Citation
Genetic analysis of intraspecific variation in pathovars of Xanthomonas campestris

Material Information

Title:
Genetic analysis of intraspecific variation in pathovars of Xanthomonas campestris
Creator:
Lazo, Gerard Raymond, 1957-
Publication Date:
Language:
English
Physical Description:
xi, 106 leaves : ill. ; 28 cm.

Subjects

Subjects / Keywords:
Cotton ( jstor )
DNA ( jstor )
DNA probes ( jstor )
Gels ( jstor )
Host range ( jstor )
Inoculation ( jstor )
Pathogens ( jstor )
Plasmids ( jstor )
Species ( jstor )
Xanthomonas campestris ( jstor )
Dissertations, Academic -- Plant Pathology -- UF
Plant Pathology thesis Ph. D
Xanthomonas campestris ( lcsh )
Genre:
bibliography ( marcgt )
theses ( marcgt )
non-fiction ( marcgt )

Notes

Thesis:
Thesis (Ph. D.)--University of Florida, 1987.
Bibliography:
Includes bibliographical references (leaves 97-105).
Additional Physical Form:
Also available online.
General Note:
Typescript.
General Note:
Vita.
Statement of Responsibility:
by Gerard Raymond Lazo.

Record Information

Source Institution:
University of Florida
Rights Management:
All applicable rights reserved by the source institution and holding location.
Resource Identifier:
0030389561 ( ALEPH )
16929126 ( OCLC )

Downloads

This item has the following downloads:


Full Text












GENETIC ANALYSIS OF INTRASPECIFIC VARIATION IN
PATHOVARS OF Xanthanonas campestris








BY

GERARD RAYMOND LAZO




















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 1987

















































Copyright 1987

by

Gerard Raymond Lazo















ACKNOiLEDGMENTS



I wish to express my gratitude to Dr. D. W. Gabriel, chairman of

the advisory committee, for his friendship, help and encouragement during the course of this research. Thanks are also expressed to all personnel associated with the laboratory, who were very generous and cooperative in supporting the research efforts which made this presentation possible. Special thanks are extended to A. Williams who

assisted with the race-specificity studies on cotton; G. V. Minsavage who assisted with the SDS-PAGE analysis; R. Pof fey, who assisted with the RFLP analysis; and A. Burges who assisted with various other aspects of this study. Thanks are also expressed to Drs. R. E. Stall,

D. R. Pring, and M. J. Bassett for serving on the advisory cormittee and for their support. Appreciation is also acknowledged for

financial support from the Oklahoma and Florida Agricultural Experiment Stations, and United States Department of Agriculture grant USD.A-58-7B30-3-465. Anid finally, I am grateful to my loving wife, Maria, for supporting and encouraging me throughout this degree program, for she tolerated my late hours and comforted me in the most difficult of times.















TABLE OF CONTENTS

Page

ACKNOWLEDGMENTS .................................................. iii

LIST OF TABLES ................................................... vi

LIST OF FIGURES .................................................. vii

ABSTRACT ......................................................... ix

CHAPTER

ONE INTRODUCTION ........................................... 1

TWO LITERATURE REVIEW ...................................... 4

THREE HOST RANGE OF PATHOVARS OF XANTHOMONAS CAMPESTRIS ...... 9

Introduction ........................................... 9
Materials and Methods .................................. 10
Bacterial Strains and Host Plants ................. 10
Plant Inoculations ................................ 12
Physiological Differentiation ..................... 12
Polyacrylamide Gel Electrophoresis ................ 13
Results ................................................ 13
Pathogenicity Tests ............................... 13
Physiological Differentiation ..................... 20
Polyacrylamide Gel Electrophoresis ................ 22
Discussion ............................................. 22

FOUR CONSERVATION OF PLASMID DNA SEQUENCES AND PATHOVAR
IDENTIFICATION OF STRAINS OF XANTHOMONAS CAMPESTRIS .... 27

Introduction ........................................... 27
Materials and Methods .................................. 28
Bacterial Strains ................................. 28
Plasmid Extraction and Visualization .............. 31
Cloning of Plasmid
Restriction Endonuclease Fragments ................ 32
DNA/DNA Hybridization ............................. 32
Results ................................................ 33
Detection of Plasmid DNA .......................... 33
Restriction Endonuclease Profiles ................. 35
DNA/DNA Hybridization ............................. 40
Dot-blot Hybridization ............................ 42
Discussion ............................................. 45

iv









FIVE ARE RACE-SPECIFIC AVIRULENCE GENES ENCODED BY PLASMIDS
OF XANTHOMONAS CAMPESTRIS PV. MALVACEARUM?...............50

Introduction ............................................50
Materials and Methods ....................................51
Bacterial Strains and Host Plants ...................51
Plant Inoculations ..................................51
Plasmid Analysis ....................................51
Plasmid Curing ......................................53
Race-Specificity Genes ..............................53
Plasrnid origin of Replication and Mobilization ...54
Results .................................................55
Plasmids and Race-Specificity .......................55
Plasmid Curing ......................................58
Race-Specificity Genes ..............................58
Plasmid Origin of Replication and Mobilization .... 61
Discussion ..............................................65

six PATH~OVARS OF XANTHOMONAS CAMPESTRIS ARE DISTINGUISHABLE
BY RESTRICTION FRAGMENT LENGTH POLYMORPHISM ..............70

Introduction ............................................70
Materials and Methods ....................................72
Bacterial Strains ...................................72
DNA Extraction .....................................72
Agarose Gel Electrophoresis.........................75
DNA Probes ..........................................75
DNA Hybridization...................................76
Restriction Fragment Patterns and Densitornetry ...77
Results .................................................77
Agarose Gel Electrophoresis .........................77
DNA Probes .........................................80
DNA Hybridization ...................................80
Discussion ..............................................88

SEVEN SUMMARY .................................................92

Intraspecific Variation ..................................92
Plasrids .........................o.......................92
Restriction Fragment Length Polymorphism .................93
Conclusions ...........................o.......... &........95

LITERATURE CITED ..................................................97

BIOGRAPHICAL SKETCH .............................................. 106











V















LIST OF TABLES

Table Page

3-1 Strains of X. campestris used in host range investigation ... 11 3-2 Legume plant reactions to inoculation with pathovars
of X. campestris ............................................ 14

3-3 Host range observed for pathovars of X. campestris
pathogenic to legume host plants ............................ 17

3-4 Malvaceous host plant reactions to inoculation
with strains of X. campestris pv. malvacearum ............... 19

3-5 Physiological reactions of strains of X. campestris ......... 21

4-1 Strains of X. campestris used for plasmid analysis .......... 29

4-2 Detection of plasmid DNA in strains of X. campestris ........ 34 4-3 Hybridization of radiolabeled plasmid probes to
total DNA of pathovars of X. campestris ..................... 44

5-1 Strains of X. campestris pv. malvacearum from cotton
used in race-specificity investigation ...................... 52

5-2 Plasmid groupings and cotton plant reactions to strains of
X. campestris pv. malvacearum ............................... 56

5-3 Pathogenicity of X. campestris pv. malvacearum
transconjugants on cotton host differentials ................ 59

5-4 Selection of plasmid replication genes in X. campestris
pv. malvacearum strain X .................................... 62

6-1 Strains of X. campestris used for restriction fragment
length polymorphism analysis ................................ 73

6-2 Sizes of DNA fragments from X. campestris genomic digests
which hybridized to the XCT1 DNA probe. See Figure 6-3 ..... 83 6-3 Sizes of DNA fragments from X. campestris genomic digests
which hybridized to the XCT1 DNA probe. See Figure 6-5 ..... 87




vi















LIST OF FIGURES


Figure Page

3-1 SDS-Polyacrylamide gel electrophoresis of total proteins
from strains of X. campestris ............................... 23

4-1 Plasmnid DNAs from strains of X. campestris pv. malvacearum
digested with restriction endonucleases EcoRl and BamHI ..... 37

4-2 Graphic representation of plasmid EcoRI restriction fragment
profiles for pathovars of X. campestris ..................... 38

4-3 Plasmid DNAs from strains of X. campestris pv. citri
digested with restriction endonuclease EcoRI ................ 39

4-4 Plasmid DNAs from strains of X. campestris pv.
malvacearum digested with restriction endonuclease
EcoRI and hybridized to probe N4.5 .......................... 41

4-5 Plasmid DNAs from strains of X. campestris digested with
restriction endonuclease EcoRI and hybridized to probes
P2.0 and P2.3 ............................................... 43

5-1 Plasmid curing of X. campestris pv. malvacearum with
SDS treatment ............................................... 60

5-2 Transconjugants of X. campestris pv. malvacearum containing
pLXD and hybridized to the plasmid probe pSa4 ............... 64

5-3 Transformation of E. coli ED8767 with plasmid DNA
from X. campestris pv. malvacearum transconjugants
mated with pLXD ............................................. 66

5-4 Plasmid DNA of X. campestris pv. malvacearum strain X
digested with restriction endonucleasea and hybridized
to the plasmid probe pXD-1 .................................. 67

6-1 Genomic DNA of strains of X. campestris pvs. phaseoli,
alfalfae, and campestris digested with restriction
endonuclease EcoRI .......................................... 78

6-2 Genomic DNA of strains from different pathovars of
X. campestris digested with restriction endonuclease EcoRI .. 79



vii









6-3 Genomic DNA of strains of X. campestris pvs. phaseoli,
alfalfae, and campestris digested with restriction
endonuclease EcoRI and hybridized with probe XCT1 ........... 81

6-4 Genomic DNA of strains of X. campestris pvs. phaseoli,
alfalfae, and campestris digested with restriction
endonuclease EcoRI and hybridized with probe XCT11 .......... 84

6-5 Genomic DNA of strains from different pathovars of
X. camoestris digested with restriction endonuclease EcoRI
and hybridized with probe XCTI ............................ 85











































viii















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 ANALYSIS OF INTRASPECIFIC VARIATION IN
PATHOVARS OF Xanthomonas campestris

By

Gerard Raymond Lazo

May, 1987

Chairman: Dean W. Gabriel
Major Department: Plant Pathology


Xanthomonas campestris is always found in association with plants. Those strains of X. campestris which are known to be pathogenic are differentiated into over 125 pathovars (pathogenic variants), on the basis of limited pathogenicity tests. Some members of a pathovar may have a broader host range than others, since exhaustive pathogenicity testing is impractical. Other than these tests, there is no definitive means to classify an unknown X.

campestris isolate. Those strains of X. campestris which are not pathogenic are unclassifiable by the pathovar system, yet they may exhibit similar host range specificity to those which are pathogenic. This work was conducted in an effort to better understand the variability of X. campestris.

Over 115 strains of X. campestris were examined for plasmid content and restriction endonuclease profiles. Of the 26 pathovars examined, only 13 were found to contain plasmids. Restriction

ix









endonuclease digested plasmid DNA from strains within a given plasmid-containing pathovar gave surprisingly similar, but not always identical, digestion profiles by agarose gel electrophoresis. All 60 strains tested of X. campestris pvs. glycines, malvacearum, phaseoli, and vignicola could be accurately identified by pathovar from

determination of the restriction fragment profile and/or by Southern hybridizations of that profile with a selected plasmid DNA probe. In

no instance was the same plasmid profile seen in more than one pathovar. The apparent stability of the plasmids provides a natural genetic marker that can be strain specific and perhaps useful in epidemiological investigations.

Cloned DNA fragments, derived from a cosmid library of a Florida isolate of X. campestris pv. citri strain 3041, were used to detect

restriction fragment length polymorphism (RFLP) of total DNA from 87 strains of X. campestris, comprising 23 different pathovars.

Autoradiographs of Southern transfers of genomic DNA probed with cosmid-sized clones revealed hybridization profiles which appeared to be highly conserved. The hybridization patterns observed between

different pathovars suggested that some DNA fragments were conserved at the species level, others were conserved at the pathovar level, and

still others were variable. The degree of variability appeared to depend on the DNA probe used. By using more than one DNA probe, or by digesting the genomic DNAs with different restriction endonucleases, all of the strains of X. campestris described as belonging to a given pathovar could be differentiated. All strains of X. campestris were readily grouped by RFLP phenotypes, and the classification based on


x









RFLP patterns correlated well with the classification based on pathogenicity. Although certain pathovars may need to be redefined, this work supports and helps validate the natural taxonomic groupings provided by the pathovar naming system.















































xi















CHAPTER ONE
INTRODUCTION



The genus Xanthomonas Dowson consists of bacteria described as being gram-negative, obligately aerobic, non-fermentative rods which are motile by a single polar flagellum and can produce a brominated yellow pigment, called xanthomonadin (6). All reported strains of this genus have been described as plant associated, and most are reported as being pathogenic to a particular plant host. Based on

microbiological classification, this genus can be separated into at

least five separate species: X. campestris (Pammnel) Dowson, X. albilineans (Ashby) Dowson, X. ampelina Panagopoulos, X. axonopodis Starr and Garces, and X. fragariae Kennedy and King (and possibly X. populi (Ride) Ride and Ride). Over 125 variants, or pathovars, of X. campestris, are essentially indistinguishable from each other, except for their plant host range (6,89).

To maintain order in differentiating among strains of X. campestris by host range, the rank of pathovar has been assigned in

addition to the species name (24,98). The primary means by which

strains can be differentiated as pathovars is by inoculation of the plants which serve as susceptible hosts to a given pathovar. The

convention for naming pathovars of X. campestris has generally been to

name it for the host from which it was first isolated (89) A

particular pathovar designation suggests a limited host range for a

1









2
given strain. The actual host range may be much broader, but a

complete host range description of a given strain would require testing on all plants known to support growth of the genus. This

would be a monumental task, and therefore the potential host range of a given strain of X. campestris is unknown. Obviously, any

information regarding pathogenicity is of value to pathologists. However, without knowledge of the potential host range variation of strains within a pathovar, the taxonomic value of the pathovar system is questionable.

Attempts to devise alternative means to differentiate pathovars

of X. campestris have included serology (1,94), membrane protein profiles (70), phage-typing (37), rRNA-DNA and DNA-DNA hybridization (21,73), and gas chromatography of fatty acids (65,81). Although most of these methods have been useful to differentiate given sets of strains of X. campestris, there is little evidence that the groupings

distinguished by these methods have any correlation to the host range groupings observed for given pathovars of X. campestris. None of

these techniques have been successful in replacing pathogenicity tests to identify X. campestris to the pathovar level. Because differences in host range primarily determine the pathogenic variability observed

in X. campestris, it is of interest to know more about the nature of this microorganism and the mechanisms which determine host-range specificity.

Genetic analyses of pathogenicity can easily be done with bacteria, for they can be quickly cloned and assayed, and studied using current recombinant DNA technologies (50). X. campestris is









3

useful because it is a facultative parasite with a high level of natural variation and has been shown to follow similar gene-for-gene patterns of interactions with plants as found with fungal pathogens

(33).

The objective of this research was to develop a better understanding of the intraspecific variation and specificity present in the species X. campestris. This was conducted by first verifying

the pathogenicity of the working strains as pathovars and, where appropriate, as races within selected pathovars of X. campestris through pathogenicity testing. This work was followed by physical

characterization of the plasmids and chromosomes of X. campestris races and pathovars. These DNA analyses were designed to determine the extent of physical variation within the species, pathovars, and races of X. campestris. Finally, genetic variation was analyzed at the race level by using cloned genes and complementation analyses to identify specific avirulence genes which contribute to the race phenotypes. By conducting experiments in a multi-faceted approach, it was hoped that a better understanding of the interactions of strains of X. campestris with their host plant(s) could be appreciated.














CHAPTER TWO
LITERATURE REVIEW



At least 124 monocotyledonous and 268 dicotyledonous plants species are susceptible to infection by bacterial strains of the genus

Xanthomonas (57). Compiled listings indicate that the genus

Xanthomonas has a wide host range extending over nine monocotyledonous families comprising 66 genera, and 49 dicotyledonous families comprising 160 genera. Pathogenicity toward gymnosperms or lower plants has not been reported.

Although pathovars are generally assumed to have a restricted host range, some strains of X. campestris have extended host ranges to more than one plant species. In these instances, host range usually

extends toward other members of the same plant family (with few exceptions). For example, the plant families Compositae, Cruciferae,

Graminae, and Leguminosae each contain different species susceptible to the same X. campestris pathovar (X. c. pv.). An example of a broad

host range pathovar of graminaceous hosts is X. c. pv. translucens (Jon., John., and Redd.) Dye, which is pathogenic on barley, rye, and wheat. Other strains pathogenic on graminaceous hosts have been described, but their interrelationships among the other pathovars were

poorly defined (6). In another example, X. c. pv. alfalfae, is

pathogenic on the legumes alfalfa, bean, and pea. Another special relationship is that of X. c. pv. vesicatoria (Doidge) Dye, which is a

4











pathogen of Capsicurm annuum L. (pepper) and Lycopersicon esculentum (tomato). While some strains of X. campestris appear to have a host

range to more than one plant species, most others appear to have an extremely narrow host range (6,57). The host range characteristic is considered to be a stable trait, although there is one report of altered host range by artificial selection (23). This report has been

refuted, with no change in host-range specificity observed for X. campestris (84). Within at least some pathovars, variability among strains exists as race-specific plant interactions (8,17,20). These are recognized when resistant plants of an otherwise susceptible plant species are discovered. The ability to differentiate races of X. campestris pathovars is dependent on the discovery of plant hosts, called host differentials, which exhibit variability in their response to different pathogenic strains. Pathogenic races have been described for X. c. pv. malvacearum (Smith) Dye, X. c. pv. oryzae, X. c. pv. translucens, X. c. pv. vesicatoria, and possibly others. This racespecific variation in X. campestris was suggestive of gene-for-gene interactions between pathogenicity determinants in the pathogen with resistance genes in the host (8,20).

Gene-for-gene relationships can only be established by genetic crosses of the host and the parasite. Because the bacterial genetics

can only be done with recombinant DNA techniques, gene-for-gene interactions could only be recently established. Proof of

gene-for-gene interactions with bacterial pathogens was documented for X. c. pv. malvacearum (33).









6

The high level of host-range and race specificity described above

for X. campestris is observed with some other plant pathogenic bacteria. For example, there are more than forty pathovars of Pseudncmonas syringae which are primarily distinguished by host-range specificity (75,86). Some P. syringae pathovars have host ranges which are narrowly restricted to specific plant species; others have a wider range of hosts. Strains of Erwinia chrysanthemi are currently

being considered for differentiation at the pathovar level (56). Strains of Agrobacterium spp. and Rhizobium spp. are not

differentiated using the pathovar system; however, host-specific interactions analogous to those observed in X. campestris exist with these bacteria. Species within each genus can be differentiated

biochemically and by plant inoculations. These two genera, which cause very different symptoms, are both in the family Rhizobiaceae and are genetically highly related (25,42). In Rhizobium spp. and

Agrobacterium spp., host range appears to be specified by the bacterium by genes which are expressed as a positive function (32). In several species of Rhizobium, host range function has been identified as being plasmid borne (4). The introduction of one of these plasmids may extend the host range of a limited host range species of

Rhizobium.

Similarly, A. tumefaciens tumor inducing (Ti) plasmids carry host range determining genes (64). Ti plasmids from wide-host-range

strains, when moved to A. tumefaciens strains with limited hostranges, confer a wide host range to the recipient (10,43,48). The pTAR plasmid, found in A. tumefaciens strains from grapevine, carries









7

a gene for tartaric acid catabolism (35). The expression of pTAR

genes appears to function more like a host range determinant rather than an oncogenic determinant, as found on tumor inducing plasmids.

The proof of plasmid involvement in virulence and host range with species of Agrobacterium and Rhizobium apparently stimulated research of plasmid involvement among other phytopathogenic bacteria. Plasmids

have been found to exist in many other phytopathogenic bacteria, but few have been associated with pathogenicity (76). Examples of plasmid functions in pathogenicity on plants include tumor induction in A. tumefaciens (97), nodulation in Rhizobium spp. (62), indoleacetic acid production by P. syringae pv. savastanoi (90), cytokinin production by

Corynebacterium fascians (syn. Rhodococcus fascians) (72), virulence of Pseudomonas solanacearum (5) and race-specific avirulence in X. c. pv. vesicatoria (88), among others. A general conclusion from these studies is that plasmids from a wide range of plant pathogenic genera can carry a variety of genes which determine the outcome of plantpathogen interactions.

Plasmids have been shown to be physically similar among strains within pathovars of P. syringae (77). The similarity is based on

homology studies using restriction endonuclease digestion patterns and

DNA-DNA hybridization experiments (15). Additionally, there are instances where similar plasmids, or even identical ones, may be found

in closely related pathovars of P. syringae (77). Although the

functional utility of these plasmids is unknown, they often cannot be eliminated or "cured" from the bacterial strain, possibly because such

an event would be lethal. For example, in P. syringae pv.











phaseolicola, a plasmid, pMC7105, can be forced to integrate into the host chromosome, but cannot be cured (79,92). The plasmid pMC7105 has some homology with other P. syringae pv. phaseolicola strains, and

even P. svringae pv. glycinea strains. Homology between these

plasmids may be due to transpositional events, the presence of

repetitive sequences, variability due to integration and excision of the plasmid, plasmid origin of replication, or other plasmid

maintenance function (79,92). Another possibility is that genes essential for pathogenicity, host-range, or race specificity may be encoded on them. The association of plasmid homology with host-range specificity may indicate the involvement of plasmids in host range determination in these cases.














CHAPTER THREE
HOST RANGE OF PATHOVARS OF XANTHOMONAS CAMPESTRIS



Introducti on

There are over 125 pathovars of X. campestris described, and the only means to differentiate between them is by pathogenicity testing

(6). Same strains of X. campestris have extended host ranges to more than one host, usually to other members of the same plant family, with few exceptions (57). The host range can sometimes overlap onto

another host for which a different X. campestris pathovar has been described. For instance, X. c. pv. alfalfae is a pathogen of the legumes alfalfa, bean, and pea, but strains exist of X. c. pv. phaseoli and X. c. pv. pisi which have host ranges limited to bean and pea, respectively. The relatedness between these strains at the

subspecific level is unknown, except for their host ranges. In

another instance, strains of X. c. pv. malvacearum have been isolated from the malvaceous hosts, cotton and hibiscus (7,12). Although given the same pathovar designation because they were each isolated from plants of the Malvaceae family, their relatedness is unknown. Similar

situations, where the relationship between strains is unclear, exist among other pathovars of X. campestris (6). The purpose of this

investigation was to verify the pathogenicity and host range of all

stock strains used in this dissertation, to examine the relationships between apparently related strains of X. campestris in terms of host9









10

range specificity, and to find a physiological test diagnostic for one or more pathovars used in these studies.

Materials and Methods

Bacterial Strains and Host Plants

Bacteria used in this investigation consisted of pathovars of X. campestris which were pathogenic to members of either the Leguminosae or Malvaceae plant families. The pathogens of leguminous host plants

consisted of X. campestris pvs. alfalfae, cyamopsidis, glycines, phaseoli, pisi, and vignicola isolated from alfalfa, guar, soybean, kidney bean, pea, and cowpea, respectively. The pathogens of

malvaceous host plants consisted of strains of X. c. pv. malvacearum isolated from cotton or hibiscus. The strains of X. campestris used in this study are shown in Table 3-1. The legume host plants used in this investigation included Medicago sativa cv. "FL-77", Glycines max cv. "Evans", Phaseolus vulgaris cv. "California Light Red", and Vigna unguiculata cv. "California Blackeye #5". The malvaceous hosts

included three cultivars of Hibiscus rosa-sinensis and eight cultivars of Gossypium hirsutum. One of the cultivars of H. rosa-sinensis was

cv. Brilliant Red; the other two were not named, but chosen to represent diversity in the species. Eight cotton cultivars were

selected for differentiating races of X. c. pv. malvacearum, two of which were cv. 101-102B, and cv. Gregg (45). The other cultivars were cv. Acala 44 (no resistance to cotton strains), and five cultivars derived from cv. Acala 44 crossed to obtain single gene resistance to the cotton strains of X. c. pv. malvacearum, the cultivars being Acala Bl, B2, B3, B5, and BIN (7).











Table 3-1. Strains of X. campestris used in host range investigation.


Pathovar Strain Plant Host Location


alfalfa KS Medicaao sativa Kansas
FL M. sativa Florida
cyamopsidis 13D5 Cyamopsis tetragonoloba
X002,XO05,XO16,XO17 C. tetragonoloba Arizona
glySines B-9-3 Glycine max Brazil
1717 G. max Africa
17915 -. max
S-9-8 G. max Wisconsin
malvacearum DMNOUVXYZTX84 osT Tium hirsutan Texas
ABEFGH G. hirsutum Oklahoma
OhlCh2 G. hirsutum Chad
HV25 -. hirsutum Upper Volta
Su2,Su3 G. hirsutum Sudan
FL79 G. hirsutum Florida
083-4244,M84-11 R-ibiscus rosa-sinensis Florida
XlOX27,X52,XlO2,XlO8 H. rosa-sinensis Florida
phaseoli EK11,Xph25,Xpfll Thaiie-olus vulgaris Nebraska
XpaXpll P. vulqaris Wisconsin
82-1,82-2 P. vulgaris Florida
LB-2,SC-3B P. vulgaris Nebraska
XP2 P. vulgaris New York
XP-JL P. vulgaris Kansas
XP-JF T. vulgaris Missouri
XP-DPIB5B P. vulgaris
RiL XP1 Pisum sativum Japan
vignicola A81-331,C-1,CB5-1, Vigna ungiuculata Georgia
Xvl9,SN2,432,82-38 V. unguiculata Georgia









12

Plant Inoculations

Pathogenicity tests were conducted by pressure infiltrating bacterial suspensions into leaf tissue with a blunt-end syringe and incubating the plants in 28 C or 30 C growth chambers until symptoms were expressed. The leguminous host plants were incubated at 28 C and

symptoms were recorded after one week. The Inalvaceous hosts were incubated at 30 C and symptoms were recorded after a two week period. Pressure infiltration facilitated rapid screening of plant reactions, but was not representative of natural inoculation. Because alf alf a

leaves were small in size, infection was by spray inoculation. Bacterial suspensions were prepared by centrifuging overnight cultures of X. campestris, resuspending them in 0.7% NaCl to an approximate optical density (OD600nm) of 0.3 for plant inoculation. These

pathogenicity tests were repeated at least once. The results were recorded to determine compatible (pathogenic) or incompatible (nonpathogenic or hypersensitive) plant reactions to inoculations. Physiological Differentiation

As X. campestris is defined (6), the physiological characteristics for gelatin and starch hydrolysis may be variable for this species. Other physiological characteristics which are consistent

with this species includes xanthomonadin production, mucoid growth, and esculin and casein hydrolysis. These above mentioned

characteristics were determined using standard methods (36,82). Additionally, physiological tests for production of cellulase (96), lecithinase, lipase, and pectinases were included. Hildebrand's

medium prepared at three pH's (4.5, 7.0, and 8.5) was used to detect pectolytic enzyme activity. In each of these three media, sodium









13

polypectate was used as the carbon source. The pH 5 medium included only pectin as the sole carbon source. These tests were each repeated once.

Polyacrylamide Gel Electrophoresis

Bacteria were grown overnight at 30 C in a peptone-glycerol broth. Cells were adjusted to a 0.3 optical density (OD600rm), and 1.5 mls of cells were collected by centrifugation, washed once in water, and resuspended in 50 ul 10% sorbitol. Then, 50 ul

solubilization buffer (90 mM Tris (pH 6.8), 20% glycerol, 4% SDS, 10%

B-mercaptoethanol, and .002% bromophenol blue) was added to the suspension and the mixture was boiled for 5 minutes. Approximately 10 mg protein (10 ul solution) was added to a 10% resolving acrylamide gel (51), and samples were electrophoresed in a 20 cm x 17 cm x 1.5 mm vertical gel unit at 10 V/cm and 15 C until the dye reached the bottom of the gel. Biorad low-range SDS-PAGE standards were used as

molecular weight markers. Gels were stained with 0.1% Coomassie blue in 42% methanol and 17% glacial acetic acid. The gel was destained in

30% methanol and 10% glacial acetic acid. Electrophoresis was only performed once. A photograph of the gel was taken using Polaroid type .55 film.

Results

Pathogenicity Tests

All of the strains of X. c. pv. phaseoli, with one exception, gave the expected disease reactions on the legume hosts tested. The exception, strain B5B, was found to be non-pathogenic to all four

hosts tested. The remaining six strains were pathogenic on kidney bean (Table 3-2), giving watersoaked lesions on inoculated leaves









14

Table 3-2. Legume plant reactions to inoculation with pathovars of X. campestris.

Host ReactionA
Inoculation Strain bean cowpea soybean alfalfa

X.campestris pv.

phaseoli 82-1 +B C D 0E
Xpa + 0
Xpfll + 0
XP-JF + 0
EK11 + 0
XP2 + _F 0
B5BG 0

vignicola CB5-1 -H +I _J 0
Xvl9 + 0
432 + 0
A81-331 + 0
C-1 + 0
82-38 +/-K + 0
SN2 +/- + 0

glycines B-9-3 +L _C +M 0
1717 + + 0
17915 + + 0
S-9-8G- 0

alfalfae FL +L C _D +N

malvacearum N _F 0 _F 0

control 0 0 0 0


A = + is compatible, is incom- G = strain appeared nonpatible, +/- is intermediate, pathogenic.
and 0 is a null reaction. H = dry necrotic lesion with
B = compatible lesions were slight watersoaking at
watersoaked and appeared to periphery of inoculation site.
be spreading. I = dry necrotic lesion with
C = dry necrotic lesion with wine shothole effect.
red reaction. J = dry necrotic lesion.
D = dry necrotic lesion with K = as described for H, but
chlorosis. slight shothole effect present.
E = no reaction seen with spray L = watersoaked lesion.
inoculation. M = watersoaked chlorotic lesion.
F = slight tissue discoloration N = watersoaked leaf spots.
at inoculation site.









15

after four days. The lesions extended from the inoculation sites

giving the appearance that the bacterium was spreading throughout the leaf.

The strains of X. c. pv. phaseoli which were found pathogenic on kidney bean were not found to be pathogenic on cowpea, soybean, or alfalfa. In cowpea and soybean, a hypersensitive reaction was apparently elicited, resulting in dry necrotic lesions at the

inoculation sites. In soybean the lesion was slightly chlorotic, whereas for cowpea, a wine red lesion occurred. However, X. c. pv. phaseoli strain XP2 did not elicit the wine red color on cowpea (Table 3-2). No response was observed on alfalfa from the spray inoculations with X. c. pv. phaseoli.

The strains of X. c. pv. vignicola were pathogenic on cowpea, resulting in irregularly shaped lesions which had a tearing, or "shothole" appearance at the inoculation site. On soybean, the

incompatible response appeared dry and collapsed. Again, no response was observed from spray inoculation on alfalfa (Table 3-2).

On kidney bean, a range of different responses occurred from inoculations with X. c. pv. vignicola. For two of the strains, A81331 and Cl, an incompatible response appeared as a dry collapsed lesion. For the other strains of X. c. pv. vignicola, there appeared to be some slight water-soaking in the tissue about the periphery of the inoculation site. In two of these instances, for strains 8238 and SN2, the "shothole" effect as seen on cowpea was observed.

The strains of X. c. pv. glycines appeared to be pathogenic on both soybean and kidney bean, but not on cowpea or alfalfa (Table 32). The pathogenic responses appeared as watersoaked lesions, with








16
the lesion being slightly chlorotic in soybean. The incompatible

response in cowpea appeared as wine red in color like that seen with X. c. pv. phaseoli.

From spray inoculations on alfalfa, only X. c. pv. alfalfa, resulted in a pathogenic response. After 7 days, small water-soaked leaf spots which later turned necrotic appeared. X. c. pv. Jlfalfa was also pathogenic on kidney bean, and appeared so slightly on soybean. There were small water-soaked spots which occurred at the inoculation sites on soybean. The wine red incompatible response was observed on cowpea.

A strain of X. c. pv. malvacearum, which was not a pathogen of legumes, resulted in only a slight tissue discoloration at the inoculation site on bean and soybean. A null reaction, as observed with the water-only control, was observed on cowpea. The water-only control also resulted in a null reaction on the other hosts tested. A summary of the plant reactions observed for the inoculation experiments is given in Table 3-3.

In separate inoculation tests, a strain of X. c. pv. cyamopsidis"'''" was not found pathogenic on alfalfa, kidney bean, cowpea, or soybean and a X. c. pv. Disi strain was not found pathogenic on kidney bean. Overall, most of the reactions observed conformed to those reported in the literature, with some exceptions. Variation was evident among strains of a given pathovar in addition to that between pathovars. The X. c. pv. alfalfae strains appeared to have overlapping host ranges, which extended to bean and pea in addition to alfalfa. The kidney bean cultivar, California Light Red, appeared susceptible to a









17

Table 3-3. Host range observed for pathovars of Xanthomonas campestris pathogenic to legume host plants.


Inoculation reaction on host plantsa Pathovar G.max M.sativa P.sativum P.vulgaris V.unguiculata


alfalfaeb + +c + _d
glycines + nt +
phaseoli -e ntf +
pisi +c
vignicola -e nt +9 +
cyamopsidish nt

a + = compatible, = incompatible, nt = not tested. Host plants were
Glycines max (soybean) cv. Evans; Medicago sativa (alfalfa) cv.
FL-77; Pisum sativum (pea) cultivar not known; Phaseolus vulgaris (bean) cv. California Light Red; and Vigna unguiculata (cowpea) cv.
California Blackeye #5.

b Other susceptible hosts reported are Trigonella and Melilotus (6).

c Reported as susceptible, but not tested.

d Reported as positive in the literature (6). e Reaction appears negative for most strains, but occasional limited
water soaking is evident.

f There is a report of pathovar phaseoli on Pisum lunatus (6).

g Symptoms are similar to those seen in V. unguiculata. h The natural host belongs to the genus Cyamopsis.









18

representative strain from each of the X. campestris pathovars of legumes tested, with the exception of X. c. pv. cyamopsidis. Various phenotypic responses were observed on the cotton and hibiscus cultivars with the X. c. pv. malvacearum strains tested. In general, it appeared that those strains of X. c. pv. malvacearum derived as pathogens from cotton were pathogenic to one of the hibiscus cultivars tested. None of the X. c. pv. malvacearum strains derived as pathogens from hibiscus were pathogenic on the cotton cultivars in these tests.

From the inoculation tests on the cotton host differentials with

the cotton derived strains of X. c. pv. malvacearum, it appeared that three races of the pathogen were being used. All of these strains were pathogenic on Acala 44 (susceptible host), but could be differentiated into races by the other cotton lines containing different resistance gene backgrounds. The X. c. pv. malvacearum

strain N was pathogenic on all the cotton lines tested, while strain H was only pathogenic on Acala 44. Strains FL79 and TX84 appeared to be

the same race because they were pathogenic on the same six out of eight cotton lines tested (Table 3-4). However, these two strains

differed in reactions on the three hibiscus cultivars inoculated. Strain FL79 appeared pathogenic on the three cultivars, but TX84 gave a null response similar to the water-only control. The two other X. c. pv. malvacearum strains (N and H) appeared identical in reaction with the three hibiscus cultivars, giving pathogenic responses to 2 cultivars, and a null response on the remaining cultivar (Table 3-4).









19

Table 3-4. Malvaceous host plant reactions to inoculation with strains of X. campestris pv. malvacearuma


Cotton Hibiscus
Acala
Strain 44 B1 B2 B3 B5 BIN 101 Gregg Hlb H2 H3


N + + + + + + + + + 0 +
H + + 0 +
FL79 + + + + + + + + +
TX84 + + + + + + 0 0 0
Xl0 +/- + +
X27 0 0 0 0 0 0 0 0 0 0 0
X52 + +
X102 + +
X103 + +
X108 + + +
83-4244 + +
M84-ll 0 0 0 0 0 0 0 0 0 0 0
84-1093c0 0 0 0 0 0 0 +/- 0

a + = compatible reaction, = incompatible reaction, 0 = null
response, and +/- = intermediate reaction.

b H. rosa-sinensis cv. "Brilliant Red".

c Strain 84-1093 is X. c. pv. esculenti.









20

With the eight X. c. pv. malvacearum strains derived from hibiscus, two gave null reactions on all of the cotton and hibiscus cultivars tested (Table 3-4). The other strains derived from hibiscus

were pathogenic on at least two of the three hibiscus cultivars tested, but were not pathogenic on any of the cotton cultivars. In a

separate experiment, one of the strains (M84-11) did appear to give pathogenic symptons on Acala 44, while another strain (83-4244) gave a hypersensitive response. In this instance the mean temperature was at 25 C, rather than 30 C.

An additional strain tested, X. c. pv. esculenti strain 84-1093, which was a pathogen derived from okra, gave a null reaction on seven of eight cotton cultivars and one of three hibiscus cultivars. The

other cotton or hibiscus cultivars gave hypersensitive responses (Table 3-4).

Physiological Differentiation

All of the strains tested were mucoid on NYGA medium, produced xanthomonadin, and hydrolyzed esculin and casein. All of the strains tested also appeared to be positive for starch and gelatin hydrolysis, and production of lipase (Table 3-5). Six of the seven X. c. pv.

vignicola strains, and one strains of X. c. pv. phaseoli appeared to hydrolyze gelatin slower than the other X. campestris strains. Cellulase activity, indicated by pitting of the solid medium, was observed for all seven of the pathovars tested. However, not all

strains of X. c. pv. phaseoli appeared to have cellulase activity, and X. c. pv. alfalfae strains were weaker in this activity. None of the strains tested exhibited pectolytic activity at pH 4.5, suggesting a









21

Table 3-5. Physiological reactions of strains of X. campestris.


Physiological Test Mediuma
Na polypectate Pectin
Pathovar Starch Gelatin Cellulose 4.5 7.0 8.5 5.0 Lecithinb


alfalfae
KS + + w +
FL + + w +
cyamopsidis
13D5 + + + + + +
glycines
B-9-3 + + + + + +
1717 + + + + + +
17915 + + + + + +
S-9-8 + + + + + +
malvacearum
H + + +
N + + +
phaseoli
EK11 + + +
Xph25 + + + +
Xpfll + + +
Xpa + w +
Xpll + + +
82-1 + + +
82-2 + + +
LB-2 + + +
SC-3B + + + + +
XP2 + + +
XP-JL + + +
XP-JF + + +
XP-DPI + + +
pisi
XP1 + + + + + +
vignicola
A81-331 + + + + + +
C-1 + w + + + +
CB5-1 + w + + + +
Xvl9 + w + + + +
SN2 + w + + + +
432 + w + + + +
82-38 + w + + + +


a All strains were mucoid, esculin positive, milk proteolytic, and
lipase positive. + = positive, = negative, and w = weak reaction.

b A lecithinase positive reaction was observed as a clearing zone around
the colony plus precipitation in the medium.









22
lack of polygalacturonase activity. At pH 7.0 and 8.5, pectolytic activity was observed for all strains of X. c. pv. cyamopsidis, X. c. pv. glycines, X. c. pv. _isi, and X. c. pv. vignicola. Only one of the

13 X. c. pv. phaseoli strains tested (SC-3B) had pectolytic activity. None of the strains degraded pectin as a carbon source, only sodium

polypectate.

Polyacrylamide Gel Electrophoresis

Total protein was extracted from 18 strains, representing seven different pathovars of X. campestris. The pathovars included were X. c. pv. alfalfae, X. c. pv. cyamopsidis, X. c. pv. glycines, X. c. pv.

malvacearum, X. c. pv. phaseoli, X. c. pv. pisi, and X. c. pv. vignicola. All of the pathovars, except X. c. pv. malvacearum (Fig. 31, lane P), are known pathogens of legumes. One of the X. c. pv.

phaseoli strains consisted of a biochemical variant of this pathovar, X. c. pv. phaseoli var. fuscans (Fig. 3-1, lane J). One of the X. c. pv. malvacearum strains was derived from hibiscus, rather than from cotton (not shown).

In general, there were few observable differences in the protein banding patterns for the X. campestris strains. Some minor differences in banding patterns between different pathovars were observed, but variation to the same extent was also present within a given pathovar.

Discussion

The plant reactions observed from the X. campestris host range study conformed to expectations, in most cases. However, it was evident

that pathogenic variation in strains associated with a given pathovar could occur, as shown by the race-specific interactions with the X. c.









23
















A B C D E F G iJ K L M N 0 P
93,

45



22


14








Figure 3-1. Polyacrylamide gel electrophoresis of total proteins from strains of X. campestris. Lanes A and B, X. c. pv. alfalfae; lanes CF, X. c. pv. glycines; lanes G-I, X. c. pv. phaseoli; lane J, X. c. pv. phaseoli var. fuscans; lane K, X. c. pv. pisi, lane L, X. c. pv. cyamopsidis; lanes M-O, X. c. pv. vignicola; and lane P, X. c. pv. malvacearun (cotton). Protein size is labeled in kilodaltons.









24

pv. malvacearum strains. In the legume study, it was interesting to note that different hosts gave different phenotypic responses to different incompatible pathovars of X. camnpestris. These different incompatible phenotypes suggest that different host resistance genes may be involved in each of these incompatible interactions. Although the hypersensitive reaction is commonly considered to be the general resistance mechanism of plants against bacteria, these data suggest a more specific response, unique to each incompatible reaction (33,49). No formal studies on the hypersensitive reaction were implemented, so

the significance of this observation will require further investigation. Differences in the phenotypes which may account for this are time of recognition, cell number, electrolyte leakage, or other bacteria- or plant-conditioned responses. Likewise, a

hypersensitive reaction may have occurred for instances where a null response was observed (27).

The results from inoculation tests on the malvaceous hosts suggested that those strains of X. c. pv. malvacearum derived from cotton are potential pathogens of hibiscus, whereas those derived from

hibiscus are generally restricted to hibiscus. However, in one

experiment, it was found that one of the hibiscus strains caused watersoaked lesions on Acala 44 when the night temperatures were lowered. This observation indicates that some of the interactions may be temperature sensitive. Some resistance genes in cotton are known to be temperature sensitive (9).

Race-specific incompatibility may account for the varied X. c. pv. malvacearum (cotton strains) reactions on hibiscus. I t was









25
interesting to note that strains FL79 and TX84, both race 16 of X. c. pv. rnalvacearum cotton strains, each differed in reactions on the hibiscus differential lines. Since Acala 44 elicited a hypersensitive

response from 6 of 8 hibiscus strains, possibly Acala 44 contains an undiscovered resistance gene to the hibiscus derived strains. Those

strains for which no hypersensitive reaction was induced may have escaped host recognition, but lack the necessary pathogenicity determinants to incite disease. Avirulence genes do not give

phenotypic expression in non-parasites (88). Bacterial growth

kinetics would help determine if the bacteria are multiplying on these

hosts. This would be useful for the case of X. c. pv. esculenti, for which 1 of 8 cotton hosts were incompatible, and 2 of 3 hibiscus hosts were incompatible.

The data from the protein and physiological tests demonstrated differences between strains, but were not useful for typing strains by pathovar. There was significant variation among strains within a given pathovar. Although the use of SDS-PAGE to study total protein profiles did not differentiate the pathovars, some work has suggested limiting the analysis to just outer membrane proteins to improve resolution of strain differences (70).

These experiments confirmed pathogenicity of the stock strains and clarified inconsistencies in the literature concerning host range. The host range studies indicated that conclusions reached may often be

strain-dependent, and that examination of a large number of strains will reveal a different overall picture than an examination of a few strains. For example, the plant source of X. c. pv. malvacearum









26

determines whether a strain will have the host range listed for the pathovar (hibiscus and cotton) or a more limited host range (hibiscus). However, the plant source of X. c. pv. alfalfae, does not determine whether a strain will have the host range listed. A strain which attacks both alfalfa and bean is X. c. pv. alfalfae. A strain with a host range limited to bean is X. c. pv. phaseoli. Finally,

several biochemical methods were evaluated for their potential to classify a strain at the pathovar level. Although standard

microbiological tests and protein gels were not adequate for such classifications, an examination of plasmid DNAs (Chapter Four) and chromosomal DNAs (Chapter Six) yielded more rewarding results.















CHAPTER FOUR
CONSERVATION OF PLASMID DNA SEQUENCES AND
PATHOVAR IDENTIFICATION OF STRAINS OF XANTHOMONAS CAMPESTRIS



Introduction

More than 125 different pathovars of Xanthomonas campestris (Pammel 1895) Dowson 1939 are currently recognized (6,24), and the primary means for differentiating them is by inoculation of the plant host(s) of that pathovar. It would be a difficult task to inoculate every plant that could serve as a host to an X. campestris isolate, and therefore the potential host range of a given isolate is unknown. Most often, the pathovar name assigned to a strain of X. campestris is determined by the host it was isolated from. Such designations may be artifactual since the primary host may be different from the one the strain was isolated from; some X. campestris pathovars are known to be pathogenic on more than one host. Epiphytes cannot be classified in this pathovar identification system. Possible taxonomic relationships among pathovars are also elusive. It would be helpful if alternative

means to differentiate among X. campestris pathovars were available. Some suggested approaches for differentiating X. campestris pathovars

have included serology (1,94), membrane protein profiles (70), phage-typing (37), and gas chromatography of fatty acids (65,81). These approaches suffer because they are often strain specific,




This chapter contains copyrighted material from the journal Phytopathology. It is reprinted here with permission of the publisher.
27









28

dependent on constant environmental parameters, and/or so cumbersome that no extensive evaluative tests have been performed.

Plasmid DNA has been identified in several pathovars of X.

campestris (13,16,30,39,46,52,53,60,80). It is relatively simple to extract large numbers of strains and visualize their plasmids with

standardized alkaline lysis procedures (46,66). To characterize the plasmids, restriction endonucleases are used to digest the DNA into distinct fragments that can be separated by size, resulting in fragment patterns visualized by agarose gel electrophoresis. To date

there have been no systematic attempts to examine the extent of plasmid variation among a large number of strains involving a large number of X. campestris pathovars. Our preliminary studies on

selected pathovars of X. campestris suggested that there was a surprisingly high degree of plasmid sequence conservation within some pathovars (31,52,53). These studies further suggested that plasmids of purified strains were quite stable and hence useful in epidemiological studies (31), similar to those used to monitor the

spread of selected human pathogens (28). The purpose of this study was to survey the extent of variation of plasmid DNAs within a large number of X. campestris pathovars and to determine whether the plasmid

content of strains based on restriction fragment polymorphism and Southern hybridization could be used in differentiating the pathovars of X. campestris.

Materials and Methods

Bacterial Strains

The X. campestris strains used in this study, their pathovar designations, geographic origin, and sources are listed in Table 4-1.









29
Table 4-1. Strains of X. campestris used for plasmid analysis.

Bacterium Strain designation
(number of strains) (geographic origin) Sourcea

Xanthomonas campestris pathovar

alfalfae (2) KS (Kansas); D.L.Stuteville
FL (Florida); R.E.Stall
argemones (1) 084-1052 (Florida); DPI
begoniae (1) 084-155 (Florida); DPI
campestris (4) XCl (Oklahoma); this study
084-720,084-809,
084-1318 (Florida); DPI
carotae (3) G1 (Idaho),G5,G7 (California); R.E.Stall
citri (5+) X59 (Brazil),X62 (Japan),
X69 (Argentina), X70 (Brazil); E.L.Civerolo Fil (Florida); DPI
cyamopsidis (5) 13D5; C.I.Kado
X002,X005,X0016,
X0017 (Arizona); J.Mihail
dieffenbachiae (2) 084-729,084-1373 (Florida); DPI
esculenti (1) 084-1093 (Florida); DPI
glycines (3) B-9-3 (Brazil),1717 (Africa),
17915; W.F.Fett
hederae (3) 084-1789,084-3928,
251G (Florida); DPI
holcicola (2) Xh66 (Kansas); L.Claflin
XH1; this study
maculifoliigardeniae(2) 084-6006,084-6166 (Florida); DPI malvacearum-cotton(32) A,B,E,F,G,H (Oklahoma); M.Essenberg
HV25 (Upper Volta),
Chl,Ch2 (Chad),
Su2,Su3 (Sudan); L.S.Bird
FL79 (Florida); DPI
D,M,N,O,U,V,W,X,Y,Z,TX84 (Texas),
I,Q,R,S,T (Oklahoma),
C,J,K,L (Upper Volta); this study
malvacearum-hibiscus(8) X10 ,X27,X52,X102, X103,X108 (Florida); A.R.Chase
083-4344,M84-ll (Florida); DPI mangiferaeindicae (1) 084-166 (Florida); DPI
pelargonii (1) 084-190 (Florida); DPI
phaseoli (13) EKll,Xph25,Xpfll (Nebraska); M.Schuster
Xpa,Xpll (Wisconsin); A.W.Saettler
82-1,82-2 (Florida); R.E.Stall
LB-2,SC-3B (Nebraska); A.K.Vidaver
XP2 (New York); J.A.Laurence
XP-JL (Kansas); J.L.Leach
XP-JF (Missouri),XP-DPI; this study









30

(Table 4-1 continued.)


Bacterium Strain designation
(number of strains) (geographic origin) Sourcea


si (1) XPl (Japan); M.Goto
poinsettiicola (1) 083-6248 (Florida); DPI
pruni (3) 068-1008,084-1793 (Florida); DPI
82-1 (Florida); R.E.Stall
translucens (2) 82-1 (Florida); R.E.Stall
XT1; this study
vesicatoria (5) E-3,69-13,71-21,
82-8,82-23 (Florida); R.E.Stall
vignicola (7) A81-331,C-1,CB5-1,
Xvl9,SN2,432,82-38 (Georgia); R.D.Gitaitis vitians (3) 084-2057,084-2848,
084-4348 (Florida); DPI
zinniae (1) 084-1944 (Florida); DPI

unknown (1) G65 this study

X. albilineans (1) Xalb (Florida); M.J.Davis
X. fragariae (1) Xfra (Florida); R.E.Stall

a DPI = Florida Department of Agricultural and Consumer Services,
Division of Plant Industry, Gainesville, Florida.








31

Some of the stock cultures of X. c. malvacearum were mixtures of different strains, maintained and described as "races" for purposes of screening cotton host differentials and breeding lines for disease

resistance. Described "races" of X. c. malvacearum may or may not be purified strains; as many as seven different strains have been derived from a single race 1 isolate (7). All strains used in this study were repeatedly purified from single colonies and confirmed to be pathogenic on their designated hosts. Broth cultures of bacteria were

grown in a peptone-glycerol medium (per L: 10.0 ml glycerol, 20.0 g peptone, and 1.5 g K2HPO4). Bacterial strains were co only stored at

-80 C in the same medium containing 15% glycerol. Plasmid Extraction and Visualization

Cultures were grown to mid- to late-logarithmic growth phase and extracted by either of two small-scale alkaline lysis extraction procedures (46,66). Extracted DNA was resuspended in TE (10 mM

Tris(hydroxymethyl)aminomethane (Tris), 1 mM sodium

ethylenediaminetetraacetate (Na2EDTA), and 20 ug/ml DNase-free pancreatic RNase; pH 7.6) and digested with either of two restriction

endonucleases, EcoRI or BamHI, using manufacturer (Bethesda Research Laboratories, Gaithersburg, MD) specifications. Plasmid DNA fragments were separated by size using agarose gel electrophoresis (0.6% agarose

(Sigma Type I:low EEO), 2-5 V/cm) in Tris-acetate buffer (40 mM Tris, 1 mM Na2EDTA, adjusted to pH 7.6 with glacial acetic acid). Fragments were visualized by ultraviolet irradiation (302 rrn) after staining

agarose gels in ethidium bromide (0.5 ug/ml). Photographs were taken with Polaroid Type 55 (or Type 57) film using a yellow filter (Tiffen









32

no. 12). All restriction fragment size estimates were based on the relative mobilities of linear DNA fragments using lambda phage DNA digested with HindIII as molecular size standards. Plasmid sizes were

estimated by addition of the sized restriction digested plasmid DNA fragments. All plasmid experiments were repeated at least twice for each strain examined.

Cloning of Plasmid Restriction Endonuclease Fragments

Plasmid DNA isolation from X. campestris was by a modification of either of two alkaline lysis extraction procedures (46,68). The

extracted plasmids were purified on CsCl-ethidium bromide gradients by

centrifugation at 55,000 rpm in a Beckman VTi65.2 rotor for 17 hr at 20 C. The purified plasmids were digested with the restriction enzyme EcoRI. The cloning vector, pUCD5 (14), was digested with EcoRI, treated with calf intestinal alkaline phosphatase (BoehringerMannheim, Indianapolis, IN), and ligated to the EcoRI digested X. campestris plasmid fragments with T4 DNA ligase (Bethesda Research Laboratories). The ligation products were transformed into E. coli strain ED8767, selecting transformed colonies on Luria-Bertani medium

containing ampicillin (50 ug/ml) or kanamycin (30 ug/ml). Selected colonies were analyzed for the vector containing desired cloned DNA fragments. These general cloning procedures are outlined in Maniatis et al. (66).

DNA/DNA Hybridization

Plasmid DNAs were transferred from agarose gels to nitrocellulose

membranes by the method of Southern as described by Maniatis et al.

(66) and hybridized against radioactively-labeled DNA probes. The DNA









33

probes derived from plasmid DNA of X. campestris pathovars were either cloned restriction digested DNA fragments of plasmid DNA in the cosmid vector pUCDS, or of the complete X. campestris plasmid. DNA probes were labeled in vitro with use of a nick translation kit (Bethesda Research Laboratories) using 32P-deoxycytidine triphosphate and

hybridized against DNA bound to nitrocellulose membranes. The

membranes were pre-hybridized and hybridized in plastic bags at 68 C. The pre-hybridization and hybridization solutions were those described by Maniatis et al. Following hybridization, membranes were washed once

in 2X SSC, 0.5% SDS and washed once in 2X SSC, 0.1% SDS at ambient temperature, and washed two times in 0.1X SSC, 0.5% SDS at 68 C as described by Maniatis et al. (66) for stringent conditions. The

membranes were then air dried and exposed at -80 C to X-ray film (Kodak X-0mat AR) using intensifier screens. Similar methods were used to probe DNA transferred to nitrocellulose using a dot-blot manifold (Schleicher and Schuell Inc., Keene, NH). All hybridization experiments were repeated at least once.

Results

Detection of Plasmid DNA

Indigenous, cryptic plasmids were detected in all strains of the following X. campestris pathovars: cyamopsidis, dieffenbachiae, glycines, malvacearum (cotton), pelargonii, phaseoli, pruni,

vesicatoria, vignicola, and vitians (Table 4-2). Plasmids were not detected in any strains of X. campestris pathovars alfalfae, argemones, begoniae, carotae, esculenti, holcicola, maculifoliigardeniae, malvacearum (hibiscus), mangiferaeindicae, pisi,









34

Table 4-2. Detection of plasmid DNA in strains of X. campestris.


No. of strains
containing plasmids/
Bacterium No. of strains tested Pathogenicitya

X. campestris pathovar

alfalfae 0/ 2 P
argemones 0/ 1 I
begoniae 0/1 I
campestris 2/ 4 R,I
carotae 0/ 3 R
citri 17/44 P,R,I
cyamopsidis 5/ 5 R
dieffenbachiae 2/ 2 I
esculenti 0/ 1 I
glycines 3/ 3 P
hederae 2/ 3 I
holcicola 0/ 2 R
maculifoliigardeniae 0/ 2 I
malvacearum cotton 32/32 P
malvacearum hibiscus 0/ 8 P
mangiferaeindicae 0/ 1 I
pelargonii 1/ 1 R
phaseoli 13/13 P
pisi 0/ 1 R
poinsettiicola 0/ 1 I
pruni 3/ 3 I
translucens 0/ 2 R
vesicatoria 5/ 5 P
vignicola 7/ 7 P
vitians 3/ 3 I
zinniae 0/ 1 I
X. albilineans 0/ 1 R
X. fragariae 0/ 1 I

a p = Pathogenicity of strains confirmed on appropriate host, conforms
to current available information for particular pathovar.

R = Received as named pathogen, appropriate host specificity and pathovar designation of strain assumed.

I = Isolated as a pathogen on host appropriate for designated pathovar; characterization of strain(s) incomplete.










poinsettiicola, translucens, and zinniae. Plasmids were found insome,

but not all strains of X. campestris pathovars campestris and

hederae. Similarly, plasmids were found in all type strains of X. c. pv. citri (A, B, and C types); this is in agreement with a previous plasmid study of this pathovar (13). However, not in all strains of X. campestris isolated from leaf spots of citrus in Florida contained

plasmids.

It appeared that plasmid-containing strains of X. campestris carried from one to three plasmids based on electrophoresis of extracted plasmid DNA. For example, a majority of the

plasmid-containing X. c. malvacearum strains contained only one plasmid, but some carried two or more. When plasmid-containing

strains of X. campestris were purified by repeated single colony isolation, the plasmid DNA content appeared to be stable. However, variation was present in bacterial stocks which were known to have been serially transferred in agar medium over a period of years. Restriction Endonuclease Profiles

Plasmid profiles for X. campestris were variable in over 60 different strains tested. Plasmids were placed into classes based on the variability of restriction endonuclease (EcoRI) digestion profiles on agarose gels. When a different restriction endonuclease (BamHI) was used, the plasmid profiles were placed into the same classes. In all cases, strains which belonged to the same plasmid class also belonged to the same pathovar. There was obvious variability within

classes, but there also appeared to be conservation of some DNA fragments of identical sizes (Fig. 4-1). Undigested plasmids were not









36

reliable for strain classification because several strains had plasmids of apparently identical size, but they were clearly different

after digesting the plasmid DNAs with restriction enzymes. By adding up the DNA fragment sizes yielded by restriction digests, plasmids in X. campestris were estimated as ranging from about 3 to 200 kb (kilobase pairs) in size. Estimation of some of these sizes were

difficult for some strains due to the presence of more than one plasmid.

Plasmid profiles of strains of X. c. cyamopsidis, X. c. glycines,

X. c. malvacearum, X. c. phaseoli, and X. c. vignicola were compared. Restriction fragment length polymorphism was evident within each of these pathovars. Although more than one plasmid was present in some of these strains, a subset of restriction fragments of similar length and overall pattern appeared to be consistent for strains within a given pathovar (Figure 4-2).

Four highly polymorphic plasmid variants were found in 17 out of 44 Florida strains tested, but Southern hybridization revealed no homology between some of the plasmids (31). Furthermore, there were no similarities in plasmid digestion patterns between X. c. pv. citri type A, type B and any of the Florida citrus leaf spot strains that carried plasmids (Figure 4-3). These Florida isolates are presumed to be X. c. citri, but they grow well and also cause disease symptoms on

kidney bean and alfalfa, thus making their pathovar status questionable (34).









37








A B C D E F G H I J K L M N OP Q R




23.1


9.4

6.7 4.4




2.3
2.0 kb







Figure 4-1. Plasmid DNAs from strains of Xanthomonas campestris pv. malvacearum (Xcm) digested with restriction endonucleases EcoRl (lanes
B-I) and BamHI (lanes J-Q). Lanes shown above contain: A and R, lambda HindIII; B and J; Xcm J; C and K, Xcm N; D and L, Xcm H; E and M, Xcmn V; F and N, Xcm Z; G and O, Xcm Q; H and P, Xcm X; and I and Q, Xcm D.









38







111111 1 11122 2 2 2 22233333333334444444
123456789012345 6789012345678901 234567890123456
f I I I I E I I I I I I I,7T7 7 I

30

25-





20





kb
________I___I____,_,________i__F J I II I I I i J






Figure 4-2. Graphic representation of plasmid restriction fragment profiles for Xanthomonas campestris pvs. cyamopsidis (Xcc; lane 1), vignicola (Xcv; lanes 2-8), phaseoli (Xcp; lanes 9-17), phaseoli var. fuscans (Xcpf; lanes 18-19), glycines (Xcg; lanes 20-22), and
malvacearum (Xcm; lanes 23-46) digested with EcoRI. Lanes shown above contain 1, Xcc 13D5; 2, Xcv SN2; 3, Xcv A81-331; 4, Xcv C-1; 5, Xcv CB5-1; 6, Xcv Xv19; 7, Xcv 82-38; 8, Xcv 432; 9, Xc EK11; 10, Xcp 82-2; 11, Xcp Xpa; 12, Xcp Xpll; 13, Xcp XP2; 14, Xcp XP-JF; 15, Xcp XP-DPI; 16, Xcp Xph25; 17, Xcp 82-1; 18, Xcpf Xpfll; 19, Xcpf SC-3B; 20, Xcg B-9-3; 21, Xcg 17915; 22, Xcg 1717; 23, Xcm J; 24, Xcm L; 25,
Xcm C; 26, Xcm O; 27, Xcm K; 28, Xcm N; 29, Xcm A; 30, Xcm B; 31, Xcm E; 32, Xcm F; 33, Xcm G; 34, Xcm H; 35, Xcm S; 36, Xcm W; 37, Xcm I; 38, Xcm V; 39, Xcm D; 40, Xcm M; 41, Xcm X; 42, Xcm U; 43, Xcm Q; 44, Xcm R; 45, Xcmn Y; and 46, Xcm Z. Calculated DNA fragment sizes are represented here by a linear scale whereas migration of DNA fragments under electrophoresis conditions approximates a logarithmic scale which is inversely proportional to the molecular weight of the DNA fragment.








39 A B C D








23



9

7

4




2






Figure 4-3. Plasmid DNAs from strains of X. campestris pv. citri digested with restriction endonuclease EcoRI. Lane A, strain F11 (Florida type); lane B, strain X59 (A type); lane C; strain X62 (A type); and lane D, strain X69 (B type). Sizes shown in kilobase pairs.









40
DNA/DNA Hybridization

Initial plasmid comparisons were done on strains of X. c. malvacearumn. Whole purified plasmid DNA from X. c. malvacearum strain

X, which contains only one plasmid, was hybridized against EcoRI digested plasmid DNAs of other strains of the same pathovar (not shown) This initial comparison demonstrated that the plasmids,although differing slightly in digestion patterns, were quite

homologous as the radiolabeled plasmid hybridized to almost all EcoRI fragments of the other strains.

A 4.5 kb EcoRI plasmid fragment of X. c. malvacearum strain N was cloned into the vector pUCD5 and used as a hybridization probe against

plasmid DNA from other strains of the same pathovar (Fig. 4-4). This plasmid DNA fragment hybridized to plasmids from all but one strain of X. c. malvacearum. This probe hybridized to EcoRI fragments of 4.5 kb

size (lanes B-J, M-N, Fig. 4-4) in several other strains of X. c. malvacearumn. Additionally, the probe hybridized to more than one of the EcoRI plasmid fragments in these strains, suggesting that some sequences on the cloned DNA are repeated in other parts of the plasmid DNA. The pUCD5 vector alone did not hybridize to any X. c. malvacearum plasmid fragments. Because pUCD5 is a cosmid vector and contains the cos site of lambda phage DNA, hybridization of the vector

to the corresponding DNA fragment of the molecular weight marker containing the cos site was observed. Plasmid DNA from X. c.

malvacearum strain Su2 did not hybridize to the 4.5 kb probe, and did not hybridize to any other plasmid fragments from X. c. malvacearum strain N. Another X. c. malvacearum strain (Ch2), which did not have









41













A BC D E F G H I J K L M N A B C D E F G H I J K L M N











2W
13CW


kb







Figure 4-4. Plasmid DNAs from strains of Xanthomonas campestris pv. malvacearum (Xcm) digested with restriction endonuclease EcoRI (left) and autoradiograph of plasmid DNAs probed with a clone containing a 4.5 kb EcoRI plasmid fragment from Xcm strain N (right). Lanes shown above contain: A, lambda HindIII B, Xcm H; C, Xcm W; D, Xcm V; E, Xcm X; F, Xcm Q; G, Xcm Y; H, Xcm L; I, Xcm N; J, Xcm HV25; K, Xcm Ch2; L, Xcm Su2; M, Xcm FL79; and N, Xcm TX84.









42

a 4.5 kb EcoRI plasmid DNA fragment, did have two other fragments (ca. 8 and 10 kb) which hybridized strongly to the 4.5 kb probe. In two other X. c. malvacearum strains (FL79 and TX84), which appeared to have multiple plasmids, the hybridization signal was weak for the 4.5

kb fragments as compared to larger EcoRI fragments (ca. 23 kb). Similar hybridization studies were done with cloned plasmid fragments from other pathovars. When the plasmid fragments selected were

smaller, they were much more specific. Two different probes were

constructed from 2.0 kb and 2.3 kb EcoRI plasmid fragments derived from X. c. phaseoli (strain XP2). When hybridized against X. c.

cyamopsidis, X. c. glycines, X. c. phaseoli, and X. c. vignicola, each of these probes only hybridized to X. c. phaseoli (Fig. 4-5). The 2.3

kb plasmid probe hybridized to similar sized fragments in other X. c. phaseoli strains, including X. c. phaseoli var. fuscans (strains SC3-B (not shown) and Xpf11). These X. c. phaseoli var. fuscans strains differ from typical X. c. phaseoli strains in that they produce an extracellular dark brown, melanin-like pigment in culture; otherwise,

they are considered similar. However, the 2.0 kb plasmid probe did not hybridize to the X. c. phaseoli var. fuscans strains, which did not have the corresponding 2.0 kb EcoRI fragment in their plasmid profile (lane C, Fig. 4-5, and strain SC3-B (not shown)). Repeated hybridizations with Southern transfers containing these strains had the same results.

Dot-blot Hybridization

DNA probes were also hybridized against total DNA of other X. campestris pathovars fixed onto a nitrocellulose membrane by use of a dot-blot manifold apparatus. Radiolabeled total plasmid DNA from









43















A B C D E F G H I J K L A B C D E F G HI J K L ABC D E F G H I J K L





9.4

6.7
4.4

2.3 MAW 4-- bI4

kb






Figure 4-5. Plasmid DNAs from strains of Xanthomonas campestris pvs. phaseoli (Xcp; lanes B, D-I), phaseoli var. fuscans (Xcpf; lane C), and cyamopsidis (Xcc; lane K) digested with restriction endonuclease EcoRI (left) and autoradiographs of plasmid DNAs probed with a clones containing a 2.0 kb EcoRI fragment (center) and a 2.3 kb EcoRI plasmid fragment (right) from Xcp strain XP2. Lane J contains chromosomal DNA of X. campestris pv. alfalfae (Xca) digested with EcoRI. Lanes shown above contains: A and L, lambda HindIII; B, Xcp Xph25; C, Xcpf Xpfll; D, Xcp EK11; E, Xcp 82-1; F, Xcp Xpa; G, Xc G65; H, Xcp Xpll; I, Xcp XP-JF; J, Xca FL; and K, Xcc 13D5. Xc G65 is a strain isolated from bean which was determined to be nonpathogenic and contains no plasmid.









44

Table 4-3. Hybridization of radiolabeled plasmid probes to total DNA of pathovars of Xanthomonas campestris and one other Xanthomonas species.


Probesa
Bacterium (No. tested) N80 N4.5 V2.3 P2.3

X. campestris pv.

alfalfae (1) +
argemones (1)
begoniae (1)
carmpestris (1)
carotae (1) +
citri (3) + +/_b +/-b +/_b
cyamopsidis (1) + + +
dieffenbachiae (1)
esculenti (1)
glycines (1) + +
hederae (1) +
holcicola (1) +
maculifoliigardeniae (1)
malvacearum cotton (6) +/-c +/_c +
malvacearum hibiscus (2)
mangiferaeindicae (1)
phaseoli (1) + + +
poinsettiicola (1)
pruni (1) + +
translucens (1) +
vesicatoria (1) + + +
vignicola (1) + + +
vitians (1)
zinniae (1)

X. albilineans (1)


a + = hybridization observed, = no hybridization observed.
N80 = plasmid DNA derived from X. c. pv. malvacearum strain N
(about 80 kb); N4.5 = cloned EcoRI plasmid fragment (4.5 kb) from strain N; V2.3 = cloned EcoRI plasmid fragment (2.3 kb) from X. c.
pv. vignicola strain SN2; P2.3 = cloned EcoRI plasmid fragment
(2.3 kb) from X. c. pv. phaseoli strain XP2

b Strain FL-11 was negative, only plasmid DNA used.

c Strain S2 was negative.









45
X. c. malvacearum strain N, which carries two plasmids, hybridized to DNA of thirteen out of twenty-three X. campestris pathovars tested (Table 4-3). Of these thirteen, seven cross-hybridized to the 4.5 kb

subcloned fragment of X. c. malvacearum strain N. Plasmids were not detected in some of the pathovars which hybridized to the probe. A 2.3 kb subcloned plasmid fragment from X. c. vignicola hybridized to only six of the 23 pathovars tested. Plasmids were present in all six

of those pathovars which hybridized to the probe. A 2.3 kb cloned plasmid fragment of X. c. phaseoli hybridized strongly to other strains of the same pathovar, and weakly to 2 of 3 different X. c. citri strains tested. The X. c. phaseoli probe appears to have

hybridized to chromosomal DNA of X. c. citri in this case, as the probe did not hybridize against Southern transfers of EcoRI digested plasmid fragments of X. c. citri strains (not shown).

Discussion

It is suggested that there is extensive conservation of plasmid

DNA sequences (as represented by conserved restriction fragments) within, but not usually among, pathovars of X. campestris. Plasmid DNA

fragment patterns will be identical if there is no rearrangement of the DNA sequence at the restriction enzyme recognition site (a six base pair sequence for EcoRI and BamHI), if no new restriction fragments are created within the fragment, and if there are no major additions or deletions causing a change in fragment size. Given these possibilities, it was surprising to find so little restriction fragment length polymorphism of plasmids within a pathovar, especially when strains obtained from different continents were compared.









46
Southern hybridization analyses confirmed that plasmid DNA fragments of similar size were in fact highly homologous. For example, X. c. malvacearum strain N, isolated in North America, had a restriction digest pattern identical to one of the African strains (K) (Fig. 4-2).

All North American strains except strain N form a distinctive pattern subgroup and the African strains form a somewhat different subgroup. It is possible that either all the African strains are derived from

strain N, or that strain N was introduced to the North America from Africa. The latter possibility seems more likely, since the African strains were isolated from more than one location in Africa. The

conservation of overall restriction fragment profiles of plasmids from geographically isolated populations and the extent of homology seen in Southern hybridizations strongly suggests that plasmid sequences are both highly conserved and stable.

Conversely, it was surprising to find so much polymorphism of plasmids between pathovars. Homology among plasmids in different strains of one pathovar of Pseudomonas syringae (pv. glycinea) has

been reported (15). However, identical plasmid profiles were found present in more than one pathovar of P. syringae (77). In the present study, similar plasmid profiles were not found in more than one pathovar. In addition, cloned plasmid fragments were identified which failed to hybridize to plasmids of different pathovars. For example,

a cloned 2.3 kb plasmid fragment of X. c. phaseoli, which hybridized to all strains of that pathovar, failed to hybridize to total DNAs of

22 other pathovars. This DNA fragment is highly conserved and is apparently pathovar-specific, with one exception (Table 4-3).









47
Plasmid homology between X. c. phaseoli and X. c. phaseoli var. fuscans was revealed by hybridization that was not apparent by restriction digest patterns. There were distinct differences in

plasmid digestion patterns of X. c. phaseoli var. fuscans in comparison to typical X. c. phaseoli strains. The fact that both X. c. phaseoli and X. c. phaseoli var. fuscans have an identical host range suggests that the homologous plasmid DNA regions that are conserved within some pathovars may encode host range specificity functions. Such functions have been described on plasmids within the

Rhizobiaceae (62,64) This is a testable hypothesis that needs further experimental support.

In X. c. malvacearum, plasmids were found in all 32 strains isolated from cotton, and no plasmids were found in the 8 strains isolated from hibiscus. Cotton and hibiscus each belong to the same plant family (Malvaceae), hence the X. c. malvacearum designation. Atypical symptoms of cotton blight could be artificially produced in cotton using syringe inoculations with hibiscus strains, and in hibiscus using cotton strains. The ability of these strains to be pathogenic on both hosts under natural conditions has not been clearly established. Plasmid DNAs from cotton strains cross-hybridized with one another in Southern analyses, indicating extensive homology was evident between the plasmids. As with X. c. phaseoli, the cotton strains of X. c. malvacearum appeared to carry highly conserved

plasmid DNA sequences (similar in restriction digest sizes and by Southern analyses) which are unique to strains which have a host range on cotton. Plasmid DNA from the cotton strain (N), when radiolabeled,









48
did not hybridize to chromosomal DNA derived from the hibiscus strain.

Based on our limited pathogenicity tests and on the absence of plasmid DNA sequences in the hibiscus strains, there may be justification to differentiate the cotton and hibiscus strains into different pathovars.

Although some cross-hybridization between plasmids of strains

front different pathovars was detected by dot-blot analyses (Table 43), strains were readily differentiated by restriction digest profiles

of the plasmid DNAs and by hybridization with selected DNA probes to identifiable restriction digested DNA fragments. Cross-hybridization

may be the result of repetitive DNA sequences, insertion elements, or of conserved DNA sequences that are important for the stable

maintenance of plasmids in X. campestris. Examples of plasmid

functions which might be conserved are those involved with replication, incompatibility, or other host dependent factors. in

some instances plasmid DNAs hybridized to total DNA of pathovars in which no plasmids were detected (Tables 4-2 and 4-3). It is suggested

that some sequences encoded on plasmids in some pathovars are located on chromosomes in other pathovars. It is also possible that those plasmids may integrate into the bacterial chromosome and excise again, in a manner similar to those in P. syringae pv. phaseolicola (79,92).

With interest in developing rapid diagnostic methods to identify bacterial pathogens, it is possible that the combined usage of plasmid restriction digest profiles and of plasmid DNA probes may be

sufficient for the identification of some pathovars of X. campestris. This would require that a plasmid be stably associated with a given









49
pathovar, that plasmid profiles for specific pathovars were known, and

that an appropriate DNA probe consisting of conserved and unique DNA sequences were available. Of the few DNA probes constructed, it was apparent that plasmid DNA sequences were highly conserved within the pathovars studied. In some instances the DNA probes may prove sufficient for pathovar identification, provided they are extensively tested. This apparent stability of the plasmids provides a natural

genetic marker that can be strain specific and perhaps useful in epidemiological investigations. In addition to aiding the

identification for some pathovars of X. campestris, these observations may have taxonomic significance to differentiate these pathogens.















CHAPTER FIVE
ARE RACE-SPECIFIC AVIRULENCE GENES ENCODED BY PLASMIDS
OF XANTHOMONAS CAMPESTRIS PV. MALVACEARUM?



Introduction
Bacterial blight of cotton, caused by the pathogen X. c. pv. malvacearum (Snith) Dye, is a destructive disease of cotton, which occurs throughout the cotton-producing areas of the world. The

development of cotton cultivars resistant to bacterial blight is the primary means by which this pathogen is controlled. At least 16 major genes have been identified which condition resistance to bacterial

blight (8). A gene-for-gene pattern of interaction between the plant resistance genes and bacterial avirulence genes has been demonstrated in this host-parasite interaction (33). In screening for resistance to bacterial blight in breeding lines, races of the pathogen have been maintained as mixed cultures, originally derived from infected leaf samples. These mixed cultures have been mass transferred on agar media to prevent the appearance of new race phenotypes. The stability of the race phenotypes of these mixed cultures is periodically checked by inoculating the host differentials.

Plasmids have been detected in several phytopathogenic prokaryotes and have been associated with functions such as pathogenicity (5), resistance to antibiotics (3,88), avirulence (88), and host range (64). With the recent discovery of plasmids in several


50









51
pathovars in X. campestris (13,16,30,39,46,52,53,80), it is of interest to understand the function of these autonomously replicating,

covalently closed circular DNAs of the bacterial genome. The purpose of this investigation was to analyze the plasmids in X. c. pv. malvacearum and determine if they influenced race-specificity in cotton.

Materials and Methods

Bacterial Strains and Host Plants

The strains of X. c. pv. malvacearum used for this investigation

are listed in Table 5-1. All of the strains were from single colony isolation of either mixed, or pure cultures. The cotton differentials used included those recommended for differentiating the races of X. c. pv. malvacearum (45); also included were six congenic cotton lines of Gossypium hirsutum cv. Acala 44, which contained single known resistance genes (B genes) to X. c. pv. malvacearum (7). The congenic Acala series included cotton with resistance genes B2, B3, B5, B6, B7,

and BN. The other host differentials were cvs. Acala 44, 1-10B, 101102B, 20-3, Mebane Bl, Stoneville 20, 2B-S9, and Gregg. Plant Inoculations

Pathogenicity tests were conducted by pressure infiltration of abaxial leaf surfaces with bacterial suspensions using a blunt end of

a syringe, and incubating the plants in 30 C growth chambers until symptom were expressed. Symptoms development was monitored over a two week period.

Plasmid Analysis

Cultures were grown to mid- to late-logarithmic growth phase and extracted by either of two small-scale alkaline lysis extraction









52

Table 5-1. Strains of X. campestris pv. malvacearxn from cotton used in race-specificity investigation.


Strain Race Location Source


K HVlb tipper Volta L.S.Bird
N 2a Texas L.S.Bird
J HV3 Upper Volta L.S.Bird
C HVla Upper Volta L.S.Bird
0 2b Texas L.S.Bird
L HV7 Upper Volta L.S.Bird
A 2bSr Oklahoma M.Essenberg
B 2aSr Oklahoma M.Essenberg
E 4-1 Oklahoma M.Essenberg
F laSr Oklahoma M. Essenberg
G lbSr Oklahoma M. Essenberg
5 3-22 Oklahoma M.Essenberg
H 4-2 Oklahoma M.Essenberg
W 6 Texas L.S.Bird
1 3 Oklahoma M.Essenberg
V 3 Texas L.S.Bird
D 2 Texas L.S.]Bird
M 15 Texas L.S.Bird
X 18 Texas L.S.Bird
U 1 Texas L.S.B3ird
Q la Oklahoma M. Essenberg
R lb Oklahoma M. Essenberg
Z 7b Texas L.S.Bird
y 7a Texas L.S.Bird
T 18 Oklahoma M. Essenberg
Chl -Chad L.S.Bird
Ch2 -Chad L.S.Bird
H-V25 -Upper Volta L.S.Bird
Su2 -Sudan L.S.Bird
Su3 -Sudan L.S.Bird
FL79 16 Florida this study








53

procedures (46,66) as described in Chapter Four. Plasmids were

digested with either EcoRI or BamHI and DNA fragment patterns

visualized by agarose gel electrophoresis. All plasmid experiments were repeated at least twice for each strain examined. Additionally, EcoRI plasmid fragments of X. c. pv. malvacearum strains were cloned into the vector pUCD5 (14) and maintained in E. coli ED8767 as described in Chapter Four. These cloned fragments were used for subsequent plasmid studies.

Plasmid Curing

To determine if plasmid encoded functions were involved in pathogenicity, attempts were made to expel, or cure, plasmids from the

wild-type strains by including ethidium-bromide or sodium lauryl sulfate (SDS) in the broth culture. Cultures were grown in peptoneglycerol broth for two 48 hr cycles, being adjusted to an optical density (OD600nm) of 0.03 at the beginning of each cycle. A dilution

series from the broth cultures were plated onto peptone-glycerol agar and single colonies were selected for inoculation on cotton host differentials.

Race-Specificity Genes

Cloned genes encoding race-specific avirulence in X. c. pv. malvacearum have been identified and isolated (33), and it was of interest to determine if these genes were plasmid-borne. Using E. coli ED8767 containing the cloned plasmid fragments in pUCD5 as donors, a tri-parental mating scheme was used to move the cloning

vector into strains of X. c. pv. malvacearum by conjugation (22,69). Into the recipient, X. c. pv. malvacearum strain N (virulent strain),

were mated cloned plasmid fragments (in pUCD5) from X. c. pv.









54

malvacearum strain H (avirulent strain). As a control, plasmids

fragments from strain N were also moved into strain H. The helper

plasmid used for mobilization of the plasmids from E. coli into X. campestris were pSa322 or pRK2013 (22,93). The donor and helper

strains were grown overnight at 37 C in LB broth with appropriate antibiotic selection pressure. The antibiotic was washed from the cells prior to mating by centrifuging and resuspending the cells in 0.7% NaCl. The X. campestris recipients were grown overnight at 30 C in MOPS minimal medium broth (69). Donors and helpers at a

concentration of 107 cells and recipients at 107 cells were mixed in a small volume onto sterile 25 mm cellulose-acetate filter membranes and placed onto peptone-glycerol medium for 6 hours. The membranes were then transferred to MOPS minimal medium with 25 ug/ml kanamycin and incubated for 72 hours at 30 C. Bacterial growth from filters was

diluted in 0.7% NaCl and plated for single colony isolation on MOPS minimal medium with 25 ug/ml kanamycin. Colonies were selected and inoculated on the Acala cotton differentials containing known resistance genes.

Plasmid Origin of Replication and Mobilization

An assay was included to determine if X. c. pv. malvacearum contained self-mobilizing plasmids. The E. coli clones containing

cloned plasmid DNA fragments from X. c. pv. malvacearum strain X were mated into spectinomycin resistant X. c. pv. malvacearum strain N in the presence, or absence, of the E. coli helper plasmid strain with pRK2013. Transconjugants were selected on MOPS minimal medium with kanamycin (25 ug/ml), or directly on POPS (33) containing kanamycin (30 ug/ml) and spectinomycin (100 ug/ml).









55
Results

Plasmids and Race-Specificity

All of the strains of X. c. pv. malvacearum tested contained at least one plasmid. The plasmid sizes ranged front about 50 to 100 kb. A majority of the American strains of X. c. pv. malvacearum contained

4.8, 4.7, and 4.5 kb EcoRI DNA fragments which appeared to be conserved (Fig. 4-2). The entire 4.5 kb EcoRI DNA fragment appeared to be conserved in all but 1 of the 25 strains tested, regardless of geographic origin.

Comparisons were made between the physical restriction fragment patterns (Fig. 4-2), the original race designations, and reactions on the 14 cotton differentials tested (Table 5-2). The strains of X. c.

pv. malvacearum were grouped into seven plasmid types based on the restriction profiles and the conservation of related restriction fragments. In the plasmid groupings I, III, and IV, a limited level of polymorphism in the restriction fragment profiles for each group were evident, but a majority of the fragments appeared to be

conserved. In plasmid groupings II, V, VI, and VII, no polymorphisms were observed.

The strains which contained plasmids of group I appeared to be pathogenic on a majority of the cotton host differentials. X. c. pv. malvacearum strain J was an exception for it was pathogenic to only 4

of the cotton differentials, whereas the other group I strains were pathogenic on at least 11 of the cotton lines. Analysis of the EcoRI restriction fragment pattern of strain J revealed it was missing a 4.5 kb fragment, and possibly had rearrangements which resulted in a shift of two other EcoRI fragments. With few exceptions, the 4.5 kb EcoRI









56
Table 5-2. Plasmid groupings and cotton plant reactions to strains of X. campestris pv. malvacearum.


Inoculation reaction of cottonV
A B C D E F G H I J K L M N 0 P Q R S T U

K HVlb I 80.7 2 + + + + + + + + + + + + + + 14 14
N 2a I 84.8 2 + + + + v + v + + + + + + + 12 14
J HV3 Ia 59.5 1 + - + v - + - 3 4
C HVla Ib 59.0 1 + + + v v v + + v v + v v + 7 14
0 2b Ib 59.0 1 + v v + v + v v + v v v 4 12
L HV7 Ic 57.9 1 + - v + + v v v + v v v 4 11
A 2bSr II 82.2 1 + + - + - - v - 3 4
B 2aSr II 83.3 1 + + - + - - v - 3 4
E 4-1 II 83.3 1+- - - v - - - 1 2
F laSr II 83.3 1 + + - + - - v - 3 4
G lbSr II 83.3 1 + + - - - v - 3 4
S 3-22 11 83.3 1+- - - - - - - 1 1
H 4-2 II 80.4 1 + - - - - - - 1 1
W 6 IIIa 88.7 1 + - v - - - - - 1 2
I 3 IIIa 88.4 1 + - v v - - - v 1 4
V 3 IIIb 87.0 1 + - v v v - v - 1 5
D 2 IVa 94.2 1 + + - + - - v + - 4 5
M 15 IVb 93.2 1 + + - + v - - v - 3 5
X 18 V 57.5 1 + v v v + - v v v v 2 9
U 1 V 62.9 1 + v v + v v v v v v 2 10
Q la VI 100.4 2+- - - - - - - 1 1
R ib VI 91.6 2 + - v v - - v - 1 4
Z 7b VII 93.0 1 + v + v v v - v v v v 2 10
Y 7a VII 98.0 1 + - v v - - - 1 3
T 18 1 + + - - - +- 4 4


A = Strain designation. P = Cultivar Mebane B1 (B2 +
B = Original strain, or race, polygenes).
designation. Q = Cultivar Stoneville 20 (B7
C = EcoRI plasmid group. + polygenes).
D = Kilobase pairs of plasmid DNA. R = Cultivar Polygenes (2B-S9) E = Number of plasmids. S = Cultivar Gregg (resistance
F = Cultivar Acala 44. genes unknown).
G = Cultivar Acala B2. T = Sum of cultivars which gave 4
H = Cultivar Acala B3. out of 4 compatible reactions
I = Cultivar Acala B5. (+).
J = Cultivar Acala B6. U = Total of cultivars which gave
K = Cultivar Acala B7. at least 1 compatible reaction
L = Cultivar Acala BN. (H).
M = Cultivar 1-10B BIN V = Plant reactions: +, compatible
(+ polygenes). reaction; -, incompatible
N = Cultivar 101-102B (B2B3 reaction; v, reaction variable
+ polygenes). over 4 replications.
0 = Cultivar 20-3 (BN + polygenes).









57

fragment appeared to be conserved in all group I strains of X. c. pv. malvacearun. The group I strains K and N each contained two plasmids and also appeared to be the most virulent of all the strains tested in any group, each being pathogenic on all of the cotton differentials tested (Table 5-2). Strain K was isolated in Upper Volta, Africa and strain N was isolated from Texas. Although the plasmids in group I were highly conserved and apparently widespread in the world, there was no correlation between the presence of these plasmids with race specificity.

The strains which were classified into plasrnid group II could be divided into two basically different pathogenicity patterns on the 14 cotton differentials. The strains which were derived from X. c. pv. malvacearun races 1 or 2, and strains derived from races 3 and 4 appeared to have related pathogenicity patterns. The plasmid EcORI

restriction fragments patterns for all seven of these strains appeared identical. Similar variability was observed in the pathogenicity patterns for the other strains placed within groups III, IV, V, VI, and VII. Again, no correlation between the pathogenicity pattern and the plasmid group was apparent. The original race designations of the

strain mixtures of X. c. pv. malvacearu~m did not correspond to the race phenotypes exhibited by the strains purified from the mixtures. For instance, of 5 strains derived from nominal race 1 (F, G, Q, R, and U), there were basically three different pathogenicity patterns observed, none of which corresponded to the reported pattern for race 1. The expected race 1 reaction would have been pathogenic on Acala 44 and Stoneville 2B-S9 (45).








58
Plasmid Curing

In preliminary tests with the curing agents, it was determined that

0.3% SDS and 20 ug/ml ethidium bromide were the concentrations which allowed growth of X. campestris. Strains did not grow at concentrations of 0.5% SDS or 30 ug/ml ethidium bromide. The

bacterial colonies isolated from ethidium bromide treatments appeared phenotypically identical to the wild-type strains. Several colorless colonies were observed with the SDS treatment; however, over a 48 hr period, color appeared to be restored. Of 11 strains selected from the SDS treatment, all were found to be pathogenic on Acala 44. From plasmid analyses it appeared that 7 of 11 strains were missing the indigenous plasmids (Fig. 5-1).

Race-Specificity Genes

Several clones containing cloned X. campestris plasmid fragments in

the vector pUCD5 were obtained and transformed into E. coli ED8767. Based upon restriction analyses, only 3 of the 8 EcoRI fragments of strain H, and 6 of 9 EcoRI fragments of strain N were cloned. These

clones carried 30 kb (36%) of the 84 kb strain H plasmid, and 70 kb (88%) of 85 kb strain N plasmids. Each of the E. coli clones were mated into X. c pv. malvacearum. The transconjugants were then selected and inoculated onto the 14 cotton differentials. The results of the inoculations are seen in Table 5-3.

None of the clones derived from plasmid DNA of X. c. pv. malvacearum changed the expected disease reactions of X. c. pv. malvacearum strain H. One clone of X. c. pv. malvacearum strain H, originally thought to carry a large plasmid insert, did carry an









59

Table 5-3. Pathogenicity of X. campestris pv. malvacearum (Xcm) transconjugantsa on cotton host differentials.Plant reEC-dic&b
Stains,~1



wild-type:
N + + + + + + + + + + + + +
H +

Plasid fragamts of X=n H in Xan N: FEA-H + + + + + + + + + + + + +
g~',5 + + + + + + + + + + + +- +

Plasriid fragrmts of Xan N in Xcm H:
gTA~- +
g-NA3 +
FEF-N4 +
p(HA4N5 +
UEA-* +- --

O-ransnal fra~et of Xcm H in Xan N: gJF.-1. + + +- + + + + + + + + +


a Cloned EcORI fragment sizes within each clone are: ptJFA-Hl, 4.9 kb;
pUFA-H2, 19.2 and 4.6 kb; pUFA-H5, 22.4 and 4.0 kb; pUFA-NI, 2.3 kb; pUFA-N3, 14.5; ptJFA-N4, 23.5 and 13.4 kb; pUFA-N5, 15.6 kb; pUFA-N9,
13.4 and 9.6 kb.

b + =compatible (pathogenic) and = in-cmpatible (hypersensitive)
plant reactions.









60


















A B C D E F G H I J K L















Figure 5-1. Plasmid curing of X. campestris pv. malvacearum with SDS treatment. Lanes A-L are plasmId extractions of strains which were
exposed to 0.3% SDS. Lanes A, E, I, and J appear to contain the indigenous plasmid. Lanes B-D, F-H, and K-L appear to be missing the indigenous plasmid.









61

avirulence gene. Transconjugants of this clone, pUFA-H1, in strain N

caused incompatibility on the Acala BN line (Table 5-3). Upon further examination of the pUFA-H1 clone by Southern hybridization analysis, it appeared that the cloned insert was not of plasmid origin. The

radiolabeled clone did not hybridize to Southern transfers of plasmid DNA derived from strain H, or 16 other strains of X. c. pv. malvacearum. However, this 4.9 kb cloned insert of pUFA-H1 did hybridize to a 23 kb EcoRI fragment of the plasmid found in strain W. Additionally, the radiolabeled pUFA-H1 clone hybridized to a 4.9 kb EcoRI chromosomal fragment of 18 strains of X. c. pv. malvacearum, and also to another clone, pUFA-702 (33). pUFA-702 was independently

derived and appeared to encode gene-for-gene avirulence on Acala BN. Thus, it was concluded that the pUFA-H1 clone was of chromosomal origin.

Plasmid Origin of Replication and Mobilization

Seven of the eight EcoRI fragments of the single 59.5 kb plasmid found in X. c. pv. malvacearum strain X were cloned into pUCD5 (Table 5-4). All of the fragments were successfully mobilized into a spectinomycin resistant strain of X. c. pv. malvacearum strain N (NSpR) in the presence of the helper plasmid, pRK2013, using MOPS minimal medium selection. In general, it appeared that the larger cloned fragments transferred at a slightly lower rate than the smaller cloned fragments, however the transfer rate was not quantified. One of these clones, pLXF, carrying a 4.7 kb fragment, was found to encode

plasmid transfer function (tra), missing on the vector pUCD5 (14). That is, pXLF could be mated into X. campestris without aid of the









62

Table 5-4. Selection of plasmid replication genes in X. campestris pv. malvacearum strain X.


Matingsa
Size MOPS K30 POPS K30SP100
Plasmid Insert no helper pRK2013 pRK2013


pLXA 17.0 kb few +
pLXB 14.0 kb NT NT NT
pLXC 9.0 kb + few
pLXD 5.0 kb ++ ++ (ori)
pLXE 4.8 kb +
pLXF 4.7 kb ++ +++ (tra)
pLXG 4.5 kb +++
pLXH 0.5 kb +++ few


a is no growth, + = about 100 colonies, ++ = 100-300 colonies, +++ =
swamped plate, NT = not tested. MOPS K30 is the MOPS minimal medium containing kanamycin (30 ug/ml) and POPS K30SP100 is the POPS complete medium containing kanamycin (30 ug/ml) and spectinomycin (100 ug/ml) pRK2013 is a helper plasmid used in tri-parental matings (22). pLXD contains the putative plasmid origin of
replication (ori) and pLXF contains a putative plasmid transfer (tra) function of the X. campestris pv. malvacearum strain X
plasmid.








63
helper plasmid. Since the vector pUCD5 is known to carry a

mobilization function, it could not be concluded that the strain X plasmid is self-mobilizable, but it must carry at least some functions required for self-mobilization.

pUCD5 is a broad host range vector with an origin of replication (ori) that functions poorly in X. c. pv. malvacearum. Matings on MOPS minimal medium provide a lengthy period of selection in which plasmid integration can take place, provided a homologous X. c. pv.

malvacearum DNA fragment is cloned on the vector to allow recombination to occur. Rapid selection procedures using complete media containing antibiotics only rarely allows this integration to occur before the recipients die. When transconjugants with X. c. pv.

malvacearum strain X plasmid DNA were selected on POPS complete medium with antibiotics only the clone containing a 5.0 kb EcoRI fragment, pLXD, mated at a high frequency. All other clones either did not grow on POPS with antibiotics, or grew poorly. Because pLXD grew rapidly

on selective medium, whereas other clones did not, it was proposed that the pXLD clone contained a functional origin of replication from the strain X plasmid.

Transconjugants containing pXLD were further analyzed because their growth rate was highly variable. Eleven of the pLXD

transconjugants were selected by colony size and grouped as large, medium or small. Plasmids from the transconjugants were extracted and

analyzed for plasmid content. All, but possibly one transconjugant, were missing a 4.5 kb EcoRI fragment associated with the indigenous plasmid of X. c. pv. malvacearum strain N-SPR (Fig 5-2). It was









64




ABC D F 1HI J K L M




23

9
87 4

2




23 L
9
8>
7










Figure 5-2. Transconjugants of X. campestris pv. malvacearum N-SpR containing pLXD (top) and hybridized to the plasmid probe pSa4 (bottom) The transconjugants were digested with the restriction endonuclease EcoRI (top). Lanes A and M, Lamda HindII; lanes B-E, large colony-size; lanes F-I, medium colony-size, and lanes J-L, small size transconjugants. All of the transconjugants, except in lane J, appear to have lost the 4.5 kb plasmid fragment seen in wild-type strains. Plasmids in lanes F-I and K-L appear to have had other plasmid rearrangements occur. All strains had an additional 8 kb EcoRI fragment present. The autoradiograph (bottom) shows the
transconjugants probed with a pUCD5 related plasmid, pSa4 (93). The 8 kb fragment hybridized to the probe, but so did other plasmid fragments corresponding to those of X. campestris pv. malvacearum.









65

presumed that the homologous recombination occurred between the pLXD clone and the indigenous plasmid. As a result, there was no EcoRI fragment which corresponded to the 13 kb pUCD5 vector, or the 5 kb pXLD insert. However, a new 8 kb EcoRI fragment was present.

The plasmid DNA from the X. campestris transconjugants with pXLD was subsequently extracted and transformed into E. coli ED8767. The transformants were selected on Luria-Bertani medium containing kanamycin (25 ug/ml), or ampicillin (30 ug/ml). Resistance genes for

these antibiotics border the EcoRI cloning site of the pUCD5 vector

(14). All of the transformed plasmids were 8 kb in size, which indicated that the new band seen in the X. c. pv. malvacearum

transconjugants was, in fact, a new autonomously replicating plasmid (Fig. 5-3). There was obvious rearrangement in the original pXLD clone which led to a 10 kb deletion. Southern hybridization analyses

revealed the presence of DNA fragments from both pUCD5 and the insert of pLXD in this new vector, named pXD-1 (Figures 5-2 and 5-4).

Discussion

Of the twenty-five clonal isolates of X. c. pv. malvacearum derived from 11 different nominal races, none of the clonal isolates corresponded to the expected disease reactions on the host differentials for that race. Likewise, there was no correlation between a given plasmid group and the observed pathogenic reactions on the 14 cotton differential lines. Although avirulence genes have been

found on plasmids in other phytopathogenic bacteria (88), no evidence has been found to link plasmids in X. c. pv. malvacearum with racespecific interactions. In these studies, it appears the plasmids









66










A B C D E F G H I J K L M






23



87

4


2






Figure 5-3. Transformation of E. coli ED8767 with plasmid DNA from X. campestris pv. malvacearum transconjugants mated with pLXD. Lane A, lamda HindIII; lanes B-M, E. coli ED8767 transformed with DNA extracted from transconjugants mated with pLXD and selected on kanamycin Luria-Bertani plates (see Fig. 5-2). The transformants were digested with restriction endonuclease EcoRI. Only the 8 kb EcoRI fragment was present; the smaller band was undigested DNA.









67











ABC D E F G H I A BC D E F G HI
MAP "M mmam
















Figure 5-4. Plasmid DNA of X. camrpestris pv. malvacearum strain X digested with restriction endonucleases (left) and hybridized to the plasmid probe pXD-1 (right). Lanes A and I, lamda HindIII; lanes B-H, plasmid DNA from X. campestris pv. malvacearum strain X digested with
BamHI (lane B), BglI (lane C), EcoRI (lane D), HindIII (lane E), KpnI (lane F), PstI (lane G), and SalI (lane H). The 5.0 kb EcoRI fragment seen in lane D is the fragment thought to contain the origin of replication (ori).









68

served as useful markers for the genomic background. Plasmids in X. campestris generally appear to be stable and have in some instances been used as epidemiological markers, such as with the recent Florida outbreak of X. c. pv. citri (31).

However, five of the six strains with group I plasmids were more virulent than the other strains. All of the Upper Volta strains fell into this class, including some North American strains. The Upper

Volta strains were derived from recently discovered races which were

found to be virulent against all the known major genes for bacterial blight resistance (29). The X. c. pv. malvacearum strain J, although

an Upper Volta strain, appeared to be the exception and varied from the other plasmids by the loss, or rearrangement, of a 4.5 kb EcoRI fragment found in all of the other 24 strains tested. Since the other group I strains are highly virulent strains, and known to be lacking a number of cultivar specific avirulence genes (33), it may be possible that this 4.5 kb EcoRI fragment may serve some positive function in pathogenicity.

Nine avirulence genes have been reported cloned from X. c. pv. malvacearum, at least some of which are chromosomally encoded (33). With further testing, it is possible that plasmid-borne avirulence genes may be uncovered. In this study, approximately 50% of the

plasmid sequences derived from an avirulent strain were tested, leaving it still possible to uncover avirulence genes in X. campestris pv. malvacearum.

A high level of race-specific variability has been described for X. c. pv. malvacearum (8). Plasmids may be involved in genome









69

rearrangement as plasmids in P. syringae pv. phaseolicola have been found to integrate and excise from the chromosome causing rearrangement (79,92). In these studies, pUFA-H!, carrying a

chromosomally encoded avirulence gene, was found to hybridize with a plasmid DNA from X. c. pv. malvacearum strain W. The evidence that plasmids in X. c. pv. malvacearum may be self-mobilizing points to their potential role in variability of the X. campestris species. It is believed that the self-mobilizing, copper resistance plasmid identified in X. c. pv. vesicatoria facilitated the rapid build up of X. campestris strains resistant to field applied copper-containing compounds (88). If avirulence genes are on self-mobilizing plasmids,

rapid shifts in pathogenic races may occur, and the appearance of new races would be facilitated.

The cloning of the X. c. pv. malvacearum plasmid origin of replication may allow for the future construction of other cloning vectors designed specifically for the study of genes in X. campestris. Some of the currently used broad-host-range vectors appear unstable in

X. campestris and are useful because cloned inserts integrate into the chromosome. Although preliminary experiments with the X. campestris ori resulted in vector rearrangement, possibly the resolved form may be applied to the construction of future vectors.














CHAPTER SIX
PATHOVARS OF XANTHOMONAS CAMPESTRIS ARE DISTINGUISHABLE
BY RESTRICTION FRAGMENT LENGTH POLYMORPHISM*



Introduction

The species Xanthomonas campestris (Parmnel) Dowson, a member of

the family Pseudomonadaceae which is always found in association with living plants, comprises over 125 different pathovars (6). The

pathovars have been named by "the plant from which first isolated"

convention. Unfortunately, strains of some pathovars can be

pathogenic on host species in different plant families (57) ; thus possible relationships between pathovars may go undetected, since there is no convenient way to determine pathogenicity for all possible host plants, nor is there a useful systematic method for narrowing the possibilities. Further, and despite a widely held view that all xanthanonads are plant pathogens (85), non-pathogenic members of X.

campestris are often found as ectoparasitic leaf colonizers (epiphytes) (26) and occasionally as endoparasites (65,71). Their inability to provoke a pathogenic plant response renders them unclassifiable in this system. The attention given to damaging plant pathogenic strains may not be taxonomically or ecologically warranted. Genes which function to provoke a pathogenic response may be entirely

different from those which confer host selectivity and parasitic ability (32). Epiphytes and nonpathogenic endoparasites are known to

* This chapter contains copyrighted material from International Journal of Systematic Bacteriology. It is reprinted here with permission of the publisher.
70









71

be host selective (19,65). Host selectivity is thought to be a stable

characteristic (84) Since xanthomonads are always found in association with living plants, host selectivity should result in genetic isolation of a pathovar population. over time such isolation and random genetic drift should produce distinctive and stable phenotypic characteristics and therefore, establish conserved genetic markers.

Attempts to differentiate pathovars by methods other than pathogenicity have included rRNA-DNA and DNA-DNA hybridization (21,73), serology (1,94,95), phage-typing (37,59), arnd comparisons of profiles resulting from plasmid and chromosomal DNA restriction enzyme

digests (40,54), protein electrophoresis (70), and gas-chromatography of fatty acids (65). Although all of these methods are useful for specific purposes, few have demonstrated utility to replace, clarify,

or indicate further pathogenicity tests.

We report here the use of restriction fragment length

polymorphism (RFLP) analysis to differentiate pathovars of X. campestris. RFLP analyses have been widely used in the medical field to identify DNA fingerprints specific for inherited diseases or other

genetic loci (2,11,61). These analyses have also been applied to plants (41), bacteria (38), and eukaryotic organelles (67,91). RFLP analyses allow the observance of genetic variation in organisms within defined regions of the genome due to DNA rear rangemen ts or mutations which affect recognition sites for restriction endonucleases. Variation may be examined over a small (a few base pairs) or large (30-40 kb) stretch of DNA depending on the desired level of









72

polymorphism detection. This variation can be observed for random or selected DNA target sequences within a species against a background of other genomic DNA fragments. In this study, RFLP analyses were applied to investigate the degree of genetic variation among 87 strains of X. campestris, comprising 23 different pathovars. Some of these results have appeared in abstracts (34,55).

Materials and Methods

Bacterial Strains

The bacterial strains used in this study are shown in Table 6-1. These strains were isolated as plant pathogens, identified as X. campestris, and classified into pathovars according to the susceptible host plants involved. Strains were tested for pathogenicity after single colony purification. For some strains, no pathovar assignments were made due to the lack of known plant pathogenic responses. Broth cultures of bacteria were grown in a peptone-glycerol medium (per L: 10.0 ml glycerol, 20.0 g peptone, and 1.5 g K2HP04; pH 7.2). The strains were commonly stored and maintained at -80 C in the same medium containing 15% glycerol.

DNA Extraction

DNA was extracted from bacterial cultures at mid- to late-logarithmic growth phase. Extraction of bacterial DNA for cosmid library construction was by a modification of the method of Silhavy et al. (87). A critical modification was to wash cells in 50 mM TRIS, 50

mM Na2EDTA, 150 mM NaCl, resuspend in the same buffer containing 150 ug/ml proteinase K, add SDS (sodium dodecyl sulfate) to 1% (w/v), and









73
Table 6-1. Strains of X. campestris used for RFrAJP analysis.


Pathamr Strain Host Location Sourcea

Xant=mas cmpestris pabiwar

alfalfa ES madicago sativa Famas D.L.Stiteville
EL M. sativa Florida R.E.Stal I
084-1052 Azamm-- mexicEra Florida DPI
begonia 084-155 a3gonia sp. Florida DPI
LT& L Oes txis "Cl Brassica oleracea (cabbage) CkLahma t-ds sb--dy
084-809,084-1236 B. oleracea (cabbage) Florida t-ds sbxly
084-720 TT. cleracea (br. sprouts) Florida tIds sbaly
084--131-8 B. cleracea (broccoli) Florida DPI
carotae 13 Cai-= carota California R.E.Stal I
citri Y59,X70 Citrus Sp. B=il E.L.Civerolo
)62 t= S P. Japan E.L.Civerolo
X69 Citrus so' Argentina E.L.Civerclo
084-3401 citrus SP. Florida DPI
'dis 13D5 tetrapomIcba C.I.Kw3o
X002,XO05, C. t t Arizcna J.Mihail
X016,XD17 C. tet olcim ArizCr3a J.-Mihail
dieffenbachiae 084-729 Anthurim sp. Florida DPI
068-1163 Dieffenbachia sp. Florida DPI
esm1enti 084-1093 Abah s esa. lentas Florica DPI
gl cinas B-9-3 Glycine nax Brazil W.F.Rett
G. max Africa W.F.Flatt
17915 G. max W.F.Flett
S-9-8 E. Wisca-isin W.F.Fett
hs5arz e 084-1789 Ti Inlix Florida DPI
nlcioala &a Sort= vulgare Kansas L.Claflin
maculifohigardeniae 084-6166 (3ardmia sp. Florida DPI
malvacearm DMNOUV, Goss piun 1-drsutLm TMOS ii stpy
XYZUS4 G. hirsitin Tbos tiis sbjJy
ABE,,FGH G. drsqalan Od*Xra M.Es
ChlCh2 G. hdrsatn Chad L.S.Bird
M725 G. himitin Upper Volta L.S.Bird
S 9.13an
U2,9-2 G. hir-Rib-m L.S.Bird
FL79 G'. hdrs-l'= Florida ti stxly
mm-eibmaeindicae 084 -116 Fki lfem in ica Florida DPI
miaraTecalans 084-1984 Arctim lappe Florida DPI
pelargonil 084-190,084-1370 u sp. Florida r1pi
lf i EICI,)Zh25,) :M P eolus w1garis Nebraska Mdtister
P. vulgaris Vdsconsin A.W.Saettler
82-1,82-2 P. vulqaris Florida R.E.Stall
LB-2,SC-M P. vulgaris Nebraska A.K.Vidmer
XP2 P. vugaris New York J.A.Lamerre
XP-JL P. vulgaris Kmsas J.L.Leach
)GLJF P. vulgaris llissazi this sb-xty
XP-DPT P. vulgaris this sb--dy









74

Table 6-1 (continued).


pathaVar Strain Host Lccation SaZCea


pisi Xpi Pisan sativun J--PM nGDto
poirret:tiicc1a 083--6248 El#x)rbia palcherrima Florida CpI
pard 084-1793 Prunis sp. Florida DPI
tonsluoa-z M05 Hcrdan Ep. Mat:zna D.Smds
vesicatcria F,3 C.-psic= arn-in Florida R.E.Stall
75-3 Lw-persiccn esculenbn Florida R.E.Stall
vigniccla A81-331,0-1, Vigna =uicilata Gxxgia R.D.Gitaitis
C25-11 V. Lrxjiumlata Gt=gia R.D.Gitaitis
XV19 qv, V. =juiculata Gam-gia R.D.Gitaitis
432,82-38 V. UTuiculata GB=gia R.D.Gitaitis
vitia-Z ICVa64 Lab-ra Sp. R.E.StaLl
zinnias 084-1944 Zinnia elegant Florida DPI

uial)iln 084-1373 Aiiloder&cn sp. (dieffen.) Florida DPI
084--3928 Fatsia sp. Cha3arae) Florida DPI
084-4348 Alocasia. sp. (vitians) Florida DPI
083-2057 Swrjoniu-n sp. (vitians) Florida DPI
084-2B48 CiSS.3S Sp. Florida DPI
084-1590 an us sp. Florida DPI
251G,084-40 hTpatiem S O- Elcrida DPI
064-6006 Jasrdnium sp. Florida ME

a-agariae xfrai Fragaria sp. Florida R.E.Stall


a DPI = Florida Department of Agricultural and Consumer Services,
Division of Plant Industry, Gainesville, Florida.









75

heat for 1 hr at 50 C. The sample was then extracted 2 times with phenol/chloroform/isoamyl alcohol (25:24:1; phenol equilibrated to pH 7.8 with 0.1 M TRIS), and the DNA spooled out from the aqueous phase after adding sodium acetate at 30 mM and 2 volumes 95% ethanol. This DNA was then washed in 70% ethanol. Extracted DNA was resuspended in TE (10 mM Tris(hydroxymethyl)aminomethane (TRIS), 1 mM disodium ethylenediaminetetraacetate (Na2EDTA) containing 20 ug/ml DNase-free pancreatic RNase; pH 7.6).

Agarose Gel Electrophoresis

Approximately 5 ug of DNA was digested with EcoRI or BamHI as specified by the manufacturer (Bethesda Research Laboratories). Digested DNA samples were run in a 20x25 can agarose gel (0.6%; type II, Sigma) in TRIS-acetate buffer (40 mM TRIS-acetate, 1 mM Na2EDTA; pH 7.6) with electrophoresis of gels set at 35 V for 14-15 hrs. Fragments were visualized by ultraviolet irradiation (302 nm) after staining agarose gels in ethidium bromide (0.5 ug/ml). Photographs were taken using Polaroid Type 55 (or Type 57) film and a yellow filter (Tiffen no. 12). After the gels were photographed, the DNA was transferred to nitrocellulose by the method of Southern as described by Maniatis et al. (66). Restriction fragment sizes were estimated

using DNA molecular size standards of lambda phage DNA digested with HindIII.

DNA Probes

The DNA probes used in this study were derived from a genomic library of X. c. pv. citri strain 3401 constructed into the modified cosmnid cloning vector pUCD5B, which was the vector pUCD5 (14) with a 2









76

kb Bam I fragment deleted from it. DNA of strain 3401 was partially digested with the restriction enzyme Mbol and fractionated by size on a 10-40% (w/v) sucrose step gradient (66). The pUCD5B vector was

digested with the restriction enzyme BamHI and treated with calf intestinal alkaline phosphatase (Boehringer-Mannheim). Vector DNA and

large fragment size fractions (25-40 kb) from strain 3401 were mixed and treated with T4 DNA ligase (Bethesda Research Laboratories) and recombinant cosmids were packaged in vitro with extracts prepared and utilized according to Scalenghe and Hohn's protocol II as described by

Maniatis et al. (66). Transduction with Escherichia coli HB101 was performed using top agar on Luria-Bertani medium containing kanamycin

(35 ug/ml). Cloned DNA fragments of strain 3401 in the vector averaged 27-38 kb.

DNA Hybridization

DNA clones used as probes were radiolabeled using 30-60 uCi of 32P-deoxycytidine (Du Pont NEN) with use of a nick translation kit (Bethesda Research Laboratories) and separated from low-molecular weight nucleotides on a mini-column of Sephadex G50-100. Southern

blots were pre-hybridized in plastic bags for 4 hr and hybridized with

the DNA probe for 16-18 hrs at 68 C. The pre-hybridization and

hybridization solutions were those described in Maniatis et al. (66).

Following hybridization, membranes were washed once in 2X SSC, 0.5% SDS (lX SSC is 0.15 M NaCl, 0.15 M sodium citrate, pH 7.0) and washed once in 2X SSC, 0.1% SDS at ambient temperature, and washed two times in 0.!X SSC, 0.5% SDS at 68 C as described by Maniatis et al. (66) for 'stringent' conditions. Nitrocellulose membranes were then air-dried









77

and exposed to Kodak X-Omat AR film at -80 C in cassettes with intensifying screens. Hybridization of the probes to individual strains of X. campestris was repeated at least three times. Restriction Fracment Patterns and Densitometr

From developed autoradiographs, DNA fragment sizes and profiles were determined and hybridization signals measured using a Gilford

Response spectrophotometer equipped with an autoradiograph scanner. Routine scans were done at 600 nm wavelength with 0.5 m aperture and 0.5 nm bandwidth setting. Scanning data was stored by computer. Using above information, comparison were made among strains of a given

pathovar and also strains derived from different pathovars of X. campest ris.

Results

Agarose Gel Electrophoresis

For a given pathovar, the banding patterns appeared very similar

and polymorphism in these strains was apparently limited (Fig. 6-1). In all, DNA from 87 strains of X. campestris, representing 23 different pathovars and including some with no known pathovar status were digested with restriction enzymes EcoRI and BamHI and separated by size on agarose gels. Based on restriction fragment patterns

alone, it was possible to visualize DNA variability among the strains,

but recognition of particular DNA fragment banding patterns was difficult due to the numerous DNA fragments involved (Fig. 6-2). Some of the apparent variation was due to the presence of plasmid bands, which appear brighter against the chromosomal bands due to their relatively higher copy number within each cell.









78







AB C D E F G H I JK LM NO P Q R S T







12.2 11.2

7.9

S.6
5.1



3.2


2.2

1.6





Figure 6-1. Genomic DNA of strains of X. campestris digested with the restriction endonuclease EcoRI. Lane A, probe XCT1; lanes B-K, X. c. pv. phaseoli; lane B, JL; lane C, LB-2; lane D, Xph25; lane E, EKll; lane F, 82-1; lane G, 82-2; lane H, Xpa; lane I, XP2; lane J, XP2-1; lane K, JF; lanes L-M, X. c. pv. phaseoli var. fuscans; lane L, SC-3B; lane M, Xpfll; lanes N-O, X. c. pv. alfalfae; lane N, KS; lane O, FL;lanes P-R, X. c. pv. campestris; lane P, XCl (cabbage); lane Q, 084-1318 (broccoli); lane R, 084-720 (brussels sprouts); lane S, unknown X. c. G65; lane T, probe XCT11.









79


















12.2







F11r.2.enncDNoftrisoX.cmetsdietdwhte resritin ndnulese coI.Lae roe X~l lneBX.C.pZ














FLigule LX. mc.DN pvf srarnsi 084-.90; lane M, Xigcstpd wphastol vla.fuan SL3; lane X. . 04-1X55; lane D, X. C. pv. tanpsuces 080; lane X. c. pv. vscataa 73; lane Cv. ..pv. igca S--; lane X. c. pv. itiansl ICP6; lane X. C pv. zinniae 084-1944; lane T, probe XOT11.









80

DNA Probes

Two cosmid DNA clones, XCTl and XCTl1 were randomly selected from a genomic library of strain 3401 for use as DNA probes. XCT1 carried

a 30 kb insert, and XCTl1 carried a 37 kb insert, as determined by adding up the cloned DNA inserts from restriction endonuclease digests. There were no detectable plasmids in strain 3401, thus the cosmid clones are assumed to contain chromosomal fragments. Assuming a 3333 kb genome for X. campestris (47), each cosmid probe represents approximately 1% of the total genome. DNA Hybridization

Autoradiographs of Southern blots hybridized against either of the two DNA probes revealed conserved DNA fragments within each pathovar. For example, in figure 6-3 (lanes B K) are shown the RFLP pattern from 10 different strains of X. c. pv. phaseoli from different geographic locations. At least five EcoRI cut DNA fragments which hybridized to XCT1 appeared to be conserved in this pathovar. In

lanes L and M are the RFLP patterns of two strains of X. c. pv. phaseoli var. fuscans. These X. c. pv. phaseoli var. fuscans strains also attack beans (and hence have the pv. phaseoli designation), but are biochemically distinct (6). They produce a dark, melanin-like pigment in nutrient media and in these tests are clearly genetically distinguishable from the other X. c. pv. phaseoli strains. Conserved DNA fragments were also seen for X. c. pv. alfalfae (Fig. 6-3, lanes N and 0) in which six of the up to 15 fragments appeared identical. Likewise, in four different strains of X. c. pv. campestris (Fig. 6-3, lanes P Q, Fig. 6-5, lane D) isolated from three different crucifer


















A B C D E F G H I J K LM NO PQ RST




12.2
11.2

7.9

5.6
5.1



3.2



2.2


1.6





Figure 6-3. Genomic DNA of strains of X. campestris digested with the restriction endonuclease EcoRI and probed with cosmid clone XCT1. Lane A, probe XCTl; lanes B-K, X. c. pv. phaseoli; lane B, JL; lane C, LB2; lane D, Xph25; lane E, EK11; lane F, 82-1; lane G, 82-2; lane H, Xpa; lane I, XP2; lane J, XP2-1; lane K, JF; lanes L-M, X. c. pv.
phaseoli var. fuscans; lane L, SC-3B; lane M, Xpfll; lanes N-O, X. c.
pv. alfalfae; lane N, KS; lane O, FL;lanes P-R, X. c. pv. campestris; lane P, XCl (cabbage); lane Q, 084-1318 (broccoli); lane R, 084-720
(brussels sprouts); lane S, unknown X. c. G65; lane T, probe XCT11.









82
sources (cabbage, broccoli, and brussels sprouts) at least four of over 10 fragments appeared to be identical. Sizes of hybridizing DNA

fragments are given in Table 6-2. In figure 6-4 is shown the RFLP patterns revealed using clone XCT11. In each case, a different RFLP

pattern is shown, but the same genetic relationship is revealed. Other sets of conserved DNA fragments within a pathovar were seen among seven strains of X. c. pv. vignicola (identical pattern), seven strains of X. c. pv. cyamopsidis (nearly identical), and at least three strains each for X. campestris pvs. citri, glycines, malvacearum, pelargonii, and vesicatoria (not shown).

DNA of strains representing other pathovars of X. campestris were also digested with either EcoRI or BamHI, and hybridized with either XCT1 or XCT11. The pathovars of X. campestris included in these

experiments were X. campestris pvs. alfalfae, begoniae, campestris, carotae, citri, cyamopsidis, dieffenbachiae, esculenti, glycines,

hederae, holcicola, maculifoliigardeniae, malvacearum, mangiferaeindicae, nigromaculans, pelargonii, phaseoli, ,

translucens, vesicatoria, vignicola, vitians, and zinniae. Also

included were some strains isolated as pathogens of an Alocasia sp., Cissus sp., Impatiens sp., Fatsia sp., Jasmine sp., Argemone sp., and a Euonymus species for which no pathovar designations were available.

The results of hybridization of probe XCT1 to DNA of different pathovars of X. campestris digested with EcoRI are shown in Figure 65. The clone XCTI appeared to contain DNA fragments of strain 3401 which were able to demonstrate variability of restriction fragment patterns among different pathovars of X. campestris. Variability in









83

Table 6-2. Sizes of DNA fragments from. Xanthanonas campestris genomic digests (EcoRI) which hybridized to the XCT1 DNA probe. See Figure 63.


A 12.2, 11.2, 7.9, 5.6, 5.1, 3.2, 2.2, 1.6, 1.0 D 14.1, 13.3, 10.4, 7.6, 6.4, 4.6, 4.3, 4.0, 3.7, 3.3, 3.1
C 14.4, 13.3, 10.4, 7.6, 6.4, 4.6, 4.3, 4.0, 3.7, 3.3, 3.1
D 14.1, 13.1, 10.4, 7.5, 6.4, 5.2, 4.6, 4.3, 4.0, 3.7, 3.1
E 13.9, 11.6, 10.4, 8.9, 5.2, 5.0, 4.6, 4.3, 4.0, 3.7, 3.1
F 13.7, 13.1, 11.6, 10.4, 8.9, 5.2, 5.0, 4.6, 4.3, 4.0, 3.7, 3.1,
1.9
G 13.7, 13.1, 11.6, 10.4, 8.9, 5.2, 5.0, 4.6, 4.3, 4.0, 3.7, 3.1,
1.9
H 13.1, 10.4, 7.5, 5.2, 5.0, 4.6, 4.3, 4.0, 3.7, 3.1, 1.9
1 13.1, 10.4, 7.5, 5.2, 5.0, 4.6, 4.3, 4.0, 3.7, 3.1, 1.9
J 13.1, 10.4, 7.5, 5.2, 5.0, 4.6, 4.3, 4.0, 3.7, 3.1, 1.9
K 13.1, 11.6, 10.4, 8.9, 5.2, 5.0, 4.6, 4.3, 4.0, 3.7, 3.1
L 11.9, 8.4, 7.1, 6.7, 4.5
M 17.0, 15.3, 10.0, 8.4, 4.5 N 19.7, 19.1, 15.5, 14.9, 12.9, 12.1, 8.0, 4.6, 3.8, 3.1, 2.7, 1.7
0 19.4, 18.0, 14.7, 13.7, 13.1, 11.7, 9.7, 7.3, 6.2, 5.2, 4.6, 3.7,
3.4, 3.1
P 20.3, 15.3, 10.9, 10.5, 10.2, 9.3, 8.4, 6.9, 5.9, 5.2, 4.4, 3.9,
2.6, 2.2, 1.9
Q 12.9, 10.8, 10.5, 9.1, 8.4, 7.7, 7.2, 6.3, 4.6, 4.4, 3.9, 3.3,
2.4, 2.2, 1.9
R 18.6, 15.8, 12.7, 10.8, 10.2, 9.1, 8.4, 7.7, 6.7, 5.6, 4.4, 3.3,
2.2, 1.9
S 11.3, 8.0, 6.9, 3.4, 2.5, 1.6 T 17.2, 15.3, 14.3, 10.5, 8.6, 7.2, 6.8, 5.8









84






A B C D E F G H I J K LM NO P 0 RST




12.2
11.2

73


5A
5.1










1.6






Figure 6-4. Genomic DNA of strains of X. campestris digested with the restriction endonuclease EcoRI and probed with cosmid clone XCT11.
Lane A, probe XCT1; lanes B-K, X. c. pv. phaseoli; lane B, JL; lane C, LB-2; lane D, Xph25; lane E, EKII; lane F, 82-1; lane G, 82-2; lane H, Xpa; lane I, XP2; lane J, XP2-1; lane K, JF; lanes L-M, X. c. pv.
phaseoli var. fuscans; lane L, SC-3B; lane M, Xpfll; lanes N-O, X. c.
pv. alfalfae; lane N, KS; lane O, FL;lanes P-R, X. c. pv. campestris; lane P, XCl (cabbage); lane Q, 084-1318 (broccoli); lane R, 084-720
(brussels sprouts); lane S, unknown X. c. G65; lane T, probe XCT11.









85







AB C DE FG H I J K LM NOP Q R ST





12.2
11.2

7.9

5.6
5.1



3.2












Figure 6-5. Genornic DNA of strains of X. campestris digested with the restriction endonuclease EcoRI and probed with cosmid clone XCT1. Lane
A, probe XCTl; lane B, 7. c. pv. alfalfae FL; lane C, X. c. pv. begoniae 084-155; lane D, X. c. pv. caxnpestris 084-809; lane E, X. c. pv. carotae 13; lane F, X. c. pv. cyamopsidis X002; lane G, X. c. pv. dieffenbachiae 068-1163; lane H, X. c. pv. glycines S-9-3; lane I, X. c. pv. holcicola Xh6G; lane J, T. c. pv. maculifoliigardeniae 0846-166; lane K, X. c. pv. rnalvacearum FL79; lane L, X. c. pv. pelargonii 084-190; lane MX.c. pv. phaseoli var. fuscans S-C-3; lane N, X. c. pv. pisi XP1; lane 05, X. c. pv. translucens X1105; lane P, X. c. p. vesicatoria 75-3; lane Q, X. c. pv. vignicola, SN2; lane R, T. c. pv.* vitians ICPBl64; lane S, X. c. pv. zinniae 084-1944; lane T, probe XCT11.









86

RFLP pattern appeared as qualitative and quantitative differences among hybridization profiles of various sized DNA fragments in the

strains. For instance, the weak hybridization signal seen from X. c. pv. translucens (fig. 6-5, lane 0) suggests that this strain is distinct from the other pathovars compared, at least over the 1% of the genome contained on the clone XCTI. In other pathovars such as X. c_. pv. alfalfae (fig. 6-5, lane B), X. c. pv. begoniae (lane C), X. c.

pv. cyamopsidis (lane F), X. c. pv. malvacearum (lane K), X. c. pv. vignicola (lane Q), and X. c. pv. vitians (lane R) where some DNA fragments had strong hybridization signals, a close relatedness with portions of DNA in the cosmid clone is suggested. Some of the DNA fragments from different pathovars which hybridized most strongly to

the probe were also of identical size with the EcoRI fragments of the XCT1 clone. As expected, DNA of X. c. pv. citri strain 3401 digested with EcoRI, or BamHI (34,55) contained fragments which corresponded to

DNA fragments of the XCT1 probe (Fig. 6-5, lane A). Sizes of

hybridizing DNA fragments are given in Table 6-3.

From observations using different enzymes and different probes,

the same patterns emerged, indicating that the basic chromosome structure of X. campestris can be used to differentiate strains into specific RFLP types. Although a pathovar may contain more than one type of variant (e.g. X. c. pv. phaseoli), all strains of a given type appear to exhibit the same host selectivity. The RFLP pattern is highly distinctive for each type, and can be used to unambiguously assign an unknown sample to a pathovar, using simple visual

comparisons of pattern against a set of known samples, such as those given in Figure 6-5.









87

Table 6-3. Sizes of DNA fragnents from Xanthomonas campestris genomic digests (EcoRI) which hybridized to the XCTl DNA probe. See Figure 65.


A 12.2, 11.2, 7.9, 5.6, 5.1, 3.2, 2.2, 1.6, 1.0 B 20.5, 18.8, 15.3, 14.2, 13.5, 12.0, 7.3, 6.2, 5.1, 4.6, 3.7, 3.4,
3.1
C 18.7, 12.1, 9.5, 8.6, 8.2, 7.7, 6.9, 6.7, 6.2, 6.0, 4.8, 4.2,
4.0, 3.4, 3.0, 2.9, 2.5, 2.3
D 19.4, 16.4, 14.4, 13.1, 11.1, 10.5, 9.2, 8.5, 7.7, 6.7, 5.6, 4.3,
4.0, 2.2, 1.9
E 17.9, 12.9, 4.1, 3.5, 3.0, 2.3, 1.5 F 16.2, 11.5, 9.5, 8.7, 5.7, 4.0, 3.6, 3.1, 3.0, 1.4 G 14.2, 11.1, 4.5, 3.9, 3.5, 3.0 H 12.2, 10.2, 8.3, 7.91 6.5, 5.5, 4.2, 3.1 1 12.9, 12.0, 7.6, 7.1, 6.5, 4.7, 3.0, 1.7 J 15.6, 14.2, 9.4, 7.9, 6.3, 5.0, 4.6, 4.0, 3.1, 2.5, 2.2
K 20 .9, 17.1, 16.2, 15.5, 14.2, 11.0, 10.4, 9.2, 8.2, 7.5, 6.7,
4.2, 4.0, 3.3, 3.1, 3.0
L 19.4, 12.7, 10.4, 9.0, 7.9, 7.7, 6.8, 5.7, 4.2, 3.9, 3.0, 2.7,
2.3, 1.9, 1.5, 1.4
M 12.0, 8.4, 7.0, 6.8, 4.5 N 15.9, 8.4, 4.9, 3.4, 2.1, 1.9
0 9.2, 4.4
P 13.3, 9.2, 7.2, 6.9, 6.2, 4.6, 3.8, 3.1, 2.1
Q 11.0, 8.5, 7.0, 6.3, 4.2, 3.2 R 16.7, 14.2, 12.9, 9.9, 7.4, 6.9, 6.3, 4.8, 3.8, 3.5, 3.2, 2.6,
2.4, 2.2, 1.6
S 12.9, 12.5, 9.5, 7.7, 6.5, 4.4, 4.0, 3.8
T 17.6, 15.6, 14.7, 10.5, 8.6, 7.2, 1.4









88

Discussion

The only widely accepted and most practical method for differentiating pathovars of X. campestris is to inoculate a plant suspected as the host for that pathovar. This practice can sometimes be tedious, time consuming, subjective and subject to a surprising number of artifactual influences. It is not known whether host selectivity is unstable as suggested by Dye (23); stable as suggested

by Schnathorst (84) or even taxonomically significant. Although the classification is thought to be useful, it can be highly misleading since emphasis is placed upon one characteristic pathogenicity. If

only one or few gene differences were involved in host selection, then differentiation by pathovar could be highly misleading, at least in the sense that a given group of strains might be capable of attacking more than one host in some cases. Alternatively, two or more strains of relatively unrelated groups could be cataloged together because they happen to attack the same host. This latter situation appears to be the case with X. c. pv. phaseoli and X. c. pv. phaseoli var. fuscans (see Fig. 6-3), and with Florida strains of X. c. pv. citri

(34).

To address these problems, we evaluated the potential of using DNA sequence variation of X. campestris for strain classification purposes. DNA sequence variation was first examined by digesting DNA

with restriction enzymes and visualizing directly the resulting fragments on ethidium bromide stained gels. These stained gels were useful for side-by-side comparisons of restriction fragments for samples run on the same gel, but comparisons between different gels









89

were more difficult. Subsequently, visualizing and comparing

variation in bacterial genomes with cosmid clones carrying 30-38 kb cloned X. campestris genome fragments was simplified. In both cases variation was revealed by alterations in the sizes of visualized DNA restriction fragments (RFLPs). The use of DN4A probes derived from chromosomal DNA fragments also alleviated some of the difficulty in comparing stained gels with the sometimes present polymorphism of the higher copy number plasmid DNA fragments which can occur in the background.

Some of the selected clones tested as DNA probes appeared to be useful for identifying DNA sequences conserved within a given pathovar, while others appeared to identify DNA sequences which were highly conserved at the species level. The DNA which was considered conserved at the species level was represented as banding patterns which were nearly identical among all strains over the several pathovars tested. Examples of DNA sequences which are known to be highly conserved are rP1NA encoding genes (38,83). If smaller DNA probes containing known genetic loci such as those associated with genes for pectate lyase and protease (18), or avirulence activity (33,88) were tested, these smaller, more defined DNA probes could be used to determine if such genetic loci were highly conserved or variable. With larger, randomly selected probes, the likelihood was increased for detecting strain- or pathovar-specific variation. The large (greater than 30 kb) size of the probes used in this study allowed detection of both highly conserved and variable regions.




Full Text
I certify that I have read this study and that in my opinion it
conforms to acceptable standards of scholarly presentation and is
fully adequate, in scope and quality, as a dissertation for the degree
of Doctor of Philosophy.
Dean W. Gabriel, Chairman
Assistant Professor of Plant
Pathology
I certify that I have read this study and that in my opinion it
conforms to acceptable standards of scholarly presentation and is
fully adequate, in scope and quality, as a dissertation for the degree
of Doctor of Philosophy.
Robert E. Stall
Professor of Plant Pathology
I certify that I have read this study and that in my opinion it
conforms to acceptable standards of scholarly presentation and is
fully adequate, in scope and quality, as a dissertation for the degree
of Doctor of Philosophy.
Daryl R. Prind
Professor of Plant Pathology
I certify that I have read this study and that in my opinion it
conforms to acceptable standards of scholarly presentation and is
fully adequate, in scope and quality, as a dissertation for the degree
of Doctor of Philosophy.
')y] oaA rB ci-vj cp\
Mark J. Bassett/
Professor of Horticultural
Science
This dissertation was submitted to the Graduate Faculty of the College
of Agriculture and to the Graduate School and was accepted as partial
fulfillment of the requirements for the degree of Doctor of
Philosophy.
May, 1987
Dean,/(College of Agriculture
Dean, Graduate School


46
Southern hybridization analyses confirmed that plasmid DNA fragments
of similar size were in fact highly homologous. For example, X. c.
malvacearum strain N, isolated in North America, had a restriction
digest pattern identical to one of the African strains (K) (Fig. 4-2).
All North American strains except strain N form a distinctive pattern
subgroup and the African strains form a somewhat different subgroup.
It is possible that either all the African strains are derived iron
strain N, or that strain N was introduced to the North America from
Africa. The latter possibility seems more likely, since the African
strains were isolated from more than one location in Africa. The
conservation of overall restriction fragment profiles of plasmids from
geographically isolated populations and the extent of homology seen in
Southern hybridizations strongly suggests that plasmid sequences are
both highly conserved and stable.
Conversely, it was surprising to find so much polymorphism of
plasmids between pathovars. Homology among plasmids in different
strains of one pathovar of Pseudomonas syringae (pv. glycinea) has
been reported (15). However, identical plasmid profiles were found
present in more than one pathovar of P. syringae (77). In the present
study, similar plasmid profiles were not found in more than one
pathovar. In addition, cloned plasmid fragments were identified which
failed to hybridize to plasmids of different pathovars. For example,
a cloned 2.3 kb plasmid fragment of X. c. phaseoli, which hybridized
to all strains of that pathovar, failed to hybridize to total DNAs of
22 other pathovars. This DNA fragment is highly conserved and is
apparently pathovar-specific, with one exception (Table 4-3) .


43
Figure 4-5. Plasmid DNAs from strains of Xanthamonas campestris pvs.
phaseoli (Xcp; lanes B, D-I), phaseoli var. fuscans (Xcpf; lane C) ,
and cyamopsidis (Xcc; lane K) digested with restriction endonuclease
EcoRI (left) and autoradiographs of plasmid DNAs probed with a clones
containing a 2.0 kb EcoRI fragment (center) and a 2.3 kb EcoRI plasmid
fragment (right) from Xcp strain XP2. Lane J contains chromosomal DNA
of X. campestris pv. alfalfae (Xca) digested with EcoRI. Lanes shown
above contains: A and L, lambda HindIII; B, Xcp Xph25; C, Xcpf Xpfll;
D, Xcp EKll; E, Xcp 82-1; F, Xcp Xpa; G, Xc G65; H, Xcp Xpll; I, Xcp
XP-JF; J, Xca FL; and K, Xcc 13D5. Xc G65 is a strain isolated from
bean which was determined to be nonpathogenic and contains no plasmid.


after four days. The lesions extended from the inoculation sites
giving the appearance that the bacterium was spreading throughout the
leaf.
15
The strains of X. c. pv. phasedi which were found pathogenic on
kidney bean were not found to be pathogenic on cowpea, soybean, or
alfalfa. In cowpea and soybean, a hypersensitive reaction was
apparently elicited, resulting in dry necrotic lesions at the
inoculation sites. In soybean the lesion was slightly chlorotic,
whereas for cowpea, a wine red lesion occurred. However, X. c. pv.
phasedi strain XP2 did not elicit the wine red color on cowpea (Table
3-2). No response was observed on alfalfa from the spray inoculations
with X. c. pv. phasedi.
The strains of X. c. pv. vignicola were pathogenic on cowpea,
resulting in irregularly shaped lesions which had a tearing, or
"shothole" appearance at the inoculation site. On soybean, the
incompatible response appeared dry and collapsed. Again, no response
was observed from spray inoculation on alfalfa (Table 3-2).
On kidney bean, a range of different responses occurred from
inoculations with X. c. pv. vignicola. For two of the strains, A81-
331 and Cl, an incompatible response appeared as a dry collapsed
lesion. For the other strains of X. c. pv. vignicola, there appeared
to be sane slight water-soaking in the tissue about the periphery of
the inoculation site. In two of these instances, for strains 8238 and
SN2, the "shothole" effect as seen on cowpea was observed.
The strains of X. c. pv. glycines appeared to be pathogenic on
both soybean and kidney bean, but not on cowpea or alfalfa (Table 3-
2). The pathogenic responses appeared as watersoaked lesions, with


23
Figure 3-1. Polyacrylamide gel electrophoresis of total proteins from
strains of X. campestris. Lanes A and B, X. c. pv. alfalfae; lanes C-
F, X. c. pv. glycines; lanes G-I, X. c. pv. phaseoli; lane J, X. c.
pv. phaseoli var. fuscans; lane K, X. c. pv. pisi, lane L, X. c. pv.
cyamopsidis; lanes M-0, X. c. pv. vignicola; and lane P, X. c. pv.
malvacearum (cotton). Protein size is labeled in kilodaltons.


CHAPTER ONE
INTRODUCTION
The genus Xanthanonas Dowson consists of bacteria described as
being gram-negative, obligately aerobic, non-fermentative rods which
are motile by a single polar flagellum and can produce a brominated
yellow pigment, called xanthcmonadin (6) All reported strains of
this genus have been described as plant associated, and most are
reported as being pathogenic to a particular plant host. Based on
microbiological classification, this genus can be separated into at
least five separate species: X. campestris (Pairmel) Dowson, X.
albilineans (Ashby) Dowson, X. ampelina Panagopoulos, X. axonopodis
Starr and Garces, and X. fragariae Kennedy and King (and possibly X.
populi (Ride) Ride and Ride) Over 125 variants, or pathovars, of X.
campestris, are essentially indistinguishable from each other, except
for their plant host range (6,89).
To maintain order in differentiating among strains of X.
campestris by host range, the rank of pathovar has been assigned in
addition to the species name (24,98). The primary means by which
strains can be differentiated as pathovars is by inoculation of the
plants which serve as susceptible hosts to a given pathovar. The
convention for naming pathovars of X. campestris has generally been to
name it for the host from which it was first isolated (8 9) A
particular pathovar designation suggests a limited host range for a
1


CHAPTER TWO
LITERATURE REVIEW
At least 124 monocotyledonous and 268 dicotyledonous plants
species are susceptible to infection by bacterial strains of the genus
Xanthomonas (57). Compiled listings indicate that the genus
Xanthanonas has a wide host range extending over nine monocotyledonous
families comprising 66 genera, and 49 dicotyledonous families
comprising 160 genera. Pathogenicity toward gymnosperms or lower
plants has not been reported.
Although pathovars are generally assumed to have a restricted
host range, seme strains of X. campestris have extended host ranges to
more than one plant species. In these instances, host range usually
extends toward other members of the same plant family (with few
exceptions). For example, the plant families Ccmpositae, Cruciferae,
Graminae, and Leguminosae each contain different species susceptible
to the same X. campestris pathovar (X. c. pv.). An example of a broad
host range pathovar of graminaceous hosts is X. c. pv. translucens
(Jon., John., and Redd.) Dye, which is pathogenic on barley, rye, and
wheat. Other strains pathogenic on graminaceous hosts have been
described, but their interrelationships among the other pathovars were
poorly defined (6). In another example, X. c. pv. alfalfae, is
pathogenic on the legumes alfalfa, bean, and pea. Another special
relationship is that of X. c. pv. vesicatoria (Doidge) Dye, which is a
4


polymorphism detection. This variation can be observed for random or
selected DNA target sequences within a species against a background of
72
other genomic DNA fragments. In this study, RFLP analyses were
applied to investigate the degree of genetic variation among 87
strains of X. campestris, comprising 23 different pathovars. Seme of
these results have appeared in abstracts (34,55).
Materials and Methods
Bacterial Strains
The bacterial strains used in this study are shown in Table 6-1.
These strains were isolated as plant pathogens, identified as X.
campestris, and classified into pathovars according to the susceptible
host plants involved. Strains were tested for pathogenicity after
single colony purification. For some strains, no pathovar assignments
were made due to the lack of known plant pathogenic responses. Broth
cultures of bacteria were grown in a peptone-glycerol medium (per L:
10.0 ml glycerol, 20.0 g peptone, and 1.5 g K2HPO4; pH 7.2). The
strains were commonly stored and maintained at -80 C in the same
medium containing 15% glycerol.
DNA Extraction
DNA was extracted from bacterial cultures at mid- to
late-logarithmic growth phase. Extraction of bacterial DNA for cosmid
library construction was by a modification of the method of Silhavy et
al. (87). A critical modification was to wash cells in 50 mM TRIS, 50
mM Na2EDTA, 150 mM NaCl, resuspend in the same buffer containing 150
ug/ml proteinase K, add SDS (sodium dodecyl sulfate) to 1% (w/v), and


BIOGRAPHICAL SKETCH
Gerard Raymond Lazo was born in Dallas, Texas on August 25, 1957,
to Francisco Gerardo and Suzanne Marie Lazo. He completed his primary
and secondary education in Weslaco, Texas, where he graduated from
Weslaco High School in 1975. He received the degree of Bachelor of
Science in microbiology in 1979 from Texas A&M University. He
continued his education under the supervision of Luther S. Bird
conducting research on fungal and bacterial diseases of cotton and
received a Master of Science degree in plant pathology frcm Texas A&M
University in 1984. Gerard was married to Maria Alicia Gonzales in
1983 while attending Texas A&M University. In the fall of 1983 he
began to pursue the degree of Doctor of Philosophy in plant pathology
at Oklahoma State University and later continued his education at the
University of Florida under the supervision of Dean W. Gabriel. Upon
completing his degree requirements he plans to serve as a postdoctoral
research associate with Robert A. Ludwig at Thimann Laboratories,
University of California, Santa Cruz. Gerard's permanent address is
1807 Briarcrest Lane, Arlington, Texas 76012.
106


93
host-range specifying functions may be plasmid encoded. Strains of X.
c. pv. phaseoli and X. c_. pv phaseoli var. fuscans have the same host
range, but differ significantly in colony appearance, physiological
tests, and chromosomal RFLP analyses. However, plasmids from these
strains appeared highly related by restriction fragments profiles and
Southern hybridization. Thus, these strains are quite distinct
biochemically and chromosomally, but similar as characterized by the
plasmids ana by host range. This correlation between host range and
plasmids allowed the use of plasmids and cloned plasmid fragments as
epidemiological markers for detection and rapid identification of X.
c. pv. citri strains in Florida. Plasmid RFLP analyses are limited to
those strains which carry plasmids; only 95 of 151 strains of X.
campestris tested, or 11 of 26 pathovars tested had plasmids in all
strains.
Restriction Fragment Length Polymorphism
Chromosomal DNA RFLP analyses proved to be a reliable method to
differentiate X. campestris by pathovar, at least for those pathovars
tested. This method provided a means to resolve taxonomic
ambiguities. For example, X. c. pv. phaseoli and X. c. pv. phaseoli
var. fuscans, appear quite different by chromosomal RFLP analyses, and
share a common host. By contrast, nearly all 100 strains tested from
24 pathovars had RFLP patterns characteristic for the pathovar, and
very little intrapathovar variation was present.
The use of RFLP methods has already been useful in other studies
as an applied tool in uncovering the origin of strains of the citrus
canker pathogen found in Florida (Gabriel et al., unpubl.). An RFLP


30
(Table 4-1 continued.)
Bacterium
Strain designation
(number of strains)
(geographic origin)
Source9
pisi (1)
XP1 (Japan);
M.Goto
poinsettiicola (1)
083-6248 (Florida);
DPI
pruni (3)
068-1008,084-1793 (Florida);
DPI
82-1 (Florida);
R.E.Stall
translucens (2)
82-1 (Florida);
R.E.Stall
XTl;
this study
vesicatoria (5)
E-3,69-13,71-21,
82-8,82-23 (Florida);
R.E.Stall
vignicola (7)
A81-331,C-1,CB5-1,
Xvl9,SN2,432,82-38 (Georgia);
R.D.Gitaitis
vitians (3)
084-2057,084-2848,
084-4348 (Florida);
DPI
zinniae (1)
084-1944 (Florida);
DPI
unknown (1)
G65
this study
X. albilineans (1)
Xalb (Florida);
M. J.Davis
X. fragariae (1)
Xfra (Florida);
R.E.Stall
a DPI = Florida Department of Agricultural and Consumer Services,
Division of Plant Industry, Gainesville, Florida.


CHAPTER SIX
PATHOVARS OF XANTHOMONAS CAMPESTRIS ARE DISTINGUISHABLE
BY RESTRICTION FRAGMENT LENGTH POLYMORPHISM*
Introduction
The species Xanthomonas campestris (Pammel) Dowson, a member of
the family Pseudcmonadaceae which is always found in association with
living plants, comprises over 125 different pathovars (6). The
pathovars have been named by "the plant from which first isolated"
convention. Unfortunately, strains of some pathovars can be
pathogenic on host species in different plant families (57); thus
possible relationships between pathovars may go undetected, since
there is no convenient way to determine pathogenicity for all possible
host plants, nor is there a useful systematic method for narrowing the
possibilities. Further, and despite a widely held view that all
xanthcmonads are plant pathogens (85), non-pathogenic members of X.
campestris are often found as ectoparasitic leaf colonizers
(epiphytes) (26) and occasionally as endoparasites (65,71). Their
inability to provoke a pathogenic plant response renders them
unclassifiable in this system. The attention given to damaging plant
pathogenic strains may not be taxonomically or ecologically warranted.
Genes which function to provoke a pathogenic response may be entirely
different from those which confer host selectivity and parasitic
ability (32) Epiphytes and nonpathogenic endoparasites are known to
_
This chapter contains copyrighted material from International Journal
of Systematic Bacteriology. It is reprinted here with permission of
the publisher.
70


useful because it is a facultative parasite with a high level of
natural variation and has been shown to follow similar gene-for-gene
patterns of interactions with plants as found with fungal pathogens
(33).
The objective of this research was to develop a better
understanding of the intraspecific variation and specificity present
in the species X. campestris. This was conducted by first verifying
the pathogenicity of the working strains as pathovars and, where
appropriate, as races within selected pathovars of X. campestris
through pathogenicity testing. This work was followed by physical
characterization of the plasmids and chromosomes of X. campestris
races and pathovars. These DNA analyses were designed to determine
the extent of physical variation within the species, pathovars, and
races of X. campestris. Finally, genetic variation was analyzed at
the race level by using cloned genes and complementation analyses to
identify specific avirulence genes which contribute to the race
phenotypes. By conducting experiments in a multi-faceted approach, it
was hoped that a better understanding of the interactions of strains
of X. campestris with their host plant(s) could be appreciated.


104
84. Schnathorst, W. C. 1966. Unaltered specificity in several
xanthomonads after repeated passage through Phaseolus vulgaris.
Phytopathology 56:58-60.
85. Schroth, M. N., and Hildebrand, D. C. 1983. Toward a sensible
taxonomy of bacterial plant pathogens. Plant Dis. 67:128.
86. Schroth, M. N., Hildebrand, D. C., and Vitanza, V. 1970.
Pathogenic variation and overlapping host ranges in Pseudomonas
phaseolicola, P. glycinea, and P. mori. (Abstr.). Phytopathology
60:1313.
87. Silhavy, T. J., Berman, M. L., and Enquist, L. W. 1984.
Experiments with Gene Fusions. Cold Spring Harbor Laboratory,
Cold Spring Harbor, NY.
88. Stall, R. E., Loschke, D. C., and Jones, J. B. 1986. Linkage of
copper resistance and avirulence loci on a self-transmissible
plasmid in Xanthomonas campestris pv. vesicatoria. Phytopathology
76:240-243.
89. Starr, M. P. 1983. The genus Xanthomonas. pp. 742-763 in: The
Prokaryotes, Vol. 2. M. P. Starr, H. Stolp, H. G. Truper, A.
Balows, and H. G. Schlegel, eds. Springer-Verlag, New York, NY.
90. Surico, G., Comai, L., and Kosuge, T. 1984. Pathogenicity of
strains of Pseudomonas syringae pv. savastanoi and their
indoleacetic acid-deficient mutants on olive and oleander.
Phytopathology 74:490-493.
91. Sytsma, K. J., and Gottlieb, L. D. 1986. Chloroplast DNA evidence
for the origin of the genus Heterogaura from a species of Clarkia
(Onagraceae). Proc. Natl. Acad. Sci. USA 83:5554-5557.
92. Szabo, L. J., and Mills, D. 1984. Characterization of eight
excision plasmids of Pseudomonas syringae pv. phaseolicola. Mol.
Gen. Genet. 195:90-95.
93. Tait, R. C., Close, T., Lundquist, R., Hagiya, M., Rodriquez, R.,
and Kado, C. I. 1983. Construction and characterization of a
versatile broad host range DNA cloning system for gram-negative
bacteria. Biotechnology 1:269-275.
94. Thaveechai, N., and Schaad, N. W. 1984. Comparison of different
immunogen preparations for serological identification of
Xanthomonas campestris pv. campestris. Phytopathology
74:1065-1070.
95. Thaveechai, N., and Schaad, N. W. 1986. Immunochemical
characterization of a subspecies-specific antigenic determinant
of a membrane protein extract of Xanthanonas campestris pv.
campestris. Phytopathology 76:148-153.


88
Discussion
The only widely accepted and most practical method for
differentiating pathovars of X. campestris is to inoculate a plant
suspected as the host for that pathovar. This practice can sometimes
be tedious, time consuming, subjective and subject to a surprising
number of artifactual influences. It is not known whether host
selectivity is unstable as suggested by Dye (23); stable as suggested
by Schnathorst (84) or even taxonomically significant. Although the
classification is thought to be useful, it can be highly misleading
since emphasis is placed upon one characteristic pathogenicity. If
only one or few gene differences were involved in host selection, then
differentiation by pathovar could be highly misleading, at least in
the sense that a given group of strains might be capable of attacking
more than one host in some cases. Alternatively, two or more strains
of relatively unrelated groups could be cataloged together because
they happen to attack the same host. This latter situation appears to
be the case with X. c. pv. phaseoli and X. c. pv. phaseoli var.
fuscans (see Fig. 6-3) and with Florida strains of X. c. pv. citri
(34).
To address these problems, we evaluated the potential of using
DNA sequence variation of X. campestris for strain classification
purposes. DNA sequence variation was first examined by digesting DNA
with restriction enzymes and visualizing directly the resulting
fragments on ethidium bromide stained gels. These stained gels were
useful for side-by-side comparisons of restriction fragments for
samples run on the same gel, but comparisons between different gels


Results
Plasmids and Race-Specificity
All of the strains of X. c. pv. malvacearum tested contained at
least one plasmid- The plasmid sizes ranged from about 50 to 100 kb.
A majority of the American strains of X. c_. pv. malvacearum contained
4.8, 4.7, and 4.5 kb EcoRI DNA fragments which appeared to be
conserved (Fig. 4-2). The entire 4.5 kb EcoRI DNA fragment appeared
to be conserved in all but 1 of the 25 strains tested, regardless of
geographic origin.
Comparisons were made between the physical restriction fragment
patterns (Fig. 4-2), the original race designations, and reactions on
the 14 cotton differentials tested (Table 5-2). The strains of X. c.
pv. malvacearum were grouped into seven plasmid types based on the
restriction profiles and the conservation of related restriction
fragments. In the plasmid groupings I, III, and IV, a limited level
of polymorphism in the restriction fragment profiles for each group
were evident, but a majority of the fragments appeared to be
conserved. In plasmid groupings II, V, VI, and VII, no polymorphisms
were observed.
The strains which contained plasmids of group I appeared to be
pathogenic on a majority of the cotton host differentials. X. c. pv.
malvacearum strain J was an exception for it was pathogenic to only 4
of the cotton differentials, whereas the other group I strains were
pathogenic on at least 11 of the cotton lines. Analysis of the EcoRI
restriction fragment pattern of strain J revealed it was missing a 4.5
kb fragment, and possibly had rearrangements which resulted in a shift
of two other EcoRI fragments. With few exceptions, the 4.5 kb EcoRI


be host selective (19,65). Host selectivity is thought to be a stable
characteristic (84) Since xanthomonads are always found in
association with living plants, host selectivity should result in
genetic isolation of a pathovar population. Over time such isolation
and random genetic drift should produce distinctive and stable
phenotypic characteristics and therefore, establish conserved genetic
markers.
Attempts to differentiate pathovars by methods other than
pathogenicity have included rRNA-DNA and DNA-DNA hybridization
(21,73), serology (1,94,95), phage-typing (37,59), and comparisons of
profiles resulting from plasmid and chromosomal DNA restriction enzyme
digests (40,54), protein electrophoresis (70), and gas-chromatography
of fatty acids (65). Although all of these methods are useful for
specific purposes, few have demonstrated utility to replace, clarify,
or indicate further pathogenicity tests.
We report here the use of restriction fragment length
polymorphism (RFLP) analysis to differentiate pathovars of X.
campestris. RFLP analyses have been widely used in the medical field
to identify DNA fingerprints specific for inherited diseases or other
genetic loci (2,11,61). These analyses have also been applied to
plants (41), bacteria (38), and eukaryotic organelles (67,91). RFLP
analyses allow the observance of genetic variation in organisms within
defined regions of the genome due to DNA rearrangements or mutations
which affect recognition sites for restriction endonucleases.
Variation may be examined over a small (a few base pairs) or large
(30-40 kb) stretch of DNA depending on the desired level of


64
Figure 5-2. Transconjugants of X. campestris pv. maivacearum N-Sp^
containing pLXD (top) and hybrrdized to the plasmid probe pSa4
(bottom) The transconjugants were digested with the restriction
endonuclease EcoRI (top) Lanes A and M, Lamda Hindu; lanes B-E,
large colony-size; lanes F-I, medium colony-size, and lanes J-L, small
size transconjugants. All of the transconjugants, except in lane J,
appear to have lost the 4.5 kb plasmid fragment seen in wild-type
strains. Plasmids in lanes F-I and K-L appear to have had other
plasmid rearrangements occur. All strains had an additional 8 kb EcoRI
fragment present. The autoradiograph (bottom) shows the
transconjugants probed with a pUCD5 related plasmid, pSa4 (93) The 8
kb fragment hybridized to the probe, but so did other plasmid
fragments corresponding to those of X. campestris pv. maivacearum.


CHAPTER THREE
HOST RANGE OF PATHOVARS OF XANTHOMONAS CAMPESTRIS
Introduction
There are over 125 pathovars of X. campestris described, and the
only means to differentiate between them is by pathogenicity testing
(6). Seme strains of X. campestris have extended host ranges to more
than one host, usually to other members of the same plant family, with
few exceptions (57) The host range can sometimes overlap onto
another host for which a different X. campestris pathovar has been
described. For instance, X. c. pv. alfalfae is a pathogen of the
legumes alfalfa, bean, and pea, but strains exist of X. c_. pv.
phaseoli and X. c. pv. pisi which have host ranges limited to bean and
pea, respectively. The relatedness between these strains at the
subspecific level is unknown, except for their host ranges. In
another instance, strains of X. c_. pv. malvacearum have been isolated
from the malvaceous hosts, cotton and hibiscus (7,12). Although given
the same pathovar designation because they were each isolated from
plants of the Malvaceae family, their relatedness is unknown. Similar
situations, where the relationship between strains is unclear, exist
among other pathovars of X. campestris (6) The purpose of this
investigation was to verify the pathogenicity and host range of all
stock strains used in this dissertation, to examine the relationships
between apparently related strains of X. campestris in terms of host-
9


95
strains. Surprisingly, differences could also be detected among the
strains assumed to be X. c. pv. campestris, where sane of the more
virulent strains belonged to a separate RFLP grouping.
Because RFLP groupings of X. campestris correlated well with host
range, it seems possible that this association could also be valid
with other bacterial systems. There is a great interest in the use of
microorganisns as biocontrol agents. In many cases, attempts to
exploit microorganisms which exhibit strong in vitro antagonism have
failed due to the poor survivability of the agent in plants. For
instance, P_. fluorescens has been used in developing rhizosphere
competent biocontrol agents against fungi (44,63,78,100). Strains of
P_. fluorescens, also a member of the same bacterial family as X.
campestris, is poorly differentiated; and as part of the soil
rhizosphere is also poorly defined. RFLP studies could be used to
identify members of P. fluorescens which have the desired host range,
thus, facilitating the development of more effective biocontrol
agents.
Conclusions
The extent to which we devise taxonomic distinctions to
differentiate among organisms remains a function of their utility to
humankind. Their relavance to "natural groupings" is in the minds of
pathologists or microbiologists whose perception may be somewhat
different from their Creator. The variation which is perceived as
significant by pathologists to differentiate strains of X. campestris
is host-specific pathogenicity. The variation which is perceived as
significant by geneticists may be chromosomal RFLP patterns. The


were more difficult. Subsequently, visualizing and comparing
variation in bacterial genomes with cosmid clones carrying 30-38 kb
89
cloned X. campestris genome fragments was simplified. In both cases
variation was revealed by alterations in the sizes of visualized DNA
restriction fragments (RFLPs). The use of DNA probes derived from
chromosomal DNA fragments also alleviated seme of the difficulty in
comparing stained gels with the sometimes present polymorphism of the
higher copy number plasmid DNA fragments which can occur in the
background.
Some of the selected clones tested as DNA probes appeared to be
useful for identifying DNA sequences conserved within a given
pathovar, while others appeared to identify DNA sequences which were
highly conserved at the species level. The DNA which was considered
conserved at the species level was represented as banding patterns
which were nearly identical among all strains over the several
pathovars tested. Examples of DNA sequences which are known to be
highly conserved are rRNA encoding genes (38,83). If smaller DNA
probes containing known genetic loci such as those associated with
genes for pectate lyase and protease (18), or avirulence activity
(33,88) were tested, these smaller, more defined DNA probes could be
used to determine if such genetic loci were highly conserved or
variable. With larger, randomly selected probes, the likelihood was
increased for detecting strain- or pathovar-specific variation. The
large (greater than 30 kb) size of the probes used in this study
allowed detection of both highly conserved and variable regions.


38
lilil
123456789012345
1 11 12222 2 2222233333333334444444
67 8 90123456789012345 678901 23456
30-
25
20H
15
10
5-
0-
kb
iii
J_L
i i i i i i
Figure 4-2. Graphic representation of plasmid restriction fragment
profiles for Xanth anonas campestris pvs. cyamopsidis (Xcc; lane 1) ,
vignicola (Xcv; lanes 2-8), pnaseoli (Xcp; lanes 9-17), phaseoli var.
fuscans (Xcpf; lanes 18-19), glycines (Xcg; lanes 20-22), and
malvacearum (Xcm; lanes 23-46) digested with EcoRI. Lanes shown above
contain 1, Xcc 13D5; 2, Xcv SN2; 3, Xcv A81-331; 4, Xcv C-l; 5, Xcv
CB5-1; 6, Xcv Xvl9; 7, Xcv 82-38; 8, Xcv 432; 9, Xcp EKll; 10, Xcp
82-2; 11, Xcp Xpa; 12, Xcp Xpll; 13, Xcp XP2; 14, Xcp XP-JF; 15, Xcp
XP-DPI; 16, Xcp Xph25; 17, Xcp 82-1; 18, Xcpf Xpfll; 19, Xcpf SC-3B;
20, Xcg B-9-3; 21, Xcg 17915; 22, Xcg 1717? 23, Xan J; 24, Xan L; 25,
Xcm C; 26, Xcm 0; 27, Xcm K; 28, Xan N; 29, Xcm A; 30, Xcm B; 31, Xcm
E; 32, Xan F; 33, Xan G; 34, Xan H; 35, Xan S; 36, Xan W; 37, Xan I;
38, Xan V; 39, Xan D; 40, Xan M; 41, Xcm X; 42, Xan U; 43, Xan Q; 44,
Xcm R; 45, Xcm Y; and 46, Xcm Z. Calculated DNA fragment sizes are
represented here by a linear scale whereas migration of DNA fragments
under electrophoresis conditions approximates a logarithmic scale
which is inversely proportional to the molecular weight of the DNA
fragment.


fragment appeared to be conserved in all group I strains of X. c. pv.
malvacearum. The group I strains K and N each contained two plasmids
and also appeared to be the most virulent of all the strains tested in
any group, each being pathogenic on all of the cotton differentials
tested (Table 5-2). Strain K was isolated in Upper Volta, Africa and
strain N was isolated from Texas. Although the plasmids in group I
were highly conserved and apparently widespread in the world, there
was no correlation between the presence of these plasmids with race
specificity.
The strains which were classified into plasmid group II could be
divided into two basically different pathogenicity patterns on the 14
cotton differentials. The strains which were derived from X. c. pv.
malvacearum races 1 or 2, and strains derived from races 3 and 4
appeared to have related pathogenicity patterns. The plasmid EcoRI
restriction fragments patterns for all seven of these strains appeared
identical. Similar variability was observed in the pathogenicity
patterns for the other strains placed within groups III, IV, V, VI,
and VII. Again, no correlation between the pathogenicity pattern and
the plasmid group was apparent. The original race designations of the
strain mixtures of X. c. pv. malvacearum did not correspond to the
race phenotypes exhibited by the strains purified from the mixtures.
For instance, of 5 strains derived from nominal race 1 (F, G, Q, R,
and U) there were basically three different pathogenicity patterns
observed, none of which corresponded to the reported pattern for race
1. The expected race 1 reaction would have been pathogenic on Acala
44 and Stoneville 2B-S9 (45).


GENETIC ANALYSIS OF INTRASPECIFIC VARIATION IN
PA1HOVARS OF Xanthanonas campestris
BY
GERARD RAYMOND LAZO
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
1987

Copyright 1987
by
Gerard Raymond Lazo

ACKNOWLEDGMENTS
I wish to express my gratitude to Dr. D. W. Gabriel, chairman of
the advisory committee, for his friendship, help and encouragement
during the course of this research. Thanks are also expressed to all
personnel associated with the laboratory, who were very generous and
cooperative in supporting the research efforts which made this
presentation possible. Special thanks are extended to A. Williams who
assisted with the race-specificity studies on cotton; G. V. Minsavage
who assisted with the SDS-PAGE analysis; R. Roffey, who assisted with
the RFLP analysis; and A. Burges who assisted with various other
aspects of this study. Thanks are also expressed to Drs. R. E. Stall,
D. R. Pring, and M. J. Bassett for serving on the advisory committee
and for their support. Appreciation is also acknowledged for
financial support from the Oklahoma and Florida Agricultural
Experiment Stations, and United States Department of Agriculture grant
USDA-58-7B30-3-465. And finally, I am grateful to my loving wife,
Maria, for supporting and encouraging me throughout this degree
program, for she tolerated my late hours and comforted me in the most
difficult of times.

TABLE OF CONTENTS
Page
ACKNOWLEDGMENTS iii
LIST OF TABLES vi
LIST OF FIGURES vii
ABSTRACT ix
CHAPTER
ONE INTRODUCTION 1
TWO LITERATURE REVIEW 4
THREE HOST RANGE OF PATHOVARS OF XANTHOMONAS CAMPESTRIS 9
Introducid on 9
Materials and Methods 10
Bacterial Strains and Host Plants 10
Plant Inoculations 12
Physiological Differentiation 12
Polyacrylamide Gel Electrophoresis 13
Results 13
Pathogenicity Tests 13
Physiological Differentiation 20
Polyacrylamide Gel Electrophoresis 22
Discussion 22
FOUR CONSERVATION OF PLASMID DNA SEQUENCES AND PATHOVAR
IDENTIFICATION OF STRAINS OF XANTHOMONAS CAMPESTRIS .... 27
Introduction 27
Materials and Methods 28
Bacterial Strains 28
Plasmid Extraction and Visualization 31
Cloning of Plasmid
Restriction Endonuclease Fragments 32
DNA/DNA Hybridization 32
Results 33
Detection of Plasmid DNA 33
Restriction Endonuclease Profiles 35
DNA/DNA Hybridization 40
Dot-blot Hybridization 42
Discussion 45
IV

FIVE ARE RACE-SPECIFIC AVIRULENCE GENES ENCODED BY PLASMIDS
OF XANTHOMONAS CAMPESTRIS PV. MALVACEARUM? 50
Introduction 50
Materials and Methods 51
Bacterial Strains and Host Plants 51
Plant Inoculations 51
Plasmid Analysis 51
Plasmid Curing 53
Race-Specificity Genes 53
Plasmid Origin of Replication and Mobilization 54
Results 55
Plasmids and Race-Specificity 55
Plasmid Curing 58
Race-Specificity Genes 58
Plasmid Origin of Replication and Mobilization .... 61
Discussion 65
SIX PATHOVARS OF XANTHOMONAS CAMPESTRIS ARE DISTINGUISHABLE
BY RESTRICTION FRAGMENT LENGTH POLYMORPHISM 70
Introduction 70
Materials and Methods 72
Bacterial Strains 72
DNA Extraction 72
Agarose Gel Electrophoresis 75
DNA Probes 75
DNA Hybridization 76
Restriction Fragment Patterns and Densitometry .... 77
Results 77
Agarose Gel Electrophoresis 77
DNA Probes 80
DNA Hybridization 80
Discussion 88
SEVEN SUMMARY 92
Intraspecific Variation 92
Plasmids 92
Restriction Fragment Length Polymorphism 93
Conclusions 95
LITERATURE CITED 97
BIOGRAPHICAL SKETCH 106
v

LIST OF TABLES
Table Page
3-1 Strains of X. campestris used in host range investigation ... 11
3-2 Legume plant reactions to inoculation with pathovars
of X. campestris 14
3-3 Host range observed for pathovars of X. campestris
pathogenic to legume host plants 17
3-4 Malvaceous host plant reactions to inoculation
with strains of X. campestris pv. malvacearum 19
3-5 Physiological reactions of strains of X. campestris 21
4-1 Strains of X. campestris used for plasmid analysis 29
4-2 Detection of plasmid DNA in strains of X. campestris 34
4-3 Hybridization of radiolabeled plasmid probes to
total DNA of pathovars of X. campestris 44
5-1 Strains of X. campestris pv. malvacearum from cotton
used in race-specificity investigation 52
5-2 Plasmid groupings and cotton plant reactions to strains of
X. campestris pv. malvacearum 56
5-3 Pathogenicity of X. campestris pv. malvacearum
transconjugants on cotton host differentials 59
5-4 Selection of plasmid replication genes in X. campestris
pv. malvacearum strain X 62
6-1 Strains of X. campestris used for restriction fragment
length polymorphism analysis 73
6-2 Sizes of DNA fragments from X. campestris genomic digests
which hybridized to the XCTl DMA probe. See Figure 6-3 83
6-3 Sizes of DNA fragments from X. campestris genomic digests
which hybridized to the XCTl DNA probe. See Figure 6-5 87
vi

LIST OF FIGURES
Figure Page
3-1 SDS-Polyacrylamide gel electrophoresis of total proteins
from strains of X. campestris 23
4-1 Plasmid DNAs from strains of X. campestris pv. malvacearum
digested with restriction endonucleases EcoRl and BamHI 37
4-2 Graphic representation of plasmid EcoRl restriction fragment
profiles for pathovars of X. campestris 38
4-3 Plasmid DNAs from strains of X. campestris pv. citri
digested with restriction endonuclease EcoRl 39
4-4 Plasmid DNAs from strains of X. campestris pv.
malvacearum digested with restriction endonuclease
EcoRl and hybridized to probe N4.5 41
4-5 Plasmid DNAs from strains of X. campestris digested with
restriction endonuclease EcoRl and hybridized to probes
P2.0 and P2.3 43
5-1 Plasmid curing of X. campestris pv. malvacearum with
SDS treatment 60
5-2 Transconjugants of X. campestris pv. malvacearum containing
pLXD and hybridized to the plasmid probe pSa4 64
5-3 Transformation of E. coli ED8767 with plasmid DNA
frcm X. campestris pv. malvacearum transconjugants
mated with pLXD 66
5-4 Plasmid DNA of X. campestris pv. malvacearum strain X
digested with restriction endonucleasea and hybridized
to the plasmid probe pXD-1 67
6-1 Genomic DNA of strains of X. campestris pvs. phaseoli,
alfalfae, and campe stris digested with restriction
endonuclease EcoRl 78
6-2 Genomic DNA of strains from different pathovars of
X. campestris digested with restriction endonuclease EcoRl .. 79
vii

6-3 Genomic DNA of strains of X. campestris pvs. phaseoli,
alfalfae, and campestris digested with restriction
endonuclease EcoRI and hybridized with probe XCTl 81
6-4 Genomic DNA of strains of X. campestris pvs. phaseoli,
alfalfae, and campestris digested with restriction
endonuclease EcoRI and hybridized with probe XCTll 84
6-5 Genomic DNA of strains from different pathovars of
X. campestris digested with restriction endonuclease EcoRI
and hybridized with probe XCTl 85
viii

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 ANALYSIS OF INTRASPECIFIC VARIATION IN
PATHOVARS OF Xanthanonas campestris
By
Gerard Raymond Lazo
May, 1987
Chairman: Dean W. Gabriel
Major Department: Plant Pathology
Xanthomonas campestris is always found in association with
plants. Those strains of X. campestris which are known to be
pathogenic are differentiated into over 125 pathovars (pathogenic
variants), on the basis of limited pathogenicity tests. Sane members
of a pathovar may have a broader host range than others, since
exhaustive pathogenicity testing is impractical. Other than these
tests, there is no definitive means to classify an unknown X.
campestris isolate. Those strains of X. campestris which are not
pathogenic are unclassifiable by the pathovar system, yet they may
exhibit similar host range specificity to those which are pathogenic.
This work was conducted in an effort to better understand the
variability of X. campestris.
Over 115 strains of X. campestris were examined for plasmid
content and restriction endonuclease profiles. Of the 26 pathovars
examined, only 13 were found to contain plasmids. Restriction
ix

endonuclease digested plasmid DNA from strains within a given
plasmid-containing pathovar gave surprisingly similar, but not always
identical, digestion profiles by agarose gel electrophoresis. All 60
strains tested of X. campestris pvs. glycines, malvacearum, phaseoli,
and vignicola could be accurately identified by pathovar iron
determination of the restriction fragment profile and/or by Southern
hybridizations of that profile with a selected plasmid DNA probe. In
no instance was the same plasmid profile seen in more than one
pathovar. The apparent stability of the plasmids provides a natural
genetic marker that can be strain specific and perhaps useful in
epidemiological investigations.
Cloned DNA fragments, derived from a cosmid library of a Florida
isolate of X. campestris pv. citri strain 3041, were used to detect
restriction fragment length polymorphism (RFLP) of total DNA from 87
strains of X. campestris, comprising 23 different pathovars.
Autoradiographs of Southern transfers of genomic DNA probed with
cosmid-sized clones revealed hybridization profiles which appeared to
be highly conserved. The hybridization patterns observed between
different pathovars suggested that some DNA fragments were conserved
at the species level, others were conserved at the pathovar level, and
still others were variable. The degree of variability appeared to
depend on the DNA probe used. By using more than one DNA probe, or by
digesting the genomic DNAs with different restriction endonucleases,
all of the strains of X. campestris described as belonging to a given
pathovar could be differentiated. All strains of X. campestris were
readily grouped by RFLP phenotypes, and the classification based on
x

RFLP patterns correlated well with the classification based on
pathogenicity. Although certain pathovars may need to be redefined,
this work supports and helps validate the natural taxonomic groupings
provided by the pathovar naming system.
xi

CHAPTER ONE
INTRODUCTION
The genus Xanthanonas Dowson consists of bacteria described as
being gram-negative, obligately aerobic, non-fermentative rods which
are motile by a single polar flagellum and can produce a brominated
yellow pigment, called xanthcmonadin (6) All reported strains of
this genus have been described as plant associated, and most are
reported as being pathogenic to a particular plant host. Based on
microbiological classification, this genus can be separated into at
least five separate species: X. campestris (Pairmel) Dowson, X.
albilineans (Ashby) Dowson, X. ampelina Panagopoulos, X. axonopodis
Starr and Garces, and X. fragariae Kennedy and King (and possibly X.
populi (Ride) Ride and Ride) Over 125 variants, or pathovars, of X.
campestris, are essentially indistinguishable from each other, except
for their plant host range (6,89).
To maintain order in differentiating among strains of X.
campestris by host range, the rank of pathovar has been assigned in
addition to the species name (24,98). The primary means by which
strains can be differentiated as pathovars is by inoculation of the
plants which serve as susceptible hosts to a given pathovar. The
convention for naming pathovars of X. campestris has generally been to
name it for the host from which it was first isolated (8 9) A
particular pathovar designation suggests a limited host range for a
1

given strain. The actual host range may be much broader, but a
complete host range description of a given strain would require
testing on all plants known to support growth of the genus. This
would be a monumental task, and therefore the potential host range of
a given strain of X. campestris is unknown. Obviously, any
information regarding pathogenicity is of value to pathologists.
However, without knowledge of the potential host range variation of
strains within a pathovar, the taxonomic value of the pathovar system
is questionable.
Attempts to devise alternative means to differentiate pathovars
of X. campestris have included serology (1,94) membrane protein
profiles (70), phage-typing (37) rRNA-DNA and DNA-DNA hybridization
(21,73), and gas chromatography of fatty acids (65,81). Although most
of these methods have been useful to differentiate given sets of
strains of X. campestris, there is little evidence that the groupings
distinguished by these methods have any correlation to the host range
groupings observed for given pathovars of X. campestris. None of
these techniques have been successful in replacing pathogenicity tests
to identify X. campestris to the pathovar level. Because differences
in host range primarily determine the pathogenic variability observed
in X. campestris, it is of interest to know more about the nature of
this microorganism and the mechanisms which determine host-range
specificity.
Genetic analyses of pathogenicity can easily be done with
bacteria, for they can be quickly cloned and assayed, and studied
using current recombinant DNA technologies (50). X. campestris is

useful because it is a facultative parasite with a high level of
natural variation and has been shown to follow similar gene-for-gene
patterns of interactions with plants as found with fungal pathogens
(33).
The objective of this research was to develop a better
understanding of the intraspecific variation and specificity present
in the species X. campestris. This was conducted by first verifying
the pathogenicity of the working strains as pathovars and, where
appropriate, as races within selected pathovars of X. campestris
through pathogenicity testing. This work was followed by physical
characterization of the plasmids and chromosomes of X. campestris
races and pathovars. These DNA analyses were designed to determine
the extent of physical variation within the species, pathovars, and
races of X. campestris. Finally, genetic variation was analyzed at
the race level by using cloned genes and complementation analyses to
identify specific avirulence genes which contribute to the race
phenotypes. By conducting experiments in a multi-faceted approach, it
was hoped that a better understanding of the interactions of strains
of X. campestris with their host plant(s) could be appreciated.

CHAPTER TWO
LITERATURE REVIEW
At least 124 monocotyledonous and 268 dicotyledonous plants
species are susceptible to infection by bacterial strains of the genus
Xanthomonas (57). Compiled listings indicate that the genus
Xanthanonas has a wide host range extending over nine monocotyledonous
families comprising 66 genera, and 49 dicotyledonous families
comprising 160 genera. Pathogenicity toward gymnosperms or lower
plants has not been reported.
Although pathovars are generally assumed to have a restricted
host range, seme strains of X. campestris have extended host ranges to
more than one plant species. In these instances, host range usually
extends toward other members of the same plant family (with few
exceptions). For example, the plant families Ccmpositae, Cruciferae,
Graminae, and Leguminosae each contain different species susceptible
to the same X. campestris pathovar (X. c. pv.). An example of a broad
host range pathovar of graminaceous hosts is X. c. pv. translucens
(Jon., John., and Redd.) Dye, which is pathogenic on barley, rye, and
wheat. Other strains pathogenic on graminaceous hosts have been
described, but their interrelationships among the other pathovars were
poorly defined (6). In another example, X. c. pv. alfalfae, is
pathogenic on the legumes alfalfa, bean, and pea. Another special
relationship is that of X. c. pv. vesicatoria (Doidge) Dye, which is a
4

5
pathogen of Capsicum annuum L. (pepper) and Lycopersicon esculentum
(tomato). While some strains of X. campestris appear to have a host
range to more than one plant species, most others appear to have an
extremely narrow host range (6,57). The host range characteristic is
considered to be a stable trait, although there is one report of
altered host range by artificial selection (23). This report has been
refuted, with no change in host-range specificity observed for X.
campestris (84) WTithin at least seme pathovars, variability among
strains exists as race-specific plant interactions (8,17,20). These
are recognized when resistant plants of an otherwise susceptible plant
species are discovered. The ability to differentiate races of X.
campestris pathovars is dependent on the discovery of plant hosts,
called host differentials, which exhibit variability in their response
to different pathogenic strains. Pathogenic races have been described
for X. c_. pv. malvacearum (Smith) Dye, X. c. pv. oryzae, X. c. pv.
translucens, X. c. pv. vesicatoria, and possibly others. This race-
specific variation in X. campestris was suggestive of gene-for-gene
interactions between pathogenicity determinants in the pathogen with
resistance genes in the host (8,20).
Gene-for-gene relationships can only be established by genetic
crosses of the host and the parasite. Because the bacterial genetics
can only be done with recombinant DNA techniques, gene-for-gene
interactions could only be recently established. Proof of
gene-for-gene interactions with bacterial pathogens was documented for
X. c. pv. malvacearum (33).

The high level of host-range and race specificity described above
for X. campestris is observed with some other plant pathogenic
bacteria. For example, there are more than forty pathovars of
Pseudomonas syringae which are primarily distinguished by host-range
specificity (75,86). Sane P. syringae pathovars have host ranges
which are narrowly restricted to specific plant species; others have a
wider range of hosts. Strains of Erwinia chrysanthemi are currently
being considered for differentiation at the pathovar level (56) .
Strains of Agrobacterium spp. and Rhizobium spp. are not
differentiated using the pathovar system; however, host-specific
interactions analogous to those observed in X. campestris exist with
these bacteria. Species within each genus can be differentiated
biochenically and by plant inoculations. These two genera, which
cause very different symptoms, are both in the family Rhizobiaceae and
are genetically highly related (25,42) In Rhizobium spp. and
Agrobacterium spp., host range appears to be specified by the
bacterium by genes which are expressed as a positive function (32). In
several species of Rhizobium, host range function has been identified
as being plasmid borne (4). The introduction of one of these plasmids
may extend the host range of a limited host range species of
Rhizobium.
Similarly, A. tumefaciens tumor inducing (Ti) plasmids carry host
range determining genes (64) Ti plasmids from wide-host-range
strains, when moved to A. tumefaciens strains with limited host-
ranges, confer a wide host range to the recipient (10,43,48). The
pTAR plasmid, found in A. tumefaciens strains from grapevine, carries

a gene for tartaric acid catabolism (35) The expression of pTAR
genes appears to function more like a host range determinant rather
than an oncogenic determinant, as found on tumor inducing plasmids.
The proof of plasmid involvement in virulence and host range with
species of Agrobacterium and Rhizobium apparently stimulated research
of plasmid involvement among other phytopathogenic bacteria. Plasmids
have been found to exist in many other phytopathogenic bacteria, but
few have been associated with pathogenicity (76). Examples of plasmid
functions in pathogenicity on plants include tumor induction in A.
tumefaciens (97), nodulation in Rhizobium spp. (62), indoleacetic acid
production by P. syringae pv. savastanoi (90), cytokinin production by
Corynebacterium fascians (syn. Rhodococcus fascians) (72), virulence
of Pseudomonas solanacearum (5) and race-specific avirulence in X. c.
pv. vesicatoria (88), among others. A general conclusion from these
studies is that plasmids from a vade range of plant pathogenic genera
can carry a variety of genes which determine the outcome of plant-
pathogen interactions.
Plasmids have been shown to be physically similar among strains
within pathovars of P_. syringae (77) The similarity is based on
homology studies using restriction endonuclease digestion patterns and
DNA-DNA hybridization experiments (15) Additionally, there are
instances where similar plasmids, or even identical ones, may be found
in closely related pathovars of P. syringae (77) Although the
functional utility of these plasmids is unknown, they often cannot be
eliminated or "cured" from the bacterial strain, possibly because such
an event would be lethal. For example, in P. syringae pv.

8
phaseolicola, a plasmid, pMC7105, can be forced to integrate into the
host chromosome, but cannot be cured (79,92). The plasmid pM27105 has
seme hcmology with other P. svringae pv. phaseolicola strains, and
even P. svringae pv. glycinea strains. Homology between these
plasmids may be due to transpositional events, the presence of
repetitive sequences, variability due to integration and excision of
the plasmid, plasmid origin of replication, or other plasmid
maintenance function (79,92) Another possibility is that genes
essential for pathogenicity, host-range, or race specificity may be
encoded on them. The association of plasmid homology with host-range
specificity may indicate the involvement of plasmids in host range
determination in these cases.

CHAPTER THREE
HOST RANGE OF PATHOVARS OF XANTHOMONAS CAMPESTRIS
Introduction
There are over 125 pathovars of X. campestris described, and the
only means to differentiate between them is by pathogenicity testing
(6). Seme strains of X. campestris have extended host ranges to more
than one host, usually to other members of the same plant family, with
few exceptions (57) The host range can sometimes overlap onto
another host for which a different X. campestris pathovar has been
described. For instance, X. c. pv. alfalfae is a pathogen of the
legumes alfalfa, bean, and pea, but strains exist of X. c_. pv.
phaseoli and X. c. pv. pisi which have host ranges limited to bean and
pea, respectively. The relatedness between these strains at the
subspecific level is unknown, except for their host ranges. In
another instance, strains of X. c_. pv. malvacearum have been isolated
from the malvaceous hosts, cotton and hibiscus (7,12). Although given
the same pathovar designation because they were each isolated from
plants of the Malvaceae family, their relatedness is unknown. Similar
situations, where the relationship between strains is unclear, exist
among other pathovars of X. campestris (6) The purpose of this
investigation was to verify the pathogenicity and host range of all
stock strains used in this dissertation, to examine the relationships
between apparently related strains of X. campestris in terms of host-
9

range specificity, and to find a physiological test diagnostic for one
or more pathovars used in these studies.
10
Materials and Methods
Bacterial Strains and Host Plants
Bacteria used in this investigation consisted of pathovars of X.
campestris which were pathogenic to members of either the Leguminosae
or Malvaceae plant families. The pathogens of leguminous host plants
consisted of X. campestris pvs. alfalfae, cyamopsidis, glycines,
phaseoli, pisi, and vignicola isolated iron alfalfa, guar, soybean,
kidney bean, pea, and cowpea, respectively. The pathogens of
malvaceous host plants consisted of strains of X. c_. pv. malvacearum
isolated from cotton or hibiscus. The strains of X. campestris used
in this study are shown in Table 3-1. The legume host plants used in
this investigation included Medicago sativa cv. "FL-77", Glycines max
cv. "Evans", Phaseolus vulgaris cv. "California Light Red", and Vigna
unguiculata cv. "California Blackeye #5". The malvaceous hosts
included three cultivars of Hibiscus rosa-sinensis and eight cultivars
of Gossypium hirsutum. One of the cultivars of H. rosa-sinensis was
cv. Brilliant Red; the other two were not named, but chosen to
represent diversity in the species. Eight cotton cultivars were
selected for differentiating races of X. c. pv. malvacearum, two of
which were cv. 101-102B, and cv. Gregg (45). The other cultivars were
cv. Acala 44 (no resistance to cotton strains), and five cultivars
derived from cv. Acala 44 crossed to obtain single gene resistance to
the cotton strains of X. c. pv. malvacearum, the cultivars being Acala
B]_, B2, B3, B5, and Bj^j (7).

11
Table 3-1. Strains of X. campestris used in host range investigation.
Pathovar Strain
Plant Host
Location
alfalfae KS
Medicago sativa
Kansas
FL
M. sativa
Florida
cyamopsidis 13D5
Cyamopsis tetragonoloba
-
X002,X005,X016,X017
C. tetragonoloba
Arizona
glycines R-9-3
Glycine max
Brazil
1717
G. max
Africa
17915
G. max
-
S-9-8
G. max
Wisconsin
malvacearum D,M,N,0,U,V,X,Y,Z,TX84
Gossypium hirsutum
Texas
A,B,E,F,G,H
G. hirsutum
Oklahoma
Chl,Ch2
G. hirsutum
Chad
HV25
G. hirsutum
Upper Volta
Su2,Su3
G. hirsutum
Sudan
FL79
G. hirsutum
Florida
083-4244,M84-11
Hibiscus rosa-sinensis
Florida
X10,X27,X52,X102,X108
H. rosa-sinensis
Florida
phaseoli EKll,Xph25,Xpfll
Phaseolus vulgaris
Nebraska
Xpa,Xpll
P. vulgaris
Wisconsin
82-1,82-2
P. vulqaris
Florida
LB-2,SC-3B
P. vulgaris
Nebraska
XP2
P. vulgaris
New York
XP-JL
P. vulqaris
Kansas
XP-JF
P. vulgaris
Missouri
XP-DPI,B5B
P. vulgaris
-
pisi XPl
Pisum sativum
Japan
vignicola A81-331,C-1,CB5-1,
Vigna ungiuculata
Georgia
Xvl9,SN2,432,82-38
V. unguiculata
Georgia

Plant Inoculations
Pathogenicity tests were conducted by pressure infiltrating
bacterial suspensions into leaf tissue with a blunt-end syringe and
incubating the plants in 28 C or 30 C growth chambers until symptoms
were expressed. The leguminous host plants were incubated at 28 C and
symptoms were recorded after one week. The malvaceous hosts were
incubated at 30 C and symptoms were recorded after a two week period.
Pressure infiltration facilitated rapid screening of plant reactions,
but was not representative of natural inoculation. Because alfalfa
leaves were small in size, infection was by spray inoculation.
Bacterial suspensions were prepared by centrifuging overnight cultures
of X. campestris, resuspending them in 0.7% NaCl to an approximate
optical density (ODggonm) f 0*3 fr plant inoculation. These
pathogenicity tests were repeated at least once. The results were
recorded to determine compatible (pathogenic) or incompatible (non-
pathogenic or hypersensitive) plant reactions to inoculations.
Physiological Differentiation
As X. c ampe s t r i s is defined (6), the physiological
characteristics for gelatin and starch hydrolysis may be variable for
this species. Other physiological characteristics which are consistent
with this species includes xanthcmonadin production, mucoid growth,
and esculin and casein hydrolysis. These above mentioned
characteristics were determined using standard methods (36,82).
Additionally, physiological tests for production of ceilulase (96) ,
lecithinase, lipase, and pectinases were included. Hildebrand's
medium prepared at three pH's (4.5, 7.0, and 8.5) was used to detect
pectolytic enzyme activity. In each of these three media, sodium

polypectate was used as the carbon source. The pH 5 medium included
only pectin as the sole carbon source. These tests were each repeated
once.
Polyacrylamide Gel Electrophoresis
Bacteria were grown overnight at 30 C in a peptone-glycerol
broth. Cells were adjusted to a 0.3 optical density (ODggonm)' and
1.5 mis of cells were collected by centrifugation, washed once in
water, and resuspended in 50 ul 10% sorbitol. Then, 50 ul
solubilization buffer (90 mM Tris (pH 6.8), 20% glycerol, 4% SDS, 10%
B-mercaptoethanol, and .002% bromophenol blue) was added to the
suspension and the mixture was boiled for 5 minutes. Approximately 10
mg protein (10 ul solution) was added to a 10% resolving acrylamide
gel (51), and samples were electrophoresed in a 20 cm x 17 cm x 1.5 mm
vertical gel unit at 10 V/cm and 15 C until the dye reached the bottom
of the gel. Biorad low-range SDS-PAGE standards were used as
molecular weight markers. Gels were stained with 0.1% Cocmassie blue
in 42% methanol and 17% glacial acetic acid. The gel was destained in
30% methanol and 10% glacial acetic acid. Electrophoresis was only
performed once. A photograph of the gel was taken using Polaroid type
55 film.
Results
Pathogenicity Tests
All of the strains of X. c_. pv. phaseoli, with one exception,
gave the expected disease reactions on the legume hosts tested. The
exception, strain B5B, was found to be non-pathogenic to all four
hosts tested. The remaining six strains were pathogenic on kidney
bean (Table 3-2), giving watersoaked lesions on inoculated leaves

14
Table 3-2. Legume plant reactions to inoculation with pathovars of X.
campestris.
Inoculation
Strain
Host Reaction^-
bean
cowpea
soybean
alfalfa
X.campestris
pv.
phaseoli
82-1
+B
C
_D
0E
Xpa
+
-
-
0
Xpfll
4-
-
-
0
XP-JF
+
-
-
0
EKll
+
-
-
0
XP2
+
_F
__
0
B5Bg
-
-
-
0
vignicola
CB5-1
_H
+ 1
0
Xvl9
-
+
-
0
432
-
+
-
0
A81-331
-
+
-
0
C-l
-
+
-
0
82-38
+/-K
+

0
SN2
+/-
+
-
0
glycines
B-9-3
+L
_C
+M
0
1717
+
-
+
0
17915
+
-
+
0
S-9-8g
-
-
-
0
alfalfae
FL
+L
_c
_D
+N
malvacearum
N
_F
0
_F
0
control
0
0
0
0
A = + is compatible, is incom
patible, +/- is intermediate,
and 0 is a null reaction.
B = compatible lesions were
watersoaked and appeared to
be spreading.
C = dry necrotic lesion with wine
red reaction.
D = dry necrotic lesion with
chlorosis.
E = no reaction seen with spray
inoculation.
F = slight tissue discoloration
at inoculation site.
G = strain appeared non-
pa thogenic.
H = dry necrotic lesion with
slight watersoaking at
periphery of inoculation site.
I = dry necrotic lesion with
shothole effect.
J = dry necrotic lesion.
K = as described for H, but
slight shothole effect present.
L = watersoaked lesion.
M = watersoaked chlorotic lesion.
N = watersoaked leaf spots.

after four days. The lesions extended from the inoculation sites
giving the appearance that the bacterium was spreading throughout the
leaf.
15
The strains of X. c. pv. phasedi which were found pathogenic on
kidney bean were not found to be pathogenic on cowpea, soybean, or
alfalfa. In cowpea and soybean, a hypersensitive reaction was
apparently elicited, resulting in dry necrotic lesions at the
inoculation sites. In soybean the lesion was slightly chlorotic,
whereas for cowpea, a wine red lesion occurred. However, X. c. pv.
phasedi strain XP2 did not elicit the wine red color on cowpea (Table
3-2). No response was observed on alfalfa from the spray inoculations
with X. c. pv. phasedi.
The strains of X. c. pv. vignicola were pathogenic on cowpea,
resulting in irregularly shaped lesions which had a tearing, or
"shothole" appearance at the inoculation site. On soybean, the
incompatible response appeared dry and collapsed. Again, no response
was observed from spray inoculation on alfalfa (Table 3-2).
On kidney bean, a range of different responses occurred from
inoculations with X. c. pv. vignicola. For two of the strains, A81-
331 and Cl, an incompatible response appeared as a dry collapsed
lesion. For the other strains of X. c. pv. vignicola, there appeared
to be sane slight water-soaking in the tissue about the periphery of
the inoculation site. In two of these instances, for strains 8238 and
SN2, the "shothole" effect as seen on cowpea was observed.
The strains of X. c. pv. glycines appeared to be pathogenic on
both soybean and kidney bean, but not on cowpea or alfalfa (Table 3-
2). The pathogenic responses appeared as watersoaked lesions, with

the lesion being slightly chlorotic in soybean. The incompatible
response in cowpea appeared as wine red in color like that seen with
X. c. pv. phased i.
From spray inoculations on alfalfa, only X. c. pv. alfalfa,
resulted in a pathogenic response. After 7 days, small water-soaked
leaf spots which later turned necrotic appeared. X. c. pv. alfalfa
was also pathogenic on kidney bean, and appeared so slightly on
soybean. There were small water-soaked spots which occurred at the
inoculation sites on soybean. The wine red incompatible response was
observed on cowpea.
A strain of X. c. pv. malvacearum, which was not a pathogen of
legumes, resulted in only a slight tissue discoloration at the
inoculation site on bean and soybean. A null reaction, as observed
with the water-only control, was observed on cowpea. The water-only
control also resulted in a null reaction on the other hosts tested. A
summary of the plant reactions observed for the inoculation
experiments is given in Table 3-3.
In separate inoculation tests, a strain of X. c. pv. cyamopsidis
was not found pathogenic on alfalfa, kidney bean, cowpea, or soybean
and a X. c_. pv. pisi strain was not found pathogenic on kidney bean.
Overall, most of the reactions observed conformed to those reported in
the literature, with some exceptions. Variation was evident among
strains of a given pathovar in addition to that between pathovars.
The X. c_. pv. alfalfae strains appeared to have overlapping host
ranges, which extended to bean and pea in addition to alfalfa. The
kidney bean cultivar, California Light Red, appeared susceptible to a

17
Table 3-3. Host range observed for pathovars of Xanthanonas campestris
pathogenic to legume host plants.
Pathovar
Inoculat
ion reaction
on host plants9
G.max
M.sativa
P.sativum
P.vulgaris V.unguiculata
alfalfaeb
+
+c
+ -d
glycines
+
-
nt
+
phaseoli
_e
-
nt^
+
pisi
-
-
+c
-
vignicola
_e
-
nt
+9 +
cyamopsidish

nt

a + = compatible, = incompatible, nt = not tested. Host plants were
Glycines max (soybean) cv. Evans; Medicago sativa (alfalfa) cv.
FL-77; Pisum sativum (pea) cultivar not known; Phaseolus vulgaris
(bean) cv. California Light Red; and Vigna unguiculata (cowpea) cv.
California Blackeye #5.
k Other susceptible hosts reported are Trigonella and Melilotus (6).
c Reported as susceptible, but not tested,
d Reported as positive in the literature (6).
e Reaction appears negative for most strains, but occasional limited
water soaking is evident.
f There is a report of pathovar phaseoli on Pisum lunatus (6).
9 Symptoms are similar to those seen in V. unguiculata.
h The natural host belongs to the genus Cyamopsis.

18
representative strain from each of the X. campestris pathovars of
legumes tested, with the exception of X. c. pv. cyamopsidis. Various
phenotypic responses were observed on the cotton and hibiscus
cultivars with the X. c. pv. malvacearum strains tested. In general,
it appeared that those strains of X. c. pv. malvacearum derived as
pathogens from cotton were pathogenic to one of the hibiscus cultivars
tested. None of the X. c_. pv. malvacearum strains derived as
pathogens from hibiscus were pathogenic on the cotton cultivars in
these tests.
From the inoculation tests on the cotton host differentials with
the cotton derived strains of X. c. pv. malvacearum, it appeared that
three races of the pathogen were being used. All of these strains
were pathogenic on Acala 44 (susceptible host), but could be
differentiated into races by the other cotton lines containing
different resistance gene backgrounds. The X. c_. pv. malvacearum
strain N was pathogenic on all the cotton lines tested, while strain H
was only pathogenic on Acala 44. Strains FL79 and TX84 appeared to be
the same race because they were pathogenic on the same six out of
eight cotton lines tested (Table 3-4). However, these two strains
differed in reactions on the three hibiscus cultivars inoculated.
Strain FL79 appeared pathogenic on the three cultivars, but TX84 gave
a null response similar to the water-only control. The two other X.
c. pv. malvacearum strains (N and H) appeared identical in reaction
with the three hibiscus cultivars,; giving pathogenic responses to 2
cultivars, and a null response on the remaining cultivar (Table 3-4).

Table 3-4. Malvaceous host plant reactions to inoculation with strains
of X. camoestris pv. malvacearuma.
Strain
Cotton
Hibiscus
44
B1
b2
Acala
B3
b5
bin
101
Gregg
Hlb
H2
H3
N
+
+
+
+
+
+
+
+
+
0
+
H
+
-
-
-
-
-
-
-
+
0
+
FL79
+
+
+
+
+
-
-
+
+
+
+
TX84
+
+
+
+
+
-
-
+
0
0
0
X10
-
-
-
-
-
-
-
-
V-
+
+
X27
0
0
0
0
0
0
0
0
0
0
0
X52
-
-
-
-
-
-
-
-
-
+
+
XI02
-
-
-
-
-
-
-
-
-
+
+
X103
-
-
-
-
-
-
-
-
-
+
+
X108
-
-
-
-
-
-
-
-
+
+
+
83-4244
-
-
-
-
-
-
-
-
-
+
+
M84-11
0
0
0
0
0
0
0
0
0
0
0
84-1093G0
0

0
0
0
0
0

+/-
0
5 + = compatible reaction, = incompatible reaction, 0 = null
response, and +/- = intermediate reaction.
b H. rosa-sinensis cv. "Brilliant Red".
c Strain 84-1093 is X. c. pv. esculenti.

With the eight X. c. pv. malvacearum strains derived from
hibiscus, two gave null reactions on all of the cotton and hibiscus
cultivars tested (Table 3-4). The other strains derived from hibiscus
were pathogenic on at least two of the three hibiscus cultivars
tested, but were not pathogenic on any of the cotton cultivars. In a
separate experiment, one of the strains (M84-11) did appear to give
pathogenic symptans on Acala 44, while another strain (83-4244) gave a
hypersensitive response. In this instance the mean temperature was at
25 C, rather than 30 C.
An additional strain tested, X. c. pv. esculenti strain 84-1093,
which was a pathogen derived from okra, gave a null reaction on seven
of eight cotton cultivars and one of three hibiscus cultivars. The
other cotton or hibiscus cultivars gave hypersensitive responses
(Table 3-4).
Physiological Differentiation
All of the strains tested were mucoid on NYGA medium, produced
xanthomonadin, and hydrolyzed esculin and casein. All of the strains
tested also appeared to be positive for starch and gelatin hydrolysis,
and production of lipase (Table 3-5) Six of the seven X. c. pv.
vignicola strains, and one strains of X. c. pv. phaseoli appeared to
hydrolyze gelatin slower than the other X. campestris strains.
Cellulase activity, indicated by pitting of the solid medium, was
observed for all seven of the pathovars tested. However, not all
strains of X. c. pv. phaseoli appeared to have cellulase activity, and
X. c_. pv. alfalfae strains were weaker in this activity. None of the
strains tested exhibited pectolytic activity at pH 4.5, suggesting a

21
Table 3-5. Physiological reactions of strains of X. campestris.
Physiological Test Medium3
Pathovar Starch
Gelatin
Cellulose
Na
4.
polypectate
5 7.0 8.5
Pectin
5.0
Lecithin0
alfalfae
KS
+
+
w
-
-
-
-
+
FL
+
+
w
-
-
-
-
+
cyamopsidis
13D5
+
+
+
-
+
+
-
+
glycines
B-9-3
+
+
+
-
+
+
-
+
1717
+
+
+
-
+
+
-
+
17915
+
+
+
-
+
+
-
+
S-9-8
+
+
+
-
+
+
-
+
malvacearum
H
+
+
+
-
-
-
-
-
N
+
+
+
-
-
-
-
-
phased i
EK11
+
+
-
-
-
-
-
+
Xph25
+
+
+
-
-
-
-
+
Xpfll
+
+
-
-
-
-
-
Xpa
+
w
-
-
-
-
-
+
Xpll
+
+
-
-
-
-
-
+
82-1
+
+
-
-
-
-
-
+
82-2
+
+
-
-
-
-
-
+
LB-2
+
+
-
-
-
-
-
+
SC-3B
+
+
+
-
+
+
-
-
XP2
+
+
-
-
-
-
-
+
XP-JL
+
+
-
-
-
-
-
+
XP-JF
T
+
-
-
-
-
-
-L
XP-DPI
+
+
-
-
-
-
-
+
pisi
XP1
+
+
+
-
+
+
-
+
vignicola
A81-331
+
-f
+
-
+
+
-
+
C-l
+
w
+
-
+
+
-
+
CB5-1
+
w
+
-
+
+
-
+
Xvl9
+
w
4.
-
+
+
-
+
SN2
+
w
+
-
+
+
-
+
432
+
w
+
-
+
+
-
+
82-38
+
w
+

+
+
_
+
a All strains were mucoid, esculin positive, milk proteolytic, and
lipase positive. + = positive, = negative, and w = weak reaction.
k A lecithinase positive reaction was observed as a clearing zone around
the colony plus precipitation in the medium.

lack of polygalacturonase activity. At pH 7.0 and 8.5, pectolytic
activity was observed for all strains of X. c. pv. cyamopsidis, X. c.
pv. glycines, X. c. pv. pisi, and X. c. pv. vignicola. Only one of the
13 X. c. pv. phaseoli strains tested (SC-3B) had pectolytic activity.
None of the strains degraded pectin as a carbon source, only sodium
polypectate.
Polyacrylamide Gel Electrophoresis
Total protein was extracted from 18 strains, representing seven
different pathovars of X. campestris. The pathovars included were X. c.
pv. alfalfae, X. c_. pv. cyamopsidis, X. c. pv. glycines, X. c. pv.
malvacearum, X. c_. pv. phaseoli, X. c. pv. pisi, and X. c. pv.
vignicola. All of the pathovars, except X. c_. pv. malvacearum (Fig. 3-
1, lane P), are known pathogens of legumes. One of the X. c. pv.
phaseoli strains consisted of a biochemical variant of this pathovar, X.
c_. pv. phaseoli var. fuscans (Fig. 3-1, lane J) One of the X. c_. pv.
malvacearum strains was derived from hibiscus, rather than from cotton
(not shown).
In general, there were few observable differences in the protein
banding patterns for the X. campestris strains. Seme minor differences
in banding patterns between different pathovars were observed, but
variation to the same extent was also present within a given pathovar.
Discussion
The plant reactions observed from the X. campestris host range
study conformed to expectations, in most cases. However, it was evident
that pathogenic variation in strains associated with a given pathovar
could occur, as shown by the race-specific interactions with the X. c.

23
Figure 3-1. Polyacrylamide gel electrophoresis of total proteins from
strains of X. campestris. Lanes A and B, X. c. pv. alfalfae; lanes C-
F, X. c. pv. glycines; lanes G-I, X. c. pv. phaseoli; lane J, X. c.
pv. phaseoli var. fuscans; lane K, X. c. pv. pisi, lane L, X. c. pv.
cyamopsidis; lanes M-0, X. c. pv. vignicola; and lane P, X. c. pv.
malvacearum (cotton). Protein size is labeled in kilodaltons.

24
pv. malvacearum strains. In the legume study, it was interesting to
note that different hosts gave different phenotypic responses to
different incompatible pathovars of X. campestris. These different
incompatible phenotypes suggest that different host resistance genes
may be involved in each of these incompatible interactions. Although
the hypersensitive reaction is commonly considered to be the general
resistance mechanism of plants against bacteria, these data suggest a
more specific response, unique to each incompatible reaction (33,49).
No formal studies on the hypersensitive reaction were implemented, so
the significance of this observation will require further
investigation. Differences in the phenotypes which may account for
this are time of recognition, cell number, electrolyte leakage, or
other bacteria- or plant-conditioned responses. Likewise, a
hypersensitive reaction may have occurred for instances where a null
response was observed (27).
The results from inoculation tests on the malvaceous hosts
suggested that those strains of X. c. pv. malvacearum derived from
cotton are potential pathogens of hibiscus, whereas those derived from
hibiscus are generally restricted to hibiscus. However, in one
experiment, it was found that one of the hibiscus strains caused
watersoaked lesions on Acala 44 when the night temperatures were
lowered. This observation indicates that some of the interactions may
be temperature sensitive. Seme resistance genes in cotton are known
to be temperature sensitive (9).
Race-specific incompatibility may account for the varied X. c.
pv. malvacearum (cotton strains) reactions on hibiscus. It was

interesting to note that strains FL79 and TX84, both race 16 of X. c.
pv. malvacearum cotton strains, each differed in reactions on the
hibiscus differential lines. Since Acala 44 elicited a hypersensitive
response from 6 of 8 hibiscus strains, possibly Acala 44 contains an
undiscovered resistance gene to the hibiscus derived strains. Those
strains for which no hypersensitive reaction was induced may have
escaped host recognition, but lack the necessary pathogenicity
determinants to incite disease. Avirulence genes do not give
phenotypic expression in non-parasites (88). Bacterial growth
kinetics would help determine if the bacteria are multiplying on these
hosts. This would be useful for the case of X. c_. pv. esculenti, for
which 1 of 8 cotton hosts were incompatible, and 2 of 3 hibiscus hosts
were incompatible.
The data from the protein and physiological tests demonstrated
differences between strains, but were not useful for typing strains by
pathovar. There was significant variation among strains within a
given pathovar. Although the use of SDS-PAGE to study total protein
profiles did not differentiate the pathovars, seme work has suggested
limiting the analysis to just outer membrane proteins to improve
resolution of strain differences (70).
These experiments confirmed pathogenicity of the stock strains
and clarified inconsistencies in the literature concerning host range.
The host range studies indicated that conclusions reached may often be
strain-dependent, and that examination of a large number of strains
will reveal a different overall picture than an examination of a few
strains. For example, the plant source of X. c. pv. malvacearum

determines whether a strain will have the host range listed for the
pathovar (hibiscus and cotton) or a more limited host range
(hibiscus). However, the plant source of X. c. pv. alfalfae, does not
determine whether a strain will have the host range listed. A strain
which attacks both alfalfa and bean is X. c. pv. alfalfae. A strain
with a host range limited to bean is X. c. pv. phaseoli. Finally,
several biochemical methods were evaluated for their potential to
classify a strain at the pathovar level. Although standard
microbiological tests and protein gels were not adequate for such
classifications, an examination of plasmid DNAs (Chapter Four) and
chromosomal DHAs (Chapter Six) yielded more rewarding results.

CHAPTER FOUR
CONSERVATION OF PLASMID DNA SEQUENCES AND
PATHOVAR IDENTIFICATION OF STRAINS OF XANTHOMONAS CAMPESTRI^
Introduction
More than 125 different pathovars of Xanthanonas campestris
(Pammel 1895) Dowson 1939 are currently recognized (6,24), and the
primary means for differentiating them is by inoculation of the plant
host(s) of that pathovar. It would be a difficult task to inoculate
every plant that could serve as a host to an X. campestris isolate,
and therefore the potential host range of a given isolate is unknown.
Most often, the pathovar name assigned to a strain of X. campestris is
determined by the host it was isolated frcm. Such designations may be
artifactual since the primary host may be different from the one the
strain was isolated from; sane X. campestris pathovars are known to be
pathogenic on more than one host. Epiphytes cannot be classified in
this pathovar identification system. Possible taxonomic relationships
among pathovars are also elusive. It would be helpful if alternative
means to differentiate among X. campestris pathovars were available.
Seme suggested approaches for differentiating X. campestris pathovars
have included serology (1,94), membrane protein profiles (70),
phage-typing (37), and gas chromatography of fatty acids (65,81).
These approaches suffer because they are often strain specific,
This chapter
Phytopathology.
contains copyrighted material from the journal
It is reprinted here with permission of the publisher.
27

dependent on constant environmental parameters, and/or so cumbersome
that no extensive evaluative tests have been performed.
28
Plasmid DNA has been identified in several pathovars of X.
campestris (13,16,30,39,46,52,53,60,80). It is relatively simple to
extract large numbers of strains and visualize their plasmids with
standardized alkaline lysis procedures (46,66). To characterize the
plasmids, restriction endonucleases are used to digest the DNA into
distinct fragments that can be separated by size, resulting in
fragment patterns visualized by agarose gel electrophoresis. To date
there have been no systematic attempts to examine the extent of
plasmid variation among a large number of strains involving a large
number of X. campestris pathovars. Our preliminary studies on
selected pathovars of X. campestris suggested that there was a
surprisingly high degree of plasmid sequence conservation within sane
pathovars (31,52,53). These studies further suggested that plasmids
of purified strains were quite stable and hence useful in
epidemiological studies (31), similar to those used to monitor the
spread of selected human pathogens (28). The purpose of this study
was to survey the extent of variation of plasmid DNAs within a large
number of X. campestris pathovars and to determine whether the plasmid
content of strains based on restriction fragment polymorphism and
Southern hybridization could be used in differentiating the pathovars
of X. campestris.
Materials and Methods
Bacterial Strains
The X. campestris strains used in this study, their pathovar
designations, geographic origin, and sources are listed in Table 4-1.

Table 4-1. Strains of X. campestris used for plasmid analysis.
Bacterium Strain designation
(number of strains) (geographic origin) Source3
Xanthanonas campestris pathovar
alfalfae (2)
KS (Kansas);
D.L.Stuteville
FL (Florida);
R.E.Stall
argemones (1)
084-1052 (Florida);
DPI
begoniae (1)
084-155 (Florida);
DPI
campestris (4)
XC1 (Oklahoma) ;
084-720,084-809,
this study
084-1318 (Florida);
DPI
carotae (3)
G1 (Idaho),G5,G7 (California);
R.E.Stall
citri (5+)
X59 (Brazil),X62 (Japan),
X69 (Argentina), X70 (Brazil);
E.L.Civerolo
Fll (Florida);
DPI
cyamopsidis (5)
13 D5;
X002,X005,X0016,
C.I.Kado
X0017 (Arizona);
J.Mihail
dieffenbachiae (2)
084-729,084-1373 (Florida);
DPI
esculenti (1)
084-1093 (Florida);
DPI
glycines (3)
B-9-3 (Brazil),1717 (Africa),
17915;
W.F.Fett
hederae (3)
084-1789,084-3928,
251G (Florida);
DPI
holcicola (2)
Xh66 (Kansas);
L.Claflin
XH1;
this study
maculifoliigardeniae(2)
084-6006,084-6166 (Florida);
DPI
malvacearum-cotton(32)
A,B,E,F,G,H (Oklahoma);
HV25 (Upper Volta),
Chl,Ch2 (Chad),
M. Essenberg
Su2,Su3 (Sudan);
L.S.Bird
FL79 (Florida);
DPI
D,M,N,0,U,V,W,X,Y,Z,TX84 (Texas),
I,Q,R,S,T (Oklahoma),
C,J,K,L (Upper Volta);
this study
malvacearum-hibiscus(8)
XI0,X27,X52,X102,
X103,X108 (Florida);
A.R.Chase
083-4344,M84-11 (Florida);
DPI
mangiferaeindicae (1)
084-166 (Florida);
DPI
pelargonii (1)
084-190 (Florida);
DPI
phased i (13)
EK11,Xph25,Xpf11 (Nebraska);
M. Schuster
Xpa,Xpll (Wisconsin);
A.W.Saettler
82-1,82-2 (Florida);
R.E.Stall
LB-2,SC-3B (Nebraska);
A.K.Vidaver
XP2 (New York);
J.A.Laurence
XP-JL (Kansas);
J.L.Leach
XP-JF (Missouri),XP-DPI;
this study

30
(Table 4-1 continued.)
Bacterium
Strain designation
(number of strains)
(geographic origin)
Source9
pisi (1)
XP1 (Japan);
M.Goto
poinsettiicola (1)
083-6248 (Florida);
DPI
pruni (3)
068-1008,084-1793 (Florida);
DPI
82-1 (Florida);
R.E.Stall
translucens (2)
82-1 (Florida);
R.E.Stall
XTl;
this study
vesicatoria (5)
E-3,69-13,71-21,
82-8,82-23 (Florida);
R.E.Stall
vignicola (7)
A81-331,C-1,CB5-1,
Xvl9,SN2,432,82-38 (Georgia);
R.D.Gitaitis
vitians (3)
084-2057,084-2848,
084-4348 (Florida);
DPI
zinniae (1)
084-1944 (Florida);
DPI
unknown (1)
G65
this study
X. albilineans (1)
Xalb (Florida);
M. J.Davis
X. fragariae (1)
Xfra (Florida);
R.E.Stall
a DPI = Florida Department of Agricultural and Consumer Services,
Division of Plant Industry, Gainesville, Florida.

31
Some of the stock cultures of X. c. malvacearum were mixtures of
different strains, maintained and described as "races" for purposes of
screening cotton host differentials and breeding lines for disease
resistance. Described "races" of X. c. malvacearum may or may not be
purified strains; as many as seven different strains have been derived
from a single race 1 isolate (7). All strains used in this study were
repeatedly purified from single colonies and confirmed to be
pathogenic on their designated hosts. Broth cultures of bacteria were
grown in a peptone-glycerol medium (per L: 10.0 ml glycerol, 20.0 g
peptone, and 1.5 g K2HPO4). Bacterial strains were commonly stored at
-80 C in the same medium containing 15% glycerol.
Plasmid Extraction and Visualization
Cultures were grown to mid- to late-logarithmic growth phase and
extracted by either of two small-scale alkaline lysis extraction
procedures (46,66). Extracted DNA was resuspended in TE (10 mM
Tris(hydroxymethy1)aminomethane (Tris) 1 mM sodium
ethylenediaminetetraacetate (N32EDTA), and 20 ug/ml DNase-free
pancreatic RNase; pH 7.6) and digested with either of two restriction
endonucleases, EcoRI or BamHI, using manufacturer (Bethesda Research
Laboratories, Gaithersburg, MD) specifications. Plasmid DNA fragments
were separated by size using agarose gel electrophoresis (0.6% agarose
(Sigma Type I:low EEO), 2-5 V/cm) in Tris-acetate buffer (40 mM Tris,
1 mM Na2EDTA, adjusted to pH 7.6 with glacial acetic acid). Fragments
were visualized by ultraviolet irradiation (302 nm) after staining
agarose gels in ethidium bromide (0.5 ug/ml). Photographs were taken
with Polaroid Type 55 (or Type 57) film using a yellow filter (Tiffen

no. 12). All restriction fragment size estimates were based on the
relative mobilities of linear DNA fragments using lambda phage DNA
digested with HindiII as molecular size standards. Plasmid sizes were
32
estimated by addition of the sized restriction digested plasmid DNA
fragments. All plasmid experiments were repeated at least twice for
each strain examined.
Cloning of Plasmid Restriction Endonuclease Fragments
Plasmid DNA isolation from X. campestris was by a modification of
either of two alkaline lysis extraction procedures (46,68). The
extracted plasmids were purified on CsCl-etnidium bromide gradients by
centrifugation at 55,000 rpm in a Beckman VT65.2 rotor for 17 hr at
20 C. The purified plasmids were digested with the restriction enzyme
EcoRI. The cloning vector, pUCD5 (14), was digested with EcoRI,
treated with calf intestinal alkaline phosphatase (Boehringer-
Mannheim, Indianapolis, IN), and ligated to the EcoRI digested X.
campestris plasmid fragments with T4 DNA ligase (Bethesda Research
Laboratories). The ligation products were transformed into E. coli
strain ED8767, selecting transformed colonies on Luria-Bertani medium
containing ampicillin (50 ug/ml) or kanamycin (30 ug/ml). Selected
colonies were analyzed for the vector containing desired cloned DNA
fragments. These general cloning procedures are outlined in Maniatis
et al. (66).
DNA/DNA Hybridization
Plasmid DNAs were transferred from agarose gels to nitrocellulose
membranes by the method of Southern as described by Maniatis et al.
(66) and hybridized against radioactively-labeled DNA probes. The DNA

probes derived iron plasmid DNA of X. campestris pathovars were either
cloned restriction digested DNA fragments of plasmid DNA in the cosmid
vector pUCD5, or of the complete X. campestris plasmid. DNA probes
were labeled in vitro with use of a nick translation kit (Bethesda
Research Laboratories) using 32P-deoxycytidine triphosphate and
hybridized against DNA bound to nitrocellulose membranes. The
membranes were pre-hybridized and hybridized in plastic bags at 68 C.
The pre-hybridization and hybridization solutions were those described
by Maniatis et al. Following hybridization, membranes were washed once
in 2X SSC, 0.5% SDS and washed once in 2X SSC, 0.1% SDS at ambient
temperature, and washed two times in 0.1X SSC, 0.5% SDS at 68 C as
described by Maniatis et al. (66) for stringent conditions. The
membranes were then air dried and exposed at -80 C to X-ray film
(Kodak X-Ctnat AR) using intensifier screens. Similar methods were
used to probe DNA transferred to nitrocellulose using a dot-blot
manifold (Schleicher and Schuell Inc., Keene, NH) All hybridization
experiments were repeated at least once.
Results
Detection of Plasmid DNA
Indigenous, cryptic plasmids were detected in all strains of the
following X. campestris pathovars: cyamopsidis, dieffenbachiae,
glycines, malvacearum (cotton), pelargonii, phaseoli, pruni,
vesicatoria, vignicola, and vitians (Table 4-2). Plasmids were not
detected in any strains of X. campestris pathovars alfalfae,
argemones begoniae carotae, esculenti, holcicola,
maculifoliigardeniae, malvacearum (hibiscus), manqiferaeindicae, pisi

34
Table 4-2. Detection of plasmid DNA in strains of X. campestris.
Bacterium
No. of strains
containing plasmids/
No. of strains tested
Pathogenicity3
X. campestris pathovar
alfalfae
0/ 2
P
argemones
0/ 1
I
begoniae
0/ 1
I
campestris
2/ 4
R,I
carotae
0/ 3
R
citri
17/44
P,R,I
cyamopsidis
5/ 5
R
dieffenbachiae
2/ 2
I
esculenti
0/ 1
I
glycines
3/ 3
P
hederae
2/ 3
I
holeicola
0/ 2
R
maculifoliigardeniae
0/ 2
I
malvacearum cotton
32/32
P
malvacearum hibiscus
0/ 8
P
mangiferaeindicae
0/ 1
I
pelargonii
1/ 1
R
phaseoli
13/13
P
pisi
0/ 1
R
poinsettiicola
0/ 1
I
pruni
3/ 3
I
translucens
0/ 2
R
vesicatoria
5/ 5
P
vignicola
7/ 7
P
vitians
3/ 3
I
zinniae
0/ 1
I
X. albilineans
0/ 1
R
X. fragariae
0/ 1
I
3 P = Pathogenicity of strains confirmed on appropriate host, conforms
to current available information for particular pathovar.
R = Received as named pathogen, appropriate host specificity and
pathovar designation of strain assumed.
I = Isolated as a pathogen on host appropriate for designated pathovar;
characterization of strain(s) incomplete.

poinsettiicola, translucens, and zinniae. Plasmids were found insome,
but not all strains of X. campestris pathovars campestris and
hederae. Similarly, plasmids were found in all type strains of X. c.
pv. citri (A, B, and C types); this is in agreement with a previous
plasmid study of this pathovar (13). However, not in all strains of X.
campestris isolated from leaf spots of citrus in Florida contained
plasmids.
It appeared that plasmid-containing strains of X. campestris
carried from one to three plasmids based on electrophoresis of
extracted plasmid DNA. For example, a majority of the
plasmid-containing X. c. malvacearum strains contained only one
plasmid, but seme carried two or more. When plasmid-containing
strains of X. campestris were purified by repeated single colony
isolation, the plasmid DNA content appeared to be stable. However,
variation was present in bacterial stocks which were known to have
been serially transferred in agar medium over a period of years.
Restriction Endonuclease Profiles
Plasmid profiles for X. campestris were variable in over 60
different strains tested. Plasmids were placed into classes based on
the variability of restriction endonuclease (EcoRI) digestion profiles
on agarose gels. When a different restriction endonuclease (BamHI)
was used, the plasmid profiles were placed into the same classes. In
all cases, strains which belonged to the same plasmid class also
belonged to the same pathovar. There was obvious variability within
classes, but there also appeared to be conservation of some DNA
fragments of identical sizes (Fig. 4-1). Undigested plasmids were not

36
reliable for strain classification because several strains had
plasmids of apparently identical size, but they were clearly different
after digesting the plasmid DNAs with restriction enzymes. By adding
up the DNA fragment sizes yielded by restriction digests, plasmids in
X. campestris were estimated as ranging from about 3 to 200 kb
(kilobase pairs) in size. Estimation of some of these sizes were
difficult for seme strains due to the presence of more than one
plasmid.
Plasmid profiles of strains of X. c. cyamopsidis, X. c. glycines,
X. c. malvacearum, X. c. phaseoli, and X. c. vignicola were compared.
Restriction fragment length polymorphism was evident within each of
these pathovars. Although more than one plasmid was present in seme
of these strains, a subset of restriction fragments of similar length
and overall pattern appeared to be consistent for strains within a
given pathovar (Figure 4-2).
Four highly polymorphic plasmid variants were found in 17 out of
44 Florida strains tested, but Southern hybridization revealed no
homology between seme of the plasmids (31). Furthermore, there were
no similarities in plasmid digestion patterns between X. c. pv. citri
type A, type B and any of the Florida citrus leaf spot strains that
carried plasmids (Figure 4-3). These Florida isolates are presumed to
be X. c. citri, but they grow well and also cause disease symptoms on
kidney bean and alfalfa, thus making their pathovar status
questionable (34).

37
Figure 4-1. Plasmid DNAs from strains of Xanthomonas campestris pv.
malvacearum (Xcm) digested with restriction endonucleases EcoRl (lanes
B-I) and BamHI (lanes J-Q). Lanes shown above contain: A and R, lambda
HindIII; B and J; Xcm J; C and K, Xcm N; D and L, Xcm H; E and M, Xcm
V; F and N, Xcm Z; G and 0, Xcm Q; H and P, Xcm X; and I and Q, Xcm D.

38
lilil
123456789012345
1 11 12222 2 2222233333333334444444
67 8 90123456789012345 678901 23456
30-
25
20H
15
10
5-
0-
kb
iii
J_L
i i i i i i
Figure 4-2. Graphic representation of plasmid restriction fragment
profiles for Xanth anonas campestris pvs. cyamopsidis (Xcc; lane 1) ,
vignicola (Xcv; lanes 2-8), pnaseoli (Xcp; lanes 9-17), phaseoli var.
fuscans (Xcpf; lanes 18-19), glycines (Xcg; lanes 20-22), and
malvacearum (Xcm; lanes 23-46) digested with EcoRI. Lanes shown above
contain 1, Xcc 13D5; 2, Xcv SN2; 3, Xcv A81-331; 4, Xcv C-l; 5, Xcv
CB5-1; 6, Xcv Xvl9; 7, Xcv 82-38; 8, Xcv 432; 9, Xcp EKll; 10, Xcp
82-2; 11, Xcp Xpa; 12, Xcp Xpll; 13, Xcp XP2; 14, Xcp XP-JF; 15, Xcp
XP-DPI; 16, Xcp Xph25; 17, Xcp 82-1; 18, Xcpf Xpfll; 19, Xcpf SC-3B;
20, Xcg B-9-3; 21, Xcg 17915; 22, Xcg 1717? 23, Xan J; 24, Xan L; 25,
Xcm C; 26, Xcm 0; 27, Xcm K; 28, Xan N; 29, Xcm A; 30, Xcm B; 31, Xcm
E; 32, Xan F; 33, Xan G; 34, Xan H; 35, Xan S; 36, Xan W; 37, Xan I;
38, Xan V; 39, Xan D; 40, Xan M; 41, Xcm X; 42, Xan U; 43, Xan Q; 44,
Xcm R; 45, Xcm Y; and 46, Xcm Z. Calculated DNA fragment sizes are
represented here by a linear scale whereas migration of DNA fragments
under electrophoresis conditions approximates a logarithmic scale
which is inversely proportional to the molecular weight of the DNA
fragment.

39
A B C D
Figure 4-3. Plasmid DNAs from strains of X. campestris pv. citri
digested with restriction endonuclease EcoRI. Lane A, strain Fll
(Florida type) ; lane B, strain X59 (A type) ; lane C; strain X62 (A
type); and lane D, strain X69 (B type). Sizes shown in kilobase pairs.

DNA/DNA Hybridization
Initial plasmid comparisons were done on strains of X. c.
malvacearum. Whole purified plasmid DNA iron X. c. malvacearum strain
X, which contains only one plasmid, was hybridized against EcoRI
digested plasmid DMAs of other strains of the same pathovar (not
shown). This initial comparison demonstrated that the
plasmids,although differing slightly in digestion patterns, were quite
homologous as the radiolabeled plasmid hybridized to almost all EcoRI
fragments of the other strains.
A 4.5 kb EcoRI plasmid fragment of X. c_. malvacearum strain N was
cloned into the vector pUCD5 and used as a hybridization probe against
plasmid DNA from other strains of the same pathovar (Fig. 4-4). This
plasmid DNA fragment hybridized to plasmids from all but one strain of
X. c. malvacearum. This probe hybridized to EcoRI fragments of 4.5 kb
size (lanes B-J, M-N, Fig. 4-4) in several other strains of X. c.
malvacearum. Additionally, the probe hybridized to more than one of
the EcoRI plasmid fragments in these strains, suggesting that seme
sequences on the cloned DNA are repeated in other parts of the plasmid
DNA. The pUCD5 vector alone did not hybridize to any X. c.
malvacearum plasmid fragments. Because pUCD5 is a cosmid vector and
contains the cos site of lambda phage DNA, hybridization of the vector
to the corresponding DNA fragment of the molecular weight marker
containing the cos site was observed. Plasmid DNA from X. c.
malvacearum strain Su2 did not hybridize to the 4.5 kb probe, and did
not hybridize to any other plasmid fragments from X. c. malvacearum
strain N. Another X. c. malvacearum strain (Ch2), which did not have

41
Figure 4-4. Plasmid DNAs from strains of Xanthomonas campestris pv.
malvacearum (Xcm) digested with restriction endonuclease EcoRI (left)
and autoradiograph of plasmid DNAs probed with a clone containing a
4.5 kb EcoRI plasmid fragment from Xcm strain N (right). Lanes shown
above contain: A, lambda HindIII B, Xcm H; C, Xcm W; D, Xcm V; E, Xcm
X; F, Xcm Q; G, Xcm Y; H, Xcm L; I, Xcm N; J, Xcm HV25; K, Xan Ch2; L,
Xcm Su2; M, Xcm FL79; and N, Xcm TX84.

42
a 4.5 kb EcoRI plasmid DNA fragment, did have two other fragments (ca.
8 and 10 kb) which hybridized strongly to the 4.5 kb probe. In two
other X. c. malvacearum strains (FL79 and TX84), which appeared to
have multiple plasmids, the hybridization signal was weak for the 4.5
kb fragments as compared to larger EcoRI fragments (ca. 23 kb) .
Similar hybridization studies were done with cloned plasmid fragments
from other pathovars. When the plasmid fragments selected were
smaller, they were much more specific. Two different probes were
constructed from 2.0 kb and 2.3 kb EcoRI plasmid fragments derived
from X. c. phaseoli (strain XP2). When hybridized against X. c.
cyamopsidis, X. c. glycines, X. c. phasedi, and X. c. vignicola, each
of these probes only hybridized to X. c. phaseoli (Fig. 4-5) The 2.3
kb plasmid probe hybridized to similar sized fragments in other X. c.
phaseoli strains, including X. c. phaseoli var. fuscans (strains SC3-B
(not shown) and Xpfll). These X. c. phaseoli var. fuscans strains
differ from typical X. c. phaseoli strains in that they produce an
extracellular dark brown, melanin-like pigment in culture; otherwise,
they are considered similar. However, the 2.0 kb plasmid probe did
not hybridize to the X. c. phasedi var. fuscans strains, which did
not have the corresponding 2.0 kb EcoRI fragment in their plasmid
profile (lane C, Fig. 4-5, and strain SC3-B (not shown)). Repeated
hybridizations with Southern transfers containing these strains had
the same results.
Dot-blot Hybridization
DNA probes were also hybridized against total DNA of other X.
campestris pathovars fixed onto a nitrocellulose membrane by use of a
dot-blot manifold apparatus. Radiolabeled total plasmid DNA from

43
Figure 4-5. Plasmid DNAs from strains of Xanthamonas campestris pvs.
phaseoli (Xcp; lanes B, D-I), phaseoli var. fuscans (Xcpf; lane C) ,
and cyamopsidis (Xcc; lane K) digested with restriction endonuclease
EcoRI (left) and autoradiographs of plasmid DNAs probed with a clones
containing a 2.0 kb EcoRI fragment (center) and a 2.3 kb EcoRI plasmid
fragment (right) from Xcp strain XP2. Lane J contains chromosomal DNA
of X. campestris pv. alfalfae (Xca) digested with EcoRI. Lanes shown
above contains: A and L, lambda HindIII; B, Xcp Xph25; C, Xcpf Xpfll;
D, Xcp EKll; E, Xcp 82-1; F, Xcp Xpa; G, Xc G65; H, Xcp Xpll; I, Xcp
XP-JF; J, Xca FL; and K, Xcc 13D5. Xc G65 is a strain isolated from
bean which was determined to be nonpathogenic and contains no plasmid.

Table 4-3. Hybridization of radiolabeled plasmid probes to total DNA of
pathovars of Xanthanonas campestris and one other Xanthanonas species.
44
Bacterium (No. tested)
N80
Probes3
N4.5 V2.3
P2.3
X. campestris pv.
alfalfae (1)
+


_
argemones (1)
-
-
-
-
begoniae (1)
-
-
-
-
campestris (1)
-
-
-
-
carotae (1)
+

-

citri (3)
+
+/-b
+/-b
+/-b
cyamopsidis (1)
+
+
+
-
dieffenbachiae (1)
-
-
-
-
esculenti (1)
-
-
-
-
cjlycines (1)
+
-
+
-
hederae (1)
+
-
-
-
holcicola (1)
+
-
-
-
maculifoliigardeniae (1)
-
-
-
-
malvacearum cotton (6)
+/-c
+/-c
+

malvacearum hibiscus (2)
-
-
-
mangiferaeindicae (1)
-
-
-
-
phaseoli (1)
+
+
-
+
poinsettiicola (1)
-
-
-
-
pruni (1)
+
+
-
-
translucens (1)
+
-
-
-
vesicatoria (1)
+
+
+
-
vignicola (1)
+
+
+
-
vitians (1)
-
-
-
-
zinniae (1)
-
-
-
-
X. albilineans (1)
-
-
-
-
3 + = hybridization observed, = no hybridization observed.
N80 = plasmid DNA derived from X. c. pv. malvacearum strain N
(about 80 kb); N4.5 = cloned EcoRI plasmid fragment (4.5 kb) from
strain N; V2.3 = cloned EcoRI plasmid fragment (2.3 kb) iron X. c.
pv. vignicola strain SN2; P2.3 = cloned EcoRI plasmid fragment
(2.3 kb) from X. c. pv. phaseoli strain XP2
b Strain FL-11 was negative, only plasmid DNA used.
c Strain S2 was negative.

45
X. c. malvacearum strain N, which carries two plasmids, hybridized to
ENA of thirteen out of twenty-three X. campestris pathovars tested
(Table 4-3). Of these thirteen, seven cross-hybridized to the 4.5 kb
subcloned fragment of X. c. malvacearum strain N. Plasmids were not
detected in sane of the pathovars which hybridized to the probe. A
2.3 kb subcloned plasmid fragment from X. c. vignicola hybridized to
only six of the 23 pathovars tested. Plasmids were present in all six
of those pathovars which hybridized to the probe. A 2.3 kb cloned
plasmid fragment of X. c. phaseoli hybridized strongly to other
strains of the same pathovar, and weakly to 2 of 3 different X. c.
citri strains tested. The X. c. phaseoli probe appears to have
hybridized to chromosomal DNA of X. c_. citri in this case, as the
probe did not hybridize against Southern transfers of EcoRI digested
plasmid fragments of X. c. citri strains (not shown).
Discussion
It is suggested that there is extensive conservation of plasmid
DNA sequences (as represented by conserved restriction fragments)
within, but not usually among, pathovars of X. campestris. Plasmid DNA
fragment patterns will be identical if there is no rearrangement of
the DNA sequence at the restriction enzyme recognition site (a six
base pair sequence for EcoRI and BamHI) if no new restriction
fragments are created within the fragment, and if there are no major
additions or deletions causing a change in fragment size. Given these
possibilities, it was surprising to find so little restriction
fragment length polymorphism of plasmids within a pathovar, especially
when strains obtained from different continents were compared.

46
Southern hybridization analyses confirmed that plasmid DNA fragments
of similar size were in fact highly homologous. For example, X. c.
malvacearum strain N, isolated in North America, had a restriction
digest pattern identical to one of the African strains (K) (Fig. 4-2).
All North American strains except strain N form a distinctive pattern
subgroup and the African strains form a somewhat different subgroup.
It is possible that either all the African strains are derived iron
strain N, or that strain N was introduced to the North America from
Africa. The latter possibility seems more likely, since the African
strains were isolated from more than one location in Africa. The
conservation of overall restriction fragment profiles of plasmids from
geographically isolated populations and the extent of homology seen in
Southern hybridizations strongly suggests that plasmid sequences are
both highly conserved and stable.
Conversely, it was surprising to find so much polymorphism of
plasmids between pathovars. Homology among plasmids in different
strains of one pathovar of Pseudomonas syringae (pv. glycinea) has
been reported (15). However, identical plasmid profiles were found
present in more than one pathovar of P. syringae (77). In the present
study, similar plasmid profiles were not found in more than one
pathovar. In addition, cloned plasmid fragments were identified which
failed to hybridize to plasmids of different pathovars. For example,
a cloned 2.3 kb plasmid fragment of X. c. phaseoli, which hybridized
to all strains of that pathovar, failed to hybridize to total DNAs of
22 other pathovars. This DNA fragment is highly conserved and is
apparently pathovar-specific, with one exception (Table 4-3) .

47
Plasmid homology between X. c. phaseoli and X. c. phaseoli var.
fuscans was revealed by hybridization that was not apparent by
restriction digest patterns. There were distinct differences in
plasmid digestion patterns of X. c. phaseoli var. fuscans in
comparison to typical X. c. phaseoli strains. The fact that both X.
c_. phaseoli and X. c. phaseoli var. fuscans have an identical host
range suggests that the homologous plasmid DNA regions that are
conserved within sane pathovars may encode host range specificity
functions. Such functions have been described on plasmids within the
Rhizobiaceae (62,64) This is a testable hypothesis that needs
further experimental support.
In X. c. malvacearum, plasmids were found in all 32 strains
isolated from cotton, and no plasmids were found in the 8 strains
isolated from hibiscus. Cotton and hibiscus each belong to the same
plant family (Malvaceae), hence the X. c_. malvacearum designation.
Atypical symptoms of cotton blight could be artificially produced in
cotton using syringe inoculations with hibiscus strains, and in
hibiscus using cotton strains. The ability of these strains to be
pathogenic on both hosts under natural conditions has not been clearly
established. Plasmid DNAs from cotton strains cross-hybridized with
one another in Southern analyses, indicating extensive homology was
evident between the plasmids. As with X. c_. phaseoli, the cotton
strains of X. c_. malvacearum appeared to carry highly conserved
plasmid DNA sequences (similar in restriction digest sizes and by
Southern analyses) which are unique to strains which have a host range
on cotton. Plasmid DNA from the cotton strain (N), when radiolabeled,

did not hybridize to chromosomal DNA derived from the hibiscus strain.
Based on our limited pathogenicity tests and on the absence of plasmid
DNA sequences in the hibiscus strains, there may be justification to
differentiate the cotton and hibiscus strains into different
pathovars.
Although some cross-hybridization between plasmids of strains
iron different pathovars was detected by dot-blot analyses (Table 4-
3), strains were readily differentiated by restriction digest profiles
of the plasmid DNAs and by hybridization with selected DNA probes to
identifiable restriction digested DNA fragments. Cross-hybridization
may be the result of repetitive DNA sequences, insertion elements, or
of conserved DNA sequences that are important for the stable
maintenance of plasmids in X. campestris. Examples of plasmid
functions which might be conserved are those involved with
replication, incompatibility, or other host dependent factors. In
seme instances plasmid DNAs hybridized to total DNA of pathovars in
which no plasmids were detected (Tables 4-2 and 4-3). It is suggested
that some sequences encoded on plasmids in some pathovars are located
on chromosomes in other pathovars. It is also possible that those
plasmids may integrate into the bacterial chromosome and excise again,
in a manner similar to those in p. syringae pv. phaseolicola (79,92).
With interest in developing rapid diagnostic methods to identify
bacterial pathogens, it is possible that the combined usage of plasmid
restriction digest profiles and of plasmid DNA probes may be
sufficient for the identification of seme pathovars of X. campestris.
This would require that a plasmid be stably associated with a given

pathovar, that plasmid profiles for specific pathovars were known, and
that an appropriate DNA probe consisting of conserved and unique DNA
sequences were available. Of the few DNA probes constructed, it was
apparent that plasmid DNA sequences were highly conserved within the
pathovars studied. In some instances the DNA probes may prove
sufficient for pathovar identification, provided they are extensively
tested. This apparent stability of the plasmids provides a natural
genetic marker that can be strain specific and perhaps useful in
epidemiological investigations. In addition to aiding the
identification for some pathovars of X. campestris, these observations
may have taxonomic significance to differentiate these pathogens.

CHAPTER FIVE
ARE RACE-SPECIFIC AVIRULENCE GENES ENCODED BY PLASMIDS
OF XANTHOMONAS CAMPESTRIS PV. MALVACEARUM?
Introduction
Bacterial blight of cotton, caused by the pathogen X. c. pv.
malvacearum (Snith) Dye, is a destructive disease of cotton, which
occurs throughout the cotton-producing areas of the world. The
development of cotton cultivars resistant to bacterial blight is the
primary means by which this pathogen is controlled. At least 16 major
genes have been identified which condition resistance to bacterial
blight (8). A gene-for-gene pattern of interaction between the plant
resistance genes and bacterial avirulence genes has been demonstrated
in this host-parasite interaction (33). In screening for resistance
to bacterial blight in breeding lines, races of the pathogen have been
maintained as mixed cultures, originally derived from infected leaf
samples. These mixed cultures have been mass transferred on agar
media to prevent the appearance of new race phenotypes. The stability
of the race phenotypes of these mixed cultures is periodically checked
by inoculating the host differentials.
Plasmids have been detected in several phytopathogenic
prokaryotes and have been associated with functions such as
pathogenicity (5), resistance to antibiotics (3,88), avirulence (88),
and host range (64). With the recent discovery of plasmids in several
50

51
pathovars in X. campestris (13,16,30,39,46,52,53,80), it is of
interest to understand the function of these autonomously replicating,
covalently closed circular DNAs of the bacterial genome. The purpose
of this investigation was to analyze the plasmids in X. c. pv.
malvacearum and determine if they influenced race-specificity in
cotton.
Materials and Methods
Bacterial Strains and Host Plants
The strains of X. c_. pv. malvacearum used for this investigation
are listed in Table 5-1. All of the strains were from single colony
isolation of either mixed, or pure cultures. The cotton differentials
used included those recommended for differentiating the races of X. c.
pv. malvacearum (45); also included were six congenie cotton lines of
Gossypium hirsutum cv. Acala 44, which contained single known
resistance genes (B genes) to X. c. pv. malvacearum (7). The congenie
Acala series included cotton with resistance genes B2, B3, B5, Bg, By,
and Bn. The other host differentials were cvs. Acala 44, 1-10B, 101-
102B, 20-3, Mebane Bl, Stoneville 20, 2B-S9, and Gregg.
Plant Inoculations
Pathogenicity tests were conducted by pressure infiltration of
abaxial leaf surfaces with bacterial suspensions using a blunt end of
a syringe, and incubating the plants in 30 C growth chambers until
symptom were expressed. Symptoms development was monitored over a two
week period.
Plasmid Analysis
Cultures were grown to mid- to late-logarithmic growth phase and
extracted by either of two small-scale alkaline lysis extraction

52
Table 5-1. Strains of X. campestris pv. malvacearum from cotton used
in race-specificity investigation.
Strain
Race
Location
Source
K
HVlb
Upper Volta
L.S.Bird
N
2a
Texas
L.S.Bird
J
HV3
Upper Volta
L.S.Bird
C
HVla
Upper Volta
L.S.Bird
0
2b
Texas
L.S.Bird
L
HV7
Upper Volta
L.S.Bird
A
2bSr
Oklahoma
M. Essenberg
B
2aSr
Oklahoma
M.Essenberg
E
4-1
Oklahoma
M. Essenberg
F
laSr
Oklahoma
M.Essenberg
G
lbSr
Oklahoma
M.Essenberg
S
3-22
Oklahoma
M.Essenberg
H
4-2
Oklahoma
M.Essenberg
W
6
Texas
L.S.Bird
I
3
Oklahoma
M.Essenberg
V
3
Texas
L.S.Bird
D
2
Texas
L.S.Bird
M
15
Texas
L.S.Bird
X
18
Texas
L.S.Bird
U
1
Texas
L.S.Bird
Q
la
Oklahoma
M.Essenberg
R
lb
Oklahoma
M. Essenberg
Z
7b
Texas
L.S.Bird
Y
7a
Texas
L.S.Bird
T
18
Oklahoma
M.Essenberg
Chi
-
Chad
L.S.Bird
Ch2
-
Chad
L.S.Bird
HV25
-
Upper Volta
L.S.Bird
Su2
-
Sudan
L.S.Bird
Su3
-
Sudan
L.S.Bird
FL79
16
Florida
this study

procedures (46,66) as described in Chapter Four. Plasmids were
digested with either EcoRI or BamHI and DNA fragment patterns
53
visualized by agarose gel electrophoresis. All plasmid experiments
were repeated at least twice for each strain examined. Additionally,
EcoRI plasmid fragments of X. c. pv. malvacearum strains were cloned
into the vector pUCD5 (14) and maintained in E. coli ED8767 as
described in Chapter Four. These cloned fragments were used for
subsequent plasmid studies.
Plasmid Curing
To determine if plasmid encoded functions were involved in
pathogenicity, attempts were made to expel, or cure, plasmids from the
wild-type strains by including ethidium-bromide or sodium lauryl
sulfate (SDS) in the broth culture. Cultures were grown in peptone-
glycerol broth for two 48 hr cycles, being adjusted to an optical
density (ODgQOnm) f 0.03 at the beginning of each cycle. A dilution
series from the broth cultures were plated onto peptone-glycerol agar
and single colonies were selected for inoculation on cotton host
differentials.
Race-Specificity Genes
Cloned genes encoding race-specific avirulence in X. c. pv.
malvacearum have been identified and isolated (33) and it was of
interest to determine if these genes were plasmid-borne. Using E.
coli ED8 767 containing the cloned plasmid fragments in pUCD5 as
donors, a tri-parental mating scheme was used to move the cloning
vector into strains of X. c. pv. malvacearum by conjugation (22,69).
Into the recipient, X. c. pv. malvacearum strain N (virulent strain),
were mated cloned plasmid fragments (in pUCD5) from X. c. pv.

ntalvacearum strain H (avirulent strain). As a control, plasmids
fragments from strain N were also moved into strain H. The helper
plasmid used for mobilization of the plasmids from E. coli into X.
campestris were pSa322 or pRK2013 (22,53). The donor and helper
strains were grown overnight at 37 C in LB broth with appropriate
antibiotic selection pressure. The antibiotic was washed from the
cells prior to mating by centrifuging and resuspending the cells in
0.7% NaCl. The X. campestris recipients were grown overnight at 30 C
in MOPS minimal medium broth (69). Donors and helpers at a
concentration of lO? cells and recipients at 10^ cells were mixed in a
small volume onto sterile 25 mm cellulose-acetate filter membranes and
placed onto peptone-glycerol medium for 6 hours. The membranes were
then transferred to MOPS minimal medium with 25 ug/ml kanamycin and
incubated for 72 hours at 30 C. Bacterial growth from filters was
diluted in 0.7% NaCl and plated for single colony isolation on MOPS
minimal medium with 25 ug/ml kanamycin. Colonies were selected and
inoculated on the Acala cotton differentials containing known
resistance genes.
Plasmid Origin of Replication and Mobilization
An assay was included to determine if X. cn. pv. malvacearum
contained self-mobilizing plasmids. The E. coli clones containing
cloned plasmid DNA fragments from X. c. pv. malvacearum strain X were
mated into spectinomycin resistant X. c. pv. malvacearum strain N in
the presence, or absence, of the E. coli helper plasmid strain with
pRK2013. Transconjugants were selected on MOPS minimal medium with
kanamycin (25 ug/ml), or directly on POPS (33) containing kanamycin
(30 ug/ml) and spectinomycin (100 ug/ml).

Results
Plasmids and Race-Specificity
All of the strains of X. c. pv. malvacearum tested contained at
least one plasmid- The plasmid sizes ranged from about 50 to 100 kb.
A majority of the American strains of X. c_. pv. malvacearum contained
4.8, 4.7, and 4.5 kb EcoRI DNA fragments which appeared to be
conserved (Fig. 4-2). The entire 4.5 kb EcoRI DNA fragment appeared
to be conserved in all but 1 of the 25 strains tested, regardless of
geographic origin.
Comparisons were made between the physical restriction fragment
patterns (Fig. 4-2), the original race designations, and reactions on
the 14 cotton differentials tested (Table 5-2). The strains of X. c.
pv. malvacearum were grouped into seven plasmid types based on the
restriction profiles and the conservation of related restriction
fragments. In the plasmid groupings I, III, and IV, a limited level
of polymorphism in the restriction fragment profiles for each group
were evident, but a majority of the fragments appeared to be
conserved. In plasmid groupings II, V, VI, and VII, no polymorphisms
were observed.
The strains which contained plasmids of group I appeared to be
pathogenic on a majority of the cotton host differentials. X. c. pv.
malvacearum strain J was an exception for it was pathogenic to only 4
of the cotton differentials, whereas the other group I strains were
pathogenic on at least 11 of the cotton lines. Analysis of the EcoRI
restriction fragment pattern of strain J revealed it was missing a 4.5
kb fragment, and possibly had rearrangements which resulted in a shift
of two other EcoRI fragments. With few exceptions, the 4.5 kb EcoRI

56
Table 5-2. Plasmid groupings and cotton plant reactions to strains of X.
campestris pv. malvacearum.
Inoculation reaction of cotton^
A
B
C
D
E
F
G
H
X
J
K
L
M
N
0
p
Q
R
s
T
U
K
HVlb
I
80.7
2
+
+
+
+
+
4-
+
+
4-
+
+
+
+
+
14
14
N
2a
I
84.8
2
+
+
+
+
V
+
V
+
+
+
+
+
+
+
12
14
J
HV3
la
59.5
1
+
-
-
-
-
+
V
-
-
-
+
-
-
-
3
4
n
HVla
lb
59.0
1
+
+
+
V
V
V
+
+
V
V
+
V
V
+
7
14
0
2b
lb
59.0
1
+
-
V
-
V
+
V
+
V
V
+
V
V
V
4
12
L
HV7
Ic
57.9
1
+
-
-
-
V
+
+
V
V
V
+
V
V
V
4
11
A
2bSr
II
82.2
1
+

4-


+





V


3
4
B
2aSr
II
83.3
1
+

+

-
+
-




V


3
4
E
4-1
II
83.3
1
+
-
-
-
-
-
V
-
-
-
-
-
-
-
1
2
F
laSr
II
83.3
1
+

+
-
-
+

-

-

V


3
4
G
lbSr
II
83.3
1
+
-
+

-
+
-
-
-
-
-
V
-
-
3
4
S
3-22
II
83.3
1
+
1
1
H
4-2
II
80.4
1
+
1
1
W
6
Ilia
88.7
1
4*
-
-
V
-
-
-
-
-
-
-
-
-
-
1
2
I
3
Ilia
88.4
1
+
-
-
-
V
-
V
-
-
-
-
-
-
V
1
4
V
3
mb
87.0
1
+
-
-
-
V
V
V
-
-
-
V
-
-
-
1
5
D
2
IVa
94.2
1
+
-
+
-
-
+
-
-
-
-
V
+
-
-
4
5
M
15
IVb
93.2
1
+
-
+
-
-
+
V
-
-
-
-
V
-
-
3
5
X
18
V
57.5
1
+
-
V
-
V
V
+
-
-
-
V
V
V
V
2
9
u
1
V
62.9
1
+
-
V
-
V
+
V
V
-
-
V
V
V
V
2
10
Q
la
VI
100.4
2
+
1
1
R
lb
VI
91.6
2
+
-
-
V
V
-
-
-
-
-
V
-
-

1
4
Z
7b
VII
93.0
1
+
-
V
-
4-
V
V
V
-
-
V
V
V
V
2
10
Y
7a
VII
98.0
1
+
-
-
-
V
-
V
-
-
-
-
-
-
-
1
3
T
18
*
1
+

+
-
-
+
-
-
-
-
-
+
-

4
4
A = Strain designation. P
B = Original strain, or race,
designation. Q
C = EcoRI plasmid group.
D = Kilobase pairs of plasmid DNA. R
E = Number of plasmids. S
F = Cultivar Acala 44.
G = Cultivar Acala B2. T
H = Cultivar Acala B3.
I = Cultivar Acala B5.
J = Cultivar Acala Bg. U
K = Cultivar Acala B7.
L = Cultivar Acala Bj^.
M = Cultivar 1-10B BIN
(+ polygenes) .
N = Cultivar 101-102B (B2B3
+ polygenes).
0 = Cultivar 20-3 (BN + polygenes).
= Cultivar Mebane B1 (B2 +
polygenes).
= Cultivar Stoneville 20 (Bg
+ polygenes).
= Cultivar Polygenes (2B-S9)
= Cultivar Gregg (resistance
genes unknown).
= Sum of cultivars which gave 4
out of 4 compatible reactions
(+)
= Total of cultivars which gave
at least 1 compatible reaction
(+)
= Plant reactions: +, compatible
reaction; -, incompatible
reaction; v, reaction variable
over 4 replications.
V

fragment appeared to be conserved in all group I strains of X. c. pv.
malvacearum. The group I strains K and N each contained two plasmids
and also appeared to be the most virulent of all the strains tested in
any group, each being pathogenic on all of the cotton differentials
tested (Table 5-2). Strain K was isolated in Upper Volta, Africa and
strain N was isolated from Texas. Although the plasmids in group I
were highly conserved and apparently widespread in the world, there
was no correlation between the presence of these plasmids with race
specificity.
The strains which were classified into plasmid group II could be
divided into two basically different pathogenicity patterns on the 14
cotton differentials. The strains which were derived from X. c. pv.
malvacearum races 1 or 2, and strains derived from races 3 and 4
appeared to have related pathogenicity patterns. The plasmid EcoRI
restriction fragments patterns for all seven of these strains appeared
identical. Similar variability was observed in the pathogenicity
patterns for the other strains placed within groups III, IV, V, VI,
and VII. Again, no correlation between the pathogenicity pattern and
the plasmid group was apparent. The original race designations of the
strain mixtures of X. c. pv. malvacearum did not correspond to the
race phenotypes exhibited by the strains purified from the mixtures.
For instance, of 5 strains derived from nominal race 1 (F, G, Q, R,
and U) there were basically three different pathogenicity patterns
observed, none of which corresponded to the reported pattern for race
1. The expected race 1 reaction would have been pathogenic on Acala
44 and Stoneville 2B-S9 (45).

Plaanid Curing
In preliminary tests with the curing agents, it was determined that
0.3% SDS and 20 ug/ml ethidium bromide were the concentrations which
allowed growth of X. campe stris. Strains did not grow at
concentrations of 0.5% SDS or 30 ug/ml ethidium bromide. The
bacterial colonies isolated from ethidium bromide treatments appeared
phenotypically identical to the wild-type strains. Several colorless
colonies were observed with the SDS treatment; however, over a 48 hr
period, color appeared to be restored. Of 11 strains selected from
the SDS treatment, all were found to be pathogenic on Acala 44. From
plasmid analyses it appeared that 7 of 11 strains were missing the
indigenous plasmids (Fig. 5-1).
Race-Specificity Genes
Several clones containing cloned X. campestris plasmid fragments in
the vector pUCD5 were obtained and transformed into E. coli ED8767.
Based upon restriction analyses, only 3 of the 8 EcoRI fragments of
strain H, and 6 of 9 EcoRI fragments of strain N were cloned. These
clones carried 30 kb (36%) of the 84 kb strain H plasmid, and 70 kb
(88%) of 85 kb strain N plasmids. Each of the E. coli clones were
mated into X. c_ pv. malvacearum. The transconjugants were then
selected and inoculated onto the 14 cotton differentials. The results
of the inoculations are seen in Table 5-3.
None of the clones derived from plasmid DNA of X. c. pv.
malvacearum changed the expected disease reactions of X. c. pv.
malvacearum strain H. One clone of X. c. pv. malvacearum strain H,
originally thought to carry a large plasmid insert, did carry an

Table 5-3. Pathogenicity of X. campestris pv. malvacearum (Xcm)
transconjugantsa on cotton host differentials.
Plant reactions^
Strains Asala
44 &| Bj Bj B4 Ej % By % 101 Gregg BJ5
wild-type:
N + + + + + + + + + + + + +
H
Plasnid fragnents of Xian H in Xcm N:
JSIEA-H2 + + + + + + + + + + + + +
pUEA-H5 + + + + + + + + + + + + +
Plasnid fragrents of Xian N in Xon H:
rOEA-Nl +
PJEA-N3 +
PEA-N4 +
PEA-N5 +
pSV^S + -
Chranosanal fragment cf Xan H in Xon N:
pOEA-PH + + + + + + + + + + + +
a Cloned EcoRI fragment sizes within each clone are: pUFA-Hl, 4.9 kb;
PFA-H2, 19.2 and 4.6 kb; pUFA-H5, 22.4 and 4.0 kb; pUFA-Nl, 2.3 kb;
PUFA-N3, 14.5; PFA-N4, 23.5 and 13.4 kb; pUFA-N5, 15.6 kb; pUFA-N9,
13.4 and 9.6 kb.
k + = compatible (pathogenic) and = incompatible (hypersensitive)
plant reactions.

60
Figure 5-1. Plasmid curing of X. campestris pv. malvacearum with SDS
treatment. Lanes A-L are plasmid extractions of strains which were
exposed to 0.3% SDS. Lanes A, E, I, and J appear to contain the
indigenous plasmid. Lanes B-D, F-H, and K-L appear to be missing the
indigenous plasmid.

avirulence gene. Transconjugants of this clone, pUFA-Hl, in strain N
caused incompatibility on the Acala line (Table 5-3). Upon further
61
examination of the pUFA-Hl clone by Southern hybridization analysis,
it appeared that the cloned insert was not of plasmid origin. The
radiolabeled clone did not hybridize to Southern transfers of plasmid
DNA derived from strain H, or 16 other strains of X. c. pv.
malvacearum. However, this 4.9 kb cloned insert of pUFA-Hl did
hybridize to a 23 kb EcoRI fragment of the plasmid found in strain W.
Additionally, the radiolabeled pUFA-Hl clone hybridized to a 4.9 kb
EcoRI chromosomal fragment of 18 strains of X. c_. pv. malvacearum, and
also to another clone, pUFA-702 (33). pUFA-702 was independently
derived and appeared to encode gene-for-gene avirulence on Acala BN.
Thus, it was concluded that the pUFA-Hl clone was of chromosomal
origin.
Plasnid Origin of Replication and Mobilization
Seven of the eight EcoRI fragments of the single 59.5 kb plasmid
found in X. c_. pv. malvacearum strain X were cloned into pUCD5 (Table
5-4) All of the fragments were successfully mobilized into a
spectinomycin resistant strain of X. c. pv. malvacearum strain N (N-
SPR) in the presence of the helper plasmid, pRK2013, using MOPS
minimal medium selection. In general, it appeared that the larger
cloned fragments transferred at a slightly lower rate than the smaller
cloned fragments, however the transfer rate was not quantified. One
of these clones, pLXF, carrying a 4.7 kb fragment, was found to encode
plasmid transfer function (tra), missing on the vector pUCD5 (14) .
That is, pXLF could be mated into X. campestris without aid of the

Table 5-4. Selection of plasmid replication genes in X. campestris pv.
malvacearum strain X.
62
Matings3
Size
MOPS
K30 pops K^nSPmn
Plaanid Insert
no helper
PRK2013 PRK2013
pLXA
17.0 kb
few
+
-
pLXB
14.0 kb
NT
NT
NT
pLXC
9.0 kb
-
+
few
pLXD
5.0 kb
-
++
++
(ori)
pLXE
4.8 kb
-
+
-
PLXF
4.7 kb
++
+++
-
(tra)
pLXG
4.5 kb
-
+++
-
pLXH
0.5 kb
-
+++
few
a is no growth, + = about 100 colonies, ++ = 100-300 colonies, +++ =
swamped plate, NT = not tested. MOPS K30 is the MOPS minimal medium
containing kanamycin (30 ug/ml) and POPS ^qSP^oq is the POPS
complete medium containing kanamycin (30 ug/ml) and spectinomycin
(100 ug/ml) pRK2013 is a helper plasmid used in tri-parental
matings (22). pLXD contains the putative plasmid origin of
replication (ori) and pLXF contains a putative plasmid transfer
(tra) function of the X. campestris pv. malvacearum strain X
plasmid.

63
helper plasmid. Since the vector pUCD5 is known to carry a
mobilization function, it could not be concluded that the strain X
plasmid is self-mobilizable, but it must carry at least some functions
required for self-mobilization.
pUCD5 is a broad host range vector with an origin of replication
(ori) that functions poorly in X. c. pv. malvacearum. Matings on MOPS
minimal medium provide a lengthy period of selection in which plasmid
integration can take place, provided a homologous X. c. pv.
malvacearum DNA fragment is cloned on the vector to allow
recombination to occur. Rapid selection procedures using complete
media containing antibiotics only rarely allows this integration to
occur before the recipients die. When transconjugants with X. c. pv.
malvacearum strain X plasmid DNA were selected on POPS complete medium
with antibiotics only the clone containing a 5.0 kb EcoRI fragment,
pLXD, mated at a high frequency. All other clones either did not grow
on POPS with antibiotics, or grew poorly. Because pLXD grew rapidly
on selective medium, whereas other clones did not, it was proposed
that the pXLD clone contained a functional origin of replication from
the strain X plasmid.
Transconjugants containing pXLD were further analyzed because
their growth rate was highly variable. Eleven of the pLXD
transconjugants were selected by colony size and grouped as large,
medium or small. Plasmids from the transconjugants were extracted and
analyzed for plasmid content. All, but possibly one transconjugant,
were missing a 4.5 kb EcoRI fragment associated with the indigenous
plasmid of X. c. pv. malvacearum strain N-SpR (Fig 5-2). It was

64
Figure 5-2. Transconjugants of X. campestris pv. maivacearum N-Sp^
containing pLXD (top) and hybrrdized to the plasmid probe pSa4
(bottom) The transconjugants were digested with the restriction
endonuclease EcoRI (top) Lanes A and M, Lamda Hindu; lanes B-E,
large colony-size; lanes F-I, medium colony-size, and lanes J-L, small
size transconjugants. All of the transconjugants, except in lane J,
appear to have lost the 4.5 kb plasmid fragment seen in wild-type
strains. Plasmids in lanes F-I and K-L appear to have had other
plasmid rearrangements occur. All strains had an additional 8 kb EcoRI
fragment present. The autoradiograph (bottom) shows the
transconjugants probed with a pUCD5 related plasmid, pSa4 (93) The 8
kb fragment hybridized to the probe, but so did other plasmid
fragments corresponding to those of X. campestris pv. maivacearum.

presumed that the homologous recombination occurred between the pLXD
clone and the indigenous plasmid. As a result, there was no EcoRI
fragment which corresponded to the 13 kb pUCD5 vector, or the 5 kb
pXLD insert. However, a new 8 kb EcoRI fragment was present.
The plasmid DNA from the X. campestris transconjugants with pXLD
was subsequently extracted and transformed into E. coli ED8767. The
transformants were selected on Luria-Bertani medium containing
kanamycin (25 ug/ml), or ampicillin (30 ug/ml). Resistance genes for
these antibiotics border the EcoRI cloning site of the pUCD5 vector
(14) All of the transformed plasmids were 8 kb in size, which
indicated that the new band seen in the X_. c_. pv. malvacearum
transconjugants was, in fact, a new autonomously replicating plasmid
(Fig. 5-3). There was obvious rearrangement in the original pXLD
clone which led to a 10 kb deletion. Southern hybridization analyses
revealed the presence of DNA fragments from both pUCD5 and the insert
of pLXD in this new vector, named pXD-1 (Figures 5-2 and 5-4).
Discussion
Of the twenty-five clonal isolates of X. c. pv. malvacearum
derived from 11 different nominal races, none of the clonal isolates
corresponded to the expected disease reactions on the host
differentials for that race. Likewise, there was no correlation
between a given plasmid group and the observed pathogenic reactions on
the 14 cotton differential lines. Although avirulence genes have been
found on plasmids in other phytopathogenic bacteria (88), no evidence
has been found to link plasmids in X. c. pv. malvacearum with race-
specific interactions. In these studies,
it appears the plasmids

66
Figure 5-3. Transformation of E. coli ED8767 with plasmid DNA from X.
campestris pv. malvacearum transconjugants mated with pLXD. Lane A,
iamda Hindlll; lanes B-M, E. coli ED8767 transformed with DNA
extracted from transconjugants mated with pLXD and selected on
kanamycin Luria-Bertani plates (see Fig. 5-2). The transformants were
digested with restriction endonuclease EcoRI. Only the 8 kb EcoRI
fragment was present; the smaller band was undigested DNA.

67
Figure 5-4. Plasmid DNA of X. campestris pv. malvacearum strain X
digested with restriction endonucleases (left) and hybridized to the
plasmid probe pXD-1 (right). Lanes A and I, lamda HinduI; lanes B-H,
plasmid DNA from X. campestris pv. malvacearum strain X digested with
BamHI (lane B), Boll (lane C), BcoRI (lane D) Hindi11 (lane E), Kpnl
(lane F), PstI (lane G), and Sail (lane H). The 5.0 kb EcoRI fragment
seen in lane D is the fragment thought to contain the origin of
replication (ori).

served as useful markers for the genomic background. Plasmids in X.
campestris generally appear to be stable and have in sane instances
68
been used as epidemiological markers, such as with the recent Florida
outbreak of X. c. pv. citri (31).
However, five of the six strains with group I plasmids were more
virulent than the other strains. All of the Upper Volta strains fell
into this class, including sane North American strains. The Upper
Volta strains were derived iron recently discovered races which were
found to be virulent against all the known major genes for bacterial
blight resistance (29) The X. c_. pv. malvacearum strain J, although
an Upper Volta strain, appeared to be the exception and varied iron
the other plasmids by the loss, or rearrangement, of a 4.5 kb EcoRI
fragment found in all of the other 24 strains tested. Since the other
group I strains are highly virulent strains, and known to be lacking a
number of cultivar specific avirulence genes (33), it may be possible
that this 4.5 kb EcoRI fragment may serve sane positive function in
pathogenicity.
Nine avirulence genes have been reported cloned from X. c. pv.
malvacearum, at least sane of which are chromosamally encoded (33) .
With further testing, it is possible that plasmid-borne avirulence
genes may be uncovered. In this study, approximately 50% of the
plasmid sequences derived from an avirulent strain were tested,
leaving it still possible to uncover avirulence genes in X. campestris
pv. malvacearum.
A high level of race-specific variability has been described for
X. c_. pv. malvacearum (8) Plasmids may be involved in genome

69
rearrangement as plasmids in P. syringae pv. phaseolicola have been
found to integrate and excise from the chromosome causing
rearrangement (79,92). In these studies, pUFA-Hl, carrying a
chromosomally encoded avirulence gene, was found to hybridize with a
plasmid DNA from X. c. pv. malvacearum strain W. The evidence that
plasmids in X. c. pv. malvacearum may be self-mobilizing points to
their potential role in variability of the X. campestris species. It
is believed that the self-mobilizing, copper resistance plasmid
identified in X. c. pv. vesicatoria facilitated the rapid build up of
X. campestris strains resistant to field applied copper-containing
compounds (88). If avirulence genes are on self-mobilizing plasmids,
rapid shifts in pathogenic races may occur, and the appearance of new
races would be facilitated.
The cloning of the X. c_. pv. malvacearum plasmid origin of
replication may allow for the future construction of other cloning
vectors designed specifically for the study of genes in X. campestris.
Seme of the currently used broad-host-range vectors appear unstable in
X. campestris and are useful because cloned inserts integrate into the
chromosome. Although preliminary experiments with the X. campestris
ori resulted in vector rearrangement, possibly the resolved form may
be applied to the construction of future vectors.

CHAPTER SIX
PATHOVARS OF XANTHOMONAS CAMPESTRIS ARE DISTINGUISHABLE
BY RESTRICTION FRAGMENT LENGTH POLYMORPHISM*
Introduction
The species Xanthomonas campestris (Pammel) Dowson, a member of
the family Pseudcmonadaceae which is always found in association with
living plants, comprises over 125 different pathovars (6). The
pathovars have been named by "the plant from which first isolated"
convention. Unfortunately, strains of some pathovars can be
pathogenic on host species in different plant families (57); thus
possible relationships between pathovars may go undetected, since
there is no convenient way to determine pathogenicity for all possible
host plants, nor is there a useful systematic method for narrowing the
possibilities. Further, and despite a widely held view that all
xanthcmonads are plant pathogens (85), non-pathogenic members of X.
campestris are often found as ectoparasitic leaf colonizers
(epiphytes) (26) and occasionally as endoparasites (65,71). Their
inability to provoke a pathogenic plant response renders them
unclassifiable in this system. The attention given to damaging plant
pathogenic strains may not be taxonomically or ecologically warranted.
Genes which function to provoke a pathogenic response may be entirely
different from those which confer host selectivity and parasitic
ability (32) Epiphytes and nonpathogenic endoparasites are known to
_
This chapter contains copyrighted material from International Journal
of Systematic Bacteriology. It is reprinted here with permission of
the publisher.
70

be host selective (19,65). Host selectivity is thought to be a stable
characteristic (84) Since xanthomonads are always found in
association with living plants, host selectivity should result in
genetic isolation of a pathovar population. Over time such isolation
and random genetic drift should produce distinctive and stable
phenotypic characteristics and therefore, establish conserved genetic
markers.
Attempts to differentiate pathovars by methods other than
pathogenicity have included rRNA-DNA and DNA-DNA hybridization
(21,73), serology (1,94,95), phage-typing (37,59), and comparisons of
profiles resulting from plasmid and chromosomal DNA restriction enzyme
digests (40,54), protein electrophoresis (70), and gas-chromatography
of fatty acids (65). Although all of these methods are useful for
specific purposes, few have demonstrated utility to replace, clarify,
or indicate further pathogenicity tests.
We report here the use of restriction fragment length
polymorphism (RFLP) analysis to differentiate pathovars of X.
campestris. RFLP analyses have been widely used in the medical field
to identify DNA fingerprints specific for inherited diseases or other
genetic loci (2,11,61). These analyses have also been applied to
plants (41), bacteria (38), and eukaryotic organelles (67,91). RFLP
analyses allow the observance of genetic variation in organisms within
defined regions of the genome due to DNA rearrangements or mutations
which affect recognition sites for restriction endonucleases.
Variation may be examined over a small (a few base pairs) or large
(30-40 kb) stretch of DNA depending on the desired level of

polymorphism detection. This variation can be observed for random or
selected DNA target sequences within a species against a background of
72
other genomic DNA fragments. In this study, RFLP analyses were
applied to investigate the degree of genetic variation among 87
strains of X. campestris, comprising 23 different pathovars. Seme of
these results have appeared in abstracts (34,55).
Materials and Methods
Bacterial Strains
The bacterial strains used in this study are shown in Table 6-1.
These strains were isolated as plant pathogens, identified as X.
campestris, and classified into pathovars according to the susceptible
host plants involved. Strains were tested for pathogenicity after
single colony purification. For some strains, no pathovar assignments
were made due to the lack of known plant pathogenic responses. Broth
cultures of bacteria were grown in a peptone-glycerol medium (per L:
10.0 ml glycerol, 20.0 g peptone, and 1.5 g K2HPO4; pH 7.2). The
strains were commonly stored and maintained at -80 C in the same
medium containing 15% glycerol.
DNA Extraction
DNA was extracted from bacterial cultures at mid- to
late-logarithmic growth phase. Extraction of bacterial DNA for cosmid
library construction was by a modification of the method of Silhavy et
al. (87). A critical modification was to wash cells in 50 mM TRIS, 50
mM Na2EDTA, 150 mM NaCl, resuspend in the same buffer containing 150
ug/ml proteinase K, add SDS (sodium dodecyl sulfate) to 1% (w/v), and

Table 6-1. Strains of X. campestris used for RFLP analysis
73
Fatixvar
Strain
Host
Location
Xantburcnas canpestris pathcvar
alfalfe
KS
hfedicago sativa
Kansas
EL
M. sativa
Florida
argemores
084-1052
Arcsnons mexicana
Elorid
begonias
084-155
Begonia sp.
Florida
carpestris
Xd
Brassica deracea (cabbage) Cklahora
084-809,084-1136
B. olercea (cabbage)
Elorida
084-720
B. deracea (br. grouts)
Florid
084-1318
B. deraoea (broccdi)
Elorida
carcfe
13
Daucus carota
Chlifamia
citri
X59,X70
dtrus sp.
Brazil
X52
dtrus sp.
Japan
X69
dtrus 3?.
ArgerrtirB
084-3401
dtrus ^o.
Florid
cyaropsidis
13D5
Cysrrccsis tetragonolcba
-
X002,X005,
C. tetragonoiabe
Arizona
X016,XD17
C. tetragonolcba
Arizona
dieffehbachiae
084-729
Anthurium sp.
Florid
068-1163
Diefferbadoia sp.
Florid
eseulenti
084-1093
Aoelmosduus esculentus
Florid
glycines
B-9-3
Glycine max
Brazil
1717
G. max
Africa
17915
G. max
-
S-9-8
G. max
Wisconsin
hederae
084-1789
Hadara helix
Florid
bdcioola
Xh66
Scaxrxm vulgare
Kansas
maculifoliigardenfe 084-6166
drcenia sp.
Florid
irelvacearum
D,M,N,0,U,V,
Qossvpium hirsutum
Tdxas
X,Y,Z,TX34
G. hirsutum
Ifexas
A,B,E,F,G,H
G. hirsutum
Cklahcma
Chl,Ch2
G. hirsutum
Gfe
Hv25
G. hirsutum
EJcrer Volta
Su2,Su3
G. hirsutum
Sdn
EL79
G. hirsutum
Elorid
mangiferaeindicae
084-116
Mangifera indica
Elorid
nigrcrnaaLans
084-1984
Arctium latee
Elorid
pelargonii
084-190,084-1370
Geranium sp.
Florid
phased!
EKil,J$h25,}££ll
Ehaseolus vulgaris
Nebraska
Xpa,Xpll
P. vulgaris
Wisconsin
82-1,82-2
P. vulgaris
Elorid
LB-2,SD-3B
P. vulgaris
Nebraska
XP2
P. vulgaris
New York
XP-JL
P. vulgaris
Kansas
XP-JF
P. vuiqaris
Missouri
XP-DPI
P. vulgaris
-
Source3
D.L.Stuteville
R.E.Stall
EPI
DPI
this study
tiis study
this study
DPI
R.E.Stall
E.L.dvsrdo
E.L.dvsrdo
E.L.d\erdo
DPI
dl.Kado
J.Mihail
J.Mihail
DPI
DPI
DPI
W.F.Efett
W.F.Etett
W.F.Ftett
W.F.Efett
EPI
L.daflin
DPI
this study
this study
M-Essaierg
L.S.Bird
L.S.Bird
L.S.Bird
this study
DPI
DPI
DPI
M. Schuster
A.W.Saettler
R.E.Stall
A.K.Vidavsr
J.A.Laurence
J.L.Ieach
this study
this study

74
Table 6-1 (continued).
I&thovar
Strain
Host
Location
Source3
pisi
XP1
Pisun sativum
Japan
M-Goto
poinsettiicnLa
083-6248
Eupxirbia pulcrerrima
Elorida
DPI
pruni
084-1793
Prunus
Florida
CPI
translunens
XLLQ5
Hordaun sp.
Montana
D.Sands
\esicatoria
E-3
Capsicum annum
Florida
R.E.Stall
75-3
Lvocpersioon esculentrm
Floricfe
R.E.Stall
vignioola
A81-331,C-1,
Vigna urguiculata
Gsorgia
R.D.Gitaitis
OB5-1,
V. urgiuoulata
(feacgia
R.D.Gitaitis
X/19,312,
V. urguiculata
Cfeorgia
R.D.Gitaitis
432,82-38
V. unguiculata
Gfexgia
R.D.Gitaitis
vitians
IOB164
Latrca S3.
-
R-E.Stall
zinnia
084-1944
Zinnia elegans
Florid
DPI
unknown
084-1373
Ihilodandrcn sp. (dieffei.)
Florida
DPI
084-3928
Fbtsia sp. (hadsrae)
Florida
DPI
084-4348
Alocasia sp. (vitians)
Florich
CPI
083-2057
Syngcniun si. (vitians)
Florida
DPI
084-2848
Cissus S3.
Florich
CPI
084-1590
Eucryrus
Florida
DPI
251G,084-480
Impatiens sp.
Florida
DPI
084-6006
Jasninium sp.
Florida
DPI
X. fragarias
Xfral
Fragaria sp.
Florida
R.E.Stall
a DPI = Flor
ida Department
of Agricultural and
Consumer
Services,
Division of
Plant Industry,
Gainesville, Florida.

heat for 1 hr at 50 C. The sample was then extracted 2 times with
phenol/chloroform/isoamyl alcohol (25:24:1; phenol equilibrated to pH
7.8 with 0.1 M TRIS), and the DNA spooled out from the aqueous phase
after adding sodium acetate at 30 mM and 2 volumes 95% ethanol. This
DNA was then washed in 70% ethanol. Extracted DNA was resuspended in
TE (10 mM Tris (hydroxymethyl) aminomethane (TRIS), 1 mM disodium
ethylenediaminetetraacetate (Na2EDTA) containing 20 ug/ml DNase-free
pancreatic RNase; pH 7.6).
Agarose Gel Electrophoresis
Approximately 5 ug of DNA was digested with EcoRI or BamHI as
specified by the manufacturer (Bethesda Research Laboratories).
Digested DNA samples were run in a 20x25 cm agarose gel (0.6%; type
II, Sigma) in TRIS-acetate buffer (40 mM TRIS-acetate, 1 mM Na2EDTA;
pH 7.6) with electrophoresis of gels set at 35 V for 14-15 hrs.
Fragments were visualized by ultraviolet irradiation (302 nm) after
staining agarose gels in ethidium bromide (0.5 ug/ml). Photographs
were taken using Polaroid Type 55 (or Type 57) film and a yellow
filter (Tiffen no. 12). After the gels were photographed, the DNA was
transferred to nitrocellulose by the method of Southern as described
by Maniatis et al. (66). Restriction fragment sizes were estimated
using DNA molecular size standards of lambda phage DNA digested with
HindiII.
DNA Probes
The DNA probes used in this study were derived from a genomic
library of X. c. pv. citri strain 3401 constructed into the modified
cosnid cloning vector pUCD5B, which was the vector pUCD5 (14) with a 2

76
kb BamHI fragment deleted from it. DNA of strain 3401 was partially
digested with the restriction enzyme Mbol and fractionated by size on
a 10-40% (w/v) sucrose step gradient (66). The pUCD5B vector was
digested with the restriction enzyme BamHI and treated with calf
intestinal alkaline phosphatase (Boehringer-Mannheim). Vector DNA and
large fragment size fractions (25-40 kb) from strain 3401 were mixed
and treated with T4 DNA ligase (Bethesda Research Laboratories) and
recombinant cosmids were packaged in vitro with extracts prepared and
utilized according to Scalenghe and Hohn's protocol II as described by
Maniatis et al. (66). Transduction with Escherichia coli HB101 was
performed using top agar on Luria-Bertani medium containing kanamycin
(3 5 ug/ml) Cloned DNA fragments of strain 3401 in the vector
averaged 27-38 kb.
DNA Hybridization
ENA clones used as probes were radiolabeled using 30-60 uCi of
32p_deoxycytidine (Du Pont NEN) with use of a nick translation kit
(Bethesda Research Laboratories) and separated from low-molecular
weight nucleotides on a mini-column of Sephadex G50-100. Southern
blots were pre-hybridized in plastic bags for 4 hr and hybridized with
the DNA probe for 16-18 hrs at 68 C. The pre-hybridization and
hybridization solutions were those described in Maniatis et al. (66).
Following hybridization, membranes were washed once in 2X SSC, 0.5%
SDS (IX SSC is 0.15 M NaCl, 0.15 M sodium citrate, pH 7.0) and washed
once in 2X SSC, 0.1% SDS at ambient temperature, and washed two times
in 0.1X SSC, 0.5% SDS at 68 C as described by Maniatis et al. (66) for
'stringent* conditions. Nitrocellulose membranes were then air-dried

and exposed to Kodak X-Qnat AR film at -8 0 C in cassettes with
intensifying screens. Hybridization of the probes to individual
strains of X. campestris was repeated at least three times.
77
Restriction Fragment Patterns and Densitometry
From developed autoradiographs, DNA fragment sizes and profiles
were determined and hybridization signals measured using a Gilford
Response spectrophotometer equipped with an autoradiograph scanner.
Routine scans were done at 600 nm wavelength with 0.5 mm aperture and
0.5 nm bandwidth setting. Scanning data was stored by computer.
Using above information, comparison were made among strains of a given
pathovar and also strains derived from different pathovars of X.
campestris.
Results
Agarose Gel Electrophoresis
For a given pathovar, the banding patterns appeared very similar
and polymorphism in these strains was apparently limited (Fig. 6-1).
In all, DNA from 87 strains of X. campestris, representing 23
different pathovars and including scene with no known pathovar status
were digested with restriction enzymes EcoRI and BamHl and separated
by size on agarose gels. Based on restriction fragment patterns
alone, it was possible to visualize DNA variability among the strains,
but recognition of particular DNA fragment banding patterns was
difficult due to the numerous DNA fragments involved (Fig. 6-2). Some
of the apparent variation was due to the presence of plasmid bands,
which appear brighter against the chromosomal bands due to their
relatively higher copy number within each cell.

Figure 6-1. Genomic DNA of strains of X. campestris digested with the
restriction endonuclease EcoRI. Lane A, probe XCT1; lanes B-K, X. c.
pv. phaseoli; lane B, JL; lane C, LB-2; lane D, Xph25; lane E, EKll;
lane F, 82-1; lane G, 82-2; lane H, Xpa; lane I, XP2; lane J, XP2-1;
lane K, JF; lanes L-M, X. c. pv. phaseoli var. fuscans; lane L, SC-3B;
lane M, Xpfll; lanes N-0, X. c. pv. alfalfae; lane N, KS; lane 0,
FL;lanes P-R, X. c_. pv. campestris; lane P, XC1 (cabbage); lane Q,
084-1318 (broccoli); lane R, 084-720 (brussels sprouts); lane S,
unknown X. c. G65; lane T, probe XCTll.

79
Figure 6-2. Genomic DNA of strains of X. campestris digested with the
restriction endonuclease EcoRI. Lane A, probe XCT1; lane B, X. c. pv.
alfalfae FL; lane C, X. c. pv. begoniae 084-155; lane D, X. c. pv.
campestris 084-809; lane E, X. c. pv. carotae 13; lane F, X. c. pv.
cyamopsidis X002; lane G, X. c. pv. dieffenbachiae 068-1163; lane H,
X. c. pv. glycines S-9-3; lane I, X. c. pv. holcicola Xh66; lane J, X.
£_ pv. maculifoliigardeniae 084-6166; lane K, X. c. pv. malvacearum
FL79; lane L, X. c. pv. pelargonii 084-190; lane M, X. c. pv. phasedi
var. fuscans SC-3B; lane N, X. c. pv. pisi XPl; lane 0, X. c. pv.
translucens X1105; lane P, X. c. pv. vesicatoria 75-3; lane Q, X. c.
pv. vignicola SN2; lane R, X. c. pv. vitians ICPB164; lane S, X. c.
pv. zinniae 084-1944; lane T, probe XCTll.

DNA Probes
Two cosmid DNA clones, XCT1 and XCT11 were randomly selected from
a gencmic library of strain 3401 for use as DNA probes. XCT1 carried
a 30 kb insert, and XCTll carried a 37 kb insert, as determined by
adding up the cloned DNA inserts from restriction endonuclease
digests. There were no detectable plasmids in strain 3401, thus the
cosmid clones are assumed to contain chromosomal fragments. Assuming
a 3333 kb genome for X. campestris (47), each cosmid probe represents
approximately 1% of the total genome.
DNA Hybridization
Autoradiographs of Southern blots hybridized against either of
the two DNA prcbes revealed conserved DNA fragments within each
pathovar. For example, in figure 6-3 (lanes B K) are shown the EFLP
pattern from 10 different strains of X. c. pv. phaseoli from different
geographic locations. At least five EcoRI cut DNA fragments which
hybridized to XCT1 appeared to be conserved in this pathovar. In
lanes L and M are the RFLP patterns of two strains of X. c. pv.
phaseoli var. fuscans. These X. c. pv. phaseoli var. fuscans strains
also attack beans (and hence have the pv. phaseoli designation), but
are biochemically distinct (6) They produce a dark, melanin-like
pigment in nutrient media and in these tests are clearly genetically
distinguishable from the other X. c. pv. phaseoli strains. Conserved
DNA fragments were also seen for X. c. pv. alfalfae (Fig. 6-3, lanes N
and 0) in which six of the up to 15 fragments appeared identical.
Likewise, in four different strains of X. c. pv. campestris (Fig. 6-3,
lanes P Q, Fig. 6-5, lane D) isolated from three different crucifer

81
ABCDE FGH I J KLMNOPQRST
Figure 6-3. Genomic DNA of strains of X. campestris digested with the
restriction endonuclease EcoRI and probed with cosmid clone XCTl. Lane
A, probe XCTl; lanes B-K, X. c. pv. phaseoli; lane B, JL; lane C, LB-
2; lane D, Xph25; lane E, EK11; lane F, 82-1; lane G, 82-2; lane K,
Xpa; lane I, XP2; lane J, XP2-1; lane K, JF; lanes L-M, X. c. pv.
phaseoli var. fuscans; lane L, SC-3B; lane M, Xpfll; lanes N-0, X. c.
pv. alfalfae; lane N, KS; lane 0, FL; lanes P-R, X. c_. pv. campe stris;
lane P, XC1 (cabbage) ; lane Q, 084-1318 (broccoli) ; lane R, 084-720
(brussels sprouts); lane S, unknown X. c. G65; lane T, probe XCTll.

sources (cabbage, broccoli, and brussels sprouts) at least four of
over 10 fragments appeared to be identical. Sizes of hybridizing DNA
fragments are given in Table 6-2. In figure 6-4 is shown the RFLP
patterns revealed using clone XCTll. In each case, a different RFLP
pattern is shown, but the same genetic relationship is revealed.
Other sets of conserved DNA fragments within a pathovar were seen
among seven strains of X. c. pv. vignicola (identical pattern), seven
strains of X. c. pv. cyamopsidis (nearly identical) and at least
three strains each for X. campestris pvs. citri, glycines,
malvacearum, pelargonli, and vesicatoria (not shown).
ENA of strains representing other pathovars of X. campestris were
also digested with either EcoRI or BamHI, and hybridized with either
XCTl or XCTll. The pathovars of X. campestris included in these
experiments were X. campestris pvs. alfalfae, begoniae, campestris,
carotae, citri, cyamopsidis, dieffenbachiae, esculenti, glycines,
hederae, holcicola, maculifoliigardeniae, malvacearum,
mangiferae indicae, nigromaculans, pelargonii, phaseoli pisi,
translucens, vesicatoria, vignicola, vitians, and zinniae. Also
included were some strains isolated as pathogens of an Alocasia sp.,
Cissus sp., Impatiens sp., Fatsia sp., Jasmine sp., Argemone sp., and
a Euonymus species for which no pathovar designations were available.
The results of hybridization of probe XCTl to DNA of different
pathovars of X. campestris digested with EcoRI are shown in Figure 6-
5. The clone XCTl appeared to contain DNA fragments of strain 3401
which were able to demonstrate variability of restriction fragment
patterns among different pathovars of X. campestris. Variability in

83
Table 6-2. Sizes of DNA fragments from Xanthanonas campestris genomic
digests (EcoRI) which hybridized to the XCT1 DNA probe. See Figure 6-
3.
A
12.2,
11.2,
7.9, 5.6, 5.1, 3.2, 2.2,
1.6,
1.0
B
14.1,
13.3,
10.4, 7.6, 6.4, 4.6, 4.3,
4.0,
3.7,
3.3,
3.1
C
14.4,
13.3,
10.4, 7.6, 6.4, 4.6, 4.3,
4.0,
3.7,
3.3,
3.1
D
14.1,
13.1,
10.4, 7.5, 6.4, 5.2, 4.6,
4.3,
4.0,
3.7,
3.1
E
13.9,
11.6,
10.4, 8.9, 5.2, 5.0, 4.6,
4.3,
4.0,
3.7,
3.1
F
13.7, 13.1,
1.9
11.6,
10.4,
8.9,
5.2,
5.0,
4.6,
4.3, 4.0,
3.7, 3.1,
G
13.7, 13.1,
11.6,
10.4,
8.9,
5.2,
5.0,
4.6,
4.3, 4.0,
3.7, 3.1,
1.9
H
13.1,
10.4,
7.5,
5.2,
5.0,
4.6,
4.3,
4.0,
3.7,
3.1,
1.9
I
13.1,
10.4,
7.5,
5.2,
5.0,
4.6,
4.3,
4.0,
3.7,
3.1,
1.9
J
13.1,
10.4,
7.5,
5.2,
5.0,
4.6,
4.3,
4.0,
3.7,
3.1,
1.9
K 13.1, 11.6, 10.4, 8.9, 5.2, 5.0, 4.6, 4.3, 4.0, 3.7, 3.1
L 11.9, 8.4, 7.1, 6.7, 4.5
M 17.0, 15.3, 10.0, 8.4, 4.5
N
19.7, 19.1,
15.5,
14.9,
12.9, 12.1, 8.0, 4.6, 3.8, 3.1,
2.7,
1.7
0
19.4, 18.0,
3.4, 3.1
14.7,
13.7,
13.1, 11.7, 9.7, 7.3, 6.2, 5.2,
4.6,
3.7
P
20.3, 15.3,
10.9,
10.5,
10.2, 9.3, 8.4, 6.9, 5.9, 5.2,
4.4,
3.9
2.6, 2.2, 1,
.9
Q
12.9, 10.8,
K
in

o
ii
9.1,
8.4, 7.7, 7.2, 6.3, 4.6, 4.4,
3.9,
3.3
2.4, 2.2, 1.
.9
R
18.6, 15.8,
12.7,
10.8,
10.2, 9.1, 8.4, 7.7, 6.7, 5.6,
4.4,
3.3
2.2, 1.9
11.3, 8.0, 6.9, 3.4, 2.5, 1.6
17.2, 15.3, 14.3, 10.5, 8.6, 7.2, 6.8, 5.8
S
T

84
Figure 6-4. Genomic DNA of strains of X. campestris digested with the
restriction endonuclease EcoRI and probed with cosmid clone XCT11.
Lane A, probe XCT1; lanes B-K, X. c. pv. phaseoli; lane B, JL; lane C,
LB-2; lane D, Xpn25; lane E, EK11; lane F, 82-1; lane G, 82-2; lane H,
Xpa; lane I, XP2; lane J, XP2-1; lane K, JF; lanes L-M, X. c. pv.
phaseoli var. fuscans; lane L, SC-3B; lane M, Xpfll; lanes N-0, X. c.
pv. alfalfae; lane N, KS; lane 0, FL;lanes P-R, X. c. pv. campestris;
lane P, XC1 (cabbage) ; lane Q, 084-1318 (broccoli) ; lane R, 084-720
(brussels sprouts); lane S, unknovm X. c. G65; lane T, probe XCTll.

85
12.2
11.2
7.9
5.6
5.1
3.2
2.2
1A
Figure 6-5. Genomic DNA of strains of X. campestris digested with the
restriction endonuclease EcoRI and probed with cosmid clone XCTl. Lane
A, probe XCTl; lane B, X. c. pv. alfalfae FL; lane C, X. c. pv.
begonlae 084-155; lane D, X. c. pv. campestris 084-809; lane E, X. c.
pv. carotae 13; lane F, X. c. pv. cyamopsidis X002; lane G, X. c. pv.
dieffenbachiae 068-1163; lane H, X. c. pv. glycines S-9-3; lane I, X.
c. pv. holcicola Xh66; lane J, X. c_. pv. maculifoliigardeniae 084-
6166; lane K, X. c. pv. malvacearum FL79; lane L, X. c. pv. pelargonii
084-190; lane M, X. c. pv. phased i var. fuscans SC-3B; lane N, X. c_.
pv. pisi XPl; lane 0, X. c. pv. translucens X1105; lane P, X. c. pv.
vesicatoria 75-3; lane Q, X. c_. pv. vignicola SN2; lane R, X. c. pv.
vitians ICPB164; lane S, X. c. pv. zinniae 084-1944; lane T, probe
XCTll.

86
RFLP pattern appeared as qualitative and quantitative differences
among hybridization profiles of various sized DNA fragments in the
strains. For instance, the weak hybridization signal seen from X. c.
pv. translucens (fig. 6-5, lane 0) suggests that this strain is
distinct from the other pathovars compared, at least over the 1% of
the genome contained on the clone XCTl. In other pathovars such as X.
c_. pv. alfalfae (fig. 6-5, lane B) X. c_. pv. begoniae (lane C), X. c.
pv. cyamopsidis (lane F) X. c. pv. malvacearum (lane K) X. c. pv.
vigncola (lane Q) and X. c. pv. vitians (lane R) where sane DNA
fragments had strong hybridization signals, a close relatedness with
portions of DNA in the cosmid clone is suggested. Some of the DNA
fragments from different pathovars which hybridized most strongly to
the probe were also of identical size with the EcoRI fragments of the
XCTl clone. As expected, ENA of X. c. pv. citri strain 3401 digested
with EcoRI, or BamHI (34,55) contained fragments which corresponded to
DNA fragments of the XCTl probe (Fig. 6-5, lane A). Sizes of
hybridizing DNA fragments are given in Table 6-3.
From observations using different enzymes and different probes,
the same patterns emerged, indicating that the basic chromosome
structure of X. campestris can be used to differentiate strains into
specific RFLP types. Although a pathovar may contain more than one
type of variant (e.g. X. c_. pv. phasedi), all strains of a given type
appear to exhibit the same host selectivity. The RFLP pattern is
highly distinctive for each type, and can be used to unambiguously
assign an unknown sample to a pathovar, using simple visual
comparisons of pattern against a set of known samples, such as those
given in Figure 6-5.

87
Table 6-3. Sizes of DNA fragments from Xanthomonas campestris genomic
digests (EcoRI) which hybridized to the XCTl DNA probe. See Figure 6-
5.
A
12.2,
11.2, 7.9, 5.6, 5.1, 3.2, 2.2, 1.6, 1.0
B
20.5,
3.1
18.8, 15.3, 14.2, 13.5, 12.0, 7.3, 6.2, 5.1,
4.6,
3.7,
3.4
C
18.7,
12.1, 9.5, 8.6, 8.2, 7.7, 6.9, 6.7, 6.2,
6.0,
4.8,
4.2
4.0,
3.4, 3.0, 2.9, 2.5, 2.3
D
19.4,
16.4, 14.4, 13.1, 11.1, 10.5, 9.2, 8.5, 7.7,
6.7,
5.6,
4.3
4.0,
2.2, 1.9
E
17.9,
12.9, 4.1, 3.5, 3.0, 2.3, 1.5
F
16.2,
11.5, 9.5, 8.7, 5.7, 4.0, 3.6, 3.1, 3.0, 1.4
G
14.2,
11.1, 4.5, 3.9, 3.5, 3.0
H
12.2,
10.2, 8.3, 7.9, 6.5, 5.5, 4.2, 3.1
I
12.9,
12.0, 7.6, 7.1, 6.5, 4.7, 3.0, 1.7
J
15.6,
14.2, 9.4, 7.9, 6.3, 5.0, 4.6, 4.0, 3.1, 2.5
, 2.2
K
20.9,
17.1, 16.2, 15.5, 14.2, 11.0, 10.4, 9.2,
8.2,
7.5,
6.7
4.2,
4.0, 3.3, 3.1, 3.0
L
19.4,
12.7, 10.4, 9.0, 7.9, 7.7, 6.8, 5.7, 4.2,
3.9,
3.0,
2.7
2.3,
1.9, 1.5, 1.4
M
12.0,
8.4, 7.0, 6.8, 4.5
N
15.9,
8.4, 4.9, 3.4, 2.1, 1.9
0
9.2,
4.4
P
13.3,
9.2, 7.2, 6.9, 6.2, 4.6, 3.8, 3.1, 2.1
Q
11.0,
8.5, 7.0, 6.3, 4.2, 3.2
R
16.7,
14.2, 12.9, 9.9, 7.4, 6.9, 6.3, 4.8, 3.8,
3.5,
3.2,
2.6
2.4,
2.2, 1.6
S
12.9,
12.5, 9.5, 7.7, 6.5, 4.4, 4.0, 3.8
T
17.6,
15.6, 14.7, 10.5, 8.6, 7.2, 1.4

88
Discussion
The only widely accepted and most practical method for
differentiating pathovars of X. campestris is to inoculate a plant
suspected as the host for that pathovar. This practice can sometimes
be tedious, time consuming, subjective and subject to a surprising
number of artifactual influences. It is not known whether host
selectivity is unstable as suggested by Dye (23); stable as suggested
by Schnathorst (84) or even taxonomically significant. Although the
classification is thought to be useful, it can be highly misleading
since emphasis is placed upon one characteristic pathogenicity. If
only one or few gene differences were involved in host selection, then
differentiation by pathovar could be highly misleading, at least in
the sense that a given group of strains might be capable of attacking
more than one host in some cases. Alternatively, two or more strains
of relatively unrelated groups could be cataloged together because
they happen to attack the same host. This latter situation appears to
be the case with X. c. pv. phaseoli and X. c. pv. phaseoli var.
fuscans (see Fig. 6-3) and with Florida strains of X. c. pv. citri
(34).
To address these problems, we evaluated the potential of using
DNA sequence variation of X. campestris for strain classification
purposes. DNA sequence variation was first examined by digesting DNA
with restriction enzymes and visualizing directly the resulting
fragments on ethidium bromide stained gels. These stained gels were
useful for side-by-side comparisons of restriction fragments for
samples run on the same gel, but comparisons between different gels

were more difficult. Subsequently, visualizing and comparing
variation in bacterial genomes with cosmid clones carrying 30-38 kb
89
cloned X. campestris genome fragments was simplified. In both cases
variation was revealed by alterations in the sizes of visualized DNA
restriction fragments (RFLPs). The use of DNA probes derived from
chromosomal DNA fragments also alleviated seme of the difficulty in
comparing stained gels with the sometimes present polymorphism of the
higher copy number plasmid DNA fragments which can occur in the
background.
Some of the selected clones tested as DNA probes appeared to be
useful for identifying DNA sequences conserved within a given
pathovar, while others appeared to identify DNA sequences which were
highly conserved at the species level. The DNA which was considered
conserved at the species level was represented as banding patterns
which were nearly identical among all strains over the several
pathovars tested. Examples of DNA sequences which are known to be
highly conserved are rRNA encoding genes (38,83). If smaller DNA
probes containing known genetic loci such as those associated with
genes for pectate lyase and protease (18), or avirulence activity
(33,88) were tested, these smaller, more defined DNA probes could be
used to determine if such genetic loci were highly conserved or
variable. With larger, randomly selected probes, the likelihood was
increased for detecting strain- or pathovar-specific variation. The
large (greater than 30 kb) size of the probes used in this study
allowed detection of both highly conserved and variable regions.

Using RFLP genomic blots, it appeared that phylogenetic
relationships between the full spectrum of described pathovars of X.
campestris might be determinable. Some mathematical approaches toward
90
determining phylogeny based on restriction cleavage sites has been
proposed (58,74). DNA probes have been used to make phylogenetic
comparisons among the relatively conserved mitochondrial (67) and
chloroplast (91) genomes. The variation seen among the different
pathovars of X. campestris appeared within the range that is usefully
distinguishable with this test. Only limited amounts of variation can
be usefully distinguished, such as that occurring within a species.
Significantly, the RFLP groupings closely corresponded with the
pathovar groupings, strengthening the taxonomic significance of this
classification. All strains tested were readily grouped by RFLP
phenotypes, and the classification based on RFLP patterns correlated
very well with the classification based on pathogenicity. This
technique may provide a more convenient means of classifying these
bacteria. In addition, unexpected taxonomic relationships between
pathovars might be revealed. The potential pathogenic range of each
RFLP group would be of obvious value.
By comparing observed RFLPs among strains of X. campestris using
selected DNA probes, it was possible to identify unknown strains when
known standards were included. Additionally, strains previously
undescribed could be classified as being related to known pathovars.
For instance, similarities were found between undescribed strains
isolated from a Alocasia sp. and Argemone sp.; and a Cissus sp. and
Jasmine species. The strains isolated from a Philodendron sp. and

91
Dief fenbachia sp. both corresponded to that of X. c. pv.
dieffenbachiae. The other unknown strains tested appeared different
from one another, and did not appear similar to any of the other X.
campestris pathovars as characterized by the DNA probes. Finally,
this technique may provide a basis for the classification of
nonpathogenic and epiphytic xanthomonads. Experiments can now be
conducted to determine whether epiphytes are all basically similar or
form diverse groups, whether they are restricted in host range or if
they are nonpathogenic, but closely related to described pathovars.
Although this approach provides another classification scheme for
strains, it appears to be compatible with the currently recognized and
useful pathovar naming scheme, which relies primarily on plant-
specificity. Although certain pathovars may need to be redefined,
this work supports and helps validate the natural taxonomic groupings
provided by the pathovar naming system.

CHAPTER SEVEN
SUMMARY
Intraspecific Variation
Analyses of intraspecific variation were conducted using common
microbiological techniques, including SDS-PAGE of total proteins.
These methods revealed strain-specific, but not pathovar-specific,
variation. Attempts to differentiate strains into pathovars of X.
campestris were not successful by these means. The physical primary
structure of plasmids and chromosomes, as revealed by restriction
fragment length polymorphism (RFLP), was useful in revealing pathovar-
specif ic variation. Strains of unknown plant origin can be at least
tentatively classified by pathovar. Additionally, non-pathogenic
epiphytes may be classifiable (epivars?) using these methods.
Plasmids
Plasmids appear useful to differentiate among some of the
pathovars of X. campestris. In comparing strains of X. c. pv.
maivacearum with race-specific interactions, no correlation was
observed between plasmids grouped by RFLP patterns and cotton host
differentials. Furthermore, plasmid DNA fragments were cloned in
broad-host-range vectors, mobilized into virulent strains and tested
for race-specific avirulence. Although not all of the possible
plasmid sequences were tested, it is still possible that avirulence
genes may reside on plasmids of X. c_. pv. maivacearum. However, sane
92

93
host-range specifying functions may be plasmid encoded. Strains of X.
c. pv. phaseoli and X. c_. pv phaseoli var. fuscans have the same host
range, but differ significantly in colony appearance, physiological
tests, and chromosomal RFLP analyses. However, plasmids from these
strains appeared highly related by restriction fragments profiles and
Southern hybridization. Thus, these strains are quite distinct
biochemically and chromosomally, but similar as characterized by the
plasmids ana by host range. This correlation between host range and
plasmids allowed the use of plasmids and cloned plasmid fragments as
epidemiological markers for detection and rapid identification of X.
c. pv. citri strains in Florida. Plasmid RFLP analyses are limited to
those strains which carry plasmids; only 95 of 151 strains of X.
campestris tested, or 11 of 26 pathovars tested had plasmids in all
strains.
Restriction Fragment Length Polymorphism
Chromosomal DNA RFLP analyses proved to be a reliable method to
differentiate X. campestris by pathovar, at least for those pathovars
tested. This method provided a means to resolve taxonomic
ambiguities. For example, X. c. pv. phaseoli and X. c. pv. phaseoli
var. fuscans, appear quite different by chromosomal RFLP analyses, and
share a common host. By contrast, nearly all 100 strains tested from
24 pathovars had RFLP patterns characteristic for the pathovar, and
very little intrapathovar variation was present.
The use of RFLP methods has already been useful in other studies
as an applied tool in uncovering the origin of strains of the citrus
canker pathogen found in Florida (Gabriel et al., unpubl.). An RFLP

94
survey of the known type strains of X. c. pv. citri and those isolated
in Florida allowed for the separation of these bacteria into four
identifiable RFLP groups. Strains belonging to the "A group" isolated
frcm Brazil and Japan were related by RFLP analysis. The strains
isolated in 1986 from citrus in backyards of the Tampa-Bradenton area
were also of the "A group". Strains belonging to the "B group" and "C
group" (isolated from Argentina and Brazil, respectively) appeared to
be related by RFLP analysis, yet could be differentiated. A Mexican
cancrosis strain was indistinguishable from the "B group" based on
RFLP analysis. Other strains of X. c. pv. citri isolated in Florida
did not appear to be related to those strains placed into the "A
group" or "B/C group". The Florida nursery strains appeared to
constitute yet a third RFLP grouping. This group was highly
polymorphic, and some strains appeared to be related to other
pathovars of X. campestris, strengthening the ,lmutation/endemic"
theory for some occurrences of citrus canker (Gabriel et al.,
unpubl.).
RFLP methods are powerful taxonomic tools to distinguish major
differences in strains assigned to the same pathovar simply because
they share a common host. For instance, the differentiation of
strains of X. campestris pathogenic to crucifers, such as X. c. pv.
campestris and X. c. pv. armoraciae, is at times difficult. X. c_. pv.
campestris is generally a systemic pathogen, whereas X. c. pv.
armoraciae is usually localized, but several strains showing
intermediate symptoms have been identified (99). In preliminary
experiments the RFLP methods were able to differentiate between the

95
strains. Surprisingly, differences could also be detected among the
strains assumed to be X. c. pv. campestris, where sane of the more
virulent strains belonged to a separate RFLP grouping.
Because RFLP groupings of X. campestris correlated well with host
range, it seems possible that this association could also be valid
with other bacterial systems. There is a great interest in the use of
microorganisns as biocontrol agents. In many cases, attempts to
exploit microorganisms which exhibit strong in vitro antagonism have
failed due to the poor survivability of the agent in plants. For
instance, P_. fluorescens has been used in developing rhizosphere
competent biocontrol agents against fungi (44,63,78,100). Strains of
P_. fluorescens, also a member of the same bacterial family as X.
campestris, is poorly differentiated; and as part of the soil
rhizosphere is also poorly defined. RFLP studies could be used to
identify members of P. fluorescens which have the desired host range,
thus, facilitating the development of more effective biocontrol
agents.
Conclusions
The extent to which we devise taxonomic distinctions to
differentiate among organisms remains a function of their utility to
humankind. Their relavance to "natural groupings" is in the minds of
pathologists or microbiologists whose perception may be somewhat
different from their Creator. The variation which is perceived as
significant by pathologists to differentiate strains of X. campestris
is host-specific pathogenicity. The variation which is perceived as
significant by geneticists may be chromosomal RFLP patterns. The

96
taxonomic validity of grouping strains by pathovar was strengthened by
the observation that, in general, strains within a pathovar fall into
a single RFLP group. Although plant tests are still necessary as a
confirmation of host pathogenicity, mutants which are no longer
pathogenic and non-pathogenic epiphytes may now be classified in one
classification system. The analyses conducted using common
microbiological techniques, including SDS-PAGE of total proteins,
revealed the relatedness of strains at the species level. Attempts to
differentiate strains into pathovars of X. campestris were not as
successful by these means, as variation was unresolvable at this
level. With the incorporation of additional plasmid and RFLP
analyses, the ability to resolve these strains into pathovars was
enhanced, even to the extent that these differences were correlated
with strains of a given host range.

LITERATURE CITED
1. Alvarez, A. M. Benedict, A. A., and Mizumoto, C. Y. 1985.
Identification of xanthomonads and grouping of strains of
Xanthanonas campestris pv. campestris with monoclonal antibodies.
Phytopathology 75:722-728.
2. Barker, D., Schafer, M., and White, R. 1984. Restriction sites
containing CpG show a higher frequency of polymorphism in human
ENA. Cell 36:131-138.
3. Bender, C. L., and Cooksey, D. A. 1986. Indigenous plasmids in
Pseud anonas syringae pv. tomato: conjugative transfer and role in
copper resistance. J. Bacteriol. 165:534-541.
4. Beynon, J. L., Beringer, J. E., and Johnston, A. W. B. 1980.
Plasmids and host-range in Rhizobium leguminosarum and Rhizobium
phaseoli. J. Gen. Microbiol. 120:421-429.
5. Boucher, C., Martinel, A., Barberis, P. Alloing, G., and
Zischek, C. 1986. Virulence genes are carried by a megaplasmid of
the plant pathogen Pseudononas solanacearum. Mol. Gen. Genet.
205:270-275.
6. Bradbury, J. F. 1984. Xanth anonas Dowson 1939. pp. 199-210 in:
Bergys Manual of Systematic Bacteriology, Vol. 1. N.R Krieg, and
J.G Holt, eds. Williams and Wilkins, Baltimore. 964 pp.
7. Brinkerhoff, L. A. 1963. Variability of Xanth anonas malvacearum:
the cotton bacterial blight pathogen. Oklahoma State University
Agricultural Experiment Station Bulletin T-98. 95 pp.
8. Brinkerhoff, L. A. 1970. Variation in Xanthomonas malvacearum and
its relation to control. Annu. Rev. Phytopathol. 8:85-110.
9. Brinkerhoff, L. A., and Presley, J. T. 1967. Effect of four day
and night temperature regimes on bacterial blight reactions of
immune, resistant, and susceptible strains of upland cotton.
Phytopathology 57:47-51.
10. Buchholz, W. G., and Thonashow, M. F. 1984. Comparison of T-DNA
complements of Agrobacterium tumefaciens tumor-inducing plasmids
with limited and wide host ranges. J. Bacteriol. 160:319-326.
97

98
11. Bufton, L., Bruns, G. A. P., Magenis, R. E., Temar, D., Shaw, D.,
Brook, D., and Litt, M. 1986. Four restriction fragment length
polymorphisms revealed by probes from a single cosmid map to
chromosome 19. Am. J. Hum. Genet. 38:447-460.
12. Chase, A. R. 1986. Cctnparisons of three bacterial leaf spots of
Hibiscus rosa-sinensis. Plant Dis. 70:334-336.
13. Civerolo, E. L. 1985. Indigenous plasmids in Xanthomonas
campestris pv. citri. Phytopathology 75:524-528.
14. Close, T. J., Zaitlin, D., and Kado, C. I. 1984. Design and
development of amplifiable broad-host-range cloning vectors:
analysis of the vir region of Agrobacterium tumefaciens plasmid
pTiC58. Plasmid 12:111-118.
15. Curiale, M. S., and Mills, D. 1983. Molecular relatedness among
cryptic plasmids in Pseudomonas syringae pv. glycinea.
Phytopathology 73:1440-1444.
16. Dahlbeck, D., Pring, D. R., and Stall, R. E. 1977. Detection of
covalently closed circular DNAs frcm Xanthanonas vesicatoria.
(Abstr.). Proc. Amer. Phytopathol. Soc. 4:176.
17. Dahlbeck, D., and Stall, R. E. 1979. Mutations for change of race
in cultures of Xanthomonas vesicatoria. Phytopathology
69:634-636.
18. Daniels, M. J., Barber, C. E., Turner, P. C., Sawczyc, M. K.,
Byrde, R. J. W., and Fielding, A. H. 1985. Cloning of genes
involved in pathogenicity of Xanthomonas campestris pv.
campe stris using the broad host range cosmid pLAFRi. EM30 J.
13:3323-3328.
19. Daub, M. E., and Hagedorn, D. J. 1981. Epiphytic populations of
Pseudomonas syringae on susceptible and resistant bean lines.
Phytopathology 71:547-550.
20. Day, P. R. 1974. Genetics of Host-Parasite Interaction. W. H.
Freeman and Co., San Francisco, CA. 238 pp.
21. De Vos, P., Goor, M., Gillis, M., and De Ley, J. 1985. Riboscmal
ribonucleic acid cistron similarities of phytopathogenic
Pseudomonas species. J. Sys. Bacteriol. 35:169-184.
22. Ditta, G., Stanfield, S., Corbin, D., and Helinski, D. R. 1980.
Broad host range DNA cloning system for Gram-negative bacteria:
Construction of a gene bank of Rhizobium meliloti. Proc. Natl.
Acad. Sci. USA 77:7347-7351.
23.
Dye, D. W. 1958. Host specificity in Xanthomonas. Nature
182:1813-1814.

99
24. Dye, D. W., Bradbury, J. F., Goto, M., Hayward, A. C., Lelliott,
R. A., and Schroth, M. N. 1980. International standards for
naming pathovars of phytopathogenic bacteria and a list of
pathovar names and pathotype strains. Rev. Plant Pathol.
59:153-168.
25. Dylan, T., Ielpi, L., Stanfield, S., Kashyap, L., Douglas, C.,
Yanofsky, M., Nester, E., Helinski, D. R., and Ditta, G. 1986.
Rhizobium meliloti genes required for nodule development are
related to chromosomal virulence genes in Agrobacterium
tumefaciens. Proc. Natl. Acad. Sci. USA 83:4403-4407.
26. Ercolani, G. L. 1978. Pseudomonas savastanoi and other bacteria
colonizing the surface of olive leaves in the field. J. Gen.
Microbiol. 109:245-257.
27. Essenberg, M., Cason, E. T., Jr., Hamilton, B., Brinkerhoff, L.
A., and Richardson, R. K. 1979. Single-colonies of Xanthamonas
malvacearum in susceptible and inmune cotton leaves and the local
resistance response to colonies in immune leaves. Physiol. Plant
Pathol. 15:53-68.
28. Farrar, W. E. Jr. 1983. Molecular analysis of plasmids in
epidemiologic investigation. J. Infect. Dis. 148:1-6.
29. Follin, J.-C. 1983. Races of Xanthomonas campestris pv.
malvacearum (Smith) Dye in western and cenral Africa. Cot. Fib.
Trop. 38:277-279.
30. Fujimoto, D. K., and Vidaver, A. K. 1985. Analysis of strain
variation in Xanth anonas campestris pv. phaseoli. (Abstr.) Proc.
Sixth Int. Conf. Plant Pathogenic Bact., College Park, MD.
31. Gabriel, D. W. 1985. Four plasmid DNA variants distinguished in
1984 Florida citrus canker epiphytotic. (Abstr.) Phytopathology
75:1320.
32. Gabriel, D. W. 1986. Specificity and gene function in
plant-pathogen interactions. ASM News 52:19-25.
33. Gabriel, D. W., Burges, A., and Lazo, G. R. 1986. Gene-for-gene
interactions of five cloned avirulence genes from Xanth anonas
campestris pv. malvacearum with specific resistance genes in
cotton. Proc. Natl. Acad. Sci. USA. 83:6415-6419.
Gabriel, D. W., Burges, A. R., Lazo, G. R., and Roffey, R. 1986.
Xanthomonas campestris pvs. citri, alfalfae, and phaseoli are
genetically and pathologically related. (Abstr.). Phytopathology
76:1076.
34.

100
35. Gallie, D. R., Zaitlin, D., Perry, K. L., and Kado, C. I. 1984.
Characterization of the replication and stability regions of
Agrobacterium tumefaciens plasmid pTAR. J. Bacteriol.
157:739-745.
36. Gerhardt, P., Murray, R. E. E., Costilow, R. N., Nester, E. W.,
Wood, W. A., Krieg, N. R., and Phillips, G. B. 1981. Manual of
Methods for General Bacteriology. American Society for
Microbiology, Washington, DC. 524 pp.
37. Goto, M., and Starr, M. P. 1972. Phage-host relationships of
Xanthcmonas citri compared with those of other Xanthanonas. Ann.
Phytopatol. Soc. Japan. 38:226-248.
38. Gottlieb, P., and Rudner, R. 1985. Restriction site polymorphism
of ribosomal ribonucleic acid gene sets in members of the genus
Bacillus. Int. J. Sys. Bacteriol. 35:244-252.
39. Haas, J. M., Fett, W. F., and Fleming, D. J. 1985. Detection and
initial characterization of plasmids in Xanthomonas campestris
pv. glycines (Abstr.). Proc. Sixth Int. Conf. Plant Pathogenic
Bact., College Park, MD.
40. Hartung, J. S., and Civerolo, E. L. 1986. Genomic fingerprints of
Xanthomonas campestris pv. citri strains from Asia, South
America, and Florida. (Abstr.). Phytopathology 76:1137.
41. Helentjaris, T., King, G., Slocum, M., Siedenstang, C., and
Wegman, S. 1985. Restriction fragment polymorphisms as probes for
plant diversity and their development as tools for applied plant
breeding. Plant Mol. Biol. 5:109-118.
42. Heumann, W., Rosch, A., Springer, R., Wagner, E., and Winkler, K.
P. 1984. In rhizobiaceae five different species are produced by
rearrangements of one genome, induced by DNA-damaging agents.
Mol. Gen. Genet. 197:425-436.
43. Hoekema, A., De Pater, B. S., Felinger, A. J., Hooykaas, P. J.
J., and Schilperoort, R. A. 1984. The limited host range of an
Agrobacterium tumefaciens strain extended by a cytokinin gene
from a wide host range T-region. EMBO J. 3:3043-3047.
44. Howie, W., and Suslow, T. 1986. Effect of antifungal compound
biosynthesis on cotton root colonization and Pythium suppression
by a strain of Pseudomonas fluoreseens and its antifungal minus
isogenic mutant. (Abstr.). Phytopathology 76:1069.
45. Hunter, R. E., Brinkerhoff, L. A., and Bird, L. S. 1968. The
development of a set of upland cotton lines for differentiating
races of Xanthcmonas malvacearum. Phytopathology 58:830-832.

101
46. Kado, C. I., and Liu, S. T. 1981. Rapid procedure for detection
and isolation of large and small plasmids. J. Bacteriol.
145:1365-1373.
47. Kamper, S. M., French, W. J., and deKloet, S. R. 1985. Genetic
relationships of some fastidious xylem-limited bacteria. Int. J.
Sys. Bacteriol. 35:185-188.
48. Kao, J. C., Perry, K. L., and Kado, C. I. 1982. Indoleacetic acid
complementation and its relation to host range specifying genes
on the Ti plasmid of Agrobacterium tumefaciens. Mol. Gen. Genet.
188:425-432.
49. Klement, Z. 1983. Hypersensitivity, pp. 150-177 in:
Phytopathogenic Prokaryotes, Vol. 2. M. S. Mount, and G. H. Lacy,
eds. Academic Press, New York, NY. 506 pp.
50. Lacy, G. H., and Patil, S. S. 1982. Why genetics?, pp. 221-228
in: Phytopathogenic Prokaryotes, Vol 2. M. S. Mount, and G. H.
Lacy, eds. Academic Press, Inc., New York, NY. 506 pp.
51. Laemmli, U. K. 1970. Cleavage of structural proteins during the
assembly of the head of bacteriophage T4. Nature 227:680-685.
52. Lazo, G. R. and Gabriel, D. W. 1984. Described 'races' of
Xanthcmonas campestris pv. malvacearum are mixtures. (Abstr.).
Phytopathology 74:837.
53. Lazo, G. R., and Gabriel, D. W. 1985. Use of plasmid DNAs to
differentiate pathovars of Xanthcmonas campe stris. (Abstr.).
Phytopathology 75:1320.
54. Lazo, G. R. and Gabriel, D. W. Conservation of plasmid DNA
sequences and pathovar identification of strains of Xanthcmonas
campestris. Phytopathology (in press).
55. Lazo, G. R., Gabriel, D. W., and Roffey, R. 1986. Differentiating
pathovars of Xanthcmonas campestris without pathogenicity tests.
(Abstr.). Phytopathology 76:1076.
56. Lelliott, R. A., and Dickey, R. S. 1984. Erwinia Winslow,
Broadhurst, Buchanan, Krumwiede, Rogers and Smith 1920. pp.
469-476 in: Bergy's Manual of Systematic Bacteriology, Vol. 1.
N. R. Krieg, and J. G. Holt, eds. Williams and Wilkins,
Baltimore, MD. 964 pp.
57. Leyns, F., De Cleene, M., Swing, J., and De Ley, J. 1984. The
host range of the genus Xanthcmonas. Bot. Rev. 50:308-356.
58. Li, WT.-H. 1986. Evolutionary change of restriction cleavage sites
and phylogenetic inference. Genetics 113:187-213.

102
59. Liew, K. W., and Alvarez, A. M. 1981. Phage typing and lysotype
distribution of Xanthomonas campestris. Phytopathology
71:274-275.
60. Lin, B. and Chen, S. 1978. Multi-plasmid in Xanthomonas
manihotis (Abstr.). Third Int. Congress Plant Pathol., Mnchen,
Germany.
61. Litt, M., and White, R. L. 1985. A highly polymorphic locus in
human DNA revealed by cosmid-derived probes. Proc. Natl. Acad.
Sci. USA 82:6206-6210.
62. Long, S. R., Buikema, W., and Ausebel, F. M. 1982. Cloning of
Rhizobium meliloti nodulation genes by direct complementation of
Nod mutants. Nature 298:485-488.
63. Loper, J. E. 1986. Role of fluorescent siderophore production in
biocontrol of Pythium ultimum by Pseudomonas fluorescens strain
3551. (Abstr.). Phytopathology 76:1069.
64. Loper, J. E., and Kado, C. I. 1979. Host range conferred by the
virulence-specifying plasmid of Agrobacterium tumefaciens. J.
Bacteriol. 139:591-596.
65. Maas, J. L., Finney, M. M., Civerolo, E. L., and Sasser, M. 1985.
Association of an unusual strain of Xanthanonas campestris with
apple. Phytopathology 75:438-445.
66. Maniatis, T., Fritsch, E. F., and Sambrook, J. 1982. Molecular
Cloning: A Laboratory Manual. Cold Spring Harbor Laboratory, Cold
Spring Harbor, N.Y. 545 pp.
67. McClean, P. E. and Hanson, M. R. 1986. Mitochondrial DNA
sequence divergence among Lycopersicon and related Solanum
species. Genetics 112:649-667.
68. McMaster, G. K., Samulski, R. J., Stein, J. L., and Stein, G. S.
1980. Rapid perification of covalently closed circular DNAs of
bacterial plasmids and animal tumor viruses. Anal. Biochem.
109:47-54.
69. McNally, K., and Gabriel, D. W7. 1984. Useful minimal culture
media for Xanthomonas campestris pv. malvacearum. (Abstr.)
Phytopathology 74:875.
70. Minsavage, G. V., and Schaad, N. W. 1983. Characterization of
membrane proteins of Xanthomonas campestris pv. campestris.
Phytopathology 73:747-755.
71. Mulrean, E. N., and Schroth, M. N. 1982. Ecology of Xanth anonas
campestris pv. juglandis on Persian (English) walnuts.
Phytopathology 72:434-438.

103
72. Murai, N., Skcx>g, F., Doyle, M. E., and Hanson, R. S. 1980.
Relationships between cytokinin production, presence of plasmids,
and fasciation caused by strains of Corynebacterium fascians.
Proc. Natl. Acad. Sci. USA 77:619-623.
73. Murata, N. and Starr, M. P. 1973. A concept of the genus
Xanthanonas and its species in the light of segmental homology of
deoxyribonucleic acids. Phytopath. Z. 77:285-323.
74. Nei, M., and Li, W.-H. 1979. Mathematical model for studying
genetic variation in terms of restriction endonucleases. Proc.
Natl. Acad. Sci. USA 76:5269-5273.
75. Palleroni, N. J. 1984. Family I. Pseudomonadaceae Winslow,
Broadhurst, Buchanan, Krumwiede, Roger and Smith 1957. pp.
140-199 in: Bergy's Manual of Systematic Bacteriology, Vol 1. N.
R. Krieg, and J. G. Holt, eds. Williams and Wilkins, Baltimore,
MD.
76. Panopoulos, N. J., and Peet, R. C. 1985. The molecular genetics
of plant pathogenic bacteria and their plasmids. Annu. Rev.
Phytopathol. 23:381-419.
77. Piwowarski, J. and Shaw, P. D. 1982. Characterization of
plasmids from plant pathogenic pseudomonads. Plasmid 7:85-94.
78. Poplawsky, A. R. Peng, Y. F. and Ellingboe, A. H. 1986.
Bacterial Tn5 mutants affected in antibiosis to Gaeumanrnonyces
graminis var. tritici. (Abstr.). Phytopathology 76:1069.
79. Quant, R. L., and Mills, D. 1984. An integrative plasmid and
multiple-sized plasmids of Pseud anonas syringae pv. phaseolicola
have extensive homology. Mol. Gen. Genet. 193:459-466.
80. Randhawa, P. S., and Civerolo, E. L. 1985. Plasmids in
Xanthononas campestris pv. pruni (Abstr.). Proc. Sixth Int. Conf.
Plant Pathogenic Bact., College Park, MD.
81. Sasser, M. 1983. Fatty acid analysis of the genus Xanthononas.
in: First Fallen Leaf Lake Conf. On the Genus Xanthononas. Fallen
Leaf Lake, CA.
82. Schaad, N. W. 1980. Laboratory Guide for Identification of Plant
Pathogenic Bacteria. American Phytopathological Society, St.
Paul, MN. 72 pp.
Schleifer, K. H., Ludwig, W., Kraus, J., and Festl, H. 1985.
Cloned ribosomal ribonucleic acid genes from Pseudomonas
aeruginosa as probes for conserved deoxyribonucleic acid
sequences. Int. J. Sys. Bacterid. 35:231-236.
83.

104
84. Schnathorst, W. C. 1966. Unaltered specificity in several
xanthomonads after repeated passage through Phaseolus vulgaris.
Phytopathology 56:58-60.
85. Schroth, M. N., and Hildebrand, D. C. 1983. Toward a sensible
taxonomy of bacterial plant pathogens. Plant Dis. 67:128.
86. Schroth, M. N., Hildebrand, D. C., and Vitanza, V. 1970.
Pathogenic variation and overlapping host ranges in Pseudomonas
phaseolicola, P. glycinea, and P. mori. (Abstr.). Phytopathology
60:1313.
87. Silhavy, T. J., Berman, M. L., and Enquist, L. W. 1984.
Experiments with Gene Fusions. Cold Spring Harbor Laboratory,
Cold Spring Harbor, NY.
88. Stall, R. E., Loschke, D. C., and Jones, J. B. 1986. Linkage of
copper resistance and avirulence loci on a self-transmissible
plasmid in Xanthomonas campestris pv. vesicatoria. Phytopathology
76:240-243.
89. Starr, M. P. 1983. The genus Xanthomonas. pp. 742-763 in: The
Prokaryotes, Vol. 2. M. P. Starr, H. Stolp, H. G. Truper, A.
Balows, and H. G. Schlegel, eds. Springer-Verlag, New York, NY.
90. Surico, G., Comai, L., and Kosuge, T. 1984. Pathogenicity of
strains of Pseudomonas syringae pv. savastanoi and their
indoleacetic acid-deficient mutants on olive and oleander.
Phytopathology 74:490-493.
91. Sytsma, K. J., and Gottlieb, L. D. 1986. Chloroplast DNA evidence
for the origin of the genus Heterogaura from a species of Clarkia
(Onagraceae). Proc. Natl. Acad. Sci. USA 83:5554-5557.
92. Szabo, L. J., and Mills, D. 1984. Characterization of eight
excision plasmids of Pseudomonas syringae pv. phaseolicola. Mol.
Gen. Genet. 195:90-95.
93. Tait, R. C., Close, T., Lundquist, R., Hagiya, M., Rodriquez, R.,
and Kado, C. I. 1983. Construction and characterization of a
versatile broad host range DNA cloning system for gram-negative
bacteria. Biotechnology 1:269-275.
94. Thaveechai, N., and Schaad, N. W. 1984. Comparison of different
immunogen preparations for serological identification of
Xanthomonas campestris pv. campestris. Phytopathology
74:1065-1070.
95. Thaveechai, N., and Schaad, N. W. 1986. Immunochemical
characterization of a subspecies-specific antigenic determinant
of a membrane protein extract of Xanthanonas campestris pv.
campestris. Phytopathology 76:148-153.

105
96. Thayer, D. W., Lowther, S. V., and Phillips, J. G. 1984.
Cellulolytic activities of strains of the genus Cellulomonas.
Int. J. Sys. Bacteriol. 34:432-438.
97. Watson, B., Currier, T. C., Gordon, M. P., Chilton, M.-D., and
Nester, E. W. 1975. Plasmid required for virulence of
Agrobacterium tumefaciens. J. Bacteriol. 123:255-264.
98. Young, J. M., Dye, D. W., Bradbury, J. F., Panagopoulos, C. G.,
and Robbs, C. F. 1978. A proposed nomenclature and classification
for plant pathogenic bacteria. N. Z. J. Agrie. Res. 21:153-177.
99. Yuen, G. Y. K., and Alvarez, A. M. 1985. Aberrant symptoms on
cabbage caused by strains of Xanthomonas campestris. (Abstr.).
Phytopathology 73:1382.
100. Yuen, G. Y., and Schroth, M. N. 1986. Interactions of Pseudomonas
fluoreseens strain E6 with ornamental plants and its effect on
the composition of root-colonizing microflora. Phytopathology
76:176-180.

BIOGRAPHICAL SKETCH
Gerard Raymond Lazo was born in Dallas, Texas on August 25, 1957,
to Francisco Gerardo and Suzanne Marie Lazo. He completed his primary
and secondary education in Weslaco, Texas, where he graduated from
Weslaco High School in 1975. He received the degree of Bachelor of
Science in microbiology in 1979 from Texas A&M University. He
continued his education under the supervision of Luther S. Bird
conducting research on fungal and bacterial diseases of cotton and
received a Master of Science degree in plant pathology frcm Texas A&M
University in 1984. Gerard was married to Maria Alicia Gonzales in
1983 while attending Texas A&M University. In the fall of 1983 he
began to pursue the degree of Doctor of Philosophy in plant pathology
at Oklahoma State University and later continued his education at the
University of Florida under the supervision of Dean W. Gabriel. Upon
completing his degree requirements he plans to serve as a postdoctoral
research associate with Robert A. Ludwig at Thimann Laboratories,
University of California, Santa Cruz. Gerard's permanent address is
1807 Briarcrest Lane, Arlington, Texas 76012.
106

I certify that I have read this study and that in my opinion it
conforms to acceptable standards of scholarly presentation and is
fully adequate, in scope and quality, as a dissertation for the degree
of Doctor of Philosophy.
Dean W. Gabriel, Chairman
Assistant Professor of Plant
Pathology
I certify that I have read this study and that in my opinion it
conforms to acceptable standards of scholarly presentation and is
fully adequate, in scope and quality, as a dissertation for the degree
of Doctor of Philosophy.
Robert E. Stall
Professor of Plant Pathology
I certify that I have read this study and that in my opinion it
conforms to acceptable standards of scholarly presentation and is
fully adequate, in scope and quality, as a dissertation for the degree
of Doctor of Philosophy.
Daryl R. Prind
Professor of Plant Pathology
I certify that I have read this study and that in my opinion it
conforms to acceptable standards of scholarly presentation and is
fully adequate, in scope and quality, as a dissertation for the degree
of Doctor of Philosophy.
')y] oaA rB ci-vj cp\
Mark J. Bassett/
Professor of Horticultural
Science
This dissertation was submitted to the Graduate Faculty of the College
of Agriculture and to the Graduate School and was accepted as partial
fulfillment of the requirements for the degree of Doctor of
Philosophy.
May, 1987
Dean,/(College of Agriculture
Dean, Graduate School



8
phaseolicola, a plasmid, pMC7105, can be forced to integrate into the
host chromosome, but cannot be cured (79,92). The plasmid pM27105 has
seme hcmology with other P. svringae pv. phaseolicola strains, and
even P. svringae pv. glycinea strains. Homology between these
plasmids may be due to transpositional events, the presence of
repetitive sequences, variability due to integration and excision of
the plasmid, plasmid origin of replication, or other plasmid
maintenance function (79,92) Another possibility is that genes
essential for pathogenicity, host-range, or race specificity may be
encoded on them. The association of plasmid homology with host-range
specificity may indicate the involvement of plasmids in host range
determination in these cases.


and exposed to Kodak X-Qnat AR film at -8 0 C in cassettes with
intensifying screens. Hybridization of the probes to individual
strains of X. campestris was repeated at least three times.
77
Restriction Fragment Patterns and Densitometry
From developed autoradiographs, DNA fragment sizes and profiles
were determined and hybridization signals measured using a Gilford
Response spectrophotometer equipped with an autoradiograph scanner.
Routine scans were done at 600 nm wavelength with 0.5 mm aperture and
0.5 nm bandwidth setting. Scanning data was stored by computer.
Using above information, comparison were made among strains of a given
pathovar and also strains derived from different pathovars of X.
campestris.
Results
Agarose Gel Electrophoresis
For a given pathovar, the banding patterns appeared very similar
and polymorphism in these strains was apparently limited (Fig. 6-1).
In all, DNA from 87 strains of X. campestris, representing 23
different pathovars and including scene with no known pathovar status
were digested with restriction enzymes EcoRI and BamHl and separated
by size on agarose gels. Based on restriction fragment patterns
alone, it was possible to visualize DNA variability among the strains,
but recognition of particular DNA fragment banding patterns was
difficult due to the numerous DNA fragments involved (Fig. 6-2). Some
of the apparent variation was due to the presence of plasmid bands,
which appear brighter against the chromosomal bands due to their
relatively higher copy number within each cell.


TABLE OF CONTENTS
Page
ACKNOWLEDGMENTS iii
LIST OF TABLES vi
LIST OF FIGURES vii
ABSTRACT ix
CHAPTER
ONE INTRODUCTION 1
TWO LITERATURE REVIEW 4
THREE HOST RANGE OF PATHOVARS OF XANTHOMONAS CAMPESTRIS 9
Introducid on 9
Materials and Methods 10
Bacterial Strains and Host Plants 10
Plant Inoculations 12
Physiological Differentiation 12
Polyacrylamide Gel Electrophoresis 13
Results 13
Pathogenicity Tests 13
Physiological Differentiation 20
Polyacrylamide Gel Electrophoresis 22
Discussion 22
FOUR CONSERVATION OF PLASMID DNA SEQUENCES AND PATHOVAR
IDENTIFICATION OF STRAINS OF XANTHOMONAS CAMPESTRIS .... 27
Introduction 27
Materials and Methods 28
Bacterial Strains 28
Plasmid Extraction and Visualization 31
Cloning of Plasmid
Restriction Endonuclease Fragments 32
DNA/DNA Hybridization 32
Results 33
Detection of Plasmid DNA 33
Restriction Endonuclease Profiles 35
DNA/DNA Hybridization 40
Dot-blot Hybridization 42
Discussion 45
IV


given strain. The actual host range may be much broader, but a
complete host range description of a given strain would require
testing on all plants known to support growth of the genus. This
would be a monumental task, and therefore the potential host range of
a given strain of X. campestris is unknown. Obviously, any
information regarding pathogenicity is of value to pathologists.
However, without knowledge of the potential host range variation of
strains within a pathovar, the taxonomic value of the pathovar system
is questionable.
Attempts to devise alternative means to differentiate pathovars
of X. campestris have included serology (1,94) membrane protein
profiles (70), phage-typing (37) rRNA-DNA and DNA-DNA hybridization
(21,73), and gas chromatography of fatty acids (65,81). Although most
of these methods have been useful to differentiate given sets of
strains of X. campestris, there is little evidence that the groupings
distinguished by these methods have any correlation to the host range
groupings observed for given pathovars of X. campestris. None of
these techniques have been successful in replacing pathogenicity tests
to identify X. campestris to the pathovar level. Because differences
in host range primarily determine the pathogenic variability observed
in X. campestris, it is of interest to know more about the nature of
this microorganism and the mechanisms which determine host-range
specificity.
Genetic analyses of pathogenicity can easily be done with
bacteria, for they can be quickly cloned and assayed, and studied
using current recombinant DNA technologies (50). X. campestris is


did not hybridize to chromosomal DNA derived from the hibiscus strain.
Based on our limited pathogenicity tests and on the absence of plasmid
DNA sequences in the hibiscus strains, there may be justification to
differentiate the cotton and hibiscus strains into different
pathovars.
Although some cross-hybridization between plasmids of strains
iron different pathovars was detected by dot-blot analyses (Table 4-
3), strains were readily differentiated by restriction digest profiles
of the plasmid DNAs and by hybridization with selected DNA probes to
identifiable restriction digested DNA fragments. Cross-hybridization
may be the result of repetitive DNA sequences, insertion elements, or
of conserved DNA sequences that are important for the stable
maintenance of plasmids in X. campestris. Examples of plasmid
functions which might be conserved are those involved with
replication, incompatibility, or other host dependent factors. In
seme instances plasmid DNAs hybridized to total DNA of pathovars in
which no plasmids were detected (Tables 4-2 and 4-3). It is suggested
that some sequences encoded on plasmids in some pathovars are located
on chromosomes in other pathovars. It is also possible that those
plasmids may integrate into the bacterial chromosome and excise again,
in a manner similar to those in p. syringae pv. phaseolicola (79,92).
With interest in developing rapid diagnostic methods to identify
bacterial pathogens, it is possible that the combined usage of plasmid
restriction digest profiles and of plasmid DNA probes may be
sufficient for the identification of seme pathovars of X. campestris.
This would require that a plasmid be stably associated with a given


CHAPTER SEVEN
SUMMARY
Intraspecific Variation
Analyses of intraspecific variation were conducted using common
microbiological techniques, including SDS-PAGE of total proteins.
These methods revealed strain-specific, but not pathovar-specific,
variation. Attempts to differentiate strains into pathovars of X.
campestris were not successful by these means. The physical primary
structure of plasmids and chromosomes, as revealed by restriction
fragment length polymorphism (RFLP), was useful in revealing pathovar-
specif ic variation. Strains of unknown plant origin can be at least
tentatively classified by pathovar. Additionally, non-pathogenic
epiphytes may be classifiable (epivars?) using these methods.
Plasmids
Plasmids appear useful to differentiate among some of the
pathovars of X. campestris. In comparing strains of X. c. pv.
maivacearum with race-specific interactions, no correlation was
observed between plasmids grouped by RFLP patterns and cotton host
differentials. Furthermore, plasmid DNA fragments were cloned in
broad-host-range vectors, mobilized into virulent strains and tested
for race-specific avirulence. Although not all of the possible
plasmid sequences were tested, it is still possible that avirulence
genes may reside on plasmids of X. c_. pv. maivacearum. However, sane
92


45
X. c. malvacearum strain N, which carries two plasmids, hybridized to
ENA of thirteen out of twenty-three X. campestris pathovars tested
(Table 4-3). Of these thirteen, seven cross-hybridized to the 4.5 kb
subcloned fragment of X. c. malvacearum strain N. Plasmids were not
detected in sane of the pathovars which hybridized to the probe. A
2.3 kb subcloned plasmid fragment from X. c. vignicola hybridized to
only six of the 23 pathovars tested. Plasmids were present in all six
of those pathovars which hybridized to the probe. A 2.3 kb cloned
plasmid fragment of X. c. phaseoli hybridized strongly to other
strains of the same pathovar, and weakly to 2 of 3 different X. c.
citri strains tested. The X. c. phaseoli probe appears to have
hybridized to chromosomal DNA of X. c_. citri in this case, as the
probe did not hybridize against Southern transfers of EcoRI digested
plasmid fragments of X. c. citri strains (not shown).
Discussion
It is suggested that there is extensive conservation of plasmid
DNA sequences (as represented by conserved restriction fragments)
within, but not usually among, pathovars of X. campestris. Plasmid DNA
fragment patterns will be identical if there is no rearrangement of
the DNA sequence at the restriction enzyme recognition site (a six
base pair sequence for EcoRI and BamHI) if no new restriction
fragments are created within the fragment, and if there are no major
additions or deletions causing a change in fragment size. Given these
possibilities, it was surprising to find so little restriction
fragment length polymorphism of plasmids within a pathovar, especially
when strains obtained from different continents were compared.


66
Figure 5-3. Transformation of E. coli ED8767 with plasmid DNA from X.
campestris pv. malvacearum transconjugants mated with pLXD. Lane A,
iamda Hindlll; lanes B-M, E. coli ED8767 transformed with DNA
extracted from transconjugants mated with pLXD and selected on
kanamycin Luria-Bertani plates (see Fig. 5-2). The transformants were
digested with restriction endonuclease EcoRI. Only the 8 kb EcoRI
fragment was present; the smaller band was undigested DNA.


105
96. Thayer, D. W., Lowther, S. V., and Phillips, J. G. 1984.
Cellulolytic activities of strains of the genus Cellulomonas.
Int. J. Sys. Bacteriol. 34:432-438.
97. Watson, B., Currier, T. C., Gordon, M. P., Chilton, M.-D., and
Nester, E. W. 1975. Plasmid required for virulence of
Agrobacterium tumefaciens. J. Bacteriol. 123:255-264.
98. Young, J. M., Dye, D. W., Bradbury, J. F., Panagopoulos, C. G.,
and Robbs, C. F. 1978. A proposed nomenclature and classification
for plant pathogenic bacteria. N. Z. J. Agrie. Res. 21:153-177.
99. Yuen, G. Y. K., and Alvarez, A. M. 1985. Aberrant symptoms on
cabbage caused by strains of Xanthomonas campestris. (Abstr.).
Phytopathology 73:1382.
100. Yuen, G. Y., and Schroth, M. N. 1986. Interactions of Pseudomonas
fluoreseens strain E6 with ornamental plants and its effect on
the composition of root-colonizing microflora. Phytopathology
76:176-180.


no. 12). All restriction fragment size estimates were based on the
relative mobilities of linear DNA fragments using lambda phage DNA
digested with HindiII as molecular size standards. Plasmid sizes were
32
estimated by addition of the sized restriction digested plasmid DNA
fragments. All plasmid experiments were repeated at least twice for
each strain examined.
Cloning of Plasmid Restriction Endonuclease Fragments
Plasmid DNA isolation from X. campestris was by a modification of
either of two alkaline lysis extraction procedures (46,68). The
extracted plasmids were purified on CsCl-etnidium bromide gradients by
centrifugation at 55,000 rpm in a Beckman VT65.2 rotor for 17 hr at
20 C. The purified plasmids were digested with the restriction enzyme
EcoRI. The cloning vector, pUCD5 (14), was digested with EcoRI,
treated with calf intestinal alkaline phosphatase (Boehringer-
Mannheim, Indianapolis, IN), and ligated to the EcoRI digested X.
campestris plasmid fragments with T4 DNA ligase (Bethesda Research
Laboratories). The ligation products were transformed into E. coli
strain ED8767, selecting transformed colonies on Luria-Bertani medium
containing ampicillin (50 ug/ml) or kanamycin (30 ug/ml). Selected
colonies were analyzed for the vector containing desired cloned DNA
fragments. These general cloning procedures are outlined in Maniatis
et al. (66).
DNA/DNA Hybridization
Plasmid DNAs were transferred from agarose gels to nitrocellulose
membranes by the method of Southern as described by Maniatis et al.
(66) and hybridized against radioactively-labeled DNA probes. The DNA


76
kb BamHI fragment deleted from it. DNA of strain 3401 was partially
digested with the restriction enzyme Mbol and fractionated by size on
a 10-40% (w/v) sucrose step gradient (66). The pUCD5B vector was
digested with the restriction enzyme BamHI and treated with calf
intestinal alkaline phosphatase (Boehringer-Mannheim). Vector DNA and
large fragment size fractions (25-40 kb) from strain 3401 were mixed
and treated with T4 DNA ligase (Bethesda Research Laboratories) and
recombinant cosmids were packaged in vitro with extracts prepared and
utilized according to Scalenghe and Hohn's protocol II as described by
Maniatis et al. (66). Transduction with Escherichia coli HB101 was
performed using top agar on Luria-Bertani medium containing kanamycin
(3 5 ug/ml) Cloned DNA fragments of strain 3401 in the vector
averaged 27-38 kb.
DNA Hybridization
ENA clones used as probes were radiolabeled using 30-60 uCi of
32p_deoxycytidine (Du Pont NEN) with use of a nick translation kit
(Bethesda Research Laboratories) and separated from low-molecular
weight nucleotides on a mini-column of Sephadex G50-100. Southern
blots were pre-hybridized in plastic bags for 4 hr and hybridized with
the DNA probe for 16-18 hrs at 68 C. The pre-hybridization and
hybridization solutions were those described in Maniatis et al. (66).
Following hybridization, membranes were washed once in 2X SSC, 0.5%
SDS (IX SSC is 0.15 M NaCl, 0.15 M sodium citrate, pH 7.0) and washed
once in 2X SSC, 0.1% SDS at ambient temperature, and washed two times
in 0.1X SSC, 0.5% SDS at 68 C as described by Maniatis et al. (66) for
'stringent* conditions. Nitrocellulose membranes were then air-dried


presumed that the homologous recombination occurred between the pLXD
clone and the indigenous plasmid. As a result, there was no EcoRI
fragment which corresponded to the 13 kb pUCD5 vector, or the 5 kb
pXLD insert. However, a new 8 kb EcoRI fragment was present.
The plasmid DNA from the X. campestris transconjugants with pXLD
was subsequently extracted and transformed into E. coli ED8767. The
transformants were selected on Luria-Bertani medium containing
kanamycin (25 ug/ml), or ampicillin (30 ug/ml). Resistance genes for
these antibiotics border the EcoRI cloning site of the pUCD5 vector
(14) All of the transformed plasmids were 8 kb in size, which
indicated that the new band seen in the X_. c_. pv. malvacearum
transconjugants was, in fact, a new autonomously replicating plasmid
(Fig. 5-3). There was obvious rearrangement in the original pXLD
clone which led to a 10 kb deletion. Southern hybridization analyses
revealed the presence of DNA fragments from both pUCD5 and the insert
of pLXD in this new vector, named pXD-1 (Figures 5-2 and 5-4).
Discussion
Of the twenty-five clonal isolates of X. c. pv. malvacearum
derived from 11 different nominal races, none of the clonal isolates
corresponded to the expected disease reactions on the host
differentials for that race. Likewise, there was no correlation
between a given plasmid group and the observed pathogenic reactions on
the 14 cotton differential lines. Although avirulence genes have been
found on plasmids in other phytopathogenic bacteria (88), no evidence
has been found to link plasmids in X. c. pv. malvacearum with race-
specific interactions. In these studies,
it appears the plasmids


84
Figure 6-4. Genomic DNA of strains of X. campestris digested with the
restriction endonuclease EcoRI and probed with cosmid clone XCT11.
Lane A, probe XCT1; lanes B-K, X. c. pv. phaseoli; lane B, JL; lane C,
LB-2; lane D, Xpn25; lane E, EK11; lane F, 82-1; lane G, 82-2; lane H,
Xpa; lane I, XP2; lane J, XP2-1; lane K, JF; lanes L-M, X. c. pv.
phaseoli var. fuscans; lane L, SC-3B; lane M, Xpfll; lanes N-0, X. c.
pv. alfalfae; lane N, KS; lane 0, FL;lanes P-R, X. c. pv. campestris;
lane P, XC1 (cabbage) ; lane Q, 084-1318 (broccoli) ; lane R, 084-720
(brussels sprouts); lane S, unknovm X. c. G65; lane T, probe XCTll.


LIST OF FIGURES
Figure Page
3-1 SDS-Polyacrylamide gel electrophoresis of total proteins
from strains of X. campestris 23
4-1 Plasmid DNAs from strains of X. campestris pv. malvacearum
digested with restriction endonucleases EcoRl and BamHI 37
4-2 Graphic representation of plasmid EcoRl restriction fragment
profiles for pathovars of X. campestris 38
4-3 Plasmid DNAs from strains of X. campestris pv. citri
digested with restriction endonuclease EcoRl 39
4-4 Plasmid DNAs from strains of X. campestris pv.
malvacearum digested with restriction endonuclease
EcoRl and hybridized to probe N4.5 41
4-5 Plasmid DNAs from strains of X. campestris digested with
restriction endonuclease EcoRl and hybridized to probes
P2.0 and P2.3 43
5-1 Plasmid curing of X. campestris pv. malvacearum with
SDS treatment 60
5-2 Transconjugants of X. campestris pv. malvacearum containing
pLXD and hybridized to the plasmid probe pSa4 64
5-3 Transformation of E. coli ED8767 with plasmid DNA
frcm X. campestris pv. malvacearum transconjugants
mated with pLXD 66
5-4 Plasmid DNA of X. campestris pv. malvacearum strain X
digested with restriction endonucleasea and hybridized
to the plasmid probe pXD-1 67
6-1 Genomic DNA of strains of X. campestris pvs. phaseoli,
alfalfae, and campe stris digested with restriction
endonuclease EcoRl 78
6-2 Genomic DNA of strains from different pathovars of
X. campestris digested with restriction endonuclease EcoRl .. 79
vii


18
representative strain from each of the X. campestris pathovars of
legumes tested, with the exception of X. c. pv. cyamopsidis. Various
phenotypic responses were observed on the cotton and hibiscus
cultivars with the X. c. pv. malvacearum strains tested. In general,
it appeared that those strains of X. c. pv. malvacearum derived as
pathogens from cotton were pathogenic to one of the hibiscus cultivars
tested. None of the X. c_. pv. malvacearum strains derived as
pathogens from hibiscus were pathogenic on the cotton cultivars in
these tests.
From the inoculation tests on the cotton host differentials with
the cotton derived strains of X. c. pv. malvacearum, it appeared that
three races of the pathogen were being used. All of these strains
were pathogenic on Acala 44 (susceptible host), but could be
differentiated into races by the other cotton lines containing
different resistance gene backgrounds. The X. c_. pv. malvacearum
strain N was pathogenic on all the cotton lines tested, while strain H
was only pathogenic on Acala 44. Strains FL79 and TX84 appeared to be
the same race because they were pathogenic on the same six out of
eight cotton lines tested (Table 3-4). However, these two strains
differed in reactions on the three hibiscus cultivars inoculated.
Strain FL79 appeared pathogenic on the three cultivars, but TX84 gave
a null response similar to the water-only control. The two other X.
c. pv. malvacearum strains (N and H) appeared identical in reaction
with the three hibiscus cultivars,; giving pathogenic responses to 2
cultivars, and a null response on the remaining cultivar (Table 3-4).


42
a 4.5 kb EcoRI plasmid DNA fragment, did have two other fragments (ca.
8 and 10 kb) which hybridized strongly to the 4.5 kb probe. In two
other X. c. malvacearum strains (FL79 and TX84), which appeared to
have multiple plasmids, the hybridization signal was weak for the 4.5
kb fragments as compared to larger EcoRI fragments (ca. 23 kb) .
Similar hybridization studies were done with cloned plasmid fragments
from other pathovars. When the plasmid fragments selected were
smaller, they were much more specific. Two different probes were
constructed from 2.0 kb and 2.3 kb EcoRI plasmid fragments derived
from X. c. phaseoli (strain XP2). When hybridized against X. c.
cyamopsidis, X. c. glycines, X. c. phasedi, and X. c. vignicola, each
of these probes only hybridized to X. c. phaseoli (Fig. 4-5) The 2.3
kb plasmid probe hybridized to similar sized fragments in other X. c.
phaseoli strains, including X. c. phaseoli var. fuscans (strains SC3-B
(not shown) and Xpfll). These X. c. phaseoli var. fuscans strains
differ from typical X. c. phaseoli strains in that they produce an
extracellular dark brown, melanin-like pigment in culture; otherwise,
they are considered similar. However, the 2.0 kb plasmid probe did
not hybridize to the X. c. phasedi var. fuscans strains, which did
not have the corresponding 2.0 kb EcoRI fragment in their plasmid
profile (lane C, Fig. 4-5, and strain SC3-B (not shown)). Repeated
hybridizations with Southern transfers containing these strains had
the same results.
Dot-blot Hybridization
DNA probes were also hybridized against total DNA of other X.
campestris pathovars fixed onto a nitrocellulose membrane by use of a
dot-blot manifold apparatus. Radiolabeled total plasmid DNA from


Table 5-3. Pathogenicity of X. campestris pv. malvacearum (Xcm)
transconjugantsa on cotton host differentials.
Plant reactions^
Strains Asala
44 &| Bj Bj B4 Ej % By % 101 Gregg BJ5
wild-type:
N + + + + + + + + + + + + +
H
Plasnid fragnents of Xian H in Xcm N:
JSIEA-H2 + + + + + + + + + + + + +
pUEA-H5 + + + + + + + + + + + + +
Plasnid fragrents of Xian N in Xon H:
rOEA-Nl +
PJEA-N3 +
PEA-N4 +
PEA-N5 +
pSV^S + -
Chranosanal fragment cf Xan H in Xon N:
pOEA-PH + + + + + + + + + + + +
a Cloned EcoRI fragment sizes within each clone are: pUFA-Hl, 4.9 kb;
PFA-H2, 19.2 and 4.6 kb; pUFA-H5, 22.4 and 4.0 kb; pUFA-Nl, 2.3 kb;
PUFA-N3, 14.5; PFA-N4, 23.5 and 13.4 kb; pUFA-N5, 15.6 kb; pUFA-N9,
13.4 and 9.6 kb.
k + = compatible (pathogenic) and = incompatible (hypersensitive)
plant reactions.


74
Table 6-1 (continued).
I&thovar
Strain
Host
Location
Source3
pisi
XP1
Pisun sativum
Japan
M-Goto
poinsettiicnLa
083-6248
Eupxirbia pulcrerrima
Elorida
DPI
pruni
084-1793
Prunus
Florida
CPI
translunens
XLLQ5
Hordaun sp.
Montana
D.Sands
\esicatoria
E-3
Capsicum annum
Florida
R.E.Stall
75-3
Lvocpersioon esculentrm
Floricfe
R.E.Stall
vignioola
A81-331,C-1,
Vigna urguiculata
Gsorgia
R.D.Gitaitis
OB5-1,
V. urgiuoulata
(feacgia
R.D.Gitaitis
X/19,312,
V. urguiculata
Cfeorgia
R.D.Gitaitis
432,82-38
V. unguiculata
Gfexgia
R.D.Gitaitis
vitians
IOB164
Latrca S3.
-
R-E.Stall
zinnia
084-1944
Zinnia elegans
Florid
DPI
unknown
084-1373
Ihilodandrcn sp. (dieffei.)
Florida
DPI
084-3928
Fbtsia sp. (hadsrae)
Florida
DPI
084-4348
Alocasia sp. (vitians)
Florich
CPI
083-2057
Syngcniun si. (vitians)
Florida
DPI
084-2848
Cissus S3.
Florich
CPI
084-1590
Eucryrus
Florida
DPI
251G,084-480
Impatiens sp.
Florida
DPI
084-6006
Jasninium sp.
Florida
DPI
X. fragarias
Xfral
Fragaria sp.
Florida
R.E.Stall
a DPI = Flor
ida Department
of Agricultural and
Consumer
Services,
Division of
Plant Industry,
Gainesville, Florida.


87
Table 6-3. Sizes of DNA fragments from Xanthomonas campestris genomic
digests (EcoRI) which hybridized to the XCTl DNA probe. See Figure 6-
5.
A
12.2,
11.2, 7.9, 5.6, 5.1, 3.2, 2.2, 1.6, 1.0
B
20.5,
3.1
18.8, 15.3, 14.2, 13.5, 12.0, 7.3, 6.2, 5.1,
4.6,
3.7,
3.4
C
18.7,
12.1, 9.5, 8.6, 8.2, 7.7, 6.9, 6.7, 6.2,
6.0,
4.8,
4.2
4.0,
3.4, 3.0, 2.9, 2.5, 2.3
D
19.4,
16.4, 14.4, 13.1, 11.1, 10.5, 9.2, 8.5, 7.7,
6.7,
5.6,
4.3
4.0,
2.2, 1.9
E
17.9,
12.9, 4.1, 3.5, 3.0, 2.3, 1.5
F
16.2,
11.5, 9.5, 8.7, 5.7, 4.0, 3.6, 3.1, 3.0, 1.4
G
14.2,
11.1, 4.5, 3.9, 3.5, 3.0
H
12.2,
10.2, 8.3, 7.9, 6.5, 5.5, 4.2, 3.1
I
12.9,
12.0, 7.6, 7.1, 6.5, 4.7, 3.0, 1.7
J
15.6,
14.2, 9.4, 7.9, 6.3, 5.0, 4.6, 4.0, 3.1, 2.5
, 2.2
K
20.9,
17.1, 16.2, 15.5, 14.2, 11.0, 10.4, 9.2,
8.2,
7.5,
6.7
4.2,
4.0, 3.3, 3.1, 3.0
L
19.4,
12.7, 10.4, 9.0, 7.9, 7.7, 6.8, 5.7, 4.2,
3.9,
3.0,
2.7
2.3,
1.9, 1.5, 1.4
M
12.0,
8.4, 7.0, 6.8, 4.5
N
15.9,
8.4, 4.9, 3.4, 2.1, 1.9
0
9.2,
4.4
P
13.3,
9.2, 7.2, 6.9, 6.2, 4.6, 3.8, 3.1, 2.1
Q
11.0,
8.5, 7.0, 6.3, 4.2, 3.2
R
16.7,
14.2, 12.9, 9.9, 7.4, 6.9, 6.3, 4.8, 3.8,
3.5,
3.2,
2.6
2.4,
2.2, 1.6
S
12.9,
12.5, 9.5, 7.7, 6.5, 4.4, 4.0, 3.8
T
17.6,
15.6, 14.7, 10.5, 8.6, 7.2, 1.4


100
35. Gallie, D. R., Zaitlin, D., Perry, K. L., and Kado, C. I. 1984.
Characterization of the replication and stability regions of
Agrobacterium tumefaciens plasmid pTAR. J. Bacteriol.
157:739-745.
36. Gerhardt, P., Murray, R. E. E., Costilow, R. N., Nester, E. W.,
Wood, W. A., Krieg, N. R., and Phillips, G. B. 1981. Manual of
Methods for General Bacteriology. American Society for
Microbiology, Washington, DC. 524 pp.
37. Goto, M., and Starr, M. P. 1972. Phage-host relationships of
Xanthcmonas citri compared with those of other Xanthanonas. Ann.
Phytopatol. Soc. Japan. 38:226-248.
38. Gottlieb, P., and Rudner, R. 1985. Restriction site polymorphism
of ribosomal ribonucleic acid gene sets in members of the genus
Bacillus. Int. J. Sys. Bacteriol. 35:244-252.
39. Haas, J. M., Fett, W. F., and Fleming, D. J. 1985. Detection and
initial characterization of plasmids in Xanthomonas campestris
pv. glycines (Abstr.). Proc. Sixth Int. Conf. Plant Pathogenic
Bact., College Park, MD.
40. Hartung, J. S., and Civerolo, E. L. 1986. Genomic fingerprints of
Xanthomonas campestris pv. citri strains from Asia, South
America, and Florida. (Abstr.). Phytopathology 76:1137.
41. Helentjaris, T., King, G., Slocum, M., Siedenstang, C., and
Wegman, S. 1985. Restriction fragment polymorphisms as probes for
plant diversity and their development as tools for applied plant
breeding. Plant Mol. Biol. 5:109-118.
42. Heumann, W., Rosch, A., Springer, R., Wagner, E., and Winkler, K.
P. 1984. In rhizobiaceae five different species are produced by
rearrangements of one genome, induced by DNA-damaging agents.
Mol. Gen. Genet. 197:425-436.
43. Hoekema, A., De Pater, B. S., Felinger, A. J., Hooykaas, P. J.
J., and Schilperoort, R. A. 1984. The limited host range of an
Agrobacterium tumefaciens strain extended by a cytokinin gene
from a wide host range T-region. EMBO J. 3:3043-3047.
44. Howie, W., and Suslow, T. 1986. Effect of antifungal compound
biosynthesis on cotton root colonization and Pythium suppression
by a strain of Pseudomonas fluoreseens and its antifungal minus
isogenic mutant. (Abstr.). Phytopathology 76:1069.
45. Hunter, R. E., Brinkerhoff, L. A., and Bird, L. S. 1968. The
development of a set of upland cotton lines for differentiating
races of Xanthcmonas malvacearum. Phytopathology 58:830-832.


98
11. Bufton, L., Bruns, G. A. P., Magenis, R. E., Temar, D., Shaw, D.,
Brook, D., and Litt, M. 1986. Four restriction fragment length
polymorphisms revealed by probes from a single cosmid map to
chromosome 19. Am. J. Hum. Genet. 38:447-460.
12. Chase, A. R. 1986. Cctnparisons of three bacterial leaf spots of
Hibiscus rosa-sinensis. Plant Dis. 70:334-336.
13. Civerolo, E. L. 1985. Indigenous plasmids in Xanthomonas
campestris pv. citri. Phytopathology 75:524-528.
14. Close, T. J., Zaitlin, D., and Kado, C. I. 1984. Design and
development of amplifiable broad-host-range cloning vectors:
analysis of the vir region of Agrobacterium tumefaciens plasmid
pTiC58. Plasmid 12:111-118.
15. Curiale, M. S., and Mills, D. 1983. Molecular relatedness among
cryptic plasmids in Pseudomonas syringae pv. glycinea.
Phytopathology 73:1440-1444.
16. Dahlbeck, D., Pring, D. R., and Stall, R. E. 1977. Detection of
covalently closed circular DNAs frcm Xanthanonas vesicatoria.
(Abstr.). Proc. Amer. Phytopathol. Soc. 4:176.
17. Dahlbeck, D., and Stall, R. E. 1979. Mutations for change of race
in cultures of Xanthomonas vesicatoria. Phytopathology
69:634-636.
18. Daniels, M. J., Barber, C. E., Turner, P. C., Sawczyc, M. K.,
Byrde, R. J. W., and Fielding, A. H. 1985. Cloning of genes
involved in pathogenicity of Xanthomonas campestris pv.
campe stris using the broad host range cosmid pLAFRi. EM30 J.
13:3323-3328.
19. Daub, M. E., and Hagedorn, D. J. 1981. Epiphytic populations of
Pseudomonas syringae on susceptible and resistant bean lines.
Phytopathology 71:547-550.
20. Day, P. R. 1974. Genetics of Host-Parasite Interaction. W. H.
Freeman and Co., San Francisco, CA. 238 pp.
21. De Vos, P., Goor, M., Gillis, M., and De Ley, J. 1985. Riboscmal
ribonucleic acid cistron similarities of phytopathogenic
Pseudomonas species. J. Sys. Bacteriol. 35:169-184.
22. Ditta, G., Stanfield, S., Corbin, D., and Helinski, D. R. 1980.
Broad host range DNA cloning system for Gram-negative bacteria:
Construction of a gene bank of Rhizobium meliloti. Proc. Natl.
Acad. Sci. USA 77:7347-7351.
23.
Dye, D. W. 1958. Host specificity in Xanthomonas. Nature
182:1813-1814.


24
pv. malvacearum strains. In the legume study, it was interesting to
note that different hosts gave different phenotypic responses to
different incompatible pathovars of X. campestris. These different
incompatible phenotypes suggest that different host resistance genes
may be involved in each of these incompatible interactions. Although
the hypersensitive reaction is commonly considered to be the general
resistance mechanism of plants against bacteria, these data suggest a
more specific response, unique to each incompatible reaction (33,49).
No formal studies on the hypersensitive reaction were implemented, so
the significance of this observation will require further
investigation. Differences in the phenotypes which may account for
this are time of recognition, cell number, electrolyte leakage, or
other bacteria- or plant-conditioned responses. Likewise, a
hypersensitive reaction may have occurred for instances where a null
response was observed (27).
The results from inoculation tests on the malvaceous hosts
suggested that those strains of X. c. pv. malvacearum derived from
cotton are potential pathogens of hibiscus, whereas those derived from
hibiscus are generally restricted to hibiscus. However, in one
experiment, it was found that one of the hibiscus strains caused
watersoaked lesions on Acala 44 when the night temperatures were
lowered. This observation indicates that some of the interactions may
be temperature sensitive. Seme resistance genes in cotton are known
to be temperature sensitive (9).
Race-specific incompatibility may account for the varied X. c.
pv. malvacearum (cotton strains) reactions on hibiscus. It was


range specificity, and to find a physiological test diagnostic for one
or more pathovars used in these studies.
10
Materials and Methods
Bacterial Strains and Host Plants
Bacteria used in this investigation consisted of pathovars of X.
campestris which were pathogenic to members of either the Leguminosae
or Malvaceae plant families. The pathogens of leguminous host plants
consisted of X. campestris pvs. alfalfae, cyamopsidis, glycines,
phaseoli, pisi, and vignicola isolated iron alfalfa, guar, soybean,
kidney bean, pea, and cowpea, respectively. The pathogens of
malvaceous host plants consisted of strains of X. c_. pv. malvacearum
isolated from cotton or hibiscus. The strains of X. campestris used
in this study are shown in Table 3-1. The legume host plants used in
this investigation included Medicago sativa cv. "FL-77", Glycines max
cv. "Evans", Phaseolus vulgaris cv. "California Light Red", and Vigna
unguiculata cv. "California Blackeye #5". The malvaceous hosts
included three cultivars of Hibiscus rosa-sinensis and eight cultivars
of Gossypium hirsutum. One of the cultivars of H. rosa-sinensis was
cv. Brilliant Red; the other two were not named, but chosen to
represent diversity in the species. Eight cotton cultivars were
selected for differentiating races of X. c. pv. malvacearum, two of
which were cv. 101-102B, and cv. Gregg (45). The other cultivars were
cv. Acala 44 (no resistance to cotton strains), and five cultivars
derived from cv. Acala 44 crossed to obtain single gene resistance to
the cotton strains of X. c. pv. malvacearum, the cultivars being Acala
B]_, B2, B3, B5, and Bj^j (7).


5
pathogen of Capsicum annuum L. (pepper) and Lycopersicon esculentum
(tomato). While some strains of X. campestris appear to have a host
range to more than one plant species, most others appear to have an
extremely narrow host range (6,57). The host range characteristic is
considered to be a stable trait, although there is one report of
altered host range by artificial selection (23). This report has been
refuted, with no change in host-range specificity observed for X.
campestris (84) WTithin at least seme pathovars, variability among
strains exists as race-specific plant interactions (8,17,20). These
are recognized when resistant plants of an otherwise susceptible plant
species are discovered. The ability to differentiate races of X.
campestris pathovars is dependent on the discovery of plant hosts,
called host differentials, which exhibit variability in their response
to different pathogenic strains. Pathogenic races have been described
for X. c_. pv. malvacearum (Smith) Dye, X. c. pv. oryzae, X. c. pv.
translucens, X. c. pv. vesicatoria, and possibly others. This race-
specific variation in X. campestris was suggestive of gene-for-gene
interactions between pathogenicity determinants in the pathogen with
resistance genes in the host (8,20).
Gene-for-gene relationships can only be established by genetic
crosses of the host and the parasite. Because the bacterial genetics
can only be done with recombinant DNA techniques, gene-for-gene
interactions could only be recently established. Proof of
gene-for-gene interactions with bacterial pathogens was documented for
X. c. pv. malvacearum (33).


63
helper plasmid. Since the vector pUCD5 is known to carry a
mobilization function, it could not be concluded that the strain X
plasmid is self-mobilizable, but it must carry at least some functions
required for self-mobilization.
pUCD5 is a broad host range vector with an origin of replication
(ori) that functions poorly in X. c. pv. malvacearum. Matings on MOPS
minimal medium provide a lengthy period of selection in which plasmid
integration can take place, provided a homologous X. c. pv.
malvacearum DNA fragment is cloned on the vector to allow
recombination to occur. Rapid selection procedures using complete
media containing antibiotics only rarely allows this integration to
occur before the recipients die. When transconjugants with X. c. pv.
malvacearum strain X plasmid DNA were selected on POPS complete medium
with antibiotics only the clone containing a 5.0 kb EcoRI fragment,
pLXD, mated at a high frequency. All other clones either did not grow
on POPS with antibiotics, or grew poorly. Because pLXD grew rapidly
on selective medium, whereas other clones did not, it was proposed
that the pXLD clone contained a functional origin of replication from
the strain X plasmid.
Transconjugants containing pXLD were further analyzed because
their growth rate was highly variable. Eleven of the pLXD
transconjugants were selected by colony size and grouped as large,
medium or small. Plasmids from the transconjugants were extracted and
analyzed for plasmid content. All, but possibly one transconjugant,
were missing a 4.5 kb EcoRI fragment associated with the indigenous
plasmid of X. c. pv. malvacearum strain N-SpR (Fig 5-2). It was


34
Table 4-2. Detection of plasmid DNA in strains of X. campestris.
Bacterium
No. of strains
containing plasmids/
No. of strains tested
Pathogenicity3
X. campestris pathovar
alfalfae
0/ 2
P
argemones
0/ 1
I
begoniae
0/ 1
I
campestris
2/ 4
R,I
carotae
0/ 3
R
citri
17/44
P,R,I
cyamopsidis
5/ 5
R
dieffenbachiae
2/ 2
I
esculenti
0/ 1
I
glycines
3/ 3
P
hederae
2/ 3
I
holeicola
0/ 2
R
maculifoliigardeniae
0/ 2
I
malvacearum cotton
32/32
P
malvacearum hibiscus
0/ 8
P
mangiferaeindicae
0/ 1
I
pelargonii
1/ 1
R
phaseoli
13/13
P
pisi
0/ 1
R
poinsettiicola
0/ 1
I
pruni
3/ 3
I
translucens
0/ 2
R
vesicatoria
5/ 5
P
vignicola
7/ 7
P
vitians
3/ 3
I
zinniae
0/ 1
I
X. albilineans
0/ 1
R
X. fragariae
0/ 1
I
3 P = Pathogenicity of strains confirmed on appropriate host, conforms
to current available information for particular pathovar.
R = Received as named pathogen, appropriate host specificity and
pathovar designation of strain assumed.
I = Isolated as a pathogen on host appropriate for designated pathovar;
characterization of strain(s) incomplete.


Table 5-4. Selection of plasmid replication genes in X. campestris pv.
malvacearum strain X.
62
Matings3
Size
MOPS
K30 pops K^nSPmn
Plaanid Insert
no helper
PRK2013 PRK2013
pLXA
17.0 kb
few
+
-
pLXB
14.0 kb
NT
NT
NT
pLXC
9.0 kb
-
+
few
pLXD
5.0 kb
-
++
++
(ori)
pLXE
4.8 kb
-
+
-
PLXF
4.7 kb
++
+++
-
(tra)
pLXG
4.5 kb
-
+++
-
pLXH
0.5 kb
-
+++
few
a is no growth, + = about 100 colonies, ++ = 100-300 colonies, +++ =
swamped plate, NT = not tested. MOPS K30 is the MOPS minimal medium
containing kanamycin (30 ug/ml) and POPS ^qSP^oq is the POPS
complete medium containing kanamycin (30 ug/ml) and spectinomycin
(100 ug/ml) pRK2013 is a helper plasmid used in tri-parental
matings (22). pLXD contains the putative plasmid origin of
replication (ori) and pLXF contains a putative plasmid transfer
(tra) function of the X. campestris pv. malvacearum strain X
plasmid.


determines whether a strain will have the host range listed for the
pathovar (hibiscus and cotton) or a more limited host range
(hibiscus). However, the plant source of X. c. pv. alfalfae, does not
determine whether a strain will have the host range listed. A strain
which attacks both alfalfa and bean is X. c. pv. alfalfae. A strain
with a host range limited to bean is X. c. pv. phaseoli. Finally,
several biochemical methods were evaluated for their potential to
classify a strain at the pathovar level. Although standard
microbiological tests and protein gels were not adequate for such
classifications, an examination of plasmid DNAs (Chapter Four) and
chromosomal DHAs (Chapter Six) yielded more rewarding results.


37
Figure 4-1. Plasmid DNAs from strains of Xanthomonas campestris pv.
malvacearum (Xcm) digested with restriction endonucleases EcoRl (lanes
B-I) and BamHI (lanes J-Q). Lanes shown above contain: A and R, lambda
HindIII; B and J; Xcm J; C and K, Xcm N; D and L, Xcm H; E and M, Xcm
V; F and N, Xcm Z; G and 0, Xcm Q; H and P, Xcm X; and I and Q, Xcm D.


DNA/DNA Hybridization
Initial plasmid comparisons were done on strains of X. c.
malvacearum. Whole purified plasmid DNA iron X. c. malvacearum strain
X, which contains only one plasmid, was hybridized against EcoRI
digested plasmid DMAs of other strains of the same pathovar (not
shown). This initial comparison demonstrated that the
plasmids,although differing slightly in digestion patterns, were quite
homologous as the radiolabeled plasmid hybridized to almost all EcoRI
fragments of the other strains.
A 4.5 kb EcoRI plasmid fragment of X. c_. malvacearum strain N was
cloned into the vector pUCD5 and used as a hybridization probe against
plasmid DNA from other strains of the same pathovar (Fig. 4-4). This
plasmid DNA fragment hybridized to plasmids from all but one strain of
X. c. malvacearum. This probe hybridized to EcoRI fragments of 4.5 kb
size (lanes B-J, M-N, Fig. 4-4) in several other strains of X. c.
malvacearum. Additionally, the probe hybridized to more than one of
the EcoRI plasmid fragments in these strains, suggesting that seme
sequences on the cloned DNA are repeated in other parts of the plasmid
DNA. The pUCD5 vector alone did not hybridize to any X. c.
malvacearum plasmid fragments. Because pUCD5 is a cosmid vector and
contains the cos site of lambda phage DNA, hybridization of the vector
to the corresponding DNA fragment of the molecular weight marker
containing the cos site was observed. Plasmid DNA from X. c.
malvacearum strain Su2 did not hybridize to the 4.5 kb probe, and did
not hybridize to any other plasmid fragments from X. c. malvacearum
strain N. Another X. c. malvacearum strain (Ch2), which did not have


procedures (46,66) as described in Chapter Four. Plasmids were
digested with either EcoRI or BamHI and DNA fragment patterns
53
visualized by agarose gel electrophoresis. All plasmid experiments
were repeated at least twice for each strain examined. Additionally,
EcoRI plasmid fragments of X. c. pv. malvacearum strains were cloned
into the vector pUCD5 (14) and maintained in E. coli ED8767 as
described in Chapter Four. These cloned fragments were used for
subsequent plasmid studies.
Plasmid Curing
To determine if plasmid encoded functions were involved in
pathogenicity, attempts were made to expel, or cure, plasmids from the
wild-type strains by including ethidium-bromide or sodium lauryl
sulfate (SDS) in the broth culture. Cultures were grown in peptone-
glycerol broth for two 48 hr cycles, being adjusted to an optical
density (ODgQOnm) f 0.03 at the beginning of each cycle. A dilution
series from the broth cultures were plated onto peptone-glycerol agar
and single colonies were selected for inoculation on cotton host
differentials.
Race-Specificity Genes
Cloned genes encoding race-specific avirulence in X. c. pv.
malvacearum have been identified and isolated (33) and it was of
interest to determine if these genes were plasmid-borne. Using E.
coli ED8 767 containing the cloned plasmid fragments in pUCD5 as
donors, a tri-parental mating scheme was used to move the cloning
vector into strains of X. c. pv. malvacearum by conjugation (22,69).
Into the recipient, X. c. pv. malvacearum strain N (virulent strain),
were mated cloned plasmid fragments (in pUCD5) from X. c. pv.


With the eight X. c. pv. malvacearum strains derived from
hibiscus, two gave null reactions on all of the cotton and hibiscus
cultivars tested (Table 3-4). The other strains derived from hibiscus
were pathogenic on at least two of the three hibiscus cultivars
tested, but were not pathogenic on any of the cotton cultivars. In a
separate experiment, one of the strains (M84-11) did appear to give
pathogenic symptans on Acala 44, while another strain (83-4244) gave a
hypersensitive response. In this instance the mean temperature was at
25 C, rather than 30 C.
An additional strain tested, X. c. pv. esculenti strain 84-1093,
which was a pathogen derived from okra, gave a null reaction on seven
of eight cotton cultivars and one of three hibiscus cultivars. The
other cotton or hibiscus cultivars gave hypersensitive responses
(Table 3-4).
Physiological Differentiation
All of the strains tested were mucoid on NYGA medium, produced
xanthomonadin, and hydrolyzed esculin and casein. All of the strains
tested also appeared to be positive for starch and gelatin hydrolysis,
and production of lipase (Table 3-5) Six of the seven X. c. pv.
vignicola strains, and one strains of X. c. pv. phaseoli appeared to
hydrolyze gelatin slower than the other X. campestris strains.
Cellulase activity, indicated by pitting of the solid medium, was
observed for all seven of the pathovars tested. However, not all
strains of X. c. pv. phaseoli appeared to have cellulase activity, and
X. c_. pv. alfalfae strains were weaker in this activity. None of the
strains tested exhibited pectolytic activity at pH 4.5, suggesting a


11
Table 3-1. Strains of X. campestris used in host range investigation.
Pathovar Strain
Plant Host
Location
alfalfae KS
Medicago sativa
Kansas
FL
M. sativa
Florida
cyamopsidis 13D5
Cyamopsis tetragonoloba
-
X002,X005,X016,X017
C. tetragonoloba
Arizona
glycines R-9-3
Glycine max
Brazil
1717
G. max
Africa
17915
G. max
-
S-9-8
G. max
Wisconsin
malvacearum D,M,N,0,U,V,X,Y,Z,TX84
Gossypium hirsutum
Texas
A,B,E,F,G,H
G. hirsutum
Oklahoma
Chl,Ch2
G. hirsutum
Chad
HV25
G. hirsutum
Upper Volta
Su2,Su3
G. hirsutum
Sudan
FL79
G. hirsutum
Florida
083-4244,M84-11
Hibiscus rosa-sinensis
Florida
X10,X27,X52,X102,X108
H. rosa-sinensis
Florida
phaseoli EKll,Xph25,Xpfll
Phaseolus vulgaris
Nebraska
Xpa,Xpll
P. vulgaris
Wisconsin
82-1,82-2
P. vulqaris
Florida
LB-2,SC-3B
P. vulgaris
Nebraska
XP2
P. vulgaris
New York
XP-JL
P. vulqaris
Kansas
XP-JF
P. vulgaris
Missouri
XP-DPI,B5B
P. vulgaris
-
pisi XPl
Pisum sativum
Japan
vignicola A81-331,C-1,CB5-1,
Vigna ungiuculata
Georgia
Xvl9,SN2,432,82-38
V. unguiculata
Georgia


52
Table 5-1. Strains of X. campestris pv. malvacearum from cotton used
in race-specificity investigation.
Strain
Race
Location
Source
K
HVlb
Upper Volta
L.S.Bird
N
2a
Texas
L.S.Bird
J
HV3
Upper Volta
L.S.Bird
C
HVla
Upper Volta
L.S.Bird
0
2b
Texas
L.S.Bird
L
HV7
Upper Volta
L.S.Bird
A
2bSr
Oklahoma
M. Essenberg
B
2aSr
Oklahoma
M.Essenberg
E
4-1
Oklahoma
M. Essenberg
F
laSr
Oklahoma
M.Essenberg
G
lbSr
Oklahoma
M.Essenberg
S
3-22
Oklahoma
M.Essenberg
H
4-2
Oklahoma
M.Essenberg
W
6
Texas
L.S.Bird
I
3
Oklahoma
M.Essenberg
V
3
Texas
L.S.Bird
D
2
Texas
L.S.Bird
M
15
Texas
L.S.Bird
X
18
Texas
L.S.Bird
U
1
Texas
L.S.Bird
Q
la
Oklahoma
M.Essenberg
R
lb
Oklahoma
M. Essenberg
Z
7b
Texas
L.S.Bird
Y
7a
Texas
L.S.Bird
T
18
Oklahoma
M.Essenberg
Chi
-
Chad
L.S.Bird
Ch2
-
Chad
L.S.Bird
HV25
-
Upper Volta
L.S.Bird
Su2
-
Sudan
L.S.Bird
Su3
-
Sudan
L.S.Bird
FL79
16
Florida
this study


served as useful markers for the genomic background. Plasmids in X.
campestris generally appear to be stable and have in sane instances
68
been used as epidemiological markers, such as with the recent Florida
outbreak of X. c. pv. citri (31).
However, five of the six strains with group I plasmids were more
virulent than the other strains. All of the Upper Volta strains fell
into this class, including sane North American strains. The Upper
Volta strains were derived iron recently discovered races which were
found to be virulent against all the known major genes for bacterial
blight resistance (29) The X. c_. pv. malvacearum strain J, although
an Upper Volta strain, appeared to be the exception and varied iron
the other plasmids by the loss, or rearrangement, of a 4.5 kb EcoRI
fragment found in all of the other 24 strains tested. Since the other
group I strains are highly virulent strains, and known to be lacking a
number of cultivar specific avirulence genes (33), it may be possible
that this 4.5 kb EcoRI fragment may serve sane positive function in
pathogenicity.
Nine avirulence genes have been reported cloned from X. c. pv.
malvacearum, at least sane of which are chromosamally encoded (33) .
With further testing, it is possible that plasmid-borne avirulence
genes may be uncovered. In this study, approximately 50% of the
plasmid sequences derived from an avirulent strain were tested,
leaving it still possible to uncover avirulence genes in X. campestris
pv. malvacearum.
A high level of race-specific variability has been described for
X. c_. pv. malvacearum (8) Plasmids may be involved in genome


RFLP patterns correlated well with the classification based on
pathogenicity. Although certain pathovars may need to be redefined,
this work supports and helps validate the natural taxonomic groupings
provided by the pathovar naming system.
xi


Table 6-1. Strains of X. campestris used for RFLP analysis
73
Fatixvar
Strain
Host
Location
Xantburcnas canpestris pathcvar
alfalfe
KS
hfedicago sativa
Kansas
EL
M. sativa
Florida
argemores
084-1052
Arcsnons mexicana
Elorid
begonias
084-155
Begonia sp.
Florida
carpestris
Xd
Brassica deracea (cabbage) Cklahora
084-809,084-1136
B. olercea (cabbage)
Elorida
084-720
B. deracea (br. grouts)
Florid
084-1318
B. deraoea (broccdi)
Elorida
carcfe
13
Daucus carota
Chlifamia
citri
X59,X70
dtrus sp.
Brazil
X52
dtrus sp.
Japan
X69
dtrus 3?.
ArgerrtirB
084-3401
dtrus ^o.
Florid
cyaropsidis
13D5
Cysrrccsis tetragonolcba
-
X002,X005,
C. tetragonoiabe
Arizona
X016,XD17
C. tetragonolcba
Arizona
dieffehbachiae
084-729
Anthurium sp.
Florid
068-1163
Diefferbadoia sp.
Florid
eseulenti
084-1093
Aoelmosduus esculentus
Florid
glycines
B-9-3
Glycine max
Brazil
1717
G. max
Africa
17915
G. max
-
S-9-8
G. max
Wisconsin
hederae
084-1789
Hadara helix
Florid
bdcioola
Xh66
Scaxrxm vulgare
Kansas
maculifoliigardenfe 084-6166
drcenia sp.
Florid
irelvacearum
D,M,N,0,U,V,
Qossvpium hirsutum
Tdxas
X,Y,Z,TX34
G. hirsutum
Ifexas
A,B,E,F,G,H
G. hirsutum
Cklahcma
Chl,Ch2
G. hirsutum
Gfe
Hv25
G. hirsutum
EJcrer Volta
Su2,Su3
G. hirsutum
Sdn
EL79
G. hirsutum
Elorid
mangiferaeindicae
084-116
Mangifera indica
Elorid
nigrcrnaaLans
084-1984
Arctium latee
Elorid
pelargonii
084-190,084-1370
Geranium sp.
Florid
phased!
EKil,J$h25,}££ll
Ehaseolus vulgaris
Nebraska
Xpa,Xpll
P. vulgaris
Wisconsin
82-1,82-2
P. vulgaris
Elorid
LB-2,SD-3B
P. vulgaris
Nebraska
XP2
P. vulgaris
New York
XP-JL
P. vulgaris
Kansas
XP-JF
P. vuiqaris
Missouri
XP-DPI
P. vulgaris
-
Source3
D.L.Stuteville
R.E.Stall
EPI
DPI
this study
tiis study
this study
DPI
R.E.Stall
E.L.dvsrdo
E.L.dvsrdo
E.L.d\erdo
DPI
dl.Kado
J.Mihail
J.Mihail
DPI
DPI
DPI
W.F.Efett
W.F.Etett
W.F.Ftett
W.F.Efett
EPI
L.daflin
DPI
this study
this study
M-Essaierg
L.S.Bird
L.S.Bird
L.S.Bird
this study
DPI
DPI
DPI
M. Schuster
A.W.Saettler
R.E.Stall
A.K.Vidavsr
J.A.Laurence
J.L.Ieach
this study
this study


The high level of host-range and race specificity described above
for X. campestris is observed with some other plant pathogenic
bacteria. For example, there are more than forty pathovars of
Pseudomonas syringae which are primarily distinguished by host-range
specificity (75,86). Sane P. syringae pathovars have host ranges
which are narrowly restricted to specific plant species; others have a
wider range of hosts. Strains of Erwinia chrysanthemi are currently
being considered for differentiation at the pathovar level (56) .
Strains of Agrobacterium spp. and Rhizobium spp. are not
differentiated using the pathovar system; however, host-specific
interactions analogous to those observed in X. campestris exist with
these bacteria. Species within each genus can be differentiated
biochenically and by plant inoculations. These two genera, which
cause very different symptoms, are both in the family Rhizobiaceae and
are genetically highly related (25,42) In Rhizobium spp. and
Agrobacterium spp., host range appears to be specified by the
bacterium by genes which are expressed as a positive function (32). In
several species of Rhizobium, host range function has been identified
as being plasmid borne (4). The introduction of one of these plasmids
may extend the host range of a limited host range species of
Rhizobium.
Similarly, A. tumefaciens tumor inducing (Ti) plasmids carry host
range determining genes (64) Ti plasmids from wide-host-range
strains, when moved to A. tumefaciens strains with limited host-
ranges, confer a wide host range to the recipient (10,43,48). The
pTAR plasmid, found in A. tumefaciens strains from grapevine, carries


Table 4-1. Strains of X. campestris used for plasmid analysis.
Bacterium Strain designation
(number of strains) (geographic origin) Source3
Xanthanonas campestris pathovar
alfalfae (2)
KS (Kansas);
D.L.Stuteville
FL (Florida);
R.E.Stall
argemones (1)
084-1052 (Florida);
DPI
begoniae (1)
084-155 (Florida);
DPI
campestris (4)
XC1 (Oklahoma) ;
084-720,084-809,
this study
084-1318 (Florida);
DPI
carotae (3)
G1 (Idaho),G5,G7 (California);
R.E.Stall
citri (5+)
X59 (Brazil),X62 (Japan),
X69 (Argentina), X70 (Brazil);
E.L.Civerolo
Fll (Florida);
DPI
cyamopsidis (5)
13 D5;
X002,X005,X0016,
C.I.Kado
X0017 (Arizona);
J.Mihail
dieffenbachiae (2)
084-729,084-1373 (Florida);
DPI
esculenti (1)
084-1093 (Florida);
DPI
glycines (3)
B-9-3 (Brazil),1717 (Africa),
17915;
W.F.Fett
hederae (3)
084-1789,084-3928,
251G (Florida);
DPI
holcicola (2)
Xh66 (Kansas);
L.Claflin
XH1;
this study
maculifoliigardeniae(2)
084-6006,084-6166 (Florida);
DPI
malvacearum-cotton(32)
A,B,E,F,G,H (Oklahoma);
HV25 (Upper Volta),
Chl,Ch2 (Chad),
M. Essenberg
Su2,Su3 (Sudan);
L.S.Bird
FL79 (Florida);
DPI
D,M,N,0,U,V,W,X,Y,Z,TX84 (Texas),
I,Q,R,S,T (Oklahoma),
C,J,K,L (Upper Volta);
this study
malvacearum-hibiscus(8)
XI0,X27,X52,X102,
X103,X108 (Florida);
A.R.Chase
083-4344,M84-11 (Florida);
DPI
mangiferaeindicae (1)
084-166 (Florida);
DPI
pelargonii (1)
084-190 (Florida);
DPI
phased i (13)
EK11,Xph25,Xpf11 (Nebraska);
M. Schuster
Xpa,Xpll (Wisconsin);
A.W.Saettler
82-1,82-2 (Florida);
R.E.Stall
LB-2,SC-3B (Nebraska);
A.K.Vidaver
XP2 (New York);
J.A.Laurence
XP-JL (Kansas);
J.L.Leach
XP-JF (Missouri),XP-DPI;
this study


101
46. Kado, C. I., and Liu, S. T. 1981. Rapid procedure for detection
and isolation of large and small plasmids. J. Bacteriol.
145:1365-1373.
47. Kamper, S. M., French, W. J., and deKloet, S. R. 1985. Genetic
relationships of some fastidious xylem-limited bacteria. Int. J.
Sys. Bacteriol. 35:185-188.
48. Kao, J. C., Perry, K. L., and Kado, C. I. 1982. Indoleacetic acid
complementation and its relation to host range specifying genes
on the Ti plasmid of Agrobacterium tumefaciens. Mol. Gen. Genet.
188:425-432.
49. Klement, Z. 1983. Hypersensitivity, pp. 150-177 in:
Phytopathogenic Prokaryotes, Vol. 2. M. S. Mount, and G. H. Lacy,
eds. Academic Press, New York, NY. 506 pp.
50. Lacy, G. H., and Patil, S. S. 1982. Why genetics?, pp. 221-228
in: Phytopathogenic Prokaryotes, Vol 2. M. S. Mount, and G. H.
Lacy, eds. Academic Press, Inc., New York, NY. 506 pp.
51. Laemmli, U. K. 1970. Cleavage of structural proteins during the
assembly of the head of bacteriophage T4. Nature 227:680-685.
52. Lazo, G. R. and Gabriel, D. W. 1984. Described 'races' of
Xanthcmonas campestris pv. malvacearum are mixtures. (Abstr.).
Phytopathology 74:837.
53. Lazo, G. R., and Gabriel, D. W. 1985. Use of plasmid DNAs to
differentiate pathovars of Xanthcmonas campe stris. (Abstr.).
Phytopathology 75:1320.
54. Lazo, G. R. and Gabriel, D. W. Conservation of plasmid DNA
sequences and pathovar identification of strains of Xanthcmonas
campestris. Phytopathology (in press).
55. Lazo, G. R., Gabriel, D. W., and Roffey, R. 1986. Differentiating
pathovars of Xanthcmonas campestris without pathogenicity tests.
(Abstr.). Phytopathology 76:1076.
56. Lelliott, R. A., and Dickey, R. S. 1984. Erwinia Winslow,
Broadhurst, Buchanan, Krumwiede, Rogers and Smith 1920. pp.
469-476 in: Bergy's Manual of Systematic Bacteriology, Vol. 1.
N. R. Krieg, and J. G. Holt, eds. Williams and Wilkins,
Baltimore, MD. 964 pp.
57. Leyns, F., De Cleene, M., Swing, J., and De Ley, J. 1984. The
host range of the genus Xanthcmonas. Bot. Rev. 50:308-356.
58. Li, WT.-H. 1986. Evolutionary change of restriction cleavage sites
and phylogenetic inference. Genetics 113:187-213.


GENETIC ANALYSIS OF INTRASPECIFIC VARIATION IN
PA1HOVARS OF Xanthanonas campestris
BY
GERARD RAYMOND LAZO
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
1987


dependent on constant environmental parameters, and/or so cumbersome
that no extensive evaluative tests have been performed.
28
Plasmid DNA has been identified in several pathovars of X.
campestris (13,16,30,39,46,52,53,60,80). It is relatively simple to
extract large numbers of strains and visualize their plasmids with
standardized alkaline lysis procedures (46,66). To characterize the
plasmids, restriction endonucleases are used to digest the DNA into
distinct fragments that can be separated by size, resulting in
fragment patterns visualized by agarose gel electrophoresis. To date
there have been no systematic attempts to examine the extent of
plasmid variation among a large number of strains involving a large
number of X. campestris pathovars. Our preliminary studies on
selected pathovars of X. campestris suggested that there was a
surprisingly high degree of plasmid sequence conservation within sane
pathovars (31,52,53). These studies further suggested that plasmids
of purified strains were quite stable and hence useful in
epidemiological studies (31), similar to those used to monitor the
spread of selected human pathogens (28). The purpose of this study
was to survey the extent of variation of plasmid DNAs within a large
number of X. campestris pathovars and to determine whether the plasmid
content of strains based on restriction fragment polymorphism and
Southern hybridization could be used in differentiating the pathovars
of X. campestris.
Materials and Methods
Bacterial Strains
The X. campestris strains used in this study, their pathovar
designations, geographic origin, and sources are listed in Table 4-1.


96
taxonomic validity of grouping strains by pathovar was strengthened by
the observation that, in general, strains within a pathovar fall into
a single RFLP group. Although plant tests are still necessary as a
confirmation of host pathogenicity, mutants which are no longer
pathogenic and non-pathogenic epiphytes may now be classified in one
classification system. The analyses conducted using common
microbiological techniques, including SDS-PAGE of total proteins,
revealed the relatedness of strains at the species level. Attempts to
differentiate strains into pathovars of X. campestris were not as
successful by these means, as variation was unresolvable at this
level. With the incorporation of additional plasmid and RFLP
analyses, the ability to resolve these strains into pathovars was
enhanced, even to the extent that these differences were correlated
with strains of a given host range.


69
rearrangement as plasmids in P. syringae pv. phaseolicola have been
found to integrate and excise from the chromosome causing
rearrangement (79,92). In these studies, pUFA-Hl, carrying a
chromosomally encoded avirulence gene, was found to hybridize with a
plasmid DNA from X. c. pv. malvacearum strain W. The evidence that
plasmids in X. c. pv. malvacearum may be self-mobilizing points to
their potential role in variability of the X. campestris species. It
is believed that the self-mobilizing, copper resistance plasmid
identified in X. c. pv. vesicatoria facilitated the rapid build up of
X. campestris strains resistant to field applied copper-containing
compounds (88). If avirulence genes are on self-mobilizing plasmids,
rapid shifts in pathogenic races may occur, and the appearance of new
races would be facilitated.
The cloning of the X. c_. pv. malvacearum plasmid origin of
replication may allow for the future construction of other cloning
vectors designed specifically for the study of genes in X. campestris.
Seme of the currently used broad-host-range vectors appear unstable in
X. campestris and are useful because cloned inserts integrate into the
chromosome. Although preliminary experiments with the X. campestris
ori resulted in vector rearrangement, possibly the resolved form may
be applied to the construction of future vectors.


99
24. Dye, D. W., Bradbury, J. F., Goto, M., Hayward, A. C., Lelliott,
R. A., and Schroth, M. N. 1980. International standards for
naming pathovars of phytopathogenic bacteria and a list of
pathovar names and pathotype strains. Rev. Plant Pathol.
59:153-168.
25. Dylan, T., Ielpi, L., Stanfield, S., Kashyap, L., Douglas, C.,
Yanofsky, M., Nester, E., Helinski, D. R., and Ditta, G. 1986.
Rhizobium meliloti genes required for nodule development are
related to chromosomal virulence genes in Agrobacterium
tumefaciens. Proc. Natl. Acad. Sci. USA 83:4403-4407.
26. Ercolani, G. L. 1978. Pseudomonas savastanoi and other bacteria
colonizing the surface of olive leaves in the field. J. Gen.
Microbiol. 109:245-257.
27. Essenberg, M., Cason, E. T., Jr., Hamilton, B., Brinkerhoff, L.
A., and Richardson, R. K. 1979. Single-colonies of Xanthamonas
malvacearum in susceptible and inmune cotton leaves and the local
resistance response to colonies in immune leaves. Physiol. Plant
Pathol. 15:53-68.
28. Farrar, W. E. Jr. 1983. Molecular analysis of plasmids in
epidemiologic investigation. J. Infect. Dis. 148:1-6.
29. Follin, J.-C. 1983. Races of Xanthomonas campestris pv.
malvacearum (Smith) Dye in western and cenral Africa. Cot. Fib.
Trop. 38:277-279.
30. Fujimoto, D. K., and Vidaver, A. K. 1985. Analysis of strain
variation in Xanth anonas campestris pv. phaseoli. (Abstr.) Proc.
Sixth Int. Conf. Plant Pathogenic Bact., College Park, MD.
31. Gabriel, D. W. 1985. Four plasmid DNA variants distinguished in
1984 Florida citrus canker epiphytotic. (Abstr.) Phytopathology
75:1320.
32. Gabriel, D. W. 1986. Specificity and gene function in
plant-pathogen interactions. ASM News 52:19-25.
33. Gabriel, D. W., Burges, A., and Lazo, G. R. 1986. Gene-for-gene
interactions of five cloned avirulence genes from Xanth anonas
campestris pv. malvacearum with specific resistance genes in
cotton. Proc. Natl. Acad. Sci. USA. 83:6415-6419.
Gabriel, D. W., Burges, A. R., Lazo, G. R., and Roffey, R. 1986.
Xanthomonas campestris pvs. citri, alfalfae, and phaseoli are
genetically and pathologically related. (Abstr.). Phytopathology
76:1076.
34.


ACKNOWLEDGMENTS
I wish to express my gratitude to Dr. D. W. Gabriel, chairman of
the advisory committee, for his friendship, help and encouragement
during the course of this research. Thanks are also expressed to all
personnel associated with the laboratory, who were very generous and
cooperative in supporting the research efforts which made this
presentation possible. Special thanks are extended to A. Williams who
assisted with the race-specificity studies on cotton; G. V. Minsavage
who assisted with the SDS-PAGE analysis; R. Roffey, who assisted with
the RFLP analysis; and A. Burges who assisted with various other
aspects of this study. Thanks are also expressed to Drs. R. E. Stall,
D. R. Pring, and M. J. Bassett for serving on the advisory committee
and for their support. Appreciation is also acknowledged for
financial support from the Oklahoma and Florida Agricultural
Experiment Stations, and United States Department of Agriculture grant
USDA-58-7B30-3-465. And finally, I am grateful to my loving wife,
Maria, for supporting and encouraging me throughout this degree
program, for she tolerated my late hours and comforted me in the most
difficult of times.


heat for 1 hr at 50 C. The sample was then extracted 2 times with
phenol/chloroform/isoamyl alcohol (25:24:1; phenol equilibrated to pH
7.8 with 0.1 M TRIS), and the DNA spooled out from the aqueous phase
after adding sodium acetate at 30 mM and 2 volumes 95% ethanol. This
DNA was then washed in 70% ethanol. Extracted DNA was resuspended in
TE (10 mM Tris (hydroxymethyl) aminomethane (TRIS), 1 mM disodium
ethylenediaminetetraacetate (Na2EDTA) containing 20 ug/ml DNase-free
pancreatic RNase; pH 7.6).
Agarose Gel Electrophoresis
Approximately 5 ug of DNA was digested with EcoRI or BamHI as
specified by the manufacturer (Bethesda Research Laboratories).
Digested DNA samples were run in a 20x25 cm agarose gel (0.6%; type
II, Sigma) in TRIS-acetate buffer (40 mM TRIS-acetate, 1 mM Na2EDTA;
pH 7.6) with electrophoresis of gels set at 35 V for 14-15 hrs.
Fragments were visualized by ultraviolet irradiation (302 nm) after
staining agarose gels in ethidium bromide (0.5 ug/ml). Photographs
were taken using Polaroid Type 55 (or Type 57) film and a yellow
filter (Tiffen no. 12). After the gels were photographed, the DNA was
transferred to nitrocellulose by the method of Southern as described
by Maniatis et al. (66). Restriction fragment sizes were estimated
using DNA molecular size standards of lambda phage DNA digested with
HindiII.
DNA Probes
The DNA probes used in this study were derived from a genomic
library of X. c. pv. citri strain 3401 constructed into the modified
cosnid cloning vector pUCD5B, which was the vector pUCD5 (14) with a 2


Plaanid Curing
In preliminary tests with the curing agents, it was determined that
0.3% SDS and 20 ug/ml ethidium bromide were the concentrations which
allowed growth of X. campe stris. Strains did not grow at
concentrations of 0.5% SDS or 30 ug/ml ethidium bromide. The
bacterial colonies isolated from ethidium bromide treatments appeared
phenotypically identical to the wild-type strains. Several colorless
colonies were observed with the SDS treatment; however, over a 48 hr
period, color appeared to be restored. Of 11 strains selected from
the SDS treatment, all were found to be pathogenic on Acala 44. From
plasmid analyses it appeared that 7 of 11 strains were missing the
indigenous plasmids (Fig. 5-1).
Race-Specificity Genes
Several clones containing cloned X. campestris plasmid fragments in
the vector pUCD5 were obtained and transformed into E. coli ED8767.
Based upon restriction analyses, only 3 of the 8 EcoRI fragments of
strain H, and 6 of 9 EcoRI fragments of strain N were cloned. These
clones carried 30 kb (36%) of the 84 kb strain H plasmid, and 70 kb
(88%) of 85 kb strain N plasmids. Each of the E. coli clones were
mated into X. c_ pv. malvacearum. The transconjugants were then
selected and inoculated onto the 14 cotton differentials. The results
of the inoculations are seen in Table 5-3.
None of the clones derived from plasmid DNA of X. c. pv.
malvacearum changed the expected disease reactions of X. c. pv.
malvacearum strain H. One clone of X. c. pv. malvacearum strain H,
originally thought to carry a large plasmid insert, did carry an


91
Dief fenbachia sp. both corresponded to that of X. c. pv.
dieffenbachiae. The other unknown strains tested appeared different
from one another, and did not appear similar to any of the other X.
campestris pathovars as characterized by the DNA probes. Finally,
this technique may provide a basis for the classification of
nonpathogenic and epiphytic xanthomonads. Experiments can now be
conducted to determine whether epiphytes are all basically similar or
form diverse groups, whether they are restricted in host range or if
they are nonpathogenic, but closely related to described pathovars.
Although this approach provides another classification scheme for
strains, it appears to be compatible with the currently recognized and
useful pathovar naming scheme, which relies primarily on plant-
specificity. Although certain pathovars may need to be redefined,
this work supports and helps validate the natural taxonomic groupings
provided by the pathovar naming system.


51
pathovars in X. campestris (13,16,30,39,46,52,53,80), it is of
interest to understand the function of these autonomously replicating,
covalently closed circular DNAs of the bacterial genome. The purpose
of this investigation was to analyze the plasmids in X. c. pv.
malvacearum and determine if they influenced race-specificity in
cotton.
Materials and Methods
Bacterial Strains and Host Plants
The strains of X. c_. pv. malvacearum used for this investigation
are listed in Table 5-1. All of the strains were from single colony
isolation of either mixed, or pure cultures. The cotton differentials
used included those recommended for differentiating the races of X. c.
pv. malvacearum (45); also included were six congenie cotton lines of
Gossypium hirsutum cv. Acala 44, which contained single known
resistance genes (B genes) to X. c. pv. malvacearum (7). The congenie
Acala series included cotton with resistance genes B2, B3, B5, Bg, By,
and Bn. The other host differentials were cvs. Acala 44, 1-10B, 101-
102B, 20-3, Mebane Bl, Stoneville 20, 2B-S9, and Gregg.
Plant Inoculations
Pathogenicity tests were conducted by pressure infiltration of
abaxial leaf surfaces with bacterial suspensions using a blunt end of
a syringe, and incubating the plants in 30 C growth chambers until
symptom were expressed. Symptoms development was monitored over a two
week period.
Plasmid Analysis
Cultures were grown to mid- to late-logarithmic growth phase and
extracted by either of two small-scale alkaline lysis extraction


Table 4-3. Hybridization of radiolabeled plasmid probes to total DNA of
pathovars of Xanthanonas campestris and one other Xanthanonas species.
44
Bacterium (No. tested)
N80
Probes3
N4.5 V2.3
P2.3
X. campestris pv.
alfalfae (1)
+


_
argemones (1)
-
-
-
-
begoniae (1)
-
-
-
-
campestris (1)
-
-
-
-
carotae (1)
+

-

citri (3)
+
+/-b
+/-b
+/-b
cyamopsidis (1)
+
+
+
-
dieffenbachiae (1)
-
-
-
-
esculenti (1)
-
-
-
-
cjlycines (1)
+
-
+
-
hederae (1)
+
-
-
-
holcicola (1)
+
-
-
-
maculifoliigardeniae (1)
-
-
-
-
malvacearum cotton (6)
+/-c
+/-c
+

malvacearum hibiscus (2)
-
-
-
mangiferaeindicae (1)
-
-
-
-
phaseoli (1)
+
+
-
+
poinsettiicola (1)
-
-
-
-
pruni (1)
+
+
-
-
translucens (1)
+
-
-
-
vesicatoria (1)
+
+
+
-
vignicola (1)
+
+
+
-
vitians (1)
-
-
-
-
zinniae (1)
-
-
-
-
X. albilineans (1)
-
-
-
-
3 + = hybridization observed, = no hybridization observed.
N80 = plasmid DNA derived from X. c. pv. malvacearum strain N
(about 80 kb); N4.5 = cloned EcoRI plasmid fragment (4.5 kb) from
strain N; V2.3 = cloned EcoRI plasmid fragment (2.3 kb) iron X. c.
pv. vignicola strain SN2; P2.3 = cloned EcoRI plasmid fragment
(2.3 kb) from X. c. pv. phaseoli strain XP2
b Strain FL-11 was negative, only plasmid DNA used.
c Strain S2 was negative.


60
Figure 5-1. Plasmid curing of X. campestris pv. malvacearum with SDS
treatment. Lanes A-L are plasmid extractions of strains which were
exposed to 0.3% SDS. Lanes A, E, I, and J appear to contain the
indigenous plasmid. Lanes B-D, F-H, and K-L appear to be missing the
indigenous plasmid.


6-3 Genomic DNA of strains of X. campestris pvs. phaseoli,
alfalfae, and campestris digested with restriction
endonuclease EcoRI and hybridized with probe XCTl 81
6-4 Genomic DNA of strains of X. campestris pvs. phaseoli,
alfalfae, and campestris digested with restriction
endonuclease EcoRI and hybridized with probe XCTll 84
6-5 Genomic DNA of strains from different pathovars of
X. campestris digested with restriction endonuclease EcoRI
and hybridized with probe XCTl 85
viii


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 ANALYSIS OF INTRASPECIFIC VARIATION IN
PATHOVARS OF Xanthanonas campestris
By
Gerard Raymond Lazo
May, 1987
Chairman: Dean W. Gabriel
Major Department: Plant Pathology
Xanthomonas campestris is always found in association with
plants. Those strains of X. campestris which are known to be
pathogenic are differentiated into over 125 pathovars (pathogenic
variants), on the basis of limited pathogenicity tests. Sane members
of a pathovar may have a broader host range than others, since
exhaustive pathogenicity testing is impractical. Other than these
tests, there is no definitive means to classify an unknown X.
campestris isolate. Those strains of X. campestris which are not
pathogenic are unclassifiable by the pathovar system, yet they may
exhibit similar host range specificity to those which are pathogenic.
This work was conducted in an effort to better understand the
variability of X. campestris.
Over 115 strains of X. campestris were examined for plasmid
content and restriction endonuclease profiles. Of the 26 pathovars
examined, only 13 were found to contain plasmids. Restriction
ix


xml version 1.0 encoding UTF-8
REPORT xmlns http:www.fcla.edudlsmddaitss xmlns:xsi http:www.w3.org2001XMLSchema-instance xsi:schemaLocation http:www.fcla.edudlsmddaitssdaitssReport.xsd
INGEST IEID ECFF40GG0_XBTSD5 INGEST_TIME 2014-10-13T20:02:19Z PACKAGE AA00025833_00001
AGREEMENT_INFO ACCOUNT UF PROJECT UFDC
FILES


17
Table 3-3. Host range observed for pathovars of Xanthanonas campestris
pathogenic to legume host plants.
Pathovar
Inoculat
ion reaction
on host plants9
G.max
M.sativa
P.sativum
P.vulgaris V.unguiculata
alfalfaeb
+
+c
+ -d
glycines
+
-
nt
+
phaseoli
_e
-
nt^
+
pisi
-
-
+c
-
vignicola
_e
-
nt
+9 +
cyamopsidish

nt

a + = compatible, = incompatible, nt = not tested. Host plants were
Glycines max (soybean) cv. Evans; Medicago sativa (alfalfa) cv.
FL-77; Pisum sativum (pea) cultivar not known; Phaseolus vulgaris
(bean) cv. California Light Red; and Vigna unguiculata (cowpea) cv.
California Blackeye #5.
k Other susceptible hosts reported are Trigonella and Melilotus (6).
c Reported as susceptible, but not tested,
d Reported as positive in the literature (6).
e Reaction appears negative for most strains, but occasional limited
water soaking is evident.
f There is a report of pathovar phaseoli on Pisum lunatus (6).
9 Symptoms are similar to those seen in V. unguiculata.
h The natural host belongs to the genus Cyamopsis.


85
12.2
11.2
7.9
5.6
5.1
3.2
2.2
1A
Figure 6-5. Genomic DNA of strains of X. campestris digested with the
restriction endonuclease EcoRI and probed with cosmid clone XCTl. Lane
A, probe XCTl; lane B, X. c. pv. alfalfae FL; lane C, X. c. pv.
begonlae 084-155; lane D, X. c. pv. campestris 084-809; lane E, X. c.
pv. carotae 13; lane F, X. c. pv. cyamopsidis X002; lane G, X. c. pv.
dieffenbachiae 068-1163; lane H, X. c. pv. glycines S-9-3; lane I, X.
c. pv. holcicola Xh66; lane J, X. c_. pv. maculifoliigardeniae 084-
6166; lane K, X. c. pv. malvacearum FL79; lane L, X. c. pv. pelargonii
084-190; lane M, X. c. pv. phased i var. fuscans SC-3B; lane N, X. c_.
pv. pisi XPl; lane 0, X. c. pv. translucens X1105; lane P, X. c. pv.
vesicatoria 75-3; lane Q, X. c_. pv. vignicola SN2; lane R, X. c. pv.
vitians ICPB164; lane S, X. c. pv. zinniae 084-1944; lane T, probe
XCTll.


81
ABCDE FGH I J KLMNOPQRST
Figure 6-3. Genomic DNA of strains of X. campestris digested with the
restriction endonuclease EcoRI and probed with cosmid clone XCTl. Lane
A, probe XCTl; lanes B-K, X. c. pv. phaseoli; lane B, JL; lane C, LB-
2; lane D, Xph25; lane E, EK11; lane F, 82-1; lane G, 82-2; lane K,
Xpa; lane I, XP2; lane J, XP2-1; lane K, JF; lanes L-M, X. c. pv.
phaseoli var. fuscans; lane L, SC-3B; lane M, Xpfll; lanes N-0, X. c.
pv. alfalfae; lane N, KS; lane 0, FL; lanes P-R, X. c_. pv. campe stris;
lane P, XC1 (cabbage) ; lane Q, 084-1318 (broccoli) ; lane R, 084-720
(brussels sprouts); lane S, unknown X. c. G65; lane T, probe XCTll.


endonuclease digested plasmid DNA from strains within a given
plasmid-containing pathovar gave surprisingly similar, but not always
identical, digestion profiles by agarose gel electrophoresis. All 60
strains tested of X. campestris pvs. glycines, malvacearum, phaseoli,
and vignicola could be accurately identified by pathovar iron
determination of the restriction fragment profile and/or by Southern
hybridizations of that profile with a selected plasmid DNA probe. In
no instance was the same plasmid profile seen in more than one
pathovar. The apparent stability of the plasmids provides a natural
genetic marker that can be strain specific and perhaps useful in
epidemiological investigations.
Cloned DNA fragments, derived from a cosmid library of a Florida
isolate of X. campestris pv. citri strain 3041, were used to detect
restriction fragment length polymorphism (RFLP) of total DNA from 87
strains of X. campestris, comprising 23 different pathovars.
Autoradiographs of Southern transfers of genomic DNA probed with
cosmid-sized clones revealed hybridization profiles which appeared to
be highly conserved. The hybridization patterns observed between
different pathovars suggested that some DNA fragments were conserved
at the species level, others were conserved at the pathovar level, and
still others were variable. The degree of variability appeared to
depend on the DNA probe used. By using more than one DNA probe, or by
digesting the genomic DNAs with different restriction endonucleases,
all of the strains of X. campestris described as belonging to a given
pathovar could be differentiated. All strains of X. campestris were
readily grouped by RFLP phenotypes, and the classification based on
x


41
Figure 4-4. Plasmid DNAs from strains of Xanthomonas campestris pv.
malvacearum (Xcm) digested with restriction endonuclease EcoRI (left)
and autoradiograph of plasmid DNAs probed with a clone containing a
4.5 kb EcoRI plasmid fragment from Xcm strain N (right). Lanes shown
above contain: A, lambda HindIII B, Xcm H; C, Xcm W; D, Xcm V; E, Xcm
X; F, Xcm Q; G, Xcm Y; H, Xcm L; I, Xcm N; J, Xcm HV25; K, Xan Ch2; L,
Xcm Su2; M, Xcm FL79; and N, Xcm TX84.


47
Plasmid homology between X. c. phaseoli and X. c. phaseoli var.
fuscans was revealed by hybridization that was not apparent by
restriction digest patterns. There were distinct differences in
plasmid digestion patterns of X. c. phaseoli var. fuscans in
comparison to typical X. c. phaseoli strains. The fact that both X.
c_. phaseoli and X. c. phaseoli var. fuscans have an identical host
range suggests that the homologous plasmid DNA regions that are
conserved within sane pathovars may encode host range specificity
functions. Such functions have been described on plasmids within the
Rhizobiaceae (62,64) This is a testable hypothesis that needs
further experimental support.
In X. c. malvacearum, plasmids were found in all 32 strains
isolated from cotton, and no plasmids were found in the 8 strains
isolated from hibiscus. Cotton and hibiscus each belong to the same
plant family (Malvaceae), hence the X. c_. malvacearum designation.
Atypical symptoms of cotton blight could be artificially produced in
cotton using syringe inoculations with hibiscus strains, and in
hibiscus using cotton strains. The ability of these strains to be
pathogenic on both hosts under natural conditions has not been clearly
established. Plasmid DNAs from cotton strains cross-hybridized with
one another in Southern analyses, indicating extensive homology was
evident between the plasmids. As with X. c_. phaseoli, the cotton
strains of X. c_. malvacearum appeared to carry highly conserved
plasmid DNA sequences (similar in restriction digest sizes and by
Southern analyses) which are unique to strains which have a host range
on cotton. Plasmid DNA from the cotton strain (N), when radiolabeled,


56
Table 5-2. Plasmid groupings and cotton plant reactions to strains of X.
campestris pv. malvacearum.
Inoculation reaction of cotton^
A
B
C
D
E
F
G
H
X
J
K
L
M
N
0
p
Q
R
s
T
U
K
HVlb
I
80.7
2
+
+
+
+
+
4-
+
+
4-
+
+
+
+
+
14
14
N
2a
I
84.8
2
+
+
+
+
V
+
V
+
+
+
+
+
+
+
12
14
J
HV3
la
59.5
1
+
-
-
-
-
+
V
-
-
-
+
-
-
-
3
4
n
HVla
lb
59.0
1
+
+
+
V
V
V
+
+
V
V
+
V
V
+
7
14
0
2b
lb
59.0
1
+
-
V
-
V
+
V
+
V
V
+
V
V
V
4
12
L
HV7
Ic
57.9
1
+
-
-
-
V
+
+
V
V
V
+
V
V
V
4
11
A
2bSr
II
82.2
1
+

4-


+





V


3
4
B
2aSr
II
83.3
1
+

+

-
+
-




V


3
4
E
4-1
II
83.3
1
+
-
-
-
-
-
V
-
-
-
-
-
-
-
1
2
F
laSr
II
83.3
1
+

+
-
-
+

-

-

V


3
4
G
lbSr
II
83.3
1
+
-
+

-
+
-
-
-
-
-
V
-
-
3
4
S
3-22
II
83.3
1
+
1
1
H
4-2
II
80.4
1
+
1
1
W
6
Ilia
88.7
1
4*
-
-
V
-
-
-
-
-
-
-
-
-
-
1
2
I
3
Ilia
88.4
1
+
-
-
-
V
-
V
-
-
-
-
-
-
V
1
4
V
3
mb
87.0
1
+
-
-
-
V
V
V
-
-
-
V
-
-
-
1
5
D
2
IVa
94.2
1
+
-
+
-
-
+
-
-
-
-
V
+
-
-
4
5
M
15
IVb
93.2
1
+
-
+
-
-
+
V
-
-
-
-
V
-
-
3
5
X
18
V
57.5
1
+
-
V
-
V
V
+
-
-
-
V
V
V
V
2
9
u
1
V
62.9
1
+
-
V
-
V
+
V
V
-
-
V
V
V
V
2
10
Q
la
VI
100.4
2
+
1
1
R
lb
VI
91.6
2
+
-
-
V
V
-
-
-
-
-
V
-
-

1
4
Z
7b
VII
93.0
1
+
-
V
-
4-
V
V
V
-
-
V
V
V
V
2
10
Y
7a
VII
98.0
1
+
-
-
-
V
-
V
-
-
-
-
-
-
-
1
3
T
18
*
1
+

+
-
-
+
-
-
-
-
-
+
-

4
4
A = Strain designation. P
B = Original strain, or race,
designation. Q
C = EcoRI plasmid group.
D = Kilobase pairs of plasmid DNA. R
E = Number of plasmids. S
F = Cultivar Acala 44.
G = Cultivar Acala B2. T
H = Cultivar Acala B3.
I = Cultivar Acala B5.
J = Cultivar Acala Bg. U
K = Cultivar Acala B7.
L = Cultivar Acala Bj^.
M = Cultivar 1-10B BIN
(+ polygenes) .
N = Cultivar 101-102B (B2B3
+ polygenes).
0 = Cultivar 20-3 (BN + polygenes).
= Cultivar Mebane B1 (B2 +
polygenes).
= Cultivar Stoneville 20 (Bg
+ polygenes).
= Cultivar Polygenes (2B-S9)
= Cultivar Gregg (resistance
genes unknown).
= Sum of cultivars which gave 4
out of 4 compatible reactions
(+)
= Total of cultivars which gave
at least 1 compatible reaction
(+)
= Plant reactions: +, compatible
reaction; -, incompatible
reaction; v, reaction variable
over 4 replications.
V


14
Table 3-2. Legume plant reactions to inoculation with pathovars of X.
campestris.
Inoculation
Strain
Host Reaction^-
bean
cowpea
soybean
alfalfa
X.campestris
pv.
phaseoli
82-1
+B
C
_D
0E
Xpa
+
-
-
0
Xpfll
4-
-
-
0
XP-JF
+
-
-
0
EKll
+
-
-
0
XP2
+
_F
__
0
B5Bg
-
-
-
0
vignicola
CB5-1
_H
+ 1
0
Xvl9
-
+
-
0
432
-
+
-
0
A81-331
-
+
-
0
C-l
-
+
-
0
82-38
+/-K
+

0
SN2
+/-
+
-
0
glycines
B-9-3
+L
_C
+M
0
1717
+
-
+
0
17915
+
-
+
0
S-9-8g
-
-
-
0
alfalfae
FL
+L
_c
_D
+N
malvacearum
N
_F
0
_F
0
control
0
0
0
0
A = + is compatible, is incom
patible, +/- is intermediate,
and 0 is a null reaction.
B = compatible lesions were
watersoaked and appeared to
be spreading.
C = dry necrotic lesion with wine
red reaction.
D = dry necrotic lesion with
chlorosis.
E = no reaction seen with spray
inoculation.
F = slight tissue discoloration
at inoculation site.
G = strain appeared non-
pa thogenic.
H = dry necrotic lesion with
slight watersoaking at
periphery of inoculation site.
I = dry necrotic lesion with
shothole effect.
J = dry necrotic lesion.
K = as described for H, but
slight shothole effect present.
L = watersoaked lesion.
M = watersoaked chlorotic lesion.
N = watersoaked leaf spots.


Copyright 1987
by
Gerard Raymond Lazo


21
Table 3-5. Physiological reactions of strains of X. campestris.
Physiological Test Medium3
Pathovar Starch
Gelatin
Cellulose
Na
4.
polypectate
5 7.0 8.5
Pectin
5.0
Lecithin0
alfalfae
KS
+
+
w
-
-
-
-
+
FL
+
+
w
-
-
-
-
+
cyamopsidis
13D5
+
+
+
-
+
+
-
+
glycines
B-9-3
+
+
+
-
+
+
-
+
1717
+
+
+
-
+
+
-
+
17915
+
+
+
-
+
+
-
+
S-9-8
+
+
+
-
+
+
-
+
malvacearum
H
+
+
+
-
-
-
-
-
N
+
+
+
-
-
-
-
-
phased i
EK11
+
+
-
-
-
-
-
+
Xph25
+
+
+
-
-
-
-
+
Xpfll
+
+
-
-
-
-
-
Xpa
+
w
-
-
-
-
-
+
Xpll
+
+
-
-
-
-
-
+
82-1
+
+
-
-
-
-
-
+
82-2
+
+
-
-
-
-
-
+
LB-2
+
+
-
-
-
-
-
+
SC-3B
+
+
+
-
+
+
-
-
XP2
+
+
-
-
-
-
-
+
XP-JL
+
+
-
-
-
-
-
+
XP-JF
T
+
-
-
-
-
-
-L
XP-DPI
+
+
-
-
-
-
-
+
pisi
XP1
+
+
+
-
+
+
-
+
vignicola
A81-331
+
-f
+
-
+
+
-
+
C-l
+
w
+
-
+
+
-
+
CB5-1
+
w
+
-
+
+
-
+
Xvl9
+
w
4.
-
+
+
-
+
SN2
+
w
+
-
+
+
-
+
432
+
w
+
-
+
+
-
+
82-38
+
w
+

+
+
_
+
a All strains were mucoid, esculin positive, milk proteolytic, and
lipase positive. + = positive, = negative, and w = weak reaction.
k A lecithinase positive reaction was observed as a clearing zone around
the colony plus precipitation in the medium.


39
A B C D
Figure 4-3. Plasmid DNAs from strains of X. campestris pv. citri
digested with restriction endonuclease EcoRI. Lane A, strain Fll
(Florida type) ; lane B, strain X59 (A type) ; lane C; strain X62 (A
type); and lane D, strain X69 (B type). Sizes shown in kilobase pairs.


the lesion being slightly chlorotic in soybean. The incompatible
response in cowpea appeared as wine red in color like that seen with
X. c. pv. phased i.
From spray inoculations on alfalfa, only X. c. pv. alfalfa,
resulted in a pathogenic response. After 7 days, small water-soaked
leaf spots which later turned necrotic appeared. X. c. pv. alfalfa
was also pathogenic on kidney bean, and appeared so slightly on
soybean. There were small water-soaked spots which occurred at the
inoculation sites on soybean. The wine red incompatible response was
observed on cowpea.
A strain of X. c. pv. malvacearum, which was not a pathogen of
legumes, resulted in only a slight tissue discoloration at the
inoculation site on bean and soybean. A null reaction, as observed
with the water-only control, was observed on cowpea. The water-only
control also resulted in a null reaction on the other hosts tested. A
summary of the plant reactions observed for the inoculation
experiments is given in Table 3-3.
In separate inoculation tests, a strain of X. c. pv. cyamopsidis
was not found pathogenic on alfalfa, kidney bean, cowpea, or soybean
and a X. c_. pv. pisi strain was not found pathogenic on kidney bean.
Overall, most of the reactions observed conformed to those reported in
the literature, with some exceptions. Variation was evident among
strains of a given pathovar in addition to that between pathovars.
The X. c_. pv. alfalfae strains appeared to have overlapping host
ranges, which extended to bean and pea in addition to alfalfa. The
kidney bean cultivar, California Light Red, appeared susceptible to a


avirulence gene. Transconjugants of this clone, pUFA-Hl, in strain N
caused incompatibility on the Acala line (Table 5-3). Upon further
61
examination of the pUFA-Hl clone by Southern hybridization analysis,
it appeared that the cloned insert was not of plasmid origin. The
radiolabeled clone did not hybridize to Southern transfers of plasmid
DNA derived from strain H, or 16 other strains of X. c. pv.
malvacearum. However, this 4.9 kb cloned insert of pUFA-Hl did
hybridize to a 23 kb EcoRI fragment of the plasmid found in strain W.
Additionally, the radiolabeled pUFA-Hl clone hybridized to a 4.9 kb
EcoRI chromosomal fragment of 18 strains of X. c_. pv. malvacearum, and
also to another clone, pUFA-702 (33). pUFA-702 was independently
derived and appeared to encode gene-for-gene avirulence on Acala BN.
Thus, it was concluded that the pUFA-Hl clone was of chromosomal
origin.
Plasnid Origin of Replication and Mobilization
Seven of the eight EcoRI fragments of the single 59.5 kb plasmid
found in X. c_. pv. malvacearum strain X were cloned into pUCD5 (Table
5-4) All of the fragments were successfully mobilized into a
spectinomycin resistant strain of X. c. pv. malvacearum strain N (N-
SPR) in the presence of the helper plasmid, pRK2013, using MOPS
minimal medium selection. In general, it appeared that the larger
cloned fragments transferred at a slightly lower rate than the smaller
cloned fragments, however the transfer rate was not quantified. One
of these clones, pLXF, carrying a 4.7 kb fragment, was found to encode
plasmid transfer function (tra), missing on the vector pUCD5 (14) .
That is, pXLF could be mated into X. campestris without aid of the


DNA Probes
Two cosmid DNA clones, XCT1 and XCT11 were randomly selected from
a gencmic library of strain 3401 for use as DNA probes. XCT1 carried
a 30 kb insert, and XCTll carried a 37 kb insert, as determined by
adding up the cloned DNA inserts from restriction endonuclease
digests. There were no detectable plasmids in strain 3401, thus the
cosmid clones are assumed to contain chromosomal fragments. Assuming
a 3333 kb genome for X. campestris (47), each cosmid probe represents
approximately 1% of the total genome.
DNA Hybridization
Autoradiographs of Southern blots hybridized against either of
the two DNA prcbes revealed conserved DNA fragments within each
pathovar. For example, in figure 6-3 (lanes B K) are shown the EFLP
pattern from 10 different strains of X. c. pv. phaseoli from different
geographic locations. At least five EcoRI cut DNA fragments which
hybridized to XCT1 appeared to be conserved in this pathovar. In
lanes L and M are the RFLP patterns of two strains of X. c. pv.
phaseoli var. fuscans. These X. c. pv. phaseoli var. fuscans strains
also attack beans (and hence have the pv. phaseoli designation), but
are biochemically distinct (6) They produce a dark, melanin-like
pigment in nutrient media and in these tests are clearly genetically
distinguishable from the other X. c. pv. phaseoli strains. Conserved
DNA fragments were also seen for X. c. pv. alfalfae (Fig. 6-3, lanes N
and 0) in which six of the up to 15 fragments appeared identical.
Likewise, in four different strains of X. c. pv. campestris (Fig. 6-3,
lanes P Q, Fig. 6-5, lane D) isolated from three different crucifer


poinsettiicola, translucens, and zinniae. Plasmids were found insome,
but not all strains of X. campestris pathovars campestris and
hederae. Similarly, plasmids were found in all type strains of X. c.
pv. citri (A, B, and C types); this is in agreement with a previous
plasmid study of this pathovar (13). However, not in all strains of X.
campestris isolated from leaf spots of citrus in Florida contained
plasmids.
It appeared that plasmid-containing strains of X. campestris
carried from one to three plasmids based on electrophoresis of
extracted plasmid DNA. For example, a majority of the
plasmid-containing X. c. malvacearum strains contained only one
plasmid, but seme carried two or more. When plasmid-containing
strains of X. campestris were purified by repeated single colony
isolation, the plasmid DNA content appeared to be stable. However,
variation was present in bacterial stocks which were known to have
been serially transferred in agar medium over a period of years.
Restriction Endonuclease Profiles
Plasmid profiles for X. campestris were variable in over 60
different strains tested. Plasmids were placed into classes based on
the variability of restriction endonuclease (EcoRI) digestion profiles
on agarose gels. When a different restriction endonuclease (BamHI)
was used, the plasmid profiles were placed into the same classes. In
all cases, strains which belonged to the same plasmid class also
belonged to the same pathovar. There was obvious variability within
classes, but there also appeared to be conservation of some DNA
fragments of identical sizes (Fig. 4-1). Undigested plasmids were not


ntalvacearum strain H (avirulent strain). As a control, plasmids
fragments from strain N were also moved into strain H. The helper
plasmid used for mobilization of the plasmids from E. coli into X.
campestris were pSa322 or pRK2013 (22,53). The donor and helper
strains were grown overnight at 37 C in LB broth with appropriate
antibiotic selection pressure. The antibiotic was washed from the
cells prior to mating by centrifuging and resuspending the cells in
0.7% NaCl. The X. campestris recipients were grown overnight at 30 C
in MOPS minimal medium broth (69). Donors and helpers at a
concentration of lO? cells and recipients at 10^ cells were mixed in a
small volume onto sterile 25 mm cellulose-acetate filter membranes and
placed onto peptone-glycerol medium for 6 hours. The membranes were
then transferred to MOPS minimal medium with 25 ug/ml kanamycin and
incubated for 72 hours at 30 C. Bacterial growth from filters was
diluted in 0.7% NaCl and plated for single colony isolation on MOPS
minimal medium with 25 ug/ml kanamycin. Colonies were selected and
inoculated on the Acala cotton differentials containing known
resistance genes.
Plasmid Origin of Replication and Mobilization
An assay was included to determine if X. cn. pv. malvacearum
contained self-mobilizing plasmids. The E. coli clones containing
cloned plasmid DNA fragments from X. c. pv. malvacearum strain X were
mated into spectinomycin resistant X. c. pv. malvacearum strain N in
the presence, or absence, of the E. coli helper plasmid strain with
pRK2013. Transconjugants were selected on MOPS minimal medium with
kanamycin (25 ug/ml), or directly on POPS (33) containing kanamycin
(30 ug/ml) and spectinomycin (100 ug/ml).


polypectate was used as the carbon source. The pH 5 medium included
only pectin as the sole carbon source. These tests were each repeated
once.
Polyacrylamide Gel Electrophoresis
Bacteria were grown overnight at 30 C in a peptone-glycerol
broth. Cells were adjusted to a 0.3 optical density (ODggonm)' and
1.5 mis of cells were collected by centrifugation, washed once in
water, and resuspended in 50 ul 10% sorbitol. Then, 50 ul
solubilization buffer (90 mM Tris (pH 6.8), 20% glycerol, 4% SDS, 10%
B-mercaptoethanol, and .002% bromophenol blue) was added to the
suspension and the mixture was boiled for 5 minutes. Approximately 10
mg protein (10 ul solution) was added to a 10% resolving acrylamide
gel (51), and samples were electrophoresed in a 20 cm x 17 cm x 1.5 mm
vertical gel unit at 10 V/cm and 15 C until the dye reached the bottom
of the gel. Biorad low-range SDS-PAGE standards were used as
molecular weight markers. Gels were stained with 0.1% Cocmassie blue
in 42% methanol and 17% glacial acetic acid. The gel was destained in
30% methanol and 10% glacial acetic acid. Electrophoresis was only
performed once. A photograph of the gel was taken using Polaroid type
55 film.
Results
Pathogenicity Tests
All of the strains of X. c_. pv. phaseoli, with one exception,
gave the expected disease reactions on the legume hosts tested. The
exception, strain B5B, was found to be non-pathogenic to all four
hosts tested. The remaining six strains were pathogenic on kidney
bean (Table 3-2), giving watersoaked lesions on inoculated leaves


CHAPTER FOUR
CONSERVATION OF PLASMID DNA SEQUENCES AND
PATHOVAR IDENTIFICATION OF STRAINS OF XANTHOMONAS CAMPESTRI^
Introduction
More than 125 different pathovars of Xanthanonas campestris
(Pammel 1895) Dowson 1939 are currently recognized (6,24), and the
primary means for differentiating them is by inoculation of the plant
host(s) of that pathovar. It would be a difficult task to inoculate
every plant that could serve as a host to an X. campestris isolate,
and therefore the potential host range of a given isolate is unknown.
Most often, the pathovar name assigned to a strain of X. campestris is
determined by the host it was isolated frcm. Such designations may be
artifactual since the primary host may be different from the one the
strain was isolated from; sane X. campestris pathovars are known to be
pathogenic on more than one host. Epiphytes cannot be classified in
this pathovar identification system. Possible taxonomic relationships
among pathovars are also elusive. It would be helpful if alternative
means to differentiate among X. campestris pathovars were available.
Seme suggested approaches for differentiating X. campestris pathovars
have included serology (1,94), membrane protein profiles (70),
phage-typing (37), and gas chromatography of fatty acids (65,81).
These approaches suffer because they are often strain specific,
This chapter
Phytopathology.
contains copyrighted material from the journal
It is reprinted here with permission of the publisher.
27


102
59. Liew, K. W., and Alvarez, A. M. 1981. Phage typing and lysotype
distribution of Xanthomonas campestris. Phytopathology
71:274-275.
60. Lin, B. and Chen, S. 1978. Multi-plasmid in Xanthomonas
manihotis (Abstr.). Third Int. Congress Plant Pathol., Mnchen,
Germany.
61. Litt, M., and White, R. L. 1985. A highly polymorphic locus in
human DNA revealed by cosmid-derived probes. Proc. Natl. Acad.
Sci. USA 82:6206-6210.
62. Long, S. R., Buikema, W., and Ausebel, F. M. 1982. Cloning of
Rhizobium meliloti nodulation genes by direct complementation of
Nod mutants. Nature 298:485-488.
63. Loper, J. E. 1986. Role of fluorescent siderophore production in
biocontrol of Pythium ultimum by Pseudomonas fluorescens strain
3551. (Abstr.). Phytopathology 76:1069.
64. Loper, J. E., and Kado, C. I. 1979. Host range conferred by the
virulence-specifying plasmid of Agrobacterium tumefaciens. J.
Bacteriol. 139:591-596.
65. Maas, J. L., Finney, M. M., Civerolo, E. L., and Sasser, M. 1985.
Association of an unusual strain of Xanthanonas campestris with
apple. Phytopathology 75:438-445.
66. Maniatis, T., Fritsch, E. F., and Sambrook, J. 1982. Molecular
Cloning: A Laboratory Manual. Cold Spring Harbor Laboratory, Cold
Spring Harbor, N.Y. 545 pp.
67. McClean, P. E. and Hanson, M. R. 1986. Mitochondrial DNA
sequence divergence among Lycopersicon and related Solanum
species. Genetics 112:649-667.
68. McMaster, G. K., Samulski, R. J., Stein, J. L., and Stein, G. S.
1980. Rapid perification of covalently closed circular DNAs of
bacterial plasmids and animal tumor viruses. Anal. Biochem.
109:47-54.
69. McNally, K., and Gabriel, D. W7. 1984. Useful minimal culture
media for Xanthomonas campestris pv. malvacearum. (Abstr.)
Phytopathology 74:875.
70. Minsavage, G. V., and Schaad, N. W. 1983. Characterization of
membrane proteins of Xanthomonas campestris pv. campestris.
Phytopathology 73:747-755.
71. Mulrean, E. N., and Schroth, M. N. 1982. Ecology of Xanth anonas
campestris pv. juglandis on Persian (English) walnuts.
Phytopathology 72:434-438.


Figure 6-1. Genomic DNA of strains of X. campestris digested with the
restriction endonuclease EcoRI. Lane A, probe XCT1; lanes B-K, X. c.
pv. phaseoli; lane B, JL; lane C, LB-2; lane D, Xph25; lane E, EKll;
lane F, 82-1; lane G, 82-2; lane H, Xpa; lane I, XP2; lane J, XP2-1;
lane K, JF; lanes L-M, X. c. pv. phaseoli var. fuscans; lane L, SC-3B;
lane M, Xpfll; lanes N-0, X. c. pv. alfalfae; lane N, KS; lane 0,
FL;lanes P-R, X. c_. pv. campestris; lane P, XC1 (cabbage); lane Q,
084-1318 (broccoli); lane R, 084-720 (brussels sprouts); lane S,
unknown X. c. G65; lane T, probe XCTll.


probes derived iron plasmid DNA of X. campestris pathovars were either
cloned restriction digested DNA fragments of plasmid DNA in the cosmid
vector pUCD5, or of the complete X. campestris plasmid. DNA probes
were labeled in vitro with use of a nick translation kit (Bethesda
Research Laboratories) using 32P-deoxycytidine triphosphate and
hybridized against DNA bound to nitrocellulose membranes. The
membranes were pre-hybridized and hybridized in plastic bags at 68 C.
The pre-hybridization and hybridization solutions were those described
by Maniatis et al. Following hybridization, membranes were washed once
in 2X SSC, 0.5% SDS and washed once in 2X SSC, 0.1% SDS at ambient
temperature, and washed two times in 0.1X SSC, 0.5% SDS at 68 C as
described by Maniatis et al. (66) for stringent conditions. The
membranes were then air dried and exposed at -80 C to X-ray film
(Kodak X-Ctnat AR) using intensifier screens. Similar methods were
used to probe DNA transferred to nitrocellulose using a dot-blot
manifold (Schleicher and Schuell Inc., Keene, NH) All hybridization
experiments were repeated at least once.
Results
Detection of Plasmid DNA
Indigenous, cryptic plasmids were detected in all strains of the
following X. campestris pathovars: cyamopsidis, dieffenbachiae,
glycines, malvacearum (cotton), pelargonii, phaseoli, pruni,
vesicatoria, vignicola, and vitians (Table 4-2). Plasmids were not
detected in any strains of X. campestris pathovars alfalfae,
argemones begoniae carotae, esculenti, holcicola,
maculifoliigardeniae, malvacearum (hibiscus), manqiferaeindicae, pisi


67
Figure 5-4. Plasmid DNA of X. campestris pv. malvacearum strain X
digested with restriction endonucleases (left) and hybridized to the
plasmid probe pXD-1 (right). Lanes A and I, lamda HinduI; lanes B-H,
plasmid DNA from X. campestris pv. malvacearum strain X digested with
BamHI (lane B), Boll (lane C), BcoRI (lane D) Hindi11 (lane E), Kpnl
(lane F), PstI (lane G), and Sail (lane H). The 5.0 kb EcoRI fragment
seen in lane D is the fragment thought to contain the origin of
replication (ori).


83
Table 6-2. Sizes of DNA fragments from Xanthanonas campestris genomic
digests (EcoRI) which hybridized to the XCT1 DNA probe. See Figure 6-
3.
A
12.2,
11.2,
7.9, 5.6, 5.1, 3.2, 2.2,
1.6,
1.0
B
14.1,
13.3,
10.4, 7.6, 6.4, 4.6, 4.3,
4.0,
3.7,
3.3,
3.1
C
14.4,
13.3,
10.4, 7.6, 6.4, 4.6, 4.3,
4.0,
3.7,
3.3,
3.1
D
14.1,
13.1,
10.4, 7.5, 6.4, 5.2, 4.6,
4.3,
4.0,
3.7,
3.1
E
13.9,
11.6,
10.4, 8.9, 5.2, 5.0, 4.6,
4.3,
4.0,
3.7,
3.1
F
13.7, 13.1,
1.9
11.6,
10.4,
8.9,
5.2,
5.0,
4.6,
4.3, 4.0,
3.7, 3.1,
G
13.7, 13.1,
11.6,
10.4,
8.9,
5.2,
5.0,
4.6,
4.3, 4.0,
3.7, 3.1,
1.9
H
13.1,
10.4,
7.5,
5.2,
5.0,
4.6,
4.3,
4.0,
3.7,
3.1,
1.9
I
13.1,
10.4,
7.5,
5.2,
5.0,
4.6,
4.3,
4.0,
3.7,
3.1,
1.9
J
13.1,
10.4,
7.5,
5.2,
5.0,
4.6,
4.3,
4.0,
3.7,
3.1,
1.9
K 13.1, 11.6, 10.4, 8.9, 5.2, 5.0, 4.6, 4.3, 4.0, 3.7, 3.1
L 11.9, 8.4, 7.1, 6.7, 4.5
M 17.0, 15.3, 10.0, 8.4, 4.5
N
19.7, 19.1,
15.5,
14.9,
12.9, 12.1, 8.0, 4.6, 3.8, 3.1,
2.7,
1.7
0
19.4, 18.0,
3.4, 3.1
14.7,
13.7,
13.1, 11.7, 9.7, 7.3, 6.2, 5.2,
4.6,
3.7
P
20.3, 15.3,
10.9,
10.5,
10.2, 9.3, 8.4, 6.9, 5.9, 5.2,
4.4,
3.9
2.6, 2.2, 1,
.9
Q
12.9, 10.8,
K
in

o
ii
9.1,
8.4, 7.7, 7.2, 6.3, 4.6, 4.4,
3.9,
3.3
2.4, 2.2, 1.
.9
R
18.6, 15.8,
12.7,
10.8,
10.2, 9.1, 8.4, 7.7, 6.7, 5.6,
4.4,
3.3
2.2, 1.9
11.3, 8.0, 6.9, 3.4, 2.5, 1.6
17.2, 15.3, 14.3, 10.5, 8.6, 7.2, 6.8, 5.8
S
T


LIST OF TABLES
Table Page
3-1 Strains of X. campestris used in host range investigation ... 11
3-2 Legume plant reactions to inoculation with pathovars
of X. campestris 14
3-3 Host range observed for pathovars of X. campestris
pathogenic to legume host plants 17
3-4 Malvaceous host plant reactions to inoculation
with strains of X. campestris pv. malvacearum 19
3-5 Physiological reactions of strains of X. campestris 21
4-1 Strains of X. campestris used for plasmid analysis 29
4-2 Detection of plasmid DNA in strains of X. campestris 34
4-3 Hybridization of radiolabeled plasmid probes to
total DNA of pathovars of X. campestris 44
5-1 Strains of X. campestris pv. malvacearum from cotton
used in race-specificity investigation 52
5-2 Plasmid groupings and cotton plant reactions to strains of
X. campestris pv. malvacearum 56
5-3 Pathogenicity of X. campestris pv. malvacearum
transconjugants on cotton host differentials 59
5-4 Selection of plasmid replication genes in X. campestris
pv. malvacearum strain X 62
6-1 Strains of X. campestris used for restriction fragment
length polymorphism analysis 73
6-2 Sizes of DNA fragments from X. campestris genomic digests
which hybridized to the XCTl DMA probe. See Figure 6-3 83
6-3 Sizes of DNA fragments from X. campestris genomic digests
which hybridized to the XCTl DNA probe. See Figure 6-5 87
vi


Table 3-4. Malvaceous host plant reactions to inoculation with strains
of X. camoestris pv. malvacearuma.
Strain
Cotton
Hibiscus
44
B1
b2
Acala
B3
b5
bin
101
Gregg
Hlb
H2
H3
N
+
+
+
+
+
+
+
+
+
0
+
H
+
-
-
-
-
-
-
-
+
0
+
FL79
+
+
+
+
+
-
-
+
+
+
+
TX84
+
+
+
+
+
-
-
+
0
0
0
X10
-
-
-
-
-
-
-
-
V-
+
+
X27
0
0
0
0
0
0
0
0
0
0
0
X52
-
-
-
-
-
-
-
-
-
+
+
XI02
-
-
-
-
-
-
-
-
-
+
+
X103
-
-
-
-
-
-
-
-
-
+
+
X108
-
-
-
-
-
-
-
-
+
+
+
83-4244
-
-
-
-
-
-
-
-
-
+
+
M84-11
0
0
0
0
0
0
0
0
0
0
0
84-1093G0
0

0
0
0
0
0

+/-
0
5 + = compatible reaction, = incompatible reaction, 0 = null
response, and +/- = intermediate reaction.
b H. rosa-sinensis cv. "Brilliant Red".
c Strain 84-1093 is X. c. pv. esculenti.


a gene for tartaric acid catabolism (35) The expression of pTAR
genes appears to function more like a host range determinant rather
than an oncogenic determinant, as found on tumor inducing plasmids.
The proof of plasmid involvement in virulence and host range with
species of Agrobacterium and Rhizobium apparently stimulated research
of plasmid involvement among other phytopathogenic bacteria. Plasmids
have been found to exist in many other phytopathogenic bacteria, but
few have been associated with pathogenicity (76). Examples of plasmid
functions in pathogenicity on plants include tumor induction in A.
tumefaciens (97), nodulation in Rhizobium spp. (62), indoleacetic acid
production by P. syringae pv. savastanoi (90), cytokinin production by
Corynebacterium fascians (syn. Rhodococcus fascians) (72), virulence
of Pseudomonas solanacearum (5) and race-specific avirulence in X. c.
pv. vesicatoria (88), among others. A general conclusion from these
studies is that plasmids from a vade range of plant pathogenic genera
can carry a variety of genes which determine the outcome of plant-
pathogen interactions.
Plasmids have been shown to be physically similar among strains
within pathovars of P_. syringae (77) The similarity is based on
homology studies using restriction endonuclease digestion patterns and
DNA-DNA hybridization experiments (15) Additionally, there are
instances where similar plasmids, or even identical ones, may be found
in closely related pathovars of P. syringae (77) Although the
functional utility of these plasmids is unknown, they often cannot be
eliminated or "cured" from the bacterial strain, possibly because such
an event would be lethal. For example, in P. syringae pv.


36
reliable for strain classification because several strains had
plasmids of apparently identical size, but they were clearly different
after digesting the plasmid DNAs with restriction enzymes. By adding
up the DNA fragment sizes yielded by restriction digests, plasmids in
X. campestris were estimated as ranging from about 3 to 200 kb
(kilobase pairs) in size. Estimation of some of these sizes were
difficult for seme strains due to the presence of more than one
plasmid.
Plasmid profiles of strains of X. c. cyamopsidis, X. c. glycines,
X. c. malvacearum, X. c. phaseoli, and X. c. vignicola were compared.
Restriction fragment length polymorphism was evident within each of
these pathovars. Although more than one plasmid was present in seme
of these strains, a subset of restriction fragments of similar length
and overall pattern appeared to be consistent for strains within a
given pathovar (Figure 4-2).
Four highly polymorphic plasmid variants were found in 17 out of
44 Florida strains tested, but Southern hybridization revealed no
homology between seme of the plasmids (31). Furthermore, there were
no similarities in plasmid digestion patterns between X. c. pv. citri
type A, type B and any of the Florida citrus leaf spot strains that
carried plasmids (Figure 4-3). These Florida isolates are presumed to
be X. c. citri, but they grow well and also cause disease symptoms on
kidney bean and alfalfa, thus making their pathovar status
questionable (34).


pathovar, that plasmid profiles for specific pathovars were known, and
that an appropriate DNA probe consisting of conserved and unique DNA
sequences were available. Of the few DNA probes constructed, it was
apparent that plasmid DNA sequences were highly conserved within the
pathovars studied. In some instances the DNA probes may prove
sufficient for pathovar identification, provided they are extensively
tested. This apparent stability of the plasmids provides a natural
genetic marker that can be strain specific and perhaps useful in
epidemiological investigations. In addition to aiding the
identification for some pathovars of X. campestris, these observations
may have taxonomic significance to differentiate these pathogens.


interesting to note that strains FL79 and TX84, both race 16 of X. c.
pv. malvacearum cotton strains, each differed in reactions on the
hibiscus differential lines. Since Acala 44 elicited a hypersensitive
response from 6 of 8 hibiscus strains, possibly Acala 44 contains an
undiscovered resistance gene to the hibiscus derived strains. Those
strains for which no hypersensitive reaction was induced may have
escaped host recognition, but lack the necessary pathogenicity
determinants to incite disease. Avirulence genes do not give
phenotypic expression in non-parasites (88). Bacterial growth
kinetics would help determine if the bacteria are multiplying on these
hosts. This would be useful for the case of X. c_. pv. esculenti, for
which 1 of 8 cotton hosts were incompatible, and 2 of 3 hibiscus hosts
were incompatible.
The data from the protein and physiological tests demonstrated
differences between strains, but were not useful for typing strains by
pathovar. There was significant variation among strains within a
given pathovar. Although the use of SDS-PAGE to study total protein
profiles did not differentiate the pathovars, seme work has suggested
limiting the analysis to just outer membrane proteins to improve
resolution of strain differences (70).
These experiments confirmed pathogenicity of the stock strains
and clarified inconsistencies in the literature concerning host range.
The host range studies indicated that conclusions reached may often be
strain-dependent, and that examination of a large number of strains
will reveal a different overall picture than an examination of a few
strains. For example, the plant source of X. c. pv. malvacearum


LITERATURE CITED
1. Alvarez, A. M. Benedict, A. A., and Mizumoto, C. Y. 1985.
Identification of xanthomonads and grouping of strains of
Xanthanonas campestris pv. campestris with monoclonal antibodies.
Phytopathology 75:722-728.
2. Barker, D., Schafer, M., and White, R. 1984. Restriction sites
containing CpG show a higher frequency of polymorphism in human
ENA. Cell 36:131-138.
3. Bender, C. L., and Cooksey, D. A. 1986. Indigenous plasmids in
Pseud anonas syringae pv. tomato: conjugative transfer and role in
copper resistance. J. Bacteriol. 165:534-541.
4. Beynon, J. L., Beringer, J. E., and Johnston, A. W. B. 1980.
Plasmids and host-range in Rhizobium leguminosarum and Rhizobium
phaseoli. J. Gen. Microbiol. 120:421-429.
5. Boucher, C., Martinel, A., Barberis, P. Alloing, G., and
Zischek, C. 1986. Virulence genes are carried by a megaplasmid of
the plant pathogen Pseudononas solanacearum. Mol. Gen. Genet.
205:270-275.
6. Bradbury, J. F. 1984. Xanth anonas Dowson 1939. pp. 199-210 in:
Bergys Manual of Systematic Bacteriology, Vol. 1. N.R Krieg, and
J.G Holt, eds. Williams and Wilkins, Baltimore. 964 pp.
7. Brinkerhoff, L. A. 1963. Variability of Xanth anonas malvacearum:
the cotton bacterial blight pathogen. Oklahoma State University
Agricultural Experiment Station Bulletin T-98. 95 pp.
8. Brinkerhoff, L. A. 1970. Variation in Xanthomonas malvacearum and
its relation to control. Annu. Rev. Phytopathol. 8:85-110.
9. Brinkerhoff, L. A., and Presley, J. T. 1967. Effect of four day
and night temperature regimes on bacterial blight reactions of
immune, resistant, and susceptible strains of upland cotton.
Phytopathology 57:47-51.
10. Buchholz, W. G., and Thonashow, M. F. 1984. Comparison of T-DNA
complements of Agrobacterium tumefaciens tumor-inducing plasmids
with limited and wide host ranges. J. Bacteriol. 160:319-326.
97


94
survey of the known type strains of X. c. pv. citri and those isolated
in Florida allowed for the separation of these bacteria into four
identifiable RFLP groups. Strains belonging to the "A group" isolated
frcm Brazil and Japan were related by RFLP analysis. The strains
isolated in 1986 from citrus in backyards of the Tampa-Bradenton area
were also of the "A group". Strains belonging to the "B group" and "C
group" (isolated from Argentina and Brazil, respectively) appeared to
be related by RFLP analysis, yet could be differentiated. A Mexican
cancrosis strain was indistinguishable from the "B group" based on
RFLP analysis. Other strains of X. c. pv. citri isolated in Florida
did not appear to be related to those strains placed into the "A
group" or "B/C group". The Florida nursery strains appeared to
constitute yet a third RFLP grouping. This group was highly
polymorphic, and some strains appeared to be related to other
pathovars of X. campestris, strengthening the ,lmutation/endemic"
theory for some occurrences of citrus canker (Gabriel et al.,
unpubl.).
RFLP methods are powerful taxonomic tools to distinguish major
differences in strains assigned to the same pathovar simply because
they share a common host. For instance, the differentiation of
strains of X. campestris pathogenic to crucifers, such as X. c. pv.
campestris and X. c. pv. armoraciae, is at times difficult. X. c_. pv.
campestris is generally a systemic pathogen, whereas X. c. pv.
armoraciae is usually localized, but several strains showing
intermediate symptoms have been identified (99). In preliminary
experiments the RFLP methods were able to differentiate between the


lack of polygalacturonase activity. At pH 7.0 and 8.5, pectolytic
activity was observed for all strains of X. c. pv. cyamopsidis, X. c.
pv. glycines, X. c. pv. pisi, and X. c. pv. vignicola. Only one of the
13 X. c. pv. phaseoli strains tested (SC-3B) had pectolytic activity.
None of the strains degraded pectin as a carbon source, only sodium
polypectate.
Polyacrylamide Gel Electrophoresis
Total protein was extracted from 18 strains, representing seven
different pathovars of X. campestris. The pathovars included were X. c.
pv. alfalfae, X. c_. pv. cyamopsidis, X. c. pv. glycines, X. c. pv.
malvacearum, X. c_. pv. phaseoli, X. c. pv. pisi, and X. c. pv.
vignicola. All of the pathovars, except X. c_. pv. malvacearum (Fig. 3-
1, lane P), are known pathogens of legumes. One of the X. c. pv.
phaseoli strains consisted of a biochemical variant of this pathovar, X.
c_. pv. phaseoli var. fuscans (Fig. 3-1, lane J) One of the X. c_. pv.
malvacearum strains was derived from hibiscus, rather than from cotton
(not shown).
In general, there were few observable differences in the protein
banding patterns for the X. campestris strains. Seme minor differences
in banding patterns between different pathovars were observed, but
variation to the same extent was also present within a given pathovar.
Discussion
The plant reactions observed from the X. campestris host range
study conformed to expectations, in most cases. However, it was evident
that pathogenic variation in strains associated with a given pathovar
could occur, as shown by the race-specific interactions with the X. c.


79
Figure 6-2. Genomic DNA of strains of X. campestris digested with the
restriction endonuclease EcoRI. Lane A, probe XCT1; lane B, X. c. pv.
alfalfae FL; lane C, X. c. pv. begoniae 084-155; lane D, X. c. pv.
campestris 084-809; lane E, X. c. pv. carotae 13; lane F, X. c. pv.
cyamopsidis X002; lane G, X. c. pv. dieffenbachiae 068-1163; lane H,
X. c. pv. glycines S-9-3; lane I, X. c. pv. holcicola Xh66; lane J, X.
£_ pv. maculifoliigardeniae 084-6166; lane K, X. c. pv. malvacearum
FL79; lane L, X. c. pv. pelargonii 084-190; lane M, X. c. pv. phasedi
var. fuscans SC-3B; lane N, X. c. pv. pisi XPl; lane 0, X. c. pv.
translucens X1105; lane P, X. c. pv. vesicatoria 75-3; lane Q, X. c.
pv. vignicola SN2; lane R, X. c. pv. vitians ICPB164; lane S, X. c.
pv. zinniae 084-1944; lane T, probe XCTll.


103
72. Murai, N., Skcx>g, F., Doyle, M. E., and Hanson, R. S. 1980.
Relationships between cytokinin production, presence of plasmids,
and fasciation caused by strains of Corynebacterium fascians.
Proc. Natl. Acad. Sci. USA 77:619-623.
73. Murata, N. and Starr, M. P. 1973. A concept of the genus
Xanthanonas and its species in the light of segmental homology of
deoxyribonucleic acids. Phytopath. Z. 77:285-323.
74. Nei, M., and Li, W.-H. 1979. Mathematical model for studying
genetic variation in terms of restriction endonucleases. Proc.
Natl. Acad. Sci. USA 76:5269-5273.
75. Palleroni, N. J. 1984. Family I. Pseudomonadaceae Winslow,
Broadhurst, Buchanan, Krumwiede, Roger and Smith 1957. pp.
140-199 in: Bergy's Manual of Systematic Bacteriology, Vol 1. N.
R. Krieg, and J. G. Holt, eds. Williams and Wilkins, Baltimore,
MD.
76. Panopoulos, N. J., and Peet, R. C. 1985. The molecular genetics
of plant pathogenic bacteria and their plasmids. Annu. Rev.
Phytopathol. 23:381-419.
77. Piwowarski, J. and Shaw, P. D. 1982. Characterization of
plasmids from plant pathogenic pseudomonads. Plasmid 7:85-94.
78. Poplawsky, A. R. Peng, Y. F. and Ellingboe, A. H. 1986.
Bacterial Tn5 mutants affected in antibiosis to Gaeumanrnonyces
graminis var. tritici. (Abstr.). Phytopathology 76:1069.
79. Quant, R. L., and Mills, D. 1984. An integrative plasmid and
multiple-sized plasmids of Pseud anonas syringae pv. phaseolicola
have extensive homology. Mol. Gen. Genet. 193:459-466.
80. Randhawa, P. S., and Civerolo, E. L. 1985. Plasmids in
Xanthononas campestris pv. pruni (Abstr.). Proc. Sixth Int. Conf.
Plant Pathogenic Bact., College Park, MD.
81. Sasser, M. 1983. Fatty acid analysis of the genus Xanthononas.
in: First Fallen Leaf Lake Conf. On the Genus Xanthononas. Fallen
Leaf Lake, CA.
82. Schaad, N. W. 1980. Laboratory Guide for Identification of Plant
Pathogenic Bacteria. American Phytopathological Society, St.
Paul, MN. 72 pp.
Schleifer, K. H., Ludwig, W., Kraus, J., and Festl, H. 1985.
Cloned ribosomal ribonucleic acid genes from Pseudomonas
aeruginosa as probes for conserved deoxyribonucleic acid
sequences. Int. J. Sys. Bacterid. 35:231-236.
83.


86
RFLP pattern appeared as qualitative and quantitative differences
among hybridization profiles of various sized DNA fragments in the
strains. For instance, the weak hybridization signal seen from X. c.
pv. translucens (fig. 6-5, lane 0) suggests that this strain is
distinct from the other pathovars compared, at least over the 1% of
the genome contained on the clone XCTl. In other pathovars such as X.
c_. pv. alfalfae (fig. 6-5, lane B) X. c_. pv. begoniae (lane C), X. c.
pv. cyamopsidis (lane F) X. c. pv. malvacearum (lane K) X. c. pv.
vigncola (lane Q) and X. c. pv. vitians (lane R) where sane DNA
fragments had strong hybridization signals, a close relatedness with
portions of DNA in the cosmid clone is suggested. Some of the DNA
fragments from different pathovars which hybridized most strongly to
the probe were also of identical size with the EcoRI fragments of the
XCTl clone. As expected, ENA of X. c. pv. citri strain 3401 digested
with EcoRI, or BamHI (34,55) contained fragments which corresponded to
DNA fragments of the XCTl probe (Fig. 6-5, lane A). Sizes of
hybridizing DNA fragments are given in Table 6-3.
From observations using different enzymes and different probes,
the same patterns emerged, indicating that the basic chromosome
structure of X. campestris can be used to differentiate strains into
specific RFLP types. Although a pathovar may contain more than one
type of variant (e.g. X. c_. pv. phasedi), all strains of a given type
appear to exhibit the same host selectivity. The RFLP pattern is
highly distinctive for each type, and can be used to unambiguously
assign an unknown sample to a pathovar, using simple visual
comparisons of pattern against a set of known samples, such as those
given in Figure 6-5.


Plant Inoculations
Pathogenicity tests were conducted by pressure infiltrating
bacterial suspensions into leaf tissue with a blunt-end syringe and
incubating the plants in 28 C or 30 C growth chambers until symptoms
were expressed. The leguminous host plants were incubated at 28 C and
symptoms were recorded after one week. The malvaceous hosts were
incubated at 30 C and symptoms were recorded after a two week period.
Pressure infiltration facilitated rapid screening of plant reactions,
but was not representative of natural inoculation. Because alfalfa
leaves were small in size, infection was by spray inoculation.
Bacterial suspensions were prepared by centrifuging overnight cultures
of X. campestris, resuspending them in 0.7% NaCl to an approximate
optical density (ODggonm) f 0*3 fr plant inoculation. These
pathogenicity tests were repeated at least once. The results were
recorded to determine compatible (pathogenic) or incompatible (non-
pathogenic or hypersensitive) plant reactions to inoculations.
Physiological Differentiation
As X. c ampe s t r i s is defined (6), the physiological
characteristics for gelatin and starch hydrolysis may be variable for
this species. Other physiological characteristics which are consistent
with this species includes xanthcmonadin production, mucoid growth,
and esculin and casein hydrolysis. These above mentioned
characteristics were determined using standard methods (36,82).
Additionally, physiological tests for production of ceilulase (96) ,
lecithinase, lipase, and pectinases were included. Hildebrand's
medium prepared at three pH's (4.5, 7.0, and 8.5) was used to detect
pectolytic enzyme activity. In each of these three media, sodium


Using RFLP genomic blots, it appeared that phylogenetic
relationships between the full spectrum of described pathovars of X.
campestris might be determinable. Some mathematical approaches toward
90
determining phylogeny based on restriction cleavage sites has been
proposed (58,74). DNA probes have been used to make phylogenetic
comparisons among the relatively conserved mitochondrial (67) and
chloroplast (91) genomes. The variation seen among the different
pathovars of X. campestris appeared within the range that is usefully
distinguishable with this test. Only limited amounts of variation can
be usefully distinguished, such as that occurring within a species.
Significantly, the RFLP groupings closely corresponded with the
pathovar groupings, strengthening the taxonomic significance of this
classification. All strains tested were readily grouped by RFLP
phenotypes, and the classification based on RFLP patterns correlated
very well with the classification based on pathogenicity. This
technique may provide a more convenient means of classifying these
bacteria. In addition, unexpected taxonomic relationships between
pathovars might be revealed. The potential pathogenic range of each
RFLP group would be of obvious value.
By comparing observed RFLPs among strains of X. campestris using
selected DNA probes, it was possible to identify unknown strains when
known standards were included. Additionally, strains previously
undescribed could be classified as being related to known pathovars.
For instance, similarities were found between undescribed strains
isolated from a Alocasia sp. and Argemone sp.; and a Cissus sp. and
Jasmine species. The strains isolated from a Philodendron sp. and


31
Some of the stock cultures of X. c. malvacearum were mixtures of
different strains, maintained and described as "races" for purposes of
screening cotton host differentials and breeding lines for disease
resistance. Described "races" of X. c. malvacearum may or may not be
purified strains; as many as seven different strains have been derived
from a single race 1 isolate (7). All strains used in this study were
repeatedly purified from single colonies and confirmed to be
pathogenic on their designated hosts. Broth cultures of bacteria were
grown in a peptone-glycerol medium (per L: 10.0 ml glycerol, 20.0 g
peptone, and 1.5 g K2HPO4). Bacterial strains were commonly stored at
-80 C in the same medium containing 15% glycerol.
Plasmid Extraction and Visualization
Cultures were grown to mid- to late-logarithmic growth phase and
extracted by either of two small-scale alkaline lysis extraction
procedures (46,66). Extracted DNA was resuspended in TE (10 mM
Tris(hydroxymethy1)aminomethane (Tris) 1 mM sodium
ethylenediaminetetraacetate (N32EDTA), and 20 ug/ml DNase-free
pancreatic RNase; pH 7.6) and digested with either of two restriction
endonucleases, EcoRI or BamHI, using manufacturer (Bethesda Research
Laboratories, Gaithersburg, MD) specifications. Plasmid DNA fragments
were separated by size using agarose gel electrophoresis (0.6% agarose
(Sigma Type I:low EEO), 2-5 V/cm) in Tris-acetate buffer (40 mM Tris,
1 mM Na2EDTA, adjusted to pH 7.6 with glacial acetic acid). Fragments
were visualized by ultraviolet irradiation (302 nm) after staining
agarose gels in ethidium bromide (0.5 ug/ml). Photographs were taken
with Polaroid Type 55 (or Type 57) film using a yellow filter (Tiffen


CHAPTER FIVE
ARE RACE-SPECIFIC AVIRULENCE GENES ENCODED BY PLASMIDS
OF XANTHOMONAS CAMPESTRIS PV. MALVACEARUM?
Introduction
Bacterial blight of cotton, caused by the pathogen X. c. pv.
malvacearum (Snith) Dye, is a destructive disease of cotton, which
occurs throughout the cotton-producing areas of the world. The
development of cotton cultivars resistant to bacterial blight is the
primary means by which this pathogen is controlled. At least 16 major
genes have been identified which condition resistance to bacterial
blight (8). A gene-for-gene pattern of interaction between the plant
resistance genes and bacterial avirulence genes has been demonstrated
in this host-parasite interaction (33). In screening for resistance
to bacterial blight in breeding lines, races of the pathogen have been
maintained as mixed cultures, originally derived from infected leaf
samples. These mixed cultures have been mass transferred on agar
media to prevent the appearance of new race phenotypes. The stability
of the race phenotypes of these mixed cultures is periodically checked
by inoculating the host differentials.
Plasmids have been detected in several phytopathogenic
prokaryotes and have been associated with functions such as
pathogenicity (5), resistance to antibiotics (3,88), avirulence (88),
and host range (64). With the recent discovery of plasmids in several
50


FIVE ARE RACE-SPECIFIC AVIRULENCE GENES ENCODED BY PLASMIDS
OF XANTHOMONAS CAMPESTRIS PV. MALVACEARUM? 50
Introduction 50
Materials and Methods 51
Bacterial Strains and Host Plants 51
Plant Inoculations 51
Plasmid Analysis 51
Plasmid Curing 53
Race-Specificity Genes 53
Plasmid Origin of Replication and Mobilization 54
Results 55
Plasmids and Race-Specificity 55
Plasmid Curing 58
Race-Specificity Genes 58
Plasmid Origin of Replication and Mobilization .... 61
Discussion 65
SIX PATHOVARS OF XANTHOMONAS CAMPESTRIS ARE DISTINGUISHABLE
BY RESTRICTION FRAGMENT LENGTH POLYMORPHISM 70
Introduction 70
Materials and Methods 72
Bacterial Strains 72
DNA Extraction 72
Agarose Gel Electrophoresis 75
DNA Probes 75
DNA Hybridization 76
Restriction Fragment Patterns and Densitometry .... 77
Results 77
Agarose Gel Electrophoresis 77
DNA Probes 80
DNA Hybridization 80
Discussion 88
SEVEN SUMMARY 92
Intraspecific Variation 92
Plasmids 92
Restriction Fragment Length Polymorphism 93
Conclusions 95
LITERATURE CITED 97
BIOGRAPHICAL SKETCH 106
v


sources (cabbage, broccoli, and brussels sprouts) at least four of
over 10 fragments appeared to be identical. Sizes of hybridizing DNA
fragments are given in Table 6-2. In figure 6-4 is shown the RFLP
patterns revealed using clone XCTll. In each case, a different RFLP
pattern is shown, but the same genetic relationship is revealed.
Other sets of conserved DNA fragments within a pathovar were seen
among seven strains of X. c. pv. vignicola (identical pattern), seven
strains of X. c. pv. cyamopsidis (nearly identical) and at least
three strains each for X. campestris pvs. citri, glycines,
malvacearum, pelargonli, and vesicatoria (not shown).
ENA of strains representing other pathovars of X. campestris were
also digested with either EcoRI or BamHI, and hybridized with either
XCTl or XCTll. The pathovars of X. campestris included in these
experiments were X. campestris pvs. alfalfae, begoniae, campestris,
carotae, citri, cyamopsidis, dieffenbachiae, esculenti, glycines,
hederae, holcicola, maculifoliigardeniae, malvacearum,
mangiferae indicae, nigromaculans, pelargonii, phaseoli pisi,
translucens, vesicatoria, vignicola, vitians, and zinniae. Also
included were some strains isolated as pathogens of an Alocasia sp.,
Cissus sp., Impatiens sp., Fatsia sp., Jasmine sp., Argemone sp., and
a Euonymus species for which no pathovar designations were available.
The results of hybridization of probe XCTl to DNA of different
pathovars of X. campestris digested with EcoRI are shown in Figure 6-
5. The clone XCTl appeared to contain DNA fragments of strain 3401
which were able to demonstrate variability of restriction fragment
patterns among different pathovars of X. campestris. Variability in