Pathogenic and genomic characterization of strains of Xanthomonas campestris causing diseases of citrus

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Pathogenic and genomic characterization of strains of Xanthomonas campestris causing diseases of citrus
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viii, 132 leaves : ill., photos ; 28 cm.
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Egel, Daniel Scott, 1958-
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Xanthomonas campestris   ( lcsh )
Citrus -- Diseases and pests   ( lcsh )
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Thesis:
Thesis (Ph. D.)--University of Florida, 1991.
Bibliography:
Includes bibliographical references (leaves 121-130).
Statement of Responsibility:
by Daniel Scott Egel.
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Vita.

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PATHOGENIC AND GENOMIC
CHARACTERIZATION OF STRAINS OF XANTHOMONAS
CAMPESTRIS CAUSING DISEASES OF CITRUS















By

DANIEL SCOTT EGEL


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


1991


































To my wife
Denise














ACKNOWLEDGEMENTS

Although there are innumerable individuals to whom I

owe a great deal, space permits me to list just a few. I

would not have entered this graduate program and could not

have completed it without the support and encouragement of

my advisor, Dr. Jim Graham. Similarly, Dr. Robert Stall

gave generously of his time and vast expertise. I would

also like to thank the other individuals who served on my

committee, Dr. Ann Chase, Dr. Jim Preston, and Dr. Daryl

Pring, who all spent considerable time with me.

The nature of the research I conducted required me to

work in the quarantine facilities of the Department of Plant

Industry where Dr. John Miller and Dr. Tim Schubert as well

as many others were accommodating and helpful. I also

worked at the Agricultural Research and Education Center in

Hastings, FL, and the quarantine laboratory at Plymouth, FL,

where the help of Mr. Mark Bruce and Mr. Tim Riley were

invaluable.

I was assisted in methods development in DNA

reassociation by Dr. John Johnson at the Virginia State

University and Polytechnic Institute in whose laboratory I

spent a week learning techniques. I also had several

valuable conversations with Dr. Don Hildebrand of the


iii








University of California, Berkeley, and Dr. Noberto

Palleroni of the New York University Medical School. Dr.

Carole Baeulieu assisted with techniques for pulsed field

gel electrophoresis while she was a postdoctoral associate

in Dr. Stall's laboratory. Dr. John Hartung, Beltsville,

MD, isolated DNA for me of Xanthomonas campestris pv. citri

group B since quarantine regulations restricted my access to

that pathogen. Jerry Minsavage was always able and willing

to offer excellent technical assistance as well as a kind

word.

Many individuals among the faculty, staff and students

of the Department of Plant Pathology aided by lending

equipment or supplies or offering advice. Especially

helpful were the other members of Dr. Stall's laboratory.

Finally, I was buoyed by my wife's constant optimism

and her selfless dedication to me, my work and to our son

Sam who has been an inspiration to both of us. I am forever

indebted to my own parents who have consistently offered

whatever support was needed.
















TABLE OF CONTENTS
page

ACKNOWLEDGEMENTS ............................iii

ABSTRACT........................... ... ....... vi

CHAPTERS

1 INTRODUCTION....................... ......1

2 REVIEW OF LITERATURE....................5

Disease Comparisons..................... 5
Epidemiological Comparisons............. 10
Phenotypic Comparisons...................11
Serological Comparisons................12
Genetic Comparisons...................14
Fatty Acid Comparisons................ 16
Isozyme Comparisons.................... 17


3 PATHOGENIC CHARACTERIZATION..............18

Materials and Methods..................20
Results........................... ....26
Discussion....... ..................... 35

4 GENOMIC CHARACTERIZATION ..............40

Materials and Methods..................42
Results.............. .................. 50
Discussion ... ........ .................. 70

5 CHARACTERIZATION WITH AN hrp GENE
CLUSTER.................................79

Materials and Methods...................81
Results..... ........ .......... .....83
Discussion ... ...........................92


















6 CARBOHYDRATE UTILIZATION.. .......................96

Materials and Methods.................. 97
Results................................99
Discussion.............................104

7 DISCUSSION............................. 107

APPENDIX.....................................118

LITERATURE CITED............................... 121

BIOGRAPHICAL SKETCH.......................... 131














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

PATHOGENIC AND GENOMIC CHARACTERIZATION
OF STRAINS OF XANTHOMONAS CAMPESTRIS
CAUSING DISEASES OF CITRUS

By

Daniel Scott Egel

May 1991

Chairman: Robert E. Stall
Major Department: Plant Pathology

Strains of Xanthomonas campestris which cause diseases

of citrus were characterized for their pathogenicity and

their genomic relatedness. Strains of X. camDestris pv.

citrumelo, which cause Citrus bacterial spot, vary widely in

their lesion expansion capabilities and their bacterial

population development in and on citrus leaves. A host-

strain interaction was observed among strains on Swingle

citrumelo and Duncan grapefruit.

Restriction endonuclease patterns of infrequently

occurring recognition sites in genomic DNA fragments

separated by pulsed field gel electrophoresis were diverse

for strains of X. campestris pv. citrumelo, whereas for

strains of X. campestris pv. citri group A which causes

Asiatic citrus canker and X. campestris pv. citri group B

which causes Cancrosis B the restriction patterns were

vii








relatively homogeneous. Strains of X. campestris pv.

citrumelo, X. campestris pv. citri group A, and X.

campestris pv. citri group B were all about 60% related to

one another as determined by DNA reassociation. Strains of

X. campestris pv. citrumelo averaged 88% similar to each

other. Pathovars from hosts other than citrus, but known to

cause lesions on citrus, were similar to strains of X.

campestris pv. citrumelo by DNA reassociation. Pathovars

from hosts other than citrus which do not cause lesions on

citrus were not as related to X. campestris pv. citrumelo.

Similarity values generated by DNA reassociation and

Restriction Fragment Length Polymorphism (RFLP) analyses

were not necessarily equivalent in comparisons of DNA

reassociation values obtained here with RFLP analyses of

other investigators.

All strains of X. campestris pv. citrumelo and X.

campestris pv. citri group A examined possessed an hrP gene

cluster which is interpreted to mean that even weakly

aggressive strains of X. campestris pv. citrumelo are true

pathogens. Strains of X. campestris pv. citrumelo were

relatively similar in restriction patterns revealed by

hybridization of an hrp probe as well as in carbohydrate

utilization patterns. Comparisons of characterization

methods cited here are illustrative of the advantages of

using several techniques in the characterization of

bacterial strains.


viii














CHAPTER 1
INTRODUCTION


The possible repercussions of the introduction of

Citrus canker have been understood since the disease was

eradicated from Florida in 1933 (Stall and Seymour, 1983).

Scientists, growers and regulators were aware of the losses

Asiatic citrus canker could cause. Asiatic citrus canker

can cause abscission of fruit and leaves and the lesions

result in unmarketable fruit. The introduction of citrus

canker would inevitably lead to an expensive eradication

effort and shipment of fruit would be restricted.

Most citrus growing regions of the world are affected

by one or more canker diseases of citrus (Koizumi, 1985).

Four of these diseases are caused by bacteria classified as

Xanthomonas campestris pv. citri (Civerolo, 1984). The

diseases caused by X. campestris pv. citri have similar

symptoms on citrus and citrus relatives, but differ in host

range and geographical locations. The similarities among

these diseases have resulted in the name citrus canker as an

umbrella term for all diseases caused by X. camDestris pv.

citri. These diseases are Asiatic citrus canker, Cancrosis

B, Mexican lime cancrosis and Citrus bacteriosis. For the

purposes of this work, these diseases will all be referred








2
to as citrus canker diseases and the causal bacteria will be

known as X. campestris pv. citri group A, group B, group C

and group D, respectively.

In September 1984, a new disease of citrus, Citrus

bacterial spot (Graham and Gottwald, 1988), was found in a

central Florida citrus nursery (Schoulties et al., 1987).

Research led to a basic understanding of this disease, but

questions remained as to the ecology and epidemiology of its

causal bacteria. Information regarding the population

development of the causal agent was needed particularly to

understand the potential for spread of the pathogen in the

field. Little was known concerning the origin of this group

of pathogens or the relationship of these strains to related

pathogens in Florida. The genetic relatedness among strains

of the causal organism and between these bacteria and

strains of X. campestris pv. citri remains unclear.

The classification of the causal agent of Citrus

bacterial spot has been the subject of controversy. The

symptomatology of Citrus bacterial spot is similar to Citrus

canker, but it can be differentiated from Citrus canker by

the type of lesion formed. Therefore, it has been proposed

that the causal bacteria be named X. campestris pv.

citrumelo (Gabriel et al., 1989). Clear differences in

symptomatology are justification for pathovar status (Dye et

al. 1980) and therefore X. campestris pv. citrumelo

terminology will be used here for the pathogen of Citrus










bacterial spot although it is recognized that a formal

description of this pathovar has not been published. The

elevation of the causal agent of Citrus bacterial spot to

pathovar status raises questions regarding the classifi-

cation of the groups of strains that cause Citrus canker.

Justification for placing at least some of the groups of X.

campestris pv. citri into separate pathovars may exist as

was suggested by Gabriel et al. (1989), but more research

needs to be done to define similarities and differences

within these strains. Any study of the pathogen causing

Citrus bacterial spot should, if possible, also include

pathogens causing Citrus canker for comparison.

The primary objective of this work is to characterize

bacterial strains responsible for Citrus canker and Citrus

bacterial spot. Experiments were undertaken to assess the

aggressiveness of strains of X. campestris pv. citri group A

and X. campestris pv. citrumelo in growth chamber and

greenhouse. Lesion expansion and development of external

and internal populations of bacteria were evaluated on

Duncan grapefruit and Swingle citrumelo to study host-strain

interactions and relate leaf population dynamics to the

ability of these strains to survive and spread in citrus

nurseries. The genomic variation within strains of X.

campestris pv. citrumelo and X. campestris pv. citri groups

A and B and the differences between these groups is

addressed. Techniques used to determine genomic relatedness








4

among strains include DNA reassociation, restriction

endonuclease analysis, plasmid profiles, and hybridization

analysis. These strains are also compared for differences

in carbohydrate utilization patterns.














CHAPTER 2
REVIEW OF LITERATURE


Citrus canker diseases are an important factor in

citrus production worldwide. Symptoms have been observed on

all above ground parts of the citrus tree (Fawcett, 1936)

with young tissue most susceptible (Stall et al., 1981;

Stall et al., 1982). Citrus canker diseases vary in host

range, symptomatology and geographical distribution

(Civerolo, 1984). The causal organisms involved are all

pathovars of Xanthomonas campestris (Civerolo, 1984). A

similar disease, Citrus bacterial spot caused by X.

campestris pv. citrumelo, affects nursery trees in Florida

(Graham and Gottwald, 1988; Schoulties et al., 1987).

Disease Comparisons

The most important Citrus canker disease is Asiatic

citrus canker, also known as Canker A, which probably

originated in Asia (Fawcett, 1936). Today, however, Asiatic

citrus canker is known in most areas where citrus is grown

(Koizumi, 1985). The causal organism was originally

described as Pseudomonas citri by Hasse (1915). The

organism is now named X. campestris pv. citri, although

recently a proposal to elevate pv. citri to species status

has been published (Gabriel et al., 1989). Infection by X.










campestris pv. citri results in erumpent lesions on leaves,

stems and fruits (Whiteside et al., 1988). Upon invasion of

susceptible citrus tissue by strains of X. campestris pv.

citri group A, swelling and degeneration of host cell walls

occur, finally resulting in dissolution of plasmalemmas

(Koizumi, 1979; Koizumi, 1988). The erumpent nature of

Asiatic citrus canker lesions results from hypertrophy of

parenchyma cells at the center of each lesion (Koizumi,

1976; Koizumi, 1977). After cell hypertrophy, bacterial

multiplication increases and bacteria begin to ooze from

lesions (Koizumi, 1976). Severe outbreaks of Asiatic citrus

canker can lead to abscission of both fruit and leaves

(Stall and Seymour, 1983). Abscission is probably due to

ethylene production by X. campestris pv. citri multiplying

within leaf tissue (Goto et al., 1980b). Asiatic citrus

canker affects all commercial varieties although to varying

degrees (Leite, 1990; Stall and Seymour, 1983).

Cancrosis B, first described as false canker, causes

symptoms similar to Asiatic citrus canker, but is limited to

South America where it was first observed in Argentina in

1928 (Ducharme, 1951). Cancrosis B affects primarily C.

limon (lemon) in the field, although a number of citrus

hosts can be affected if in close proximity to infected

lemons (DuCharme, 1951). In side-by-side inoculations on

grapefruit leaves, a strain of X. campestris pv. citri group

B caused fewer lesions per cm2 than a strain of X.










camDestris pv. citri group A (Stall et al., 1981).

Xanthomonas campestris pv. citri group B, varies physio-

logically from strains of X. campestris pv. citri group A

and has been referred to as X. citri f. atypica (Rossetti,

1977). Due to a limited host range, X. campestris pv. citri

group B is not considered as important a pathogen as X.

campestris pv. citri group A (Stall et al., 1981).

In 1963, a citrus canker-like disease was observed in

the state of SAo Paulo, Brazil (Rossetti, 1977). Although

originally feared to be Asiatic citrus canker, the disease

affected only Mexican lime. Mexican lime cancrosis,

sometimes called Canker C, has only been observed in the

state of Sao Paulo, Brazil, on Mexican lime. Symptoms on

Mexican lime are similar to both Asiatic citrus canker and

Cancrosis B. On grapefruit, hypersensitive-like symptoms

were observed upon artificial inoculations with a Mexican

lime cancrosis strain (Stall et al., 1981). The bacterium

was originally classified as X. citri f. sp. aurantifolia

(Namekata and Oliveira, 1971) but is now referred to as

group C of X. campestris pv. citri (Civerolo, 1984). It is

of limited occurrence and thus is not considered of economic

importance.

A disease known as citrus bacteriosis or leaf-and twig-

spot disease was first observed on Mexican lime trees in

Mexico in 1981 (Rodriguez G. et al., 1985). Symptoms were

similar to Asiatic citrus canker, but no lesions have been










observed on fruit. Citrus bacteriosis appears to be

concentrated in the Pacific coastal regions of Mexico

(Rodriguez G. et al., 1985). Strains of X. campestris have

been inconsistently isolated from lesions (Rodriguez G. et

al., 1985) and based on pathogenicity were classified as X.

campestris pv. citri. The disease is most prevalent during

the dry season, unlike most citrus canker diseases

(Stapleton and Garza-Lopez, 1988). It has been suggested

that Alternaria sp. may be the causal agent of this disease

(Stapleton and Garza-Lopez, 1988). Recently, it has been

proposed that strains of X. campestris pv. citri groups B, C

and D be placed in X. campestris pv. aurantifolia (Gabriel

et al., 1989).

Canker-like symptoms were discovered on trees in a

Florida citrus nursery in September, 1984 (Schoulties et

al., 1987). Lesions occurred on leaves and stems and in

artificial inoculations, fruit were susceptible (Graham et

al., 1990b) and the host range seemed to be similar to that

of Asiatic citrus canker (Schoulties et al., 1987).

Symptoms differed from citrus canker diseases in that

lesions on leaves and fruit were flat with very prominent

watersoaked margins (Schoulties et al., 1987). Hypertrophic

tissue has not been associated with these lesion types

(Dienelt and Lawson, 1989; Lawson et al., 1989). These

symptom differences have led to the proposed name Citrus

bacterial spot for the disease (Graham and Gottwald, 1988).










Genetic differences, based on RFLP analysis, from X.

campestris pv. citri resulted in the proposal of the name X.

campestris pv. citrumelo for the causal organism (Gabriel et

al., 1989). Citrus bacterial spot has been restricted to

Florida citrus nurseries occurring mainly on Swingle

citrumelo and grapefruit varieties (Graham and Gottwald,

1990). Citrus cultivars derived from hybrids with Poncirus

trifoliata are susceptible to Citrus bacterial spot in

artificial inoculations (Graham et al., 1990a).

There exist two additional xanthomonad strains that

have been reported to cause disease on rutaceous hosts.

Xanthomonas campestris pv. bilvae causes disease on the

following hosts: Aeqle marmelos (L.) Correa; C. aurantifolia

(Christm.) Swingle, Feronia elephantum and Limonia

acidissima L. (Dye and Lelliote, 1974; Leyns et al., 1984).

The name X. bilvae was not included on the approved lists of

pathovar names (Dye et al., 1980) and is not listed by

Bradbury (1984). Also not included by either Bradbury

(1984) or Dye and Lelliote (1974), is X. campestris pv.

feronia which exists in the American Type Culture

Collection. This is a pathogen of Feronia spp. but, there

is no record of a list of hosts for this strain (E. L.

Civerolo, personal communication). The relationship of

these strains to any group of X. campestris pv. citri or X.

campestris pv. citrumelo is not known.

In addition to symptomatology, host range, and

geographical distribution, the causal agents of the Citrus








10

canker diseases and Citrus bacterial spot differ in a number

of other ways. They differ in their epidemiology,

phenotypic characteristics, serological determinants,

genetic variation, isozyme variation and fatty acid

profiles. Collectively, these methods indicate these

pathogens are distinct (Civerolo, 1985b; Civerolo, 1988).

Epidemiological Comparisons

Citrus canker diseases are generally more frequent in

climates where rainy periods and warm weather coincide

(Peltier and Frederick, 1926). Wind-driven rain is critical

to the spread of strains of X. campestris pv. citri group A

(Gottwald et al., 1989; Stall et al., 1980) and allows

stomatal entry of the causal bacteria by watersoaking

leaves. Short distance spread of the bacteria may be due to

wind and rain which cause the spread of leaf surface

bacteria (Dan6s et al., 1984; Gottwald et al., 1988b),

especially those bacteria exuded onto leaf surfaces after

rain events (Stall et al., 1980). The spread of X.

campestris pv. citri group A is often directional, most

often according to the prevailing winds (Gottwald et al.,

1989). Thus, wind breaks have been effectively used as a

method of control (Civerolo, 1981; Leite, 1990).

In contrast to Asiatic citrus canker, X. campestris pv.

citrumelo is most often spread mechanically (Gottwald and

Graham, 1990). Weakly aggressive strains of X. campestris

pv. citrumelo were spread mechanically down rows, while a










highly aggressive strain was apparently capable of wind

driven spread (Gottwald and Graham, 1990; Gottwald and

Graham, unpublished). Citrus bacterial spot appears not to

be spread directionally in response to windblown rain in

both simulated grove and nursery conditions (Gottwald et

al., 1988a). However, a highly aggressive strain did spread

in response to artificially generated wind (Gottwald and

Graham, unpublished data). Outbreaks of Citrus bacterial

spot have been primarily limited to citrus nurseries, in

contrast with Asiatic citrus canker which also affects

mature trees (Schoulties et al., 1987). As with citrus

canker diseases, Citrus bacterial spot has been observed

primarily during warm, rainy months (Graham and Gottwald,

1990).

Phenotypic Comparisons

Although all strains of X. campestris share the

physiological characters of the species, other properties

such as carbohydrate utilization and phage sensitivity vary

among strains of X. campestris pv. citri and X. campestris

pv. citrumelo. Strains of X. campestris pv. citri group B

are difficult to grow on common bacteriological media, but a

few colonies from such isolations appear that are capable of

growth on nutrient agar (Canteros de Echenique et al., 1985;

Stall et al., 1981). All other strains of X. campestris pv.

citri and X. campestris pv. citrumelo grow well on nutrient

agar. Strains of 5. campestris pv. citri group B capable of








12
growth on nutrient agar were not able to utilize lactose or

maltose in contrast with strains of X. campestris pv. citri

group A (Goto et al., 1980a). Other investigators noted the

differential carbon utilization of strains of X. campestris

pv. citri group A and B (Alcaraz, 1977; Gotuzzo and Rossi,

1968) and proposed the name X. citri f. atvyica for the

latter strains (Gotuzzo and Rossi, 1968). Comparisons of

strains of X. campestris pv. citri group A and X. campestris

pv. citrumelo with physiological traits have been reported,

but the tests did not differentiate between these pathovars

(Gabriel et al., 1989). Susceptibility of X. campestris pv.

citri group B to phage Cp3 separated those strains from

strains of _. campestris group A (Goto et al., 1980a).

Although most strains of X. campestris pv. citri group A

were susceptible to phages Cpl and Cp2, these phages did not

attack strains of X. campestris pv. citri group B and C

(Namekata, 1975).

Seroloqical Comparisons

Strains of the different groups of X. campestris pv.

citri have also been distinguished by serological

techniques. Using indirect ELISA (Enzyme Linked

Immunosorbent Assay) tests, strains of X. campestris pv.

citri group A could be differentiated from strains of X.

campestris pv. citri groups B and C, although the difference

between strains of X. campestris pv. citri groups B and C

was not conclusive (Civerolo and Helkie, 1981). Bach (1981)










distinguished strains of X. campestris pv. citri group A

from strains of X. campestris pv. citri group C with ELISA.

Strains of X. campestris pv. citri groups A and D may be

related since five of six strains of X. campestris pv. citri

group D reacted positively in ELISA assays using anitsera to

X. campestris pv. citri group A (Rodriguez G. et al., 1987).

Strains of X. campestris pv. citri group A and B were

differentiated serologically with immunodiffusion and

immunoelectrophoresis (Messina, 1980; Goto et al., 1980a).

Strains of X. campestris pv. citri group C differed from

strains of X. campestris pv. citri group A in agglutination,

precipitation, gel-diffusion and immunoelectrophoresis tests

(Namekata and Oliveira, 1971). Based on these results, as

well as physiological and pathogenicity tests, Namekata and

Oliveira (1971) suggested the name X. citri f. sp.

aurantifolia as the causal agent of Mexican lime cancrosis.

Strains of X. campestris pv. citri groups A, B, and C

were differentiated by monoclonal antibodies, and few other

xanthomonads reacted to any of the monoclonal antibodies

tested (Benedict et al., 1985). Monoclonal antibodies (from

unstable hybridomas) specific for strains of X. campestris

pv. citri group C have been obtained (Alvarez et al., 1987).

These investigators found monoclonal antibodies that

distinguished X. campestris pv. citri groups A and B.

However, a monoclonal antibody that reacted with slow

growing X. campestris pv. citri group B also reacted with X.








14
campestris pv. citri group C and M3 strain of X. campestris

pv. citri group D from Mexico (Alvarez et al., 1987).

Strains of X. campestris pv. citri group A shared a common

epitope and were distinct from strains of X. campestris pv.

citrumelo (Alvarez et al., 1990). Strains of the latter

pathovar are diverse and sometimes share epitopes with

several other pathovars of X. campestris (Alvarez, 1990). A

monoclonal antibody made to strain X-4600 of X. campestris

pv. citrumelo did not react with any groups of X. campestris

pv. citri and only half of the strains of X. campestris pv.

citrumelo tested (Permar and Gottwald, 1989). Thus, strains

of X. campestris pv. citrumelo are serologically distinct

from groups of X. campestris pv. citri (Alvarez et al.,

1990; Permar and Gottwald et al., 1989).

Genetic Comparisons

Xanthomonas campestris pv. citrumelo apparently differs

from X. campestris pv. citri, and the three groups of the

latter pathovar can be separated on the basis of pathogenic,

physiological and serological characteristics. All of these

characteristics have a genetic basis, but the amount of

genetic variation can not be ascertained by the above

techniques. Several techniques have been used to examine

genetic differences among these groups. At least seven

distinct plasmids exist for X. campestris pv. citri which

include separate profiles for strains of each group

(Civerolo, 1985a). Some strains of X. campestris pv.










citrumelo were reported to have plasmids of 41 or 67

kilobase pairs (Gabriel et al., 1989). In the latter

study, plasmid profiles were not compared with profiles of

strains in groups of X. campestris pv. citri, but at least

one plasmid size class (67 kilobase pairs) was found in

strains of both X. campestris pv. citrumelo and strains of

X. campestris pv. citri group A (Civerolo, 1985a; Gabriel et

al., 1989).

Restriction endonuclease analysis of frequently

occurring recognition sites qualitatively distinguished

between groups of X. campestris pv. citri and X. campestris

pv. citrumelo (Hartung and Civerolo, 1987). This technique

also revealed the heterogeneity of strains of X. campestris

pv. citrumelo and the uniformity of each group of X.

campestris pv. citri.

Whereas the restriction endonuclease analysis cited

above yielded qualitative differences, restriction fragment

length polymorphism (RFLP) analysis allows quantitative

comparisons to be made. Based on RFLP data, strains of each

group of X. campestris pv. citri were relatively uniform,

whereas strains of X. campestris pv. citrumelo were more

diverse (Gabriel et al., 1988, 1989; Graham et al., 1990c;

Hartung and Civerolo, 1989). Strains of X. campestris pv.

citri group A and B were readily differentiated; however,

the degree of similarity between these groups depended upon

the individual study (Gabriel et al., 1988; Hartung and








16
Civerolo, 1989). Strains of X. campestris pv. citri groups

B and D were similar in RFLP comparisons (Gabriel et al.,

1989; Hartung and Civerolo, 1989). Gabriel et al. (1989)

placed X. campestris pv. citri groups B, C, and D together

as X. campestris pv. aurantifolia; however, Hartung and

Civerolo (1989) found a significant difference between the

strain of X. campestris pv. citri group C and the B and D

groups. In all three investigations, the groups of X.

campestris pv. citri were separated from strains of X.

campestris pv. citrumelo by RFLP analysis (Gabriel et al.,

1988, 1989; Hartung and Civerolo, 1989).

Strains of X. campestris isolated from citrus and

noncitrus hosts, including strains of X. campestris pv.

citrumelo, have been compared by RFLP analysis (Graham et

al., 1990c). Those strains capable of eliciting a necrotic

response on citrus, whether of citrus origin or not, were

more related than strains which did not elicit a necrotic

reaction. This study emphasized the diversity of X.

campestris pv. citrumelo and the host range and genetic

overlap between these strains and other pathovars.

Fatty Acid Comparisons

Graham et al. (1990c) also compared fatty acid profiles

of strains of X. campestris of citrus and noncitrus origin.

As with RFLPs, those strains capable of lesion formation on

citrus were more similar to X. campestris pv. citrumelo than

nonlesion-forming strains, regardless of whether they were

isolated from citrus.










Isozvme Comparisons

The three groups of X. campestris pv. citri and strains

of X. campestris pv. citrumelo were also investigated using

isozyme analysis (Kubicek et al., 1989). The results of

this study are in general agreement with the above

discussion of genetic variation. Isozyme analysis revealed

that strains of X. campestris pv. citri group A and B were

relatively homogeneous, whereas strains of X. campestris pv.

citrumelo were diverse. In addition, isozyme analysis

clearly differentiated among the groups of X. campestris pv.

citri and strains of X. campestris pv. citrumelo.

Although Asiatic citrus canker, Cancrosis B and Mexican

lime cancrosis cause similar leaf, stem and fruit spots on

citrus, the diseases and their causal agents differ in

several characteristics. The current situation whereby

three diseases of citrus exist within X. campestris pv.

citri leads to confusion. Perhaps sufficient differences in

host range and/or aggressiveness exist to justify placing

the causal agents of these three diseases in separate

pathovars. The demonstration of pathogenicity through

artificial inoculation is often difficult; therefore,

pathogenicity characters should be complemented with

physiological tests, bacteriophage sensitivities or genetic

determinants which correspond to pathogenicity characters.














CHAPTER 3
PATHOGENIC CHARACTERIZATION


In Florida, two diseases of citrus are caused by

xanthomonads. Asiatic citrus canker, caused by Xanthomonas

campestris pv. citri group A Hasse (Syn. Xanthomonas citri

Hasse) strains, which has a worldwide distribution, causes

erumpent lesions on leaves, stems, and fruits of many citrus

cultivars (Civerolo, 1984). Economic losses from citrus

canker may result from fruit lesions which decrease fresh

fruit value, abscission of fruits or leaves, and regulatory

measures (e.g., shipping restrictions, eradication) designed

to halt the spread of the disease (Civerolo, 1984; Stall and

Seymour, 1983). Citrus bacterial spot, caused by X.

campestris pv. citrumelo (Gabriel et al., 1989) (syn. X.

campestris pv. citri strain E), has only been associated

with nursery plants in Florida (Schoulties et al., 1987).

The majority of outbreaks have occurred on Swingle citrumelo

(Poncirus trifoliata (L.)Raf. x Citrus paradisi Macf.) or

grapefruit varieties (C. paradisi). Lesions on stems and

leaves are flat, variously watersoaked and/or necrotic

(Graham and Gottwald, 1990). Although X. campestris pv.

citrumelo has not caused severe disease loss, regulatory

actions to eradicate citrus canker have been applied to










citrus bacterial spot due to uncertainty surrounding the

biological relationships of these two diseases and their

causal bacteria (Schoulties et al., 1987).

The epidemiological significance of strains of X.

campestris pv. citrumelo compared to strains of X.

campestris pv. citri group A is not fully resolved. The

strains causing citrus bacterial spot have been classified

into aggressiveness types based on the extensiveness of

watersoaking and necrosis on wound-inoculated leaves (Graham

and Gottwald, 1990) and by different interactions on Swingle

citrumelo, Duncan grapefruit and other citrus cultivars

(Graham and Gottwald, 1990; Graham et al., 1990a). The most

severe reactions are associated with the highly aggressive

strain on Swingle citrumelo. Graham et al. (1990c)

suggested, based on host reactions and the genetic

uniformity of the highly aggressive strains, that they are

the only strains that should be classified as X. campestris

pv. citrumelo.

Less aggressive strains, however, have been found on

several citrus cultivars in field nurseries (Graham and

Gottwald, 1990). When leaves of Swingle citrumelo and

Duncan grapefruit are inoculated, lesions are readily formed

and undergo limited expansion (Graham and Gottwald, 1990;

Graham et al., 1990a). While the highly aggressive strains

appear to be spread by wind blown rain in nurseries, the

less aggressive strains are spread only mechanically down










nursery rows on wounded plants (Gottwald and Graham, 1990;

Graham and Gottwald, 1990). Although differences in the

internal populations of different aggressiveness types on

citrus cultivars have been identified in the greenhouse

(Graham et al., 1990a), such differences have not been

demonstrated in the field. In addition, it is not clear how

external populations differ among aggressiveness types.

Leaf surface populations are important in the spread and

development of citrus canker (Dan6s et al., 1984; Gottwald,

et al., 1988b; Stall et al., 1980).

The relationships were investigated among internal and

external bacterial populations and lesion expansion on

leaves for X. campestris pv. citri group A and X. campestris

pv. citrumelo on Swingle citrumelo and grapefruit, the

cultivars most commonly affected by citrus bacterial spot in

Florida nurseries. Different inoculation and sampling

methods were used in an attempt to relate aggressivess of

the two pathovars and strains of X. campestris pv. citrumelo

to differences in population dynamics in leaves and the

availability of leaf surface populations for spread. A

preliminary report of a portion of this study has been

published (Egel et al., 1988).

Materials and Methods

Bacterial strains. All strains were isolated by the

Florida Department of Agriculture and Consumer Services,

Division of Plant Industry (DPI) except for X. campestris










pv. citri group A strain MF23P which was isolated by T.

Riley. Three strains of X. campestris pv. citrumelo Fl (DPI

no. 84-3048), F6 (DPI no. 84-3401), and F100 (DPI no. 85-

12869) were previously determined to be highly, moderately,

and weakly aggressive on Swingle citrumelo and Duncan

grapefruit respectively (Graham and Gottwald, 1990). Based

on morphological and physiological tests which identified

strains to the species X. campestris (Schoulties et al.,

1987) and their reaction on host to determine pathovar

(Gabriel et al., 1989; Graham and Gottwald, 1990; Graham et

al., 1990a), strains were classified as either X. campestris

pv. citri group A or X. campestris pv. citrumelo. The two

strains of X. campestris pv. citri group A, MF23P and 9771,

were equally aggressive in wound inoculations like those

previously reported (Graham and Gottwald, 1990). For

inoculation, bacteria were cultured 12-15 hr in Difco

(Detroit, MI) nutrient broth (NB), harvested by

centrifugation at 8000 g for 15 min, and resuspended in

sterile tap-water. Bacteria were adjusted turbidimetrically

to 1 x 108 colony forming units (cfu)/ml (0.1 absorbance at

600 nm) and appropriate dilutions made. Final populations

were determined by plating on Difco nutrient agar or

nutrient-glucose agar (Difco nutrient agar amended with 0.1

g glucose/L).

Population dynamics after injection-infiltration of

leaves. Experiments were conducted with strain 9771 of X.










campestris pv. citri group A and the 3 strains of X.

campestris pv. citrumelo Fl, F6 and F100 on Swingle

citrumelo and Duncan grapefruit in a quarantine greenhouse

at DPI, Gainesville, FL. Seedlings were cut back to produce

uniformly susceptible immature leaves (3/4 to fully

expanded). These were inoculated by injecting 104 cfu/ml

into the mesophyll of the leaf with a 26 gauge needle which

resulted in initial populations of 102 to 103 cfu/cm2 of leaf

area. Populations of bacteria per cm2 were estimated at 0,

1, 5, 10, 20, 30 and 40 days in a leaf disk (0.6 or 0.28

cm2) harvested from a randomly chosen site within the

inoculated area and grinding the tissue in 1 ml phosphate

buffer (0.075 M, pH 7.0) in a glass tissue homogenizer.

Dilutions of the extracts were plated on nutrient agar

amended with chlorothalonil (Bravo 720, a.i., 12 mg/L).

Populations were expressed as the log transformation of

cfu/cm2 of leaf area. Each treatment was replicated five

times and each replicate was represented by one leaf per

seedling. Each experiment involving a particular host was

repeated at least once.

Population dynamics under growth chamber conditions.

Replicate experiments were conducted with Swingle citrumelo

seedlings in a Percival Dew Chamber (Model 1-35 DL,

Percival, Boone IA) at USDA quarantine facilities in

Plymouth, FL. Photoperiods were 10 hr light (28 C, 92%

relative humidity) and 14 hr dark (30 C, 96% relative








23
humidity). Although dew formed daily, mist was applied for

4 hr prior to sampling thereby augmenting moisture on the

leaf surface. The experimental design was a randomized

complete block with five replications per treatment;

seedlings were placed randomly on chamber shelves and

rearranged every third day.

Six-month old seedlings were cut back to produce

uniformly susceptible immature leaves which were inoculated

by puncturing each side of the midvein with a 26 gauge

syringe needle and applying a 10 gl drop of a 108 cfu/ml

suspension to the adaxial side of the puncture wounds. All

four strains (MF23P, Fl, F6, and F100) were inoculated on to

each leaf with a minimum of five leaves treated.

Internal populations in leaves were determined by

removing lesions with a cork borer. Lesion diameter was

measured to the nearest 0.5 mm with a micrometer. Tissue

was ground in 2 ml of phosphate buffer, and the suspension

plated on KCB semiselective medium (NA plus kasugamycin 16.0

mg/L, cephalexin 16.0 mg/L, and chlorothalonil (Bravo 720)

12.0 mg/L) (Graham and Gottwald, 1990). Lesions were

sampled at 10, 20, 32, and 41 days after inoculation.

Populations were expressed as the log cfu/lesion.

External populations on leaves were evaluated by

absorbing the moisture from the adaxial surface of

individual lesions with a sterile, cotton swab after an

overnight dew cycle. Swabs were placed in 5 ml of phosphate










buffer, sonicated for 3 min, and incubated for 30 min on a

rotary shaker at room temperature. Sonication did not

adversely affect the viability of bacteria. The solution

was plated onto KCB media. External bacterial populations

were expressed as log cfu/lesion and sampling times were as

above.

Population dynamics under simulated nursery

conditions. Field experiments included X. campestris pv.

citrumelo strains Fl, F6, and F100 on Swingle citrumelo and

Duncan grapefruit at a quarantine facility in Hastings, FL.

The use of X. campestris pv. citri group A strains in field

experiments was prohibited by federal and DPI quarantine

regulations.

Simulated nurseries consisted of 4 rows of 25 seedlings

(20-30 cm tall) of each cultivar spaced 10 cm apart within

rows and 30 cm between rows with 10 m between plots. Each

plot was separated by nylon screening as wind breaks to

prevent spread of bacteria among plots. Plants were

inoculated by mechanically rubbing a 108 cfu/ml mixture of

each strain with carborundum onto the upper and lower

surface of leaves. The experiment was a 3 x 2 factorial

where each treatment consisted of a strain-cultivar

combination. Daily minimum temperatures during the

experiment (spring 1989) ranged from 8 C to 23 C and maximum

temperatures ranged from 25 C to 35 C. Total rainfall for

the experimental period was 117 mm. Seedlings were










inoculated on 1 September 1988 and 10 May 1989. Although

conducted during different times of the year, the results of

the two experiments reinforced each other and data from the

second experiment are reported.

After symptoms appeared (in ca. 14 days), seven

seedlings were chosen as replicates in each treatment with

five lesions chosen on each seedling. These lesions were

measured to the nearest 0.5 mm with a micrometer at

approximately 7 day intervals until 56 days post-

inoculation. To estimate external populations, lesions on

the adaxial surface of leaves were swabbed at about 8-9 am

when dew was present. The moisture present on all five

lesions of a single plant was absorbed onto a single sterile

cotton swab. On some dates dew formation was too low to

supply a sample, in which case it was augmented by overhead

irrigation for 30 min prior to sampling (on days 15 and 36).

Each swab absorbed, on average, 25 pl moisture per seedling.

Swabs were placed in 5 ml of phosphate buffer and held at 4

C (not more than 24 hr) until samples could be plated.

Vials were vortexed 10-20 sec, 0.5 ml removed and plated at

the appropriate dilutions on KCB medium.

Internal populations in leaf lesions were estimated

from seven randomly chosen lesions from each treatment at 7

day intervals until 70 days post-inoculation. Lesions were

removed with a cork borer and held at 4 C until processed.

Lesions were measured to the nearest 0.5 mm with a








26
micrometer and then ground in 2 ml of phosphate buffer in a

tissue homogenizer and the extract plated onto KCB media.

Statistical analysis. Population and lesion diameter

data for each date were compared by ANOVA; if the F test was

significant at the 0.05 level, means were compared by

Tukey's HSD-procedure (a=0.05) for each date. Both GLM and

Tukey procedures were run using SAS (Statistical Analysis

Systems, Cary, NC). In the simulated nursery experiment,

significant interactions were present at the a=0.05 level

for cultivar x strain on several dates; therefore, strains

were compared separately on each cultivar.

Results

Population dynamics after iniection-infiltration of

leaves. Strains Fl and F6 of X. campestris pv. citrumelo

caused indistinguishable flat lesions with watersoaked,

necrotic centers, and chlorotic halos 5-7 days after

inoculation by injection-infiltration. Lesions elicited by

X. campestris pv. citrumelo strain F100 developed slowly (8-

10 days after inoculation) as small, reddish, raised spots

and expanded into slightly raised necrotic areas with little

watersoaking or chlorosis. In contrast, lesions caused by

X. campestris pv. citri group A strain 9771 appeared as

raised green spots (5-7 days after inoculation), expanded

quickly and eventually became erumpent, necrotic lesions

with marginal watersoaking and chlorosis.










On both hosts, internal leaf populations of strains

9771, Fl, and F6 increased rapidly up to 5 days, peaked by

20 days, and slowly declined thereafter (Fig. 3-1). Except

on day five for Swingle citrumelo, the populations of these

three strains were not different on either cultivar. Nor

were cultivar-strain interactions detected. Strain F100

reached populations that were ca. 2 log units lower than any

other strain; this difference was generally significant for

all dates and both cultivars (Fig. 3-1). In Duncan

grapefruit, strain F100 was not detected at 30 and 40 days

after inoculation (Fig. 3-1B).

Population dynamics under growth chamber conditions.

More differences among strains in bacterial population

dynamics in and on lesions were detected using the pin-prick

inoculation method than were detected by the injection-

infiltration method (Fig. 3-1, 3-2). Internal leaf

populations of X. campestris pv. citri group A strain MF23P

and X. campestris pv. citrumelo strain Fl in Swingle

citrumelo were generally not different (Fig. 3-2A) but were

significantly higher than populations of strain F6 by day

30. As previously indicated by injection-infiltration,

strain F100 generally produced lower populations than any

other strain (Fig. 3-2A).

There were fewer differences among strains in external

populations on leaves sampled by absorbing dew off of the

lesions with swabs. At 10 and 20 days, populations of X.










campestris pv. citri group A strain MF23P were higher than

strains of X. campestris pv. citrumelo, but populations of

strains F1 and F6 were similar (Fig. 3-2B). Moreover,

internal populations of strain F100 significantly differed

from strains F1 and F6 only on days 20 and 32 (Fig. 3-2B).

Thus, under dew-forming conditions in the growth chamber,

external populations among the strains were not as readily

distinguishable as internal populations.

Expansion of the erumpent lesions elicited by X.

campestris pv. citri group A strain MF23P was not comparable

to the flat lesions produced by the strains of X. campestris

pv. citrumelo (Fig. 3-2C). Strains Fl and F6 produced

larger lesions than strain F100 and strain MF23P. Lesion

expansion by strains F1 and F6 was indistinguishable as was

that of strains F100 and MF23P.

Considering both X. campestris pv. citri group A and X.

campestris pv. citrumelo strains, external populations were

well correlated with internal populations (r=0.64) at 41

days but were less correlated with lesion diameter (r=0.14).

If X. campestris pv. citri group A, which did not form a

comparable lesion type, was excluded from the analysis, then

internal and external populations and lesion diameters of

strains of X. campestris pv. citrumelo were all well

correlated with each other at 41 days (Table 3-1). Those

correlations which included external populations tended to

be lower.








29

Population dynamics under simulated nursery conditions.

There were significant host-strain interactions among the

three strains of X. campestris pv. citrumelo on the two

cultivars when populations in lesions produced by wound

inoculation were compared (Fig. 3-3A, 3-3B). Internal

populations usually exceeded 106 cfu/lesion for strain Fl on

Swingle citrumelo and were higher than populations of all

other host-strain combinations. Internal populations in

lesions produced by Fl and F6 on Duncan grapefruit generally

did not differ significantly. Strain F100 populations were

consistently below 104 cfu/lesion and were not detectable

after day 49 and 56 on Duncan grapefruit and Swingle

citrumelo, respectively (Fig. 3-3A, 3-3B). Overall,

populations in lesions were stable for strain Fl on both

hosts, but fluctuated for strain F6 and were constantly

dropping for strain F100.

Bacterial populations sampled from the dew on the

surface of leaf lesions followed the same general trends as

internal populations (Fig. 3-3C and 3-3D). For Swingle

citrumelo, external populations of strain Fl were usually

significantly higher than populations of F6 and generally

higher than any other host-strain combination (Fig. 3-3C and

3-3D). On Duncan grapefruit, populations of strains Fl and

F6 were not significantly different throughout the sampling

period (Fig. 3-3D). External populations on lesions

produced by strain F100 were generally less than 10



















Table 3-1. Correlation of internal and external populations and
lesion diameters at 41 days for strains of Xanthomonas campestris
pv. citrumelo of different aggressiveness types on Swingle
citrumelo (SC) and Duncan grapefruit (DG) in growth chamber and
field experiments.


Growth chamber


Correlation of:

Internal vs external

Internal vs lesion


diameter


SC

0.56


0.83


Field


SC

0.79


0.87


DG

0.84


0.94


External vs lesion


0.55 0.85


diameter


0.88



























a a a b a
06 b a 6- b a
0 a


2 9771 F1 F6 F100 2

0 0
0 10 20 30 40 0 10 20 30 40
DAYS POST INOCULATION






Fig. 3-1. Populations of Xanthomonas campestris pv. citri
strain 9771 and 1. p. citrumelo strains Fl, F6, and F100 in
leaves of greenhouse-grown seedlings of Swingle citrumelo
(A) and Duncan grapefruit (B) leaves inoculated by an
injection-infiltration method in the greenhouse. Each data
point is the mean of five replications. Mean values on each
sampling date are significantly different from means for
treatments on that same date that are not accompanied by the
same letter by Tukey's HSD, a=0.05.








10
A a INTERNAL 32
8- a a


U- c

O 4
MF23P F1
2 F6 F100

0
10 20 30 40
10
B EXTERNAL
b8 a a
z
o ba a
bb a
w 6 b

a be
b c
2 4 b c

2

01
10 20 30 40

E------------------^ \
-C
E :b
Saab
E a a


0cb
b

W a

5 ^b
Z 2- b
(5
-J
0
10 20 30 40
DAYS POST INOCULATION
Figure 3-2. Internal (A) and external (B) leaf populations
of Xanthomons campestris pv. citri strain MF23P and X. c.
citrumelo strains Fl, F6, and F100 and expansion of lesions
(C) on Swingle citrumelo seedlings inoculated by a pin-prick
method under growth chamber conditions. Each data point is
the mean of five replications. Mean values on each sampling
date are significantly different from means for treatments
on that same date that are not accompanied by the same
letter by Tukey's HSD, a=0.05.



































Figure 3-3. Internal and external populations of
Xanthomonas campestris pv. citrumelo strains Fl, F6 and F100
and expansion of lesions on Swingle citrumelo (A, C, E) and
Duncan grapefruit (B, D, F) leaves inoculated by a wounding
method in field nurseries. Each data point is the mean of
seven seedlings. Mean values on each sampling date are
significantly different from means for treatments on that
same date that are not accompanied by the same letter by
Tukey's HSD, a=0.05.
























b b b
CL b b


U. C
b/

o b

82-

INTERNAL
0
10 20 30 40 50 60






8 a
4 a

2 a b b b b


EXTERNAL
0
10 20 30 40 50



-3E
E .
a


S2- b b B



b C C C C
wu b

10 20 30 40 50


DUNCAN GRAPEFRUIT B


b
EXTERNAL b
0
10 20 30 40 50


F
3 -- F1 -- F6 -- F100



2 a a
a a
1 a a a a
Sa a a


c c c c c
60 10 20 30 40 60
60 10 20 30 40 50 60


DAYS POST INOCULATION


-o
a



b
-o


iB
C


~










cfu/lesion on Duncan grapefruit but were higher on Swingle

citrumelo and similar to populations of F6 (Fig. 3-3C, 3-

3D).

The ranking of aggressiveness types by lesion expansion

after 56 days (Fig. 3-3E, 3-3F) corresponded with rankings

by bacterial populations levels in and on lesions. Lesions

produced by strain F1 on Swingle citrumelo continued to

expand up to 49 days and the final lesion diameter far

exceeded that on other host-strain combinations (Fig. 3-3E,

3-3F). These watersoaked lesions sometimes coalesced,

producing leaf abscission and stem dieback. In contrast to

strain Fl, lesions elicited by strain F6 and F100 appeared

dry and stopped expanding after 20 days on both hosts. On

Duncan grapefruit, lesions produced by strain F100 did not

appear until after 28 days and did not expand thereafter.

When all three X. campestris pv. citrumelo strains were

considered, lesion diameter at 41 days on Swingle citrumelo

and Duncan grapefruit was highly correlated with both

internal and external populations on lesions (Table 3-1).

Internal and external populations were also well correlated

with each other. The correlations derived from field were

higher than for the same comparisons on Swingle citrumelo in

the growth chamber.

Discussion

Strains of X. campestris pv. citrumelo varied in

external and internal populations and lesion diameters










produced upon wound inoculation of Swingle citrumelo and

Duncan grapefruit in simulated nurseries. The highly

aggressive strain Fl on Swingle citrumelo produced higher

external and internal populations and lesion diameters than

all other strain-cultivar combinations in the field. These

results confirm and extend similar greenhouse experiments in

which the highly aggressive F1 strain had higher populations

in lesions on Swingle citrumelo and its parent trifoliate

orange than on other citrus cultivars (Graham et al.,

1990a). In addition, external and internal populations and

lesion diameters were strongly correlated for the different

aggressiveness types of X. campestris pv. citrumelo in the

field, although less so in the growth chamber. These

findings explain why the highly aggressive strains of X.

campestris pv. citrumelo appeared to be spread by wind

driven rain in field situations, but the less aggressive

strains probably spread only by mechanical means (Gottwald

and Graham, 1990; Gottwald and Graham, unpublished data).

These data strengthen the contention that, among the

aggressiveness types, the highly aggressive strains are the

only pathogens of Swingle citrumelo and related cultivars

that should be classified as X. campestris pv. citrumelo

(Graham et al., 1990c).

Leaf surface populations, whether from lesions as in

this study or living epiphytically, serve as an important

source of inoculum for pathogen spread (Crosse, 1959;










Crosse, 1963; Crosse, 1966; Crosse and Bennett, 1955;

Ercoloni et al., 1974). Such external populations have been

related to internal populations for Xanthomonas campestris

pv. phaseoli (Cafati and Saettler, 1980b) and Pseudomonas

phaseolicola (Stadt and Saettler, 1981) on beans.

Monitoring internal populations in leaf lesions may be used

to determine the potential of a pathogen to spread and may

serve as an alternative to estimating leaf surface

populations which are highly variable and more difficult to

sample.

Using more artificial inoculation methods under

greenhouse or growth chamber conditions, there were not

clear population differences between X. campestris pv. citri

group A and X. campestris pv. citrumelo or among the X.

campestris aggressiveness types. The internal populations

of injection-infiltrated leaves were probably not an

accurate indication of the capability of these strains to

develop in lesions and become available for spread in the

field. For example, lesions produced by the highly and

moderately aggressive strains were indistinguishable on

Swingle citrumelo after injection-infiltration. Injection

into the leaf mesophyll may deliver bacteria to many

susceptible sites over a large area. In contrast, by

inoculating with a wounding technique, population growth

depended on lesion expansion from a relatively small point

of introduction. The pin-prick technique was demonstrated










previously to be more effective than either leaf spray or

injection of X. campestris pv. citrumelo to determine

susceptibility of citrus cultivars (Garran, 1988), and to

determine the host-strain interaction of X. campestris pv.

citrumelo strains (Graham and Gottwald, 1990; Graham et al.,

1990a).

Likewise, the highly conducive dew-forming conditions

in the growth chamber were inappropriate for distinguishing

leaf surface populations on Swingle citrumelo of highly

aggressive strain populations from the less aggressive

strains. O'Brien and Lindow (1989) found that differences

in epiphytic populations were more likely to be demonstrated

with wetting and drying cycles than in constantly humid

conditions. In these growth chamber experiments, humidity

was maintained at 92% or higher, which probably accounted

for the lack of differences in leaf surface populations of

the highly and moderately aggressive strains on Swingle

citrumelo. Field conditions of wetting and drying cycles

more truly demonstrated the greater potential for the highly

aggressive strains to produce bacteria on leaf surfaces of

Swingle citrumelo than in any other strain-cultivar

combination.

Furthermore, under field conditions it was possible to

ascertain whether strains of X. campestris pv. citrumelo

behave as true epiphytes in the absence of internal

populations. Internal populations of the weakly aggressive

strain F100 were not detectable by day 56 and day 49 on










Swingle citrumelo and Duncan grapefruit respectively,

whereas external populations of F100 were detected beyond

this time. Sampling by absorbing moisture off of lesions

was designed to detect bacteria under conditions conducive

for exudation and survival, i.e., mornings when dew formed

an excellent microclimate for bacterial activity on leaves.

Sampling of lesions for internal populations was conducted

at midafternoon, when leaves were dry and conditions

inhospitable for bacterial survival at least on the leaf

surface (Graham, unpublished data). Thus, bacterial

populations which existed on the lesion surface were not

detected when sampling for internal populations. The

continued presence of F100 on leaf surfaces in the absence

of internal populations may indicate that F100 is capable of

an epiphytic existence independent of lesion populations

(Leben, 1981). Capability for epiphytic survival by X.

campestris pathovars has been demonstrated on host and non-

host plants in the absence of disease (Cafati and Saettler,

1980a; Mulrean and Schroth, 1982; Timmer, et al., 1987;

Wrather et al., 1986). Multiplication of strains of X.

campestris pv. citrumelo on leaf surfaces may have played a

role as an inoculum source for the original outbreaks of

citrus bacterial spot in citrus nurseries where no obvious

source of bacteria was observed.














CHAPTER 4
GENOMIC CHARACTERIZATION


Asiatic citrus canker, caused by Xanthomonas campestris

pv. citri, is world wide in distribution and has been known

since at least 1899 (Tanaka, 1918). In 1984, strains of X.

campestris were isolated from leafspots in a central Florida

citrus nursery which seemed to cause a different disease

(Schoulties et al., 1987). This disease has been designated

Citrus bacterial spot (Graham and Gottwald, 1988), and the

causal organism named X. campestris pv. citrumelo (Gabriel

et al., 1989).

Several methods have been used in an effort to

characterize these two pathovars and the relationship

between them. Restriction endonuclease analysis of

frequently occurring recognition sites revealed that strains

of X. campestris pv. citrumelo are heterogeneous and

distinct from strains of X. campestris pv. citri group A

which are relatively homogeneous (Hartung and Civerolo,

1987). These relationships were confirmed and quantified by

isozyme (Kubicek et al., 1989) and restriction fragment

length polymorphism (RFLP) analyses (Gabriel et al., 1988,

1989; Hartung and Civerolo, 1989, Graham et al., 1990c).

Similarity values of RFLPs were relatively low among weakly








41
aggressive strains of X. campestris pv. citrumelo (Graham et

al., 1990c). Based on these similarity values and weak

pathogenicity of many of these strains, it was suggested

that some strains of X. campestris pv. citrumelo had been

only incidentally isolated from citrus (Gabriel et al.,

1989; Graham et al., 1990c). In addition, several pathovars

of X. campestris were found to be genetically related to

strains of X. campestris pv. citrumelo, and pathogenicity

tests indicated an overlap of host ranges (Gabriel et al.,

1988, 1989; Graham et al., 1990c; Hartung and Civerolo,

1989). Finally, on the basis of RFLP data, Gabriel et al.

(1989) proposed to elevate X. campestris pv. citri group A

to species status and to separate Florida nursery strains

into X. campestris pv. citrumelo.

Although the above studies have yielded a level of

understanding of the variability among these bacteria, the

strains involved have yet to be compared using DNA

reassociation. Reassociation of DNA is a standard technique

which has been useful in estimating genetic distance between

bacterial strains (Grimont, 1988). Many of these strains

have not been previously described and their taxonomic

status is uncertain; it has been recommended that final

taxonomic designations include DNA reassociation analysis

(Wayne et al., 1987).

The presence of several published RFLP analyses on

strains of X. campestris pv. citri and X. campestris pv.

citrumelo (Gabriel et al., 1988, 1989; Graham et al., 1990c;










Hartung and Civerolo, 1989) presents an excellent

opportunity to compare conventional RFLP analyses to

restriction endonuclease analysis of infrequently occurring

recognition sites in genomic DNA fragments separated by

pulsed field gel electrophoresis (Cooksey and Graham, 1989;

Grothues and Tummler, 1987; Le Bourgeois et al., 1989;

Sorbral et al., 1990; Tanskanen et al., 1990). Restriction

endonuclease analysis has been useful in discriminating

between closely related strains of Pseudomonas syringae pv.

tomato and X. campestris pv. vesicatoria (Cooksey and

Graham, 1989). In this study, restriction endonuclease

patterns were used to analyze diversity within groups of

closely related strains.

By using DNA reassociation and restriction endonuclease

analysis, the genetic distance among and between strains of

X. campestris pv. citri and X. campestris pv. citrumelo was

determined. In addition, the genetic distance between the

above pathovars and other X. campestris pathovars, espe-

cially those pathovars or strains which form lesions on

citrus was of interest. The final objective was to compare

similarities generated by DNA reassociation and restriction

endonuclease analysis to published similarities generated by

other techniques.

Materials and Methods

Culture conditions. The taxonomic designations and

sources of bacterial strains are listed in Table A-1.

Before use, all strains were streaked onto nutrient agar










(Difco, Detroit, MI) or Lima bean agar (Difco) and single

colonies selected. Nutrient broth cultures were grown 12-16

hours on a circular shaker (150 rpm) at 30 C. Strains of X.

campestris pv. citri group B were grown in a sucrose based

medium (Canteros de Echenique et al., 1985). Long term

storage of bacteria was in nutrient broth/glycerol (85/15%,

v/v) at -70 C.

DNA isolation. A procedure modified from Boucher et al.

(1987) was used to extract DNA for DNA reassociation

experiments. Bacterial cells from 300 ml nutrient broth

were pelleted by centrifuging 10 min at 8000 g. The

resulting pellet was washed in 40 ml TE8 (50 mM Tris, pH

8.0; 20 mM EDTA, pH 8.0), pelleted again and resuspended in

15 ml TE8. Deoxynucleases were inactivated by incubating

the cells at 70 C for 15 min. After allowing the

preparations to cool to room temperature, Proteinase K

(Boehringer Mannheim Indianapolis, IN) and N-lauryl sarkosyl

(Sigma, St. Louis, MO, sodium-salt) were added for a final

concentration of 200 Ag/ml and 0.5%, respectively. After

incubation at 50 C for at least 15 hours, preparations were

removed from the water bath and allowed to cool to room

temperature. Each preparation was made to 2 M ammonium

acetate. Five mls of phenol(Fisher):chloroform:isoamyl-

alcohol (25:24:1) was added, the preparations were shaken by

hand for 10 min and centrifuged 10 min at 8000 g. The

aqueous phase was removed and the extraction repeated until










there was no protein interface (at least 3X). DNA was

precipitated by the addition of 2 volumes of cold 95%

ethanol and spooled with a heat sealed pasteur pipet. After

allowing the DNA to air dry a few minutes, the DNA was

dissolved in T1,E, (10 mM Tris, pH 8.0; 1 mM EDTA, pH 8.0).

The removal of RNA was accomplished by adding 500 Ag RNase A

(Sigma, type III-A) and 500 units RNase T1 (Sigma) to each

10 ml preparation and incubating at 37 C for 60 min. After

RNase treatment, 5 ml of a chloroform:isoamyl alcohol

mixture (24:1) was added to each preparation which was

shaken 10 min by hand and centrifuged as above. This step

was repeated as needed. The aqueous layer was removed and

the DNA precipitated as above. The DNA was dissolved in 0.1

X SSC (15 mM sodium chloride; 1.5 mM sodium citrate) and

adjusted spectrophotometrically (260 nm) to 400 Ag/ml and

sheared by passing 3X through a French Press (16,000 psi).

DNA reassociation. The experimental procedure

described herein has been adapted from Johnson (1985).

Sheared DNA was radiolabelled using the random primed method

according to instructions of the manufacturer (Boehringer

Mannheim) to incorporate tritiated dCTP into genomic DNA.

Reassociation reaction mixtures consisted of 10 Al

denatured, labelled DNA (approx. 0.02 Ag), 50 pl of

denatured, unlabelled DNA (20 Mg), 25 pl of salt buffer

(5.15 M NaCl, 3 mM HEPES buffer, pH 7.0, final concentration

equivalent to 6 X SSC) and 25 Al of formamide (Boehringer










Mannheim, electrophoretic grade) (final concentration,

22.7%, deionized by mixing with amberlite MB-3 resin, 10 %

w/v) added to 500 Al microcentrifuge tubes. In this

fashion, radiolabelled DNA from one strain was compared to

DNA of several strains of interest. Each experiment

included replicate samples of each comparison and four

replicate homologous controls (labelled and unlabelled DNA

from the same strain) and four controls to measure self-

reassociation of labelled DNA using sheared, native, herring

sperm DNA (Boehringer Mannheim) for the unlabelled DNA.

The DNA was allowed to reassociate for 20 hrs submerged

in a 57 C water bath. At the end of this period, 24 Ag of

sheared, denatured herring sperm DNA and 300 units of Sl

nuclease (Bethesda Research Laboratories, Bethesda, MD) were

added to each tube, and the tubes incubated in a 50 C water

bath for 1 hour to eliminate single stranded DNA. Double

stranded DNA was precipitated by adding 30 Ag sheared,

native, herring sperm DNA and 1/5 volume of ice cold acid

solution (1 N HC1, 10% (w/v) sodium pyrophosphate and 10%

(w/v) momobasic sodium phosphate) and incubating on ice

1 hr.

The temperature of 57 C for reassociation experiments

was derived from the midpoint of the mol % GC range for the

species X. campestris (63.5-69.2, Bradbury, 1984). The

midpoint, 66.4, can be converted into a Tm value (96.5 C)

using Marmur's equation (Marmur and Doty, 1962) (Tm=69.3 +








46

0.41[% GC content]). Standard reassociation experiments are

conducted 25 C below Tm (Johnson and Ordall, 1968), so 96.5

C 25 C=71.5 C. This temperature is lowered 0.7 (Maniatus

et al., 1982) or 0.61 (Johnson, 1985) degrees for each 1%

formamide used; therefore the final figure is 57 C (using a

value midway between 0.7 and 0.61 degrees for each percent

formamide).

Labelled double stranded DNA was quantified using a

method modified from Preston et al. (1975). After

precipitation, the reassociated, double stranded DNA was

separated from unincorporated nucleotides by filtering

through Whatman GF/C glass fiber filters at 15 psi vacuum.

Each sample was washed 3X with 10 to 15 ml of the acid

solution and subsequently 3X with 10 to 15 ml 95% ethanol.

The vacuum was maintained for an additional 10 minutes to

dry the filters. Additional drying was accomplished by

incubating the filters in an oven at 50-55 C overnight.

Filters were placed in 5 ml scintillation cocktail and

radioactivity quantified by liquid scintillation counting.

Each sample contained 0.13 pci of tritiated dCTP

incorporated into linear genomic DNA. This level of

radioactivity usually generated between 20,000 and 30,000

cpm for the homologous controls and 10 to 20% of this value

for reassociation controls. Heterologous controls ranged

between these values depending on the similarity of the DNA

being compared. Counting efficiency was 17%.

Percent similarities were calculated by first










subtracting reassociation control values from all samples

and then calculating the percent of each comparison from the

homologous value. All values herein represent the mean of

at least two experiments. The similarity values generated

by each experiment were considered a replication and

standard errors were calculated based on these replications.

Restriction endonuclease analysis. The methods used

were similar to those reported earlier (Cooksey and Graham,

1989). Each strain was grown for 12-16 hrs at 30 C at 150

rpm on a rotary shaker in nutrient broth inoculated from a

single colony. Cells were pelleted in a Beckman micro-

centrifuge, washed once in SE buffer (NaCl 75 mM, EDTA 25

mM, pH 8.0) with the cells finally resuspended in 0.5 ml SE

buffer. The cell suspension was mixed with 0.5 ml melted,

cooled, low melting point agarose solution (10 mM Tris, pH

8.0; 10 mM MgC12; 0.1 mM EDTA, pH 8.0; 2% w/v LMP agarose,

Bethesda Research Laboratories, in sterile distilled water)

and pipetted into a Bio-rad (Richmond, CA) plastic mold.

The mold was then placed at 4 C for 10-15 min. After the

agarose had hardened, the inserts were removed from the mold

and transferred to lysing solution (0.5 mg/ml Proteinase K;

1% w/v N-lauryl sarkosyl; 0.5 mM EDTA, pH 9.5) in sterile

tubes. The tubes were placed in a 50 C water bath and the

cells lysed overnight (at least 15 hrs).

After lysis, the inserts were removed from the lysis

solution and placed in sterile TE buffer. After 15 min at










room temperature, the TE solution was changed and the

inserts incubated for an additional 6-8 hrs. The inserts

were then removed from the TE buffer and a 1-2 mm slice cut

from the insert and placed in a microcentrifuge tube with

200 1l restriction buffer (as obtained from the

manufacturer, Boehringer Mannheim); the remainder of the

insert was saved in 250 mM EDTA, pH 8.0, at 4 C. After 15

min incubation at room temperature, the restriction buffer

was changed and 30 units of either Xba I or Spe I

(Boehringer Mannheim) restriction enzyme was added.

Microcentrifuge tubes were then incubated at least 8 hrs at

37 C in a horizontal position. After incubation, the

restriction buffer was removed and 500 pl lysing solution

(without proteinase K) was added. Samples were incubated at

50 C in a water bath for 2 hrs, the lysing solution changed,

and the samples incubated for an additional 2 hrs at room

temperature.

The agarose slices were then ready for electrophoresis.

The slices were placed in the wells of a 1% gel made with

0.5 X TBE (44.5 mM Tris, 44.5 mM boric acid, 1 mM EDTA, pH

8.0) and the wells sealed with cooled 1% agarose. The gel

was placed in a BIO-RAD CHEF DR II (Chu et al., 1986) unit

containing approximately 1.6 L of 0.5 X TBE and run at 200

volts (16 V/cm gel). Unless otherwise indicated, pulse

times for DNA restricted with Xba I were 4 sec for 2 hr and

15 sec for 22 hrs and for DNA restricted with Spe I pulse










times were increased linearly from 4 sec to 50 sec for 22

hrs. Phage 1 concatemers from BIO-RAD were used as

molecular markers. Gels were stained in 0.5 mg ethidium

bromide per liter and photographed with type 55 Polaroid

film.

Similarity values were calculated as follows. The

number of fragments greater than 100 kb shared between

strains was recorded for strains on the same gel. The

similarity coefficient was calculated using the following

formula:

S=2ny/(nx + ny),

where nY was the number of fragments shared between two

strains and nx and ny were the total number of fragments

larger than 100 kb for strain x and strain y, respectively.

Comparisons between methods. A summary of the results

presented here was compared with published accounts dealing

with the same pathovars or groups of strains. The published

reports used for comparison purposes were: Gabriel et al.,

1988, 1989; Graham et al., 1990c; Hartung and Civerolo,

1989; and Kubicek et al., 1989. Several investigators

recognized three groups within X. campestris pv. citrumelo

based on aggressiveness and genetic relatedness (Gabriel et

al., 1989; Graham et al., 1990c; Hartung and Civerolo,

1989). These groups were called, respectively, highly,

moderately and weakly aggressive by Graham et al. (1990c);

E2, El and an unspecified group by Gabriel et al. (1989);










and subgroups E, F and G by Hartung and Civerolo (1989).

The similarity values for all comparisons in which both

strains were within a particular group (within group

comparisons) were averaged and the standard deviation

calculated for each applicable investigation. Also

presented is the overall similarity for all comparisons

where both strains were X. campestris pv. citrumelo, again

for each applicable investigation. In the same manner,

similarity values for comparisons in which both strains were

either X. campestris pv. citri group A or B were averaged

and the standard deviation calculated for each applicable

data set. Similarities for between group comparisons are

also presented. Similarity values for all relevant

comparisons were averaged and the standard deviation

calculated.

Results

DNA reassociation. The strains which comprise X.

campestris pv. citrumelo were highly related to one another

by DNA reassociation (Table 4-1). Aggressiveness groups of

X. campestris pv. citrumelo (Graham and Gottwald, 1990) were

not distinguished by DNA reassociation; all strains were ca.

80-90% related, however, there were two comparisons of 76%

(Table 4-1). It is of interest to note that both

comparisons involved strain F100.

Strains of X. campestris pv. citrumelo representing all

three aggressiveness types were ca. 60% related to strain










9771 of X. campestris pv. citri group A (Table 4-2).

Similarly, both strain 9771 and strain Fl of X. campestris

pv. citrumelo were ca. 60% related to the two strains of X.

campestris pv. citri group B (Table 4-2). Therefore,

bacterial strains representing three different diseases of

citrus were only distantly related to each other. Strain

33913 of X. campestris pv. campestris, the type strain of X.

campestris, was 34% related to strain Fl of X. campestris

pv. citrumelo, ca. 40% related to strains B64 and B94 of X.

campestris pv. citri group B and 30% similar to strain 9771

of X. campestris pv. citri group A.

In an attempt to place X. campestris pv. citri and X.

campestris pv. citrumelo within the context of noncitrus

pathovars of X. campestris, strains 9771 and Fl were

compared to several pathovars. Strains of X. campestris pv.

campestris, X. campestris pv. vesicatoria, and X. campestris

pv. phaseoli were not closely related to either strain 9771

or strain Fl (Table 4-3). Strains Xv 56 and Xv 75-3 of X.

campestris pv. vesicatoria each gave different relatedness

values to strain 9771 and Fl. Strain N of X. campestris pv.

malvacearum was only 71% similar to strain Fl, but was 90%

related to strain 9771. Strain Xp 20 of X. campestris pv.

phaseoli was ca. 40% distant to strains 9771 and Fl. Type

strain 33913 of X. campestris pv. campestris was not

closely related to any of the strains in these experiments.

Four strains of X. campestris, not isolated from citrus

but capable of lesion formation on citrus, were compared to










strains of the three aggressiveness types of X. campestris

pv. citrumelo (Table 4-4). Strain X198 of X. campestris

from Strelitzia reainae, strain X151 of X. campestris pv.

fici and strain 82-1 of X. campestris pv. alfalfa ranged

from 80 to 91% related to the strains of X. campestris pv.

citrumelo. However, strain X22j of X. campestris pv.

maculifoliigardeniae ranged from 68 to 76% related to

aggressiveness types of X. campestris pv. citrumelo.

Restriction endonuclease analysis. Strains which

shared common pathogenicity traits were compared on the same

gel. The four pathogenicity groups analyzed here include

two aggressiveness types of X. campestris pv. citrumelo

(moderately and weakly aggressive), and X. campestris pv.

citri groups A and B. The highly aggressive strains of X.

campestris pv. citrumelo could not be compared by

restriction endonuclease analysis due to excessive shearing

of genomic DNA prior to or during lysis. This problem was

also encountered with strain F100 of the weakly aggressive

group.

Seven moderately aggressive strains had few comigrating

DNA fragments when restricted with Xba I or Spe I (Figs. 4-1

and 4-2). The weakly aggressive strains restricted with Xba

I had similarly diverse restriction patterns (Fig. 4-3).

In contrast to the diversity of the moderately and

weakly aggressive strains of X. campestris pv. citrumelo, X.

campestris pv. citri groups A and B each gave characteristic

restriction patterns (Figs. 4-4, 4-5, 4-6, 4-7). The








53

restriction patterns of four of the strains of X. campestris

pv. citri group A were identical to each other as restricted

by Xba I and Spe I (Fig. 4-4 and 4-5, Table 4-5), however

strain T1 exhibited one polymorphism with each restriction

endonuclease reducing its similarity to the other strains.

Although not as homogenous as X. campestris pv. citri

group A from Florida, the strains of X. campestris pv. citri

group B are moderately to highly related as seen in Xba I

and Spe I restriction patterns (Figs. 4-6 and 4-7). These

strains ranged from 0.57 to 0.98 similarity when Xba I and

Spe I derived values were averaged (Table 4-6).

Comparisons between methods. Similarity values for

comparisons within X. campestris pv. citrumelo and X.

campestris pv. citri groups A and B varied by technique and

by investigation. However, the similarity values for

comparisons among highly aggressive strains of X. campestris

pv. citrumelo and among strains of X. campestris pv. citri

group A or X. campestris pv. citri group B tended to be

similar based on all techniques (Table 4-7). Similarity

values for moderately and weakly aggressive strains of X.

campestris pv. citrumelo were generally higher based on DNA

reassociation than for RFLP analyses. Overall similarities

for X. campestris pv. citrumelo comparisons were higher for

DNA reassociation and Hartung and Civerolo (1989) RFLP

analysis than for Gabriel et al. (1989) RFLP or isozyme

analysis (Kubicek et al. (1989). In general, similarity











Table 4-1. Similarity values generated by DNA reassociation
for strains of Xanthomonas campestris pv. citrumelo causing
citrus bacterial spot.


Aggressiveness
Type Strain Fl F6 F100


Highly

aggressive





Moderately

aggressive











Weakly

aggressive


F1

F54

F274

F361


F6

F228

F254

F311

F348

F397



F59

F86

F94

F100

F306


100b

91

84

91


89

86

102

80

91

90



85

99

96

87

100


(0.1)c

(6.9)

(2.8)


(0.9)

(4.4)

(16.0)

(2.3)

(8.1)

(18.9)



(4.2)

(11.4)

(13.3)

(3.2)

(7.9)


NDd

ND

ND


100

96

82

89

84

87



87

92

89

76

90


(4.0)

(6.2)

(6.2)

(4.5)

(3.3)



(4.9)

(4.0)

(4.5)

(7.6)

(0.5)


83

82

92

100

76


(5.9)

(6.3)

(6.6)



(4.9)


aStrains Fl, F6 and F100 represent, respectively,
highly, moderately and weakly aggressive strains of X.
campestris pv. citrumelo as rated by a detached leaf assay
(Graham and Gottwald, 1990).
value represents percentage of homologous value which was
set to 100%.
Standard error of the mean.
Not Done.





















Table 4-2. Similarity values generated by DNA reassociation
for strains of Xanthomonas campestris causing diseases of
citrus.


X. c. pv.
campestris X. c. pv. citri

33913T B64a B94a 9771a


Flb 34 (0.3)c 59 (6.6) 57 (4.8) 56 (3.3)

F6b NDd ND ND 55 (6.4)

F100b ND ND ND 61 (6.3)

9771 30 (4.2) 63 (1.8) 62 (0.1 100e

33913T 100 37 (8.0) 40 (8.7)

rType strain 33913 is the type strain of X. campestris
and causes black rot of crucifers.
aStrains B64 and B94 are X. c. pv. citri group B and cause
Cancrosis B; Strain 9771 is X. c. pv. citri group A and
causes Asiatic citrus canker;
bStrains Fl, F6 and F100 are, respectively, highly,
moderately, and weakly aggressive strains of X. c. pv.
citrumelo and cause Citrus bacterial spot.
Standard error of the mean.
dNot Done
value represents percentage of homologous value which was
set to 100%.




























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Table 4-5. Similarity matrix of strains of Xanthomonas
campestris pv. citri group A, which cause Asiatic citrus
canker, generated by restriction endonuclease analysis of
infrequently occurring recognition sites in genomic DNA
fragments separated by pulsed field gel electrophoresis.



9771 3213 3340 9760 T1



9771 -- 1.00a 1.00 1.00 0.90

3213 -- 1.00 1.00 0.90

3340 -- -- 1.00 0.90

9760 -- -- 0.90

T1


'Values are the average of similarities derived with Xba I
and Spe I.






















Table 4-6. Similarity values of strains of Xanthomonas
campestris pv. citri group B, which cause Cancrosis B,
generated by restriction endonuclease analysis of
infrequently occurring recognition sites in genomic DNA
fragments separated by pulsed field gel electrophoresis.



B84 B93 B148 B80 B64 B69 B94



B84 -- 0.85a 0.98 0.71 0.54 0.78 0.73

B93 -- 0.82 0.76 0.59 0.69 0.73

B148 -- 0.68 0.56 0.82 0.70

B80 -- -- 0.69 0.67 0.78

B64 -- -- -- 0.52 0.60

B69 -- -- -- 0.63

B94 -- -- -- -- -- -- --


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Ha)0+



I~ 02


0
I Q
o
H N


1-1

0
..-I
M


0V 4O
Q mP






,4 .4
-, 94-I 9





*q U-, 0i
o 0go r


40

o) 0) 00 0







0. M, 10
m -- l









KIr 0 -.-Il V
*0m1 1 M C 1




to o o

















r. 0 .,,-
m k l uH 0u
*H Hk 0




0 4
0 *402 *4






H10 44-44'-
W -r4 o Q) 0 0 0



4-, C% O l )
4' 0 --4 >-r-4




>C CCU 0
0' C0 4O i


OOM 4 00

()5e .pr4' a)
00c & 0(0-4'





*e lcmx


u
tp 0
0


01


Z0 ) 0'O

Sw IAmt0o


0U U



oU U
U U
0 0 U















L 2 34 5 6 7 8




388-









97-




Figure 4-1. Restriction endonuclease patterns of moderately
aggressive strains of Xanthomonas campestris pv. citrumelo
restricted with Xba I. Electrophoresis was by Pulsed field
for 1 hr at 4 sec and 22 hr at 15 sec. Lanes: L, phage A
concatamers; 2, F6; 3, F311; 4, F254; 5, F299; 6, 7222; 7,
3274; 8, 540. Molecular sizes are given in kilobases.























824












97



Figure 4-2. Restriction endonuclease patterns of moderately
aggressive strains of Xanthomonas campestris pv. citrumelo
restricted with Spe I. Electrophoresis was by Pulsed Field
for 22 hrs with a pulse time increasing linearly from 4 to
50 sec. Lanes: L, phage I concatemers; 1, F6; 2, F311; 3,
7222; 4, F254; 5, F299; 6, 3274; 7, 540. Molecular sizes
are given in kilobases.



















-340













97





Figure 4-3. Restriction endonuclease patterns of weakly
aggressive strains of Xanthomonas campestris pv. citrumelo
restricted with Xba I. Electrophoresis was by Pulsed Field
for 2 hr at 4 sec and 22 hr at 15 sec. Lanes: L, phage A
concatemers; 1, F306; 2, F59; 3, F94; 4, F86. Molecular
sizes are given in kilobases.













L 1


2 3 4


-291








97





Figure 4-4. Restriction endonuclease patterns of strains of
Xanthomonas campestris pv. citri group A restricted with Xba
I. Electrophoresis was by Pulsed Field for 22 hr with a
pulse time increasing linearly from 1 to 20 sec. Lanes: L,
phage A concatemers; 1, 9771; 2, 3213; 3, 9760; 4, 3340; 5,
Tl. Molecular sizes are given in kilobases.























S-582











-97



Figure 4-5. Restriction endonuclease patterns of strains of
Xanthomonas campestris pv. citri group A restricted with Sae
I. Electrophoresis was by Pulsed Field for 22 hr with a
pulse time increasing linearly from 4 to 50 sec. Lanes: 1,
9771; 2, 3340; 3, 3213; 4, 9760; 5, T1; L, phage X
concatemers. Molecular sizes are given in kilobases.


























388






.. 97







Figure 4-6. Restriction endonuclease patterns of strains of
Xanthomonas campestris pv. citri group B restricted with Xba
I. Electrophoresis was by Pulsed Field for 22 hr with a
pulse time increasing linearly from 2 to 18 sec. Lanes: L,
phage I concatemers; 1, B80; 2, B93; 3, B64; 4, B69; 5,
B148; 6, B84; 7, B94. Molecular sizes are given in
kilobases.


























727











-97




Figure 4-7. Restriction endonuclease patterns of strains of
Xanthomonas campestris pv. citri group B restricted with Spe
I. Electrophoresis was by Pulsed Field for 22 hr with a
pulse time increasing linearly from 4 to 50 sec. Lanes: L,
phage 1 concatemers; 1, B84; 2, B93; 3, B148; 4, B80; 5,
B64; 6, B69; 7, B94. Molecular sizes are given in
kilobases.










values for Gabriel et al. (1989) RFLP analysis values were

lower than similarities obtained by any other technique or

investigator (Table 4-7).

Comparisons of similarity values for between pathovars

are presented in Table 4-8. Similarity values for DNA

reassociation were similar to isozyme values. When X.

campestris pv. citri groups A and B were compared, DNA

reassociation and Hartung and Civerolo (1989) RFLP values

were both ca. 60%. In contrast, when X. campestris pv.

citrumelo was compared to either X. campestris pv. citri

group A or B, both sets of RFLP similarity values were lower

than similarity values generated by DNA reassociation.

Similarity values obtained by Gabriel et al. (1989) RFLP

analysis were similar to Hartung and Civerolo (1989) RFLP

values when X. campestris pv. citri group A was compared to

X. campestris pv. citrumelo, but Gabriel et al. (1989) RFLP

values were much lower than Hartung and Civerolo (1989) RFLP

values (0.16 vs. 0.60, respectively) when strains of X.

campestris pv. citri group A and B were compared.

Discussion

Although the bacterial strains responsible for Citrus

bacterial spot, Asiatic citrus canker and Cancrosis B all

belong to the species X. campestris and cause similar

diseases on related hosts, X. campestris pv. citrumelo and

the two groups of X. campestris pv. citri are only ca. 60%

related to each other by DNA reassociation. The ability to








71

cause these similar diseases on citrus is either represented

by a small portion of the genome and/or these pathogens have

independently evolved means to cause similar diseases.

These three groups of pathogens also differ in the diversity

of strains within each group as revealed by restriction

endonuclease analysis. The strains of X. campestris pv.

citri groups A and B are closely related within each group,

whereas, strains of X. campestris pv. citrumelo responsible

for Citrus bacterial spot are more diverse.

Six DNA-DNA homology groups have been described within

X. campestris, including group 1 which includes several

legume pathovars and has similarity values of 50-90%

(Vauterin et al., 1990a). Strain Fl of X. campestris pv.

citrumelo may belong to DNA-DNA homology group 1 based on

its similarity to strain Xv 75-3 of X. campestris pv.

vesicatoria and strain Xp 20 of X. campestris pv. phaseoli.

Strain 9771 of X. campestris pv. citri group A is similarly

related to strain Xp 20, but is less related than strain Fl

to strain Xv 75-3. Nevertheless, it is possible that both

X. campestris pv. citrumelo and X. campestris pv. citri

belong to DNA-DNA homology group 1.

Strain Xv 56 may belong to a separate X. campestris pv.

vesicatoria group with little similarity to the Xv 75-3

group (Stall et al., unpublished) or any of the described

DNA-DNA homology groups (Vauterin et al., 1990a). Little

similarity was observed between X. campestris pv.










campestris, which belongs to a separate DNA-DNA homology

group (Vauterin et al., 1990a), and any of the other strains

analyzed here. Strain 9771 of X. campestris pv. citri was

similar to X. campestris pv. malvacearum which was not

included in any of the described DNA-DNA homology groups

previously (Vauterin et al., 1990a). Fatty acid profiles of

these two pathogens are also similar (Stall and Hodge,

1989), although X. campestris pv. malvacearum does not

exhibit pathogenicity to citrus (Graham et al., 1990c).

Strain X198 of X. campestris, strain X151 of pathovar

fici, and strain 82-1 of pathovar alfalfa have genomic

similarities to X. campestris pv. citrumelo as shown by DNA

reassociation data presented here as well as RFLP and fatty

acid profiles (Graham et al., 1990c) and share the ability

to cause lesions on citrus (Graham et al., 1990c).

Collectively, these strains may form a group with a broad

host range. If such a population exists, it may have

implications for the origin of Citrus bacterial spot in

Florida and the relatively diverse genetic nature of these

pathogens.

As previously reported (Graham et al., 1990c), X.

campestris pv. maculifoliiqardeniae does not appear to be as

related to X. campestris pv. citrumelo as strain X198 from

Strelitzia sp. or X. campestris pv. fici. However, X.

campestris pv. maculifoliiqardeniae is capable of causing

internal population growth and lesion development on citrus








73

(Graham et al., 1990c). As discussed above, ability to grow

in planta in this pathosystem may constitute a small portion

of the genome and/or may be superimposed on diverse genetic

backgrounds.

The diverse nature of X. campestris pv. citrumelo has

been emphasized previously (Gabriel et al., 1988, 1989;

Graham et al., 1990c; Hartung and Civerolo, 1987; Hartung

and Civerolo, 1989; Kubicek et al., 1989). Although the

diverse nature of this group has been confirmed here with

restriction endonuclease analysis, DNA reassociation

similarity values within the moderately and weakly

aggressive strains of X. campestris pv. citrumelo are

considerably higher than previously published RFLP values

(Gabriel et al., 1988, 1989; Graham et al., 1990c; Hartung

and Civerolo, 1989). Similarly, in a characterization of

pathovars of Pseudomonas syringae, DNA reassociation

similarity values tended to be higher than corresponding

RFLP values (Denny et al., 1988). Whereas similarities

generated by RFLPs provide a relative measure of the genetic

difference between strains, DNA reassociation is valuable in

estimating the total genomic distance between bacterial

strains. By combining both approaches, a more accurate

picture of the relationships among strains emerges. The

RFLP analyses indicate differences between aggressiveness

types; however the DNA reassociation data is interpreted

here to indicate that the total genetic distance among these










strains is relatively small. In contrast, highly related

strains, e.g. of highly aggressive strains of X. campestris

pv. citrumelo, had high similarities regardless of technique

employed. Although diverse as illustrated with RFLP

analyses and restriction endonuclease analysis, X.

campestris pv. citrumelo does not appear to be a collection

of diverse pathovars. Other pathovars of X. campestris may

have been identified as X. campestris pv. citrumelo based on

pathogenicity; if so, however, these pathovars all have

similar genetic backgrounds.

Restriction endonuclease analysis of infrequently

occurring recognition sites and conventional RFLPs generated

by southern blotting and probing, both generate

polymorphisms based on sequence changes, deletions,

insertions, and genomic rearrangements. Similarity values

generated by these two techniques might be expected to be

similar. Similarities for comparisons between X. campestris

pv. citri group A are slightly higher for restriction

endonuclease analysis than for either RFLP analyses. One

reason for this was that the author only had access to

strains of X. campestris pv. citri group A from Florida,

while strains from many geographical regions were available

for the RFLP analyses. The slightly different restriction

pattern of strain Tl of X. campestris pv. citri group A is

probably because it was isolated from a single citrus tree

in Gainesville, FL, and probably had a different origin than










the other strains. All other strains of X. campestris pv.

citri group A were isolated from citrus groves and dooryard

trees south of Tampa, Florida. In contrast, similarity

values for comparisons between strains of X. campestris pv.

citri group B were lower for restriction endonuclease

analysis than for either RFLP analyses. Restriction

endonuclease analysis of infrequently occurring recognition

sites as performed here can detect genomic differences at 12

to 15 sites with a single enzyme. Interlocus variation can

best be overcome by sampling many areas within the genome.

The great power of this type of restriction endonuclease

analysis is the ability to detect large scale genomic

rearrangements. At present, however, determining whether

comigrating bands of diverse strains represent analogous DNA

is difficult (Cooksey and Graham, 1989). For closely

related strains, restriction endonuclease analysis of

infrequently occurring recognition sites is a relatively

simple technique that gives results similar to conventional

RFLP analysis.

In general, the trends noted for characterization

techniques used for within group comparisons were valid for

between group comparisons. Comparisons between stains of X.

campestris pv. citrumelo and either strains of X. campestris

pv. citri group A or B yielded higher similarities with DNA

reassociation than the corresponding RFLP analyses. However,

when the X. campestris pv. citri groups A and B were








76

compared, Hartung and Civerolo (1989) RFLP comparisons were

very similar to the DNA reassociation values, ca. 60%. It

is possible that the latter values were similar because both

X. campestris pv. citri groups are relatively homogeneous,

whereas comparisons involving the heterogenous strains of X.

campestris pv. citrumelo yielded lower RFLP values. As in

group comparisons, Gabriel et al. (1989) RFLP comparisons

between groups tended to be lower than any other comparison.

Isozyme analysis of between strain similarities

indicates a correspondence of isozyme and DNA reassociation

values. Similarities between the values generated by these

techniques has been noted previously (Gilmour et al., 1987).

It is also of interest to note the isozyme variation within

the strains of X. campestris pv. citrumelo is similar to the

difference between the X. campestris pv. citri group A and

strains of X. campestris pv. citrumelo. The large number of

isomorphs observed within the X. campestris pv. citrumelo

group led to a high degree of variability within X.

campestris pv. citrumelo although sequence divergence is not

great as measured by DNA reassociation.

The presence of published characterizations of X.

campestris pv. citrumelo and X. campestris pv. citri offered

an excellent opportunity to compare several bacterial

characterization techniques. One of the difficulties in

making these comparisons was that the published accounts

dealt with different numbers of comparisons and, at times,








77

different strains. All investigators placed strains in the

same groups (three groups of X. campestris pv. citrumelo and

X. campestris pv. citri groups A and B), however, lending

validity to comparisons between investigations. At the very

least, perhaps the above discussion will encourage further

investigations into the relationships between techniques.

The techniques used in this study, DNA reassociation

and restriction endonuclease analysis of infrequently

occurring recognition sites, complement each other well in

the characterization of bacterial strains. The total

genomic distance between bacterial strains or groups of

strains may be estimated by DNA reassociation, while

restriction endonuclease analysis indicates the relatedness

between similar strains. Several techniques are ultimately

necessary in the characterization of any bacterial group.

Relationships derived by the use of just one technique could

be misleading, yet upon consideration of several techniques,

each can be put into its proper context.

It has been proposed that Xanthomonas campestris pv.

citri be elevated to species status solely on the basis of

unique RFLP patterns using the authors probes (Gabriel et

al., 1989). Such a proposal was premature since

insufficient data was presented (Vauterin et al., 1990a).

In addition, DNA reassociation values have been recommended

for such a proposal (Wayne et al., 1987). RFLP values can

be combined with DNA reassociation studies as well as other








78

techniques (Vauterin et al., 1990a). The comparison of the

data presented here with RFLP data of others indicates that

RFLP and DNA reassociation data are not necessarily

synonymous.

Although it has been shown here that strains of X.

campestris pv. citri group A are only ca. 60% related to the

type strain of X. campestris pv. campestris, this is not

sufficient for the elevation of the former strains to

species status (Wayne et al., 1987). The work presented

herein, however, may facilitate the understanding of the

relationships among strains of X. campestris pv. citrumelo

and X. campestris pv. citri as well as other pathovars of X.

campestris. It is hoped that by combining the data

presented here with additional data generated through

different techniques, a stable classification of these

important pathogens may result.












CHAPTER 5
CHARACTERIZATION WITH AN hrV GENE CLUSTER

Xanthomonas campestris pv. citrumelo causes Citrus

bacterial spot primarily on Swingle citrumelo and grapefruit

varieties. Three aggressiveness groups have been identified

among strains of X. campestris pv. citrumelo (Graham and

Gottwald, 1990). The least aggressive strains cause small

(< 1 mm), slightly raised lesions with little or no

watersoaking or chlorosis (Graham et al., 1990a; Graham and

Gottwald, 1990). Both weakly and moderately aggressive

strains are usually associated with injury to the plant

(Gottwald and Graham, 1990; Graham and Gottwald, 1990).

One possible explanation for the limited aggressiveness

of many strains of X. campestris pv. citrumelo is that these

strains are opportunistic. Opportunistic xanthomonads have

been isolated from decayed vegetables (Liao and Wells, 1977)

and from tomato and pepper transplants (Gitaitis et al.,

1977). These strains, like many strains of X. campestris

pv. citrumelo, are limited in pathogenicity and are pectate

positive (Gitaitis et al., 1977).

Opportunistic xanthomonads differ from phytopathogenic

xanthomonads by lacking an intact hrp gene cluster. The hBr

gene cluster is required by plant pathogens to produce

symptoms on susceptible hosts and a hypersensitive reaction

on nonhosts (U. Bonas et al., in press; Boucher et al.,

79










1987; Lindgren et al., 1986). An hrp gene cluster has been

discovered in Pseudomonas syringae pv. phaseolicola

(Lindgren et al., 1986), Pseudomonas solanacearum (Boucher

et al., 1987) and Xanthomonas campestris pv. vesicatoria (U.

Bonas et al., in press). Genomic DNA of opportunistic

xanthomonads hybridized weakly or not at all to the hrp gene

cluster from X. campestris pv. vesicatoria, whereas genomic

DNA from 33 pathovars of X. campestris did hybridize, but

fragments were polymorphic (Stall and Minsavage, 1991).

Strains of X. campestris from citrus, which lacked

pathogenicity on that host, also lacked an hrp gene cluster

homologous to the hrp gene cluster from X. campestris pv.

vesicatoria (J. Graham unpublished). If the less aggressive

strains of X. campestris pv. citrumelo are opportunistic on

citrus, the hrp region might be absent from these strains.

This hypothesis might help explain the origin and the

pathogenicity of many strains of X. campestris pv.

citrumelo.

Alternatively, the less aggressive strains of X.

campestris pv. citrumelo may be representatives of other X.

campestris pathovars that have incidentally been isolated

from citrus (Gabriel et al., 1989; Graham et al., 1990c).

These strains are diverse and some can cause symptoms on

noncitrus hosts (Gabriel et al., 1989; Graham et al.,

1990c). In this case, the hrp gene cluster should be

present but display polymorphisms between pathovars (Stall

and Minsavage, 1991).








81

Plasmid profiles or plasmid sequence analyses have been

used to distinguish pathovars of X. campestris (Lazo and

Gabriel, 1987). If strains X. campestris pv. citrumelo

represent several diverse pathovars of X. campestris, these

strains may also possess diverse plasmid profiles. In

addition, plasmid profiles of strains of X. campestris pv.

citrumelo may be similar to known plasmid profiles of

strains of X. campestris pv. citri groups A and B which

cause similar symptoms on related hosts (Civerolo, 1985a).

To test these hypotheses, genomic DNA of weakly

aggressive and moderately aggressive strains of X.

campestris pv. citrumelo were probed with the hrD gene

cluster from X. campestris pv. vesicatoria. Highly

aggressive strains of X. campestris pv. citrumelo and

strains of X. campestris pv. citri group A were probed for

comparison purposes. Plasmid profiles of these strains were

studied to determine whether plasmid profiles could be used

to distinguish between the less aggressive strains of X.

campestris pv. citrumelo as might be expected if these

strains belonged to different pathovars of X. campestris.

Materials and Methods

Culture conditions. The taxonomic designations and

sources of all strains are listed in Table A-1. Strains were

cultured on nutrient agar (BBL Microbiology Systems,

Cockeysville, MD) or Lima bean agar (Difco, Detroit, MI) and

single colonies were selected for use. Cells were grown in










nutrient broth (BBL Microbiology Systems) overnight (20-24

hrs) or, for X. campestris pv. citri group B, in a sucrose

based medium (Canteros de Echenique et al., 1985) for 30-36

hrs. Long term storage was achieved in glycerol/nutrient

broth (85/15%, v/v) at -70 C.

Hybridization analysis. Genomic DNA was isolated by

the procedure of Boucher et al. (1987) and restricted with

the appropriate restriction endonuclease for 2 hr at 37 C.

Samples were then treated with RNase A (Sigma, Ribonuclease

A, type II-A) for 0.5 hr and electrophoresed in 0.5% agarose

gel containing TAE buffer (40 mM Tris-acetate, 1 mM EDTA, pH

8.2) at 2V/cm of gel. The gel was then denatured in 0.4 N

NaOH and 0.6 M NaCl for 0.5 hr and neutralized for 0.5 hr in

0.5 M Tris, 1.5 M NaCl. The denatured DNA was then

transferred by the procedure of Southern (1975) to a nylon

membrane (Gene Screen Plus, DuPont, Boston, MA).

Hybridizations were performed at 68 C (without formamide)

for 18 to 24 hrs and washed at 68 C with 0.1 X SSC, 0.1% w/v

sodium dodecyl sulfate (SDS). Probes were labelled by the

random primed method and detected by the use of the Genius

Nonradioactive DNA Labelling and Detection kit (Boerhinger

Mannheim, Indianapolis, IN) according to the manufacturers

instructions. The probe was the hrp gene cluster 993 of X.

campestris pv. vesicatoria (Stall and Minsavage, 1991) as an

insert of pLAFR-3.








83

Plasmid analysis. Plasmid profiles were determined by

a modification of a previously published procedure (Kado and

Liu, 1981). Bacterial cells (5 x 108 cfu/ml) were pelleted

in a 1.5 ml centrifuge tube in a microcentrifuge, washed in

sterile distilled water and resuspended in 50 ~l TAE buffer.

The bacterial cells were then lysed for 15 min at 30 C in

400 Al of 50 mM Tris, 0.57 M sodium chloride, 0.04 M sodium

hydroxide and 3% w/v SDS. The lysate was extracted with 2

volumes of phenol (Fisher): chloroform: isoamyl alcohol

(25:24:1). The supernatant was electrophoresised in 0.5%

agarose gels in tris-acetate buffer at 5 V/cm. Gels were

stained in 0.5 mg ethidium bromide per liter. Plasmids of

strain SW2 of Erwinia stewartii were used as molecular

markers (Coplin et al., 1981). Molecular weights were

determined by regressing the log,1 of the molecular weight

versus the log10 of the relative mobility of the plasmids as

determined from Polaroid type 55 photographs.

Results

Hybridization analysis. The hrp gene cluster

hybridized to genomic DNA of all strains of X. campestris

pv. citrumelo regardless of aggressiveness type (Fig. 5-1).

Genomic DNA of the two strains of X. campestris pv. citri

group A also hybridized with the hrp gene cluster. The hrp

gene cluster restriction patterns generated with Eco RI were

similar for strains of X. campestris pv. citrumelo of






















Lhrpl 1 A4 _6A _8_9


22- 4W





mo a -&
r rL r*,o Cu


3.5- -


Figure 5-1. Hybridization of the hrD gene cluster from
Xanthomonas campestris pv. vesicatoria to genomic DNA of
strains of X. campestris pv. citrumelo and X. campestris pv.
citri group A. Lane L, phage A restricted with Hind III and
Eco RI; lane hrp, hrp gene cluster 993 restricted with Eco
RI. Lanes 1 to 9 represent genomic DNA restricted with Eco
RI. Lanes 1 to 7 are strains of X. c. pv. citrumelo. Lane
1 is strain 540 isolated from Clausena wampi; lanes 2 and 3
are weakly aggressive strains F306 and F59; lanes 4 and 5
are moderately aggressive strains F254 and F6; lanes 6 and 7
are highly aggressive strains F54 and Fl. Lanes 8 and 9 are
strains 9771 and 3213 of X. c. citri group A. Molecular
sizes are given in kilobases.


f,1 Milo


J 3~; i'': I~.I
i ~ni.




















H
R 1 1 27901 234 567890 234562893
L r P 5 6 7 8 9 0 1 2 3 4 5 1 1 1 1 2 2 2 2 2 2





22- j





3.5-

'I'





Figure 5-2. Hybridization of the hrp gene cluster from
Xanthomonas campestris pv. vesicatoria to genomic DNA of
strains of X. campestris pv. citrumelo and X. campestris pv.
citri group A. Lane L, phage 1 restricted with Hind III and
Eco RI; lane HRP, hrp gene cluster 993 restricted with Eco
RI; lanes 4 to 12 represent genomic DNA restricted with Eco
RI. Lanes 4 and 5, are strains of X. c. pv. citri 3213 and
9771; Lanes 6 to 12 are strains of X. c. pv. citrumelo.
Lanes 6 and 7 are highly aggressive strains F1 and F361;
lanes 8 and 9 are moderately aggressive strains F6 and F254;
lanes 10 and 11 are weakly aggressive strains F100 and F306;
lane 12 is strain 534 isolated from Clausena wampi. Lanes
13 to 21 are the same as 4 to 12, except restricted with Bam
HI. Lanes 22 to 30 are the same as 4 to 12, except
restricted with Eco RV. Molecular sizes are given in
kilobases.







































Figure 5-3. Plasmid profiles of Xanthomonas campestris pv.
citrumelo from Florida. Lanes 1 and 20 are Erwinia stewartii
plasmids as molecular size markers (labelled in kilobases).
Lanes: 2, Fl; 3, F274; 4, F54; 5, F361; 6, F5; 7, 84-3166;
8, F228; 9, F6; 10, F254; 11, F299; 12, F311; 13, F348; 14,
89-3274; 15, F397; 16, F100; 17, F306; 18, F86; 19, F94.



















Es 1 2 3 4 5 s


-319

-78

-35
-26

-13



-4


Figure 5-4. Plasmid profiles of Xanthomonas campestris pv.
citri group A from Florida. Lanes Es are Erwinia stewartii
plasmids as molecular size markers (labelled in kilobases).
Lanes: 1, T1; 2, 9760; 3, 3340; 4, 3213; 5, 9771.
















Es 1 2 3 4


-319

78


35
26

13




4



Figure 5-5. Plasmid profiles of strains of Xanthomonas
campestris pv. citri group B from South America. Lanes Es
are plasmids from Erwinia stewartii used as molecular size
markers (labelled in kilobases). Lanes: 1, B84; 2, B93; 3,
B148; 4, B80; 5, B64; 6, B69; 7, B94.
















Table 5-1. Size classes of plasmids found in strains of
Xanthomonas campestris pv. citri group A from Florida.


Strain

T1 9760 3340 3218 9771


1. 190.6ab

2. 148.4 148.4 131.8

3. 31.0

4. 11.8 11.6

5. 6.2 6.1

6. 5.2 5.2


(Coplin


"Size in kilobases.
bplasmids sized by Erwinia stewartii size standards
et al., 1981).

















Table 5-2. Plasmid size classes of strains of Xanthomonas
campestris pv. citri group B from South America.


Strains

B84 B93 B148 B80 B64 B69 B94


1. 43.5ab 45.8 41.3

2. 37.5 37.5 35.7 32.5 35.7 34.2

3. 29.8 31.2 29.8 29.8 31.2

4. 28.6 27.4

5. 25.1

"Size in kilobases.
bPlasmids sized by Erwinia stewartii size standards (Coplin
et al., 1981).










different aggressiveness types and the few polymorphisms

that were observed did not correspond to aggressiveness type

(Fig. 5-1). Restriction patterns of genomic DNA of strains

of X. campestris pv. citrumelo and X. campestris pv. citri

group A were different when restricted with Eco RI and

probed with the hrD gene cluster (Fig. 5-1).

In order to investigate these relationships further, a

hybridization analysis was conducted with two additional

restriction endonucleases (Fig. 5-2). Restriction patterns

for strains of X. campestris pv. citrumelo probed with the

hrp gene cluster were similar for all three restriction

endonucleases, Eco RI, Bam HI, and Eco RV. Eco RV and Bam

HI digests of genomic DNA of X. campestris pv. citrumelo and

X. campestris pv. citri group A produced several comigrating

bands when probed with the hrp gene cluster.

Plasmid analysis. No plasmids were observed for any

strain of X. campestris pv. citrumelo (Fig. 5-3). Strains

of X. campestris pv. citri group A from Florida display at

least six plasmid size classes (Fig. 5-4, Table 5-1).

Strains Tl, 9760 and 9771 contain a plasmid ranging in size

from 131.8 to 148.4 kb, while strain 3340 contains a 190.6

kb plasmid. In contrast, the largest plasmid in strain 3213

is 31 kb. Strains 3213 and 9771 also contain several

smaller plasmids (Fig. 5-4 and Table 5-1).

The seven strains of X. campestris pv. citri group B

represent seven different plasmid profiles (Fig. 5-5).










Plasmids ranged in size from 25.1 to 45.8 kb (Table 5-2).

Some 13 different plasmids are represented on the basis of

size.

Discussion

All strains of X. campestris pv. citrumelo studied here

have an hrp region as demonstrated by hybridization of the

X. campestris pv. vesicatoria hrp gene cluster to genomic

DNA of the above strains. Strains of opportunistic

xanthomonads, exhibiting limited pathogenicity (Gitaitas et

al., 1977), were observed to lack an hrp region homologous

with the hrp gene cluster from X. campestris pv. vesicatoria

(Stall and Minsavage, 1991). Although some strains of X.

campestris pv. citrumelo are only weakly to moderately

aggressive on citrus, the presence of an hrp gene cluster is

not consistent with an opportunistic etiology for these

pathogens on citrus. It is possible that the hrp gene

cluster in strains of X. campestris pv. citrumelo, while

shown here to be present, is not expressed. However, a

hypersensitive reaction is exhibited upon inoculation of

strains of X. campestris pv. citrumelo into tobacco (Stall,

unpublished data).

Alternatively, the limited pathogenicity of some

strains of X. campestris pv. citrumelo and the presence of

an hrD gene cluster could be explained if these strains

belonged to other pathovars of X. campestris (Gabriel et

al., 1989; Graham et al., 1990c). The hrp gene cluster