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GENETIC DETERMINANTS OF HOST RANGE SPECIFICITY OF THE
WELLINGTON STRAIN OF Xanthomonas axonopodis pv. citri
MYRIAN ASUCENA RYBAK
A DISSERTATION PRESENTED TO THE GRADUATE SCHOOL
OF THE UNIVERSITY OF FLORIDA IN PARTIAL FULFILLMENT
OF THE REQUIREMENTS FOR THE DEGREE OF
DOCTOR OF PHILOSOPHY
UNIVERSITY OF FLORIDA
Myrian Asucena Rybak
To God my guide and fortitude
I wish to express my thanks and compliments to the chairman of my supervisory
committee, Dr. Jeffrey B. Jones, for his encouragement and guidance. I also want for to
thank the members of my committee, Dr. James Graham and Dr. Gloria Moore for their
critical reading of the manuscript and for their helpful suggestions
My most considerable debt is to Dr. Robert E. Stall who was generous with his
time and expertise in this research. His knowledge, patience and constant support were
equally important to the completion of this manuscript. My appreciation and gratitude go
to him now and always.
Special thanks go to Mr. Jerry Minsavage for his indispensable help; he generously
donated many hours of his valuable time with his technical assistance.
I owe gratitude to the Plant Pathology Department at the University of Florida;
especially I want to thank the members of our laboratory that I met between 2001-2005.
Sincere gratitude is extended to the faculty staff members and other people in Fifield Hall
for their assistance and friendship.
I would like to thank friends and relatives in Argentina, especially Dr. Nelly
Canteros; her guidance and useful advice will be always appreciated.
My family helped and their faith kept me going though some pretty hard times. I
would particularly like to acknowledge Raquel, my dear sister and friend, and my
wonderful mother Veronica, my sisters and brothers, nieces and nephews and thanks go
to the memory of my father for his example and integrity.
TABLE OF CONTENTS
A C K N O W L E D G M E N T S ................................................................................................. iv
LIST OF TABLES .................. .................. .................. ........... .............. vii
LIST OF FIGU RE S ................ ............................ ............ ........... .......... viii
ABSTRAC T ................. .............. ........................................ .................. x
1 IN TR O D U C TIO N ......................................................................... .... .. ........
2 L IT E R A TU R E R E V IE W .................................................................... ....................4
3 EVIDENCE FOR HYPERSENSITIVITY REACTION IN XAC-Aw STRAIN IN
G R A PEFR U IT ........................................................................... .. 10
In tro d u ctio n ...................................... ................................................ 10
M materials and M methods ........................................................................ .................. 10
R e su lts ...........................................................................................1 2
D isc u ssio n .............................................................................................................. 1 2
4 CHANGE OF HOST RANGE BY MUTATION. ................................................ 16
In tro d u ctio n ............................................................................................................ 1 6
M materials and M methods ........................................................................ .................. 16
R e su lts ...........................................................................................1 7
D isc u ssio n .............................................................................................................. 1 8
5 CHANGE OF HOST RANGE BY CONJUGATION....................................23
Intro du action ...................................... ................................................ 2 3
M materials and M methods ........................................................................ ..................23
R e su lts ...........................................................................................2 6
D isc u ssio n .............................................................................................................. 2 7
6 CLONING AN A VR GENE FROM THE XAC-Aw STRAIN...............................33
M materials and M methods ........................................................................ ..................33
R e su lts ...........................................................................................3 7
D isc u ssio n .............................................................................................................. 3 9
7 D IS C U S S IO N .............. ..... ............ ................. ............................................ 5 0
LIST OF REFERENCES .......................... ..................54
B IO G R A PH IC A L SK E T C H ........................................................................................ 61
LIST OF TABLES
4-1 Effect in vitro of NTG mutagenesis on development of white colonies and
streptomycin resistant colonies of Xac-A" strain................. ................ ............20
4-2 Total area of inoculated Key lime and grapefruit leaves in all experiments...........21
4-3 Number of lesions per cm2 that were produced in Key lime and grapefruit leaves
inoculated with 103 cfu/m l of Xac-A" ................................. .. ....... ...............21
5-1 List of bacterial strain used in chapter 5 ...................................... ............... 31
5-2 Frequency of transfer of copper resistance genes among Xanthomonas
axonopodis pv. citri (Xac-A) after conjugation in a liquid medium........................31
5-3 Frequency of transfer of copper resistance genes among strains of Xanthomonas
axonopodis pv. citri (Xac-A) after conjugation in a liquid medium........................32
5-4 Frequency of transfer of copper resistant genes from Xanthomonas campestris
pv. vesicatoria (Xcv) to copper sensitive strains of Xanthomonas axonopodis pv.
citri by conjugation on a solid medium ...................................... ...............32
6-1 List of bacterial strains used in chapter 6 ..... ......... ........................................ 49
LIST OF FIGURES
3-1 Electrolyte leakage in grapefruit leaves inoculated with Xac-Aw and Xac-A
strains at a concentration of 5x108 cells per milliliter..............................................14
3-2 Internal bacterial populations in grapefruit leaves inoculated with Xac-Aw and
Xac-A strains at various times after inoculation. ............................................ 14
3-3 Symptoms of Xac-Aw (left side of leaf) and Xac-A (right side of leaf) in old
(left) and young leaves (right) of grapefruit ........................................................15
4-1 Symptoms of Xanthomonas axonopodis pv. citri strain Xac-Aw in grapefruit
leaf after infiltration with 103 cfu/ml bacterial concentration ...............................22
4-2 Symptoms ofXanthmonas axonopodis pv. citri strain Xac-Aw in Key lime leaf
after infiltration with 103 cfu/ml bacterial concentration.........................................22
5-1 Agarose gel stained with ethidium bromide. Lanes contain plasmid extractions
from different strains .............................................................. .. ........ ........ 28
5-2 Agarose gel stained with ethidium bromide. Lanes contain plasmid extractions
from different strains:1= Xac-A (16, Cur), 2= Xac-A (26, Cur), 3= Xac-A
( 1 8 7 4 C u s)...................................................................... 2 9
5-3 Agarose gel stained with ethidium bromide. Lanes contained plasmid extractions
from different strains: 1= Xcv 75-3 (Cur) 2= Xac-A 20 (1874, GFP+, Cus), 3=
X cv 75-x X ac-A 20) ................................................................... ........ ......30
6-1 Symptoms caused by Xac-A (left) and Xac-Aw (right) strains in tobacco, tomato,
and pepper leaves. ........................ .... .................. .. .... ..... .. ............42
6-2 Symptoms in grapefruit leaves after infiltration with the bacterial suspensions.....43
6 -3 Symptoms in Key lime leaves after infiltration with different bacterial
suspensions..................................................... .......................... 43
6-4 Symptoms in grapefruit following inoculation with Xac-A 40 strain (A), Xac-Aw
12879 strain (A") and X ac-Aw Q (3)..................................... ....................... 44
6-5 Symptoms in grapefruit after inoculation by needle pricks with Xac-Aw strain
12879 (right top), Xac-Aw Q (bottom left), and Xac-A 40 (bottom right) ..............44
6-6 Bacterial populations in grapefruit leaves infiltrated with Xac-A 40; Xac-A" 0;
Xac-Aw 12879;Xac-A 40 (pU799-3), Xac-A" 0 (pU799-3)...............................45
6-7 Electrolyte leakage in grapefruit leaves infiltrated with of Xac-A 40, Xac- A"O,
Xac-Aw 12879, Xac-A 40 (pU799-3), Xac-Aw" (pU799-3) ...................................45
6-8 Hybridization of the subclone pU799-3 fragment in avrGfl to total genomic
DNA of Xanthomonas strains digested with HindI ...........................................46
6-9 Nucleotide sequence of the 2.3 kb fragment of DNA from Xac-AW containing
avrGfl nucleotide sequencing (bold letters) ................................. ................47
6-10 Alignment between the complete sequence of XCC3600 from Xanthomonas
campestris pv. campestris and the avrGfl .................................... ............... 48
Abstract of Dissertation Presented to the Graduate School
of the University of Florida in Partial Fulfillment of the
Requirements for the Degree of Doctor of Philosophy
GENETIC DETERMINANTS OF HOST RANGE SPECIFICITY OF THE
WELLINGTON STRAIN OF Xanthomonas axonopodis pv. citri
Myrian Asucena Rybak
Chair: Jeffrey B. Jones
Major Department: Plant Pathology
A new strain of the citrus canker bacterium (Xanthomonas axonopodis pv. citri)
was detected in Florida. It was significant in that it was primarily pathogenic on Key lime
trees, but not on grapefruit trees. This strain has been designated as Xac-A". This strain
has caused concern among regulators with regard to how to treat this bacterium in the
eradication program. Of major concern was the stability of the bacterium in regard to host
specificity of the strain; for example, could the strain mutate to attack grapefruit and
other citrus plants. We investigated the frequency of the development of mutants, the
transfer of genes by conjugation, and the presence of an avirulence gene to grapefruit in
the genome. We never were able to find a mutant, either natural or induced, that changed
host range. In conjugation studies, transfer of marker genes on the chromosome to and
from the Xac-Aw strain by conjugation was not successful. In an attempt to locate
possible avirulence genes, a genetic library of Xac-Aw DNA was made using the pLAFR
vector and transformed into Escherichia coli. This library was transferred to strain 91-
118 of X perforans, which is pathogenic to tomato, but causes a null reaction in
grapefruit leaves. The transconjugants were screened for HR in grapefruit leaves. The
inoculated leaves were observed for development of an HR in the infiltrated area. In the
screening of the Xac-Aw genomic library we found an avirulence gene in the genome of
Xac-Aw that interacts with grapefruit leaves to cause an HR. This is the first report of an
avirulence gene in the genome of a citrus canker bacterium that interacts with a citrus
species. This gene was designated as avrGfl, was sequenced, and then was characterized.
The possibility of a second avr gene exists in the Xac-Aw strain, because the Xac-A"w
lacking a functional avrGfl did not cause exactly the same symptoms and develop the
same population as the wild-type Xac-A strain when it was inoculated into grapefruit
Citrus production in Florida contributes greatly to the economy of the state.
Production for the 2003-04 seasons totaled 16.4 million tons from 679,000 acres of trees.
Florida accounted for 79 percent of the total U.S. citrus production, followed by
California with 18 percent; and Texas and Arizona which accounted for the remaining 3
percent. The value of the 2003-04 U.S. citrus crop was up 4 percent from the previous
season to $2.35 billion (packinghouse-door equivalent) as published by the USDA,
Statistical Service (2004).
Citrus plants are susceptible to diseases caused by bacteria. One of these diseases,
citrus canker, is caused by two pathovars of Xanthomonas axonopodis. Pathovars of this
bacterium have been distinguished based on phenotypic reactions on different hosts, i.e.,
Xanthomonas axonopodis pv. citri (Xac) and Xanthomonas axonopodis pv. aurantifoii
(Xaa). The most destructive bacterium is Xanthomonas axonopodis pv. citri which causes
Asiatic citrus canker (Xac-A).
Asiatic citrus canker was discovered in North America in 1912 and was distributed
throughout the Gulf states (Loucks, 1934; Dopson, 1964). Eradication of the disease
began in 1915 and the disease was eradicated in 1947. This disease was rediscovered
again in 1986 in Manatee County, Florida, and declared eradicated in 1994 (Schubert et
al., 1996). A new focus of this disease appeared in Miami in 1995 (Gottwald et al.,
1997a; Schubert et al., 2001), and a current eradication program exists. This disease was
also successfully eradicated from the United States, South Africa, Australia, and New
Zealand, but a new focus of citrus canker appeared in Australia in 2004 (Hibberd, A.,
personal communication). An eradication program currently exists in Brazil. The disease
is endemic to Argentina, Paraguay, and Uruguay. It is also endemic to all countries in
The symptoms of the disease are erumpent lesions on fruit, foliage, and young
stems of susceptible citrus cultivars (Schubert et al., 2001 and Gottwald et al., 2002a).
The occurrence of citrus canker lesions on the fruit rind decreases the commercial quality
and infected fruit is not accepted by the most important markets. The warm spring and
summer conditions of Florida, together with rains with strong winds that occasionally
occur, offer ideal conditions for the spread of Xac. Optimum temperatures for infection
range between 20 and 300C (Koizumi, 1985).
The introduction of the leafminer, Phyllocnistis citrella Stainton, in Florida in
1993, has increased citrus canker severity due to wounds caused by the insect that expose
leaf mesophyll tissues to splashed inoculum, thus increasing the probability of ingress by
Xac (Gottwald et al., 1997a). The leafminer adults, however, are not efficient vectors of
the pathogen (Belasque et al., 2005).
Changes have recently occurred in the eradication protocols performed by the
Division of Plant Industry (Gottwald et al., 2002b). The increased disease severity
associated with the leafminer has resulted in increased areas of tree disposal around a
focus of infection (Schubert et al., 2001 and Gottwald et al., 2002a). In addition, tornados
and hurricanes during the summer months have caused dissemination of Xac-A much
greater distances than previously thought possible. Legal questions about eradications, in
general, have hindered eradication (Gottwald et al., 2002b). Citrus canker has spread
rapidly in Florida despite changes in eradication protocols (McEver, 2005).
Schubert et al. (2001) described at least two separate introductions of Xac-A in
Florida since 1985. In one of the introductions a strain of Xac that is pathogenic to Key
lime and alemow plants, but not to grapefruit and orange was found. This strain (Xac-A")
was characterized by Sun et al. (2004) and found to be related genetically to Xac-A rather
than Xaa-B or Xaa-C, which have more similar host ranges. Regulations and eradication
protocols adopted for this strain were different from those adopted for Asiatic citrus
canker. For the Xac-Aw only Key lime and alemow plants were destroyed. Because of the
relationship of Xac-Aw to Xac-A, questions as to whether Xac-Aw could revert to an Xac-
A strain exist. If this occurred, a new focus of Xac-A could occur with the eradication
program that was adopted.
The purpose of this work was to investigate the stability of the Xac-Aw strain in
terms of host specificity. The Xac-Aw strain causes a hypersensitive reaction in
grapefruit. Such a reaction is usually the result of an avirulence gene interacting with a
resistance gene in the host (Minsavage et al., 1990b). If resistance in grapefruit to the
Xac-Aw strain is the result of the presence of a single gene in the bacterium, then the
stability of the host specificity of the Xac-Aw may be low. The change to virulence in
grapefruit of the Xac-Aw strain was studied by mutagenesis of the strain, by conjugations
of strains, and by isolation of avirulence genes in the genome of Xac-Aw.
Many reviews have been published on the citrus canker disease (Locks, 1934;
Garnsey et al., 1979; Schoulties et al., 1987; Graham and Gottwald, 1991; Stall and
Civerolo, 1991; Schubert et al., 2001 Gottwald et al., 2002a; Graham et al., 2004). An
excellent early review is an unpublished manuscript that was written by K. W. Loucks in
1934. The review is located in the library of the Division of Plant Industry, Florida
Department of Agriculture, Gainesville. It is a review of research on regulation and
eradication of this disease from the appearance of citrus canker disease in Florida until
eradication of citrus canker was declared in 1933. This manuscript contains a history and
geographical distribution of citrus canker in the world, United States, and Florida. Hosts
reported to be susceptible and the economic importance of citrus canker were included. A
description of citrus canker symptoms of leaves, twigs, thorns, branches and fruits is
given. The general appearance on an infected tree with citrus canker was described. The
morphology, physiology, cultural characteristics and taxonomy of the causal organism at
that time were described in this review. The viability and longevity of the pathogen were
described; including describing the ability of the bacterium to enter the host tissue and
remain quiescent until higher temperatures occurred. The means of dissemination,
methods of infection, and the incubation period involved with the disease cycle were
reviewed. The histology of the disease was described. Humidity and temperature
relationships for disease development, as well the seasonal development of the host and
disease, were included. Progress on the eradication and control of citrus canker and the
epidemiological situation at the time that this review was written were discussed. The last
issue that Louck considered in his review was the future danger of the disease to citrus in
An analytical bibliography of research publications relating to citrus canker was
annotated by Rossetti et al. (1981). This huge effort contains abstracts of scientific
papers, reviews, extension reports and notes from 1912 though 1981. There are 1246
abstracts included from all over the world.
Before the reintroduction of citrus canker in Florida in 1985 there were concerns
about the reintroduction of citrus canker after it was declared eradicated in 1933 from
Florida and in 1947 from the USA (Stall and Seymour, 1983). Those concerns were
based on importation of citrus fruits into the United States from Japan, where citrus
canker is endemic; an epidemic of canker occurring in South America, and the
interception of citrus canker at ports of entry. This article described the causal agent, the
disease, the history of citrus canker, information about losses due to this disease,
measures of control and finally the outlook about the real risks of the reintroduction of
citrus canker in the USA.
A bacterial disease of citrus was observed in Florida in 1984. This disease was
referred to as the nursery form of citrus canker, or sometimes as canker E (Schoulties et
al., 1987). In this review they described the nursery form of the disease, diagnosis,
symptoms, distribution, host range, eradication and regulatory program. The review also
discussed the introduction of the Asiatic form of the disease in Florida in 1985 and the
eradication policies regarding the reintroduction of Asiatic citrus canker. The article also
mentioned the costs of the eradications and regulatory programs which were $25 million
from September 1984 to July 1986.
Stall and Civerolo (1991) described Asiatic canker and ongoing research with both
types of diseases of citrus in Florida. They reviewed the history of the disease in Florida,
and the efforts on eradication, the different forms of citrus canker, and the localization of
citrus canker in the USA. Research related to regulation, and eradication problems
included the comparisons between the citrus canker and the bacterial spot disease.
Isozyme analyses, serological studies, and DNA analyses of different strains involved in
the two diseases were included in the comparison of the strains.
Two recent reviews pertain mostly to the introduction of Asiatic citrus canker in
Florida in 1995 (Schubert et al., 2001 and Gottwald et al., 2002a). These reviews provide
a history of the disease in Florida and describe a citrus canker disease cycle. Both reviews
describe the host range of the citrus canker disease, although Gottwald et al. (2002a)
provide a more expansive list. Schubert et al. (2001) list three separate introductions of
the Asiatic citrus canker from 1910 until now and describe a fourth introduction in 2000.
The pathogen associated with the 2000 introduction has a limited host range and was
designated as the Wellington strain. Both reviews treated the problems of regulation and
eradication of citrus canker in Florida. Gottwald et al. (2002b) discuss the epidemiology
of the disease in the urban setting which was not considered previously. Both reviews
discuss the role of the Asian citrus leafminer (Phyllocnistis citrella Stainton) damage in
the epidemiology of the disease.
From the above reviews it is apparent that different xanthomonads are pathogenic
to various citrus species. Only those that cause a lesion that is erumpent or pustule-like in
the early stages of symptoms on leaves are now included in the citrus canker disease
(Graham and Gottwald, 1990). These lesions become crateriform as they increase in size.
The bacteria that do not cause such lesions are not presently classed as causing citrus
canker, and this includes the xanthomonad that causes the citrus bacterial spot disease.
The nomenclature of the bacterial strains that cause citrus canker has evolved.
Gabriel et al. (1989) proposed that Xanthomonas citri be restored for the name of the
Xac-A group. They also proposed that the Xaa-B and Xaa-C strains be named
Xanthomonas campestris pv. aurantifolii. Their primary evidence for this nomenclature
was based on restriction fragment length polymorphism (RFLP) using DNA probes
hybridizing to total DNA of the strains. The change in nomenclature based on this
technique was criticized by Young et al. (1991) and Vauterin et al. (1990). Vauterin et al.
(1991) studied the xanthomonads that cause the citrus canker disease using DNA-DNA
hybridization and other techniques and concluded that they should be divided into two
Vauterin et al. (1995) revised the nomenclature of the genus Xanthomonas and
placed the bacteria that cause citrus canker into X. axonopodis that also included many
other plant disease-causing xanthomonads. The A group was given pathovar status and
named X axonopodis pv. citri. The B and C strains were placed into the pathovar
aurantifolii as was proposed by Gabriel et al. (1989) previously. This nomenclature
seems to be the most accepted at present for the bacteria that cause the citrus canker
Two groups of strains that are placed in X axonopodis pv. citri cause citrus canker
can be distinguished, based on DNA and other genetic aspects (Vemiere, 1998). The first
group is the most important group and consists of X. axonopodis pv. citri strain A (Xac-
A) that causes Asiatic citrus canker. Several subgroups have been identified. One
subgroup is referred to as A* because strains are genetically similar to the Xac-A strains
(Hartung and Civerolo, 1989; Cubero and Graham, 2004), but have different host
specificities and a few physiological differences from typical Xac-A strains (Sun et al.
2004). This subgroup of strains occurs in southeastern Asia, Iran and possibly India.
Another subgroup of strains was recently found in Florida and is referred to as Xac-Aw
strains (Sun et al., 2004). These strains are pathogenic on Key lime and alemow plants,
but not on grapefruit or orange plants. The Xac-Aw strains are also closely related
genetically to the Xac-A and Xac-A* strains, but have some physiological differences
from the latter strains.
Another group consists of strains that cause citrus canker and are significantly
different genetically from the Xac-A strains.The members within this group are closely
related genetically (Vauterin et al; in 1991). The strains that cause canker B and C, X
axonopodis pv. aurantifolii, are in this group. This group can be subdivided based on
pathogenicity and a few physiological tests. Both subgroups are not pathogenic on
grapefruit and oranges, but are pathogenic on Key lime. The B subgroup (Xaa-B) of
strains is pathogenic on lemon trees also. After the introduction of Xac-A in Argentina in
1973 the B subgroup was not isolated from the field any more and is only present in
pathogen collections (Nelly Canteros, personal communication). The Xaa-C strain was
isolated in Brazil, but does not occur in nature at this time.
Some work has been done regarding the host specificities of the strains of bacteria
that cause citrus canker. In Loucks review it was pointed out that some species of citrus
are more susceptible than others. Levels of resistance, or susceptibility, of the citrus
species and cultivars were recorded during evaluations in the field. The extreme
susceptibility of grapefruit to the Xac-A strains was attributed to the larger stomatal pores
compared to the mandarins which are more resistant and have smaller stomatal pores (Mc
Lean, 1921). This may be a factor, but Stall et al. (1982a) demonstrated that many more
lesions developed in grapefruit leaves also after infiltration with a low level of inoculum.
The infiltration technique of inoculation was used to evaluate a few cultivars of orange
and grapefruit plants in the field. There was good correlation of disease and susceptibility
in the field results. Viloria et al. (2004) used this technique to evaluate many citrus
genotypes under greenhouse conditions. There seemed to be a continuum of resistance or
susceptibility levels in the genotypes tested.
Hypersensitivity in citrus to Xac-A strains has not been reported. However, a
hypersensitivity reaction was suggested as occurring after inoculations of grapefruit
leaves with a strain of the canker C (Xaa-C) from Brazil (Stall et al., 1982b). This was
suggested because a rapid necrosis occurred in young grapefruit leaves inoculated with
high concentrations of cells (108 cfu/ ml). The rapid necrosis was compared to relatively
slow necrosis in grapefruit caused by the Xac-A strain and a strain of canker B (Xaa-B).
Further documentation of the hypersensitive reaction in grapefruit caused by the C strain
has not been reported.
EVIDENCE FOR HYPERSENSITIV REACTION IN XAC-Aw STRAIN IN
Xanthomonas axonopodis pv. citri strain AW (Xac-Aw) was compared with Xac-A
(strain that causes Asiatic citrus canker) in Duncan grapefruit and Key lime (Sun et al.,
2004). Even though rapid necrosis occurs when high concentrations of the Xac-Aw
bacterium are infiltrated into grapefruit leaves, data for the development of a
hypersensitive reaction (HR) in grapefruit were not reported. In this chapter we present
experiments on electrolyte leakage and bacterial growth after inoculations with high
concentrations of Xac-A and Xac-Aw strains into grapefruit leaves to determine if a
typical HR occurs in grapefruit leaves.
Materials and Methods
Plants. Plants of grapefruit (Citrus paradi Macf), cultivar Duncan, were kept in a
quarantine greenhouse at the Division Plant Industry at 200 to 350 C and used in this
experiment. Before inoculation the plants were pruned to stimulate new growth. Leaves
on the newly developed shoots were inoculated ca. 10 days after new shoots began
growth. The leaves chosen for inoculations were fully expanded, but soft to the touch,
and not as fully green as mature leaves. By using this procedure all the inoculated leaves
were in a similar developmental stage.
Bacterial strains and preparation of inoculum. Two Xac strains were used.
Suspensions of strains Xac-Aw 12879 and Xac-A 40 (Corrientes, Argentina) were each
transferred to nutrient agar medium (NA) from culture stored at -800C and isolated
colonies were obtained. Several colonies of each strain were transferred to nutrient broth.
After the cultures were shaken overnight, the bacterial suspensions were centrifuged and
the cells were resuspended in sterile tap-water, and standardized to an absorbance of 0.3
at a light wavelength of 600 nm in a Spectronic 20 spectrophotomer. This optical density
corresponds to a bacterial concentration of 5x108 cfu /ml.
Electrolyte leakage. Leaves of grapefruit were inoculated with 5x108 cfu/ml of
Xac-Aw or Xac-A strains (15 leaves each). The inoculations were made by infiltrating
leaves with 10 ml syringe and 27 g nettle (Klement, 1963). After 2 h and 2, 4, 6, and 8
days electrolyte leakage was measured from three leaves infiltrated with each strain.
Electrolyte leakage was determined as an increase in electrical conductivity over a 2 h
period of 3 ml of de-ionized water containing six 0.5 cm2 leaf disks of each inoculated
area (Cook and Stall, 1968; Hibberd et al., 1987). The mean of the three determinations
was used as the conductivity at each time, but each determination was used to determine
the experimental error.
Bacterial populations. The populations of the Xac-Aw and Xac-A strains in
grapefruit leaves were determined in the same leaves and the same times as electrolyte
leakage determinations. From each inoculated leaf 0.5 cm2 of leaf area was taken and
triturated in one ml of sterile tap-water, and after appropriate ten-fold dilutions, 50 il
were plated on NA medium. The colonies were counted 3 days later (Cook and Stall,
1968; Hibberd et al., 1987). Three replicates were included at each time period. The
experiment with population and electrolyte leakage was repeated three times.
The appearance of rapid necrosis in grapefruit leaves occurred only on young
leaves of grapefruit plants (Figure 3-3). Therefore, the inoculation of leaves in a young
stage of development is essential for consistent results. The inoculation of new growth
that resulted from pruning of shoots of grapefruit plants gave consistent reactions.
Electrolyte leakage of grapefruit leaves. The conductivity of water containing
leaf disks inoculated with the Xac-Aw strain was very high at day four compared to
conductivity of water containing leaf disks inoculated with the Xac-A strain (Figure 3-1).
Conductivity of water containing leaf disks inoculated with the Xac-A strain was highest
at day eight. Thus, the Xac-Aw strain caused more rapid electrolyte leakage from
grapefruit leaves than the Xac-A strain.
Growth of Xac-Aw and Xac-A strains. The populations of the Xac-A strain were
significantly higher than the Xac-Aw strain in grapefruit leaves at day four and day six.
The populations reached 109 cfu/cm2 of leaf tissue with the Xac-A strain, but the
populations of the Xac-Aw strain were slightly lower and reached only 108 cfu/cm2
The populations obtained in leaves inoculated with Xac-Aw were very similar to the
results obtained by Sun et al. (2004). Growth was slower in leaves inoculated with the
Xac-Aw strain than growth in leaves inoculated with Xac-A strain. In both studies the
Xac-Aw populations were nearly ten-fold less than the Xac-A strain. Comparisons of
growth after infiltration in Key lime leaves were not determined in this study, because we
were only interested in determination of HR in grapefruit leaves of the Xac-Aw strain.
However, Sun et al. (2004) found no differences in growth of Xac-A and Xac-Aw in
leaves of Key lime.
To determine if necrosis occurs more rapidly in leaves inoculated with strain Xac-
A" compared with Xac-A, electrolyte leakage was monitored in grapefruit leaves after
infiltration with these strains. Leakage of electrolytes from citrus leaf tissue infected by
Xac-A was determined previously (Goto, et al., 1979). In this work necrosis caused by
Xac-Aw occurred more rapidly compared with Xac-A strain (Figure 3-1). Electrolyte
leakage reflects the extent of necrosis in leaves. A rapid necrosis in plant tissue and lower
populations in leaves compared to a susceptible reaction are indications of a HR.
Therefore, it is reasonable to assume that Xac-Aw causes an HR in grapefruit leaves based
on this work. HR is the result of an avirulence (avr) gene interacting with a resistance
gene (Flor, 1955; Ellingboe, 1976; Klement, 1982; Staskawicz et al. 1984; Crute, 1985;
Gabriel et al. 1986). In future work we will determine if an avr gene is involved in this
0 2 4 6 8
Figure 3-1. Electrolyte leakage in grapefruit leaves inoculated with Xac-Aw and Xac-A
strains at a concentration of 5x108 cells per milliliter at various times after
inoculation. The leaves inoculated with Xac-Aw abscised 6 days after
inoculation. Vertical bars represent standard error.
Figure 3-2. Internal bacterial populations in grapefruit leaves inoculated with Xac-Aw and
Xac-A strains at various times after inoculation. The leaves inoculated with
Xac-Aw abscised 6 days after inoculation. Vertical bars represent standard
Figure 3-3: Symptoms of Xac-Aw (left side of leaf) and Xac-A (right side of leaf) in old
(left) and young leaves (right) of grapefruit on the abaxial face of the leaves
(Four days after inoculation).
CHANGE OF HOST RANGE BY MUTATION.
Xanthomonas axonopodis pv. citri strain AW (Xac-Aw) causes a hypersensitive
response (HR) in grapefruit leaves. The HR is correlated with the gene-for-gene
relationship in many plants. In such gene-for-gene interactions an avirulence (avr) gene
in the pathogen interacts with a corresponding resistance (R) gene in the plant
(Minsavage et al., 1990b). Mutation of an avr gene often leads to a susceptible reaction.
Such mutations can occur in high frequency in some strains of Xanthomonas (Dahlbeck
and Stall, 1979). The purpose of this chapter is to determine if a mutation in a postulated
avr gene would change the host specificity of the Xac-Aw strain after treatment with a
mutagen and if so, to determine the frequency of the mutation.
Materials and Methods
Inoculum. The Xac-Aw 12879 strain was used because it causes an HR in
grapefruit leaves and a susceptible reaction in leaves of Key lime. The preparation of
inoculum was the same as described in Chapter 3.
Mutation in vitro. The Xac-Aw cells were treated with N-methyl-nitro-N-
nitrosoguanidine (NTG), which is a strong mutagen (Gerhardt et al., 1981). A stock
solution of NTG containing 1 pg/ml in deionized water was made and 100 pl of stock
NTG were added to 900 tl of a suspension of 5 x 108 cfu/ml of Xac-Aw cells in sterile
tap-water. After 1 h at 28 C the suspension was diluted serially ten-fold, and 200 tl of a
5 x 103 cfu/ml bacterial suspension were spread onto the surface of nutrient agar in Petri
plates. In previous work (unpublished) the NTG treatment killed ca. 50 % of bacterial
cells. After 3 days the colonies were counted to determine the actual concentration of
viable cells following treatment with NTG and to observe for development of mutant
white colonies from the typical yellow colonies. In addition 200 pl of a suspension
consisting of the 105 cfu/ ml were spread onto nutrient agar containing 200 tg/ml
streptomycin sulfate to check for growth of colonies resistant to streptomycin.
About 30 young grapefruit leaves were completely infiltrated with a suspension
consisting of 5 X 103 cfu/ml of mutagenized cells. At the same time ten leaves of Key
lime were infiltrated with the same inoculum as a control. The leaves used in these
experiments were at the same stage of development as described in Chapter 3. The
technique used by Klement (1963) was used to infiltrate each leaf with inoculum. Lesions
in both Key lime and grapefruit were counted and were calculated as lesions per cm2 of
inoculated leaf area (Table 4-3). The leaf area of tissue infiltrated was determined using
the dot-counting method (Marshall, 1968). The mutagenesis experiment was repeated
Mutagenesis. The numbers of Xac-Aw cells remaining after NTG treatment ranged
from 0.5 to 3.0 x 108 cfu/ml in the eleven experiments. White colonies developed in five
of the experiments in which 103 cfu/ml were spread on NA. Streptomycin-resistant
colonies formed in which 105 cfu/ml were spread on NA containing streptomycin in only
one experiment (Table 4-1). No white colonies of Xac-Aw or streptomycin resistant
colonies developed with non-treated suspensions. These results were evidence that NTG
did cause mutations in the population of Xac-Aw cells used for inoculum.
Change of host range. The Xac-A" strain caused fleck lesions in grapefruit leaves
(Figure 4-1) and typical citrus canker lesions in Key lime (Figure 4-2). Each lesion was
probably caused by a single cell of the bacterium, because of the low inoculum level
used. Over the eleven experiments an average of 10.1 canker lesions per cm2 occurred in
Key lime and 2.7 fleck lesions per cm2 occurred in grapefruit leaves. Assuming the
intercellular spaces on grapefruit and Key lime leaves were similar, only 27 % of the
Xac-A" cells in grapefruit actually caused a visible fleck lesion.
The total area of grapefruit leaves infiltrated with inoculum in the eleven
experiments was 20,827 cm2 (Table 4-2). Using the estimate of 10.0 lesions/cm2 based on
what occurred in Key lime leaves (Table-4-3), approximately 2.0 x 105 cells of Xac-A"
were infiltrated into grapefruit leaves in the eleven experiments. Typical citrus canker
lesions were never observed in grapefruit leaves receiving the mutagenized cells of Xac-
Cells of Xac-A" were treated with NTG, a powerful mutagenic agent, to increase
the frequency of mutation. The number of white colonies and streptomycin resistant
colonies after NTG treatment reflected the development of mutants in culture.
The frequency to streptomycin resistance in non-mutagenized cultures of X.
campestris pv. vesicatoria was around 1.9 x 10-9 per cell per division (Dahlbeck and
Stall, 1979). In this work, streptomycin resistant colonies occurred rarely after NTG
treatment but did occur in one test and was determined to be approximately one per 105
cells. The frequency of mutation for white colonies was higher than for streptomycin
resistance. At least seven genes are involved in the development of the yellow pigment
(Poplawsky and Chun, 1997). Mutation in any of them will result in loss of pigmentation.
Thus, one would expect a higher frequency of white colonies than streptomycin resistant
colonies after NTG treatment. As resistance to streptomycin is encoded by a single gene,
the frequency of streptomycin resistance probably corresponds better to an avr gene
mutation, if the change of the host range depends only on a single avr gene. Thus, one
would not expect a change of host range of the Xac-Aw strain unless nearly 105 cfu were
infiltrated into the leaves of grapefruit. We inoculated grapefruit leaves with 2 x 105 cells
and, thus, one would have expected a single avirulence gene to be mutated and detected
in our work. However, this did not occur. Therefore, possibly more than one gene is
involved in the incompatibility of the grapefruit leaves to the Xac-Aw strain.
Table 4-1. Effect in vitro of NTG mutagenesis on development of white colonies and
streptomycin resistant colonies of Xac-Aw strain
x 108 cfu
a These values were based on the colonies that developed on nutrient agar after dilution to
103 cfu/ml. The values are based on three replicates per experiment.
b These values are number of colonies that developed on nutrient agar after dilution to 103
cfu/ml. A suspension of 200 ul were spread on the plate. Average of three replicates per
' There values are number of colonies that developed on nutrient agar containing 200 Ltg
of streptomycin per liter. A suspension of 200 ul of 105 cfu/ml were spread on the plate.
Average of three replicates per experiment.
Table 4-2. Total area of inoculated Key lime and grapefruit leaves in all experiments
Table 4-3. Number of lesions per cm2 that were produced in Key lime and grapefruit
leaves inoculated with 103 cfu/ml of Xac-Aw
Numbers of lesion per cm
Key lime a
a Lesions were typical of citrus canker
b Lesions were sunken and very small (typical fleck lesion caused by HR)
r -"y r 1~.
F~4~_ ~ r "ll~
Figure 4-1. Symptoms ofXanthomonas axonopodis pv. citri strain Xac-AW in grapefruit
leaf after infiltration with 103 cfu/ml bacterial concentration. Fleck lesions
occur on upper half of leaf (30 days after inoculation).
Figure 4-2. Symptoms ofXanthmonas axonopodis pv. citri strain Xac-AW in Key lime
leaf after infiltration with 103 cfu/ml bacterial concentration. Typical pustule
type lesions occur on upper half of leaf (21 days after inoculation).
CHANGE OF HOST RANGE BY CONJUGATION
More than one avr gene may be involved in the hypersensitive reaction (HR) by the
Xac-Aw strain in grapefruit leaves. If that is true, the host specificity of the Xac-Aw strain
would not change by simple mutation. However, the factor for host specificity of the
Xac-Aw strain might cause the Xac-A strain to change after conjugation of the strains,
because large amounts of DNA transfer during conjugation. Conjugation is a process in
which the DNA of a donor bacterium moves to a recipient bacterium. Plasmid or
chromosomal DNA can move to recipient cells in conjugation. Conjugation requires cell
to cell contact and the presence of transfer genes in the DNA of the donor. Many
avirulence genes involved in host and cultivar specificities (Leyns et al., 1984; Hayward,
1993), and the genes for resistance to copper (Stall et al., 1986) and streptomycin are
located on plasmids (Minsavage et al., 1990a). However, some avirulence genes are
located on the chromosome (Ronald and Staskawicz, 1988; Tamaki et al., 1988; 1988;
Minsavage et al., 1990b; Jenner et al., 1991; Mansfield et al., 1994; Whalen et al., 1993;
Lorang and Keen, 1995). Horizontal transfer of chromosomal genes among strains of X
axonopodis pv. vesicatoria was confirmed by Basim et al. (1999), but the frequency of
transfer of plasmid DNA is usually higher than for chromosomal DNA.
Materials and Methods
Bacterial strains. All bacterial strains, relevant characteristics, and origin are listed
in Table 5-1.
Change of host range by conjugation. To test for conjugation among strains,
genetic markers were incorporated into both Xac-Aw and Xac-A so that each could be
used as a donor or recipient. Some strains were marked with fluorescent proteins by G.V.
Minsavage (Plant Pathology, Department of University of Florida) using the pMODTM
antibiotic cassette used in this experiment was excised from pKRP11 plasmid (KmR) and
pKRP13 (SmR SpR). A KmR clone was marked with the EYFP gene with lacZ promoter
from pEYFP and a clone that carried the SmR SpR was marked with the ECFP gene with
the lacZ promoter from pECFP. The fluorescent-marked strains were obtained by
electroporation using EZ::TN transposase enzyme and Biorad Gene Pulser (for more
details contact G.V. Minsavage).
Strains with antibiotic resistance were selected after plating 200 pl of a culture
containing 108-109 cfu/ml onto nutrient agar containing the selective antibiotic. Colonies
that developed on the selective medium were purified by single-colony selection on the
antibiotic medium. Strains of Xac-A and Xac-Aw containing the GFP in the chromosome
or non-pigmented strains obtained in Chapter 4 were sometimes used for selection of
antibiotic resistance. Strains were stored at -800 C. The antibiotics used and final
concentrations were: rifamicyn (80 ig/ml), nalidixic acid (50 ig/ml), kanamycin (50
ig/ml), streptomycin (50 ig/ml), and spectinomycin (50 ig/ml).
For conjugation on a solid medium, donor and recipient strains were grown in
nutrient broth (NB) overnight on a rotary shaker at 280 C, after which 100 il of culture
was added to 900 il of sterile tap-water and centrifuged. The pellets obtained were
resuspended in 100 ul of sterile tap-water and donor and recipient strains were mixed in
different proportions (10 or 200 tl of donor was added to 100 tl of recipient). Then 10 tl
of each mixture was plated on nutrient-yeast-glycerol agar (NYGA), (Daniels, et al.,
1984). The same suspensions sometimes were inoculated into leaves of grapefruit or Key
lime for conjugation. After 48 h each mixture was resuspended in 800 pl of sterile tap-
water and 400 tl was transferred to each of two plates of an antibiotic selective medium.
For conjugation in liquid medium, bacteria were grown during shaking at 280 C
overnight in 4 ml of NB, then donor and recipient strains were mixed, and incubated with
very gentle shaking for 5 h. Afterward 200 ul of the mixture were plated on appropriate
selective medium and incubated at 280C for 2-3 days until conjugants developed.
Sometimes colonies grew on media containing two antibiotics. To test whether the
colonies were mutants or conjugants, cells were observed for the presence of GFP using a
UV microscope was performed and PCR with the primers specific for GFP (G.V.
insavage). Sometimes they were inoculated into grapefruit leaves for strain identification.
To determine if Xac-A" and Xac-A could act as donors, or recipients in
conjugation, experiments were designed for transfer of Cu resistance genes which were
known to be transferred by conjugation (Basim et al, 1999). Two strains ofX. campestris
pv. vesicatoria (one contained Cu resistance on a plasmid, the other on a chromosome)
were mated with Xac-A" and Xac-A recipient strains and screened for transfer of copper-
resistance genes (Cur). In addition, four Cur Xac-A strains (plasmid-borne Cur) from
Argentina and a strain of X axonopodis pv. citrumelo that was copper resistant were
mated with an Xac-A copper sensitive (Cu') strain from Florida. The conjugations using
only the strains from Florida were made on solid NYGA medium and the conjugations
among strains from Argentina and Florida were made using liquid medium. Copper
resistance was determined by growth on nutrient agar supplemented with copper sulfate
(200 [tg/ml). The frequency of Cur transfer during conjugations was determined from
dilution plating of bacteria after matings.
Plasmid DNA isolation. Plasmid DNA was extracted by a modification of the
method of Kado and Liu (1981). Detection of plasmids was performed by electrophoresis
of the plasmid extraction as described previously (Stall et al., 1986). Donors and
recipients could be identified by plasmid profiles (Figure 5-1, Lanes 2 and 4).
Selected Xac-Aw strains were mated with Xac-A strains in all combinations of the
donor and recipient. In addition, the Xac-Aw strain was mated with itself and the A strain
was also mated with itself. In these tests a marker gene on a chromosome was never
transferred by conjugation. Putative transconjugants that grew on antibiotic media all
were negative for transfer of chromosomal genes.
The copper resistance genes on a plasmid in Xcv 75-3 were transferred to Xac-A in
vitro and in plant in all tests performed; however, these copper resistant genes were
transferred to the Xac-Aw strain only one time, and that occurred in plant (Figure 5-1).
No transfer to either Xac-A or Xac-Aw of the chromosomal Cu genes was observed.
Conjugal transfer of Cur genes from copper resistant Xac-A strain from Argentina
to copper sensitive Xac-A strain from Florida also occurred (Figure 5-2). The copper
resistance genes in Xac-A were found to be plasmid-bome (Figure 5-3). The frequency
of transfer of the Cur plasmid (per donor cell) was variable depending on the donor and
recipient combination (Table 5-2). The same variability of transfer frequency was
observed when the conjugation was made with the Cur and Cus strains from Argentina
(Table 5-3). It is interesting that in this combination, the Xac-A 16 Cur strain was the best
donor and had the highest frequency of transfer with the Xac-A 40 Cus strain. However,
no transfer was observed with the Xac-A 45 Cur strain used as the donor and with any
recipient strain (Table 5-3). When Xcv 75-3 Cur was used as a plasmid donor, the
frequency of transfer of Cur genes to Xac-A Cus strains was very high (Table 5-4). When
the copper resistant donor, X axonopodis pv. citrumelo strain (2a, Cur), was mated with
Xac-A 20, (Cus), the Cu plasmid was transferred from the donor to the Xac-A recipient
strain (Lane 12 of the Figure 5-3).
The marker genes developed in these experiments were probably inserted in the
chromosome. The genes for yellow pigment had been determined to be in the
chromosome (Poplawsky and Chun, 1997). The markers were developed to primarily see
if chromosomal movement occurs in Xac-A and Xac-A" by conjugation. The
experiments were designed to have two markers in the donor strain, but only one marker
was included in the recipient strain. The development of some colonies on selective
media after mating was due to mutation for the donor selective marker in the recipient.
The mutants could be detected by the lack of movement of the other chromosomal genes,
such as the GFP or pigment genes.
Apparently chromosomal movement between strains of Xac is not a significant
factor in change of host specificities in nature. However, conjugal movement of plasmids
between strains of Xac does occur. Movement of plasmids among strains of Xac can be
particularly significant for the movement of copper resistance genes between strains.
Copper is used in the field to control citrus canker and the development of resistance
among strains would hinder control. The first report of copper resistance in strains of Xac
was by Canteros et al. (2004). In the present work we found that the copper resistance
genes in strains from Argentina were plasmid borne, which had not been demonstrated
previously. Copper resistance among strains of Xac in Florida was not detected when
over 100 random strains were screened for resistance. However, copper resistance was
detected in a strain of X axonopodis pv. citrumelo from Florida (R.E. Stall, personal
communication) and the resistance was determined to be plasmid-borne and could be
transferred by conjugation to an Xac-A Cus strain. Since X axonopodis pv. citrumelo and
Xac-A both cause disease of citrus it is expected that cell to cell contact of the two
pathovars can occur in nature. If citrus canker were to become widespread in Florida and
copper is used for control, it would be expected that copper resistance strains of Xac-A
would be selected quickly in the field.
1 2 3 4 5
Figure 5-1. Agarose gel stained with ethidium bromide. Lanes contain plasmid
extractions from different strains: 1= Xcv 75-3 (Cur), 2= Xac-A (1874, GFP+,
Cus), 3= Xcv 75-3 x Xac-A conjugantt), 4= Xac-Aw (GFP+ Cus), and 5= Xcv
x Xac-Aw conjugantt). Strains in lanes 1, 3, and 5 were resistant to copper and
contained a large plasmid (arrow). The large plasmid was transferred by
conjugation to copper sensitive strains.
I I A A
Figure 5-2. Agarose gel stained with ethidium bromide. Lanes contain plasmid
extractions from different strains: 1= Xac-A (16, Cur), 2= Xac-A (26, Cur), 3=
Xac-A (1874, Cus), 4= Xac-A (16) x Xac-A (1874) conjugant, and 5= Xac-
A (26) x Xac-A (1874) conjugant. Strains in lanes 1, 2, 4 and 5 were copper
resistant and contained a large plasmid (arrow). The large plasmid from
strains 16 and 26 were transferred to 1874 by conjugation.
1 2 3 4 5 6 7 8 9 10 11 12 13 14
Figure 5-3. Agarose gel stained with ethidium bromide. Lanes contained plasmid
extractions from different strains: 1= Xcv 75-3 (Cur) 2= Xac-A 20 (1874,
GFP+, CuS), 3= Xcv 75-x Xac-A 20) conjugant, 4=Xac-Aw (GFP+ Cus),
5=Xcv x Xac-A" conjugantt, 6=Xac-A (16 Cur), 7- Xac=A (26 Cur ), 8= Xac-
A 16 (Cur) x Xac-A 20 (Cus) conjugant, 9= Xac-A 26 x Xac-A 20, conjugant,
10= Xac-E (104, Cu'), 11= Xac-E (2a, Cur),12= Xac-E (2a x Xac-A 20 ),
conjugant, 13= Xac-E (lc Cur), 14= Xac-E (9 a Cur). Strains in lanes 1, 3, 5,
6, 7, 8, 9, 11, 12, 13 and 14 were copper resistant and contained a large
Table 5-1. List of bacterial strain used in chapter 5
Xanthomonas axonopodis pv. citri
Xac-A 20 (1874
Xac-A 20 (1874
Xac-A 16 (Xc02-1443)
Xac-A 26 (Xc01-1394-1)
Xac-A 44 (Xcc03-1338-1-1)
Xac-A 45 (Xcc03-1639-1-4)
Xac-A 40 (Xcc03-1633-1)
Xac-A 40 (Xcc03-1634)
Xac-A 42 (Xcc03)
Xanthomonas axonopodis pv. citrumelo
Xanthomonas campestris pv. vesicatoria
Rif ; Specr
Xanthomonas axonopodis pv. citri (Xac), Xanthomonas campestris pv. vesicatoria (Xcv),
Fl, Florida, provided by Robert Stall from DPI Gainesville Fl. collection and Ctes,
Corrientes, provided by Nelly Canteros from INTA, Bella Vista, Corrientes, Argentina,
Kanamycin resistant (Kanr), Streptomicyn resistant (Strepr) Rifamycin resistant (Rif ),
Spectinomycin resistant (Specr ), Copper resistance (Cur); Copper sensitive (Cus).
Table 5-2. Frequency of transfer of copper resistance genes among Xanthomonas
axonopodis pv. citri (Xac-A) after conjugation in a liquid medium
Range of frequency of transfer
(per donor cell)a
3.9 x 10-6 to 1.25 x 10-
0 to 1.12 x 10-6
0 to 8.66 x 10-6
0 to 4 x 10-7
a Based on four determinations per donor x recipient combinations.
Table 5-3.Frequency of transfer of copper resistance genes among strains of
Xanthomonas axonopodis pv. citri (Xac-A) after conjugation in a liquid
Range of frequency of transfer
(per donor cell)a
0 to 2.4 x 10-4
0 to 5 x 105
0 to 1.82 x 10-9
0 to 9.09 x 10-9
Oto 1.3 x 105
a Based on four tests of conjugation.
Table 5-4. Frequency of transfer of copper resistant genes from Xanthomonas campestris
pv. vesicatoria (Xcv) to copper sensitive strains of Xanthomonas axonopodis
pv. citri by conjugation on a solid medium
Donor strain Recipient strain
Range of frequency of transfer
(per donor cell)a
0 to 2 x 10-4
Oto 1.33 x 10-6
Oto 1.33 x 10-6
a Based on four tests of conjugation.
CLONING ANA VR GENE FROM THE XAC-Aw STRAIN
Previously we found no evidence for a genetic basis for the host specificity of Xac-
A" strains compared to the Xac-A strains. In many cases, however, hypersensitivity of a
strain in a particular host is the basis for and is the result of the interaction of an avr gene
in the pathogen and a resistance gene in a host. The purpose of this chapter is to provide
results of screening a DNA library of Xac-Aw for the presence of avr genes in Xac-Aw
that cause a hypersensitivity in grapefruit leaves.
Materials and Methods
Bacterial strains and media. Strains used in this study are listed in Table 6-1. All
strains were maintained at -800 C and subcultured, when needed, on nutrient agar (NA).
Rifamycin-resistant strains were obtained by plating 500 .il of Xac-Aw strain 12879 at
109 colony-forming units (cfu) /ml onto nutrient agar supplemented with 80 tg/ml of
rifamycin. Escherichia coli (Ec) strains were cultured on Luria-Bertani medium
(Maniatis et. al., 1982). Conjugations were performed on nutrient-yeast-glycerol agar
(NYGA, Daniels et. al., 1984). The antibiotics and concentration used in media were:
rifamycin, 75 [tg/ml; tetracycline, 12.5 [tg/ml; and kanamycin 5 [tg/ml.
Growth of plants and inoculum preparation. Growth of plants and inoculum
preparation were the same as described in Chapter 3.
Electrolyte leakage determinations. Electrolyte-leakage determinations were
made in the same way as described in Chapter 3.
Bacterial growth curves. Bacterial growth curves were obtained in the same way
as described in Chapter 3.
Recombinant DNA techniques. Techniques used for cosmid cloning, enzyme
digestion, ligation, Southern transfer, plasmid alkaline lysis, and agarose gel
electrophoresis were described by Maniatis et al. (1982).
A genomic library of Xac-A" strain 12879 was constructed in the vector pLAFR3
as previously described (Metz et al., 2005) and supplied by G. V. Minsavage (Plant
Pathology Dept., University of Florida). Individual clones were maintained in Ec DH5a
(BRL) and maintained on Luria-Bertani medium. The helper plasmid pRK2013 in Ec HB
101 was used in conjugations involving triparental matings.
Individual clones of an Xac-A" genomic library in Ec DH5a were conjugated into
strain 91-118 of X perforans (Jones et al., 2004). Transconjugants were infiltrated into a
1 cm2 area of leaf tissue with a syringe and needle. The recipient bacterium is pathogenic
to tomato, but causes a null reaction in grapefruit leaves. A clone that caused an HR
reaction in both grapefruit and tomato leaves was detected by rapid necrosis in the
infiltrated area of the leaf. Tomato leaves were inoculated as a control, because the Xac-
A" strain also elicits an HR in tomato leaves (Figure 6-1). Grapefruit leaves and tomato
leaves were inoculated with ca. 300 transconjugants individually. Each clone contained
approximately a 25-kb fragment of Xac-A" DNA.
Subcloning. A clone, pL799-1 with HR activity in grapefruit, but not tomato was
obtained and consisted of about a 25-kb insert of Xac-A" DNA. This clone was
subcloned to contain only DNA necessary for HR activity. In this subcloning, to identify
the exact location of the gene in pL799-1, the transposon pHoGus was used to knock out
the gene responsible for HR activity by the procedure described by Huguet and Bonas,
1997. About 160 kanamycin resistant clones, which contained pHoGus inserts were
screened for the lack of HR in grapefruit after they were transferred to X perforans.
Three clones were selected for lack of an HR and one clone (pL799-1) was selected from
the three for further work. The clone pL799-1 was restricted with each of several
enzymes to find a fragment that contained the Tn3 insert. A HindIII restriction fragment,
contained the Tn3 insert and about 3.0 kb of DNA. This fragment was ligated into
pBluescript II KS and labeled pBs3.0. A portion of the 3.0 kb insert in pBs3.0 was then
sequenced using forward and reverse primers from the cloning vector. Custom
oligonucleotide primers were designed to complete the sequencing of the intact HindIII
An open reading frame (ORF) was identified in the sequence of the intact HindIII
fragment. However, the ORF was at the end of the fragment and was not complete. When
the intact HindIII fragment was ligated into pLAFR6 (pL799-2) and conjugated into X.
perforans, no HR occurred in grapefruit leaves. Then primers were selected for
sequencing beyond the end of the HindIII fragment in the original clone to obtain the
sequence of the complete ORF. Primers were selected from the sequence to amplify by
PCR a 2.3-kb fragment containing the complete ORF, which was then ligated into
pGEMT Easy Vector (Promega, Madison, Wisconsin). The 2.3 kb insert was removed
from pGEMT Easy Vector with EcoRI enzyme and then ligated into the vector
pUFR043, and designated pU799-3. The pUFR043 cosmid was used as a vector because
DNA inserts in this vector could be conjugated into strains of Xac-A", whereas pLAFR
derivatives could not be transferred to strains of Xac-A". The pU799-3 in X perforans
caused an HR in grapefruit leaves. The open reading frame was labeled avrGfl.
Mutation of avrGfl in Xac-A". The avrGfl gene in Xac-Aw was mutated to
investigate the role of the gene in the host specificity of the bacterium. Mutation in
avrGfl in pGEMT Easy Vector occurred by inserting an Omega cassette into the gene
by the procedure described by Huguet et al., (1998). The inactive gene was exchanged
into the Xac-Aw strain by using the suicide vector pOK1. Eventually an Xac-Aw strain
with the mutated avr gene was obtained. This strain was labeled Xac-Aw Q.
DNA sequence analysis. Sequencing of pBluescript clone pBs3.0 was initiated at
the sequencing facility (University of Florida, Gainesville, Fl, USA) with the Applied
Biosystems model 373 system (Foster City, CA, U.S.A.). To complete sequencing of
both strands of DNA, custom primers were synthesized at the ICBR facility with an
Applied Biosystem model 394 DNA synthesizer. The computer program SeqAid II
version 3.81 was used to analyze nucleotide sequence data and predicted protein products
of the 2.3 kb region that contained avrGfl. A search for nucleotide and amino acid
sequence homology was conducted with the BLASTN and BLASTP 2.2.11 programs
(Altschul et al. 1997).
PCR procedure. Based on DNA sequence analysis of the avirulence gene
identified in Xac-A custom primers were designed to amplify a fragment from the
avirulence gene from DNA of xanthomonads that cause disease in citrus plants. The
primers used were forward 5-'CGCCGGTTTCTGTCCTGCACTTG-3' and reverse 5'-
GCCGCCTTTGCCATCGACCAG-3'. The final product was 199 bp. PCR reactions were
performed in a thermocycler (M.J. Research, Watertown, MA, U.S.A.).
Southern hybridization. All hybridization experiments were performed on
nitrocellulose membranes using the GENIUS nonradioactive DNA labeling and detection
kit according to the manufacturers instructions (Boeheringer Mannheim Biochemicals,
Indianapolis, IN, U.S.A). Genomic DNA extractions were made using the
GenomicPrepTM Cells and Tissue DNA Isolation Kit (Amersham Pharmacia Biotech,
Inc. 800 Centennial Avenue, PO Box 1327 Piscataway, NJ 08855, USA). Plasmid
extraction from Xac-A and Xac-Aw strains was as described in Chapter 5.
Selection of avirulence gene. Three clones were selected that caused rapid
necrosis in grapefruit leaves but not in tomato leaves, and three other clones caused rapid
necrosis in tomato leaves, but not in grapefruit. The first three clones were designated
pL799-1, pL22, and pL622. All three clones were successfully transferred from E. coli
by conjugation into a strain that causes Asiatic citrus canker (Xac-A) in grapefruit and
Key lime. Only one of the three clones, pL799-1, caused an HR in grapefruit leaves
when expressed in the Xac-A strain (Figure 6-2). The pL799-1 clone in the Xac-A strain
did not cause an HR in Key lime leaves, which is typical of the host range of the wild
Xac-Aw strain (Figure 6-3). This clone was further characterized in this work.
To obtain more information about the significance of the avirulence gene in the
host range of Xac-A", primer sequences were obtained for amplification of a portion of
avirulence genes by PCR from the sequence of the 2.3 fragment in pU799-3. DNA from
three Xac-A strains and four Xac-Aw strains was extracted and tested for the presence of
the avirulence gene by amplification of DNA with those primer sequences. Amplification
of a product of the expected size occurred with all of the Xac-Aw strains, but no
amplification of a fragment of the expected size occurred with any Xac-A strain. Using
Southern hybridization with the pU799-3 clone as a probe, the avirulence gene was
determined to be present in the Xac-Aw strains but not in the Xac-A strains. In addition,
the avirulence gene was not present in strains of the B and C groups of X. axonopodis pv.
aurantifolii, or in strains of the Xac-A* strains of the Xac group, or in strains of X.
axonopodis pv. citrumelo (Figure 6-8). The B strain and strains of X axonopodis pv.
citrumelo did not cause rapid necrosis in grapefruit leaves but the C strain did. Also, at
least some of the Xac-A* strains also caused rapid necrosis in grapefruit leaves (Data not
presented). Apparently, other avirulence genes may exist in those strains. The avirulence
gene (avrGfl) hybridized with Xanthomonas campestris pv. campestris strain 8004 but
the restriction pattern was different. Strain 8004 causes rapid necrosis in grapefruit
leaves. The avirulence gene (avrGfl) was located in the chromosome because the
plasmid DNA did not hybridize with the avrGfl probe (Figure 6-8).
Comparison of mutated Xac-Aw with the Xac-A and Xac-A". The strain Xac-
A" with the mutated avirulence gene was compared with the Xac-A and Xac-Aw strains
in inoculation tests in grapefruit leaves. Visually, the symptoms caused by the mutant
strain of Xac-Aw were more like the wild-type Xac-A strain than the wild-type Xac-Aw
strain (Figure 6-4 and 6-5). However, the symptoms of the mutated strain were not
identical to Xac-A strain. Nevertheless, inactivation of the avirulence gene in Xac-Aw did
alter the disease reaction in grapefruit leaves.
Growth of the strains in grapefruit leaves was compared. In addition, electrolyte
leakage from the leaves inoculated with the strains was determined to differentiate the
time to necrosis after inoculations. In these experiments cultures, of Xac-A 40, Xac-Aw
12879, Xac-A 40 (pU799-3), Xac-AwQ, and Xac-Awf (pU799-3) were compared.
Illustrations of the results are included in Figures 6-6 and 6-7. Growth of all strains was
about equal for the first four days after inoculation. However, at day six the populations
of the strains were different. At day ten the population of the Xac-A strain was the
greatest. The population of the Xac-Aw strain was about 1.5 log units lower than the Xac-
A strain. The Xac-AwQ strain reached a population intermediate between the Xac-A and
the Xac-A". The strains with the pU799-3 clone had the lowest populations.
In electrolyte leakage determinations the Xac-Aw strain and the Xac-A strains with
the clone pU799-3 began to increase at day four compared to that caused by Xac-A and
Xac-AwQ strains. At day eight the strains with the avirulence gene had significantly
greater electrolyte leakage than the strains that did not contain the avirulence gene. This
data confirmed the visual observations of necrosis caused by those strains.
DNA sequencing analysis. Sequence analysis of the 2.3 kb fragment in pU799-3
was determined. A 1599 bp nucleotide open reading frame (ORF) was found that was
sufficient for avrGfl activity. The complete sequence of the 2.3 kb, the avrGfl gene and
the primers used to amplify the avrGfl are shown in the Figure 6-9. The upstream regions
of this ORF do not contain a hrp box but do contain an imperfect PIP box TTCGT-N10-
TTCGC 80 bp upstream of the start codon (Huguet and Bonas, 1997). The GenBank
search identified significant homology with a gene in the genome of Xanthomonas
campestris pv. campestris (Xcc) str. 8004 (84 % identical). When an alignment was done
between the completely sequenced Xcc3600 gene and avrGfl using the clustal W (1.82)
multiple sequence alignment 84.99 % identity was found (Figure 6-10).
A genomic library of an Xac-Aw strain was successfully produced and incorporated
into E. coli DH5a. For successful screening of the library for avirulence genes one would
normally transfer each clone into a strain that was pathogenic on the plant in question; in
this case into a strain of Xac-A. However, in previous experiments the transfer of clones
in pLAFR3 cosmid to strains of Xac-A did not occur at high frequency by triparental
matings (G. V. Minsavage, Plant Pathology Dept., University of Florida, Gainesville). To
circumvent this problem the Xac-Aw library was transferred from E. coli to strain 91-118
of X perforans by triparental conjugations and nearly 100 % of the matings were
successful. The X perforans strain contains the hrp genes (Bonas et al., 1991) necessary
for transfer of avirulence gene proteins into host cells, so it was thought that an
avirulence gene in the genome of Xac-Aw to grapefruit could be found by this procedure.
In fact, an avirulence gene in the Xac-Aw library was found. This procedure could
possibly be used to locate other avirulence genes when transfer of clones to a pathogen
occurs very infrequently during triparental matings with E. coli.
Three clones from the Xac-Aw library were obtained that produced an HR in
grapefruit leaves when expressed in X perforans. When the three clones were transferred
to the Xac-A strain only one caused an HR in grapefruit leaves. The two clones that did
not cause an HR probably have similar DNA sequences based on restriction enzyme
digestion (data not given). One of the clones, pL22, is being investigated further to
determine the reason that no HR occurred in grapefruit when expressed in the Xac-A
The clone that was expressed in Xac-A and that caused an HR in grapefruit leaves
did not cause an HR in leaves of Key lime. This is the same reaction as the Xac-Aw strain
in the two hosts. The importance of the avirulence gene in determination of the host
specificity of the Xac-Aw strain was further investigated by determining the presence of
the gene in other bacterial strains pathogenic to citrus. This gene was only found in Xac-
A" strains by PCR and Southern hybridization techniques. When the gene was mutated in
an Xac-Aw strain the symptoms caused by the mutated strain were similar to those caused
by the Xac-A strains. However, the symptoms were not quite the same. In addition the
growth of the mutated strain in grapefruit leaves was significantly greater than the wild-
type Xac-Aw strain but lower than the Xac-A strain. Therefore, the avirulence gene
avrGfl seems to be important in delimiting the pathogenic specificity of the Xac-Aw
An assumption could be made that another avr gene exists in the wild type of Xac-
Aw that prevents the symptoms of the mutated strain to be equal to as the wild-type Xac-
A strain and also prevents the populations of the two strains from being the same. There
could be another avr gene in the genomic library that was not identified because we only
screened 300 clones. More clones should be screened to search for another avr gene or
host range limiting factor.
Clones causing an HR in tomato, but not in grapefruit were also found in the
genomic library of Xac-A". Wild-type strains of Xac-A and Xac-Aw cause an HR in
tomato, but not in leaves of tobacco or pepper (Figure 6-1). Very little was done with
these clones in this work. It would be interesting to determine if those clones contain an
avirulence gene that provides the differential reactions in the three plants.
Figure 6-1. Symptoms caused by Xac-A (left) and Xac-Aw (right) strains in tobacco,
tomato, and pepper leaves.
"" i, '* !" .*
Figure 6-2. Symptoms in grapefruit leaves after infiltration with the following bacterial
suspensions: 1= Xac-A 40; 2= Xac-A 40 (pL799-1); 3= Xac-A 40 (pL22); 4=
Xac-A 40 (pL622).
Figure 6 -3. Symptoms in Key lime leaves after infiltration with different bacterial
suspensions: 1= Xac-A 40; 2= Xac-A 40 (pL799-1); 3= Xac-A 40 (pL22); 4=
Xac-A 40 (pL622).
Figure 6-4. Symptoms in grapefruit following inoculation with Xac-A 40 strain (A), Xac-
A" 12879 strain (A") and Xac-Aw Q (3).
Figure 6-5. Symptoms in grapefruit after inoculation by needle pricks with Xac-A" strain
12879 (right top), Xac-Aw Q (bottom left), and Xac-A 40 (bottom right).
2 4 6 8
Figure 6-6. Bacterial populations in grapefruit leaves infiltrated with Xac-A 40; Xac-Aw
0; Xac-Aw 12879;Xac-A 40 (pU799-3), Xac-A" 0 (pU799-3) at various times
0 2 4 6 8 10
Figure 6-7. Electrolyte leakage in grapefruit leaves infiltrated with of Xac-A 40, Xac-
A"W, Xac-Aw 12879, Xac-A 40 (pU799-3), Xac-Aw O(pU799-3) at a
concentration of 5x108 cells (cfu/ml).
1 2 3 4 5 6 .7 8 91011 12 13 14 1 2 3 4 5 6 7 8 9 1011 1213 14
... ....... .... .. ...... -. i
Figure 6-8. Hybridization of the subclone pU799-3 fragment in avrGfl to total genomic
DNA of Xanthomonas strains digested with HindIII. Lanes 1= Xac-A"; 2=
Xac-Aw" ; 3= Xac-A 40; 4= Xac-A 306; 5=Xac- A* 1574; 6=Xac-A* 1575;
7= Xaa-B; 8=Xaa-C; 9= Xac-E 1887; 10= Xcc 8004; 11= Xac-Aw 12879; 12=
plasmid Xac-A"; 13= Plasmid Xac-A 40; 14= X Marker digested with HindIII.
Ethidium bromide stained gel on the left. Southern blot of gel on the right.
GTCCCTATTCCA CCATCAGCAC CTAGTCAACAAGCTCCGATGGCTCCGAGCATGCATTCGG
Figure 6-9. Nucleotide sequence of the 2.3 kb fragment of DNA from Xac-AW containing
avrGfl nucleotide sequencing (bold letters).The primer sequences used to
amplify a fragment of avrGfl by PCR are underlined. Imperfect PIP-box is
shaded in gray.
seq 2 ATGGCTCCGAGCATGCATTCGGCGGCGTCGCCGGTTTCTGTCCTGCACT-TGAGAGATAC
**** ********** **** *
seq 1 ATC-ATGCGAACAAAAACCTCACTGCCGTTGGCCACCGTCCAGCGGCTACTGACCCCTGG
seq 2 ATCCATGCGCACCAAAGCCCAACTCCCATTGACTGCCATTCAACGGTTTCTTGCCCATGA
seq 1 CACCTCCACCGGGTTATCCACCCCCGCGTCGGCGTCAGCCACTCCCTGTGCAGAAACGAC
seq 2 TGCAGCGTCAACGCAGGCCCCCTCTGCATCGGCATCCACATCGCTCCACAAAAATGAGAC
seq 1 GGCAGGCTTACTGGGAGCGTTGCCAACTCGAAAGAACAAGCAAAAACAGCAAAGCCAGCG
seq 2 CGCAGGCTTGCTGGCAGCCTTGCCAGCGCGAAACGCCAGGCAAGGAGCGCAGAG--GAAG
seq 1 TCCACCCAATACGCAGGACGGTACACCAAAGAATGGCAGAGACCATGGCGGACAGTGGGC
seq 2 TCCGGCGAAAAAGAAGGC--GCACGCCAAA--ACAACGGGGGCCGGGGCGGACAATGGGC
seq 1 AACACGAGCTGCCAAGTACGCTCTTGGCATTGCTGGCGCAGGCTATGTGGCAGACAACTT
seq 2 ATCTCGGGCGGCCAAGTACGCCCTGGGAATCGCCGGTGCTGGCTATGTTGCAGACAATTT
seq 1 CTTTCTTTCAACGACCTCGCTCCGCGACGGCAAGGCCGGATTTAGCAGCAATGATCGGCT
seq 2 CGTTCTGTCCACGACATCGCTGGTCGATGGCAAAGGCGGCTTTACCAGTAATGATCGTTT
seq 1 TGAGAAAGCATGCGTGAAAGCGGAGAGCTATCACGCGCGGTATCACAGCGCTACCGAAGG
seq 2 GGATAAAGCATGCGCAAAGGCCGAGACGTATTACGCCCGGTACCACAGTGCCACTGAGGA
seq 2 AGAGCGGCATCCATAGCCGCCCCTTCGTACCGATCAGAACGTGCGGTTCCAACCAGTT
seq 2 TGAGCGTGCATCCCATAGCCGCCCCTTTGTACCGATCAGAACGTGCGGTTCCAACCAGTT
seq 1 CGCCACCATGAGCGACTACCGTGCGGCGACCAAGGTTCATATCGGCCACCTCTTCGACAG
seq 2 CGCCACCATGACCGACTACCGCGCGGCGACCAAGGTCCATGTCGGTCATCTTTTCGACAG
seq 1 CCAGCACGCACGGCAATCGCTGCTCACCAACCTTGCCTGCCTCAAGGGCGAGCGCATCAG
seq 2 CCAAGCCGCCGCGGAATCGCTCGTCACCAACCTCGCCTGCCTCAAGGGCGAGCGGATCAA
seq 1 GGACGAGTGCATTGCCCAGTACGCCCCTACGCATGTCCCGGCCAATCCGGACCTGAGTAG
seq 2 GCAGGAGTGCATCATCAGGTATGCGCCTGCGCAGGTGCCAGCGGATCCGGACCTAAGCAA
seq 1 AAGCCCGCTCTACGAAACCAAGAACAAGTACTCTCTGACCGGCGTACCCAATGCTCAGAC
seq 2 GAGCGAGCTGTACGACAGGAAAAACAAGTACTCGTTGGTTGGCATGCCCAACGCCCAAAC
seq 1 CGGTGCAAGCGGATATACCTCACGATCAATCACCCAGCCCTTCATCAATCGCGGCATGCA
seq 2 CGGAGCAAGTGGATATACCTCACGCTCGATCACCCAGCCCTTCATCAACCGCGGCATGGA
*** ***** ************** ** ******************** ********* *
Figure 6-10. Alignment between the complete sequence of XCC3600 from Xanthomonas
campestris pv. campestris and the avrGfl using the clustal W (1.82) multiple
sequence alignment (seq_l xc3600 and seq_2 avrGFI). The Xcc 3600 gene
and avrGfl are 84.99 % similar.
Table 6-1. List of bacterial strains used in chapter 6
Xa pv. citri
Xac-A 40 (pL799-1)
Xac-A 40 (PL799-2)
Xac-A 40 (pL22)
Xac-A 40 (pL622)
Xa pv. citri
Xa pv. citri
Xa pv. aurantifolii
Xc pv. campestris
Original strain number Origin
Xac-A 40 (Xcc03-1633-1)
Xanthomonas axonopodis pv. citri (Xac), Xanthomonas campestris pv. vesicatoria (Xcv),
Fl, Florida, provided by Robert Stall from DPI Gainesville Fl. Collection and Ctes,
Corrientes, provided by Nelly Canteros from INTA, Bella Vista, Corrientes, Argentina.
****************************** ******** ** ****** *** *****
Many pathogenically different strains of the causal agent of citrus canker
(Xanthomonas axonopodis pv. citri and X axonopodis pv. aurantifoli) have been
described. These strains sometimes differ genetically and pathologically. The variation
within the strains involved in citrus canker disease is not unusual. For example, the
xanthomonads that cause the bacterial spot disease of tomato and pepper consist of many
strains and some have significant genetic differences, resulting in different species of
Xanthomonas (Jones et al., 2004).
The Xac-Aw strain that was characterized by Sun et al. (2004) was found to be a
close relative of Xac-A even though they had pathogenic differences (Cubero and
Graham, 2002). Contrary to the xanthomonads that cause the bacterial spot disease of
pepper and tomato, there were no reports of a hypersensitive reaction (possible exception
Xac-C strain) responsible for host range differences. The characterization of the HR
caused by Xac-Aw in grapefruit leaves is new.
The Xac-Aw strains presented a problem for the eradication and regulation program
of the Division of Plant Industry (DPI) for citrus canker (Sun et al., 2004). After careful
consideration, DPI decided to destroy only Key lime and alemow plants in a 1900 ft
radius, and not other citrus plants, near the focus of disease caused by the Xac-Aw strain.
One of the considerations in establishing the program was the lack of knowledge of the
stability of the pathogenicity of the Xac-Aw strain. To obtain information on this
problem, we chose three avenues of research. We investigated the frequency of
development of mutants for host specificity of the Xac-Aw strain, the transfer of genes by
conjugation, and the search of the genome of the Xac-Aw strain for an avirulence gene.
We never were able to find a mutant of the Xac-Aw strain that was pathogenic on
grapefruit after treatment with the mutagen NTG. However, streptomycin and pigment
production mutants of the Xac-Aw strain developed in vitro. The methods used to find
mutants pathogenic to grapefruit were used previously with X campestris pv. vesicatoria
to find mutants for virulence on pepper plants with a plant resistance gene (Dahlbeck and
Stall, 1979). Mutants for change of race 2 to race 1, which occurred very frequently in X
campestris pv. vesicatoria, was due to an insertion element that inactivated an avirulence
gene (Kearney et al., 1990). In addition, inactivation of the avrBs2 gene in X campestris
pv. vesicatoria by several ways makes the pathogen susceptible to plants with the Bs2
gene for resistance. This has occurred frequently in the field (Gassmann et al., 2000).
Chromosomal genes were also transferred from donor to recipient in X campestris
pv. vesicatoria by conjugation (Basim, 1999). The hrp genes, which are involved in
pathogenicity and hypersensitivity in bacteria, were transferred in that work. The hrp
gene cluster contains ca. 25 kb of DNA. However, it is not known if the host specificity
genes in Xac-Aw are in the chromosome or in a plasmid. We did determine that a plasmid
can move by conjugation between strains of Xac and this was demonstrated by using
copper (Cu) resistant strains. It was determined that Cu resistance genes occur on a
plasmid in Xac-A from Argentina and in X axonopodis pv. citrumelo strains from
Florida. The Cu resistance genes from both pathogens were transferred to Cu sensitive
strains. Copper resistance was not found in strains of Xac-A from Florida (R.E. Stall,
personal communication). Copper resistance was associated with transfer of a large
The most important part of this work was that we found an avirulence gene,
avrGfl, in the genome of Xac-Aw that interacts with grapefruit leaves to cause an HR.
The avr gene was found to be located in the chromosome. This is the first report of an
avirulence gene in the genome of the citrus canker bacterium that functions in citrus. The
HR depends on a gene for resistance in grapefruit, based on the gene-for-gene hypothesis
(Minsavage, 1990b). The evidence for a resistance gene in the grapefruit genome will be
very difficult to obtain, because crossing between susceptible and resistant plants is
usually required. Genetic analysis of characteristics in citrus is difficult (Novelli et al.,
Identification of the Xac-Aw strain by the division of plant industry is currently
done by inoculation of citrus plants. In addition, the Xac-Aw strain can be identified by
serological means in an ELISA test (Sun et al., 2004). The primers selected in the
avirulence gene (avrGfl) can also be used to identify the Xac-Aw strain by PCR. The
avirulence gene was not found in other strains of xanthomonads pathogenic to citrus.
The regulation and eradication procedures of the Division of Plant Industry were
probably correct for the Xac-Aw strain. The factors for host specificity of the Xac-Aw
strain seem to be quite stable based on this work. Even if the single avrGfl gene is
responsible for host specificity, inactivation of the gene in the Xac-Aw genome does not
appear to be of a high frequency as occurs with avr genes in X campestris pv. vesicatoria
(Dahlbeck and Stall, 1979). One should note, however, that the frequency of inactivation
of avrGfl could change with the introduction of an insertion sequence (Kearney et al.,
The stability of the host specificity of the Xac-Aw strain could be the result of the
presence of a second avr gene in wild Xac-A". The fact that we did not obtain mutants
that were pathogenic to grapefruit when the cells of Xac-Aw were treated with the
mutagen NTG (Chapter 4) would support this. If only one gene was present, mutants
should have been found. If two genes were present in Xac-A", each of which was
important in susceptibility to grapefruit, one would not expect to find pathogenic mutants
after treatment with a mutagen. Other evidence for another avr gene in Xac-Aw was that
there were differences in populations and symptoms for the Xac-A strain with the mutant
lacking a functional avrGfl in comparison with the wild-type Xac-A.
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Myrian Rybak was born in Leandro N. Alem, Misiones, Republica Argentina, on
June 29, 1967. She is the daughter of Veronica Kuyesen and Jose Rybak.
Myrian graduated from the Nacional University of the Northeast in Corrientes,
Argentina, in 1992 with the Agricultural Engineer degree. In 1993 she began to work in
the National Institute of Agricultural Technology (INTA). In April-May of 1995 Myrian
was awarded a fellowship of Rotary International, Exchange Study Groups (IGE),
Mexico. In 1998 she was awarded a fellowship to pursue graduate studies in the
University of La Plata, La Plata, Buenos Aires, Argentina. She graduated with the
master's degree in 2000.
In 2001 she was awarded a fellowship from INTA-FULBRIGHT to pursue
graduate studies in the United States.
Myrian enrolled at the University of Florida in Fall 2001 with Dr. Jeffrey Jones as
her adviser. She is currently a candidate for the degree of Doctor in Philosophy. At the
end of her studies she will resume her position in INTA, Argentina.
Myrian was awarded the Francis Aloysius Wood Memorial Award in recognition
of outstanding graduate student research in plant pathology at the College of Agricultural
and Life Sciences, on March, 2005.
Myrian is a member of Argentinean Society of Horticulture (ASHAO),
International Society of Citriculture (ISC), Professional Council of Agricultural Engineers
(CPIA) and American Phytopathological Society (APS)