG GATACCACGC TGAACTTAAG CATATCAATA AGC
C. ovatum -----GT-GC GCAGCACATT G-CGTCGCGA TTC-T---GG CAGACCTC-C TCCCATTAAG C-TCCTTTCT AGA
M. corni -----GT-GC GCAGCACATT G-CGTCGCGA TTC-T---GG CAGACCTC-C TCCCATTAAG C-TCCTTTCT AGT
C. palmarum CTCTC-TACA CCATAAAGTT GGCAT-CC-A T-CTT-T--- ---------- -GAA------ ---CCNNNNN NNN
C. iuckelii GGGCG-TGGC CCGCGTCCAC GAAGCAACAT TACGTCTTT- ---------- -GAA------ ---CCNNNNN NNN
ID
ID
CRY 1017
CRY 1056
CRY 1023
CRY 1057
CRY 1000 5OUTH AFRICAN
r- ~SOlATES
CRY 1047
CRY 1049
91% CRY 964
CRY 957
"\
CMW 5231
CMW 5234
100% CMW 5235
;0- ...... THAILAND
990/0 CMW 5236 ISOLATES
CMW 5233
CMW 5232
C. palmarum
100% -C. ovatum
99% ......._ M. corn;
C. fuckelii
101
Fig. 3. Dendrogram generated of Coniothyrium zuluense isolates from South Africa
(CRY numbers) and Thailand (CMW numbers), together with four related species [C.
palmarum (CBS 758.73), C. fuckelii (CBS 132.26), Massarina corni (CBS 496.64)
. and C. ovatum], based on AFLP data using UPGMA cluster analysis of pairwise
distance data. The scale represents genetic similarity obtained using the equation of
Nei & Li (1979).
C. fuckelii
C.palmarum
C.ovatum
-
Massar;na corn;
CRY 1056
CMW 5234
~ CMW 5235
.---
CMW 5236
CRY 1047
r- CRY 957
~ CRY 1049
CRY 1000
CRY 964
CRY 1057
CRY 1023
L-
CRY 1017
CMW 5233
r---
L...-- CMW 5231
CCMW 5232
I I I I
3.00 2.25 1.50 0.75 0.00
Nails Genetic Similarity
Appendix 1. Input data matrix used for UPGMA analysis of 177 AFLP markersa
Thai4.3
10000000100100000001010000100110100100000000010000000011100110101000
01001001001000101000000000010100000010000000000000000000100001010010
10000100000000000000000001000100000000000
Thai7.5
10011000100100000001010001100110100100000000010000010011010100101010
01001001001000111000000100010100000010000000010010000000100001010010
10000100000000000010000001000100000000000
Thai14.5
00010010000110000010110001101110110110000010010000001011000110001000
01001001001100011100101010010100110100000000000000010000010001010010
10001000000000000000000001001100000000000
Thai11.5
00000010000110000000010001110100100000000010011000001001000110101000
11001001001100011000100010010010010010000000000000000000000001010010
10000000000000000000000001001100000000000
Thai10.1
10000010000110000000010001100110110100000010010000001011000110001000
11101001001000111100100010010100010000000000000000011000000001010010
10001000000000000000000001000100000000000
Thai1.3
10100001000100000001010001100110100100000000010000000011000111101010
01001001001001101100000000010100000011000000000001000000000001010010
10000100000000000000000000000100000000000
Teza1.4
00101000000100000000010001100110100000000100010000001001000100101010
01011001001000011010110110010100001010000000000000000000100001010100
10001000000000000000000001000100000000000
Teza3.2
00011100110100000000010001110110100000101000010000001011000110101000
01011011001000011010110010010100001000000000000000001000000001010000
10010100000000000000000000000100000000000
Teza18.3
00001001000000000000010001110100100100000010011000001011000100001010
01011001010100011001010100010000001010000000001000011000101001010010
10001110000000100000000000000100100000000
Trus4.1
00000000000100001001010000100100000100000000010000001011001110001000
01000100001001101010100100011100000010100000000000001000000000010100
10000000010000000000000000000100000000010
Trus1.13
00000000100100001001010001101110100000000100010000001011000100001010
01010001001000011010010100011100000000000000000000000000100000010100
10000000010000000000000000000100000000000
PR8.2
01100000100100000000010001110100100000000100010000000001000110101010
0101100101010001101001011001001000101000000000000000000010001'1010100
10000100000000000000000000000100000000000
PR10.3
00000100100110000000010001100110100000000000010000101011100110101000
01101001001000011010110010010100001000000000000000010000100001110010
10001010000000000000000001000100000000000
Fut6.2
00100101001000000001010001100110100100000000010000001011000110101010
01001001001010011010100000011100000000010000010000001000001000010100
10000000110000000000000000000100100000010
Fut2.2
01100100000000000000010001100110110000000010010000001001000110101000
11110011001001011010001010010100001010000000000000001000010001010010
10000100100000000000000000000100000000000
Massarina corni (CBS 496.64)
00000100000000100000011011110101010101001011111100010011100110101110
01001000110100011011001011110010001010010001010001001001000101010100
10000000000000100100010100000001011100000
C.ovatum
10000000000000100000011011100101000101001011111100000011101110101110
01001000110100011010001011110010001010010001010001001001000101010100
10000000000000100000010100000001011100000
C. fuckelii (CBS 132.26)
01000001000011011000010100100000111000110010010010000000000110111001
01010000101000001001010000110001100100000000101100110010100000011001
00000001010110010101100110110000000010101
C. palmarum (CBS 758.73)
00000001010010000100110000100000101001011100110101110110010101001010
11101010000110101010101010010100010100001110000001101100000001011001
01100110001001001000001001000010001001000
"Each text string shows the name of different Coniothyrium zuluense isolates
together with related species, as well as, the presence (1) and absence (0) of AFLP
markers.
102
CHAPTER 5
A synerqistic relationship between the Eucalyptus canker
pathogen, Coniothyrium zuluense, and two Pantoea species
Coniothyrium zuluense is the causal agent of a serious Eucalyptus stem canker
disease in South Africa. Despite this fact, very little is known about its biology.
Bacteria commonly exude from necrotic cankers or severely infected Eucalyptus
clones in plantations. Isolation from cankers has shown that two bacterial species
commonly occur, together with C. zuluense. The objectives of this study were to
identify these bacteria and to consider whether they influence pathogenicity of C.
zuluense. Bacteria were identified using the Biolog identification system, as well as
by 16S rRNA gene sequence. Sequence data were then compared to those of
related Enterobacteriaceae. Combined and individual inoculation studies, using both
bacterial and fungal isolates, were conducted on fresh Granny Smith apples, as well
as on a susceptible Eucalyptus grandis clone. We constructed a phylogenetic tree
based on 16S rDNA sequence data and found that one bacterium is Pantoea
ananatis pv. ananatis and the other appears to be probably an undescribed Pantoea
species. Combined fungal and bacterial inoculations resulted in a significant
increase in pathogenicity as opposed to individual inoculations. Results indicate a
synergistic interaction between C. zuluense and the two Pantoea species in disease
development.
103
Coniothyrium zuluense Wingfield, Crous & Coutinho was first reported in South Africa
by Wingfield et al. (1997). The disease, Coniothyrium canker, poses a serious threat
to the local forestry industry where Eucalyptus species are cultivated extensively in
plantations. The South African forestry industry depends largely on vegetatively
propagated Eucalyptus species and, therefore, these genetically uniform stands are
at risk from diseases such as Coniothyrium canker (Wingfield et al., 1997).
Strategies to ensure that large numbers of different clones are planted and that a
high degree of genetic diversity is maintained in clonal plantations, have thus been
implemented.
Initial C. zuluense infections occur on the young, green stem tissue during the peak
of the growing season. It has been shown that once conidia germinate, the germ
tubes infect the stems directly through the epidermis of the young tissue (Wingfield et
al., unpublished data). Small necrotic lesions are then formed that coalesce to form
large necrotic spindle-shaped cankers (Coutinho et al., 1997; Wingfield et al., 1997).
It is thought that conidia are dispersed through rain that flows down the stem and
thus ultimately leads to the development of a series of stems cankers representing
annual infection events (Coutinho et al., 1997; Wingfield et al., 1997).
Considerable variation in resistance to Coniothyrium canker exists within and among
Eucalyptus species (Coutinho et al., 1997; Wingfield et al., 1997). Many E. grandis
Hill ex. Maid. clones currently available for planting show susceptibility to infection.
Some hybrid clones of E. grandis with E. urophylla S. T. Slake, E. camaldulensis
Dehnh. or E. nitens (Deane et Maid.) Maid. are highly resistant to C. zuluense
infection. Recent evidence, however, suggests that disease resistant clones are
beginning to show signs of infection (Wingfield et et., 1997). This is unexpected as C.
zuluense in South Africa is represented by a uniform population structure, indicative
of an asexually reproducing fungus (Van Zyl et al., unpublished data). Generally this
would suggest that the capacity of the pathogen to adapt and overcome tree disease
resistance mechanisms, would be limited.
In a study undertaken to collect isolates of C. zuluense from severely infected
Eucalyptus clones, it was found that bacteria commonly exude from cankers. Two
bacterial species were also frequently isolated together with single conidia of C.
104
zuluense. The objectives of this study are, therefore, to identify the bacterial species
and to consider their possible role in lesion development.
MATERIALS AND MIETHODS
Bacterial and fungal isolates
Bacterial masses exuding from cankers on severely infected Eucalyptus clones were
collected (Fig. 1). It was evident from isolations that two bacterial species were
present. Bacteria with colony morphologies similar to those obtained from
Coniothyrium cankers were also frequently isolated in association with single conidia
of C. zuluense (Fig. 2). Ten isolates of each bacterial species were used for further
investigation (Table 1).
Six C. zuluense isolates were used in this study (Table 2). Fungal isolates (CRY
1016, CRY 1023, CRY 1054, CRY 1055, CMW 1778, CMW 2100) were selected
based on their association with the two bacterial species (Table 2). Choice of these
Coniothyrium isolates was also based on the fact that they represent a range of
levels of pathogenicity including non-pathogenic, intermediately pathogenic and
highly pathogenic, as determined in a previous study (Van Zyl et al., 1997). All fungal
and bacterial isolates are maintained in the culture collection of the Forestry and
Agricultural Biotechnology Institute (FABI), University of Pretoria.
Identification of bacteria
Gram negative microplate technique. Pure bacterial cultures were obtained by
streaking isolates on the Biolog Universal Growth Medium (BUGM) (Biolog, CA).
Once pure cultures had been obtained, characteristics, such as Gram stain and
colony morphology, were determined. Identification was carried out using the Biolog
Gram negative microplate technique (GN MicroPlate™, Biolog, CA), together with
Biolog's Microlog ™ 1, Microlog 2, or Microlog 3 computer software programmes
(Biolog, CA).
105
DNA sequence comparisons. Bacterial isolates (one isolate for each bacterium)
were grown overnight in 5 ml of LB broth (10 g Tryptone (Merck); 5 g Yeast Extract
(Merck); 5 g NaCI (Merck); 1 I distilled H20) and pelleted by centrifugation. Nucleic
acid was extracted and purified as described by Hauben et al. (1997). DNA pellets
were dried and suspended in 100 1-11 H20. Nucleic acid extracts were quantified by
fluorometry and adjusted to a final concentration of 30 ng I ul.
Two primers were used for amplification of 16S rDNA (Table 2). Bacterial DNA was
added to a solution (100 ul) containing 2 units of Taq DNA polymerase (Boehringer
Mannheim, Germany), 10 x reaction buffer (Boehringer), 1.5 mM MgCI2 (Boehringer),
20 mM dNTPs and 0.5 !lI of each primer (100 pM). Amplification reactions were done
in a Hybaid Omnigene Temperature Cycler (Hybaid, Middlesex, U.K.). The samples
were processed through an initial extensive denaturation step consisting of 94°C for
2 min. This was followed by 30 amplification cycles consisting of 1 min at 92 °C, 30
sec of annealing at 54°C, and 1 min of primer extension at 72 °C. Final chain
elongation took place at 72°C for 10 min. PCR products were electrophoresed in 1.5
% agarose gels, stained with ethidium bromide, and visualised using UV light.
Amplification reactions were done in duplicate.
i
PCR products were purified using a QIAquick gel extraction kit (Qiagene GmgH,
Hilden, Germany). Purified products were then sequenced in both directions using
the Big Dye Cycle 8equencing kit with Amplitaq® DNA polymerase, F8 (Perkin-
Elmer, Warrington, UK) on a ABI PRI8M™ 377 DNA automatic sequencer (Perkin-
Elmer). Nearly complete 168 rDNA sequences were determined by constructing
internal primers (Table 3). Nucleotide sequences were manually aligned.
In order to consider the relationship between the bacterial species with related
species in the family Enterobacteriaceae, 168 rDNA sequence data for
phytopathogenic species of the genera Erwinia, Pantoea, Pectobacterium and
Brenneria were obtained from GenBank (Table 4). All of these species were
previously known as members of the genus Erwinia (Hauben et al., 1998).
106
Relationships between species were determined by the neighbour-joining method of
Satou & Nei (1987) using the neighbour-joining program of PAUP 4.0 (Phylogenetic
Analysis Using Parsimony) (Swofford, 1993). Trees were rooted to two outgroup
taxa, Buchnera aphidicola (L18927) and Proteus vulgaris (J01874), as previously
shown by Hauben et al. (1998). The stability of the relationships was assessed using
the BOOTSTRAP (Bootstrap confidence intervals on DNA parsimony) method (1000
replicates) (Felsenstein, 1993).
Pathogenicity tests
On apples. Granny Smith apples were inoculated with 10 isolates of each of the two
bacterial species. For each isolate, five apples were inoculated by removing a 5 mm
diameter disk of tissue, and replacing this with discs of agar bearing similar amounts
of bacteria (3.4 x 107 CFU / ml). All wounds were closed with masking tape to
prevent desiccation of the inoculum and wounds. Apples were then incubated at 25
°C for 7 days and lesion diameters were measured. Means were tested for
significance using Tukey's procedure (ANOVA analysis, NCSS97). The isolate of
each of the two bacterial species producing the largest lesions was then selected for
further study.
Granny Smith apples were inoculated with pure bacteria-free C. zuluense isolates
and each of the bacterial isolates selected in preliminary screening. At first, apples
were inoculated with each of six C. zuluense isolates that were free of bacteria.
Similarly, both of the pre-selected pathogenic isolates of the two bacterial species
were inoculated individually. One C. zuluense isolate that had been associated with
bacteria at the time of isolation was then selected from non-pathogenic and
intermediate pathogenic isolates, as well as isolates showing high pathogenicity. An
additional pathogenic isolate of C. zuluense was also included. Each of these four
C. zuluense isolates, CRY 1055, CRY 1016, CRY 1023 and CMW 2100, were then
inoculated alone and in combination with each of the two bacterial species. In
addition to this, each of these fungal isolates was inoculated together with both
bacterial species. In each case, five apples were inoculated for each treatment.
Inoculations were done by removing a 5 mm diameter disk of tissue, and replacing
107
these with individual discs of agar bearing either fungal or bacterial isolates. Sterile
uninoculated agar discs were used to inoculate five apples to serve as control
treatments. All wounds were closed with masking tape to prevent desiccation of the
inoculum and wounds. Apples were then incubated at 25°C for 7 days and lesion
diameters were measured. Means were tested for significance using Tukey's
procedure (ANOVA analysis, NCSS97). The entire experiment was repeated once.
On trees. Coniothyrium zuluense and the two bacterial species were inoculated
into young green stem tissue on a susceptible one-year-old E. grandis clone (ZG 14)
in the Zululand forestry region, KwaZulu-Natal. Treatments were the same as those
on apples where six C. zuluense isolates, as well as the two Pantoea species were
initially inoculated alone. The selected four C. zuluense isolates (same as those
selected for the apple experiment) were then inoculated in combination with each of
the two bacteria, followed by the inoculation of each fungal isolate together with both
Pantoea species. Twenty trees were inoculated for each treatment. Inoculations
were done by removing a 10 mm diameter disc of bark from trees at breast height,
and replacing this with individual discs of agar bearing either fungal or bacterial
growth, respectively. Sterile uninoculated agar discs were used to inoculate twenty
trees to serve as control treatments. Inoculation wounds were covered with
masking tape to prevent desiccation of the inoculum. Lesion lengths were
measured six weeks after inoculation. Means were tested for significance using
Tukey's procedure (ANOVA analysis, NCSS97). The entire experiment was
repeated once.
RESULTS
Identification of bacteria
Gram negative microplate technique. Two bacterial species were isolated from
surface lesions in the field, as well as occurring together with single conidial isolates
of C. zuluense. These species were characterised by Gram negative straight rods
varying between 0.5 - 1.0 urn in width and 1.0 - 3.0 um in length. Computer analysis
of the data using the Biolog Gram negative microplate technique showed that both
108
bacteria were Enterobacteriaceae. One bacterium was identified as Erwinia ananas
with a Biolog similarity index ranging between 0.540 and 0.543 after 24 hours.
Colonies of this bacterium have a typically yellow pigment, domed, shining, and
mucoid after 2 or 3 days at 27°C. Biolog analysis indicated that the other bacterium
was also a species of Erwinia. However, it was not possible to identify a specific
taxon for it. Colonies are characteristically white with a slight yellow pigment and
mucoid after 2 or 3 days at 27°C.
DNA sequence comparisons. Direct sequencing of the PCR-amplified 16S rDNAs,
allowed us to determine a continuous stretch of 1478 base pairs (bp) for the unknown
Erwinia species and 1472 bp for E. ananas. Manual alignment of 16S rDNA
sequences gave rise to a total alignment length of 1470 bp (Fig. 3).
The 16S rDNA sequences of the suspected Erwinia species were compared with 16S
rDNA sequences of other related Enterobacteriaceae (genera Erwinia, Pantoea,
Pectobacterium and Brenneria) obtained from GenBank. The levels of 16S rDNA
sequence similarity for species used in this study, ranged from 91 % to 99.9 %.
Sequence similarity of the bacterium tentatively identified as E. ananas was 99.9 %
homologous to Pantoea ananatis pv. ananatis, followed by P. ananatis pv. uredovora
(99.8 %) and P. agglomerans (99 %). 16S rDNA sequence similarity of the unknown
Erwinia species was found to be 98 % homologous to Pantoea stewartii subspecies
stewartii, followed by Erwinia psidii (96.6 %). The levels of 16S rDNA sequence
similarity are shown in Table 5.
A phylogenetic tree was constructed using distance matrix data obtained from the
16S rDNA sequences and the neighbour-joining method (Fig. 4). The tree was
based on 1470 pairwise aligned sequence similarities. The 16S rDNA sequences of
both Erwinia species from C. zuluense cankers clustered together with species of
Pantoea (Fig. 4). Phylogenetic analysis indicated that the purported E. ananas
isolates from South Africa are closely related to P. ananatis pv. ananatis (synonym E.
ananas), with a more distant relatedness to P. ananatis pv. uredovora (synonym, E.
uredovora) followed by P. agglomerans (synonym, E. herbicola). Sequence data,
thus supported results obtained using the Biolog identification system in that this
bacterium associated with C. zuluense, is P. ananatis pv. ananatis.
109
Phylogenetic analysis indicated a close relationship between the unknown Erwinia
species from C. zuluense cankers and that of phytopathogenic bacterium, P. stewartii
subspecies stewartii (synonym E. stewartii). A more distant phylogenetic relationship
was evident to E. psidii, E. tracheiphila and E. mollotivora, respectively. It is, thus
evident that the unknown Erwinia species from South Africa is most closely related to
P. stewartii subspecies stewartii. However, based on a somewhat low level of
similarity, this bacterium most probably represents a new species of Pantoea.
Pathogenicity tests
On apples. All bacterial isolates were able to cause tissue maceration (Table 6). No
significant differences (P = 0.05) in lesion diameter were evident among isolates of
the same bacterial species. Significant differences in lesion diameters were,
however, evident between isolates of the two different bacteria (Table 6). Based on
this, we selected one isolate of each bacterial species producing the largest lesions
(Table 6, highlighted in bold). These isolates were then used for further
investigations of a possible synergistic effect between bacteria and C. zuluense.
Inoculations of individual C. zuluense isolates showed that little tissue maceration
was evident when inoculated into Granny Smith apples (Table 7; Fig. 5A). No
significant differences (P = 0.05) were evident between C. zuluense isolates varying
in their pathogenicity to a susceptible E. grandis clone. Both Pantoea species,
however, caused severe tissue maceration that significantly (P = 0.05) distinguished
them from C. zuluense isolates (Table 7; Fig. 5B). Pantoea ananatis pv. ananatis
produced significantly larger lesions than the unknown Pantoea species (Table 7).
Significant differences (P = 0.05) in lesion diameter were evident when each of the
four selected C. zuluense isolates were inoculated in combination with each of the
two Pantoea species (Table 7; Fig. 5C). Inoculation of the non-pathogenic or
intermediately pathogenic C. zuluense isolates (CRY 1055 and CRY 1016) in
combination with either P. ananatis pv. ananatis or the unknown Pantoea species,
produced significantly larger lesions compared with those using these fungi alone.
110
This enhanced effect was especially obvious when pathogenic C. zuluense isolates
(CMW 2100 and CRY 1023) were inoculated in combination with either P. ananatis
pv. ananatis or the unknown Pantoea sp. (Table 7).
Inoculation results where each of the four selected C. zuluense isolates were
inoculated together with both Pantoea species, showed a significant (P = 0.05)
increase in lesion development as compared with those obtained when the fungi
were inoculated alone or in combination with each of the two Pantoea species (Table
7). The only exception was observed when non-pathogenic C. zuluense isolate, CRY
1055, showed no increase in lesion length when inoculated in combination with both
bacteria (Table 7).
On trees. All C. zuluense isolates were able to produce lesions on a susceptible E.
grandis clone (ZG 14) when inoculated on their own and in the absence of bacteria
(Table 7; Fig. 68). Neither of the Pantoea species were able to cause lesions alone
(Table 7; Fig. 6A). Using ANOVA, it was possible to identify significant differences (P
= 0.05) among lesion lengths produced by different C. zuluense isolates (Table 7).
Thus, previously designated pathogenic C. zuluense isolates produced significantly
larger lesions than those of the selected intermediate and non-pathogenic isolates.
Lesion lengths were the greatest for isolate, CRY 1023 which differed significantly
from isolate CMW 2100 (Table 7).
When Coniothyrium isolates were inoculated in combination with either P. ananatis
pv. ananatis or the unknown Pantoea species, significantly larger lesions developed
(P = 0.05), compared to inoculations where the fungus was used alone (Table 7).
Results showed that P. ananatis pv. ananatis inoculated in combination with each of
the C. zuluense isolates led to a significant increase in lesion size (Table 7). Similar
results were obtained when the unknown Pantoea species was used, although the
synergistic effect was less obvious (Table 7). The inoculation of both Pantoea
species together with C. zuluense produced lesions that were significantly larger (P =
0.05) than when C. zuluense isolates were inoculated alone or in combination with
either P. ananatis pv. ananatis or the unknown Pantoea sp. (Table 7, Fig. 6C).
111
Results obtained from inoculating the susceptible E. grandis clone, ZG 14, correlated
significantly (rs = 0.74) with data obtained after inoculating Granny Smith apples. The
main difference between the two inoculation trials was that both Pantoea species
were unable to cause disease symptoms on the susceptible Eucalyptus clone when
inoculated alone (Table 7; Fig. 6).
DISCUSSION
In this study, we have shown that bacteria found exuding from cankers caused by C.
zuluense and in association with single isolated conidia, represent two species of
Pantoea. Most Gram-negative bacteria in the Enterobacteriaceae colonise the
apoplast of plants and produce a range of symptoms on virtually all crop plants
(Alfano & Collmer, 1996). Phytopathogenic species of Brenneria, Enterobacter,
Erwinia, Pantoea, and Pectobacterium are particularly well known for causing blights,
cankers, die back, leaf spots, wilts and soft-rots on a wide range of plant species
(Starr & Chatterjee, 1972; Perombelon, 1980; Hauben et al., 1998). It is, therefore,
not surprising that the two Pantoea species reported in this study are associated with
disease.
Analysis of Biolog data enabled us to identify one of the two bacterial species. It
was, however, not possible to identify a specific taxon for the second bacterium.
Identification of this bacterium was made possible by sequencing the 16S rRNA
gene. One bacterium was identified as P. ananatis pv. ananatis (synonym, Erwinia
ananas) and the other has shown to be closely related to P. stewartii subsp. stewartii.
Analysis of 16S rDNA sequences has previously been used to successfully determine
phylogenetic, as well as, inter- and intrageneric relationships between bacterial
species (Bereswill et al., 1995; Leblond-Bourget et al., 1996; Hauben et al., 1997;
Kwon et al., 1997; Hauben et al., 1998; Kim et al., 1999). This is due to the existence
of conserved regions for all bacterial genera within the 16S rRNA gene, together with
smaller parts that are variable to accurately determine phylogenies (Massol-Deya,
1995; Hauben et al., 1997; Kwon et al., 1997; Hauben et al., 1998; Kim et al., 1999).
112
Pantoea ananatis pv. ananatis is the causal agent of brown rot of pineapple (Ananas
comosus Schuit. f.) fruitlets (Fahy & Persley, 1983; Mergaert et al., 1993). It has also
previously been reported to occur on sugar cane (Saccharum L. hybrids) (Elliott,
1951; Fahy & Persley, 1983). As far as we are aware, this is the first report of this
bacterial pathogen from South Africa. The second unknown Pantoea sp. was shown
to be closely related to P. stewartii subsp. stewartii (synonym, Erwinia stewartii),
however, nucleotide differences of the 16S rDNA sequences indicated that this
bacterium is probably new to science. Pantoea stewartii subsp. stewartii is the
causative agent of Stewart's bacterial wilt of maize (Zea mays L.) and sweet corn
(Zea mays var. rugosa L.) (Fahy & Persley, 1983; Mergaert et al., 1993). Further
biochemical, genotypic, as well as pathogenicity characteristics are needed before
this South African species can be fully identified.
Both Pantoea species were able to cause significant tissue maceration on Granny
Smith apples when inoculated alone. In contrast to this, no lesion development was
evident when each of the two Pantoea species was inoculated into a susceptible E.
grandis clone. Similarly, pure bacteria-free C. zuluense isolates produced almost no
tissue maceration on Granny Smith apples, whereas, significant lesions were
produced when they were inoculated into a susceptible E. grandis clone (ZG 14).
However, inoculation of C. zuluense isolates in combination with either P. ananatis
pv. ananatis or the unknown Pantoea species, or both these species on Granny
Smith apples, showed an average of a 307 % increase in lesion diameter relative to
the inoculation of fungal isolates alone. This was also true for field inoculations of a
susceptible E. grandis clone (ZG 14) where a significant increase (43 %) in lesion
length was observed. Results of this study, therefore, indicate a strong synergistic
interaction between C. zuluense and both Pantoea species.
Synergism refers to a simultaneous effect of two organisms or environmental factors
acting together, producing a change greater than either could alone (Hawksworth et
al., 1995). Results of this study indicate a strong synergistic effect between C.
zuluense and both Pantoea species. Similar synergistic interactions between fungi
and bacteria have been reported previously. Toro et al., (1996) showed that
combined fungal and bacterial inoculations of the mycorrhizal fungus, Glomus
mosseae (Nicol & Gerd) Gerd & Trappe, and species of rhizosphere calcium-
113
phosphate solubilizing bacteria, Bacillus and Pseudomonas, resulted in a 283 %
increase in growth of Pueraria phasealoides (Kudzu) relative to the uninoculated
controls. Similarly, Hodrova et al. (1995) showed that eo-culture inoculations of
anaerobic rumen fungus, Orpinomyces joyonii, together with two bacteria species,
Megasphaera elsdenii and Eubacterium limosum, resulted in an increase of between
7.96 % to 10.12 % of microcrystalline cellulose degradation.
In a previous study, Van Zyl et al. (1997), showed a very large variation in
pathogenicity among C. zuluense isolates. -It was found that 78 % of a large
collection of C. zuluense isolates (344 isolates in total) were not able to cause lesions
when inoculated into a susceptible E. grandis clone (ZG 14). At that stage, we were
unable to explain this phenomenon in that all isolates were collected from severely
infected Eucalyptus species and clones. However, the discovery of a synergistic
interaction between C. zuluense and both Pantoea species in the present study,
might provide a possible explanation for this anomaly. All C. zuluense isolates were
purified of all bacteria prior to pathogenicity studies in the field. This might be the
reason why such a large variation in virulence was evident. In the presence of the
bacteria, many non-pathogenic C. zuluense isolates might have resulted in disease
development. This question deserves further study.
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116
Table 1. Isolates of the two Pantoea species found in association with Coniothyrium
zuluense.
Bacterial species Strain no." Plantations of origin in
KwaZulu-Natal
Pantoea ananatis pv. ananatis Ea1 Fairbreeze
" Ea2 Futululu
Ea3 Teza
Ea4 Palm Ridge
Ea5 Trust
" Ea6 Honey Farm
" Ea? Teranera
" EaB Aboyni
Ea9 Palm Ridge
" Ea10 Teza
Unknown Pantoea species uE1 Futululu
" uE2 Palm Ridge
" uE3 Teza
" uE4 Teza
uE5 Honey Farm
uE6 Shire
" uE? Teranera
" uEB Palm Ridge
" uE9 Honey Farm
uE10 Teza
a Bacterial strains are stored in the culture collection of Dr. K-H Riedl,
Department of Microbiology and Biochemistry, University of the Orange Free State,
Bloemfontein.
117
Table 2. List of Coniothyrium zuluense isolates used in this study.
Culture number" OriginO Pathogenicity to an
Eucalyptus grandis clone (ZG 14)C
CRY 1055 Zululand, KZN non-pathogenic
CRY 1054 " non-pathogenic
CRY 1016 " intermediately pathogenic
CRY 1023 pathogenic
CMW 1778 intermediately pathogenic
CMW 2100 " pathogenic
aCMW and CRY numbers represent C. zuluense isolates maintained in the
culture collection of the Tree Pathology Co-operative Programme (TPCP), Forestry
and Agricultural Biotechnology Institute (FABI), University of Pretoria, South Africa.
bAil isolates were collected from diseased Eucalyptus species, clones and
hybrids in South Africa. KZN refers to the KwaZulu-Natal Province.
"Pathoqenlcity of relevant isolates was determined in a previous study (Van
Zyl et al., 1997).
Table 3: Primer sequences for the 16S rRNA gene amplification.
Primer Sequence (5'-3') Target region Reference
PA AGA err TGA TCC TGG CTC AG 1.5 kb DNA fragment from 16S rDNA region Massol-Deya et al., 1995
PH AAG GAG GTG ATC CAG CCG CA " "
OT1 GAA GM GGC err CGG GTT G Internal Current study
OT2 CAC GAC ACG AGC TGA CGA C Internal Current study
-
0-0
Table 4: List of bacterial strains considered in this study together with the Gen8ank accession numbers for their 16S rDNA
sequence.
Spedesorsubspedes Synonyms Gen8ank accession numbers
Pantoea ananatis pv. ananatis Erwinia ananas U80196
Pantoea ananatis pv. uredovora Erwinia uredovora U80209
Pantoea agglomerans Erwinia herbicola U80202
Pantoea agglomerans Erwinia milletiae U80183;AB004757
Pantoea stewartii subspecies stewartii Erwinia stewartii U80208
Erwinia tracheiphila Y13250
Erwinia psidii Z96085
Erwinia mallotivora AJ233414;Z96084
Erwinia persicinus AJ001190;Z96086U80205
Erwinia rhapontici AJ233417;Z96087U80206
Erwinia amylovora Z96088;AJ233410AJ010485; U80195
Erwinia pyrifoliae AJ009930
Pectobacterium cypripedii Erwinia cypripedii Z96094
Pectobacterium chrysanthemi Erwinia chrysanthemi AJ233412
Pectobacterium carotovorum subspecies carotovorum Erwinia carotovora subspecies carotovora Z96089
Pectobacterium carotovorum subspecies odoriferum Erwinia carotovora subspecies odoriferum AJ223407
Pectobacterium carotovorum subspecies wasabiae Erwinia carotovora subspecies wasabiae AJ223408
Pectobacterium carotovorum subspecies betavasculorum Erwinia carotovora subspecies betavasculorum U80198
Pectobectetium carotovorum subspecies atrosepticum Erwinia carotovora subspecies atrosepticum Z96090
Pectobacterium cacticidum Erwinia cacticida Z96092
8renneria selleis Erwinia salicis Z96097
8renneria rubrifaciens Erwinia rubrifaciens AJ233418
8renneria paradisiaca Erwinia paradisiaca Z96096
8renneria nigrifluens Erwinia nigrifluens AJ233415
8renneria alni Erwinia alni AJ233409
......
\Cl
Table 5: Percentages of sequence similarity based on 16S rDNA sequences.
% 165 rDNA sequence similarity
Organism 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16
1 Erwinia ananas, Zululand, KZN, SAa 100
2 Unknown Erwinia species, Zululand, KZN, SAa 96.7 100
3 Pantoea ananatis Py. ananatis (U80196) 99.911 96.5 100
4 Pantoea ananatis Py. uredovora (U80209) 99.8 96.5 99.9 100
5 Pantoea agglomerans (U80202) ,99.0 96.4 99.0 98.9 100
6 Pantoea agglomerans (AB004757) 98.8 96.6 98.7 98.6 99.0 100
-,
7 Pantoea agglomerans (U80183) 98.2 96.2 98.1 98.0 98.8 99.3 100
8 Pantoea stewartii subspecies stewartii (U80208) 97.4 98.0 97.4 97.4 97.1 96.9 96.5 100
9 Erwinia amylovora (Z96088) 95.4 95.8 95.3 95.2 95.2 95.5 95.1 95.9 100
10 Erwinia tracheiphila (Y13250) 95.7 95.1 95.6 95.5 95.6 95.3 95.2 95.4 94.8 100
11 Erwinia psidii (Z96085) 95.4 96.6 95.2 95.2 95.2 95.0 94.8 96.6 95.3 96.0 100
12 Erwinia mallotivora (Z96084) 94.5 96.1 94.4 94.3 94.4 94.9 94.7 95.9 96.3 94.8 96.8 100
13 Pectobacterium carotovorum subspecies carotovorum (Z96089) 95.2 96.3 95.1 95.0 95.1 95.5 95.4 95.4 95.2 95.0 95.2 94.7 100
14 Brenneria salicis (Z96097) 93.3 92.7 93.3 93.3 92.9 92.8 92.8 92.5 93.2 91.1 91.4 92.9 94.3 100
15 Proteus vulgaris (JO1874) 91.1 91.6 91.1 91.0 91.6 91.4 91.2 91.0 91.4 90.1 91.0 91.7 92.0 91.6 100
16 Buchnera aphidicola (L 18927) 86.8 86.5 86.7 86.6 86.3 86.5 86.6 86.4 86.8 85.4 87.3 86.2 86.8 85.8 85.1 100
"The sequences of both South African Erwinia species were determined in this study; the other sequences were obtained from the
Gen8ank database.
b The strains correspond with the strains in bold in Fig. 4.
-N
<::)
121
Table 6. Lesions produced on Granny Smith apples inoculated with each of the two
Pantoea species
Bacterial species Strain no." Lesion diameter (mm)6
Pantoea ananatis pv. ananatis Ea1 23.5 b
" Ea2 26.1 b
" Ea3 24.6 b
Ea4 23.6 b
Ea5 25.6 b
" Ea6 25.3 b
Ea7 24.9 b
Ea8 23.5 b
" Ea9 24.1 b
" Ea10 25.4 b
Unknown Pantoea species UE1 15.5 a
UE2 15.7 a
" UE3 16.3 a
" UE4 14.9 a
" UE5 16.2 a
UE6 16.5 a
UE7 18.2 a
" UE8 16.3 a
UE9 16.1 a
UE10 16.8 a
"One isolate of each bacterial species (bold) was selected for further
inoculation.
bValues are the means of two duplicate sets of five inoculated apples. CV =
10.3 %. Values followed by the same letter are not significantly different at P = 0.05.
122
Table 7. Comparison of pathogenicity after inoculation with Coniothyrium zuluense
and two Pantoea species on apples and Eucalyptus trees.
Pathogenicity study
Apples Trees C
Isolates a Lesion diam. (mm) Lesion length (mm)
Exp.#1 Exp.#2 Exp.#1 Exp.#2
Control 5.0 ad 5.0 ad 10.0 ae 10.0 ae
CRY 1055 5.9 a 5.0 a 10.0 a 10.0 a
CRY 1054 5.0 a 5.0 a 10.0 a 10.1 a
CMW 1778 7.2 a 8.1 b 27.2 cd 26.5 de
CRY 1016 7.0. a 7.6 b 27.1 cd 27.9 de
CMW 2100 11.1 b 10.8 c 37.2 ef 36.8 fg
CRY 1023 13.0 b 13.4 d 59.2 i 57.3j
P. a. pv. ananatis (Strain Ea2) 25.9 d 23.9 fg 10.7 a 10.0 a
Pantoea sp. (Strain uE7) 18.1 c 19.4 e 10.1 a 10.0 a
CRY 1055 + P.a. pv. ananatis 25.8 d 25.3 gh 18.0 b 20.5 c
CRY 1016 + P.a. pv. ananatis 31.4 ef 29.8 i 34.1 e 35.8 f
CMW 2100 + P.a. pv. ananatis 37.2 g 39.6 k 47.4 h 46.3 i
CRY 1023 + P.a. pv. ananatis 40.5 h 40.0 k 71.6 k 72.0 I
CRY 1055 + Pantoea species 20.6 c 22.4 f 14.8 b 15.8 b
CRY 1016 + Pantoea species 23.4 d 22.9 f 29.7 d 28.5 e
CMW 2100 + Pantoea species 30.7 e 32.8 j 41.0 fg 41.8 h
CRY 1023 + Pantoea species 31.8 ef 32.8j 64.9j 63.4 k
CRY 1055 + P. a. pv. ananatis + Pantoea sp. 25.7 d 26.5 h 24.8 c 24.0 cd
CRY 1016 + P. a. pv. ananatis + Pantoea sp. 33.9 f 33.4 j 42.4 g 40.6 gh
CMW 2100 + P. a. pv. ananatis + Pantoea sp. 40.0 h 40.9 k 61.0 j 58.9 j
CRY 1023 + P. a. pv. ananatis + Pantoea sp. 45.5 i 45.0 I 82.81 80.7 m
aCRY and CMW numbers refer to C. zuluense isolates used in this study. Two
Pantoea species, P. ananatis pv. ananatis and an unknown Pantoea species, are
isolated in association with C. zuluense. All isolates were collected from severely
infected Eucalyptus trees in KwaZulu-Natal.
bFresh Granny Smith apples were used in this pathogenicity study.
cA susceptible E. grandis clone, ZG 14, was used for field conditions.
dValues are the means of five inoculated apples (Exp. #1: CV = 11.3 %) (Exp.
#2: CV = 10.5 %). Values followed by the same letter are not significantly different at
P = 0.05. Exp. # 1 and 2 represents two separate inoculation studies.
"Values are the means of twenty inoculated trees (Exp. #1: CV = 18.5 %)
(Exp. #2: CV = 16.7 %). Values followed by the same letter are not significantly
different at P = 0.05. Exp. # 1 and 2 represents two separate inoculation studies.

124
Fig. 3. Aligned nucleotide sequences data for the 16S rDNA of both South African
Erwinia species, as well as species of the genera Pantoea, Erwinia, Pectobacterium,
Brenneria, Proteus and Buchnera (Table 3). All sequences, except those of the two
South African species were obtained from the GenBank database (Table 3). Gaps
that were inserted due to alignment are indicated by a dash (-). N indicates unknown
bases.
10 20 30 40 50 60 70 80
Buchnera aphidicola AACACATGCA AGTCGAGCGG CAGCGAAAAG AAAGCTTGCT TTCTTGTCGG -CGAGCGGCA AACGGGTGAG TAATATCTGG
Proteus vulgaris AACACATGCA AGTCGAGCGG TAACAGGAGA AA-GCTTGCT TTCTTGCTGA -CGAGCGGCG GACGGGTGAG TAATGTATGG
Erwinia ananas, Zululand, KZN, SA AACACATGCA AGTCGGACGG TAGCACAGA- GA-GCTTGCT CTC-GGGTGA -CGAGTGGCG GACGGGTGAG TAATGTCTGG
Pantoea a. Py. ananatis AACACATGCA AGTCGGACGG TAGCACAGA- GA-GCTTGCT CTC-GGGTGA -CGAGTGGCG GACGGGTGAG TAATGTCTGG
Pantoea a. Py. uredovora AACACATGCA AGTCGGACGG TAGCACAGA- GA-GCTTGCT CTC-GTGTGA -CGAGTGGCG GACGGGTGAG TAATGTCTGG
Pantoea agglomerans (U80202) AACACATGCA AGTCGGACGG TAGCACAGA- GA-GCTTGCT CTC-GGGTGA -CGAGTGGCG GACGGGTGAG TAATGTCTGG
Pantoea agglomerans (AB004757) AACACATGCA AGTCGGACGG TAGCACAGA- GA-GCTTGCT CTC-GGGTGA -CGAGTGGCG GACGGGTGAG TAATGTCTGG
Pantoea agglomerans (U80183) AACACATGCA AGTCGGACGG TAGCACAGAG GA-GCTTGCT CTCTGGGTGA -CGAGTGGCG GACGGGTGAG TAATGTCTGG
Unknown Erwinia sp. AACACATGCA AGTCGAGCGG TAGCACAGAG GA-GCTTGCT CTC-GCCGGA -CGAGTGGCG GACGGGTGAG TAATGTCTGG
Pantoea s. subsp. stewartii AACACATGCA AGTCGGACGG TAGCACAGAG GA-GCTTGCT CTC-GGGTGA -CGAGTGGCG GACGGGTGAG TAATGTCTGG
Erwinia tracheiphila AACACATGCA AGTCGGACGG TAGCACAGAA GA-GCGTGCT CTTTGGGTGA -CGAGTGGCG GACGGGTGAG TAATGTCTGG
Erwinia psidii AACACATGCA AGTCGAACGG TAGCGGGAAG AA-GCTTGCT TCTTTGCCGA -CGAGTGGCG GACGGGTGAG TAATGTCTGG
Erwinia mallotivora (AJ2334l4) AACACATGCA AGTCGAACGG TAGCACAGGG GA-GCTTGCT CCCTGGGTGA -CGAGTGGCG GACGGGTGAG TAATGTCTGG
Erwinia mallotivora (Z96084) AACACATCCA AGTCGAACGG TAGCACAGGG GA-GCTTCCT CCCTGGGTGA -CGAGTGGCG GACGGGTGAG TAATGTCTGG
Erwinia persicinus (AJOOl190) AACACATGCA AGTCGAACGG TAGCACAGA- GA-GCTTGCT CTC-GGGTGA -CGAGTGGCG GACGGGTGAG TAATGTCTGG
Erwinia persicinus (Z96086) AACACATGCA AGTCGAACGG TAGCACAGA- GA-GCTTGCT CTC-GGGTGA -CGAGTGGCG GACGGGTGAG TAATGTCTGG
Erwinia persicinus (U80205) AACACATGCA AGTCGAACGG TAGCACAGA- GA-GCTTGCT CTC-GTGTGA T-GAGTGGCG GACGGGTGAG TAATGTCTGG
Erwinia rhapontici (AJ233417) AACACATGCA AGTCGAACGG TAGCACAGAG GA-GCTTGCT CCTTGGGTGA -CGAGTGGCG GACGGGTGAG TAATGCCTGG
Erwinia rhapontici (Z96087) AACACATGCA AGTCGAACGG TAGCACAGAG GA-GCTTGCT CCTTGGGTGA -CGAGTGGCG GACGGGTGAG TAATGCCTGG
Erwinia rhapontici (U80206) AACACATGCA AGTCGAACGG TAGCACAGAG GA-GCTTGCT CTCTGGGTGA -CGAGTGGCG GACGGGTGAG TAATGCCTGG
Erwinia amylovora (Z96088) AACACATGCA AGTCGAACGG TAGCACAGA- GA-GCTTGCT CNT-GGGTGA -CGAGTGGCG GACGGGTGAG TAATGTCTGG
Erwinia amylovora (AJ233410) AACACATGCA AGTCGAACGG TAGCACAGA- GA-GCTTGCT CTT-GGGTGA -CGAGTGGCG GACGGGTGAG TAATGTCTGG
Erwinia amylovora (AJ010485) AACACATGCA AGTCGAACGG TAGCACAGA- GA-GCTTGCT CTT-GGGTGA -CGAGTGGCG GACGGGTGAG TAATGTCTGG
Erwinia amylovora (U80195) AACACATGCA AGTCGAACGG TAGCACAGA-' GA-GCTTGCT CTT-GGGTGA -CGAGTGGCG GACGGGTGAG TAATGTCTGG
Erwinia pyrifoliae AACACATGCA AGTCGAACGG TAGCACAGA~ GA-GCTTGCT CTC-GGGTGA -CGAGTGGCG GACGGGTGAG TAATGTCTGG
pectobacterium cypripedii AACACATGCA AGTCGGACGG TAGCACAGNG GA-GCTTNCT CCCTGGGTGA -CGAGNGGCG GACGGGTGAG TAATNNCTGG
pectobacterium chrysanthemi AACACATGCA AGTCGGGCGG TAGCACAAGG GA-GCTTGCT CCC-GGGTGA -CGAGCGGCG GACGGGTGAG TAATGTCTGG
pectobacterium c. subsp. carotovorum AACACATGCA AGTCGAGCGG TAGCACAGAG GA-GCTTGCT CCTTGGGTGA -CGAGCGGCG GACGGGTGAG TAATGTCTGG
Pectobacterium c. subsp. wasabiae AACACATGCA AGTCGAGCGG TAGCACAGGA GA-GCTTGCT CTCTGGGTGA -CGAGCGGCG GACGGGTGAG TAATGTCTGG
Pectobacterium c. subsp. betavasculorum AACACATGCA AGTCGAGCGG CAGCGGGAAG TA-GCTTGCT ACTTTGCCGG -CGAGCGGTG GACGGGTGAG TAATGTCTGG
pectobacterium c. subsp. atrosepticum AACACATGCA AGTCGAGCGG TAGCACAGAA GA-GCTTGCT CTTTGGGTGA -CGAGCGGCG GACGGGTGAG TAATGTCTGG
pectobacterium c. subsp. odoriferum AACACATGCA AGTCGAGCGG TAGCACAAGA GA-GCTTGCT CTCTGGGTGA -CGAGCGGCG GACGGGTGAG TAATGTCTGG
pectobacterium cacticidum AACACATGCA AGTCGAGCGG TAACACAGAG GA-GCTTGCT NTC-GGGTGA -CGAGCGGCG GACGGGTGAG TAATGTCTGG
Brenneria salicis AACACATGCA AGTCGGGCGG TAGCACAGAG GA-GCTTGCT CCTTGGGTGA -CGAGCGGCG GACGGGTGAG TAAAGTCTGG
Brenneria rubrifaciens AACACATGCA AGTCGAGCGG CAGCGGGAAG TA-GCTTGCT ACTTTGCCGG -CGAGCGGCG GACGGGTGAG TAATGTCTGG
Brenneria nigrifluens AACACATGCA AGTCGAGCGG TAGCACAGAG GA-GCTTGCT CCTTGGGTGA -CGAGCGGCG GACGGGTGAG TAATGTCTGG
Brenneria alni AACACATGCA AGTCGGGCGG TAGCACAGGG GA-GCTTGCT TCCT-GGTGA ACGAGCGGCG GACGGGTGAG TAAAGTCTGG
Brenneria paradisiaca AACACATGCA AGTCGAGCGG CAGCGGGGGG AA-GCTTGCT TCCCCGCCGG -CGAGCGGCG GACGGGTGAG TAATGTCTGG
.......
N
VI
90 100 110 120 130 140 150 160
Buchnera aphidicola GGAT-CTGCC CAAAAGAGGG GGATAACTAC TAGAAATGGT AGCTAATACC GCATAAAGTT GAAAAACCAA AGTGGGGGAC
Proteus vulgaris GGAT-CTGCC -GATAGAGGG GGATAACTAC TGGAAACGGT GGCTAATACC GCATGACGTC TACGGACCAA AGCAGGGGTT
Erwinia ananas. Zululand. KZN. SA GGA-TCTGCC CGATAGAGGG GGATAACCAC TGGAAACGGT GGCTAATACC GCATAACGTC GCAAGACCAA AGAGGGGGAC
Pantoea a. Py. ananatis GGA-TCTGCC CGATAGAGGG GGATAACCAC TGGAAACGGT GGCTAATACC GCATAACGTC GCAAGACCAA AGAGGGGGAC
Pantoea a. Py. uredovora GGA-TCTGCC CGATAGAGGG GGATAACCAC TGGAAACGGT GGCTAATACC GCATAACGTC GCAAGACCAA AGAGGGGGAC
Pantoea agglomerans (U80202) GGA-TCTGCC CGATAGAGGG GGATAACCAC TGGAAACGGT GGCTAATACC GCATAACGTC GCAAGACCAA AGAGGGGGAC
Pantoea agglomerans (AB004757) GGA-TCTGCC CGATAGAGGG GGATAACCAC TGGAAACGGT GGCTAATACC GCATAACGTC GCAAGACCAA AGAGGGGGAC
Pantoea agglomerans (U80183) GGA-TCTGCC CGATAGAGGG GGATAACCAC TGGAAACGGT GGCTAATACC GCATAACGTC GCAAGACCAA AGAGGGGGAC
Unknown Erwinia sp. GAAA-CTGCC CGATGGAGGG GGATAACTAC TGGAAACGGT AGCTAATACC GCATAACGTC GCAAGACCAA AGTGGGGGAC
Pantoea s. subsp. stewartii GAAA-CTGCC CGATGGAGGG GGATAACTAC TGGAAACGGT AGCTAATACC GCATAACGTC GCAAGACCAA AGTGGGGGAC
Erwinia tracheiphila GAAA-CTGCC TGATGGCGGG GGATAACCAC. TGGAAACGGT GGCTAATACC GCATAATCTC GCAAGAGCAA AGAGGGGGAC
Erwinia psidii GAAA-CTGCC CGATGGAGGG GGATAACTAC TGGAAACGGT AGCTAATACC GCATAACGTC GCAAGACCAA AGTGGGGGAC
Erwinia mallotivora (AJ233414) GAAA-CTGCC CGATGGAGGG GGATAACTAC TGGAAACGGT AGCTAATACC GCATAACGTC TTCGGACCAA AGTGGGGGAC
Erwinia mallotivora (Z96084) GAAC-CTGCC CGATGGAGGG GGATANCTAC TGGAAACGGT AGCTAATACC NCATANCGTC TTCGGACCAA AGTGGGGGAC
Erwinia persicinus (AJOOl190) GAAA-CTGCC CGATGGAGGG GGATAACTAC TGGAAACGGT AGCTAATACC GCATAACGTC TTCGGACCAA AGTGGGGGAC
Erwinia persicinus (Z96086) GAAA-CTGCC CGATGGAGGG GGATAACTAC TGGAAACGGT AGCTAATACC GCATAACGTC TTCGGACCAA AGTGGGGGAC
Erwinia persicinus (U80205) GAAA-CTGCC CGATGGAGGG GGATAACTAC TGGAAACGGT AGCTAATACC GCATAACGTC TTCGGACCAA AGTGGGGGAC
Erwinia rhapontici (AJ233417) GAAA-CTGCC CGATGGAGGG GGATAACTAC TGGAAACGGT AGCTAATACC GCATAACGTC TTCGGACCAA AGTGGGGGAC
Erwinia rhapontici (Z96087) GAAA-CTGCC CGATGGAGGG GGATAACTAC TGGAAACGGT AGCTAATACC GCATAACGTC TTCGGACCAA AGTGGGGGAC
Erwinia rhapontici (U80206) GAAA-CTGCC CGATGGAGGG GGATAACTAC TGGAAACGGT AGCTAATACC GCATAACGTC TTCGGACCAA AGTGGGGGAC
Erwinia amylovora (Z96088) GAAA-CTGCC CGATGGAGGG GGATAACTAC TGGAAACGGT AGCTAATACC GCATAACGTC TACGGACCAA AGTGGGGGAC
Erwinia amylovora (AJ233410) GAAA-CTGCC CGATGGAGGG GGATAACTAC TGGAAACGGT AGCTAATACC GCATAACGTC TACGGACCAA AGTGGGGGAC
Erwinia amylovora (AJ010485) GAAA-CTGCC CGATGGAGGG GGATAACTAC TGGAAACGGT AGCTAATACC GCATAACGTC TACGGACCAA AGTGGGGGAC
Erwinia amylovora (U80195) GAAATCTGCC CGATGGAGGG GGATAACTAC TGGAAACGGT AGCTAATACC GCATAACGTC TACGGACCAA AGTGGGGGAC
Erwinia pyrifoliae GAAA-CTGCC CGATGGAGGG GGATAACTAC TGGAAACGGT AGCTAATACC GCATAACGTC TACGGACCAA AGTGGGGGAC
pectobacterium cypripedii GNA-TCTGCC TGATGGNGGG GGATAACTAC TGGAAACGGT AGCTAATACC GCATACCGTC TNCGGNNCAA AGTGGGGGAC
pectobacterium chrysanthemi GAAA-CTGCC TGATGGAGGG GGATAACTAC TGGAAACGGT AGCTAATACC GCATAACGTC TTCGGACCAA AGAGGGGGAC
pectobacterium c. subsp. carotovorum GAAA-CTGCC TGATGGAGGG GGATAACTAC TGGAAACGGT AGCTAATACC GCATAACCTC GCAAGAGCAA AGAGGGGGAC
pectobacterium c. subsp. wasabiae GAAA-CTGCC TGATGGAGGG GGATAACTAC TGGAAACGGT AGCTAATACC GCATAACGTC TTCGGACCAA AGAGGGGGAC
pectobacterium c. subsp. betavasculorum GAAA-CTGCC TGATGGAGGG GGATAACTAC TGGAAACGGT AGCTAATACC GCATAACGTC TTCGGACCAA AGAGGGGGAC
Pectobacterium c. subsp. atrosepticum GAAA-CTGCC TGATGGAGGG GGATAACTAC TGGAAACGGT AGCTAATACC GCATAACGTC TTCGGACCAA AGAGGGGGAC
Pectobacterium c. subsp. odoriferum GAAA-CTGCC TGATGGAGGG GGATAACTAC TGGAAACGGT AGCTAATACC GCATAACCTC GCAAGAGCAA AGAGGGGGAC
pectobacterium cacticidum GAAA-CTGCC TGATGGAGGG GGATAACTAC TGGAAACGGT AGCTAATACC GCATAATGTC GCAAGACCAA AGAGGGGGAC
Brenneria salicis GGA-TCTGCC TGATGGAGGG GGATAACTAC TGGAAACGGT AGCTAATACC GCATGACGTC TTCGGACCAA AGTGGGGGAC
Brenneria rubrifaciens GGA-TCTGCC TGATGGAGGG GGATAACCAC TGGAAACGGT GGCTAATACC GCATGACGTC GCAAGACCAA AGTGGGGGAC
Brenneria nigrifluens GAAA-CTGCC TGATGGAGGG GGATAACTAC TGGAAACGGT AGCTAATACC GCATAACCTC GCAAGAGCAA AGTGGGGGAC
Brenneria alni GAAA-CTGCC TGATGGAGGG GGATAACTAC TGGAAACGGT AGCTAATACC GCATAATGTC TTCGGACCAA AGTGGGGGAC
Brenneria paradisiaca GAAA-CTGCC TGATGGAGGG GGATAACTAC TGGAAACGGT AGCTAATACC GCATAACGTC TTCGGACCAA AGTGGGGGCT
.....
N
0\
170 180 190 200 210 220 230 240
Buchnera aphidicola CTTTTTTAAA GGCCTCATGC TTT-TGGATG AACCC-AGAC GAGATTAGCT TGTTGGTAAG GTAAAAGCTT ACCAAGGCAA
Proteus vulgaris CTTCGGACCT TGCGCTATCG GATGAA---- --CCC-ATAT GGGATTAGCT AGTAGGTGAG GTAATGGCTC ACCTAGGCGA
Erwinia ananas, Zululand, KZN, SA CTTCGGGCCT CTCACTATCG GAT------G AACCC-AGAT GGGATTAGCT AGTAGGCGGG GTAACGGCCC ACCTAGGCGA
Pantoea a. Py. ananatis CTTCGGGCCT CTCACTATCG GAT------G AACCC-AGAT GGGATTAGCT AGTAGGCGGG GTAACGGCCC ACCTAGGCGA
Pantoea a. Py. uredovora CTTCGGGCCT CTCACTATCG GAT------G AACCC-AGAT GGGATTAGCT AGTAGGCGGG GTAACGGCCC ACCTAGGCGA
Pantoea agglomerans (U80202) CTTCGGGCCT CTCACTATCG GAT------G AACCC-AGAT GGGATTAGCT AGTAGGCGGG GTAATGGCCC ACCTAGGCGA
Pantoea agglomerans (AB004757) CTTCGGGCCT CTCACTATCG GAT------G AACCC-AGAT GGGATTAGCT AGTAGGCGGG GTAATGGCCC ACCTAGGCGA
Pantoea agglomerans (U80183) CTTCGGGCCT CTCACTATCG GAT------G AACCC-AGAT GGGATTAGCT AGTAGGCGGG GTAATGGCCC ACCTAGGCGA
Unknown Erwinia sp. CTTCGGGCCT CACACCATCG GATGTG---- --CCC-AGAT GGGATTAGCT AGTAGGCGGG GTAATGGCCC ACCTAGGCGA
Pantoea s. subsp. stewartii CTCCGGGCCT CACACCATCG GATGTG---- --CCC-AGAT GGGATTAGCT AGTAGGCGGG GTAACGGCCC ACCTAGGCGA
Erwinia tracheiphila CTTATGGCCT CTTGCCATCG GATGTG---- --CCC-AGAT GGGATTAGCT GGCAGGTAGG GTAACGGCCT ACCTGGGCGA
Erwinia psidii CTTAGGGCCT CACACCATCG GATGTG---- --CCC-AGAT GGGATTAGCT TGTTGGTGGG GTAAAAGCTC ACCAAGGCGA
Erwinia mallotivora (AJ233414) CTTCGGGCCT CACACCATCG GATGTG---- --CCC-AGAT GGGATTAGCT GGTTGGTGAG GTAACGGCTC ACCAAGGCGA
Erwinia mallotivora (Z96084) CTTCGGGCCT CACACCATCG GATGTG---- --CCC-AGAT GGGATTAGCT GGTTGGTGAG GTAACGGCTC ACCAAGGCGA
Erwinia persicinus (AJOOl190) CTTCGGGCCT CACACCATCG GATGTG---- --CCC-AGAT GGGATTAGCT AGTAGGTGGG GTAACGGCTC ACCTAGGCGA
Erwinia persicinus (Z96086) CTTCGGGCCT CACACCATCG GATGTG---- --CCC-AGAT GGGATTAGCT AGTAGGTGGG GTAACGGCTC ACCTAGGCGA
Erwinia persicinus (U80205) CTTCGGGCCT CACACCATCG GATGTG---- --CCC-AGAT GGGATTAGCT AGTAGGTGGG GTAACGGCTC ACCTAGGCGA
Erwinia rhapontici (AJ233417) CTTCGGGCCT CACACCATCG GATGTG---- --CCC-AGGT GGGATTAGCT AGTAGGTGGG GTAATGGCTC ACCTAGGCGA
Erwinia rhapontici (Z96087) CTTCGGGCCT CACACCATCG GATGTG---- --CCC-AGGT GGGATTAGCT AGTAGGTGGG GTAATGGCTC ACCTAGGCGA
Erwinia rhapontici (U80206) CTTCGGGCCT CACACCATCG GATGTG---- --CCC-AGGT GGGATTAGCT AGTAGGTGGG GTAATGGCTC ACCTAGGCGA
Erwinia amylovora (Z96088) CTTCGGGCCT CACACCATCG GATGTG---- --CCC-AGAT GGGATTAGCT GGTAGGTGGG GTAANGGCTC ACCTAGGCGA
Erwinia amylovora (AJ233410) CTTCGGGCCT CACACCATCG GATGTG---- --CCC-AGAT GGGATTAGCT GGTAGGTGGG GTAACGGCTC ACCTAGGCGA
Erwinia amylovora (AJ010485) CTTCGGGCCT CACACCATCG GATGTG---- --CCC-AGAT GGGATTAGCT AGTAGGTGGG GTAACGGCTC ACCTAGGCGA
Erwinia amylovora (U80195) CTTCGGGCCT CACACCATCG GATGTG---- --CCC-AGAT GGGATTAGCT GGTAGGTGAG GTAATGGCTC ACCTAGGCGA
Erwinia pyrifoliae CTTCGGGCCT CACACCATCG GATGTG---- --CCC-AGAT GGGATTAGCT GGTAGGTGGG GTAACGGCTC ACCTAGGCGA
pectobacterium cypripedii CTTCGGGCCT CATGCCATCN GATGTG---- --CCC-AGAT GGGATTAGCT AGTAGGTGGG GTAAAGGCTC ACCTAGGCGA
pectobacterium chrysanthemi CTTCGGGCCT CTTGCCATCG GATGTG---- --CCC-AGAT GGGATTAGCT AGTAGGTGGG GTAAAGGCTC ACCTAGGCGA
Pectobacterium c. subsp. carotovorum CTTNGGGCCT CTCGCCATCA GATGTG---- --CCC-AGAT GGGATTAGCT AGTAGGTGAG GTAATGGCTC ACCTAGGCGA
Pectobacterium c. subsp. wasabiae CTTCGGGCCT CTTGCCATCG GATGTG---- --CCC-AGAT GGGATTAGCT AGTAGGTGAG GTAATGGCTC ACCTAGGCGA
Pectobacterium c. subsp. betavasculorum CTTCGGGCCT CTTGCCATCG GATGTG---- --GCCCAGAT GGGATTAGCT AGTAGGCGGG GTAATGGCCC ACCTAGGCGA
pectobacterium c. subsp. atrosepticum CTTCGGGCCT CTTGCCATCA GATGTG---- --CCC-AGAT GGGATTAGCT AGTAGGCGGG GTAATGGCCC ACCTAGGCGA
pectobacterium c. subsp. odoriferum CTTCGGGCCT CTCGCCATCA GATGTG---- --CCC-AGAT GGGATTAGCT AGTAGGTGAG GTAATGGCTC ACCTAGGCGA
Pectobacterium cacticidum CTTAGGGCCT CTTGCCATCG GATGTG---- --CCC-AGAT GGGATTAGCT AGTAGGTGGG GTAAAGGCTC ACCTAGGCGA
Brenneria salicis CTTCGGGCCT CACGCCATGA GAT------G AACCC-AGAT GGGATTAGCT GGTAGGTGAG GTAACGGCTC ACCTAGGCGA
Brenneria rubrifaciens CTTCGGGCCT CACGCCATCG GAT------G AACCC-AGAT GGGATTAGCT AGTAGGCGGG GTAATGGCCC ACCTAGGCGA
Brenneria nigrifluens CTTATGGCCT CACGCCATCG GATGTG---- --CCC-AGAT GGGATTAGCT GGTAGGTGGG GTAAAGGCTC ACCTAGGCGA
Brenneria alni CTTAGGGCCT CACGCCATCG GATGTG---- --CCC-AGAT GGGATTAGCT GGTAGGTGGG GTAAAGGCTC ACCTAGGCGA
Brenneria paradisiaca CTTCGGACCT CATGCCATCG GATGTG---- --CCC-AGAT GGGATTAGCT AGTAGGCGGG GTAAAGGCCC ACCTAGGCGA
......
IV
-....l
250 260 270 280 290 300 310 320
Buchnera aphidicola CGATCTCTAG CTGGTCTGAG AGGATAACCA GCCACACTGG AACTGAGACA CGGTCCAGAC TCCTACGGGA GGCAGCAGTC
Proteus vulgaris CGATCTCTAG CTGGTCTGAG AGGATGATCA GCCACACTGG GACTGAGACA CGGCCCAGAC TCCTACGGGA GGCAGCAGTG
Erwinia ananas, Zululand, KZN, SA CGATCCCTAG CTGGTCTGAG AGGATGACCA GCCACACTGG AACTGAGACA CGGTCCAGAC TCCTACGGGA GGCAGCAGTG
Pantoea a. Py. ananatis CGATCCCTAG CTGGTCTGAG AGGATGACCA GCCACACTGG AACTGAGACA CGGTCCAGAC TCCTACGGGA GGCAGCAGTG
Pantoea a. Py. uredovora CGATCCCTAG CTGGTCTGAG AGGATGACCA GCCACACTGG AACTGAGACA CGGTCCAGAC TCCTACGGGA GGCAGCAGTG
Pantoea agglomerans (U80202) CGATCCCTAG CTGGTCTGAG AGGATGACCA GCCACACTGG AACTGAGACA CGGTCCAGAC TCCTACGGGA GGCAGCAGTG
Pantoea agglomerans (AB004757) CGATCCCTAG CTGGTCTGAG AGGATGACCA GCCACACTGG AACTGAGACA CGGTCCAGAC TCCTACGGGA GGCAGCAGTG
Pantoea agglomerans (U80183) CGATCCCTAG CTGGTCTGAG GGGATGACCA GCCACACTGG AACTGAGACA CGGTCCAGAC TC-TACGGGA GGCAGCAGTG
Unknown Erwinia sp. CGATCCCTAG CTGGTCTGAG AGGATGACCA GCCACACTGG AACTGAGACA CGGTCCAGAC TCCTACGGGA GGCAGCAGTG
Pantoea s. subsp. stewartii CGATCCCTAG CTGGTCTGAG AGGATGACCA GCCACACTGG AACTGAGACA CGGTCCAGAC TCCTACGGGA GGCAGCAGTG
Erwinia tracheiphila CGATCCCTAG CTGGTCTGAG AGGATGACCA GCCACACTGG AACTGAGACA CGGTCCAGAC TCCTACGGGA GGCAGCAGTG
Erwinia psidii CGATCCCTAG CTGGTCTGAG AGGATGACCA GCCACACTGG AACTGAGA-A CGGTCCAGAC TCCTACGGGA GGCAGCAGTG
Erwinia mallotivora (AJ233414) CGATCCCTAG CTGGTCTGAG AGGATGACCA GCCACACTGG AACTGAGACA CGGTCCAGAC TCCTACGGGA GGCAGCAGTG
Erwinia mallotivora (Z96084) CGATCCCTAG CTGGTCTGAG AGGATGACCA GCCACACTGG AACTGAGACA CGGTCCAGAC TCCTACGGGA GGCAGCAGTG
Erwinia persicinus (AJOOl190) CGATCCCTAG CTGGTCTGAG AGGATGACCA GCCACACTGG AACTGAGACA CGGTCCAGAC TCCTACGGGA GGCAGCAGTG
Erwinia persicinus (Z96086) CGATCCCTAG CTGGTCTGAG AGGATGACCA GCCACACTGG AACTGAGACA CGGTCCAGAC TCCTACGGGA GGCAGCAGTG
Erwinia persicinus (U80205) CGATCCCTAG CTGGTCTGAG AGGATGACCA GCCACACTGG AACTGAGACA CGGTCCAGAC TCCTACGGGA GGCAGCAGTG
Erwinia rhapontici (AJ233417) CGATCCCTAG CTGGTCTGAG AGGATGACCA GCCACACTGG AACTGAGACA CGGTCCAGAC TCCTACGGGA GGCAGCAGTG
Erwinia rhapontici (Z96087) CGATCCCTAG CTGGTCTGAG AGGATGACCA GCCACACTGG AACTGAGACA CGGTCCAGAC TCCTACGGGA GGCAGCAGTG
Erwinia rhapontici (U80206) CGATCCCTAG CTGGTCTGAG AGGATGACCA GCCACACTGG AACTGAGACA CGGTCCAGAC TCCTACGGGA GGCAGCAGTG
Erwinia amylovora (Z96088) CGATCCCTAG CTGGTCTGAG AGGATGACCA GCCACACTGG AACTGAGACA CGGTCCAGAC TCCTACGGGA GGCAGCAGTG
Erwinia amylovora (AJ233410) CGATCCCTAG CTGGTCTGAG AGGATGACCA GCCACACTGG AACTGAGACA CGGTCCAGAC TCCTACGGGA GGCAGCAGTG
Erwinia amylovora (AJOI0485) CGATCCCTAG CTGGTCTGAG AGGATGACCA GCCACACTGG AACTGAGACA CGGTCCAGAC TCCTACGGGA GGCAGCAGTG
Erwinia amylovora (U80195) CGATCCCTAG CTGGTCTGAG AGGATGACCA GCCACACTGG AACTGAGACA CGGTCCAGAC TCCTACGGGA GGCAGCAGTG
Erwinia pyrifoliae CGATCCCTAG CTGGTCTGAG AGGATGACCA GCCACACTGG AACTGAGACA CGGTCCAGAC TCCTACGGGA GGCAGCAGTG
Pectobacterium cypripedii CGATCCCTAG CTGGTCTGAG AGGATGACCA GCCACACTGG AACTGAGACA CGGTCCAGAC TCCTACGGGA GGCAGCAGTG
pectobacterium chrysanthemi CGATCCCTAG CTGGTCTGAG AGGATGACCA GCCACACTGG AACTGAGACA CGGTCCAGAC TCCTACGGGA GGCAGCAGTG
pectobacterium c. subsp. carotovorum CGATCCCTAG CTGGTCTGAG AGGATGACCA GCCACACTGG AACTGAGACA CGGTCCAGAC TCCTACGGGA GGCAGCAGTG
pectobacterium c. subsp. wasabiae CGATCCCTAG CTGGTCTGAG AGGATGACCA GCCACACTGG AACTGAGACA CGGTCCAGAC TCCTACGGGA GGCAGCAGTG
pectobacterium c. subsp. betavasculorum CGATCCCTAG CTGGTCTGAG AGGATGACCA GCCACACTGG AACTGAGACA CGGTCCAGAC TCCTACGGGA GGCAGCAGTG
Pectobacterium c. subsp. atrosepticum CGATCCCTAG CTGGTCTGAG AGGATGACCA GCCACACTGG AACTGAGACA CGGTCCAGAC TCCTACGGGA GGCAGCAGTG
pectobacterium c. subsp. odoriferum CGATCCCTAG CTGGTCTGAG AGGATGACCA GCCACACTGG AACTGAGACA CGGTCCAGAC TCCTACGGGA GGCAGCAGTG
pectobacterium cacticidum CGATCCCTAG CTGGTCTGAG AGGATGACCA GCCACACTGG AACTGAGACA CGGTCCAGAC TCCTACGGGA GGCAGCAGTG
Brenneria salicis CGATCCCTAG CTGGTCTGAG AGGATGACCA GCCACACTGG AACTGAGACA CGGTCCAGAC TCCTACGGGA GGCAGCAGTG
Brenneria rubrifaciens CGATCCCTAG CTGGTCTGAG AGGATGACCA GCCACACTGG AACTGAGACA CGGTCCAGAC TCCTACGGGA GGCAGCAGTG
Brenneria nigrifluens CGATCCCTAG CTGGTCTGAG AGGATGACCA GCCACACTGG AACTGAGACA CGGTCCAGAC TCCTACGGGA GGCAGCAGTG
Brenneria alni CGATCCCTAG CTGGTCTGAG AGGATGACCA GCCACACTGG AACTGAGACA CGGTCCAGAC TCCTACGGGA GGCAGCAGTG
Brenneria paradisiaca CGATCCCTAG CTGGTCTGAG AGGATGACCA GCCACACTGG AACTGAGACA CGGTCCAGAC TCCTACGGGA GGCAGCAGTG
......
N
00
330 340 350 360 370 3BO 390 400
Buchnera aphidicola GGGAATATTG CACAATGGGC GAAAGCCTG- ATGCAGCTAT GCCGCGTGTA TGAAGAAGGC CTTAGGGTTG TAAAGTACTT
Proteus vulgaris GGCAATATTG CACAATGGGC GCAAGCCTG- ATGCAGCCAT GCCGCGTGTA TGAAGAAGGC CTTAGGGTTG TAAAGTACTT
Erwinia ananas, Zululand, KZN, SA GGGAATATTG CACAATGGGC GCAAGCCTG- ATGCAGCCAT GCCGCGTGTA TGAAGAAGGC CTTCGGGTTG TAAAGTACTT
Pantoea a. Py. ananatis GGGAATATTG CACAATGGGC GCAAGCCTG- ATGCAGCCAT GCCGCGTGTA TGAAGAAGGC CTTCGGGTTG TAAAGTACTT
Pantoea a. Py. uredovora GGGAATATTG CACAATGGGC GCAAGCCTG- ATGCAGCCAT GCCGCGTGTA TGAAGAAGGC CTTCGGGTTG TAAAGTACTT
Pantoea agglomerans (UB0202) GGGAATATTG CACAATGGGC GCAAGCCTG- ATGCAGCCAT GCCGCGTGTA TGAAGAAGGC CTTCGGGTTG TAAAGTACTT
Pantoea agglomerans (AB004757) GGGAATATTG CACAATGGGC GCAAGCCTG- ATGCAGCCAT GCCGCGTGTA TGAAGAAGGC CTTCGGGTTG TAAAGTACTT
Pantoea agglomerans (UBOlB3) GGGAATATTG CACAATGGGC GCAAGCCTG- ATGCAGCCAT GCCGCGTGTA TGAAGAAGGC CTTCGGGTTG TAAAGTACTT
Unknown Erwinia sp. GGGAATATTG CACAATGGGC GCAAGCCTG- ATGCAGCCAT GCCGCGTGTA TGAAGAAGGC CTTCGGGTTG TAAAGTACTT
Pantoea s. subsp. stewartii GGGAATATTG CACAATGGGC GCAAGCCTG- ATGCAGCCAT GCCGCGTGTA TGAAGAAGGC CTTCGGGTTG TAAAGTACTT
Erwinia tracheiphila GGGAATATTG CACAATGGGC GCAAGCCTG- ATGCAGCCAT GCCGCGTGTA TGAAGAAGGC CTTCGGGTTG TAAAGTACTT
Erwinia psidii GGGAATATTG CACAATGGGC GCAAGCCTG- ATGCANCCAT GCCGCGTGTA TGAAGAAGGC CTTCGGGTTG TAAAGTACTT
Erwinia mallotivora (AJ2334l4) GGGAATATTG CACAATGGGC GCAAGCCTG- ATGCAGCCAT GCCGCGTGTA TGAAGAAGGC CTTCGGGTTG TAAAGTACTT
Erwinia mallotivora (Z960B4) GGGAATATTG CACAATGGGC GCNAGCCTG- ATGCAGCCAT GCCGCGTGTA NGAAGAAGGC CTTCGGGTTG TAAAGNACTT
Erwinia persicinus (AJOOl190) GGGAATATTG CACAATGGGC GCAAGCCTG- ATGCAGCCAT GCCGCGTGTA TGAAGAAGGC CTTCGGGTTG TAAAGTACTT
Erwinia persicinus (Z960B6) GGGAATATTG CACAATGGGC GCAAGCCTG- ATGCAGCCAT GCCGCGTGTA TGAAGAAGGC CTTCGGGTTG TAAAGTACTT
Erwinia persicinus (UB0205) GGGAATATTG CACAATGGGC GCAAGCCTG- ATGCAGCCAT GCCGCGTGTA TGAAGAAGGC CTTCGGGTTG TAAAGTACTT
Erwinia rbapontici (AJ2334l7) GGGAATATTG CACAATGGGC GCAAGCCTG- ATGCAGCCAT GCCGCGTGTA TGAAGAAGGC CTTCGGGTTG TAAAGTACTT
Erwinia rhapontici (Z960B7) GGGAATATTG CACAATGGGC GCAAGCCTG- ATGCAGCCAT GCCGCGTGTA TGAAGAAGGC CTTCGGGTTG TAAAGTACTT
Erwinia rhapontici (UB0206) GGGAATATTG CACAATGGGC GCAAGCCTG- ATGCAGCCAT GCCGCGTGTA TGAAGAAGGC CTTCGGGTTG TAAAGTACTT
Erwinia amylovora (Z960BB) GGGAATATTG CACAATGGGC GCAAGCCTG- ATGCAGCCAT GCCGCGTGTA TGAAGAAGGC CTTCGGGTTG TAAAGTACTT
Erwinia amylovora (AJ2334l0) GGGAATATTG CACAATGGGC GCAAGCCTG- ATGCAGCCAT GCCGCGTGTA TGAAGAAGGC CTTCGGGTTG TAAAGTACTT
Erwinia amylovora (AJOl04B5) GGGAATATTG CACAATGGGC GCAAGCCTG- ATGCAGCCAT GCCGCGTGTA TGAAGAAGGC CTTCGGGTTG TAAAGTACTT
Erwinia amylovora (UBOl95) GGGAATATTG CACAATGGGC GCAAGCCTG- ATGCAGCCAT GCCGCGTGTA TGAAGAAGGC CTTCGGGTTG TAAAGTACTT
Erwinia pyrifoliae GGGAATATTG CACAATGGGC GCAAGC-TG- ATGCAGCCAT GCCGCGTGTA TGAAGAAGGC CTTCGGGTTG TAAAGTACTT
Pectobacterium cypripedii GGGAATATTG CACAATGGNC GNAAGCCTG- ATGCAGCCAT GCCGCGTGTG TGAAGAAGGC CTTCGGGTTG TAAAGCACTT
pectobacterium chrysanthemi GGGAATATTG CACAATGGGC GCAAGCCTG- ATGCAGCCAT GCCGCGTGTG TGAAGAAGGC CTTCGGGTTG TAAAGCACTT
pectobacterium c. subsp. carotovorum GGGAATATTG CACAATGGGC GCAAGCCTG- ATGCAGCCAT GCCGCGTGTG TGAAGAAGGC CTTCGGGTTG TAAAGCACTT
Pectobacterium c. subsp. wasabiae GGGAATATTG CACAATGGGN GCAAGCCTG- ATGCAGCCAT GCCGCGTGTG TGAAGAAGGC CTTCGGGTTG TAAAGCACTT
Pectobacterium c. subsp. betavasculorum GGGAATATTG CACAATGGGC GCAAGCCTG- ATGCAGCCAT GCCGCGTGTG TGAAGAAGGC CTTCGGGTTG TAAAGCACTT
Pectobacterium c. subsp. atrosepticum GGGAATATTG CACAATGGGC GCAAGCCTG- ATGCAGCCAT GCCGCGTGTG TGAAGAAGGC CTTCGGGTTG TAAAGCNCTT
pectobacterium c. subsp. odoriferum GGGAATATTG CACAATGGGC GCAAGCCTG- ATGCAGCCAT GCCGCGTGTG TGAAGAAGGC CTTCGGGTTG TAAAGCACTT
pectobacterium cacticidum GGGAATATTG CACAATGGGC GCAAGCCTG- ATGCAGCCAT GCCGCGTGTG TGAAGAAGGC CTTCGGGTTG TAAAGCACTT
Brenneria salicis GGGAATATTG CACAATGGGG GAAACCCTG- ATGCAGCCAT GCCGCGTGTG TGAAGAAGGC CTTCGGGTTG TAAAGCACTT
Brenneria rubrifaciens GGGAATATTG CACAATGGGG GAAACCCTG- ATGCAGCCAT GCCGCGTGTG TGAAGAAGGC CTTCGGGTTG TAAAGCACTT
Brenneria nigrifluens GGGAATATTG CACAATGGGG GAAACCCTG- ATGCAGCCAT GCCGCGTGTG TGAAGAAGGC CTTCGGGTTG TAAAGCACTT
Brenneria alni GGGAATATTG CACAATGGGG GAAACCCTG- ATGCAGCCAT GCCGCGTGTG TGAAGAAGGC CTTCGGGTTG TAAAGCACTT
Brenneria paradisiaca GGGAATATTG CACAATGGGG GAAACCCTG- ATGCAGCCAT GCCGCGTGTG TGAAGAAGGC CTTCGGGTTG TAAAGCACTT
.....
N
\C)
410 420 430 440 450 460 470 480
Buchnera aphidicola TCAGCGGGGA GGAAAAAATT ---AAAACTA ATAA--TTTT A-TTTTGTGA CGTTACCCGC AGAAGAAGCA CCGGCTAACT
Proteus vulgaris TCAGCGGGGA GGAAGGTGAT ---AAAGTTA ATA-CCTTTG TCAA--TTGA CGTTACCCGC AGAAGAAGCA CCGGCTAACT
Erwinia ananas, Zululand, KZN, SA TCAGCGGGGA GGAAGGCGAT GT---GGTTA ATAACCGCAT T-GA--TTGA CGTTACCCGC AGAAGAAGCA CCGGCTAACT
Pantoea a. Py. ananatis TCAGCGGGGA GGAAGGCGAT GT---GGTTA ATAACCGCAT T-GA--TTGA CGTTACCCGC AGAAGAAGCA CCGGCTAACT
Pantoea a. Py. uredovora TCAGCGGGGA GGAAGGCGAT GT---GGTTA ATAACCGCAT T-GA--TTGA CGTTACCCGC AGAAGAAGCA CCGGCTAACT
Pantoea agglomerans (U80202) TCAGCGGGGA GGAAGGCGAC G----GGTTA ATAACCCT-G TCGA--TTGA CGTTACCCGC AGAAGAAGCA CCGGCTAACT
Pantoea agglomerans (AB004757) TCAGCGGGGA GGAAGGCGAT GG---GGTTA ATAACCGC-G TCGA--TTGA CGTTACCCGC AGAAGAAGCA CCGGCTAACT
Pantoea agglomerans (U80l83) TCAGCGGGGA GGAAGGCGAC G----GGTTA ATAACCCT-G TCGA--TTGA CGTTACCCGC AGAAGAAGCA CCGGCTAACT
Unknown Erwinia sp. TCAGCGAGGA GGAAGGC--T GGTG-GGTTA ATAACCTGA- TC-A-ATTGA CGTTACCCGC AGAAGAAGCA CCGGCTAACT
Pantoea s. subsp. stewartii TCAGCGGGGA GGAAGG---T GGTGAGGTTA ATAACCTCA- TC-A-ATTGA CATTACCCGC AGAAGAAGCA CCGGCTAACT
Erwinia tracheiphila TCAGCGGGGA GGAAGG-GAC GCT--GGTTA ATAACCAGCG TC-A--TTGA TGTTACCCGC ANAANAAGCA CCGGCTAACT
Erwinia psidii TCAGCGGGGA GGAAGG---T GGTGAGGTTA ATAACCTTA- TC-A-ATTGA CGTTACCCGC AGAAGAAGCA CCGGCTAACT
Erwinia mallotivora (AJ233414) TCAGCGGGGA GGAAGG---T GGTGGGGTTA ATAACCTTA- TC-A-ATTGA CGTTACCCGC AGAAGAAGCA CCGGCTAACT
Erwinia mallotivora (Z96084) TCAGCGGGGA GGAAGG---T GGTGGGGTTA ATAACCTTA- TC-A-ATTGA CGTTACCCGC AGAAGAAGCA CCGGCTAACT
Erwinia persicinus (AJOOl190) TCAGTGGGGA GGAAGGCGA- -TGAA-GTTA ATAACTTCG- TCGA--TTGA CGTTACCCGC AGAAGAAGCA CCGGCTAACT
Erwinia persicinus (Z96086) TCAGTGGGGA GGAAGGCGA- -TGAA-GTTA ATAACTTCG- TCGA--TTGA CNTTACCCGC AGAAGAAGCA CCGGCTAACT
Erwinia persicinus (U80205) TCAGTGGGGA GGAAGGCGA- -TGAA-GTTA ATAACTTCG- TCGA--TTGA CGTTACCCGC AGAAGAAGCA CCGGCTAACT
Erwinia rhapontici (AJ233417) TCAGTGGGGA GGAAGGCGA- -TGA-GGTTA ATAGCCTCG- TCGA--TTGA CGTTACCCGC AGAAGAAGCA CCGGCTAACT
Erwinia rhapontici (Z96087) TCAGTGGGGA GGAAGGCGA- -TGA-GGTTA ATAGCCTCG- NCGA--TTGA CGTTACCCGC AGAAGAAGCA CCGGCTAACT
Erwinia rhapontici (U80206) TCAGTGGGGA GGAAGGCGA- -TGA-GGTTA ATAGCCTCG- TCGA--TTGA CGTTACCCGC AGAAGAAGCA CCGGCTAACT
Erwinia amylovora (Z96088) TCAGCGGGGA GGAAGGGGAA --GA-GGTTA ATAACCTCC- TCCA--TTGA CGTTACCCGC AGAAGAAGCA CCGGCTAACT
Erwinia amylovora (AJ233410) TCAGCGGGGA GGAAGGGGAA --GA-GGTTA ATAACCTCC- TCCA--TTGA CGTTACCCGC AGAAGAAGCA CCGGCTAACT
Erwinia amylovora (AJ010485) TCAGCGGGGA GGAAGGGGAA --GA-GGTTA ATAACCTTT- TCCA--TTGA CGTTACCCGC AGAAGAAGCA CCGGCTAACT
Erwinia amylovora (U80195) TCAGCGGGGA GGAAGGGGAA --GA-GGTTA ATAACCTTT- TCCA--TTGA CGTTACCCGC AGAAGAAGCA CCGGCTAACT
Erwinia pyrifoliae TCAGCGGGGA GGAAGGGGGA ---AAGGTTA ATAACCTTTT TC-A--TTGA CGTTACCCGC AGAAGAAGCA CCGGCTAACT
Pectobacterium cypripedii TCAGCGGGGA AGAAGGGGA- -TAA-GGTTA ATACCCTTG- TNNA--TTGA CGTTACCCGC AGAAGAAGCA CCGGCTAACT
Pectobacterium cbrysanthemi TCAGCGGGGA GGAAGGGAA- --CAAGGTTA ATACCTTTGT TC-A--TTGA CGTTACCCGC AGAAGAAGCA CCGGCTAACT
pectobacterium c. subsp. carotovorum TCAGCGAGGA GGAAGGCGG- -T-AAGGTTA ATAACCTTA- TCGA--TTGA CGTTACTCGC AGAANAAGCA CCGGCTAACT
pectobacterium c. subsp. wasabiae TCAGCGGGGA GGAAGGCAG- -T-AAGGTTA ATAACCTTG- CTGA--TTGA CGTTACCCGC AGAAGAAGCA CCGGCTAACT
pectobacterium c. subsp. betavasculorum TCAGCGGGGA GGAAGGCAG- -T-AAGGCTA ATAACCTTA- TTGA--TTGA CGTTACCCGC AGAAGAAGCA CCGGCTAACT
Pectobacterium c. subsp. atrosepticum TCNGCGGGGA GGAAGGCAG- -T-AAGGTTA ATAACCTTG- CTGA--TTGA CGTTACCCGC AGAAGAAGCA CCGGCTAACT
Pectobacterium c. subsp. odoriferum TCAGCGAGGA GGAAGGCAG- -T-CNTGTTA ATANCACGG- NTGA--TTGA CGTTACTCGC AGAAGAAGCA CCGGCTAACT
pectobacterium cacticidum TCAGCGGGGA GGAAGGCN-- -TGAAAGCGA ATACCTTTCA T-GA--TTGA CNTTACCCGC AGAANAAGCA CCGGCTAACT
Brenneria salicis TCAGCGGGGA GGAAGGCGA- --TAAACTTA ATAAGTTTGT T-GA--TTGA CGTTACCCGC AGAAGAAGCA CCGGCTAACT
Brenneria rubrifaciens TCAGCGGGGA GGAAGGGGA- --AAGGTTTA AGAGACTTTT TC-A--TTGA CGTTACCCGC AGAAGAAGCA CCGGCTAACT
Brenneria nigrifluens TCAGCGGGGA GGAAGGCAA- --CAAGCTTA ATAATCTTGT TC-A--TTGA CGTTACCCGC AGAAGAAGCA CCGGCTAACT
Brenneria alni TCAGCGAGGA GGAAGGCGG- --TAAGGTTA ATAACCTTA- TCGA--TTGA CGTTACTCGC AGAAGAAGCA CCGGCTAACT
Brenneria paradisiaca TCAGCGGGGA GGAAGGGGAC ----AGGCTT AATACGTCTG TTCA--TTGA CGTTACCCGC AGAAGAAGCA CCGGCTAACT
IJJ
o
490 500 510 520 530 540 550 560
Buchnera aphidicola CCGTGCCAGC AGCCGCGGTA ATACGGAGGG TGCGAGCGTT AATCAGAATT ACTGGGCGTA AAGAGCACGT AGGTGGTTTT
Proteus vulgaris CCGTGCCAGC AGCCGCGCTA ATACGGAGGG TGCAAGCGTT AATCGGAATT ACTGGGCGTA AAGCGCACGC AGGCGG-CAA
Erwinia ananas, Zululand, KZN, SA CCGTGCCAGC AGCCGCGGTA ATACGGAGGG TGCAAGCGTT AATCGGAATT ACTGGGCGTA AAGCGCACGC AGGCGGTCTG
Pantoea a. Py. ananatis CCGTGCCAGC AGCCGCGGTA ATACGGAGGG TGCAAGCGTT AATCGGAATT ACTGGGCGTA AAGCGCACGC AGGCGGTCTG
Pantoea a. Py. uredovora CCGTGCCAGC AGCCGCGGTA ATACGGAGGG TGCAAGCGTT AATCGGAATT ACTGGGCGTA AAGCGCACGC AGGCGGTCTG
Pantoea agglomerans (U80202) CCGTGCCAGC AGCCGCGGTA ATACGGAGGG TGCAAGCGTT AATCGGAATT ACTGGGCGTA AAGCGCACGC AGGCGGTCTG
Pantoea agglomerans (AB004757) CCGTGCCAGC AGCCGCGGTA ATACGGAGGG TGCAAGCGTT AATCGGAATT ACTGGGCGTA AAGCGCACGC AGGCGGTCTG
Pantoea agglomerans (U80l83) CCGTGCCAGC AGCCGCGGTA ATACGGAGGG TGCAAGCGTT AATCGGAATT ACTGGGCGTA AAGCGCACGC AGGCGGTCTG
Unknown Erwinia sp. CCGTGCCAGC AGCCGCGGTA ATACGGAGGG TGCAAGCGTT AATCGGAATT ACTGGGCGTA AAGCGCACGC AGGCGGTCTG
Pantoea s. subsp. stewartii CCGTGCCAGC AGCCGCGGTA ATACGGAGGG TGCAAGCGTT AATCGGAATT ACTGGGCGTA AAGCGCACGC AGGCGGTCTG
Erwinia tracheiphila CCGTGCCANC ANCCGCGGTA ATACGGAGGG TGCAAGCGTT AATCGGAATT ACTGGGCGTA AAGCGCACGC AGGCGGTCTG
Erwinia psidii CCGTGCCAGC AGCCGCGGTA ATACGGAGGG TGCAAGCGTT AATCGGAATT ACTGGGCGTA AAGCGCACGC AGGCGGTCTG
Erwinia mallotivora (AJ2334l4) CCGTGCCAGC AGCCGCGGTA ATACGGAGGG TGCAAGCGTT AATCGGAATT ACTGGGCGTA AAGCGCACGC AGGCGGTCTG
Erwinia mallotivora (Z96084) CCGTGCCAGC AGCCGCGGTA ATACGGAGGG TGCAAGCGTT AATCGGAATT ACTGGGCGTA AAGCGCACGC AGGCGGTCTG
Erwinia persicinus (AJOOl190) CCGTGCCAGC AGCCGCGGTA ATACGGAGGG TGCAAGCGTT AATCGGAATT ACTGGGCGTA AAGCGCACGC AGGCGGTCTG
Erwinia persicinus (Z96086) CCGTGCCAGC AGCCGCGGTA ATACGGAGGG TGCAAGCGTT AATCGGAATT ACTGGGCGTA AAGCGCACGC AGGCGGTCTG
Erwinia persicinus (U80205) CCGTGCCAGC AGCCGCGGTA ATACGGAGGG TGCAAGCGTT AATCGGAATT ACTGGGCGTA AAGCGCACGC AGGCGGTCTG
Erwinia rhapontici (AJ2334l7) CCGTGCCAGC AGCCGCGGTA ATACGGAGGG TGCAAGCGTT AATCGGAATT ACTGGGCGTA AAGCGCACGC AGGCGGTCTG
Erwinia rhapontici (Z96087) CCGTGCCAGC AGCCGCGGTA ATACGGAGGG TGCAAGCGTT AATCGGAATT ACTGGGCGTA AAGCGCACGC AGGCGGTCTG
Erwinia rhapontici (U80206) CCGTGCCAGC AGCCGCGGTA ATACGGAGGG TGCAAGCGTT AATCGGAATT ACTGGGCGTA AAGCGCACGC AGGCGGTCTG
Erwinia amylovora (Z96088) CCGTGCCAGC AGCCGCGGTA ATACGGAGGG TGCAAGCGTT AATCGGAATT ACTGGGCGTA AAGCGCACGC AGGCGGTCTG
Erwinia amylovora (AJ2334l0) CCGTGCCAGC AGCCGCGGTA ATACGGAGGG TGCAAGCGTT AATCGGAATT ACTGGGCGTA AAGCGCACGC AGGCGGTCTG
Erwinia amylovora (AJ010485) CCGTGCCAGC AGCCGCGGTA ATACGGAGGG TGCAAGCGTT AATCGGAATT ACTGGGCGTA AAGCGCACGC AGGCGGTCTG
Erwinia amylovora (U80l95) CCGTGCCAGC AGCCGCGGTA ATACGGAGGG TGCAAGCGTT AATCGGAATT ACTGGGCGTA AAGCGCACGC AGGCGGTCTG
Erwinia pyrifoliae CCGTGCCAGC AGCCGCGGTA ATACGGAGGG TGCAAGCGTT AATCGGAATT ACTGGGCGTA AAGCGCACGC AGGCGGTCTG
pectobacterium cypripedii CCGTGCCAGC AGCCGCGGTA ATACGGAGGG TGCAAGCGTT AATCGGAATN ACTGGGCGTA AAGCGCACGC AGGCGGTCTG
pectobacterium chrysanthemi CCGTGCCAGC AGCCGCGGTA ATACGGAGGG TGCAAGCGTT AATCGGAATG ACTGGGCGTA AAGCGCACGC AGGCGGTCTG
pectobacterium c. subsp. carotovorum CCGTGCCAGC AGCCGCGGTA ATACGGAGGG TGCAAGCGTT AATCGGAATG ACTGGGCGTA AAGCGCACGC AGGCGGTCTG
pectobacterium c. subsp. wasabiae CCGTGCCAGC AGCCGCGGTA ATACGGAGGG TGCAAGCGTT AATCGGAATG ACTGGGCGTA AAGCGCACGC AGGCGGTCTG
pectobacterium c. subsp. betavasculorum CCGTGCCAGC AGCCGCGGTA ATACGGAGGG TGCAAGCGTT AATCGGAATG ACTGGGCGTA AAGCGCACGC AGGCGGTCTG
pectobacterium c. subsp. atrosepticum CCGTGCCAGC AGCCGCGGTA ATACGGAGGG TGCAAGCGTT AATCGGAATG ACTGGGCGTA AAGCGCACGC AGGCGGTCTG
pectobacterium c. subsp. odoriferum CCGTGCCAGC AGCCGCGGTA ATACGGAGGG TGCAAGCGTT AATCGGAATG ACTGGGCGTA AAGCGCACGC AGGCGGTCTG
pectobacterium cacticidum CCGTGCCAGC AGCCGCGGTA ATACGGAGGG TGCAAGCGTT AATCGGAATG ACTGGGCGTA AAGCGCACGC AGGCGGTTTG
Brenneria salicis CCGTGCCAGC AGCCGCGGTA ATACGGAGGG TGCAAGCGTT AATCGGAATG ACTGGGCGTA AAGCGCACGC AGGCGGTGTG
Brenneria rubrifaciens CCGTGCCAGC AGCCGCGGTA ATACGGAGGG TGCAAGCGTT AATCGGAATG ACTGGGCGTA AAGCGCACGC AGGCGGTCTG
Brenneria nigrifluens CCGTGCCAGC AGCCGCGGTA ATACGGAGGG TGCAAGCGTT AATCGGAATG ACTGGGCGTA AAGCGCACGC AGGCGGTCTG
Brenneria alni CCGTGCCAGC AGCCGCGGTA ATACGGAGGG TGCAAGCGTT AATCGGAATG ACTGGGCGTA AAGCGCACGC AGGCGGTCTG
Brenneria paradisiaca CCGTGCCAGC AGCCGCGGTA ATACGGAGGG TGCAAGCGTT AATCGGAATG ACTGGGCGTA AAGCGCACGC AGGCGGTCTG
.....
w
570 580 590 600 610 620 630 640
Buchnera aphidicola TTAAGTCAGA TGTGAAATCC CTAGGCTTAA CCTAGGAACT GCATTTGAAA CTGAAATGCT AGAGTATCGT AGAGGGAGGT
Proteus vulgaris TTAAGTCAGA TGTGAAAGCC CCGAGCTCAA CTTGGGAACT GCATCTGAAA CTGGCTGGCT AGAGTCTTGT AGAGGGGGGT
Erwinia ananas, Zululand, KZN, SA TTAAGTCAGA TGTGAAATCC CCGGGCTTAA CCTGGGAACT GCATTTGAAA CTGGCAGGCT TGAGTCTCGT AGAGGGGGGT
Pantoea a. Py. ananatis TTAAGTCAGA TGTGAAATCC CCGGGCTTAA CCTGGGAACT GCATTTGAAA CTGGCAGGCT TGAGTCTCGT AGAGGGGGGT
Pantoea a. Py. uredovora TTAAGTCAGA TGTGAAATCC CCGGGCTTAA CCTGGGAACT GCATTTGAAA CTGGCAGGCT TGAGTCTCGT AGAGGGGGGT
Pantoea agglomerans (U80202) TTAAGTCAGA TGTGAAATCC CCGGGCTTAA CCTGGGAACT GCATTTGAAA CTGGCAGGCT TGAGTCTTGT AGAGGGGGGT
Pantoea agglomerans (AB004757) TTAAGTCAGA TGTGAAATCC CCGGGCTTAA CCTGGGAACT GCATTTGAAA CTGGCAGGCT TGAGTCTTGT AGAGGGGGGT
Pantoea agglomerans (U80183) TTAAGTCAGA TGTGAAATCC CCGGGCTTAA CCTGGGAACT GCATTTGAAA CTGGCAGGCT TGAGTCTTGT AGAGGGGGGT
Unknown Erwinia sp. TTAAGTCAGA TGTGAAATCC CCGGGCTTAA CCTGGGAACT GCATTTGAAA CTGGCAGGCT TGAGTCTCGT AGAGGGGGGT
Pantoea s. subsp. stewartii TTAAGTCAGA TGTGAAATCC CCGGGCTTAA CCTGGGAACT GCATTTGAAA CTGGCAGGCT TGAGTCTCGT AGAGGGGGGT
Erwinia tracheiphila TTAAGTCAGA TGTGAAATCC CCGGGCTTAA CCTGGGAACT GCATTTGAAA CTGGCAGGCT GGAGTCTTGT AGAGGGGGGT
Erwinia psidii TTAAGTCAGA TGTGAAATCC CCGGGCTTAA CCTGGGAACT GCATTTGAAA CTGGCAGGCT TGAGTCTTGT AGAGGGGGGT
Erwinia mallotivora (AJ233414) TTAAGTCAGA TGTGAAATCC CCGGGCTTAA CCTGGGAACT GCATTTGAAA CTGGCAGGCT TGAGTCTTGT AGAGGGGGGT
Erwinia mallotivora (Z96084) TTAAGTCAGA TGTGAAATCC CCGGGCTTAA CCTGGGAACT GCATTTGAAA CTGGCAGGCT TGAGTCTTGT AGAGGGGGGT
Erwinia persicinus (AJOOl190) TCAAGTCGGA TGTGAAATCC CCGGGCTCAA CCTGGGAACT GCATTCGAAA CTGGCAGGCT AGAGTCTTGT AGAGGGGGGT
Erwinia persicinus (Z96086) TCAAGTCGGA TGTGAAATCC CCGGGCTCAA CCTGGGAACT GCATTCGAAA CTGGCANGCT AGAGTCTTGT AGAGGGGGGT
Erwinia persicinus (U80205) TCAAGTCGGA TGTGAAATCC CCGGGCTCAA CCTGGGAACT GCATTCGAAA CTGGCAGGCT AGAGTCTTGT AGAGGGGGGT
Erwinia rhapontici (AJ233417) TCAAGTCGGA TGTGAAATCC CCGGGCTCAA CCTGGGAACT GCATTCGAAA CTGGCAGACT AGAGTCTTGT AGAGGGGGGT
Erwinia rhapontici (Z96087) TCAAGTCGGA TGTGAAATCC CCGGGCTCAA CCTGGGAACT GCATTCGAAA CTGGCAGACT AGAGTCTTGT AGAGGGGGGT
Erwinia rhapontici (U80206) TCAAGTCGGA TGTGAAATCC CCGGGCTCAA CCTGGGAACT GCATTCGAAA CTGGCAGACT AGAGTCTTGT AGAGGGGGGT
Erwinia amylovora (Z96088) TCAAGTCGGA TGTGAAATCC CCGGGCTTAA CCTGGGAACT GCATTCGANA CTGGCAGGCT AGAGTCTCGT NGAGGGGGGT
Erwinia amylovora (AJ233410) TCAAGTCGGA TGTGAAATCC CCGGGCTTAA CCTGGGAACT GCATTCGAAA CTGGCAGGCT AGAGTCTCGT AGAGGGGGGT
Erwinia amylovora (AJ010485) TCAAGTCGGA TGTGAAATCC CCGGGCTTAA CCTGGGAACT GCATTCGAAA CTGGCAGGCT AGAGTCTCGT AGAGGGGGGT
Erwinia amylovora (U80195) TCAAGTCGGA TGTGAAATCC CCGGGCTTAA CCTGGGAACT GCATTCGAAA CTGGCAGGCT AGAGTCTCGT AGAGGGGGGT
Erwinia pyrifoliae TCAAGTCGGA TGTGAAATCC CCGGGCTTAA CCTGGGAACT GCATTCGAAA CTGGCAGGCT AGAGTCTCGT AGAGGGGGGT
Pectobacterium cypripedii TNAAGTTGGA TGTGAAATCC CCGGGCTTAA CCTGGGAACT GCATTCAAAA CTGACAGGCT AGAGTCTCGT AGAGGGGGGT
Pectobacterium chrysanthemi TTAAGTTGGA TGTGAAATCC CCGGGCTTAA CCTGGGAACT GCATTCAAAA CTGACAGGCT AGAGTCTCGT AGAGGGGGGT
pectobacterium c. subsp. carotovorum TTAAGTTGGA TGTGAAATCC CCGGGCTTAA CCTGGGAACT GCATTCAAAA CTGACAGGCT AGAGTCTTGT AGAGGGGGGT
pectobacterium c. subsp. wasabiae TTAAGTTGGA TGTGAAATCC CCGGGCTTAA CCTGGGAACT GCATTCAAAA CTGACAGGCT AGAGTCTTGT AGAGGGGGGT
pectobacterium c. subsp. betavasculorum TTAAGTTGGA TGTGAAATCC CCGGGCTTAA CCTGGGAACT GCATTCAAAA CTGACAGGCT AGAGTCTTGT AGAGGGGGGT
pectobacterium c. subsp. atrosepticum TTAAGTTGGA TGTGAAATCC CCGGGCTTAA CCTGGGAACT GCATTCAAAA CTGACAGGCT AGAGTCTTGT AGAGGGGGGT
pectobacterium c. subsp. odoriferum TTAAGTTGGA TGTGAAATCC CCGGGCTTAA CCTGGGAACT GCATTCAAAA CTGACAGGCT AGAGTCTTGT AGAGGGGGGT
pectobacterium cacticidum TTAAGTTGGA TGTGAAATCC CCGGGCTTAA CCTGGGAACT GCATTCAAAA CTGACAGGCT AGAGTCTTGT AGAGGGGGGT
Brenneria salicis TTAAGTTGGA TGTGAAATCC CCGGGCTCAA CCCGGGAACA GCATTCAAAA CTGACAGGCT AGAGTCTCGT AGAGGGGGGT
Brenneria rubrifaciens TTAAGTTGGA TGTGAAATCC CCGGGCTTAA CCCGGGAACT GCATTCAAAA CTGACAGGCT GGAGTCTCGT AGAGGGGGGT
Brenneria nigrifluens TTAAGTTGGA TGTGAAATCC CCGGGCTTAA CCTGGGAACT GCATTCAAAA CTGACAGGCT AGAGTCTCGT AGAGGGGGGT
Brenneria alni TTAAGTTGGA TGTGAAATCC CCGGGCTTAA CCTGGGAACT GCATTCAAAA CTGACAGGCT AGAGTCTTGT AGAGGGGGGT
Brenneria paradisiaca TTAAGTTGGA TGTGAAATCC CCGGGCTTAA CCTGGGAACT GCATTCAAAA CTGACAGGCT AGAGTCTCGT AGAGGGGGGT
_.
I.;J
N
650 660 670 680 690 700 710 720
Buchnera aphidicola AGAATTCTAG GTGTAGCGGT GAAATGCGTA GATATCTGGA GGAATACCCG TGGCGAAAGC GGCCTCCTAA ACGAATACTG
Proteus vulgaris AGAATTCCAC GTGTAGCGGT GAAATGCGTA GAGATGTGGA GGAATACCGG TGGCGAAGGC GGCCCCCTGG ACAAAGACTG
Erwinia ananas, Zululand, KZN, SA AGAATTCCAG GTGTAGCGGT GAAATGCGTA GAGATCTGGA GGAATACCGG TGGCGAAGGC GGCCCCCTGG ACGAAGACTG
Pantoea a. Py. ananatis AGAATTCCAG GTGTAGCGGT GAAATGCGTA GAGATCTGGA GGAATACCGG TGGCGAAGGC GGCCCCCTGG ACGAAGACTG
Pantoea a. Py. uredovora AGAATTCCAG GTGTAGCGGT GAAATGCGTA GAGATCTGGA GGAATACCGG TGGCGAAGGC GGCCCCCTGG ACGAAGACTG
Pantoea agglomerans (U80202) AGAATTCCAG GTGTAGCGGT GAAATGCGTA GAGATCTGGA GGAATACCGG TGGCGAAGGC GGCCCCCTGG ACAAAGACTG
Pantoea agglomerans (AB004757) AGAATTCCAG GTGTAGCGGT GAAATGCGTA GAGATCTGGA GGAATACCGG TGGCGAAGGC GGCCCCCTGG ACAAAGACTG
Pantoea agglomerans (U80l83) AGAATTCCAG GTGTAGCGGT GAAATGCGTA GAGATCTGGA GGAATACCGG TGGCGAAGGC GGCCCCCTGG ACAAAGACTG
Unknown Erwinia sp. AGAATTCCAG GTGTAGCGGT GAAATGCGTA GAGATCTGGA GGAATACCGG TGGCGAAGGC GGCCCCCTGG ACGAAGACTG
Pantoea s. subsp. stewartii AGAATTCCAG GTGTAGCGGT GAAATGCGTA GAGATCTGGA GGAATACCGG TGGCGAAGGC GGTCCCCTGG ACGAAGACTG
Erwinia tracheiphila GGAATTCCAG GTGTANCNGT GAAATGCGTA NAGATCTGGA GGAATACCGG TGGCGAAGGC GGCCCCCTGG ACAAAGACTG
Erwinia psidii GGAATTCCAG GTGTAGCGGT GAAATGCGTA GAGATCTGGA GGAATACCGG TGGCGAAGGC GGCCCCCTGG ACAAAGACTG
Erwinia mallotivora (AJ2334l4) AGAATTCCAG GTGTAGCGGT GAAATGCGTA GAGATCTGGA GGAATACCGG TGGCGAAGGC GGCCCCCTGG ACAAAGACTG
Erwinia mallotivora (Z96084) AGAATTCCAG GTGTAGCGGT GAAATGCGTA GAGATCTGGA GGAATACCGG TGGCGAAGGC GGCCCCCTGG ACAAAGACTG
Erwinia persicinus (AJOOl190) AGAATTCCAG GTGTAGCGGT GAAATGCGTA GAGATCTGGA GGAATACCGG TGGCGAAGGC GGCCCCCTGG ACAAAGACTG
Erwinia persicinus (Z96086) AGAATTCCAG GTGTAGCGGT GAAATGCGTA GAGATCTGGA GGAATACCGG TGGCGAAGGC GGCCCCCTGG ACAAAGACTG
Erwinia persicinus (U80205) AGAATTCCAG GTGTAGCGGT GAAATGCGTA GAGATCTGGA GGAATACCGG TGGCGAAGGC GGCCCCCTGG ACAAAGACTG
Erwinia rhapontici (AJ2334l7) AGAATTCCAG GTGTAGCGGT GAAATGCGTA GAGATCTGGA GGAATACCGG TGGCGAAGGC GGCCCCCTGG ACAAAGACTG
Erwinia rhapontici (Z96087) AGAATTCCAG GTGTAGCGGT GAAATGCGTA GAGATCTGGA GGAATACCGG TGGCGAAGGC GGCCCCCTGG ACAAAGACTG
Erwinia rhapontici (U80206) AGAATTCCAG GTGTAGCGGT GAAATGCGTA GAGATCTGGA GGAATACCGG TGGCGAAGGC GGCCCCCTGG ACAAAGACTG
Erwinia amylovora (Z96088) AGAATTCCAG GTGTAGCGGT GAAATGCGTA GAGATCTGGG AGGATAC-GG TGGCGAAGGC GGCCCCCTGG ACGAAGACTG
Erwinia amylovora (AJ2334l0) AGAATTCCAG GTGTAGCGGT GAAATGCGTA GAGATCTGGA GGAATACCGG TGGCGAAGGC GGCCCCCTGG ACGAAGACTG
Erwinia amylovora (AJ010485) AGAATTCCAG GTGTAGCGGT G~TGCGTA GAGATCTGGA GGAATACCGG TGGCGAAGGC GGCCCCCTGG ACGAAGACTG
Erwinia amylovora (U80l95) AGAATTCCAG GTGTAGCGGT GAAATGCGTA GAGATCTGGA GGAATACCGG TGGCGAAGGC GGCCCCCTGG ACGAAGACTG
Erwinia pyrifoliae AGAATTCCAG GTGTAGCGGT GAAATGCGTA GAGATCTGGA GGAATACCGG TGGCGAAGGC GGCCCCCTGG ACGAAGACTG
Pectobacterium cypripedii AGAATTCCAG GTGTAGCGGT GAAATGCGTA GAGATCTGGA GGAATACCGG TGGCGAAGGC GGCCCCCTGG ACGAAGACTG
pectobacterium chrysanthemi AGAATTCCAG GTGTAGCGGT GAAATGCGTA GAGATCTGGA GGAATACCGG TGGCGAAGGC GGCCCCCTGG ACGAAGACTG
pectobacterium c. subsp. carotovorum AGAATTCCAG GTGTAGCGGT GAAATGCGTA GAGATCTGGA GGAATACCGG TGGCGAAGGC GGCCCCCTGG ACAAANACTG
pectobacterium c. subsp. wasabiae AGAATTCCAG GTGTANCGGT G~TGCGTA NAGATCTGGA GGAATACCGG TGGCGAAGGC GGCCCCCTGG ACAAAGACTG
pectobacterium c. subsp. betavasculorum AGAATTCCAG GTGTAGCGGT GAAATGCGTA GAGATCTGGA GGAATACCGG TGGCGAAGGC GGCCCCCTGG ACAAAGACTG
Pectobacterium c. subsp. atrosepticum AGAATTCCAG GTGTAGCGGT GAAATGCGTA GAGATCTGGA GGAATACCGG TGGCGAAGGC GGCCCCCTGG ACAAAGACTG
pectobacterium c. subsp. odoriferum AGAATTCCAG GTGTAGCGGT GAAATGCGTA GAGATCTGGA GGAATACCGG TGGCGAAGGC GGCCCCCTGG ACAAAGACTG
Pectobacterium cacticidum AGAATTCCAG GTGTANCGGT GAAATGCGTA GAGATCTGGA GGAATACCGG TGGCGAAGGC GGCCCCCTGG ACAAAGACTG
Brenneria salicis AGAATTCCAG GTGTAGCGGT GAAATGCGTA GAGATCTGGA GGAATACCGG TGGCGAAGGC GGCCCCCTGG ACGAAGACTG
Brenneria rubrifaciens AGAATTCCAG GTGTAGCGGT GAAATGCGTA GAGATCTGGA GGAATACCGG TGGCGAAGGC GGCCCCCTGG ACGAAGACTG
Brenneria nigrifluens AGAATTCCAG GTGTAGCGGT GAAATGCGTA GAGATCTGGA GGAATACCGG TGGCGAAGGC GGGCCCCTGG ACGAAGACTG
Brenneria alni AGAATTCCAG GTGTAGCGGT GAAATGCGTA GAGATCTGGA GGAATACCGG TGGCGAAGGC GGCCCCCTGG ACAAAGACTG
Brenneria paradisiaca AGAATTCCAG GTGTAGCGGT GAAATGCGTA GAGATCTGGA GGAATACCGG TGGCGAAGGC GGCCCCCTGG ACGAAGACTG
......
w
w
730 740 750 760 770 7BO 790 BOO
Buchnera aphidicola ACACTGAGGT GCGAAAGCGT GGGGAGCAAA CAGGATTAGA TACCCTGGTA GTCCATGCCG TAAACGATGT CGACTTGGAG
Proteus vulgaris ACGCTCAGGT GCGAAAGCGT GGGGACCAAA CAGGATTAGA TACCCTGGTA GTCCACGCTG TAAACGATGT CGATTTAGAG
Erwinia ananas, Zululand, KZN, SA ACGCTCAGGT GCGAAAGCGT GGGGAGCAAA CAGGATTAGA TACCCTGGTA GTCCACGCCG TAAACGATGT CGACTTGGAG
Pantoea a. Py. ananatis ACGCTCAGGT GCGAAAGCGT GGGGAGCAAA CAGGATTAGA TACCCTGGTA GTCCACGCCG TAAACGATGT CGACTTGGAG
Pantoea a. Py. uredovora ACGCTCAGGT GCGAAAGCGT GGGGAGCAAA CAGGATTAGA TACCCTGGTA GTCCACGCCG TAAACGATGT CGACTTGGAG
Pantoea agglomerans (U80202) ACGCTCAGGT GCGAAAGCGT GGGGAGCAAA CAGGATTAGA TACCCTGGTA GTCCACGCCG TAGACGATGT CGACTTGGAG
Pantoea agglomerans (AB004757) ACGCTCAGGT GCGAAAGCGT GGGGAGCAAA CAGGATTAGA TACCCTGGTA GTCCACGCCG TAAACGATGT CGACTTGGAG
Pantoea agglomerans (U80l83) ACGCTCAGGT GCGAAAGCGT GGGGAGCAAA CAGGATTAGA TACCCTGGTA GTCCACGCCG TAAACGATGT CGACTTGGAG
Unknown Erwinia sp. ACGCTCAGGT GCGAAAGCGT GGGGAGCAAA CAGGATTAGA TACCCTGGTA GTCCACGCCG TAAACGATGT CGATTTGGAG
Pantoea s. subsp. stewartii ACGCTCAGGT GCGAAAGCGT GGGGAGCAAA CAGGATTAGA TACCCTGGTA GTCCACGCCG TAAACGATGT CGACTTGGAG
Erwinia tracheiphila ACGCTCATGT GCGAAAGCGT GGGGAGCAAA CAGGATTAGA TACCCTGGTA GTCCACGCTG TAAACGATGT CGATTTGGAG
Erwinia psidii ACGCTCAGGT GCGAAAGCGT GGGGAGCAAA CAGGATTAGA TACCCTGGTA GTCCACGCCG TAAACGATGT CGATTTGGAG
Erwinia mallotivora (AJ2334l4) ACGCTCAGGT GCGAAAGCGT GGGGAGCAAA CAGGATTAGA TACCCTGGTA GTCCACGCCG TAAACGATGT CGATTTGGAG
Erwinia mallotivora (Z96084) ACGCTCAGGT GCGAAAGCGT GGGGAGCAAA CAGGATTAGA TACCCTGGTA GTCCACGCCG TAAACGATGT CGATTTGGAG
Erwinia persicinus (AJOOl190) ACGCTCAGGT GCGAAAGCGT GGGGAGCAAA CAGGATTAGA TACCCTGGTA GTCCACGCCG TAAACGATGT CGACTTGGAG
Erwinia persicinus (Z96086) ACGCTCAGGT GCGAAAGCGT GGGGAGCAAA CAGGATTAGA TACCCTGGTA GTCCACGCCG TAAACGATGT CGACTTGGAG
Erwinia persicinus (U80205) ACGCTCAGGT GCGAAAGCGT GGGGAGCAAA CAGGATTAGA TACCCTGGTA GTCCACGCCG TAAACGATGT CGACTTGGAG
Erwinia rhapontici (AJ2334l7) ACGCTCAGGT GCCAAAGCGT GGGGAGCAAA CAGGATTAGA TACCCTGGTA GTCCACGCCG TAAACGATGT CGACTTGGAG
Erwinia rhapontici (Z96087) ACGCTCAGGT GCGAAAGCGT GGGGAGCAAA CAGGATTAGA TACCCTGGTA GTCCACGCCG TAAACGATGT CGACTTGGAG
Erwinia rhapontici (U80206) ACGCTCAGGT GCGAAAGCGT ~GGGAGCAAA CAGGATTAGA TACCCTGGTA GTCCACGCCG TAAACGATGT CGACTTGGAG
Erwinia amylovora (Z96088) ACGCTCAGGT GCGAAAGCGT GGGGAGCAAA CAGGATTAGA TACCCTGGTA GTCCACGCCG TAAACGATGT CGACTTGGAG
Erwinia amylovora (AJ2334l0) ACGCTCAGGT GCGAAAGCGT GGGGAGCAAA CAGGATTAGA TACCCTGGTA GTCCACGCCG TAAACGATGT CGACTTGGAG
Erwinia amylovora (AJOl0485) ACGCTCAGGT GCGAAAGCGT GGGGAGCAAA CAGGATTAGA TACCCTGGTA GTCCACGCCG TAAACGATGT CGACTTGGAG
Erwinia amylovora (U80l95) ACGCTCAGGT GCGAAAGCGT GGGGAGCAAA CAGGATTAGA TACCCTGGTA GTCCACGCCG TAAACGATGT CGACTTGGAG
Erwinia pyrifoliae ACGCTCAGGT GCGAAAGCGT GGGGAGCAAA CAGGATTAGA TACCCTGGTA GTCCACGCCG TAAACGATGT CGACTTGGAG
Pectobacterium cypripedii ACGCTCAGGT GCGAAAGCGT GGGGANCAAA CAGGATTAGA TACCCTGGTA GTCCACGCTG TAAACGATGT CGACTTGGAG
pectobacterium chrysanthemi ACGCTCAGGT GCGAAAGCGT GGGGAGCAAA CAGGATTAGA TACCCTGGTA GTCCACGCTG TAAACGATGT CGATTTGGAG
pectobacterium c. subsp. carotovorum ACGCTCAGGT GCGAAAGCGT GGGGAGCAAA CAGGATTAGA TACCCTGGTA GTCCACGCTG TAAACGATGT CGATTTGGAG
pectobacterium c. subsp. wasabiae ACGCTCAGGT GCGAAAGCGT GGGGAGCAAA CAGGATTAGA TACCCTGGTA GTCCACGCTG TAAACGATGT CGACTTGGAG
Pectobacteriiun c. aubep. betavasculorum ACGCTCAGGT GCGAAAGCGT GGGGAGCAAA CAGGATTAGA TACCCTGGTA GTCCACGCTG TAAACGATGT CGACTTGGAG
Pectobacterium c. subsp. atrosepticum ACGCTCAGGT GCGAAAGCGT GGGGAGCAAA CAGGATTAGA TACCCTGGTA GTCCACGCTG TAAACGATGT CGACTTGGAG
Pectobacterium c. subsp. odoriferum ACGCTCAGGT GCGAAAGCGT GGGGAGCAAA CAGGATTAGA TACCCTGGTA GTCCACGCTG TAAACGATGT CGATTTGGAG
pectobacterium cacticidum ACGCTCAGGT GCGAAAGCGT GGGGAGCAAA CAGGATTAGA TACCCTGGTA GTCCACGCTG TAAACGATGT CNACTTGGAG
Brenneria salicis ACGCTCAGGT GCGAAAGCGT GGGGAGCAAA CAGGATTAGA TACCCTGGTA GTCCACGCCG TAAACGATGT CGACTTGGAG
Brenneria rubrifaciens ACGCTCAGGT GCGAAAGCGT GGGGAGCAAA CAGGATTAGA TACCCTGGTA TTCCACGCCG TAAACGATGT CGACTTGGAG
Brenneria nigrifluens ACGCTCAGGT GCGAAAGCGT GGGGAGCAAA CAGGATTAGA TACCCTGGTA GTCCACGCCG TAAACGATGT CGACTTGGAG
Brenneria alni ACGCTCAGGT GCGAAAGCGT GGGGAGCAAA CAGGATTAGA TACCCTGGTA GTCCACGCTG TAAACGATGT CGACTTGAAG
Brenneria paradisiaca ACGCTCAGGT GCGAAAGCGT GGGGAGCAAA CAGGATTAGA TACCCTGGTA GTCCACGCTG TAAACGATGT CGATTTGGAG
\-~;.)
810 820 830 840 850 860 870 880
Buchnera aphidicola GTTGTTTCCA AGAGAAGTGA CTTCCGAAGC TAACGCGTTA AGTCGACCGC CTGGGGAGTA CGGCCGCAAG GCTAAAACTC
Proteus vulgaris GTTGTGGTCT TGAACTGTGG CTTCTGCAGC TAACGCGTTA AATCGACCGC CTGGGGAGTA CGGCCGCAAG GTTAAAACTC
Erwinia ananas, Zululand, KZN, SA GTTGTTCCCT TGAGGAGTGG CTTCCGGAGC TAACGCGTTA AGTCGACCGC CTGGGGAGTA CGGCCGCAAG GTTAAAACTC
Pantoea a. Py. ananatis GTTGTTCCCT TGAGGAGTGG CTTCCGGAGC TAACGCGTTA AGTCGACCGC CTGGGGAGTA CGGCCGCAAG GTTAAAACTC
Pantoea a. Py. uredovora GTTGTTCCCT TGAGGAGTGG CTTCCGGAGC TAACGCGTTA AGTCGACCGC CTGGGGAGTA CGGCCGCAAG GTTAAAACTC
Pantoea agglomerans (U80202) GTTGTTCCCT TGAGGAGTGG CTTCCGGAGC TAACGCGTTA AGTCGACCGC CTGGGGAGTA CGGCCGCAAG GTTAAAACTC
Pantoea agglomerans (AB004757) GTTGTTCCCT TGAGGAGTGG CTTCCGGAGC TAACGCGTTA AGTCGACCGC CTGGGGAGTA CGGCCGCAAG GTTAAAACTC
Pantoea agglomerans (U80l83) GTTGTTCCCT TGAGGAGTGG CTTCCGGAGC TAACGCGTTA AGTCGACCGC CTGGGGAGTA CGGCCGCAAG GTTAAAACTC
Unknown Erwinia sp. GTTGTTCCCT TGAGGCGTGG CTTCCGGAGC TAACGCGTTA AATCGACCGC CTGGGGAGTA CGGCCGCAAG GTTAAAACTC
Pantoea s. subsp. stewartii GTTGTTCCCT TGAGGAGTGG CTTCCGGAGC TAACGCGTTA AGTCGACCGC CTGGGGAGTA CGGCCGCAAG GTTAAAACTC
Erwinia tracheiphila GTTGTGCCCT TGAGGTGTGG CTTCCGTAGC TAACGCGTTA AATCGACCGC CTGGGGAGTA CGGCCGCAAG GTTAAAACTC
Erwinia psidii GTTGTGCCCT TGAGGCGTGG CTTCCGTAGC TAACGCGTTA AATCGACCGC CTGGGGAGTA CGGCCGCAAG GTTAAAACTC
Erwinia mallotivora (AJ2334l4) GTTGTGCCCT TGAGGCGTGG CTTCCGTAGC TAACGCGTTA AATCGACCGC CTGGGGAGTA CGGCCGCAAG GTTAAAACTC
Erwinia mallotivora (Z96084) GTTGTGCCCT TGAGGCGTGG NTTCNNTAGN TAACGCGTTA AATCGACCGC CTGGGGAGTA CGGCCGCAAG GTTAAAACTC
Erwinia persicinus (AJOOl190) GTTGTGCCCT TGAGGCGTGG CTTCCGGAGC TAACGCGTTA AGTCGACCGC CTGGGGAGTA CGGCCGCAAG GTTAAAACTC
Erwinia persicinus (Z96086) GTTGTGCCCT TGAGGCGTGG CTTCCGGAGC TAACGCGTTA AGTCGACCGC CTGGGGAGTA CGGCCGCAAG GTTAAAACTC
Erwinia persicinus (U80205) GTTGTGCCCT TGAGGCGTGG CTTCCGGAGC TAACGCGTTA AGTCGACCGC CTGGGGAGTA CGGCCGCAAG GTTAAAACTC
Erwinia rhapontici (AJ2334l7) GTTGTGCCCT TGAGGCGTGG CTTCCGGAGC TAACGCGTTA AGTCGACCGC CTGGGGAGTA CGGCCGCAAG GTTAAAACTC
Erwinia rhapontici (Z96087) GTTGTGCCCT TGAGGCGTGG NTTCCGGAGC TAACGCGTTA AGTCGACCGC CTGGGGAGTA CGGCCGCAAG GTTAAAACTC
Erwinia rhapontici (U80206) GTTGTGCCCT TGAGGCGTGG CTTCCGGAGC TAACGCGTTA AGTCGACCGC CTGGGGAGTA CGGCCGCAAG GTTAAAACTC
Erwinia amylovora (Z96088) GTTGTTCCCC TGAGGAGTGG NTTCNGGAGC TAACGCGTTA AGTCGACCGC CTGGGGAGTA CGGCCGCAAG GTTAAAACTC
Erwinia amylovora (AJ233410) GCTGTTCCCC TGAGGAGTGG CTTCCGGAGC TAACGCGTTA AGTCGACCGC CTGGGGAGTA CGGCCGCAAG GTTAAAACTC
Erwinia amylovora (AJ010485) GCTGTTCCCC TGAGGAGTGG CTTCCGGAGC TAACGCGTTA AGTCGACCGC CTGGGGAGTA CGGCCGCAAG GTTAAAACTC
Erwinia amylovora (U80195) GCTGTTCCCT TGAGGAGTGG CTTCCGGAGC TAACGCGTTA AGTCGACCGC CTGGGGAGTA CGGCCGCAAG GTTAAAACTC
Erwinia pyrifoliae GCTGTTCCCC TGAGGAGTGG CTTCCGGAGC TAACGCGTTA AGTCGACCGC CTGGGGAGTA CGGCCGCAAG GTTAAAACTC
Pectobacterium cypripedii GTTGTGCCCT TGAGGCGTGA CTTCCGGAGC TAACGCGTTA AGTCGACCGC CTGGGGAGTA CGGCCGCAAG GTTNAAACTC
pectobacterium chrysanthemi GTTGTGCCCT TGAGGCGTGG CTTCCGGAGC TAACGCGTTA AATCGACCGC CTGGGGAGTA CGGCCGCAAG GTTAAAACTC
pectobacterium c. subsp. carotovorum GTTGTGCCCT TGAGGCGTGG CTTCCGGAGC TAACGCGTTA AATCGACCGC CTGGGGAGTA CGGCCGCAAG GTTAAAACTC
pectobacterium c. subsp. wasabiae GTTGTGCCCT TGAGGCGTGG CTTCCGGAGC TAACGCGTTA AGTCGACCGC CTGGGGAGTA CGGCCGCAAG GTTAAAACTC
pectobacterium c. subsp. betavasculorum GTTGTGCCCT TGAGGCGTGG CTTCCGGAGC TAACGCGTTA AGTCGACCGC CTGGGGAGTA CGGCCGCAAG GTTAAAACTC
Pectobacterium c. subsp. atrosepticum GTTGTGCCCT TGAGGCGTGG CTTCCGGAGC TAACGCGTTA AGTCGACCGC CTGGGGAGTA CGGCCGCAAG GTTAAAACTC
pectobacterium c. subsp. odoriferum GTTGTGCCCT TGAGGCGTGG CTTCCGGAGC TAACGCGTTA AATCGACCGC CTGGGGAGTA CGGCCGCAAG GTTAAAACTC
pectobacterium cacticidum GTTGTGCCCT AAAAGCGTGG CTTCCGGAGC TAACGCGTTA AGTCGACCGC CTGGGGAGTA CGGCCGCAAG GTTAAAACTC
Brenneria salicis GCTGTGGTCT TGAACCGTGG CTTCCGGAGC TAACGCGTTA AGTCGACCGC CTGGGGAGTA CGGCCGCAAG GTTAAAACTC
Brenneria rubrifaciens GCTGTGGTCC AGAACCGTGG CTTCCGGAGC TAACGCGTTA AGTCGACCGC CTGGGGAGTA CGGCCGCAAG GTTAAAACTC
Brenneria nigrifluens GCTGTGGTCT TGAACCGTGG CTTCCGGAGC TAACGCGTTA AGTCGACCGC CTGGGGAGTA CGGCCGCAAG GTTAAAACTC
Brenneria alni GTTGTGGCCT TGAGCCGTGG CTTTCGGAGC AAACGCGTTA AGTCGACCGC CTGGGGAGTA CGGCCGCAAG GTTAAAACTC
Brenneria paradisiaca GTTGTGGTCT TGAACCGTGG CTTCCGGAGC TAACGCGTTA AATCGACCGC CTGGGGAGTA CGGCCGCAAG GTTAAAACTC
......
v.>
Vl
890 900 910 920 930 940 950 960
Buchnera aphidicola AAATGAATTG ACGGGGGCCC GCACAAGCGG TGGAGCATGT GGTTTAATTC GATGCAACGC GAAAAACCTT ACCTGGTCTT
Proteus vulgaris AAATGAATTG ACGGGGGCCC GCACAAGCGG TGGAGCATGT GGTTTAATTC GATGCAACGC GAAGAACCTT ACCTACTCTT
Erwinia ananas, Zululand, KZN, SA AAATGAATTG ACGGGGGCCC GCACAAGCGG TGGAGCATGT GGTTTAATTC GATGCAACGC GAAGAACCTT ACCTACTCTT
Pantoea a. pv. ananatis AAATGAATTG ACGGGGGCCC GCACAAGCGG TGGAGCATAT GGTTTAATTC GATGCAACGC GAAGAACCTT ACCTACTCTT
Pantoea a. pv. uredovora AAATGAATTG ACGGGGGCCC GCACAAGCGG TGGAGCATAT GGTTTAATTC GATGCAACGC GAAGAACCTT ACCTACTCTT
Pantoea agglomerans (U80202) AAATGAATTG ACGGGGGCCC GCACAAGCGG TGGAGCATGT GGTTTAATTC GATGCAACGC GAAGAACCTT ACCTACTCTT
Pantoea agglomerans (AB004757) AAATGAATTG ACGGGGGCCC GCACAAGCGG TGGAGCATGT GGTTTAATTC GATGCAACGC GAAGAACCTT ACCTACTCTT
Pantoea agglomerans (U80l83) AAATGAATTG ACGGGGGCCC GCACAAGCGG TGGAGCATGT GGTrTAATTC GATGCAACGC GAAGAACCTT ACCTACTCTT
Unknown Erwinia sp. AAATGAATTG ACGGGGGCCC GCACAAGCGG TGGAGCATGT GGTTTAATTC GATGCAACGC GAAGAACCTT ACCTACTCTT
Pantoea s. subsp. stewartii AAATGAATTG ACGGGGGCCC GCACAAGCGG TGGAGCATGT GGTTTAATTC GATGCAACGC GAAGAACCTT ACCTACTCTT
Erwinia tracheiphila AAATGAATTG ACGGGGGCCC GCACAAGCGG TGGAGCATGT GGTTTAATTN GATGCAACGC GAAGAACCTT ACCTGGCCTT
Erwinia psidii AAATGAATTG ACGGGGGCCC GCACAAGCGG TGGAGCATGT GGTTTAATTC GATGCAACGC GAAGAACCTT ACCTGGCCTT
Erwinia mallotivora (AJ2334l4) AAATGAATTG ACGGGGGCCC GCACAAGCGG TGGAGCATGT GGTTTAATTC GATGCAACGC GAAGAACCTT ACCTGGCCTT
Erwinia mallotivora (Z96084) AAATGAATTG ACGGGGGCCC GCACAAGCGG TGGAGCATGT GGTTTAATTC GATGCAACGG GAAGAACCTT ACCTGGCCTT
Erwinia persicinus (AJOOl190) AAATGAATTG ACGGGGGCCC GCACAAGCGG TGGAGCATGT GGTTTAATTC GATGCAACGC GAAGAACCTT ACCTGGCCTT
Erwinia persicinus (Z96086) AAATGAATTG ACGGGGGCCC GCACAAGCGG TGGAGCATGT GGTTTAATTC GATGCAACGC GAAGAACCTT ACCTGGCCTT
Erwinia persicinus (U80205) AAATGAATTG ACGGGGGCCC GCACAAGCGG TGGAGCATGT GGTTTAATTC GATGCAACGC GAAGAACCTT ACCTGGCCTT
Erwinia rhapontici (AJ2334l7) AAATGAATTG ACGGGGGCCC GCACAAGCGG TGGAGCATGT GGTTTAATTC GATGCAACGC GAAGAACCTT ACCTGGCCTT
Erwinia rhapontici (Z96087) AAATGAATTG ACGGGGGCCC GCACAAGCGG TGGAGCATGT GGTTTAATTC GATGCAACGC GAAGAACCTT ACCTGGCCTT
Erwinia rhapontici (U80206) AAATGAATTG ACGGGGGCCC GCACAAGCGG TGGAGCATGT GGTTTAATTC GATGCAACGC GAAGAACCTT ACCTGGCCTT
Erwinia amylovora (Z96088) AAATGAATTG ACGGGGGCCC GCACAAGCGG TGGAGCATGT GGTTTAATTC GATGCAACGC GAAGAACCTT ACCTGGCCTT
Erwinia amylovora (AJ2334l0) AAATGAATTG ACGGGGGCCC GCACAAGCGG TGGAGCATGT GGTTTAATTC GATGCAACGC GAAGAACCTT ACCTGGCCTT
Erwinia amylovora (AJ010485) AAATGAATTG ACGGGGGCCC GCACAAGCGG TGGAGCATGT GGTTTAATTC GATGCAACGC GAAGAACCTT ACCTGGCCTT
Erwinia amylovora (U80195) AAATGAATTG ACGGGGGCCC GCACAAGCGG TGGAGCATGT GGTTTAATTC GATGCAACGC GAAGAACCTT ACCTGGCCTT
Erwinia pyrifoiiae AAATGAATTG ACGGGGGCCC GCACAAGCGG TGGAGCATGT GGTTTAATTC GATGCAACGC GAAGAACCTT ACCTGGCCTT
pectobacterium cypripedii AAATGAATTG ACGGGGGCCC GCACAAGCGG TGGAGCATGT GGTTTAATTC GATGCAACGC GAAGAACCTT ACCTGCTCTT
pectobacterium chrysanthemi AAATGAATTG ACGGGGGCCC.GCACAAGCGG TGGAGCATGT GGTTTAATTC GATGCAACGC GAAGAACCTT ACCTACTCTT
Pectobacterium c. subsp. carotovorum AAATGAATTG ACGGGGGCCC GCACAAGCGG TGGAGCATGT GGTTTAATTC GATGCAACGC GAAGAACCTT ACCTACTCTT
Pectobacterium c. subsp. wasabiae AAATGAATTG ACGGGGGCCC GCACAAGCGG TGGAGCATGT GGTTTAATTC GATGCAACGC GAAGAACCTT ACCTACTCTT
Pectobacterium c. subsp. betavasculorum AAATGAATTG ACGGGGGCCC GCACAAGCGG TGGAGCATGT GGTTTAATTC GATGCAACGC GAAGAACCTT ACCTACTCTT
pectobacterium c. subsp. atrosepticum AAATGAATTG ACGGGGGCCC GCACAAGCGG TGGAGCATGT GGTTTAATTC GATGCAACGC GAAGAACCTT ACCTACTCTT
pectobacterium c. subsp. odoriferum AAATGAATTG ACGGGGGCCC GCACAAGCGG TGGAGCATGT GGTTTAATTC GATGCAACGC GAAGAACCTT ACCTACTCTT
pectobacterium cacticidum AAATGAATTG ACGGGGGCCC GCACAAGCGG TGGAGCATGT GGTTTAATTC GATGCAACGC GAAGAACCTT ACTAATTCTT
Brenneria salicis AAATGAATTG ACGGGGGCCC GCACAAGCGG TGGAGCATGT GGTTTAATTC GATGCAACGC GAAGAACCTT ACCTACTCTT
Brenneria rubrifaciens AAATGAATTG ACGGGGGCCC GCACAAGCGG TGGAGCATGT GGTTTAATTC GATGCAACGC GAAGAACCTT ACCTACTCTT
Brenneria nigrifluens AAATGAATTG ACGGGGGCCC GCACAAGCGG TGGAGCATGT GGTTTAATTC GATGCAACGC GAAGAACCTT ACCTACTCTT
Brenneria alni AAATGAATTG ACGGGGGCCC GCACAAGCGG TGGAGCATGT GGTTTAATTC GATGCAACGC GAAGAACCTT ACCTACTCTT
Brenneria paradisiaca AAATGAATTG ACGGGGGCCC GCACAAGCGG TGGAGCATGT GGTTTAATTC GATGCAACGC GAAGAACCTT ACCTACTCTT
(-jJ
0\
970 980 990 1000 1010 1020 1030 1040
Buchnera aphidicola GACATCCACA GAA-TT---- T--TTT-AGA AATAAAAAAG TGCCTTCGGG AACTGTGAGA CAGGTGCTGC ATGGCTGTCG
Proteus vulgaris GACATCCAGC GAA------- TCCTTT-AGA GATAGAGGAG TGCCTTCGGG AACGCTGAGA CAGGTGCTGC ATGGCTGTCG
Erwinia ananas, Zululand, KZN, SA GACATCCAGA GAACTTAGC- -------AGA GATGCTTTGG TGCCTTCGGG AACTGTGAGA CAGGTGCTGC ATGGCTGTCG
Pantoea a. Py. ananatis GACATCCAGA GAACTTAGC- -------AGA GATGCTTTGG TGCCTTCGGG AACTCTGAGA CAGGTGCTGC ATGGCTGTCG
Pantoea a. Py. uredovora GACATCCAGA GAACTTAGC- -------AGA GATGCTTTGG TGCCTTCGGG AACTCTGAGA CAGGTGCTGC ATGGCTGTCG
Pantoea agglomerans (U80202) GACATCCAGC GAACTTAGC- -------AGA GATGCTTTGG TGCCTTCGGG AACGCTGAGA CAGGTGCTGC ATGGCTGTCG
Pantoea agglomerans (AB004757) GACATCCACG GAA-TTTGGC -------AGA GATGCCTTAG TGCCTTCGGG AACCGTGAGA CAGGTGCTGC ATGGCTGTCG
Pantoea agglomerans (U80183) GACATCCACG GAA-TTTGGC -------AGA GATGCCTTAG TGCCTTCGGG GACCGTGAGA CAGGTGCTGC ATGGCTGTCG
Unknown Erwinia sp. GACATCCACG GAACTTAGCC AG-T---AGA GATGCCTTGG TGCCTTCGGG AACGGTGAGA CAGGTGCTGC ATGGCTGTCG
Pantoea s. subsp. stewartii GACATCCAGC GAACTTGGC- -------AGA GATGCCTTGG TGCCTTCGGG AACGCTGAGA CAGGTGCTGC ATGGCTGTCG
Erwinia tracheiphila GACATCCACA GAACTTAGC- -------ACA GATGCTTTGG TGCCTTCGGG AGCTGTGAGA CAGGTGCTGC ATGGCTGTCG
Erwinia psidii GACATCCACA GAACTTAGC- -------AGA GATGCTTTGG TGC-TTCGGG AACTGTGAGA CAGGTGCTGC ATGGCTGTCG
Erwinia mallotivora (AJ233414) GACATCCACG GAA------G ACCT--CAGA GATGGGGTTG TGCCTTCGGG AACCGTGAGA CAGGTGCTGC ATGGCTGTCG
Erwinia mallotivora (Z96084) GACATCCACG GAA------G ACCT--CAGA GATGGGGTTG TGCCTTCGGG AACCGTGAGA CAGGTGCTGC ATGGCTGTCG
Erwinia persicinus (AJOOl190) GACATCCACG GAA-TTCGGC -------AGA GATGCCTTAG TGCCTTCGGG AACCGTGAGA CAGGTGCTGC ATGGCTGTCG
Erwinia persicinus (Z96086) GACATCCACG GAA-TTCGGC -------AGA GATGCCTTAG TGCCTTCGGG AACCGTGAGA CAGGTGCTGC ATGGCTGTCG
Erwinia persicinus (U80205) GACATCCACG GAA-TTTGGC -------AGA GATGCCTTAG TGCCTTCGGG AACCGTGAGA CAGGTGCTGC ATGGCTGTCG
Erwinia rhapontici (AJ233417) GACATCCACG GAA-TTCGGC -------AGA GATGCCTTAG TGCCTTCGGG AACCGTGAGA CAGGTGCTGC ATGGCTGTCG
Erwinia rhapontici (Z96087) GACATCCACG GAA-TTCGGC -------AGA GATGCCTTAG TGCCTTCGGG AACCGTGAGA CAGGTGCTGC ATGGCTGTCG
Erwinia rhapontici (U80206) GACATCCACG GAA-TTCGGC -------AGA GATGCCTTAG TGCCTTCGGG AACCGTGAGA CAGGTGCTGC ATGGCTGTCG
Erwinia amylovora (Z96088) GACATCCACG GAA-TT---- --CT-GCAGA GATGCGAATG TGCCTTCGGG AACCGTGAGA CAGGTGCTGC ATGGCTGTCG
Erwinia amylovora (AJ233410) GACATCCACG GAA-TT---- --CT-GCAGA GATGCGGAAG TGCCTTCGGG AACCGTGAGA CAGGTGCTGC ATGGCTGTCG
Erwinia amylovora (AJOI0485) GACATCCACG GAA-TT---- --CT-GCAGA GATGCGGAAG TGCCTTCGGG AACCGTGAGA CAGGTGCTGC ATGGCTGTCG
Erwinia amylovora (U80195) GACATCCACG GAA-TT---- --CT-GCAGA GATGCGGAAG TGCCTTCGGG AACCGTGAGA CAGGTGCTGC ATGGCTGTCG
Erwinia pyrifoliae GACATCCACG GAA-TTTTGC -------AGA GATGCGGAAG TGC-TTCGGG AACCGTGAGA CAGGTGCTGC ATGGCTGTCG
Pectobacterium cypripedii GACATCCAGA GAA-TT---- --CT-GCAGA GATGCGNTNG TGCCTTCGGG ACCTCTGAGA CAGGTGCTGC ATGGCTGTCG
pectobacterium chrysanthemi GACATCCAGA GAA------G -CCT-GCAGA GATGCGGGTG TGCCTTCGGG AGCTCTGAGA CAGGTGCTGC ATGGCTGTCG
Pectobacterium c. subsp. carotovorum GACATCCACA GAA-TTTGG- ---T---AGA GATACCTTAG TGCCTTCGGG AACTGTGAGA CAGGTGCTGC ATGGCTGTCG
pectobacterium c. subsp. wasabiae GACATCCACA GAA-TTCGG- ---T---AGA GATACCTTAG TGCCTTCGGG AACTGTGAGA CAGGTGCTGC ATGGCTGTCG
pectobacterium c. subsp. betavasculorum GACATCCACA GAA-TTTGGC -------AGA GATGCCTTAG TGCCTTCGGG AACTGTGAGA CAGGTGCTGC ATGGCTGTCG
pectobacterium c. subsp. atrosepticum GACATCCAGA GAA-TTTGGC -------AGA GATGCCTTAG TGCCTTCGGG AACTCTGAGA CAGGTGCTGC ATGGCTGTCG
pectobacterium c. subsp. odoriferum GACATCCAGA GAA-TTAGC- ---T---AGA GATAGCTGAG TGCCTTCGGG AACTCTGAGA CAGGTGCTGC ATGGCTGTCG
Pectobacterium cacticidum GACATCCACA GAA----GGC ---TTT-AGA GATAGAGCTG TGTCTTCGGA AACTGTGAGA CAGGTGCTGC ATGGCTGTCG
Brenneria salicis GACATCCAGA GAA------G AC-TGT-AGA GATACGGTTG TGCCTTCGGG AGCTCTGAGA CAGGTGCTGC ATGGCTGTCG
Brenneria rubrifaciens GACATCCAGA GAA------G AC-T-TCAGA GATGAGGTTG TGCCTTAGGG AGCTCTGAGA CAGGTGCTGC ATGGCTGTCG
Brenneria nigrifluens GACATCCTCA GAA----GAG AC-T---GGA GACAGTCTTG TGCCTTCGGG AACTGAGAGA CAGGTGCTGC ATGGCTGTCG
Brenneria alni GACATCCTCA GAA----GAG AC-T---GGA GATAGTTTTG TGCCTTCGGG AACTGAGAGA CAGGTGCTGC ATGGCTGTCG
Brenneria paradisiaca GACATC--CA GAG---A-AG AC-T-GCAGA GATGCGGTTG TGCCTTCGGG AGCTCTGAGA CAGGTGCTGC ATGGCTGTCG
-W
-.J
r
1050 1060 1070 1080 1090 1100 1110 1120
Buchnera aphidicola TCAGCTCGT- GTTGTGAAAT GTTGGGTTAA GTCCCGCAAC GAGCGCAACC CTTATCCCCT GTTGCCAGCG GTTC--GGCC
Proteus vulgaris TCAGCTCGTT GTTGTGAAAT GTTGGGTTAA GTCCCGCAAC GAGCGCAACC CTTATCCTTT GTTGCCAGCG CGTAATGG-C
Erwinia ananas, Zululand, KZN, SA TCAGCTCGT- GTTGTGAAAT GTTGGGTTAA GTCCCGCAAC GAGCGCAACC CTTATCCTTT GTTGCCAGCG ATTC--GGTC
Pantoea a. Py. ananatis TCAGCTCGT- GTTGTGAAAT GTTGGGTTAA GTCCCGCAAC GAGCGCAACC CTTATCCTTT GTTGCCAGCG ATTC--GGTC
Pantoea a. Py. uredovora TCAGCTCGT- GTTGTGAAAT GTTGGGTTAA GTCCCGCAAC GAGCGCAACC CTTATCCTTT GTTGCCAGCG ATTC--GGTC
Pantoea agglomerans (U80202) TCAGCTCGT- GTTGTGAAAT GTTGGGTTAA GTCCCGCAAC GAGCGCAACC CTTATCCTTT GTTGCCAGCG ATTC--GGTC
Pantoea agglomerans (AB004757) TCAGCTCGT- GTTGTGAAAT GTTGGGTTAA GTCCCGCAAC GAGCGCAACC CTTATCCTTT GTTGCCAGCG ATTC--GGTC
Pantoea agglomerans (U80183) TCAGCTCGT- GTTGTGAAAT GTTGGGTTAA GTCCCGCAAC GAGCGCAACC CTTATCCTTT GTTGCCAGCG ATTC--GGTC
Unknown Erwinia sp. TCAGCTCGT- GTTGTGAAAT GTTGGGTTAA GTCCCGCAAC GAGCGCAACC CTTATCCTTT GTTGCCAGCG ATTC--GGTC
Pantoea s. subsp. stewartii TCAGCTCGT- GTTGTGAAAT GTTGGGTTAA GTCCCGCAAC GAGCGCAACC CTTATCCTTT GTTGCCAGCG ATTC--GGTC
Erwinia ·trache·iphila TCAGCTCGT- GTTGTGAAAT GTTGGGTTAA GTCCCGCAAC GAGCGCAACC CTTATCCTTT GTTGCCAGCG ATTT--GGTC
Erwinia psidii TCAGCTCGT- GTTGTGAAAT GTTGGGTTAA GTCCCGCAAC GAGCGCAACC CTTATCCTTT GTTGCCATCG ATTC--GGTC
Erwinia mallotivora (AJ233414) TCAGCTCGT- GTTGTGAAAT GTTGGGTTAA GTCCCGCAAC GAGCGCAACC CTTATCCTTT GTTGCCATCG ATTC--GGTC
Erwinia mallotivora (Z96084) TCAGCTCGT- GTTGTGAAAT GTTGGGTTAA GTCCCGCAAC GAGCGCAACC CTTATCCTTT GTTGCCATCG ATTC--GGTC
Erwinia persicinus (AJOOl190) TCAGCTCGT- GTTGTGAAAT GTTGGGTTAA GTCCCGCAAC GAGCGCAACC CTTATCCTTT GTTGCCAGCA CGTAATGGT-
Erwinia persicinus (Z96086) TCAGCTCGT- GTTGTGAAAT GTTGGGTTAA GTCCCGCAAC GAGCGCAACC CTTATCCTTT GTTGCCAGCA CGTAATGGT-
Erwinia persicinus (U80205) TCAGCTCGT- GTTGTGAAAT GTTGGGTTAA GTCCCGCAAC GAGCGCAACC CTTATCCTTT GTTGCCAGCA CGTAATGGT-
Erwinia rhapontici (AJ233417) TCAGCTCGT- GTTGTGAAAT GTTGGGTTAA GTCCCGCAAC GAGCGCAACC CTTATCCTTT GTTGCCAGCA CGTAATGGT-
Erwinia rhapontici (Z96087) TCAGCTCGT- GTTGTGAAAT GTTGGGTTAA GTCCCGCAAC GAGCGCAACC CTTATCCTTT GTTGCCAGCA CGTAATGGT-
Erwinia rhapontici (U80206) TCAGCTCGT- GTTGTGAAAT GTTGGGTTAA GTCCCGCAAC GAGCGCAACC CTTATCCTTT GTTGCCAGCG AGTAAT-GTC
Erwinia amylovora (Z96088) TCAGCTCGT- GTTGTGAAAT GTTGGGTTAA GTCCCGCAAC GAGCGCAACC CTTATCCTTT GTTGCCAGCG ATTC--GGTC
Erwinia amylovora (AJ233410) TCAGCTCGT- GTTGTGAAAT GTTGGGTTAA GTCCCGCAAC GAGCGCAACC CTTATCCTTT GTTGCCAGCG ATTC--GGTC
Erwinia amylovora (AJ010485) TCAGCTCGT- GTTGTGAAAT GTTGGGTTAA GTCCCGCAAC GAGCGCAACC CTTATCCTTT GTTGCCAGCG ATTC--GGTC
Erwinia amylovora (U80195) TCAGCTCGT- GTTGTGAAAT GTTGGGTTAA GTCCCGCAAC GAGCGCAACC CTTATCCTTT GTTGCCAGCG ATTC--GGTC
Erwinia pyrifoliae TCAGCTCGT- GTTGTGAAAT GTTGGGTTAA GTCCCGCAAC GAGCGCAACC CTTATCCTTT GTTGCCAGCG ATTC--GGTC
pectobacterium cypripedii TCAGCTCGT- GTTGTGAAAT GTTGGGTTAA GTCCCGCAAC GAGCGCAACC CTTNTCCTCT GTTGCCAGCA CGTCATGGG-
pectobacterium chrysanthemi TCAGCTCGT- GTTGTGAAAT GTTGGGTTAA GTCCCGCAAC GAGCGCAACC CTTATCCTCT GTTGCCAGCA CGTTATGGT-
pectobacterium c. subsp. carotovorum TCAGCTCGT- GTTGTGAAAT GTTGGGTTAA GTCCCGCAAC GAGCGCAACC CTTATCCTTT GTTGCCAGCG ATTC--GGTC
pectobacterium c. subsp. wasabiae TCAGCTCGT- GTTGTGAAAT GTTGGGTTAA GTCCCGCAAC GAGCGCAACC CTTATCCTTT GTTGCCAGCA AGTAA-TGTC
pectobacterium c. subsp. betavasculorum TCAGCTCGT- GTTGTGAAAT GTTGGGTTAA GTCCCGCAAC GAGCGCAACC CTTATCCTTT GTTGCCAGCG ATTC--GGTC
Pectobacterium c. subsp. atrosepticum TCAGCTCGT- GTTGTGAAAT GTTGGGTTAA GTCCCGCAAC GAGCGCAACC CTTATCCTTT GTTGCCAGCG CGTAATGG-C
pectobacterium c. subsp. odoriferum TCAGCTCGT- GTTGTGAAAT GTTGGGTTAA GTCCCGCAAC GAGCGCAACC CTTATCCTTT GTTGCCAGCG ATTC--GGTC
pectobacterium cacticidum TCAGCTCGT- GTTGTGAAAT GTTGGGTTAA GTCCCGCAAC GAGCGCAACC CTTATCCTTT GTTGCCAGCG ATTA--GGTC
Brenneria salicis TCAGCTCGT- GTTGTGAAAT GTTGGGTTAA GTCCCGCAAC GAGCGCAACC CTTATCCTTT GTTGCCAGCA CGTAATGGT-
Brenneria rubrifaciens TCAGCTCGT- GTTGTGAAAT GTTGGGTTAA GTCCCGCAAC GAGCGCAACC CTTATCCTTT GTTGCCAGCG ATTC--GGTC
Brenneria nigrifluens TCAGCTCGT- GTTGTGAAAT GTTGGGTTAA GTCCCGCAAC GAGCGCAACC CTTATCCTTT GTTGCCAGCG ATTC--GGTC
Brenneria alni TCAGCTCGT- GTTGTGAAAT GTTGGGTTAA GTCCCGCAAC GAGCGCAACC CTTATCCTTT GTTGCCAGCA CGTAATGGT-
Brenneria paradisiaca TCAGCTCGT- GTTGTGAAAT GTTGGNTTAA GTCCCGCAAC GAGCGCAACC CTTATCCTTT GTTGCCAGCA CGTGATGGT-
......
w
00
1130 1140 1150 1160 1170 1180 1190 1200
Bucnnera aphidicola GGGAACTCAG AGGAGACTGC CGGTT-ATAA ACCGGAGGAA GGTGGGGACG ACGTCAAGTC --ATCATGGC CCTTACGACC
Proteus vulgaris GGGAACTCAA AGGAGACTGC CGGTG-ATAA ACCGGAGGAA GGTGGGGATG ACGTTAAGTC GTATCATGGC CCTTACGAGT
Erwinia ananas, Zululand, KZN, SA GGGAACTCAA AGGAGACTGC CGGTG-ATAA ACCGGAGGAA GGTGGGGATG ACGTCAAGTC --ATCATGGC CCTTACGAGT
Pantoea a. Py. ananatis GGGAACTCAA AGGAGACTGC CGGTG-ATAA ACCGGAGGAA GGTGGGGATG ACGTCAAGTC --ATCATGGC CCTTACGAGT
Pantoea a. pv. uredovora GGGAACTCAA AGGAGACTGC CGGTG-ATAA ACCGGAGGAA GGTGGGGATG ACGTCAAGTC --ATCATGGC CCTTACGAGT
Pantoea agglomerans (U80202) GGGAACTCAA AGGAGACTGC CGGTG-ATAA ACCGGAGGAA GGTGGGGATG ACGTCAAGTC --ATCATGGC CCTTACGAGT
Pantoea agglomerans (AB004757) GGGAACTCAA AGGAGACTGC CGGTG-ATAA ACCGGAGGAA GGTGGGGATG ACGTCAAGTC --ATCATGGC CCTTACGAGT
Pantoea agglomerans (U80183) GGGAACTCAA AGGAGACTGC CGGTG-ATAA ACCGGAGGAA GGTGGGGATG ACGTCAAGTC --ATCATGGC CCTTACGAGT
Unknown Brwinia sp. GGGAACTCAA AGGAGACTGC CGGTG-ATAA ACCGGAGGAA GGTGGGGATG ACGTCAAGTC --ATCATGGC CCTTACGAGT
Pantoea s. subsp. stewartii GGGAACTCAA AGGAGACTGC CGGTG-ATAA ACCGGAGGAA GGTGGGGATG ACGTCAAGTC --ATCATGGC CCTTACGAGT
Erwinia tracheiphila GGGAACTCAA AGGAGACTGC CGGTG-ATAA ACCGGAGGAA GGTGGGGATG ACGTCAAGTC --ATCATGGC CCTTACGGCC
Erwinia psidii GGGAACTCAA AGGAGACTGC CGGTG-ATAA ACCGGAGGAA GGTGGGGATG ACGTCAAGTC --ATCATGGC CCTTACGGCC
Erwinia mallotivora (AJ2334l4) GGGAACTCAA AGGAGACTGC CGGTG-ATAA ACCGGAGGAA GGTGGGGATG ACGTCAAGTC --ATCATGGC CCTTACGGCC
Erwinia mallotivora (Z96084) GGGAACTCAA AGGAGACTGC CGGTG-ATAA ACCGGAGGAA GGTGGGGATG ACGTCAAGTC --ATCATGGC CCTTACGGCC
Erwinia persicinus (AJ001l90) GGGAACTCAA AGGAGACTGC CGGTGCATAA ACCGGAGGAA GGTGGGGATG ACGTCÁAGTC --ATCATGGC CCTTACGGCC
Erwinia persicinus (Z96086) GGGAACTCAA AGGAGACTGC CGGTG-ATAA ACCGGAGGAA GGTGGGGATG ACGTCAAGTC --ATCATGGC CCTTACGGCC
Erwinia persicinus (U80205) GGGAACTCAA AGGAGACTGC CGGTG-ATAA ACCGGAGGAA GGTGGGGATG ACGTCAAGTC --ATCATGGC CCTTACGGCC
Erwinia rhapontici (AJ233417) GGGAACTCAA AGGAGACTGC CGGTG-ATAA ACCGGAGGAA GGTGGGGATG ACGTCAAGTC --ATCATGGC CCTTACGGCC
Erwinia rhapontici (Z96087) GGGAACTCAA AGGAGACTGC CGGTG-ATAA ACCGGAGGAA GGTGGGGATG ACGTCAAGTC --ATCATGGC CCTTACGGCC
Erwinia rhapontici (U80206) GGGAACTCAA AGGAGACTGC CGGTG-ATAA ACCGGAGGAA GGTGGGGATG ACGTCAAGTC --ATCATGGC CCTTACGGCC
Erwinia amylovora (Z96088) GGGAACTCAA AGGAGACTGC CGGTG-ATAA ACCGGAGGAA GGTGGGGATG ACGTCAAGTC --ATCATGGC CCTTACGGCC
Erwinia amylovora (AJ2334l0) GGGAACTCAA AGGAGACTGC CGGTG-ATAA ACCGGAGGAA GGTGGGGATG ACGTCAAGTC --ATCATGGC CCTTACGGCC
Erwinia amylovora (AJ010485) GGGAACTCAA AGGAGACTGC CGGTG-ATAA ACCGGAGGAA GGTGGGGATG ACGTCAAGTC --ATCATGGC CCTTACGGCC
Erwinia amylovora (U80l95) GGGAACTCAA AGGAGACTGC CGGTG-ATAA ACCGGAGGAA GGTGGGGATG ACGTCAAGTC --ATCATGGC CCTTACGGCC
Erwinia pyrifoliae GGGAACTCAA AGGAGACTGC CGGTG-ATAA ACCGGAGGAA GGTGGGGATG ACGTCAAGTC --ATCATGGC CCTTACGGCC
pectobacterium cypripedii GGGAACTCAA GGGNGACTGC CGGNG-ATAA ACCGGAGGAA GGTGGGGATG ACGTCAAGTC --ACCATGGC CCTTACGAGN
Pectobacterium chrysanthemi GGGAACTCAG GGGAGACTGC CGGTG-ATAA ACCGGAGGAA GGTGGGGATG ACGTCAAGTC --ATCATGGC CCTTACGAGT
Pectobacterium c. subsp. carotovorum GGGAACTCAA AGGAGACTGC CGGTG-ATAA ACCGGAGGAA GGTGGGGATG ACGTCAAGTC --ATCATGGC CCTTACGAGT
pectobacterium c. subsp. wasabiae GGGAACTCAA AGGAGACTGC CGGTG-ATAA ACCGGAGGAA GGTGGGGATG ACGTCAAGTC --ATCATGGC CCTTACGAGT
Pectobacterium c. subsp. betavasculorum GGGAACTCAA AGGAGACTGC CGGTG-ATAA ACCGGAGGAA GGTGGGGATG ACGTCAAGTC --ATCATGGC CCTTACGAGT
pectobacterium c. subsp. atrosepticum GGGAACTCAA AGGAGACTGC CGGTG-ATAA ACCGGAGGAA GGTGGGGATG ACGTCAAGTC --ATCATGGC CCTTACGAGT
pectobacterium c. subsp. odori.ferum GGGAACTCAA AGGAGACTGC CGGTG-ATAA ACCGGAGGAA GGTGGGGATG ACGTCAAGTC --ATCATGGC CCTTACGAGT
pectobacterium cacticidum GGGAACTCAA AGGAGACTGC CGGTG-ATAA ACCGGAGGAA GGTGGGGATG ACGTCAAGTC --ATCATGGC CCTTACGAGT
Brenneria salicis GGGAACTCAA AGGAGACTGC CGGTG-ATAA ACCGGAGGAA GGTGGGGATG ACGTCAAGTC --ATCATGGC CCTTACGAGT
Brenneria rubrifaciens GGGAACTCAA AGGAGACTGC CGGTG-ATAA ACCGGAGGAA GGTGGGGATG ACGTCAAGTC --ATCATGGC CCTTACGAGT
Brenneria nigrifluens GGGAACTCAA AGGAGACTGC CGGTG-ATAA ACCGGAGGAA GGTGGGGATG ACGTCAAGTC --ATCATGGC CCTTACGAGT
Brenneria alni GGGAACTCAA AGGAGACTGC CGGTG-ATAA ACCGGAGGAA GGTGGGGATG ACGTCAAGTC --ATCATGGC CCTTACGAGT
Brenneria paradisiaca GGGAACTCAA GGGAGACTGC CGGTG-ATAA ACCGGAGGAA GGTGGGGATG ACGTCAAGTC --ATCATGGC CCTTACGAGT
......
W
\0
1210 1220 1230 1240 1250 1260 1270 1280
Buchnera aphidicola AGGGCTACAC ACGTGCTACA ATGGTTTATA CAAAGAGAAG CAAATCTGTA AAGA-CAAGC AAACCTCATA AAGTAAATCG
Proteus vulgaris AGGGCTACAC ACGTGCTACA ATGGCAGATA CAAAGAGAAG CGA-CCTCGC GAGAGCAAGC GGAACTCATA AAGTCTGTCG
Erwinia ananas, Zululand, KZN, SA AGGGCTACAC ACGTGCTACA ATGGCGCATA CAAAGAGAAG CGA-CCTCGC GAGAGCAAGC GGACCTCATA AAGTGCGTCG
Pantoea a. Py. ananatis AGGGCTACAC ACGTGCTACA ATGGCGCATA CAAAGAGAAG CGA-CCTCGC GAGAGCAAGC GGACCTCATA AAGTGCGTCG
Pantoea a. Py. uredovora AGGGCTACAC ACGTGCTACA ATGGCGCATA CAAAGAGAAG CGA-CCTCGC GAGAGCAAGC GGACCTCATA AAGTGCGTCG
Pantoea agglomerans (U80202) AGGGCTACAC ACGTGCTACA ATGGCGCATA CAAAGAGAAG CGA-CCTCGC GAGAGCAAGC GGACCTCACA AAGTGCGTCG
Pantoea agglomerans (AB004757) AGGGCTACAC ACGTGCTACA ATGGCGCATA CAAAGAGAAG CGA-CCTCGC GAGAGCAAGC GGACCTCACA AAGTGCGTCG
Pantoea agglomerans (U80l83) AGGGCTACAC ACGTGCTACA ATGGCGCATA CAAAGAGAAG CAA-CCTCGC GAGAGCAAGC GGACCTCACA AAGTGCGTCG
Unknown Erwinia sp. AGGGCTACAC ACGTGCTACA ATGGCGCATA CAAAGAGAAG CGA-CCTCGC GAGAGCAAGC GGACCTCATA AAGTGCGTCG
Pantoea s. subsp. stewartii AGGGCTACAC ACGTGCTACA ATGGCGCATA CAAAGAGAAG CGA-CCTCGC GAGAGCAAGC GGACCTCATA AAGTGCGTCG
Erwinia tracheiphila ANGGCTACAC ACGTGCTACA ATGGCGCATA CAAAGAGAAG CGA-CCTCGC GAGAGCATGC GGACCTCATA AAGTGCGTCG
Erwinia psidii AGGGCTACAC ACGTGCTACA ATGGCGCATA CAAAGAGAAG CGA-CCTCGC GAGAGCAAGC GGACCTCATA AAGTGCGTCG
Erwinia mallotivora (AJ233414) AGGGCTACAC ACGTGCTACA ATGGCGCATA CAAAGAGAAG CGA-CCTCGC GAGAGCAAGC GGATCTCATA AAGTGCGTCG
Erwinia mallotivora (Z96084) AGGGCTACAC ACGTGCTACA ATGGCGCATA CAAAGAGAAG CGA-CCTCGC GAGAGCAAGC GGATCTCATA AAGTGCGTCG
Erwinia persicinus (AJ001190) AGGGCTACAC ACGTGCTACA ATGGCGCATA CAAAGAGAAG CGA-CCTCGC GAGAGCAAGC GGACCTCATA AAGTGCGTCG
Erwinia persicinus (Z96086) AGGGCTACAC ACGTGCTACA ATGGCGCATA CAAAGAGAAG CGA-CCTCGC GAGAGCAAGC GGACCTCATA AAGTGCGTCG
Erwinia persicinus (U80205) AGGGCTACAC ACGTGCTACA ATGGCGCATA CAAAGAGAA~ CGA-CCTCGC GAGAGCAAGC GGACCTCATA AAGTGCGTCG
Erwinia rhapontici (AJ233417) AGGGCTACAC ACGTGCTACA ATGGCGCATA CAAAGAGAAG CGA-CCTCGC GAGAGCAAGC GGACCTCATA AAGTGCGTCG
Erwinia rhapontici (Z96087) AGGGCTACAC ACGTGCTACA ATGGCGCATA CAAAGAGAAG CGA-CCTCGC GAGAGCAAGC GGACCTCATA AAGTGCGTCG
Erwinia rhapontici (U80206) AGGGCTACAC ACGTGCTACA ATGGCGCATA CAAAGAGAAG CGA-CCTCGC GAGAGCAAGC GGACCTCATA AAGTGCGTCG
Erwinia amylovora (Z96088) AGGGCTACAC ACGTGCTACA ATGGCGCATA CAAAGAGAAG CGA-CCTCGC GAGAGCAAGC GGACCTCATA AAGTGCGTCG
Erwinia amylovora (AJ233410) AGGGCTACAC ACGTGCTACA ATGGCGCATA CAAAGAGAAG CGA-CCTCGC GAGAGCAAGC GGACCTCATA AAGTGCGTCG
Erwinia amylovora (AJ010485) AGGGCTACAC ACGTGCTACA ATGGCGCATA CAAAGAGAAG CGA-CCTCGC GAGAGCAAGC GGACCTCATA AAGTGCGTCG
Erwinia amylovora (U80195) AGGGCTACAC ACGTGCTACA ATGGCGCATA CAAAGAGAAG CGA-CCTCGC GAGAGCAAGC GGACCTCATA AAGTGCGTCG
Erwinia pyrifoliae AGGGCTACAC ACGTGCTACA ATGGCGCATA CAAAGAGAAG CGA-CCTCGC GAGAGCAAGC GGACCTCATA AAGTGCGTCG
pectobacterium cypripedii AGGGCTACAC ACGTGCTACA ATGGCGNATA CAGAGAGATG CGA-CCTCGC GAGAGCAAGC GGACCTCATA AAGNGCGTCG
pectobacterium chrysanthemi AGGGCTACAC ACGTGCTACA ATGGCGCATA CAAAGAGAAG CGA-CCTCGC GAGAGCAAGC GGACCTCATA AAGTGCGTCG
pectobacterium c. subsp. carotovorum AGGGCTACAC ACGTGCTACA ATGGCGTATA CAAAGAGAAG CGA-CCTCGC GAGAGCAAGC GGACCTCATA AAGTACGTCG
Pectobacterium c. subsp. wasabiae AGGGCTACAC ACGTGCTACA ATGGCGTATA CAAAGAGAAG CGA-CCTCGC GAGAGCAAGC GGACCTCATA AAGTACGTCG
Pectobacterium c. subsp. betavasculorum AGGGCTACAC ACGTGCTACA ATGGCGTATA CAAAGAGAAG CGA-CCTCGC GAGAGCAAGC GGACCTCATA AAGTACGTCG
Pectobacterium c. subsp. atrosepticum AGGGCTACAC ACGTGCTACA ATGGCGTATA CAAAGAGAAG CGAA-CTCGC GAGAGCCAGC GGACCTCATA AAGTACGTCG
Pectobacterium c. subsp. odoriferum AGGGCTACAC ACGTGCTACA ATGGCGTATA CAAAGAGAAG CGA-CCTCGC GAGAGCAAGC GGACCTCATA AAGTACGTCG
pectobacterium cacticidum AGGGCTACAC ACGTGCTACA ATGGCGTATA CAAAGAGAAG CGAGCCT-GC GAGGGTGAGC GGACCTCATA AAGTACGTCG
Brenneria salicis AGGGCTACAC ACGTGCTACA ATGGCGCATA CAAAGAGAAG CGAGCCT-GC GAGGGTGAGC GGACCTCATA AAGTGCGTCG
Brenneria rubrifaciens AGGGCTACAC ACGTGCTACA ATGGCGCATA CAAAGAGAAG CGAGCCT-GC GAGGGTGAGC GGACCTCATA AAGTGCGTCG
Brenneria nigrifluens AGGGCTACAC ACGTGCTACA ATGGCGCATA CAAAGAGAAG CGAACTT-GC GAGAGTAAGC GGACCTCATA AAGTGCGTCG
Brenneria alni AGGGCTACAC ACGTGCTACA ATGGCGCATA CAAAGAGAAG CGAGCCT-GC GAGGGTGAGC GGACCTCATA AAGTGCGTCG
Brenneria paradisiaca AGGGCTACAC ACGTGCTACA ATGGCGCATA CAAAGAGAAG CGA-CCTCGC GAGAGCAAGC GGATCTCATA AAGTGCGTCG
o.".
1290 1300 1310 1320 1330 1340 1350 1360
Buchnera aphidicola TAGTCCGGAC TGGAGTCTGC AACTCGACTC CACGAAGTCG GAATCGCTAG TAATCGTGGA TCA-GAATGC CACGGTGAAT
Proteus vulgaris TAGTCCGGAT TGGAGTCTGC AACTCGACTC CATGAAGTCG GAATCGCTAG TAATCGTAGA TCA-GAATGC TACGGTGAAT
Erwinia ananas, Zululand, KZN, SA TAGTCCGGAT CGGAGTCTGC AACTCGACTC CGTGAAGTCG GAATCGCTAG TAATCGTGGA TCA-GAATGC CACGGTGAAT
Pantoea a. Py. ananatis TAGTCCGGAT CGGAGTCTGC AACTCGACTC CGTGAAGTCG GAATCGCTAG TAATCGTGGA TCA-GAATGC CACGGTGAAT
Pantoea a. Py. uredovora TAGTCCGGAT CGGAGTCTGC AACTCGACTC CGTGAAGTCG GAATCGCTAG TAATCGTGGA TCA-GAATGC CACGGTGAAT
Pantoea agglomerans (U80202) TAGTCCGGAT CGGAGTCTGC AACTCGACTC CGTGAAGTCG GAATCGCTAG TAATCGTGGA TCA-GAATGC CACGGTGAAT
Pantoea agglomerans (AB004757) TAGTCCGGAT CGGAGTCTGC AACTCGACTC CGTGAAGTCG GAATCGCTAG TAATCGTGGA TCA-GAATGC CACGGTGAAT
Pantoea agglomerans (U80l83) TAGTCCGGAT CGGAGTCTGC AACTCGACTC CGTGAAGTCG GAATCGCTAG TAATCGTGGA TCA-GAATGC CACGGTGAAT
Unknown Erwinia sp. TAGTCCGGAT TGGAGTCTGC AACTCGACTC CATGAAGTCG GAATCGCTAG TAATCGTAGA TCA-GAATGC TACGGTGAAT
Pantoea s. subsp. stewartii TAGTCCGGAT CGGAGTCTGC AACTCGACTC CGTGAAGTCG GAATCGCTAG TAATCGTGGA TCA-GAATGC CACGGTGAAT
Erwinia tracbeiphila TAGTCCGGAT CGGAGTCTGC AACTCGACTC CGTGAAGTCG GAATCGCTAG TAATCGTGGA TCA-GAATGC CACGGTGAAT
Erwinia psidii TAGTCCGGAT CGGAGTCTGC AACTCGACTC CGTGAAGTCG GAATCGCTAG TAATCGTGGA TCA-GAATGC CACGGTGAAT
Erwinia mallotivora (AJ2334l4) TAGTCCGGAT CGGAGTCTGC AACTCGACTC CGTGAAGTCG GAATCGCTAG TAATCGTGGA TCA-GAATGC CACGGTGAAT
Erwinia mallotivora (Z96084) TAGTCCGGAT CGGAGTCTGC AACTCGACTC CGTGAAGTCG GAATCGCTAG TAATCGTGGA TCA-GAATGC CACGGTGAAT
Erwinia persicinus (AJOOl190) TAGTCCGGAT CGGAGTCTGC AACTCGACTC CGTGAAGTCG GAATCGCTAN TAATCGTAGA TCAAGAATGC TACGGTGAAT
Erwinia persicinus (Z96086) TAGTCCGGAT CGGAGTCTGC'AACTCGACTC CGTGAAGTCG GAATCGCTAG TAATCGTAGA TCA-GAATGC TACGGTGAAT
Erwinia persicinus (U80205) TAGTCCGGAT CGGAGTCTGC AACTCGACTC CGTGAAGTCG GAATCGCTAG TAATCGTAGA TCA-GAATGC TACGGTGAAT
Erwinia rhapontici (AJ2334l7) TAGTCCGGAT CGGAGTCTGC AACTCGACTC CGTGAAGTCG GAATCGCTAG TAATCGTAGA TCA-GAATGC TACGGTGAAT
Erwinia rhapontici (Z96087) TAGTCCGGAT CGGAGTCTGC AACTCGACTC CGTGAAGTCG GAATCGCTAG TAATCGTAGA TCA-GAATGC TACGGTGAAT
Erwinia rhapontici (U80206) TAGTCCGGAT CGGAGTCTGC AACTCGACTC CGTGAAGTCG GAATCGCTAG TAATCGTAGA TCA-GAATGC TACGGTGAAT
Erwinia amylovora (Z96088) TAGTCCGGAT CGGAGTCTGC AACTCGACTC CGTGAAGTCG GAATCGCTAG TAATCGTAGA TCA-GAATGC TACGGTGAAT
Erwinia amylovora (AJ2334l0) TAGTCCGGAT CGGAGTCTGC AACTCGACTC CGTGAAGTCG GAATCGCTAG TAATCGTAGA TCA-GAATGC TACGGTGAAT
Erwinia amylovora (AJOl0485) TAGTCCGGAT CGGAGTCTGC AACTCGACTC CGTGAAGTCG GAATCGCTAG TAATCGTAGA TCA-GAATGC TACGGTGAAT
Erwinia amylovora (U80l95) TAGTCCGGAT CGGAGTCTGC AACTCGACTC CGTGAAGTCG GAATCGCTAG TAATCGTAGA TCA-GAATGC TACGGTGAAT
Erwinia pyrifoliae TAGTCCGGAT CGGAGTCTGC AACTCGACTC CGTGAAGTCG GAATCGCTAG TAATCGTAGA TCA-GAATGC TACGGTGAAT
pectobacterium cypripedii NAGTCCGGAT TGGAGTCTGC AACTCGACTC CATGAAGTCG GAATCGCTAG TAATCGTAGA TCA-GAATGG TACGGTGAAT
Pectobacterium cbrysanthemi TAGTCCGGAT TGGAGTCTGC AACTCGACTC CATGAAGTCG GAATCGCTAG TAATCGTAGA TCA-GAATGC TACGGTGAAT
pectobacterium c. subsp. carotovorum TAGTCCGGAT TGGAGTCTGC AACTCGACTC CATGAAGTCG GAATCGCTAG TAATCGTAGA TCA-NAATGC TACGGTGAAT
pectobacterium c. subsp. wasabiae TAGTCCGGAT TGGAGTCTGC AACTCGACTC CATGAAGTCG GAATCGCTAG TAATCGTAGA TCA-GAATGC TACGGTGAAT
pectobacterium c. subsp. betavasculorum TAGTCCGGAT TGGAGTCTGC AACTCGACTC CATGAAGTCG GAATCGCTAG TAATCGTAGA TCA-GAATGC TACGGTGAAT
pectobacterium c. subsp. atrosepticum TAGTCCGGAT TGGAGTCTGC AACTCGACTC CATGAAGTCG GAATCGCTAG TAATCGTAGA TCA-GAATGC TACGGTGAAT
pectobacterium c. subsp. odoriferum TAGTCCGGAT TGGAGTCTGC AACTCGACTC CATGAAGTCG GAATCGCTAG TAATCGTAGA TCA-GAATGC TACGGTGAAT
Pectobacterium cacticidum TAGTCCGGAT TGGAGTCTGC AACTCGACTC CATGAAGTCG GAATCGCTAG TAATCGTANA TCA-GAATGC TACGGTGAAT
Brenneria salicis TAGTCCGGAT TGGAGTCTGC AACTCGACTC CATGAAGTCG GAATCGCTAG TAATCGTAGA TCA-GAATGC TACGGTGAAT
Brenneria rubrifaciens TAGTCCGGAT TGGAGTCTGC AACTCGACTC CATGAAGTCG GAATCGCTAG TAATCGTAGA TCA-GAATGC TACGGTGAAT
Brenneria nigrifluens TAGTCCGGAT TGGAGTCTGC AACTCGACTC CATGAAGTCG GAATCGCTAG TAATCGTAGA TCA-GAATGC TACGGTGAAT
Brenneria alni TAGTCCGGAT TGGAGTCTGC AACTCGACTC CATGAAGTCG GAATCGCTAG TAATCGTGGA TCA-GAATGC CACGGTGAAT
Brenneria paradisiaca TAGTCCGGAT TGGAGTCTGC AACTCGACTC CATGAAGTCG GAATCGCTAG TAATCGCGGA TCA-GAATGC CGCGGTGAAT
......
.j>.
1370 1380 1390 1400 1410 1420 1430 1440
Buchnera aphidicola ACGTTCCCGG GCCTTGTACA CACCGCCCGT CACACCATGG GAGTGGGTTG CAAAAGAAGC AGATTTCCTA ACCAC-GAAA
Proteus vulgaris ACGTTCCCGG GCCTTGTACA CACCGCCCGT CACACCATGG GAGTGGCTTG CAAAAGAAGT AGGTAGCTTA ACCTTCGGGA
Erwinia ananas, Zululand, KZN, SA ACGTTCCCGG GCCTTGTACA CACCGCCCGT CACACCATGG GAGTGGGTTG CAAAAGAAGT AGGTAGCTTA ACCTTCGGGA
Pantoea a. Py. ananatis ACGTTCCCGG GCCTTGTACA CACCGCCCGT CACACCATGG GAGTGGGTTG CAAAAGAAGT AGGTAGCTTA ACCTTCGGGA
Pantoea a. Py. uredovora ACGTTCCCGG GCCTTGTACA CACCGCCCGT CACACCATGG GAGTGGGTTG CAAAAGAAGT AGGTAGCTTA ACCTTCGGGA
Pantoea agglomerans (U80202) ACGTTCCCGG GCCTTGTACA CACCGCCCGT CACACCATGG GAGTGGGTTG CAAAAGAAGT AGGTAGCTTA ACCTTCGGGA
Pantoea agglomerans (AB004757) ACGTTCCCGG GCCTTGTACA CACCGCCCGT CACACCATGG GAGTGGGTTG CAAAAGAAGT AGGTAGCTTA ACCTTCGGGA
Pantoea agglomerans (U80l83) ACGTTCCCGG GCCTTGTACA CACCGCCCGT CACACCATGG GAGTGGGTTG CAAAAGAAGT AGGTAGCTTA ACCTTCGGGA
Unknown Erwinia sp. ACGTTCCCGG GCCTTGTACA CACCGCCCGT CACACCATGG GAGTGGGTTG CAAAAGAAGT AGGTAGCTTA ACCTTCGGGA
Pantoea s. subsp. stewartii ACGTTCCCGG GCCTTGTACA CACCGCCCGT CACACCATGG GAGTGGGTTG CAAAAGAAGT AGGTAGCTTA ACCCTCGGGA
Erwinia tracheiphila ACGTTCCCGG GCCTTGTACA CACCGCCCGT CACACCATGG GAGTGGGTTG CAAAAGAAGT ANGTANCTTA ACCTTCGGGA
Erwinia psidii ACGTTCCCGG GCCTTGTACA CACCGCCCGT CACACCATGG GAGTGGGTTG CAAAAGAAGT AGGTAGCTTA ACCTTCGGGA
Erwinia mallotivora (AJ2334l4) ACGTTCCCGG GCCTTGTACA CACCGCCCGT CACACCATGG GAGTGGGTTG CAAAAGAAGT AGGTAGCTTA ACCTTCGGGA
Erwinia mallotivora (Z96084) ACGTTCCCGG GCCTTGTACA CACCGCCCGT CACACCATGG GAGTGGGTTG CAAAAGAAGT AGGTAGCTTA ACCTTCGGGA
Erwinia persicinus (AJOOl190) ACGTTCCCGG GCCTTGTACA CACCGCCCGT CACACCATGG GAGTGGGTTG CAAAAGAAGT AGGTAGCTTA ACCTTCGGGA
Erwinia persicinus (Z96086) ACGTTCCCGG GCCTTGTACA CACCGCCCGT CACACCATGG GAGTGGGTTG CAAAAGAAGT AGGTAGCTTA ACCTTCGGGA
Erwinia persicinus (U80205) ACGTTCCCGG GCCTTGTACA CACCGCCCGT CACACCATGG GAGTGGGTTG CAAAAGAAGT AGGTAGCTTA ACCTTCGGGA
Erwinia rhapontici (AJ233417) ACGTTCCCGG GCCTTGTACA CACCGCCCGT CACACCATGG GAGTGGGTTG CAAAAGAAGT AGGTAGCTTA ACCTTCGGGA
Erwinia rhapontici (Z96087) ACGTTCCCGG GCCTTGTACA CACCGCCCGT CACACCATGG GAGTGGGTTG CAAAAGAAGT AGGTAGCTTA ACCTTCGGGA
Erwinia rhapontici (U80206) ACGTTCCCGG GCCTTGTACA CACCGCCCGT CACACCATGG GAGTGGGTTG CAAAAGAAGT AGGTAGCTTA ACCTTCGGGA
Erwinia amylovora (Z96088) ACGTTCCCGG GCCTTGTACA CACCGCCCGT CACACCATGG GAGTGGGTTG CAAAAGAAGT AGGTAGCTTA ACCTTCGGGA
Erwinia amylovora (AJ233410) ACGTTCCCGG GCCTTGTACA CACCGCCCGT CACACCATGG GAGTGGGTTG CAAAAGAAGT AGGTAGCTTA ACCTTCGGGA
Erwinia amylovora (AJ010485) ACGTTCCCGG GCCTTGTACA CACCGCCCGT CACACCATGG GAGTGGGTTG CAAAAGAAGT AGGTAGCTTA AC-TTCGGGA
Erwinia amylovora (U80195) ACGTTCCCGG GCCTTGTACA CACCGCCCGT CACACCATGG GAGTGGGTTG CAAAAGAAGT AGGTAGCTTA ACCTTCGGGA
Erwinia pyrifoliae ACGTTCCCGG GCCTTGTACA CACCGCCCGT CACACCATGG GAGTGGGTTG CAAAAGAAGT AGGTAGCTTA AC-TTCGGGA
pectobacterium cypripedii NCGTTCCCGG GCCTTGTACA CACCGCCCGT CACACCATGG GAGTGGGTTG CAAANGAAGT AGGTAGCTTA ACCNTCGGGA
pectobacterium chrysanthemi ACGTTCCCGG GCCTTGTACA CACCGCCCGT CACACCATGG GAGTGGGTTG CAAAAGAAGT AGGTAGCTTA ACCTTCGGGA
pectobacterium c. subsp. carotovorum ACGTTCCCGG GCCTTGTACA CACCGCCCGT CACACCATGG GAGTGGGTTG CAAAAGAAGT AGGTAGCTTA ACCTTCGGGA
pectobacterium c. subsp. wasabiae ACGTTCCCGG GCCTTGTACA CACCGCCCGT CACACCATGG GAGTGGGTTG CAAAAGAAGT AGGTAGCTTA ACCTTCGGGA
Pectobacterium c. subsp. betavasculorum ACGTTCCCGG GCCTTGTACA CACCGCCCGT CACACCATGG GAGTGGGTTG CAAAAGAAGT AGGTAGCTTA ACCTTCGGGA
Pectobacterium c. subsp. atrosepticum ACGTTCCCGG GCCTTGTACA CACCGCCCGT CACACCATGG GAGTGGGTTG CAAAAGAAGT AGGTAGCTTA ACCTTCGGGA
pectobacterium c. subsp. odoriferum ACGTTCCCGG GCCTTGTACA CACCGCCCGT CACACCATGG GAGTGGGTTG CAAAAGAAGT AGGTAGCTTA ACCTTCGGGA
Pectobacterium cacticidum ACGTTCCCGG GCCTTGTACA CACCGCCCGT CACACCATGG GAGTGGGTTG CAAAANAAGT AGGTAGCTTA ACCTTCGGGG
Brenneria salicis ACGTTCCCGG GCCTTGTACA CACCGCCCGT CACACCATGG GAGTGGGTTG CAAAAGAAGT AGGTAGCTTA ACCTTAGGGG
Brenneria rubrifaciens ACGTTCCCGG GCCTTGTACA CACCGCCCGT CACACCATGG GAGTGGGTTG CAAAAGAAGT AGGTAGCTTA ACCTTCGGGG
Brenneria nigrifluens ACGTTCCCGG GCCTTGTACA CACCGCCCGT CACACCATGG GAGTGGGTTG CAAAAGAAGT AGGTAGCTTA ACCTTAGGGG
Brenneria alni ACGTTCCCGG GCCTTGTACA CACCGCCCGT CACACCATGG GAGTGGGTTG CAAAAGAAGT AGGTAGCTTA ACCTCAGGGG
Brenneria paradisiaca ACGTTCCCGG GCCTTGTACA CACCGCCCGT CACACCATGG GAGTGGGTTG CAAAAGAAGT AGGTAGCTTA AC-TTCGGGA
.....
.l'>-
N
1450 1460 1470
Buchnera aphidicola GTGGAAGGCG -TCTACCACT TTGTGATTCA TGA
Proteus vulgaris G-----GGCG CT-TACCACT TTGTGATTCA TGA
Erwinia ananas, Zululand. KZN. SA G-----GGCG CT-TACCACT TTGTGATTCA TGA
Pantoea a. Py. ananatis G-----GGCG CT-TACCACT TTGTGATTCA TGA
Pantoea a. Py. uredovora G-----GGCG CT-TACCACT TTGTGATTCA TGA
Pantoea agglomerans (UB0202) G-----GGCG CT-TACCACT TTGTGATTCA TGA
Pantoea agglomerans (AB004757) G-----GGCG CT-TACCACT TTGTGATTCA TGA
Pantoea agglomerans (UBOIB3) G-----GGCG CT-TACCACT TTGTGATTCA TGA
Unknown Erwinia sp. G-----GGCG CT-TACCACT TTGTGATTCA TGA
Pantoea s. subsp. stewartii G-----GGCG CT-TACTACT TTGTGATTCA TGA
Erwinia tracheiphila G-----GGCG CT-TACCACT TTGTGATTCA TNA
Erwinia psidii G-----GGCG CT-TACCACT TTGTGATTCA TGA
Erwinia mallotivora (AJ233414) G-----GGCG CT-TACCACT TTGTGATTCA TGA
Erwinia mallotivora (Z960B4) G-----GGCG CT-TACCACT TTGTGATTCA TGA
Erwinia persicinus (AJOOl190) G-----GGCG CT-TACCACT TTGTGATTCA TGA
Erwinia persicinus (Z960B6) G-----GGCG CT-TACCACT TTGTGATTCA TGA
Erwinia persicinus (UB0205) G-----GGCG CT-TACCACT TTGTGATTCA TGA
Erwinia rhapontici (AJ233417) G-----GGCG CT-TACCACT TTGTGATTCA TGA
Erwinia rhapontici (Z960B7) G-----GGCG CT-TACCACT TTGTGATTCA TGA
Erwinia rhapontici (UB0206) G"----GGCG CT-TACCACT TTGTGATTCA TGA
Erwinia amylovora (Z96088) G-----GGCG CT-TACCACT TTGTGATTCA TGA
Erwinia amylovora (AJ233410) G-----GGCG CT-TACCACT TTGTGATTCA TGA
Erwinia amylovora (AJOI04B5) G-----GGCG CT-TACCACT TTGTGATTCA TGA
Erwinia amylovora (UB0195) G-----GGCG CT-TACCACT TTGTGATTCA TGA
Erwinia pyrifoliae G-----GGCG CT-TACCACT TTGTGATTCA TGA
pectobacterium cypripedii G-----GGCG CT-TACCACT TTGTGATTCA TGA
Pectobacterium chrysanthemi G-----GGCG CT-TACCACT TTGTGATTCA TGA
pectobacterium c. subsp. carotovorum G-----GGCG CT-TACCACT TTGTGATTCA TGA
pectobacterium c. subsp. wasabiae G-----GGCG CT-TACCACT TTGTGATTCA TGA
pectobacterium c. subsp. betavasculorum G-----GGCG CT-TACCACT TTGTGATTCA TGA
pectobacterium c. subsp. atrosepticum G-----GGCG CT-TACCACT TTGTGATTCA TGA
pectobacterium c. subsp. odoriferum G-----GGCG CT-TACCACT TTGTGATTCA TGA
pectobacterium cacticidum G-----GGCG CT-TACCACT TTGTGATTCA TGA
Brenneria salicis G-----GGCG CT-TACCACT TTGTGATTCA TGA
Brenneria rubrifaciens G-----GGCG CT-TACCACT TTGTGATTCA TGA
Brenneria nigrifluens G-----GGCG CT-TACCACT TTGTGATTCA TGA
Brenneria alni G-----GGCG CT-TACCACT TTGTGATTCA TGA
Brenneria paradisiaca G-----GGCG CT-TACCACT TTGTGATTCA TGA
.j:>.
IJJ
144
Fig. 4. Dendrogram based on 16S rDNA sequences, showing the phylogenetic
relationships of both South African Erwinia species with species of the genera
Pantoea, Erwinia, Pectobacterium and Brenneria. The tree is rooted with Proteus
vulgaris and Buchnera aphidicola and was constructed by using the neighbour-joining
method and bootstrap values calculated from 1000 trees. The tree length = 1161
steps, Cl = 0.498, HI = 0.502 and RI = 0.766. Bootstrap values are given above each
branching point.
Buchnera aphidicola (L 18927)
Proteus vulgaris (J01874)
98 Erwinia ananas Zululand, KZN, SA
r-U Pantoea ananatis Py. ananatis (U80196)
r- 1 Pantoea ananatis pv. uredovora (U80209)
100 '-- Pantoea agglomerans (U80202)
Lr Pantoea agglomerans (AB004757)
79L Pantoea agglomerans (U80183)
Unknown Erwinia sp., Zululand, KZN, SA
Pantoea stewartii subsp. stewartii (U80208)
Erwinia tracheiphila (Y13250)
53y_ Erwinia psidii (Z96085)j Erwinia mallotivora (AJ233414)
""'_---j
100 Erwinia mallotivora (Z96084)
r Erwinia persicinus (AJ001190)
94- n Erwinia persicinus (296086)
.-9-9--r-'-l- Erwinia persicinus (U80205)Erwinia rhapontici (AJ233417)
_ Erwinia rhapontici (296087)
82._ Erwinia rhapontici (U80206)
.__
80 r-- Erwinia amylovora (Z96088)
r- r Erwinia amy/ovora (AJ233410)
,___ 1'- Erwinia amy/ovora (AJ010485)79
90 ._ Erwinia amy/ovora (U80195)
- Erwinia pyrifo/iae (AJ009930)
__ Pectobacterium cypripedii (296094)~ '-- Pectobacterium chrysanthemi (AJ233412)
J Brenneria salicis (Z96097)
I Brenneria rubrifaciens (AJ233418)
~ Lt Brenneria nigrifluens (AJ233415)
85 '----- Brenneria a/ni (AJ233409)
Brennette. paradisuaca (296096)
"-
52 PC Pectobacterium carotovorum subsp. carotovorum (Z96089
Pectobacterium carotovorum subsp. odoriferum (AJ223407)
r- r-- Pectobacterium carotovorum subsp. wasabiae (AJ223408)
'- Pectobacterium carotovorum subsp. betavasculorum (U80198)
L Pectobacterium carotovorum subsp. atrosepticum (296090)
Pectobacterium cacticidum (296092)
I-- 10 changes
145
Fig. 5. Necrotic lesion development on fresh Granny Smith apples after inoculation
with C. zuluense (CRY and CMW numbers) and both Pantoea species (P. ananatis
pv. ananatis and an unknown Pantoea sp.). (A - D) Insignificant lesion development
was observed when apples were inoculated with C. zuluense isolates (A = CRY
1055; B = CRY 1016; C = CMW 2100; D = CRY 1023). (E - F) Bacteria species
caused significant tissue maceration when inoculated alone (E = unknown Pantoea
sp.; F = P. ananatis pv. ananatis). (G - H) Inoculation of both Pantoea species in
association with C. zuluense resulted in significantly larger lesions (G = Pantoea sp.
+ P. ananatis pv. ananatis + CMW 2100; H = Pantoea sp. + P. ananatis pv. ananatis
+ CRY 1023).

146
Fig. 6. Lesion development on a susceptible Eucalyptus grandis clone (ZG 14)
inoculated with isolates of C. zuluense and both Pantoea species (A) No lesion
development was observed after inoculations using each of the Pantoea species,
respectively (8) Canker development after inoculation with virulent isolates of C.
zuluense. (C) Significantly larger lesions were observed with inoculation of C.
zuluense in combination with both Pantoea species.

147
CHAPTER 6
Polygalacturonase production by the Eucalyptus canker pathogen,
Coniothyrium zuluense, and two Pantoea species
The Eucalyptus stem canker pathogen, Coniothyrium zuluense, has been shown to
be associated with two bacterial species, Pantoea ananatis pv. ananatis and an
unknown Pantoea species. A significant increase in pathogenicity occurs when these
bacteria are inoculated in combination with C. zuluense. This suggests that a
synergistic interaction may possibly exist between these micro-organisms.
Pathogenicity in some fungi has been shown to be correlated with the enzyme,
polygalacturonase (PG). In this study we determined the ability to produce PG for
pathogenic and non-pathogenic C. zuluense isolates, as well as both Pantoea
species. The level of PG production by these micro-organisms was estimated by
cup-plate and reducing sugars assays. A significant correlation was found between
PG activity and pathogenicity of C. zuluense. Experimental assays demonstrated
that levels of PG activity for both Pantoea species were significantly higher than
those obtained for C. zuluense isolates. As PG is the first enzyme produced during
disease development, it is hypothesised that the two Pantoea species may play a
significant role in the development of Coniothyrium canker. Production of PG might
also be used as an assay to evaluate pathogenicity in different isolates of C.
zuluense.
~--------------------------------------------------------------------------
148
The biotrophic canker pathogen, Coniothyrium zuluense Wingfield, Crous &
Coutinho, is the causal agent of a serious Eucalyptus stem canker disease in South
Africa (Wingfield et al., 1997). Wingfield et al. (unpublished data) showed that once
conidia of this fungus germinate, the germ tubes infect the stem directly through the
epidermis of young stem tissue. To accomplish this, a number of cell-wall-degrading
enzymes are needed to enable penetration and colonisation of the plant tissue, as
well as to release the nutrients necessary for growth (Walton, 1994; Alghisi &
Favaron, 1995; Annis & Goodwin, 1997). Enzymes that are known to be involved in
this process include pectic enzymes, cellulases, arabinases, xylanases and
galactanases (Alghisi & Favaron, 1995; Annis & Goodwin, 1997). Of these enzymes,
only the pectin-degrading group of enzymes have been positively linked to
pathogenesis (Barras et al., 1994; Alghisi & Favaron, 1995; Kombrink & Somssich,
1995; Hugouvieux-Cotte-Pattat et al., 1996).
Pectic enzymes are produced in large quantities by many plant-associated micro-
organisms and are important requirements for colonisation of plant tissue (Cooper,
1983; Colimer & Keen, 1986; Barras et al., 1994; Alghisi & Favaron, 1995; Alfano &
Collmer, 1996; Hugouvieux-Cotte-Pattat et al., 1996; Annis & Goodwin, 1997).
Pectin is the major component of the primary plant cell wall and middle lamellae. The
depolymerisation of pectin is, therefore, essential for further cell wall breakdown by
other cell-wall degrading enzymes (Karr & Albersheim, 1970). Polygalacturonase
(E.C. 3.2.1.15) (PG) is the main component of the pectic enzyme complex (Collmer &
Keen, 1986; Collmer et al., 1988). PG is the first hydrolytic enzyme produced by
many plant pathogens and is a determining factor in pathogenicity for bacterial and
fungal pathogens (Barras et al., 1994; Alghisi & Favaron, 1995; Hugouvieux-Cotte-
Pattat eta!., 1996; Annis & Goodwin, 1997).
The role that polygalacturonase production plays in bacteria, differs significantly
between bacterial species. An endo-polygalacturonase defective mutant of
Pectobacterium carotovorum subsp. carotovorum (synonym, Erwinia carotovora
subsp. carotovora), that maintained pectate-Iyase and exo-polygalacturonase
activities, kept its virulence on tissues of different plants (Willis et al., 1987).
Similarly, the endo-polygalacturonase of P. so/anacearum pv. tomato is not required
to cause tomato disease, although it accelerates its development (Denny et al.,
149
1990). On the contrary, mutation in the single polygalacturonase-encoding gene of
Agrobacterium tumefaciens, eliminates polygalacturonase activity and substantially
decreases its virulence (Rodriquez-Palenzuela et al., 1991). It is, therefore, evident
that endo - and exo - polygalacturonase production is important in diseases caused
by bacteria.
There are relatively few studies on the role of fungal polygalacturonases in plant
disease. Most studies have considered the ability of purified enzymes to reproduce
disease symptoms (Benhamou et al., 1991; Favaron et al., 1993). Convincing
evidence of polygalacturonase involvement, however, was obtained using Aspergillus
flavus Link.. Low-virulence strains lacking a major endo-polygalacturonase, caused a
reduction of disease symptoms in developing cotton boils (Cleveland & Cotty, 1991,
Brown et al., 1992). Similarly, mutants of Fusarium oxysporum f.sp. /ycopersici
Schlechtend.:Fr. [(Sacc.) W.C. Snyder & H.N. Hans.] lacking polygalacturonase
activity had reduced virulence on tomato (Mann, 1962). In contrast, a strain of
Coch/iobo/us carbonum Nelson in which the gene for endo-polygalacturonase had
been disrupted, was unaffected for pathogenicity on maize (Scott-Craig et al., 1990).
The importance of the contribution of fungal polygalacturonases towards
pathogenesis must be considered case by case. However, this enzyme together with
the rest of the depolymerases is essential for complete disease development.
Coniothyrium canker has been shown to be associated with two Pantoea species, P.
ananatis pv. ananatis and an unknown Pantoea species (Van Zyl et al., chapter 5). A
significant increase in pathogenicity to a susceptible E. grandis clone was observed
when fungal and bacterial isolates were inoculated in combination (Van Zyl et al.,
chapter 5). It is known that pathogenicity of fungal and bacterial plant pathogens is
directly influenced by the production of polygalacturonase (Yang et al., 1992; Barras
et al., 1994; Le Cam et al., 1994; Alghisi & Favaron, 1995; Alfano & Coli mer, 1996;
Annis & Goodwin, 1997). The objective of this study was, therefore, to screen strains
of C. zu/uense and the two Pantoea species for PG activity and to link this with
pathogenicity data presented in a previous study (Van Zyl et al., chapter 5).
150
MATERIALS AND METHODS
Fungal and bacterial isolates
Van Zyl et al. (Chapter 5) observed a possible synergistic interaction between C.
zuluense and both Pantoea species, Pantoea ananatis pv. ananatis and an unknown
species closely related to Pantoea stewartii subsp. stewartii. Based on this, we have
selected six C. zuluense isolates to be used in this study (Table 1). Pathogenicity
characteristics of pure bacteria-free C. zuluense isolates varied between non-
pathogenic, intermediately pathogenic and highly pathogenic as determined in a
previous study (Van Zyl et al., 1997). It was also shown that relative pathogenicity of
selected C. zuluense isolates was significantly increased when fungal and bacterial
isolates were inoculated in combination with each other (Van Zyl et al., Chapter 5).
One strain of each Pantoea sp. was included in this study (Table 1). Van Zyl et al.
(chapter 5) showed that significant differences in tissue maceration of Granny Smith
apples were evident between strains of each of the two Pantoea species (ten strains
for each species). No significant tissue maceration was, however, found among
strains of each bacterial species (Van Zyl et al., chapter 5). For the purpose of this
study we have, therefore, selected one strain for each Pantoea species, producing
most severe tissue maceration (Table 1).
All fungal and bacterial isolates used in this study were collected from the Zululand
forestry region in the KwaZulu-Natal province of South Africa and are maintained in
the culture collection of the Forestry and Agricultural Biotechnology Institute (FABI),
University of Pretoria.
Detection of PG in C. zu/uense
Polygalacturonase production was induced by growing isolates of C. zuluense in a
sterile liquid minimum salts medium (2.0 g NH4N03; 1.0 g KH2P04; 0.1 g MgS04; 0.5
g yeast extract (Merck); 1.0 g NaOH; 3.0 g DL-malic; 1 I distilled H20) supplemented
with 0.5 % w/v sodium polygalacturonic acid (PGA) (Sigma Chemical Company, St.
Louis, MO) as a carbon source (Errampalli & Kohn, 1995). Five, 4 mm plugs of
151
mycelium, from actively growing areas of 7-day-old C. zuluense colonies were
inoculated in 100 ml of liquid medium and incubated for 10 days on a shaker
incubator at 25°C in the dark. Mycelium was harvested by filtration through
Whatman no. 113 filter paper, using a Buchner funnel and suction filtration system.
Samples were collected each day for 10 days, supernatant was filter sterilised (0.22
um disposable syringe filters, Millipore, US) and stored at 4°C. All samples were
assayed for PG activity in triplicate.
Detection of PG in Pantoea spp.
Culture filtrates were prepared by growing Pantoea species in Errampalli & Kahn's
(1995) minimum salts medium with PGA as a carbon source. Bacteria were rinsed
from a 24-hr nutrient agar slant culture and diluted according to the method described
by Varvaro (1987). One ml of the suspension was then added to 100 ml of the
minimum salts medium in 250 ml Erlenmeyer flasks. Isolates were incubated at 25
"C for 10 days. Samples were collected each day, bacteria were removed by
filtration through 0.22 urn disposable syringe filters (Millipore, US), and the filtrate
stored at 4°C. All samples were then assayed for PG activity in triplicate.
152
Polygalacturonase assay
Cup-plate assay. Polygalacturonase activity was visualised using a modified
agarose diffusion assay (Cup-plate method) described by Dingle et al. (1953). Assay
medium contained 0.5 % ammonium oxalate, 0.2 % sodium azide, and 1.0 % Type II
agarose (Sigma Chemical Co.) dissolved in 100 ml of 0.2 M phosphate buffer (1 M
K2HP04; 1 M KH2P04; 1 I distilled H20, adjusted to pH 5.3). PGA (0.01 %) was used
as substrate. The medium was heated to dissolve PGA and agarase, then
transferred to Petri plates (20 ml per plate). A no. 1 cork borer was used to punch
five wells, 4.1 mm in diameter and 2.5 cm apart, in the solidified medium. Each well
was filled (30 ul) with standard, control, or unknown (filtrate) solutions. Petri dishes
were incubated overnight at 30°C. The gel was developed after incubation by
flooding the assay plate with 10 ml of 0.05 % ruthenium red (Sigma Chemical Co.) for
2 hr at 25°C. Excess dye was removed by washing the plate several times with
ddH20. A clear area around the well (Fig. 1) indicated PG activity. Two diameter
measurements (at right angles) of the cleared areas were taken for each well from
duplicate plates and averages recorded. Each isolate was tested three times. The
supernatant showing the highest activity (largest rings) was then assayed using the
reduced end-group analysis procedure, in order to determine a more accurate
enzyme activity.
A standard curve (ring diameter vs. concentration of standard) was prepared using
polygalacturonase, poly [1,4-a-D-galacturonide] glycanohydrolase: EC 3.2.1.15
(Sigma Chemical Co.) at the following concentrations: 5.0, 0.5, 0.05, 0.005, and
0.0005 mg / ml in dH20. The lyophilised polygalacturonase had an activity of 1440
units (U)/mg (Sigma Chemical Co.). Pectolytic activity was expressed as Units / ml.
Reducing sugar assay. Polygalacturonase activity was determined by measuring
the release of reducing-end groups by enzymatic hydrolysis of PGA. The reducing-
end groups were measured by the method of York et al. (1985). Galacturonic acid
was used as the standard for the assays. Activity of polygalacturonase was
measured by incubating 50 !lI of the sample to be assayed, for 60 min at 30°C, in 1
ml of a solution containing 0.25 % w/v PGA and 40 mM sodium-acetate (pH 5.0).
153
The reaction was terminated by addition of 1.5 ml of PAHBAH reagent (p-
Hydroxybenzoic acid hydrazide (Fw 152.2), Sigma #H 9882). PAHBAH reagent was
freshly prepared each time, by mixing 4 volumes of 0.5 M NaOH with 1 volume of 5
% PAHBAH in 0.5 M HCI. The tubes containing the samples were boiled for 10 min,
cooled and the absorbence was read at A410 nm. One unit of PG activity (RGU) was
defined as the amount of enzyme producing 1 urnol of reducing group per minute at
30°C with 0.5 % PGA in 40 mM of sodium acetate buffer, pH 5. Experiment was
done in triplicate. Means were tested for significance according to Tukey's procedure
for comparison of means (ANOVA analysis, NCSS97).
RESULTS
Detection of PG
All C. zuluense isolates, as well as the two Pantoea species produced
polygalacturonase in the presence of polygalacturonic acid (PGA) in a minimal salts
medium. Coniothyrium zuluense isolates reached maximum PG activity after 5 days
of incubation. Both Pantoea species reached maximum PG activity 3 days after
inoculation (Fig. 2).
Cup-plate assay. Using ANOVA (NCSS97), it was possible to identify significant
differences (P = 0.05) between C. zuluense isolates that differ in their pathogenicity
to a susceptible E. grandis clone (Table 2). The most pathogenic isolates, CMW
2100 and CRY 1023, showed significantly higher levels of PG activity (0.243 U/ml, P
= 0.05; 0.249 U/ml, P = 0.05, respectively) than intermediate and non-pathogenic
isolates (Table 2). There was no significant difference (P = 0.05, not significant, NS)
between PG activity of intermediate (CMW 1778 and CRY 1016) and non-pathogenic
(CRY 1055 and CRY 1054) isolates (Table 2).
A significant difference of PG activity (P = 0.05) between isolates of C. zuluense and
those of both Pantoea species, was evident (Table 2). Pantoea ananatis pv.
ananatis produced significantly higher PG activity (0.418 U/ml; P = 0.05) than the
isolate of the unknown Pantoea species (0.348 U/ml; P = 0.05) (Table 2). Levels of
154
PG activity from both Pantoea species were, however, significantly higher when
compared with PG levels measured for the C. zuluense isolates (Table 2).
Reducing sugar assay. The amount of reducing groups released (depolymerisation
of PGA) showed a significant correlation of 96 % (r = 0.96) with levels of PG activity
obtained in the cup-plate assay procedure. The depolymerisation of PGA differed
significantly (P = 0.05) between PG activity of pathogenic, intermediate and non-
pathogenic C. zuluense isolates. PG activity of the pathogenic isolates (CMW 2100
and CRY 1023) was significantly higher (P = 0.05) than that of the intermediate and
non-pathogenic isolates (Table 2). No significant difference was, however, evident
between PG activity of pathogenic isolates, CMW 2100 (0.136 umol ml" min"; NS)
and CRY 1023 (0.14 urnol ml" rnin": NS). A significant difference (P = 0.05) in the
level of depolymerisation of PGA was observed between intermediate (CMW 1778
and CRY 1016) and non-pathogenic (CRY 1055 and CRY 1054) isolates of C.
zuluense (Table 2).
Data indicated that the depolymerisation of PGA was more rapid and more extensive
for the two Pantoea species than that of C. zuluense isolates. There was a
significant difference in the ability of P. ananatis pv. ananatis (0.36 urnol ml" rnln': P
= 0.05) to depolymerise PGA, compared to that of the unknown Pantoea species
(0.31 urnol ml" min": P = 0.05) (Table 2). Isolates of C. zuluense had significantly
lower levels of PG activity (P = 0,05) than the two Pantoea species, thus, significantly
reducing the ability of C. zuluense to depolymerise PGA (Table 2).
155
DISCUSSION
Results of this study showed that isolates of C. zuluense were able to produce PG.
The level of PG activity was, however, low in comparison to levels of PG activity
reported for other Tungal pathogens (Collmer & Keen, 1986; Le Cam et al., 1994).
PG activity levels for C. zuluense were, however, similar to those reported for the
mycorrhizal fungus, Glomus versifarme (Karst.) Berchin (Peretto et al., 1995). These
authors concluded that biotrophic fungi are characterised by low and regulated
production of cell wall-degrading enzymes, particularly cellulases and pectinolytic
enzymes. Coniothyrium zuluense is a biotrophic fungus and low PG activity levels
are, perhaps not surprising.
There was a positive correlation between levels of PG activity and the pathogenicity
of C. zuluense isolates. Non-pathogenic C. zuluense isolates were characterised by
low levels of PG activity and vice versa. Similar results were reported for the
necrotrophic pathogen, Mycocentrospora acerina (Hartig) Deighton (Le Cam et al.,
1994). These authors reported a positive correlation between levels of PG activity
and the aggressiveness of different M. acerina isolates to carrot roots. Results of the
current study, therefore, indicate that levels of PG production might play a role in the
pathogenicity of C. zuluense isolates.
Both species of Pantoea that are associated with Coniothyrium canker in South
Africa were able to produce high levels of polygalacturonase. The production of PG
is, however, charaéteristic of soft rot bacteria belonging to the genus Pectobacterium.
Species of Pectobacterium are particularly well known for their production and
secretion of a variety of pectolytic enzymes (He & Collmer, 1990; Call mer & Keen,
1986; Barras et al., 1994; Hugouvieux-Cotte-Pattat et al., 1996). Bath Pantoea
species, used in this study, are not known to be associated with soft rot. However,
the relatively high levels of PG activity produced by these two bacteria are in
agreement with those reported for PG production by other non-soft rot bacteria
(Basham et al., 1985; Rodriquez-Palenzuela et al., 1991; Longland et al., 1992;
Magro et al., 1994; Herlache et al., 1997).
156
High levels of PG activity were observed for both P. ananatis pv. ananatis and the
unknown Pantoea species. This suggests that PG activity might play a significant
role in disease development. Pathogenicity results in a previous study (Van Zyl et
al., chapter 5) showed that both Pantoea species were able to cause severe tissue
maceration on Granny Smith apples, suggestive of a positive correlation between
levels of PG activity and the ability to cause disease. This is in agreement with
results obtained by Herlache et al. (1997) who showed that levels of PG activity
produced by Agrobacterium vitis, Pectobacterium carotovorum subsp. carotovorum
(synonym, Erwinia carotovora) and Ra/stonia solanacearum are positively correlated
with their respective ability to cause plant tissue maceration. Similar results were
also reported for PGs produced by Pseudomonas syringae pv. glycinea (Magro et al.,
1994).
Levels of PG activity were significantly higher for both Pantoea species, P. ananatis
pv. ananatis and the unknown Pantoea species, compared to PG activity levels of C.
zuluense isolates. Pathogenicity data from a previous study, using the same
isolates, showed a significant increase (43 %) in the pathogenicity of the fungus
when it was inoculated in combination with both bacteria species (Van Zyl et al.,
chapter 5). It was, therefore, concluded that a synergistic interaction occurs between
these micro-organisms. Results of this study, thus, support this hypothesis. It is,
therefore, possible that both Pantoea species, each capable of producing relatively
high levels of PG activity, might actively contribute to the acceleration of tissue
maceration. Coniothyrium zuluense isolates are characterised by low levels of PG
activity, suggestive of a limited ability to cause tissue maceration. It is, however,
known that bacteria are able to alter the rates of cellulose activity and wood
colonisation of decay fungi and simultaneously help provide growth factors and
soluble nitrogen (Shortie et al., 1978). The extensive breakdown of cell walls due to
the action of PG from both Pantoea species could, therefore, be important in
improving the ability of C. zuluense to colonise wood.
157
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161
Table 1. List of Coniothyrium zuluense isolates, as well as the two Pantoea species
from Eucalyptus clones, used in this study.
Culture number" Association with bacteria spp. b Pathogenicity C
CRY 1055 P. a. pv. ananatis and a Pantoea sp. non-pathogenic
CRY 1054 " non-pathogenic
CRY 1016 " Intermediately pathogenic
CRY 1023 pathogenic
CMW 1778 Intermediately pathogenic
CMW 2100 " pathogenic
P. a. pv. ananatisd pathogenic
Unknown Pantoea sp. pathogenic
aCMW and CRY numbers refer to C. zuluense isolates. All isolates were
collected from diseased Eucalyptus species, clones and hybrids in the Zululand
forestry region of KwaZulu-Natal.
blsolates selected for this study were isolated in association with two bacterial
species, Pantoea ananatis pv. ananatis and an unknown Pantoea sp.
"Pathoqenlcity of pure bacteria-free C. zuluense isolates to a susceptible E.
grandis clone (ZG 14) was determined in a previous study (Van Zyl et al., 1997).
Pathogenicity of both Pantoea species to Granny Smith apples was determined in a
previous study (Van Zyl et al., chapter 5).
dp. a. pv. ananatis represents Pantoea ananatis pv. ananatis.
162
Table 2. Comparison of polygalacturonase (PG) activity between isolates of
Coniothyrium zuluense and two Pantoea species.
Polygalacturonase activity a
Cup-plateD Reducing sugars Degree of
(units I ml) released? pathogenicity of
Isolates (units I mil min) isolates"
CRY 1055 0.1160ad 0.071 a non-pathogenic
CRY 1054 0.1240 a 0.052 a non-pathogenic
CRY 1016 0.1960 ab 0.1106 b intermediate
CMW 1778 0.1880 ab 0.1082 b intermediate
CMW 1200 0.2730 c 0.1355 c pathogenic
CRY 1023 0.2860 c 0.1395 c pathogenic
P. ananatis. pv. ananatis 0.4680 e 0.3596 e pathogenic
Unknown Pantoea sp. 0.3980 d 0.3070 d pathogenic
PG (EC 3.2.1.15) 0.5236 f 0.4248 f
apG production was induced when isolates were grown in a liquid minimal
salts medium, supplemented with polygalacturonic acid (PGA) as the sole carbon
source.
'Polyqalacturonase activity (units / ml) was determined according to the cup-
plate assay procedure of Dingle et al. (1953).
cReducing sugar assays for polygalacturonase activity was adapted from the
method described by York et al. (1985).
"Values are the mean of three repetitions. Within columns, values followed by
different letters differ significantly at P = 0.05.
"Pathoqenlcity of pure bacteria-free C. zuluense isolates was determined in a
previous study (Van Zyl et al., 1997). Pathogenicity of P. ananatis pv. ananatis and
the unknown Pantoea species to Granny Smith apples was determined in a previous
study (Van Zyl et al., chapter 5)
163
Fig. 1. Polygalacturonase (PG) activity using the 'cup-plate' method described by
Dingle et al. (1953). A clear zone surrounding the well indicates polygalacturonase
activity. Different letters represent PG activity produced by C. zuluense isolates
(CRY and CMW numbers) or the two Pantoea species. (A) CRY 1055, (8) Unknown
Pantoea sp., (C) CRY 1054, (D) CRY 1016, (E) P. ananatis pv. ananatis, (F)
Commercial polygalacturonase from Aspergillus niger (Sigma # P3429, Sigma
Chemical Co.), (G) CRY 1023 and (H) CMW 2100.

164
Fig. 2. Polygalacturonase (PG) activity produced by six Coniothyrium zuluense and
two Pantoea spp. (P. ananatis pv. ananatis and an unknown Pantoea sp. closely
related to P. stewertit subsp. stewartii). PG activity was measured over a period of
10 days on minimal salts medium supplemented with polygalacturonic acid (PGA).
Polygalacturonase activity (units / ml) was determined according to the cup-plate
assay procedure of Dingle et al. (1953). Coniothyrium zuluense isolates reached
maximum PG activity after five days of incubation. The Pantoea spp. used in this
study reached maximum PG activity three days after inoculation.
0.5 .,----------------,
.-.-..
.E.. 0.4..........-
..§,_.. 0.3
...~.S-.: 0.2
CJ
oC'CS 0.1
e,
O. .. I I I I I I I I I
o 1 2 3 4 5 6 7 8 9 10
Incubation period (days)
--+-CRY 1055 ---CRY1054 --.--CRY1016
~CMW1778 ---- CMW2100 _._CRY1023
--+-P. a. pv. ananatis .- Unknown Pantoea sp.
165
CHAPTER 7
Partial cloning of al possible disease resistance gell1lefrom two
Eucalyptus grandis clones
Pathogen-produced cell wall-degrading enzymes play a key role in activating plant
defence responses. Most inducible defence responses are the result of
transcriptional activation of genes. Various plant resistance (R) genes, as well as
pathogenesis-related proteins, such as polygalacturonase inhibiting proteins (PGIPs),
have been linked with resistance to various fungal and bacterial pathogens. The
objective of this study is to determine whether such genes are present in two
Eucalyptus grandis clones differing in their response to infection by Eucalyptus stem
canker pathogen, Coniothyrium zuluense. Clone TAG 5 is known to be resistant and
clone lG 14 is extremely susceptible to this pathogen. Degenerate primers for
nucleotide sequences of the pear PGIP gene were used. Amplification resulted in a
range of fragments of different size, with a major fragment of about 600 bp. Each
major PCR fragment was sub-cloned and sequenced. The nucleotide sequences for
TAG 5 and lG 14, comprised of 556 bp and 555 bp, respectively. Suggested amino
acid sequences indicated the existence of a single open reading frame for clone TAG
5. A shift in reading frame of this gene, however, was observed in the susceptible
Eucalyptus clone, lG 14. Homology analysis suggests that the partially sequenced
E. grandis gene showed very low homology to PGIPs and is most similar to a gene
hypothesised to be associated with resistance in Arabidopsis thaliana.
166
Coniothyrium zuluense Wingfield, Crous & Coutinho is an important Eucalyptus stem
canker pathogen in South Africa. The disease was initially reported to occur on a
single E. grandis clone, but has since become widespread and currently affects
various Eucalyptus species, clones and hybrids (Coutinho et al., 1997; Wingfield et
al., 1997). The rapid spread of this fungal pathogen throughout South Africa is of
considerable concern to local forestry industries and strategies to manage the impact
of this disease are currently being investigated.
The most reliable management strategy used to reduce the impact of Eucalyptus
diseases is by selecting clones and hybrids that show disease resistance. Field trials
have clearly indicated variation in the susceptibility of different Eucalyptus clones to
C. zuluense infection (Wingfield et al., 1997). However, previously selected disease
resistant clones are beginning to show signs of infection, indicating that virulence of
the pathogen is changing (Coutinho et al., 1997; Wingfield et al., 1997).
Virulence of plant pathogens to their hosts, has been shown to be influenced by the
production of several cell-wall-degrading enzymes (Barras et al., 1994; Alghisi &
Favaron, 1995; Kombrink & Somssich, 1995; Alfano & Callmer, 1996; Hugouvieux-
Cotte-Pattat et al., 1996). A study previously conducted in our laboratory showed
that the degree of pathogenicity of C. zuluense to a susceptible Eucalyptus clone,
was positively linked to the levels of polygalacturonase (PG) produced by isolates of
the fungus (Van Zyl et al., chapter 6). Polygalacturonase belongs to the pectic
enzyme group and is considered to be a determining factor in fungal pathogenicity
(Alghisi & Favaron, 1995; Annis & Goodwin, 1997).
Pectic enzymes are not only known to be important to pathogens in breaching plant
cell wall defence, but they are essential in the process of detection by which plants
detect the presence of pathogens (De Lorenzo et ai., 1994; Walton, 1994; Barras et
al., 1994; Alghisi & Favaron, 1995; Kombrink & Somssich, 1995; Huqouvieux-Cotte-
Pattat et al., 1996; Protsenko, 1996). There is also a growing body of evidence that
microbial enzymes that hydrolyse the pectic substances in plant cell walls, generate
fragments that activate the defence system (De Lorenzo et al., 1994; Walton, 1994;
Alghisi & Favaron, 1995; Kombrink & Somssich, 1995; Annis & Goodwin, 1997).
These hydrolytic enzymes are the "pre-elicitors" that release "true" elicitors from plant
167
cell walls (De Lorenzo et al., 1994; Walton, 1994; Alghisi & Favaron, 1995;
Protsenko, 1995; Annis & Goodwin, 1997). True elicitors are biologically active
oligosaccharides produced as a result of endogenous microbial enzyme hydrolysis
on larger, inactive polysaccharides (De Lorenzo et al., 1994; Walton, 1994; Alghisi &
Favaron, 1995). Induction of disease resistance in plants due to the transcriptional
activation of the corresponding genes, is closely associated with the release of active
oligogalacturonides (De Lorenzo et al., 1994; Walton, 1994; Alghisi & Favaron, 1995;
Kombrink & Somssich, 1995). The physiological mechanism by which these
processes are activated is, however, not clear.
Pathogen derived elicitors are recognised by specific plant target receptors, encoded
by major disease resistance genes (De Lorenzo et al., 1994; Walton, 1994; Kombrink
& Somssich, 1995). If recognised, plants protect themselves by activating various
defence mechanisms that include preformed, as well as induced defence responses
(Walton, 1994; Alghisi & Favaron, 1995; Kombrink & Somssich, 1995). The synthesis
of several disease resistance (R) gene products (Martin et al., 1993; Bent et al.,
1994; Jones et al., 1994; Mindrinos et al., 1994; Whitham et al., 1994; Song et al.,
1995; Salmeron et al., 1996), as well as pathogenesis-related proteins, such as
polygalacturonase inhibiting proteins (PGIPs) (Abu-Goukh et al., 1983; Lafitte et al.,
1984; Salvi et al., 1990; Bergmann et al., 1994; Gao & Shain, 1995; Caprari et al.,
1996), have been positively correlated with increased resistance to pathogen
invasion.
Results from various studies have shown that PGIPs have a high degree of
sequence similarity to various disease resistance gene products (Jones et al., 1994;
Steinmayr et al., 1994). This suggests a possible evolutionary conservation of these
proteins. The objective of this preliminary study is to determine whether PGIP or
related plant resistance genes are present in two E. grandis clones, ZG 14 and TAG
5, differing in their susceptibility to C. zuluense.
168
MATERIALS AND METHODS
Plant material and DNA isolation
Nucleic acid was isolated from E. grandis clones, ZG 14 and TAG 5. DNA extracts
were prepared using a modified rapid DNA isolation method (Edwards et a/., 1991).
Fresh leaves were cut into small sections and crushed to a powder in liquid nitrogen.
Five ml of a pre-heated CTAB isolation buffer (5 % Cytyltrimethylammonium bromide
(CTAB) (Sigma Co., USA); 1.4 M NaCI; 0.2 % (v/v) 2-Mercaptoethanol; 20 mM Tris-
HCI, pH 8.0; 1 % (w/v) polyvinylpyrolidone (PVP)) was subsequently added to the
frozen tissue. This mixture was incubated at 60°C for one hour and mixed by
inversion every 5 min. One volume of chloroform:isoamyl-alcohol (24:1) was added
before mixing vigorously. The extract was centrifuged at 12000 x g for 15 min and
the aqueous phase subsequently removed to a new tube containing 900 ul ice-cold
100 % ethanol. After 5 min, the spooled DNA was removed (Micheli et a/., 1994)
using a sterile pipette tip and washed in 500 ul buffer [76 % v/v ethanol; 10 mM
ammonium acetate]. The DNA was air-dried, dissolved in 100 ul sterile water and its
concentration determined by using a fluorometer. All DNA samples were diluted to
2.5 ng / !-lIand stored at -20°C.
peR procedure
Oligonucleotides used as primers were synthesised (Applied Biosystems) on the
basis of a published sequence of Pyrus communis L. cv Bartlett PGIP gene (Stotz et
al., 1993) and have the following sequences: Primer 1 (5' -G GAA TTC AAY CCN
GAY GAY AAR AAR GT- 3'), primer 2 (5' -GC TCT AGA TCD ATN GAN GTR AAR
TCC AT- 3'), and primer 3 (5' -G GAA TIC CAR ATH AAR AAR GCN TIY GG- 3').
Degeneracies are indicated using conventional nucleotide codes and are primarily in
the third base position. Polymerase chain reactions from diluted genomic DNA (1 :50)
were carried out in a reaction volume of 100 !-lI with 1 unit of SuperTaq DNA
Polymerase (Applied Biotechnologies), 10 X Taq PCR buffer (Applied
169
Biotechnologies), 3 pmol of each primer 1 and 2, 0.25 mM dNTPs and 1.5 mM MgCI2.
Amplification conditions were for 40 cycles at 94°C for 1 min., 48°C for 2 min., 65°C
for 3 min. and 1 cycle of 72 °C for 5 min. Reactions were carried out in a Hybaid
Omnigene Temperature Cycler (Hybaid, Middlesex, U.K.). The identity of the PCR
product was verified by amplifying 150 pg of DNA from the first PCR in a second
PCR, utilising primers 2 and 3. The reaction conditions were the same as described
above, although 25 cycles were used. Amplified products were analysed on 1.5 %
agarose gels.
Sequencing
PCR products were extracted from agarose gels and purified using QIAquick Gel
Extraction and QIAquick PCR Purification Kits (QIAGEN Inc., USA), respectively.
PCR fragments were then cloned using the pCR-Script™ Amp SK (+) Cloning Vector
System as described in the pCR-Script™ Amp SK (+) Cloning Vector Systems
technical manual (Stratagene, San Diego, CA). Screening for positive colonies
containing the insert was done by PCR using plasmid-specific primers M13U (5' -
GTA AAA CGA CGG CCA GFT- 3') and M13R (5' -GGA AAC AGC TAT GAC CAT
G- 3') (pCR-Script™ Amp SK (+) Cloning Kit, Stratagene, San Diego, CA). Cloned
products were precipitated and purified as described above and sequenced using
primers M13U and M13R.
PCR products were sequenced using the Big Dye Cycle Sequencing kit with
Amplitaq@ DNA Polymerase, FS (Perkin-Elmer, Warrington, UK) on a ABI PRISM™
377 DNA Autosequencer (Perkin-Elmer). Phylogenetic relationships from full-length
amino acid sequences of different PGIP and resistance (R) genes were obtained
from the GenBank database (Table 1). Amino acid sequences were aligned using
the Clustal (release 6.7) program (Higgins & Sharp, 1988). PAUP version 3.1.1
(Phylogenetic Analysis Using Parsimony) (Swofford, 1993) was used to analyse the
sequence data by executing Heuristic searches. Gaps were treated as missing data
(Swofford, 1993). A 1000 replicate bootstrap analysis was performed to assess the
confidence intervals of the branch points (Felsenstein, 1993).
170
RESULTS
Polymerase chain reaction analysis carried out on genomic DNA of both Eucalyptus
clones (ZG 14 and TAG 5), resulted in a range of amplification fragments, with one
major fragment of about 600 bp. Amplified products were cloned and sequenced.
However, in order to avoid incorporation of errors due to Taq DNA Polymerase
activity, nucleotide sequencing was also performed directly on the product amplified
from genomic DNA. This yielded the same nucleotide sequence as for the sub-
cloned products.
Nucleotide sequence data for amplified PCR products of both E. grandis clones, TAG
5 and ZG 14, comprised 556 bp and 555 bp, respectively (Fig. 1). Amino acid
sequences indicated the existence of a single open reading frame for clone TAG 5
(Fig. 2). However, a shift in reading frame was observed for susceptible E. grandis
clone, ZG 14 (Fig. 2). A Heuristic search option, with no branch swapping, from the
aligned amino acid sequence data (Fig. 3) produced one most parsimonious tree of
1551 steps (Fig. 4). Values for the Consistency Index (Cl), Retention Index (RI) and
Homoplasy Index (HI) were 0.879,0.121, and 0.773, respectively.
Analysis showed that PGIPs grouped separately from most resistance genes (Fig. 4).
Resistance gene, FIL 2 of Anthirrhinum majus L., however, grouped closest to
PGIPs. FIL 2 formed a sister group to PGIPs of Phaseolus vulgaris L. and Glycine
max (L.) Merr. with a confidence interval at the branching point of 55 %. Resistance
gene, CF-9 isolated "trom Lycopersicon pimpinellifolium (JusI.) MilL, also grouped
closer to PGIPs as compared to most other resistance genes used in this study (61 %
confidence interval).
Two clades were obtained from Heuristic searches of amino acid sequence data of
the different plant resistance genes (100 % confidence interval) (Fig. 4). The one
clade contained representative resistance genes isolated from Brassica napus L. (1A
and 9N), Arabidopsis thaliana (L.) Heynh. (RPM1 and RPS2) and Lycopersicon
esculentum Mill. (PRF). The second clade was comprised of two unknown resistance
genes isolated from A. thaliana (R), the partially sequenced E. grandis (R) gene, as
171
well as resistance gene N, isolated from Nicotiana glutinosa L. (Fig. 4). The function
of both genes isolated from A. thaliana is currently unknown, however, amino acid
sequence analysis revealed similarities to other known plant resistance genes. The
partially sequenced E. grandis gene grouped closest to the newly isolated A. thaliana
genes (100 % confidence interval) with tobacco N (isolated from Nicotiana glutinosa)
basal to them.
DISCUSSION
In this preliminary study, a possible pathogen resistance gene from E. grandis was
partially cloned, sequenced and characterised. Phylogenetic analysis showed a
distant relationship between the sequence data of the gene obtained from E. grandis
and sequence data of the pathogenesis-related protein (PGIP) from Pyrus communis.
This was unexpected, since primers used in this study were derived from published
P. communis PGIP sequences. Instead, PAUP analysis indicated a stronger
similarity between sequence data from E. grandis and that of disease resistance
genes in other plants. These genes are known to be very similar to PGIPs in that
they all possess leucine-rich repeats (Jones et al., 1994; Steinmayr et al., 1994;
Kombrink & Somssich, 1995).
Preliminary results from the current study indicated the existence of a possible plant
disease resistance gene in E. grandis that is closely related to two unknown
Arabidopsis thaliana resistance genes obtained from GenBank. No information is
available regarding their role in pathogenesis. It was, however, stated that these
resistance genes are similar to the tobacco mosaic virus (TMV) resistance gene N,
isolated from Nicotiana glutinosa (Whitham et al., 1994). PAUP analysis of results
supported the view that these two genes are closely related.
A deletion of a single nucleotide that causes an interruption in the open-reading
frame was detected in the partially sequenced E. grandis gene in clone ZG 14, which
is also susceptible to Coniothyrium canker. This was, however, not true for the more
disease resistant E. grandis clone TAG 5, in which the amino acid sequence was
complete. Similar findings have been reported for the Arabidopsis RPM1 gene
172
(Grant et al., 1995). Grant et al. (1995) identified a single nucleotide deletion that
caused a frame shift and resulted in a loss of function. This finding may provide a
partial explanation for disease susceptibility of E. grandis clone ZG 14. However,
gene isolation procedures such as positional cloning and transposon tagging are
needed to determine the role that this gene might play in disease resistance.
The cloning and characterisation of part of a possible E. grandis disease resistance
gene makes it possible to design primers to sequence the rest of the gene. The
complete sequences should provide further information on the relationship to other
resistance (R) genes and a more complete view of their role in fungal pathogenesis.
Results of the present study could, furthermore, provide valuable information for the
development of molecular markers to screen clones that are susceptible to
Coniothyrium canker for similar open reading frame interruptions. Such a process
could significantly speed up breeding for improved disease resistant Eucalyptus
clones.
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176
Table 1. Phylogenetic relatedness of the partially characterised E. grandis gene was
done using full-length amino acid sequences of different PGIPs and plant resistance
genes accessed from the GenBank databases.
Gene Plant species Gen8ank accession numbers
PGIP Actinidia deliciosa Siebold & Zucc. CAA88846
PGIP Citrus unshiu Marcovitch BAA31841
PGIP Forlunella margarita Swingle BAA34814
PGIP Glycine max CAA55081
PGIP Phaseolus vulgaris P35334
PGIP Poncirus trifoliata (L.) Raf BAA34813
PGlpa Pyres communis 005091; AAA33865
FIL2 Antirrhinum majus CAA54303
RPM1 Arabidopsis thaliana CAA61131
RPS2 Arabidopsis thaliana AAA50236
Unknown R Arabidopsis thaliana AC0023544.4; AC0072606
1A Brassica napus AAC99464
9N Brassica napus AAC99466
plf Lycopersicon esculentum AAC49408
Cf-9 Lycopersicon pimpinellifolium CAA05274
N Nicotiana glutinosa AAA50763
177
Fig. 1. Aligned nucleotide sequences of the partial Eucalyptus grandis gene from
two E. grandis clones, TAG 5 and ZG 14. The nucleotide sequences of TAG 5 and
ZG 14 comprises 556 bp and 555 bp, respectively. Frame shift is indicated in (:~).
178
10 20 30 40 50
TAGS GGAATTCAAC CCTGAATAAC AAGAAGGTCG AGAGGGAGAC ~~~.;' ' :.~:::[:50]
ZG14 GGAATTCAAT CCTGGATGAT AAGAAGGTCG AGAGGGAAAC ~H"':':' [49]
., ••• ,., •••••••••••• ' ••••••••• ',.,1
60 70 80 90 100
TAGS CGGGTTGAGG TACGTCACCA AGGTGCTGGT GTGCTTTTTG GTGGGGACTC [100]
ZG14 CAGGTTGAGG TACTTCACCA AGGTGCTGGT GTGCTTTTTG GTGGGGACTC [99]
110 120 130 140 150
TAGS TACTGTGGTT GATCAAAACA CTGATCATCA AGGTGCTGGC ATGGTCGTTC [150]
ZG14 TACTGTGGTT GATCAAAACA CTGATCATCA AGGTGCTGGC ATGGTCGTTC [149]
160 170 180 190 200
TAGS CATGTGAGCA CCTACTTTGA ACGGATCCAG AAGTCTCTGT TCAATCAGTA [200]
ZG14 CATGTGAGCA CCTACTTTGA ACGGATCCAG AATTCTCTGT TCAATCAGTA [199]
210 220 230 240 250
TAGS CGTGATCGAG ACGTTGTCGG GTCCCCCTGT GATTGAAATT TGGAGGAGAC [250]
ZG14 CGTGATCGAA ACGTTGTCGG GTCCCCCTGT GATTGAAATT TGGAGGAGAC [249]
260 270 280 290 300
TAGS AGGAAGAGGA GGAGGAGATT GCATGTGATC TCCAGACTTT ACAGAAAGCA [300]
ZG14 AGGAAGAGGA GGAGGAGATT GCATGTGATC TCCAGACTTT ACAGAAAGCA [299]
310 320 330 340 350
TAGS GAAGTGACTG TGCGGCCAGA TTTAAGGGCG GCGGCTTTTC CAACTGAAAG [350]
ZG14 GGCGTGACTG TGCCGCCAGA TTTAAGGGCG GCAGCTTTTC CAACTGAAAG [349]
360 370 380 390 400
TAGS TGGGATGGTA GTTGGGAGCG GAGGAGGGCT TCAGAGAAGT CCCCGAGAGA [400]
ZG14 TGGGATGGTA GTTGGGAGCG GAGGAGGGCT TCAGAGAAGT CCCCGAGAGA [399]
410 420 430 440 450
TAGS AGAACACCAA GCTCTTTCCA GGGCTGTCGG GAAACAGCGA GGGAGGGATC [450]
ZG14 AGAACACCAA GCTCTCTCCA GGGCTGTCGG GAAACAGCGA GGGAGGGATC [449]
460 470 480 490 500
TAGS ACAATGGAGC ACTTGCAAAA GATGAATCCC AAAAATGTGT TTGCCTGGAA [500]
ZG14 ACAATGGAGC ACTTGCAAAA GATGAATCCC AAAAATGTGT TTGCCTGGAA [499]
510 520 530 540 550
TAGS TATGAAAAGA TTGATTAATG TCGTGCGGCA TGGACTTCAC TCCATAGATT [550]
ZG14 TATGAAAAGA TTGATTAATA TCATGCGGCA TGGACTTCAC TCAATCGATT [549]
TAGS TAGAGC [556]
ZG14 TAGAGC [555]
179
Fig. 2. Amino acid sequences derived from nucleotide sequences of the partial
Eucalyptus grandis gene. Two E. grandis clones were used, disease resistant clone
TAG 5, and susceptible clone ZG 14. PC Gene analysis indicated an interruption in
the open reading frame of clone ZG 14, compared to the uninterrupted open-reading
frame of TAG 5.
180
TAG5:
RRSRGRRTVHGLRYVTKVL VCFL VGTLL WLI KTLlIKVLAWSFHVSTYFERIQKSLFN
QYVIETLSGPPVIEIWRRQEEEEEIACDLQTLQKAEVTVRPDLRAAAFPTESGMVVG
SGGGLQRSPREKNTKLFPGLSGNSEGGITMEHLQKMNPKNVFAWNMKRLlNVVRH
GLHSIDLE
ZG 14:
RRSRGKQTVTG-
181
Fig. 3. Comparisons of amino acid sequence data for PGIPs and plant resistance
genes isolated from several plant species. All sequences, except that of the partially
characterised E. grandis gene, were obtained from the GenBank database. Amino
acid sequences were aligned using the Clustal (release 6.7) program (Higgins &
Sharp, 1988). Gaps that were inserted due to alignment are indicated by a dash (-).
182
10 20 30 40 50
PGIP - Fortunella margarita
PGIP - Citrus unshiu
PGIP - poncirus trifoliata
PGIP - Pyrus communis
PGIP - Pyrus communis
PGIP - Actinidia deliciosa
FIL2 - Antirrhinum majus ---------- ---------- ---------- ---------- ----------
PGIP - Phaseolus vulgaris ---------- ---------- ---------- -----MTQFN ----------
PGIP - Glycine max ---------- ---------- ---------- ---------- ----------
CF-9 - Lycopersicon pimpinellifolium LSSNSLTGPI PSNISGLQNL ECLYLSSNHL NGSIPSWIFS ----------
lA - Brassica napus LVERCQGLPL AIASLGSMMS TKRLESEWKQ VYNSLNWELN ----------
9N - Brassica napus LLERCQGLPL AIASLGSMMS TKRLESEWKQ VYNSLNWELN ----------
RPMl - Arabidopsis thaliana LVERCQGLPL AIASLGSMMS TKKFESEWKK VYSTLNWELN ----------
Unknown R - Arabidopsis thaliana ---------- ---------- --LFDKKVEK ETQSD--VLL ----------
Unknown R - Arabidopsis thaliana ---------- ---------- --LFDEKVAK AANTK--ALR ----------
Eucalyptus grandis ---------- ---------N SILDDKKVER ETNSDR--LR ----------
PRF - Lycopersicon esculentum AVAIEAESAV CLHYEDNMNN NSREINQVLQ FLTVTFWLIK SEGNLMDLLK
RPS2 - Arabidopsis thaliana TTMMEQVLEF LSEEEERGII GVYGPGGVGK TTLMQSINNE LITKG-----
N - Nicotiana glutinosa LIQDMGKYIV NFQKDPGERS RLWLAKEVEE VMSNNTGTMA MEAIWVS---
60 70 80 90 100
PGIP - Fortunella margarita ---------- -------MSN TSLLSLFFFL CLCISPSLS- ----DLCN--
PGIP - Citrus unshiu ---------- -------MSN TSLLSLFFFL CLCISPSLS- ----DLCN--
PGIP - poncirus trifoliata ---------- -------MSN TSLLSLFFFL SLFTSLSLS- ----DLCN--
PGIP - Pyrus communis -----MELKF STFLSLTLLF SSVLNPALS- ----DLCN--
PGIP - Pyrus communis -----MELKF STFLSLTLLF SSVLNPALS- ----DLCN--
PGIP - Actinidia deliciosa -------MKS TTAISLLLFL S-LLSPSLS- ----DRCN--
FIL2 - Antirrhinum majus ----'- -MKI T-FLLVLSLL ALFSQPFLSQ ---AERCH--
PGIP - Phaseolus vulgaris -----IPVTM SSSLSIILVI LVSLRTALS- ----ELCN--
PGIP - Glycine max ---------- ---------- ---------- ---------- ----ELCN--
CF-9 - Lycopersicon pimpinellifolium ---------- -----LPSLV ELDLSNNTFS GKIQEFKSKT ---LSAVT--
lA - Brassica napus ---------- -----NNLEL KVVRSILLLS FSDLPYPLKR C--FLYCCLF
9N - Brassica napus ---------- -----NNLEL KVVRSILSLS FSDLPYPLKR C--FLYCCMF
RPMl - Arabidopsis thaliana ---------- -----NNHEL KIVRSIMFLS FNDLPYPLKR C--FLYCSLF
Unknown R - Arabidopsis thaliana ---------- -----LMSKI LVCFLLSTVL WLIKTLVVK- ---VLASS--
Unknown R - Arabidopsis thaliana ---------- -----VVTKI FVCLLVGFLL WLVKTLLVK- ---VLASS--
Eucalyptus grandis ---------- -----yFTKV LVCFLVGTLL WLIKTLIIK- ---VLAWS--
PRF - Lycopersicon esculentum HKSTLGNQVL DLIESAHEEL ILLRSILMDL LRKKLYRLDD L--LMHAEVT
RPS2 - Arabidopsis thaliana ------HQYD VLIWVQMSRE FGECTIQQAV GARLGLSWDE K--ETGENRA
N - Nicotiana glutinosa -----SYSST LRFSNQAVKN MKRLRVFNMG RSSTHYAIDY LPNNLRCFVC
110 120 130 140 150
PGIP - Fortunella margarita ---------- ---------- -----PNDKK VLLKFK----
PGIP - Citrus unshiu ---------- ---------- -----PNDKK VLLKFK----
PGIP - poncirus trifoliata -----PNDKR VLLNFK----
PGIP - Pyrus communis -----PDDKK VLLQIK----
PGIP - Pyrus communis -----PDDKK VLLQIK----
PGIP - Actinidia deliciosa -----PNDKK VLLRIK----
FIL2 - Antirrhinum majus -----PQDKR VLLKIK----
PGIP - Phaseolus vulgaris -----PQDKQ ALLQIK----
PGIP - Glycine max -----PQDKQ TLLQIK----
CF-9 - Lycopersicon pimpinellifolium -----LKQNK LKGRIP----
lA - Brassica napus PVNYRMKR -- -KKLVRMWMA QRFVEPIRGV KAEEVA----
9N - Brassica napus PVNYRMKR-- -KRLVRMWMA QRFVEPIRGV KAEEVA----
RPMl - Arabidopsis thaliana PVNYRMKR -- -KRLIRMWMA QRFVEPIRGV KAEEVA----
Unknown R - Arabidopsis thaliana -----FHVST YFDRIQ----
Unknown R - Arabidopsis thaliana -----FHMST YFDRIQ----
Eucalyptus grandis -----FHVST yFERIQ----
PRF - Lycopersicon esculentum AKRLAIFSGS CYEYFMNGSS TEKMRPLLSD FLQEIESVKV EFRNVCLQVL
RPS2 - Arabidopsis thaliana LKIYRALR-- ---------- QKRFLLLLDD VWEEIDLEKT G---------
N - Nicotiana glutinosa TNYPWESFP- ---------- --STFELKML VHLQLR----
183
160 170 180 190 200
PGIP - Fortunella margarita
PGIP - Citrus unshiu
PGIP - poncirus trifoliata
PGIP - Pyrus ccmmunz s
PGIP Pyrus commun.i s
PGIP - Actinidia deliciosa
FIL2 - Antirrhinum majus ---------- ---------- ---------- ---------- ----------
PGIP - Phaseolus vulgaris ---------- ---------- ---------- ---------- ----------
PGIP - Glycine max ---------- ---------- ---------- ---------- ----------
CF-9 - Lycopersicon pimpinellifolium ---------- ---------- ---------- ---------- ----------
LA - Brassica napus ---------- ---------- ---------- ---------- ----------
9N - Brassica napus ---------- ---------- ---------- ---------- ----------
RPMl - Arabidopsis thaliana ---------- ---------- ---------- ---------- ----------
Unknown R - Arabidopsis thaliana ---------- ---------- ---------- ---------- ----------
Unknown R - Arabidopsis thaliana ---------- ---------- ---------- ---------- ----------
Eucalyptus grandis ---------- ---------- ---------- ---------- ----------
PRF - Lycopersicon esculentum DISPFSLTDG EGLVNFLLKN QAKVPNDDAV SSDGSLEDAS STEKMGLPSD
RPS2 - Arabidopsis thaliana ---------- ---------- ---------- ---------- ----------
N - Nicotiana glutinosa ---------- ---------- ---------- ---------- ----------
210 220 230 240 250
PGIP - Fortunella margarita ---------- ---------- ---------- ---KALNNPY VLA-------
PGIP - Citrus unshiu ---------- ---------- ---------- ---KSLNNPY VLA-------
PGIP - poncirus trifoliata ---------- ---------- ---------- ---KALNNPY VLA-------
PGIP - Pyrus commlUlis ---KAFGDPY VLA-------
PGIP - Pyrus commlUlis ---KAFGDPY VLA-------
PGIP - Actinidia deliciosa ---QALNNPY LLA-------
FIL2 - Antirrhinum majus ---KAFNNPY HLA-------
PGIP - Phaseolus vulgaris ---KDLGNPT TLS-------
PGIP - Glycine max ---KELGNPT TLS-------
CF-9 - Lycopersicon pimpinellifolium ---NSLLNQK NLQ-------
lA - Brassica napus ---DGYLNEL VYRNM-----
9N - Brassica napus ---DGYLNEL VYRNM-----
RPMl - Arabidopsis thaliana ---DSYLNEL VYRNM-----
Unknown R - Arabidopsis thaliana ---EALFHHY LIET------
Unknown R - Arabidopsis thaliana ---------- ---------- ---ESLFTQY VIET------
Eucalyptus grandis ---------- ---------- ---NSLFNQY VIET------
PRF - Lycopersicon esculentum FLREIESVEI KEARKLYDQV LDATHCETSK TDGKSFINIM LTQQDKLPDY
RPS2 - Arabidopsis thaliana -VPRPDRENK CKVMFTTRSI ALCNNMGAEY KLRVEFLEKK
N - Nicotiana glutinosa ---------- ---------- --HNSLRHLW TETKHLPSLR
260 270 280 290 300
PGIP - Fortunella margarita
PGIP - citrus lUlshiu
PGIP - poncirus trifoliata
PGIP - Pyrus commlUlis
PGIP - Pyrus commlUlis
PGIP - Actinidia deliciosa
FIL2 - Antirrhinum majus
PGIP - Phaseolus vulgaris
PGIP - Glycine max
CF-9 - Lycopersicon pimpinellifolium
lA - Brassica napus
9N - Brassica napus
RPMl - Arabidopsis thaliana
Unknown R - Arabidopsis thaliana
Unknown R - Arabidopsis thaliana
Eucalyptus grandis
PRF - Lycopersicon esculentum DAGSVSYLLN QISVVKDKLL HIGSLLVDIV QYRNMHIELT DLAERVQDKN
RPS2 - Arabidopsis thaliana HAWELFCS-- ----------
N - Nicotiana glutinosa
184
310 320 330 340 350
PG1P - Fortunella margarita --SW------ --------NP
PG1P - Citrus unshiu --SW------ --------NP
PG1P - Poncirus trifoliata --SW------ --------NP
PG1P - Pyrus communis --SW------ --------KS
PG1P - Pyrus communis --SW------ --------KS
PG1P - Actinidia deliciosa --SW------ --------NP
FIL2 - Antirrhinum majus --SW------ --------1P
PG1P - Phaseolus vulgaris --SW------ --------LP
PG1P - Glycine max --SW------ --------HP
CF-9 - Lycopersicon pimpinellifolium --LL------ --------LL
lA - Brassica napus --------LQ V1LW------ --------NP
9N - Brassica napus --------LQ V1LW------ --------NP
RPMl - Arabidopsis thaliana --------LQ V1LW------ --------NP
Unknown R - Arabidopsis thaliana ---------- --LS------ --------GP
Unknown R - Arabidopsis thaliana --LS------ --------GP
Eucalyptus grandis --LS------ --------GP
PRF - Lycopersicon esculentum Y1CFFSVKGY 1PAWYYTLYL SDVKQLLKFV EAEVK11CLK VPDSSSYSFP
RPS2 - Arabidopsis thaliana ---------- -KVWRKDLLE SSS1RRLAE1 1VSKCG---- ---G----LP
N - Nicotiana glutinosa --------R1 DLSWSKR--- ------LTR- ---------- --------TP
360 370 380 390 400
PG1P - Fortunella margarita KTD------- ---------- ---------- ---------- ----------
PG1P - Citrus unshiu KTD------- ---------- ---------- ---------- ----------
PG1P - poncirus trifoliata KTD------- ---------- ---------- ---------- ----------
PG1P - Pyrus communis DTD-------
PG1P - Pyrus communis DTD-------
PG1P - Actinidia deliciosa DND-------
FIL2 - Antirrhinum majus DTD-------
PG1P - Phaseolus vulgaris TTD-------
PG1P - Glycine max KTD-------
CF-9 Lycopersicon pimpinellifolium SHN-------
lA - Brassica napus FGR-------
9N - Brassica napus FGR-------
RPMl - Arabidopsis thaliana FGR-------
Unknown R - Arabidopsis thaliana PML-------
Unknown R - Arabidopsis thaliana P-L-------
Eucalyptus grandis pv-------- ---------- ---------- ---------- ----------
PRF - Lycopersicon esculentum KTNGLGYLNC FLGKLEELLR SKLDL11DLK HQ1ESVKEGL LCLRSF1DHF
RPS2 - Arabidopsis thaliana LAL1TLG--- ---------- ---------- ---------- ---------G
N - Nicotiana glutinosa DFTG------ ---------M
410 420 430 440 450
PG1P - Fortunella margarita CCD--WYCVT CDLTTN---- ---------- ---------- -------R1N
PG1P - citrus unshiu CCD--WYCVT CDLTTN---- ---------- ---------- -------R1N
PG1P - poncirus trifoliata CCD--WYCVT CDLTTN---- ---------- ---------- -------R1N
PG1P - Pyrus communis CCD--WYCVT CDSTTN---- -------R1N
PG1P - Pyrus communis CCD--WYCVT CDSTTN---- -------R1N
PG1P - Actinidia deliciosa CCD--WYNVD CDLTTN---- -------R11
FIL2 - Antirrhinum majus CCS--WYVVE CDRTTN---- -------R1N
PG1P - Phaseolus vulgaris CCNRTWLGVL CDTDTQT--- ------YRVN
PG1P - Glycine max CCNNSWVGVS CDTVTPT--- ------YRVD
CF-9 - Lycopersicon pimpinellifolium N1SGH1SSA1 CNLKT----- --------L1
lA - Brassica napus PKVFKMHDV1 RE1ALS1--- ------SKAE
9N - Brassica napus PKVFKMHDV1 RE1ALS1--- ------SKAE
RPMl - _Arabidopsis thaliana PKAFKMHDV1 WE1ALSV --- ------SKLE
Unknown R - Arabidopsis thaliana -EL-SR1EEE EDRTQDE--- ---------- ---------- ------1YKM
Unknown R - _Arabidopsis thaliana 1E1QKNEEEE E-R1SVE--- ---------- ---------- ------VKKF
Eucalyptus grandis IEIWRRQEEE EE-IACD--- ---------- ---------- ------LQTL
PRF - Lycopersicon esculentum SESYDEHDEA CGL1ARVSVM AYKAEYV1DS CLAYSHPLWY KVLW1SEVLE
RPS2 - Arabidopsis thaliana AMAHRETEEE W1HASEVLTR FPAEMKG--- ---------- ----MNYVFA
N - Nicotiana glutinosa PNLEYVNLYQ CSNLEEVHHS LG-------- ---------- ----CCSKV1
185
460 470 480 490 500
PGIP - Fortunella margarita SLTIFAGDLP G-----QIPP EVGD------ --LPYLDTLM FHKLPSLTGP
PGIP - Citrus unshiu SLTIFAGDLP G-----QIPP EVGD------ --LPYLETLM FHKLPSLTGP
PGIP - poncirus trifoliata SLTIFAGDLP G-----QIPP EVGD------ --LPYLETLM FHKLPSLTGP
PGIP - Pyrus communis SLTIFAGQVS G-----QIPA LVGD------ --LPYLETLE FHKQPNLTGP
PGIP - Pyrus communis SLTIFAGQVS G-----QIPA LVGD------ --LPYLETLE FHKQPNLTGP
PGIP - Actinidia deliciosa ALTIFSGNIS G-----QIPA AVGD------ --LPYLQTLI FRKLSNLTGQ
FIL2 - Antirrhinum majus DFHLFSASVS G-----QIPE TIAE------ --LPFLESLM FRKITNLTGT
PGIP - Phaseolus vulgaris NLDLSGHNLP KP---YPIPS SLAN------ --LPYLNFLY IGGINNLVGP
PGIP - Glycine max NLDLSELNLR KP---YPIPP SVGS------ --LPCLKFLY ITNNPNIVGT
CF-9 - Lycopersicon pimpinellifolium LLDLGSNNLE G-----TIPQ CVVER----- --NEYLSHLD LSK-NRLSGT
lA - Brassica napus RFCDVNGDDD D----DDDAE TAEDHGTRHL CIQKEMRSGT LRRTNLHTLL
9N - Brassica napus RFCDVNGDDD D----DD-AE TAEDHGTRHL CIQKEMRSGT VRRTNLHTLL
RPMl - Arabidopsis thaliana RFCDVYNDDS DG---DDAAE TMENYGSRHL CIQKEMTPDS IRATNLHSLL
Unknown R - Arabidopsis thaliana QK-GGADLSP E-LCSAAFPQ EKSGSTMN-- --MKFSPIIP KTGSDN----
Unknown R - Arabidopsis thaliana QNPGGVEIQS G----AQKSP MKTGKSP--- ---FLSHVLS NGGGGG-GE-
Eucalyptus grandis QKAG-VTVPP D-LRAAAFPT E-SGMVVGSG GGLQRSPREK NTKLSPGLSG
PRF - Lycopersicon esculentum NIKLVNKVVG ETCERRNIEV TVHEVAKTTT YVAPSFSAYT QRANEEMEGF
RPS2 - Arabidopsis thaliana LLKFSYDNLE SDLLRSCFLY CALFPEEHSI EIEQLVEYWV GEGFLTSSHG
N - Nicotiana glutinosa GLYLNDCKSL KRFPCVNVES LEYLGLRSCD SLEKLPEIYG RMKPEIQIHM
510 520 530 540 550
PGIP - Fortunella margarita IQP------- AIAKLKNLKT LRISWT----
PGIP - Citrus unshiu IQP------- AIAKLKNLKT LRISWT----
PGIP - poncirus trifoliata IQP------- AIAKLKNLKM LRISWT----
PGIP - Pyrus communis IQP------- AIAKLKGLKS LRLSWT----
PGIP - Pyrus communis IQP------- AIAKLKGLKS LRLSWT----
PGIP - Actinidia deliciosa IPS------- AISKLSNLKM VRLSWT----
FIL2 - Antirrhinum majus IPH------- AITRLTRLRS LTISWT----
PGIP - Phaseolus vulgaris IPP------- AIAKLTQLHY LYITHT----
PGIP - Glycine max IPT------- TITKLTKLRE LNIRYT----
CF-9 - Lycopersicon pimpinellifolium INT------- TFSVGNILRV ISLHGN----
lA - Brassica napus VCT--KHSIE LPPSLKLLRA LDLEGS----
9N - Brassica napus VCT--KHSIE LPPSLKLLRA LDLEGS----
RPMl - Arabidopsis thaliana VCSSAKHKME LLPSLNLLRA LDLEDS---- ---------- ----------
Unknown R - Arabidopsis thaliana ---------- ---GITMDDL HKMNQK---- ---------- ----------
Unknown R - Arabidopsis thaliana NK-------- ---GITIDSL HKLNPK---- ---------- ----------
Eucalyptus grandis NSEG------ ---GITMEHL QKMNPK---- ---------- ----------
PRF - Lycopersicon esculentum QDTIDELKDK LLGGSPELDV ISIVGMPGLG KTTLAKKIYN DPEVTSRFDV
RPS2 - Arabidopsis thaliana VNTIYKG-YF LIGDLKAACL LETGDEKTQV KMHN------ ----------
N - Nicotiana glutinosa QGSGIRELPS SIFQYKTHVT KLLLWN---- ---------- ----------
560 570 580 590 600
PGIP - Fortunella margarita ---------- ---------- ---------- -----NISGP VPDFISQLTN
PGIP - Citrus unshiu ---------- ---------- ---------- -----NISGP VPDFISQLTN
PGIP - poncirus trifoliata ---------- ---------- ---------- -----NISGP VPDFISQLTN
PGIP - Pyrus communis ---------- ---------- ---------- -----NLSGS VPDFLSQLKN
PGIP - Pyrus communis ---------- ---------- ---------- -----NLSGS VPDFLSQLKN
PGIP - Actinidia deliciosa ---------- ---------- ---------- -----NLSGP VPSFFSQLKN
FIL2 - Antirrhinum majus ---------- ---------- ---------- -----NISGP VPAFLSELKN
PGIP - Phaseolus vulgaris ---------- ---------- ---------- -----NVSGA IPDFLSQIKT
PGIP - Glycine max ---------- ---------- ---------- -----NISGQ IPHFLSQIKA
CF-9 - Lycopersicon pimpinellifolium ---------- ---------- ---------- -----KLTGK VPRSMINCKY
lA - Brassica napus ---------- ---------- ---------- -----GVT-K LPDFLVTLFN
9N - Brassica napus ---------- ---------- ---------- -----GIS-K LPEILVTLFN
RPMl - Arabidopsis thaliana ---------- ---------- ---------- -----SIS-K LPDCLVTMFN
Unknown R - Arabidopsis thaliana ---------- ---------- ---------- -----NVSAW NMKRLMKIVR
Unknown R - Arabidopsis thaliana ---------- ---------- ---------- -----NVSAW KMKRLMNIIR
Eucalyptus grandis ---------- ---------- ---------- -----NVFAW NMKRLINIMR
PRF - Lycopersicon esculentum HAQCVVTQLY SWRELLLTIL NDVLEPSDRN EKEDGEIADE LRRFLLTKRF
RPS2 - Arabidopsis thaliana ---------- ---------- ---------- --VVRSFALW MASEQGTYKE
N - Nicotiana glutinosa ---------- ---------- ---------- ----MKNLVA LPSSICRLKS
186
610 620 630 640 650
PGIP - Fortunella margarita LTFLE----- --LSFNNLSG -----TIPGS
PGIP - Citrus unshiu LTFLE----- --LSFNNLSG -----TIPGS
PGIP - poncirus trifoliata LTFLE----- --LSFNNLSG -----TIPSS
PGIP - Pyrus communis LTFLD----- --LSFNNLTG -----AIPSS
PGIP - Pyrus communis LTFLD----- --LSFNNLTG -----AIPSS
PGIP - Actinidia deliciosa LTFLD----- --LSFNDLTG -----SIPSS
FIL2 - Antirrhinum majus LTSLD----- --LSFNNLSG -----SIPPS
PGIP - Phaseolus_vulgaris LVTLD----- --FSYNALSG -----TLPPS
PGIP - Glycine max LGFLD----- --LSNNKLSG -----NLPSW
CF-9 - Lycopersicon pimpinellifolium LTLLD----- --LGNNMLND -----TFPNW
lA - Brassica napus LKYLN----- --LSKTEVK- -----ELPRD
9N - Brassica napus LKYLN----- --LSKTEVK- -----ELPRD
RPMl - Arabidopsis thaliana LKYLN----- --LSKTQVK- -----ELPKN
Unknown R - Arabidopsis thaliana NVSLS----- --T-LDEQAL QNTC-EDEST
Unknown R - Arabidopsis thaliana NGSLT----- --T-LDEQLQ DPSL-DDDKG
Eucalyptus grandis HGLHS----- --IDL----- ----------
PRF - Lycopersicon esculentum LILIDDVWDY KVWDNLCMCF SDVSNRSRII LTTRLNDVAE YVKCESDPHH
RPS2 - Arabidopsis thaliana LILVEPSMG- ---------- --------HT EAPKAENWRQ ALVISLLDNR
N - Nicotiana glutinosa LVSLS----- ---------- ---------- --VSGCSKLE -----SLPEE
187
Fig. 4. Phylogram generated with PAUP analysis based on comparisons of amino
acid sequence data from PGIPs and plant resistance genes. All sequence data,
except those of the partially characterised E. grandis gene, were obtained from the
GenBank. GenBank accession numbers are indicated in brackets. The tree length =
1551 steps, Cl = 0.879, HI = 0.121 and RI = 0.773. Bootstrap values (1000
replicates) are given at the branching points.
PGIP - Forlunella margarita (BAA34814)
PGIP - Citrus unshiu (BAA31841)
PGIP - Poncirus trifoliata (BAA34813)
.....- PGIP - Pyrus communis (Q05091)
""'-
88 ~ PGIP - Pyrus communis (AAA33865)
....._ PGIP - Actinidia deliciosa (CAA88846)
100
FlL2 - Antirrhinum majus (CAA54303)
....._ ;--- PGIP - Pheseolus vulgaris (P35334)
100 ....._
.____ PGIP - Glycine max (CAA55081)
....._ CF-9 - Lycopersicon pimpinellifolium (CAA05274)
55
.....- 1A - Brassica napus (AAC99464)
,....-
100 .____- 9N - Brassica napus (AAC99466)
-61 19Q... RPM1 - Arabidopsis thaliana (CAA61131)
- RPS2 - Arabidopsis thaliana (AAA50236)
PRF - Lycopersicon esculentum (AAC49408)
....._ 52 - R - Arabidopsis thaliana (AC0023544.4)100 ,....-
100 .____ R - Eucalyptus grandis
r--
R - Arabidopsis thaliana (AC0072606)
N- Nicotiana glutinosa (AAA50763)
188
SUMMARY
In chapter one of this thesis, the literature pertaining to the genus
Coniothyrium and its importance in plant pathology, is reviewed. Special
attention is given to Coniothyrium species associated with Eucalyptus but the
focus is on Eucalyptus stem canker pathogen, C. zuluense. Coniothyrium
zuluense is an important pathogen in South Africa and has, since its
discovery, become widespread throughout plantation areas of KwaZulu-Natal.
The current means for reducing the impact of this disease is to plant disease
resistant species and clones of Eucalyptus. It is evident from this review that
very little information is available pertaining to the biology, reproductive
system, or the population structure of C. zuluense. Such information is
essential for managing the disease successfully in the future.
The strategy currently used to reduce the impact of Coniothyrium canker in
plantations is to deploy Eucalyptus species or clones that are resistant to the
disease. Considerable success has already been achieved in this regard,
but the long-term durability of resistance is of concern. Results of the study
represented in chapter two showed that there is considerable variation in
colony colour and pathogenicity of a large collection (344) of C. zuluense
isolates. Conidial morphology and growth requirements are, however, similar
for all isolates tested. The considerable variation in pathogenicity indicates
that C. zuluense has been present in South Africa for an extended period of
time, or that virulence is changing rapidly due to strong directional selection
pressure.
Results of the taxonomic and pathogenicity studies in chapter two, suggest
that the C. zuluense population is well established. In chapter three, the
population diversity of 108 C. zuluense isolates, differing in their pathogenicity
to a susceptible Eucalyptus clone, was investigated using Amplified Fragment
Length Polymorphism (AFLP) technology. Results indicated that the level of
genetic diversity is relatively low, but higher than expected for an asexually
reproducing pathogen. Genetic similarity values also indicated a significant
population differentiation between different plantation regions (sub-
189
populations), suggesting that gene flow, together with selection, might be
responsible for most of the gene diversity. New epidemics would, therefore,
not be as a result of the emergence of new aggressive strains, but would
rather be due to the introduction of susceptible Eucalyptus species, together
with environmental conditions favouring disease development.
A Coniothyrium species associated with similar symptoms to those associated
with C. zuluense in South Africa was observed on E. camaldulensis in
Thailand in 1996. It was previously thought that C. zuluense was restricted to
South Africa. In chapter four, I show using morphological and molecular
comparisons, as well as pathogenicity studies, that C. zuluense and the
Coniothyrium sp. from Thailand are the same organism. This is, thus, the first
record of this important Eucalyptus stem canker pathogen, C. zuluense,
outside South Africa.
Bacteria commonly exude from necrotic cankers on severely infected
Eucalyptus clones in plantations. In chapter five, it was shown that bacteria
associated with Coniothyrium canker in the field are species of the genus
Pantoea. These species were identified based on 16S rDNA sequence data
as P. ananatis pv. ananatis and a species closely related to P. stewartii subsp.
stewartii. It was also shown that a synergistic interaction between C.
zuluense and both Pantoea species exists. Inoculation studies, using both
Pantoea species together with C. zuluense isolates, resulted in a significant
\
increase in pathogenicity as opposed to inoculations where the bacterial and II
fungal isolates were used alone. Future studies should consider the presence
or absence of both bacteria species in disease development in Thailand.
During plant-pathogen interactions, pathogens are known to produce cell wall-
degrading enzymes, in particular pectin degrading enzymes.
Polygalacturonase (PG) is the first enzyme produced during such interactions
and is known to be a determining factor in pathogenicity. Chapter six showed
that C. zuluense isolates and both Pantoea species, P. ananatis pv. ananatis
and an unknown Pantoea sp., produce PG. Experimental assays show that
levels of PG activity for both Pantoea spp. are significantly higher than those
190
obtained for C. zuluense isolates. As PG is the first enzyme produced during
disease development it is hypothesised that the two Pantoea species might
play a significant role in the development of Coniothyrium canker. Production
of PG could also be used as an assay to evaluate pathogenicity in different
isolates of C. zuluense.
Pathogen-produced cell wall-degrading enzymes play a key role in activating
plant defence responses. Most inducible defence responses are the result of
transcriptional activation of genes. Various plant resistance (R) genes, as well
as pathogenesis-related proteins, such as polygalacturonase inhibiting
proteins (PGIPs), have been linked with resistance to various fungal and
bacterial pathogens. In chapter seven, a partially sequenced resistance gene
from disease resistant E. grandis clone, TAG 5, was shown to be similar to a
gene associated with a disease resistance gene in Arabidopsis thaliana. The
most exciting aspect of this study was, however, the discovery of a shift in
reading frame of this gene for the susceptible Eucalyptus clone, ZG 14. The
complete sequence of this gene should provide a more complete view of its
importance in disease resistance. Screening for similar interruptions in the
open reading frame of various commercially available Eucalyptus clones could
significantly speed up breeding programmes aimed at producing improved
disease resistant clones.
191
OPSOMMiNG
Die literatuur met betrekking tot die belang van die genus Coniothyrium in plant
patologie is in hoofstuk 1 in heroonskou geneem. Spesifieke aandag is gegee aan
die Eucalyptus stamkankerpatogeen, Coniothyrium zuluense. Uit die oorsig is dit
duidelik dat daar 'n tekort aan informasie rakende die basiese biologie van die fungus
soos voortplantings meganisme en populasie diversiteit bestaan. Hierdie inligting is
van kardinale belang met die oog op die ontwikkeling van siekte beheerprogramme.
Die strategie wat tans gebruik word om verspreiding van die siekte te beheer, is deur
die seleksie van siekte weerstandbiedende Eucalyptus klone. Bogenoemde praktyk
is suksesvol, alhoewel daar kommer bestaan oor die langdurigheid van weerstand in
geselekteerde klone. Resultate van hoofstuk twee het getoon dat daar 'n aansienlike
variasie in die populasie van C. zuluense is met betrekking tot koloniekleur en
patogenisiteit. Spoor morfologie en groei temperatuur voorkeure was egter dieselfde
vir al die isolate (344 isolate) bestudeer. Die vlak van variasie in patogenisiteit kan
moontlik daarop dui dat C. zuluense al 'n geruime tyd in Suid Afrika voorkom, of dat
'n verandering in virulensie bespoedig word as gevolg van seleksie druk.
Resultate van hoofstuk twee dui daarop dat die populasie van C. zuluense in Suid
Afrika relatief divers is. In hoofstuk drie is daar, dus, gefokus op die bepaling van die
populasie diversiteit van 108 C. zuluensie isolate met behulp van "Amplified
Fragment Length Polymorphism". Geselekteerde isolate het verskil ten opsigte van
hul patogenisiteit tot 'n vatbare Eucalyptus kloon. Resultate het daarop gedui dat die
vlak van genetiese diversiteit relatief laag was, maar hoër as wat verwag is van 'n
patogeen wat nie-seksueel voortplant. Genetiese similariteitswaardes het ook
aangedui dat betekenisvolle differensiasie tussen plantasie gebiede (sub-populasies)
van die patogeen voorkom. Dit is 'n aanduiding dat genetiese vloei, tesame met
seleksie, moontlik vir die geen diversiteit verantwoordelik is. Uitbreek van nuwe
epidemies sal dus nie veroorsaak word deur nuwe aggressiewe rasse van die
patogeen nie, maar eerder deur die aanplanting van vatbare Eucalyptus klone,
tesame met gunstige omgewings faktore.
192
Coniothyrium kanker was voorheen net bekend in Suid Afrika. Gedurende 1996, is
siekte simtome, soortgelyk aan die van C. zuluense in Suid Afrika, opgemerk op 'n E.
. camaldulensis kloon in Thailand. In hoofstuk vier is deur middel van morfologiese-,
molekulêre- en patogenisiteits-studies bewys dat die Coniothyrium spesie van
Thailand en C. zuluense, dieselfde organisme is. Dit is dus die eerste aanmelding
van hierdie gevreesde siekte buite Suid Afrika.
Bakterieë word alledaags opgemerk waar dit uit stam kankers van uiters vatbare
Eucalyptus klone vloei. Resultate van hoofstuk vyf het getoon dat twee bakterie
spesies van die genus Pantoea met Coniothyrium kanker geassosieer is.
Identifikasie is gedoen deur die basispaar opeenvolging van die 16S rDNA geen van
beide bakterieë te vergelyk met die basispaar opeenvolging van 16S rDNA gene van
verwante spesies. Een bakterium is geïdentifiseer as P. ananatis pv. ananatis en die
ander spesie as naby verwant aan P. stewartii subsp. stewartii. Uit die resultate was
dit ook duidelik dat daar 'n sinergistiese verwantskap tussen C. zuluense en beide
Pantoea spesies bestaan. Patogenisiteits-studies het getoon dat 'n betekenisvol
greoter letsels geproduseer is wanneer C. zuluense isolate in kombinasie met beide
Pantoea spesies in bome geinokuleer is.
Plant patogene is bekend vir die produksie van verskeie ensieme om plantselwande
af te breek, spesifiek pektien-afbreekende-ensieme. Die pektiese ensiem,
polygalacturonase (PG), is bekend as die eerste ensiem wat gedurende plant-
patogeen interaksies geproduseer word, en word beskou as 'n bepalende faktor in
siekte vorming. Resultate uit hoofstuk ses toon dat C. zuluense en beide Pantoea
spesies, P. ananatis pv. ananatis en die onbekende Pantoea spesie, PG kan
produseer. Polygalacturonase aktiwiteit vir beide Pantoea spesies was betekenisvol
hoër in vergelyking met die van C. zuluense. Dit bleik uit die resultate dat PG
produksie van beide Pantoea spesies moontlik 'n noemenswaardige rol kan speel in
letsel vorming van Coniothyrium canker.
Plantselwand-afbrekende-ensieme speelook 'n noemenswaardige rol in die
aktivering van die plant se weerstandsmeganismes. Die aktivering van verskeie PG
inhiberings-proteine (PGIPs), is voorheen direk gekoppel aan verhoogde weerstand
teen fungus- and bakteriesiektes. In hoofstuk sewe is gevind dat 'n weerstandsgeen
193
wat naby verwant is aan 'n weerstandsgeen in Arabidopsis thetiene. in 'n siekte-
tolerante E. grandis kloon, TAG 5, teenwoordig is. Die E. grandis kloon, ZG 14, wat
bekend is vir vatbaarheid vir verskeie bosbou verwante siektes, het egter 'n leesraam
verskuiwing in die bogenoemde geen gehad. Dit kan 'n moontlike verduideliking
verskaf vir die vatbaarheid van die spesifieke kloon vir siektes. Laasgenoemde
resultate kan gebruik word vir die vinnige identifisering van soortgelyke leesraam
verskuiwings in klonale materiaal, wat tot die uitskakeling van sulke vatbare klone in
die bosbou bedryf kan lei.