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AGROBACTERII.JM: PLASMIDS AND BIOVARS

Kathleen Margaret Ophel

B.Sc.(Ag¡.) Univ. of British Columbia

Departnent of Plant PathologY

Waite Agricultural Research Institute

University of Adelaide

South Australia

Thesis submitted to The University of Adelaide

in fulfrlment of the requirements for the

degree of Doctor of Philosophy.

December 1987

by

u)N *. o, ðoA \\\ ¡n'

"_¿1c1'?-¡?, '

dedicated to my father,Ivan OPhel

TABLE OF CONTENTS

SI.]MMARY

STATEMENT

ACKNOWLEDGEMENTS

ABBREVIATIONS

LIST OF TABLES

LIST OF FIGURES

GENERAL INTRODUCTION

PART A: ECOLOGY OF AGROBACTERIUM BIOVARS 2

AND 3 ON STONEFRUIT AND GRAPEVINE

IntroductionMaterials andMethodsResults:

A. I Sampling techniques

4.2 Colonization of almonds and vines by

biovars 2 and3,A..3 Construction of transconjugant strains

4.4 Colonization of almonds and vines by

constructed strains

,A'.5 Colonization of nopaline galls

Discussion

PART B: TAXONOMY OF AGROBACTERIUM ISOLATES

FROM RUBUS AND GRAPEVINE

IntroductionMaterials and Methods

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1

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Resuls:8.1 Characteristics of isolates from Rubus

and grapevine

B.2 Relatedness among Agrobacterium strains

Discussion

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PART C: OTÉIER OPINE-UTILIZING BACTERTA

IntroductionPseudomonas

Materials and Methods

Results:

L. Occurrence and abundance

2. Opine utilization

3. Biochemical tests

4.Inhibitory activityDiscussion

Fermentative isolates

Materials and Methods

Results

1. Biochemical characteristics

2. Growth rates

3. Pathogenicity

4. Microscopy5. DNA melting points

6. Plasmids

Discussion

GENERAL DISCUSSION

APPENDICES:A. CULTURE MEDIAB. BUFFERS AND SOLUTIONS

C. DATA PRESENTED IN PART A

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BIBLIOGRAPHY 113

I

AGROBACTERIUM: PLASMIDS AND BIOVARS

Two chromosomal forms of Agrobacterium, biova¡s 2 and 3, are found in

association with specific host plants. Biovar 2 is found in nature associated with

crown gall disease on almonds and biovar 3 is found almost exclusively with the

disease on grapevine. The nature of these associations was examined. Root

colonization studies performed over a l2-month period showed a specific rhizosphere

effect between biovar 2 and.almonds. Biovar 2 colonizeÀalmonds at levels of 106 to

lO7 cfutcr& root, significantly higher than biovar 3 on almonds and than biovar 2 on

vines. Biovar 3 survived poorly in the rhizosphere of both almonds and grapevine,

dropping to levels of 104 cfulcm2 root in the first few months. However, biovar 3

but not biovar 2 was isolated from the vascular system of grapevine.

Other workers have shown in vitro that the tumour-inducing (Ii)-plasmids of

Agrobacterium carry genes coding for host specificity (Loper & Kado, 1979;

Thomasho'w eta!., 1980; Knauf et al., 1982). To test the effect of Ti-plasmid makeup

on host plant colonization, reciprocal plasmid transfers were made between biovars 2

and 3. A Ti-plasmidless strain of biovar 3 was obtained by elimination of its resident

plasmid using a cloned fragment of the Agrobacterium plasmid incompatibility region.

Ti-plasmid transfer was achieved by mobilization with the wide host range plasmid

RP4. The transconjugants were then compared with wild-type strains in terms of their

ability to colonize almonds and grapevine. In all cases, the level of colonization

achieved was determined by the chromosomal background. However, specificity was

also observed between Ti-plasmids and biovars in vivo. The biovar 3 Ti-plasmid was

highly unstable in the biovar 2 background and some plasmid loss was observed in

biovar 3 strains carrying a biovar 2 Ti-plasmid. This instability was not observed in

vitro.

ll

The 'opine concept' proposes that the ability of the Ti-plasmid to catabolize

the gall-specifrc opines creates a selective advantage for Ti-plasmid carrying bacteria

(Petit et al., 1978a; Tempé eta!., 1979; Guyon g1ggl,, 1980). In vivo gall colonization

was studied using octopine and nopaline strains of biovar 3. The nopaline strain

achieved signifrcantly higher populations than did the octopine strain on nopaline

galls. This provides preliminary evidence in support of the opine concept.

Confusion exists over the taxonomic position of Agrobacterium strains from

Rubus and grapevine. Recent isolates of both groups were compared with existing A.

rubi type strains and isolates of the well-characterized biova¡s I andZ. Comparisons

were made primarily by single-tinkage cluster analysis of data obtained from standard

bacteriological tests and by DNA reassociation studies. Both approaches showed that

the Rubus strains all belong to the existing species, ¡\.ruþi, and that the grapevine

strains form a separate and new species. Changes to the nomenclatu¡e are proposed.

Two genera of Gram-negative bacteria capable of catabolizing opines were

studied. One group contained fermentative isolates found in Rubus galls. The second

group consisted of fluorescent Pseudomonas spp. which were isolated from

grapevine. The latter isolates are able to colonize the vascula¡ system of grapevine and

they produce a diffusible, non-siderophore molecule inhibitory to Aerobacterium

biovar 3 in vitro. These characteristics give the Pseudomonas isolates potential as

biological control agents for crown gall on grapevine.

iii

STATEMENT

This thesis contains no material which has been accepted

for the awa¡d of any other degree or diploma in any

university and to the best of my knowledge contains no

material previously published or written by another person,

except where due reference is made in the text. The author

consents to the thesis being made available for photocopying

and loan if applicable if accepted for the award of the degree.

Kathy Ophel

1V

ACKNOWLEDGEMENTS

I would like to thank my supervisor, Allen Kerr, for his guidance and

encogragement throughout this work. I would also like to acknowledge David Jones,

Maarten Ryder, Tom Burr and J.S. Shim for helpful discussions and suggestions on

various aspects of this study and Steve Farrand for generously allowing me to work

in his lab in Chicago and for his invaluable suggestions on the plasmid transfers.

Thanks are also due to a number of people for advice on specific procedures:

Max Tate and his lab for help with high-voltage paper electrophoresis and for

suppyling me with opines, John Randles and Nigel Scott for advice on the DNA

reassociation studies, Stuart Mclure for scanning electron microscopy, Trevor Cock

and Richard Miles for transmission elecüon microscopy, Margie Monis for statistical

advice and Brian Palk for assistance with photogaphy.

I would also like to thank my friends, in particular Tracey \Moodhead, David

Dall and Karen Gibb for moral support, and Mike Keller for advice, enthusiasm and

friendship. Finally,I wish to thank my family, especially my mother, for unfailing

encouragement right from the start

V

ABBREVIATIONS

ANOVA

AS

ATCC

bp

cfu

cv

df

dMGlu

DNA

F

GC

kb

M

MS

IvIW

LambdaHind Itr

HVPE

analysis of variance

acetosyringone

American Type Culture Collection

base pairs

colony forming units

cultivar

degrees of freedom

deoxymannityl glutamato

deoxyribonucleic acid

statistical test of equality of variance

guanine plus cytosine

lambdaDNA cut with therestriction enzyme from Haemophilusinfluenzae Rd

high voltage paper electrophoresis

region of A grobacterium Ti-plasmidcoding for plasmid incompatibility

kilobase

molar

mean square

molecular weight

National Collection of PlantPathogenic Bacteria

optical density

probability

pounds per square inch

Inc

NCPPB

OD

P

ps1

Ri-plasmid

RNA

RNase

SDS

SDV/

SEM

SS

SSC

sym

T-DNA

TE

TEM

Ti-plasmid

vt

root-inducing plasmid

ribonucleic acid

ribonuclease

sodium dodecyl sulphate

sterile distilled water

scanning electron microscoPe

sum of squares

saline sodium citrate

symbiotic

transferred-DNA

Tris-EDTA buffer

transmission electron microscoPe

tumour-inducin g plasmid

Tm

UV

vir

midpoint of the thermaldenaturation profile

ultraviolet

virulence

LIST OF TABLES

PART A

1A Strains and their origin2A Antibiotic concentrations in media

3A Pot experiments described in this study

4A Comparison of root surface area and root weight

5A Gall weights: Almond Ex. L

6A Virulence of biovar 2 strains carrying biovar 3 Ti-plasmidson tomato

7A Pathogenicity of biovar 2/blovar 3 transconjugants ongrapevine and almond

Tarrate utilization of biovar 3 strains

Root populations: Almond Ex. 4

Stem and Lateral root populations: Almond Ex. 4

Root populations: Vine Ex. 2

Gall weights: Almond Ex. 3

8A9A104114t2A

vll

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PART B

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1B

2B3B4B

5B

6B

7B

8B

9B10B

118LzB138

Strains used and their originCommon opines and their detection

Cuvette differences in DNA reassociation studies

Comparison of original and recalculated DNA degreesof bindingPathogenicity and host range of grapevine andRubus isolates

Opine catabolism and synthesis by grapevineand Rubus isolates

Biochemical cha¡acteristics of A grobacterium strainsused in this study

Growth characteristics of Agrobacterium strains innutrient brothEffect of growth factor addition on Agrobacterium gowthDNA melting points of selected Aelrobacterium strains

Motility of Agrobacterium strains

S ummary : Differentiation of A erobacterium species

Serological relationships between Rubus andgrapevine isolates

vtlt

148 Degree of DNA binding: Grapevine strains andother agrobacteria

158 Degree of DNA binding: Rubus isolates a¡rdother agrobacteria

PART C

4C5C6C

APPENDTX C

AC-l Root Populations: Almond Ex. 1

AC-2 Root Populations: Vine Ex. 1

AC-3 Root Populations: Almond Ex. 2AC-4 Gall Populations: Almond Ex.3

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tt2TT2

tt2L12

1C

2C3C

Strains and their originBiochemical characteristics of Pseudomonas isolates

Specificity of in vitro activity of inhibitoryPseudomonas isolates

Effect of iron on inhibition of biovar 3 by Pseudomonas

Relative inhibitory activity of strains K84 and P-14

Biochemical characteristics of fermentative Rubu s isolates

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PART A

8A9A104114t2A

PART B1B

5B

6B

7B8B

9B108

4C5C6C

1A2A3A4A5A6A7A

LIST OF FIGURES

Sampling sites for root colonization studies

Colonization of almond roots by biovar 2 and 3 strains

Colonization of vine roots by biovar 2 and 3 strains

Gel: Transfer of pTiK309 into K128

Scheme for transfer of pTiK27 into K377

Gel: Transfer of pTiK2l intoK377 pTi-

Pathogenicity of Biovar 2/ Biovar 3 transconjugantson tomato

Tartrate utilization by biovar 3

Colonization of almond roots by biovars L and2Plasmid loss from transconjugant strains in Almond Ex. 4

Plasmid loss from transconjugant strains in Vine Ex. 2

C-olonization of nopaline galls by biovar 2 and 3 strains

Gel: Fragmented DNA preparations for DNAreassociation studies

Absorbance profile of DNA for reassociation studies

Sample DNA reassociation plot: Closely related strains

Sample DNA reassociation plot: DifferentAgrobacterium species

TIVPE: Opines in grapevine crown gall extract

Growth of Ti-plasmidless strains on octopine and nopaline

Gel: Plasmids of Sconish Rubus isolates

Transmission EM: Rubus and biova¡ 3 isolates

Single linkage cluster analysis (negative matches not included)

Single linkage cluster analysis (negative matches included)

Octooine utilization bv Pseudomonas isolates

Inhibition of Agrobacterium spp. by Pseudomonas

Effect of i¡on on in viffo activity of inhibitoryPseudomonas isolates

FIVPE: Opines in Rubus crown gall extract

Scanning EM of fermentative Rubus isolates

Gel: Plasmids of fermentaúve Rubus isolates

Following page:

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4B

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1

INTRODUCTION/ LITERATURE REVIEV/

The plant disease crown gall is caused by soil-inhabiting bacteria belonging to

rhe genus Agrobacterium (Smith & Townsend 1907) Conn 1942. The disease is

characterized by tumorous growths on the root, crown or aerial parts of affected

plants. The host range of AÊrobacterium spp. is large but in nature confined to

dicotyledons (DeCleene & Deley, L976). Some Agrobacterium spp. cause a related

condition, known as hairy loot d"isease, fîrst described by Smith et al. (1911) and

characterized by massive root proliferation.

Aerobacterium has been well-studied, initially for its ability to cause disease

and, more recently, because of iS impact on plant genetic engineering. Its role in the

latter has been reviewed elsewhere (Depicker et a1., 1983; Caplan et al., 1983).

Crown gall disease is an economically damaging problem in many parts of the world

and in Australia is a problem on stonefruit and rose (Ken & Brisbane, 1983) and

recently on grapevine. Crown gall of grapevine has been a major problem in

European and North American vineyards for some time. Aspects of crown gall

disease and. i¡5 control have been reviewed by Schroth et al. (1971), Moore & Warren

(lg7g),Kerr (1980), Moore et al. (1980) and Kerr & Tate (1984)'

Taxonomic nomenclature

The nomenclature of Agrobacterium is somewhat confused at present, a

problem addressed in Part B of this thesis. The genus is closely related to the fast-

growing strains of Rhizobium and this relationship has been reviewed by Kersters &

Det ey (1984). At present there are four species of Aerobacterium described- A.

tumefaciens, A. radiobacter, A. rhizogenes and A. rubi (Kersters & Deley, 1984).

Confusion arises because the genes for pathogenicity in the genus are located on large

plasmids which are transferable between bacterial strains (Van Larebeke et al., 1975;

Watson et a1., 197s).These plasmids are known as Ti- (or tumour-inducing) and Ri-

(or root-inducing) and define a strain as being tumorigenic or rhizogenic respectively.

In the present classification scheme, species are defined by their pathogenicity with

')

the result that species are distinguished by a characteristic which is highly

transferable.

There are at least three chromosomal forms of Agrobacterium which can be

referred to as species (Holmes & Roberts, 1981) or biovars (Keane et al., l97O;

DeIæy et al., 1973; Kersters et al., 1973). Biovars I and2 are well-dehned and easily

separated (Keane e]!3l., 1970; Deley et 41., 1973; Kersters et al., 1973; Holmes &

Roberts, 19S1) but the taxonomic position of A. rubi and biova¡ 3 is less clear. This

is discussed extensively in Part B of this thesis. Throughout this study the biovar

nomenclature is used except when referring to isolates from Rubus cane galls, which

are refered to as'þS cane gall isolates' or as A. rubi, for isolates previously

placed in the species (Kersters & Deley, 1984).

Events in Pathogenesis

Ti-plasmids and their role in tumour formation have been the subject of

numerous reviews, including those by Nester & Kosuge (1981)' Bevan & Chilton

(L982), Zambryski er al. (1983), and Nester et al. (1984). Transfer of pathogenicity

to avirulent bacteria was fîrst shown to occur in galls (Ker, 1969). Initial

experiments demonstrating the role of the large plasmids showed that their loss by

curing ar3Tocwas correlated with loss of pathogenicity (Van Larebeke et al., 1974).

Transfer of virulence was shown to be correlated with transfer of the plasmids (Van

LarebekeS! ¡1., L975; Watson et al., L975), thus defrning them as Ti- (tumour-

inducing) plasmids. Ti-plasmids are conjugative (Kerr et al., 1977; Genetello et al.,

Lg71)and large with a molecular weight of approximatety 120 x 106 Daltons (Z'aenen

et al., 1974),corresponding to 180 to225 kb (Currier & Nester, L976).

Subsequentþ it has been demonstrated that large Ri- (root-inducing) plasmids in

some Agrobacterium strains carry the genes for hairy root formation (White & Nester,

1980).

The steps leading to tumour formation by Agrobacterium have been

intensively studied in the past 10 years and have been reviewed recentþ by

Hooykaas & Schilperoort (1986) and Stachel &Zarnbryski (1986b). Bacterial

3

anachment to the cell wall is required for virulence (Lippincott & Lippincott, 1969)

and this is encoded by two genes, chvA and chvB, which are located on the bacterial

chromosome @ouglas et al., L982; Draper et a1., 1983; Douglas et al., 1985). The

process of attachment is not clearly understood. The pectic portion of the plant cell

wall is reported to be implicated in binding (Lippincott et al., 1977).

Lipopolysaccha¡ide in the bacterial outer membrane has been suggested as the

bacterial factor involved in binding (Whatley et al., L976) but more recent evidence

suggests the involvement of a bacterial surface polypeptide (Matthyse, 1986).

However, chvB codes for the production of a ß-2-glucan (Puvanesarajah 9I-41., 1985)

and mutants which are attachment-defective do not produce ttre ß-1-2-glucan so, by

inference, chvB and the ß-l-2-glucan must be involved in attachment. Recent

evidence indicates that chvA mutants also lack the ß-l-2-glucan (J. Handelsman,

unpublished data). Bacterial motility and thus chemotaxis are also associated with the

chv loci (Bradley g!-AL, 1984) which appear to be very pleiotropic. Chemotaxis is a

response, at least ir part, to the production of exudates by the plant from the wound

site (Schroth & Ting, 1968; Shaw et al., 1986). A requirement of wounding for

Agrobacterium pathogenesis has long been realised (Riker g]!el., 1946) and recent

work has shown that the key substances are the phenolic compounds, acetosyringone

(AS) and a-hydroxy-acetosyringone (OH-AS) (Stachel et al. 1986a; 1986b). These

compounds are produced at low levels by normal plant tissue but at high levels in

wounded plant tissue (Søchel etal., 1986b).

A discrete segment of the Ti-plasmid, known as the T-DNA, is

transferred to the plant (Chilton g!, 1977;L97&\where it becomes integrated into

the plant nuclear DNA (Chilton et al., 1980; Willmitzer et al., 1980). Three T-DNA

loci essential for oncogenicity have been characterized and are involved in the

biosynthesis of plant hormones. The lpt (formerþ tmr) locus codes for an enzyme

involved in cytokinin synthesis (Akiyoshi et al., 1984; Barry et al., 1984; Buchmann

9!.-ú, 1985) and two genes, iaaM and iaaH (formerly tms-l and tms-2) (Schroder et

a1.,1984; Thomashow 9!_ú, 1984) code for enzymes involved in the conversion of

tryptophan to the auxin, indole acetic acid. The elucidation of the functions of these

4

genes explains the observation of Braun (1958) that crown gall tumours can grow in

tissue culture in the absence of phytohormones.

The incorporated r-DNA genes arso encode the synthesis of novel

compounds known as 'opines' (Bomhoff et al. , L976; Kerr & Roberts, 1976;

Montoya et al., 1977). Opines a¡e found within the plantkingdom only in plant tissue

which has been transformed by Aerobacterium. Octopine and nopaline are both

arginine derivatives and were amongst the first opines to be described @etit et al.,

1970) and a number more have since been characterized (reviewed by Tempé &Goldmann, L982). Opines can be amino acid-keto acid conjugates, such as octopine

and nopaline, or phosphorylated sugar derivatives (Eilis & Murphy, l9g1). An opine

is def,rned by its function; it is a substance whose synthesis in plants is encoded bybacterial genes and is catabolized by the inciting bacteria as a specific growth substrate

(Tempé et al., L979). Opine catabolic genes are usually located on the non-transferred

portion of the Ti-plasmid (Holsters et al., 1982) although there are strains which have

chromosomal genes for opine degradation (Montoya et al., 1978). Bacteria from other

genera, especially Pseudomonas, are also able to catabolize opines (Kohn &Beiderbeck, rgS2; Beaulieu et al., 1983; Brisbane & Kerr, 19g3; Rossignol & Dion,1985; Tremblay et al., 1987) but none of these bacteria appear to be capable ofdirecting opine synthesis.

The Ti-plasmid atso contains a region of approximately 35 kb, known as the

virulence (Vir) region. This region is required for pathogenicity but, unlike the T-DNA, is not transferred and integraæd into the plant genome (tlille et al., l9g4:Hooykaas et al., 1984). In an octopine plasmid the Vir region contains six operons

@-E, virG) (Stachel & Nester, 1986). VirA andg[¡G are expressed constitutively

but the other vir loci are expressed only in the presence of plant cells (Stachel et al.,

1986b), specifically in the presence of the phenolic inducer molecules in woundexudate. Both virA and virG are required for the induction of the other vir loci(Stachel &Z,ambryski, 1986a; Winans er al., 19g6).

The functions of virA and virG have been studied more intensively than the

other vir loci. Winans et al. (1936) showed that virG codes for a protein of 30, 000

5

daltons. The amino acid sequence of this protein is homologous to a number ofproteins which are components of two-part chemoregulatory systems in other

bacteria. Læroux et al. (1987) showed that virA encodes a92,000 dalton inner

membrane protein which has homology to receptor proteins in these other regulatory

systems. A model has been proposed where virA detects the presence of the plant

inducer molecules and transmits the signal across the cytoplasmic membrane. VirAcannot regulate the other vir loci without the presence of virG (Winans et al., 1986)

and both Leroux and Winans suggest that, by analogy with other bacterial systems,

virA probably acts to convert the virG protein, located in the cytoplasm, to an active

form, which in turn activates the remaining vir loci. The functions ofltg,C and E

have not been elucidated but the function of virD has been studied. The T-DNA is

flanked by 24 bp imperfect direct repeat sequences, known as the T-DNA bord.ers

(Yadav et al., 1982). VirD encodes an endonuclease which causes a'nicking' within

the T-DNA borders ( Yanofsky et al., 1986). This results in the generation of single-

stranded, linear molecules known as the T-strands (Stachel et al., 1986a) which are

probably transferred into the plant cell.

Comparatively little is known about T-DNA transfer and integration. Stachel

et al. (1986b) propose that the T-strand is an intermediate molecule similiar to the

linear single-stranded DNA transferred from donor to recipient during bacterial

conjugation. Recently, Buchanan-Wollaston et al. (19g7) showed functional

homology between bacterial mobilization (mqþ) genes required for plasmid transfer

and virD, as well as between origins of plasmid transfer (or[!) and the T-DNA border

repeats. Extending the comparison, the plasmid transfer (tra) genes are then

analogous to the remaining vir genes. This further strengthens a model for T-DNAtransfer which is analogous to bacterial conjugation. It remains to be understood how

the T-strand gets to the plant cell nucleus and how it then becomes integrated into the

plant genome. Stachel &z,atrbryski (1986b) suggesr rhat wounding may also

stimulate DNA replication in the plant cell and that the DNA recombination a¡rd repair

processes thus set in motion may be essential for T-DNA integration. Once integration

has occurred, T-DNA genes are transcribed and translated in the plant cells.

6

Octopine and nopaline Ti-plasmids belong to the same plasmid incompatibility

group (Hooykaas et al., 1930) and Ri-plasmids belong to a separate group

(Costantino er al., 1980). In addition Agrrobacterium isolates may contain other

compatible plasmids. There a¡e often large plasmids (Merlo & Nester, 1977;

Sheikholeslam et a1., 1979) of up to zl4Mrdin size (Casse et al., 1979) whose

functions remain cryptic. There are several well-studied smaller plasmids. One of

them resides in the economically important biological control strain, K84. It contains

a 47.7 kb conjugative plasmid, pAgK84, which encodes the production of and

immunity to the antibiotic, agocin 84 (Ellis et a1., 1979; Slota & Farand, 1982). The

use of this strain wilt be discussed later. Some biovar 1 strains isolated from

grapevine possess a Mkbplasmid, pTAR, which enables them to catabolize sodium

tartrate (Gallie etal., 1984).

The linkage of genes for opine synthesis and catabolism genes on the Ti-

plasmid and the role of the opines as a nutritional substrate for agrobacteria has led to

postulation that the opine functions are the 'raison d'etre' for the existence of the Ti-

ptasmid. This theory has been termed the'opine concept' (Petit et al., 1978a; Tempé

., L979; Guyon et al., 1980 or'genetic colonization' (Schell, L978; Schell d,lg7g).The basic tenet of the theory is that the opines create a selective advantage for

opine-catabolizing agrobacteria. The theory is strengthened by the discovery that

some opines, octopine (Petit et al., 1978b; Klapwijk et al., 1978), the agrocinopines

(Ellis et al., L982) and, more recently, cucumopine (4. Petit, pers. communication)

promote transfer of some Ti-plasmids. Thus the opines may be creating an ecological

niche for agrobacteria where the Ti-plasmids confer not only a nutritional advantage

but, in the presence of opines, promote their own propagation. There has been no

experimental proof of this theory to date. The role of the opines is further discussed in

Part A of this thesis.

A number of functions have been mapped on the non-transferred portion of

'the Ti-plasmid besides the virulence and opine catabolism genes, and functional maps

have been made of both octopine and nopaline Ti-plasmids @epickeret al., 1980;

Holsters et al., 1980; DeGreve et al., 1981; DeVos et al., 1981). Ti-plasmids contain

7

regions which code for conjugal plasmid transfer (Tra), replication (Rep) and

exclusion of the bacteriophage APl (Ape). Sensitivity to the antibiotic produced by

strain K84 is also Ti-ptasmid encoded (Engler et al., 1975) but is presont only on

some nopaline Ti-plasmids and some Ri-plasmids (Ryder, 1984). In addition, a

number of agropine strains appear to have a repressed agrocin uptake system (Ellis &

Murphy, 1981).

Host soecificitv

Not all Agrobacterium strains have the same host range (Anderson & Moore,

LgTg) and strains from grapevine in particular often have a limited hostrange (Knauf

et al., 1982). A number of studies have shown that the host range of Agrobacterium

is determined by Ti-plasmid genes (L,oper & Kado, 1979; Thomashow et al., 1980;

Knauf et al. ,1982; Unger et al., 1935). This was initially demonstrated by the

transfer of the Ti-plasmid from a more limited host lange strain into a wide host range

strain and vice versa. In both cases the host range of the donor strain was also

transferred. The host specificity of the plasmid in the new background was not the

same as the wild-type in atl cases, suggesting some modulating effect from the

bacterial chromosome or perhaps from a cryptic plasmid (fhomashow e]!31., 1980;

Knauf et a1.., 1982).

Several studies have investigated the molecular basis of host range

determination in greater detail (Yanofsþ et a1., 1985a, 1985b; Yanofsky & Nester,

1936). Wide and narow host range octopine Ti-plasmids (isolated from biovars 1

and 3 respectively) are distinct, sharing only 6 to l57o homology (Thomashow et a1.,

1930). F{owever, they are still in the same plasmid incompatibility group (Knauf &

Nester, Lg8z).Initial work with pTiAg162, a narrow host range plasmid, indicated

that the pathogenic loci were on two widely separated T-DNA regions (Knauf et al.,

1984). One region, the T¡-DNA, contains the cytokinin biosynthesis ûpt) genes and

the other, the T3-DNA, contains the auxin biosynthesis (!aa) genes (Bucholz &

Thomashow, 1984a; Yanofsky et al., 1985a). Cytokinin biosynthesis genes were

initially implicated in host range expression when it was shown that part of the T-

8

DNA from a wide host mnge strain encompassing the !E locus expanded the host

range of the limited host range strain (Knauf et al., 1983). This was confrmed when

the cytokinin biosynthesis gene alone expanded the host range (Bucholz &

Thomashow, 1984b; Hoekema er a1., 1984) although it should be noted that the wide

host range phenotype was not completely restored. Yanofsky et al. (1985b) showed

that a limited host range plasmid contained a weak or inactive !p¡ locus which further

implicated cytokinin levels as an important factor in host range determination. Other

workers (Inze et al., 1984; Klee et al., 1935) have shown that some plants require

both the introduction of the ipt ard iaa loci for gall formation and other plants require

only one. The suggestion is that endogenous plant phytohormone levels may play a

deciding role in the host specificity of the bacteria.

The evidence suggests ttrat there must be other factors involved with host

range expression. Studies by Yanofsky et al. (19S5b) show homology between the

virB, virG, virD and virE loci of wide and limited host range plasmids but no

homology between the-virA and virC loci. Introduction of virA and virC from a wide

host range plasmid restored the wide host range phenotype to a narrow host range

srrain. Mutations in the virC locus affect host range (Hille et al., 1984; Hooykaas et

al., 1984; Yanofsky et al., 1985b), so there is evidence that virC plays a role in host

range determination, possibly by controlling the number of T-DNA copies transferred

to the plant cell (Yanofsky & Nester, 1936). Other evidence implies a role for vifE

(Hirooka & Kado, 1986) and more strongly for virA (Leroux et a1.,1987; Ma et

a1.,1987). Ma et al. (1987) isolated strains from grapevine in northem China, most of

which showed little or no homology to the virA locus of a wide host range strain.

Leroux et al. (1987) showed that the virA proteins from wide- and limited- host range

strains had457o homology and that they were most divergent in the region postulated

to be a binding site for the plant inducer molecules. The virA locus from limited host

range strains did not induce the Vir region when exposed to acetosyringone, the

inducer for wide host range strains. It has been suggested that the virA gene product

from limited host range strains may recognize different plant inducer molecules

specific to grapevines ([.eroux et al., L987).

9

Crown gall of srapevine

Clown gall of grapevine has long been a serious economic problem in much

of the world, particularly Europe and North America. Until recently it was not

considered a major problem in Australia. The causal agents of the disease on vines

belong almost exclusively to the biovar 3 group (Panagopoulos & Psallidas, 1973:

Kerr & Panagopoulous, 1977; Panagopoulos g! ¿L, 1978; Bur & Hurwitz, 1981;

Bur & Katz,1983). The disease is charactenzednot only by tumours at the crown

of affected plants but often by extensive galling of aerial parts. These aerial galls may

girdle the trunk of the vine and kill the more cold-sensitive cultivars @urr, 1978).

Initial evidence that biovar 3 survives in the vascular system of grapevine

came from Iæhoczky (1963;197I). He postulated that in moist spring conditions

water flow through the xylem sweeps bacteria from the root system to aerial wounds

caused by frost injory. Other workers (Burr, 1978) have observed an association

between low-temperature injury and aerial galling.Recent shrdies conf[m the

importance of the systemic survival of biovar 3 in dissemination of the disease @urr

&Kat2,1983; Burr & Katz, L984;Tarbah & Goodman, 1986). Recently, Tarbah &

Goodman (1987) monitored the movement of antibiotic resistant mutants of biovar 3

and found that they were confined to xylem vessels. Burr et al. (1987c) detected

biovar 3 in grape shoots collected late in the growing season but not in green shoots

collected in spring and summer. This may reflect the development of secondary xylem

in the growing shoots. Early in the season the xylem of canes and new shoots are not

joined so bacteria cannot migrate to ttie growing shoots @urr et a1., 1987c).

Agrobacterium biovar 3 is found in vineyard soils (Burr &Kat2,1983), in the

grapevine rhizosphere but rarely in nonrhizosphere soils (Burr 9!-AL, 1987a). Recent

work by Burr et al. (1987b) has shown that biovar 3 is capable of forming sunken

lesions on the roots of grapevine and may be isolated with high frequency from these

lesions. This root decay is highly specifrc as it is not cauSed by other Agrobacterium

biovars and biovar 3 causes root decay only on grapevine roots, not on their shoots or

on the roots of other test plants. The root lesions can extend into the vascular system

10

and Burr et al. (1987b) suggest that this may be the mode of enury of biovar 3 into the

vascular system.

'Cane gall' on Rubus sPP.

Crown gall on Rubus spp. has been less-studied than its counterpaÍ on

gfapevine. The disease is also known as 'cane gall' because, as on grapevine, galls

are ofren formed on the aerial parts of the plant. Early work by Banfreld (1930;1935)

and pinckard (1935) showed that the causal agents of 'cane gall' and 'cro\ryn gall' on

Rubus spp. were fundamentally differenl More recent reports indicate that the crown

gall isolates usually belong to biovar 2 (Perry & Kado,1981; 1982; M. I-opez, pers'

communication). A full description of cane gall and its causal agent, A. rubi, is

provided by Hildebrand (1940). Reports of the disease come largely from North

America @anf,reld, 1935; Hildebrand, l94O McKeen, 1954) though it has been

feported elsewhere. Symptoms appeaf in the late spring and the disease is

characterized by ridges of galls which run up the side of the cane. Banfield (1930)

found, evidence for systemic infection by A. rubi and Pinckard (1935) showed that its

host range was limited to Rubus spp. although it is only weakly pathogenic on red

raspberry ß-Xlaeus). Cane gall has been reported in blackberry ßubus sp.) and

boysenberry Rubus sp. cv. 'Boysen') plantings in Canada (McKeen' 1954) and

evidence for the probable systemic nature of the pathogen was provided in that case

by the isolation of virulent A. rubi from apparently healthy floral canes.

Control of crown gall disease

Until the early 1970's, there were few effective controls for crown gall

disease. The use of antibiotics (Klemmer et al., 1955), fungicides (Helton &'Williams, 1968) and soil fumigants (Deep et al., 1968) has been attempted but these

approaches are expensive and not entirely effective. Biological control of crown gall

disease on some hcist plants has been achieved through the use of an avirulent

antibiotic-producing strain of Agrobacterium, K84 (Kerr, 1972; New &Kerr,1972;

Htay & Kerr,I974: Kerr & Htay, 1974). Strain K84 produces an antibiotic (Kerr &

11

HOy, Lg/4),now called agrocin 84 @ngler et a1., I975), whose structure was

determined by Tate et al. (1979). Agrocin 84 inhibits nopaline strains of

Agrobacterium and its production is encoded by a small plasmid, pAgK84 (Ellis et

a1.,1979; Slota & Farrand, 1982). Strain K84 has been used commercially in many

countries (Moore & Warren, 1979; Kerr, 1980). Control of crown gall of grapevine

and of Rubus spp. is not possible by strain K84 because of its specificity to strains

containing nopaline Ti-ptasmids. As a result, it has been used largely for the control

of the disease on stonefruit and rose.

problems with the biological control of crown gatl by strain K84 have been

reporred (I(err & Htay, 1974; Moore, 1978) and some pathogenic agrobacteria have

become resistant to the antibiotic (Panagopoulos et al., 1979; Ellis et al., 1979;

Cooksey & Moore, Lgïz).Recently a transfer dehcient (Trr) derivative of K84 was

shown to be an effective control agent (Shim et al., 1987) and a Tra- deletion mutant

has been engineered in this laboratory (Jones g!¿!, 1988); it is currently being tested

in vivo for its control eff,rcacy @. Jones, personal communication).

Biological control of grapevine ffown gall is not possible at present.

Inhibition of biovar 3 has been achieved in vitro by agrocin-producing Agrobacterium

strains (Webster et al., 1986; Thomson, 1986; Chen & Xiang, 1986)' but this has not

led to disease control in vivo. A more promising approach appeals to be based on the

early detection of the pathogen in planting material (Tarbah & Goodman, 1986) and

the subsequent use of Aerobacterium-free stock.

Ecolo g.v of A grobacterium

Although much is understood about the infection process by

Agrobacterium, relatively little is known about its ecology. A number of early studies

(Patel, L928; 1929; Hildebrand,l94l) report the ability of Agobacterium spp. to

survive for long periods in soil which has been confirmed by Schroth et al. (1971)

and Dickey (1961). The isolation of Agrobacterium biovars 1 and 2 from soils where

crown gall had never been observed (Bouzar & Moore, 1987) is another indication of

their ability to survive saprophytically for long periods. On the other hand, it has been

l2

reporred that A. rubi may have much less ability to survive in soil (Hildebrand,

1940). One very interesting aspect of Agrobacterium ecology is the high proportion

(as high as 100:1) of nonpathogenic to pathogenic srains found in the soil and in the

galls themselves (Kerr, 1969).

Studies have looked at the effect of pH (Siegler, 1938) and temperature

(Riker, lg26).The former study established that crown gall is more prevalent in

alkaline soils and the latter demonstrated that tumour formation is inhibited by high

temperatures. Both of these observations were confirmed by Dickey (1961). None of

these studies distinguished between biovars of Agrobacterium. Selective media for

Agrobacterium biovars have been developed by Schroth et al. (1965), New & Kerr

(Ig7l),Brisbane & Kerr (1933) and Roy & Sasser (1983). These selective media

make possible ecological studies which differentiate between Aerobacterium species

or biovars.

Scooe of this studv

This thesis is divided into tlree parts. Part A looks at aspects of the ecology of

Agrobacterium biovars 2 and3 and at the role of the opines. Although host specifrcity

is relatively well-understood at the level of the Ti-plasmid, little or no work has been

done examining the specific associations which have been observed between

chromosomal forms of agrobacteria and certain host plants. This study looks at the

association between biovar 2 and stonefruit and beween biovar 3 and grapevine. At

the onset of this study, there was little information on grapevine crown gall and

experiments concentrated on the early stages of the infection process, specifically root

colonization. Part A also examines the effect of the Ti-plasmid on this early

interaction. Much has been speculated about the ecological importance of the Ti-

plasmid but to date there has been very little in vivo data in this area. Experiments

were designed to study the contribution of the Ti-plasmid to bacterial colonization of

the plant host and to examine whether the opines provide any selective advantage to

opine-catabolizing bacteria in terrns of their colonization of the plant root surface and

of crown galls.

Part B examines ta:ronomic relationships among Agrobacterium spp. with

particular reference to the relationship berween the grapevine strains (biovar 3) and

those from Rubus spp. In the past there have been conflicting reporß conceming the

taxonomic position of isolates from these hosts so studies were performed to

determine if they were separate species. Both phenotypic and genetic methods of

comparison were used.

Part C looks at two non-Agobacterium genera of Gram-negative, soil-

inhabiting bacteria which are able to catabolize opines. Some of these a¡e fluorescent

pseudomonas spp. which are inhibitory to some Agrobacterium spp. and the possible

use of organisms such as these for control of grapevine crown gall is discussed.

13

PART A: ECOLOGY OF AGROBACTERIUM BIOVARS 2 AND 3 ONSTONEFRUIT AND GRAPEVINE

INTRODUCTION

Much work has been done to demonstrate the involvement of Ti-plasmid

encoded genes in the determination of host range of Agrobacterium species (Loper &

Kado, 1979; Thomashow g!-ù 1980; Knauf e! 3!.,1982; Unger et al., 1985). Work

on a limited host range grapevine sEain, A9162, has furttrer demonstrated that the

cytokinin biosynthetic locus on the T-DNA and two of thevirulence genes are the

regions of the Ti-plasmid involved in host specificity (Yanofsþ et a1., 1985a;

19S5b). Thus there is conclusive proof that host range is determined by the Ti-

plasmid and thar Ti-plasmids all belong to the same incompatibility group (Hooykaas

et al., 1980; Montoya et al., 1977) and, as well, that they are transferable between

strains (Van Larebeke et al., 19751, 'Watson et al., 1975). Despite this, one still

observes specific natural associations between the chromosomal forms (biovars) of

AÊrobacterium and particular plant hosts. Crown galls on stonefruit yield

predominantly biovar 2 strains (New, L972) and grapevine galls yield biovar 3 strains

(Panagopoulos & Psallidas, \973; Kerr & Panagopoulos, 1977; Panagopoulos et al.,

L978; Sule,1978; Burr & Hurwitz, 1981; Perry & Kado, 1982). It is apparent that, in

nature, there must be a more complex situation with regard to host specificity. The

primary aim of this section of the work was to experimentally determine the nature of

host specif,rc interactions between biovar 2 and stonefruit and between biovar 3 and

grapevine. Reciprocal Ti-plasmid transfers were then made between biovars 2 and3

in order to examine the effect of a strain's Ti-plasmid makeup on this early interaction

with the plant host.

The second aim of this study was to obtain ecological data to critically examine

the opine concept. The theory that opine-related functions are the'raison d'etre'for

the existence of the Ti-plasmid has been proposed (Petit et al. ,1978a; Tempe et al.,

1979; Guyon @1.., 1930) and is widely accepted. However, there has been no

ecological data to actually support the theory which proposes that the opines provide

t4

agrobacteria which are able to catabolize them with a selective advantage in the

rhizosphere. This advantage may be provided in two ways. Ti-plasmid carrying

agrobacteria can selectively catabolize the opines and thereby gain a nutritional

advantage. As well, some opines induce conjugation (Petit 9¡ 41.,1978b; Ellis et

a1.,1982), ensuring the propagation of the Ti-plasmid. This study was an attempt to

examine the nutritional role of the opines.

An understanding of the ecology of Agrrobacterium is important in developing

a strategy for strains not conEolled by present biological control organisms.

Ecological studies on Agrobacterium in general have been very limited. At the onset

of this work very little was known about the ecology of the disease on grapevine. The

work of Lehackzy (1978) indicated that Agrobacterium could be isolated from the

vascular system of grapevine. The importance of this systemic inoculum versus the

conventional model of soil inoculum was not established. An understanding of the

rhizosphere dynamics of Agrobacterium is important in the development of cultural

control strategies for grapevine crown gall and also in investigations into the use of

any biological control strains on vines. Additionally, because the molecular basis of

pathogenicity and plasmid-encoded host specifrcity have been so intensively studied

and a¡e relatively well-understood, Agrobacterium is a useful model system for the

study of the relative contributions of plasmid, chromosome and host plant in the early

stages of a pathogenic interaction.

15

PART A: MATERIALS AND METHODS

a) Bacterial strains and culture conditions

All strains used in ecological studies and their sources are listed in

Table 14. Transconjugant strains constn¡cted in this study which are not described in

Table 1A are designated by the strain number of the recipient plus the plasmid derived

from rhe donor e.g. CIRS (pTiK309). Wild-type Ti-plasmids are thus designated

pTi-X where X is the strain name of the plasmid donor, according to the system of

Scialcy et al. (1978). Bacteria were generally maintained on nutrient agar (NA) or

yeast-mannitol (YM) agar at2Soc for short-tenn storage. They were mantained as

lyophilized cultures at 4oC for long-term storage.

b) Pathogenicitv testing

For pathogenicity tests, bacteria were grown at 28oC for 48 hours on

YM slopes. A turbid suspension (about 109 ce[s/ml) of bacteria \ryas made in

buffered saline and sterile toothpicks were used to inoculate suspensions directly into

the young internodes of the plant stem. A buffered saline control was included in all

caSeS.Plantsusedinthisstudyweretomato@Mill.var.'Early Dwarf Red'), tobacco (Niçotianegauca Graham.), grapevine (Vitis vinifera L.

cv.'Cabernet Sauvignon'), almond (PrunuS¿¡qygdalus Batsch. cv.'Chellaston').

Results were recorded after 4 weeks on tomato and tobacco and after 10 weeks on

grapevine and almond. When pathogenicity was unclear, putative galls were checked

for the presence of opines by high-voltage paper elecüophoresis (as described in

Part B).

For pathogenicity tests on carrot (Oaucus carola L.) discs, whole caûots were

washed, peeled, dipped in ethanol and flamed. Discs approximately 1 cm in thickness

\ilere cut aseptically and were placed apical side facing upwards in sterile specimen

containers containing 2Vo water agar. Bacterial suspensions were prepared as

previously described and the cambial area of ttre carot discs inoculated with 50 pl of

Table 14. Strains and their origin

AGROBACTERIUM:

Strain Biovar Grouping:Desisnation:

Plasmid(s):

cryptlc

pTiK309cryptigPRP4I

pTiK305pTiK309pTiK374pTiK377pTiK252

pT1K27pRP4

Antibiotics

Km, Cb, Ap 4rif

Cm, Gm, Sm, Sprif

Km, Cb, Tc

rif

K57ClRSK57(pTiK27)

2-4x K884

K382

SF-1

Biovar 1

Biovar 1

Description

IIB101 (pDP35 :: Kpnl)2(cointegrate)

H8101 (pPHlII)3

C600 (pRP4)

H8101 rec-

Source:

potting soil, SAex. J. Schell

A. Kerr

this study

grapevine, SA

interrow soil, SApeach gall, SApeach gall, SA

Kl03K128K27

Biovar 2

K128(pTiK309) Biovar 2

K305K309K374K377K252

K377@TLK27.) Biovar 3

ilcr)?trcpTlK27

Biovar3ttil

I

lt

il grapevine, Greece(strain Ag57)

this study

this study

S.K. Farrand

1- pRP4 described by Thomas (1981)

2- pDP35 described by Pischl & Farrand (1983); cointegrate formation described inPart A Results, Section 4.3.

3- pPHIJI described by Hinch & Beringer (1984)

4-By convention, two letter antibiotic codes (e.g. Km) designate plasmid-encodedantibiotic resistance and the three letter codes (e.g. riÐ represent chromosomally-encoded resista¡ce.

16

suspension. Discs were incubated at zsocunder low intensity fluorescent light (4.2

pEm-4-1¡ and results were recorded after 4 to 6 weeks.

c) Bacterial matings

For all bacterial matings, cultures were grown overnight at 25oC on a

rotary shaker and harvested by centrifugation when cells were in the mid- to late- log

phase of growth. Cultures were grown in yeast-extract (YE) broth,with antibiotics

when appropriate. Antibiotics used for Agrobacterium and E. coli and the

concentrations used in a variety of media are listed in Table 24. Before mating, all

cells were washed several times and resuspended in 2 ml YE broth. Aliquots of 1 ml

of donor and recipient bacteria were mixed, loaded into a syringe and forced onto a

0.45 pm Millipore filter. The filter was then placed on a non-selective medium- YE

agar or TY agar (see Appendix A) and incubated at 28oC for 2 days. As a control,

donor and recipient suspensions were mixed separately with 1 ml of broth and

transfered to a filter as described. After incubation, filters were suspended in 1 ml

buffered saline, diluted as desired and 200 pl of each dilution spread onto selective

media. Transconjugants usually appeared after 2 to 7 days incubation at 28oC. These

were then purified further by streaking for single colonies on selective medium.

d) Plasmid isolation and visualization by sel electrophoresis

The method used was a modification of that of Bimboim and DoIy

(1979) devised by Dr. S.K. Farrand (personal communication). The soluúons used in

this procedure are described in Appendix B. Cells were grown to late log phase at

25ocin NB or YE broth with or without antibiotics and adjusted to ttre equivalent of

1.0 ml of a suspension with an optical density (640 nm) of 0.4 and harvested by

centrifugation. Cells were resuspended in 1 ml TE buffer with 100 pl 5M NaCl and

l0 ¡tl lOVo Na Sarkosyl. After mixing and recentrifugation, the pellet was suspended

in 100 pl Solution 1 and kept on ice 5 min. Solution 2 (200 pl) was added, the tube

inverted and left to stand at room temperature for 15 minutes before the addition of 50

Table 2A: Antibiotic Concentrations

Antibiotic3 Abbreviation4

Rifampicin Rf (riÐ

Gentamycin Gm

Streptomycin Sm (sn)

Chloramphenicol Cp

Carbenicillin Cb

Tetracycline Tc

Kanamycin Km (kan)

Concentrations Used in:

Minimalmedia:l UndefinedmeÅiaZ:Agrobætelw Agrobacterium E.coli

50 50 50

50 50 20

100 50 50

100 30 50

100 50 50

10 2.5 2.5

100 s0 50

1- Minimal media of Petit et al.(1978b); for Agrobacterium only.

2- Nutrient or YE media

3- All antibiotics used in this study wero rtxrrkele.l by Sigma Chemical Company,USA. Concentrations expressed as pørnl.

4- Two letter abbreviations refer to plasmid-encoded antibiotic resistance and threeletter abbreviations refer to chromosomally-encoded resistance.

L7

ttl 2M Tris-HCl pH 7.0. After 30 min at room temperature, 50 ¡tl 5M NaCl was

added, gently mixed and extracted with an equal volume of phenol saturated w\¡h37o

NaCl for 5 minutes. The emulsions were then centrifuged for 10 min at 4oC and the

aqueous (upper) phase transferred to a fresh tube; 0.1 volume 3M sodium acetate and

2 volumes ice-cold absolute ethanol were added to precipitate the DNA.

The DNA was collected by centrifugation (15 min at 4oC), the pellet dried

under vacuum and redissolved in 20 pl TE8 plus 10 pl tracking dye. The samples

were loaded into 2 mm wells inaD.7%o orl%o agatose (Seakem) gel; the gel was

covered in Tris-borate buffer and electrophoresis ca:ried out at 60 mA for 4 to 6

hou¡s. Gels were stained with ethidium bromide (2 ¡tg/ml) for 15-30 minutes, then

photographed on Polaroid type 665 positive/ negative film under UV (302 nm)

illumination.

e) Plasmid tran sformation

The method of Holsters et al. (1978a) was used for plasmid

transformation of Agrobacterium. Large-scale plasmid isolations for use in the

transformation procedure were done by the method of Casse et al. (1979) and purified

in ethidium bromide-cesium chloride density gradients (Maniatis eta!, 1982).

fl Hish voltase DaDer electroohoresis

High voltage paper electrophoresis (HVPE) was used to detect opines

in gall tissue and is described in Materials and Methods, Part B, in the section, "Opine

synthesis and catabolism".

Table 3A. Pot tials described in this studyl

ALMONDS:

Treatmens2

K27()K30e (B)I07 + K309 (C)

Sample Sites

I¿teral roos5Systemics

RoosStem

Galls

RootsStemIåteral roots

Rootstem5

Ex.1

Ex.2

Ex.3 6

Ex.4

Y'n (A\ B2|RSK27 ì K309 (B) (all rearnens)r<27 +K377 (C)

BIEZ (Trts. A to C)

B2 + rif5g

B2IRS CTrts. A to D)Seä fooinoæ 7 for mifiaused for Ets. E and F

r0ß5n2ß6

4ß6tÐ9ß6

5/86 ro 11/86

K3K128K377K128

VINES:

Ex.1 K27 (A)K30e (B)K27 + K30e (C)

B2lRS(all reatmens)

Rootstern5l¿teralroos5Vascular tissueS

9Æ5 to 9Æ6

Ex.2 as AlmondEx.4 RootStemIåteral rootsVascular tissue

9ß6ta6ß7

Foohotes:

1- The general method for all pot rials is described in Part A, Materials and Methods.

2- All srains used in these studies are described in Table 1A'.

3- All media are described in AFpendix À 81 = Biovar 1 medium; B2=BiovarZmedium; RS = selective medium for biovar 3 of Roy & Sasser (1983).

4- Samples for all experiments taken at 4 week intervals.

5- Siæ was not sampled at all of the sample times.

6- Galls were formed by K27 (described in Part A, Materials and Methods).

j- 2%ropaline,2%oNaClplus cblg¡ tc16'

0.2fo ætoPne Plus cblgg tc19.

18

s) Pot Exoerimentse.-

Alt pot experiments are referred to by number and are described in

Table 34.

i) Preparation oÍ inocula

All strains used as inocula for pot experiments are described in

Table 14. Fresh cultures of each srain were inoculated on to 50 ml YM slopes and

grown a¡25oCfor 48 hours before use. Bacteria were suspended in sterile distilled

water (SDUD and diluted to a concentration of approximately 1 x 108 ce[s/ml. Cell

densities were determined spectrophotometrically at 640 nm using the equation:

No. Agrobacterium cells = t 0.10 + (ODO¿O x22.93)lx 108 cells/ml (B.

Lethbridge, personal communication).

7l) Soil and growing condirtons

The soil used for all pot experiments was a 1:1 mixture (non-

sterilized) of sand and loam with no fertilizer added. A handful of bark chips was

added to each pot to improve drainage. Fertilizer (60 ammonium sulphate: 11

potassium nitrate: 9 mono-ammonium phosphate) was applied (3 g/poÐ and watered

in thoroughly every 6 to 8 weeks. All planS were grown outdoors in 8" or 10" pots

and watered regularly throughout the growing season.Initial soil levels of both biovar 2

and 3 strains were tested and found to be below 193 çfu/g soil.

ibl) Preoaration and irnctilation of planting material

Almond seeds (cv. 'Chellaston') were obtained locally. After

shelling, seeds were soaked overnight in water and placed in moistened peat at 4oC.

Captan (2gper litre) was applied weekly to inhibit fungal growth and seeds were kept

moist. After 4 weeks, seeds were transferred to 10" pots containing UC mix and

gïown under shadecloth for 3 to 4 weeks. By this time, young seedlings had emerged

and were transplanted at the initiation of each experiment.

19

For Vine Ex. 1, grapevines (cv. 'Cabernet Sauvignon'clone GV93) were

obtained from Wynn's Coonawarra estate as 1 yearrootlings and stored for a week

before use at 4oC. For Vine Ex. 2, vines (cv. 'Cabernet Sauvignon') were obtained

from Kemp's Murray Valley Nursery, Barmera as 1 year rootlings and were stored at

10oC for 10 days before use.

When inoculated, almond seedlings were dipped in the bacterial suspensions

for several minutes to a level2.5 cm above the cotyledons. Vines were dipped to 15

cm above the base of the stem and were planted to the same level. For Almond Ex. 3,

galls were induced at the time of planting by wounding the almond seedlings just

below the cotyledons with a sterile toothpick which had been dipped in a cell

suspension (109 ce[s/ml) of I(27. Seedlings 'were then inoculated with

ffeatment stains by dipping as described above'

iv) Expe rime ntal desígn

All pot experiments sha¡ed a basic randomized block design

with either 6 or 8 blocks (= replicates), each containing a complete set of treatments

and sample times. The actual treatments used and sample times for each experiment

are described in detail in Table 34.

v\ Samnlinp nrocedure

Samples were taken from all experimens at 4 week intervals.

In the experiment involving colonization of galls, the fust sample was taken 8 to 10

weeks after planting to allow time for gall formation; in all other experiments the

initial sample was taken within 2 weeks after planting.

At each sample time, plants were gently pulled from pots and roots were

shaken free of all but closely adhering soil. Sections (2.5 crn in length) were cut from

the root, stem and lateral toots as indicated in Figure 14. An effort was made to

sample from the same location on the root system each time. The diameters (top and

bottom) of each of these sections was recorded in order to estimate root surface area

and sections were suspended in l0 ml SDV/.

Figure 14. Sampling sites for root colonization studies.

1. Almond

A = 0.5 cm below soil line (stem)

B = 0.5 cm below cotyledons (roo$

C = lateral roots (no secondary ttrickening)

2. Grapevine

A = 10 cm above soil line (systemic)

B = 0.5 cm below soil line (stem)

C = on side roots 0.5 cm from stem (rooÐ

D = lateral roots (no secondary thickening)

@

c oty I edon++B

þcm

J*r

l<BcI

\c

20

Initially, where systemic samples were taken, stem sections (2.5 cm in length)

were cut approximately 10 cm above the soil line, surface-sterilised in 1:10 Milton's

solution (16.51o sodium hypochlorite) followedby 90Vo ethanol, then rinsed and

macerated thoroughly in 10 ml SDV/.In Vine E,x.2, systemic samples were taken

using a modification of the method of Tarbah and Goodman (1986). A vacuum pump

(GEC Machines, UK; Type 852208) was attached to a 1 litre vacuum flask and holes

bored in rubber stoppers to allow a snug fit of the vine cutting into the stopper.

Vascular washings were collected in plastic tubes after I to 2 ml of SDV/ was sucked

through L0 cm internodal cuttings (taken 10 cm above soil level). Vascular washings

(200 pl per plate) were spread on the selecúve medium of Roy and Sasser (1983) and

colonies counted after 5 days' incubation at 28oC.In Almond Ex. 1, systemic

samples were taken from almonds at 6 sample times by maceration in SDW. Samples

were taken from stem sections 5 to 10 cm above the soil line.

For all samples, tenfold dilutions were made in SDV/ after 30 sec. votexing;

three 5 pl aliquots of each dilution were plated on selective media and incubated at

25oC (see Table 3A for details of media used in each experiment). Enumerations were

made alter2days on nutrient agar,3 days on biovar 2 medium and after 5

days'incubation on the biovar 3 medium of Roy and Sasser (1983) or on minimal

medium.In Almond Ex. ,root weights were taken for 10 root samples in addition to

root surface areas in order to correlate surface area versus wet and dry root weights.

For Almond Ex. 3, the fresh weights, number and posiúon of galls were

recorded. For an estimate of gall surface populations, whole galls were suspended in

known volumes (dependent on gall size) of SDW and agitated on a rotary shaker for

30 minutes. Ten-fold dilutions in SDV/ were made and three 5 ¡rl aliquots plated on

selective media for each dilution. Gall surface areawas measured by recording the

average diameter of each gall and surface area was calculated on the assumption that

the galls were either spherical or hemi-spherical in shape.

For internal gall populations, galls were surface-sterilised as described above

and 0.5 cm3 sections were removed aseptically from the fresh, inner portion of the

It should be noted that for each treatment, samples wore plated both on the

medium selective for the inoculated strain as well as on media selective for the strains

inoculated in other treatments. Biovar 2 populations in the soil remained below 104

cfulcrn2on both almonds and vines and biovar 3 populations were below l'03 cfu/cnr2

footonbothhosts.Contaminationbybackgroundpopulationswasnotperceivedasa

major souÍce of error'

The identity of strains reisolated from soil was checked both by their ability to

catabolizetheappropriateopinesandbytheirplasmidprofiles.Althoughthisisnota

definitive test of stain identity, it was considered suffrcient because of the low numbers

of agrobacteria present as contaminants'

2l

gall. For very small galls, the diameter was recorded and volume estimated. Sections

were ground with a mortar and pestle in 1 ml SDW and left to sit atroom temperature

30 min to allow movement of bacteria out of the gall. Dilutions and plating were

performed as above. ( tnt"n+)

vi) AnalYsis of results

Resuls of all pot experiments were analysed on GENSTAT

(Rothamsted Experimental Station, 1977) using a one-way analysis of variance. Data

were log-transformed in order to give a normal distribution of variances. Block

effects were analysed separately and found to be non-significant in all cases.

h) Comparison of selective mediaÍor biovar 3

In a preliminary experiment, 10 known strains of biovars 1 and 2 and

12 strains of biovar 3 were streaked for single colonies on the selective media for

biovar 3 of Brisbane and Kerr (1983) and of Roy and Sasser (1983); growth was

recorded after days'incubation at 28oC. In a second experiment, overnight NB

cultures of three biovar 1 and 2 and six biovar 3 strains were diluted to 500 to 1000

cells/ml in SDW. Three 100 pl aliquots for each strain were spread on the biovar 3

medium of Brisbane and Kerr (1983), the biovar 3 medium of Roy and Sasser (1983)

and nutrient agar amended with 0.17o yeast extract. Results were enumerated after 3

days'incubation at 28oC for all media and again after 5 days for the two biovar 3

media.

22

PART A: RESULTS

4.1 Sampling Techniques

i) Comparison of selective media for biovar 3

When strains of biovars 1,2 and 3 were compared after streaking on

two biovar 3 selective media, 83 (Brisbane and Kerr,1983) and RS (Roy and

Sasser,1983), all 12 biovar 3 strains grew on both media. On 83 media, 2/10 biovar

2 andS/lQ biovar 1 strains gfow, though biovar 2 strains grew very slowly and

therefore could be read.ily distinguished from biovar 3. On RS media, 10/10 biovar 1

and 0/10 biovar 2 strains grew; biovar L strains did not display the characteristic deep

red centre of the biovar 3 strains. It was observed for some of the biovar 3 strains that

the dark red coloration was often only visible where single colonies appeared and was

not evident on the heavily streaked section of the plate.

When bacteria were diluted and plated, 83 media gave lO\Vo recovery of 5/6

biovar 3 strains andTOVo fecovery of the remaining strain G<252), in comparison

with their recovery on amended nutrient agar. Recovery of biov* 2 st ins and, of 213

of the biovar 1 strains was O7o but 607o recovery of a third biovarl strain was

observed. However, colonies of the latter were smaller than biovar 3 on the same

medium. On RS medium, there was O{uorecover! of biovar 2 andl007o recovery of

all biovar 3 strains andof.2l3 biovar L strains. However, biovar 1 colonies were

small and white in comparison with the distinctive, large, red-centred biovar 3

colonies. It was noted that both of the biovar 3 media actually gave a higher recovery

than amended nutrienr agar of 416 of the biovar 3 strains. Although neither of the

media are completely selective for biovar 3, it is easier to distinguish biovar 3 from

biovar 1 colonies on the media of Roy and Sasser because of the differential

tetrazolium dye uptake and resulting pigmentation of biovar 3. Therefore RS medium

was used for selection and recovery of biovar 3 in all ecological studies.

23

ii) Correlation of root surface area and root weight

Tabte 4A shows the correlation between root surface area and root

weight. Because there is no convention about expressing colonization data and, as

many authors express bacterial populations in terms of root weight" this correlation

serves as a rough guide to compare data presented in this study with data expressed in

terms of bacterial cfry' unit weight of rool Therefore, populations per gram fresh

weight of root are approximately twice those per cm2 root and populations per gram

of dry root aIe very roughly lO-fold higher. Obviously, these correlations are

extremely crude and would serve only as a guide on a comparable host species.

4.2 Colonization of Almonds and vines b]¡ Biovars 2 and 3

The results presented in this section were obtained from Almond Ex. 1 and

Vine Ex. 1 (Iable 3A). The purpose of these experiments tw¿ts to determine ifAerobacterium strains representing biovars 2 and3 differed in their ability to colonize

their respective hosts, stonefruit and grapevine. Almond was used to represent

stonefruits in all of the pot experiments.

i) Almonds

Figure 2A shows the panern of root colonization over 12 months of

wild-type isolates K27 a¡dK309. In a separate treatment in the same experiment, a

mixed inoculation of K27 and K309 was made in order to test for competitive effects

between the two strains. No significant differences were found betrveen single and

mixed inoculations for K27. Differences for K309 between single and mixed

inoculations were observed at 3 out of the 12 sample times but followed no consistent

pattern (Appendix C, Table AC-l). There were significant differences (P< 0.05)

between root populations of K27 andK309 at all months except February for single

inoculations and, in November, December and February, for mixed inoculations.

From August to October, during the period where the first gall formation \ilas

observed" K27 maintained levels of more than 10- to 100-fold higherthan the biovar

Table 44. Comparison of Root Surface Area and RootWeightl

Surface area(cm2)

Fresh weight Ratio 2

Root Section:

1

2

3

4

5

6

7

8

2.75

2.36

3.93

3.14

r.96

2.36

L.57

3.93

(e)

.99

2.0r

2.19

t.29

.64

L.25

.60

1.98

2.78

r.t7

1.79

2.43

3.06

1.89

2.62

1.98

.2

.43

.51

.26

.1

.37

.15

.54

10.58

5.49

7.71

12.08

t2.38

6.38

10.47

7.28

Drv weishtG)

3 Ratio 4

Fresh weight/ surface area = 2.22 +/- 0.62

Dry weighl surface area = 9.05 +l- 2.65

1 - Method of root sampling is described in Part A, Materials and Methods. All rootsections were 2.5 cm in length. samples were taken at month 4 in Ahón¿ 8.. ¿.

2 - Ratio of fresh root weight to root surface area.

3 - Dry weights taken after 3 days'incubation at 90oC.

4 - Ratio of dry root weight to surface area.

Figure 2A: Colonization of almond roots by strain K27 (brovar 2)

and strain K309 (biovar 3). These data represent the

result of single inoculations of both srains and are

presented along with data from mixed inoculations

of these strains in Appendix C, Table AC-1. Plants were

inoculated in June.

ooLì¡E()

=lo0)o

I

6

4

0

2

JJASONDJFMAMmonth of samPle

Iø

K27K309

24

3 strain, K309. In the finat 3 months of the experiment,KzT numbers were again 10-

fold higher than K309. Only in December did K309 exceed K27 following single

inoculations.

The rapid decrease of K27 numbers from October to December suggested that

the pattern of root colonization may have changed, due perhaps to secondary

thickening of the main roots which occurred during that period. This decrease in

biovar 2 populations on almonds has been observed previously (Shim et al., 1987).

However, when numbers on unthickened lateral foots were measured from February

to May, they were not signifrcantly different (P< 0.05) from numbers on roots (data

not shown). This indicates that secondary thickening is not the cause of the fall in

numbers

Bacterial numbers on stetns were measured in the latter half of the experiment

and they followed the same trend as those on roots but in general were more variable

(data not shown).

Numbers of biovar 2 in galls were low (approximately 103 "¡u7"t13

gall

tissue), indicating that even the galls were not heavily colonized after 10 to 12

months. Galls were not generally decayed and appeared to be physiologically active.

All galls sampled during the experiment contained nopaline (confumed by

IIVPE) and thus wele presumably formed byK27. Gall weights increased

throughout the experiment for both of the treaunents which included K27 Clable 5A).

Several galls were formed in the K309 only treatment near the end of the experiment

but were found by TIVPE to contain nopaline. As K309 is an octopine strain, it was

assumed that the galls were fomred as a result of contamination with K27, a nopaline

strain or possibly by biovar 2 strains present as contaminants in the soil.

Systemic samples from the almonds were taken in October to January and

again in April and May; neither K27 nor K309 was found in these samples.

Colonies of both K27 andK309 were periodically picked from the

enumerating media, purihed and checked for the presence of the colrect plasmid. A

Table 54. Gall weights: Almond Ex. 1 I

Month

June

July

August

Sept.

Oct.

Nov.

Dec.

Jan.

Feb.

March

April

May

Dry weight of galls 2(g)

Treatments3:

K309 K27 K27: K309

0a 0a 0a

0a 0a 0a

0a 0.084 0a

0 a 0.t7 a 0.38 a

0 a 0.35 a 0.13 a

0 a 0:58 a 0.02 a

0a 3.58b 0.92a

0.11 a 11.31b 236a

0 a 8.39 b 5.84 b

0a 38.10b 16.52b

0 a t7.48 b 8.59 b

9.432 ll.r1 a 19.74a

1- Almond Ex. 1 described in Table 34. Procedures for sampling etc. described inPart A, Materials andMethods.

2- Values with the same letter are not significantly different. Data was analysed byone-way ANOVA andleast squares difference (P< 0.05) between means at eachmonth were deterrnined.

3- Treatments K27 and K309 were single inoculations of each strain andK27l K309was a 1:1 mixture of the two strains.

25

grcater number of contaminants were found on RS media but not at a level which

would signifrcantþ affect results.

ii) Vines

Figure 3A shows the pattern of colonization over 12 months of K27

and K309 on vine roots. As with the comparable experiment on almonds, few

competitive effects were observed in the mixed versus the single inoculations (data

presented in Appendix C, Table AC-2). There were no signifrcant differences (P<

0.05) between single and mixed inoculations for K309 and for K27 dtffetences were

observed ar only 2 of the 12 sampling times. K27 maintained significantly higher (P <

0.05) numbers than.K309 during September to December in the single inoculations.

The actual levels of Í(27 were approximately l0-fold lower on vines than they were

on almonds. Levels of K309 dropped markedly in the fust 3 months to 103 cfulcrû

(the detection limit) but stabilized betrveen 103 to lú cfulcr& in the subsequent

samples. No galls were formed on any of the treatments, despite the fact that K309

was pathogenic in artif,rcial inoculations on the same cultivar of grapevine.

Numbers on stems at crown level were not signif,rcantly different from

numbers on roots and there were few significant differences benveen treatments.

Numbers on lateral roots were also low (103 cfu/cm2 root) during February to

August for all treatmenrs and became undetectable (<103¡ in June and July, as the

vines became dormant (data not shown).

Only K309 and never K27 was found in systemic samples. K309 was flrst

detected in systemic samples taken in October and was further detected at low but

consistent levels (101 to 102 cfu/cm3 stem) until March, after which time it was not

detected again until July, when the vines were fully dormant and August, at bud

burst.

Figure 34. Colonization of grapevine roots by strain K27

(biovar 2) andstrain K309 (biovar 3). These

data represent the result of single inoculations

and are presented along with data from mixed

inoculations in Appendix C, Table AC-Z. Plants were

inoculated in S eptember.

ooLnEofoO)o

6

5

4

3

2

1

0SONDJ FMAMJ JA

month of samPle

Iø

K27K309

26

4.3 Constmction of Transconjugant Strains

¿) Transfer of Biovar 3 Ti Plasmid into Biovar 1 and 2 Backgrounds

Purified plasmid DNA from K309 was transformed into an NTl

background, resulting in a biovar 1 strain that contained a biovar 3 plasmid. Initial

attempts to transform a plasmidless strain of biovar 2,K103, with pTiK309 were

unsuccessful and, because the conjugative opine for biovar 3 was unknown at this

time, the technique of RP4 mobilization was used @omhoff etal.,L976; Chilton et

a1.,1976; Van Larebeke et a1.,1977).

RP4 was introduced into K309 via mating with E.coli strain K382 (Table 1A).

Strain K309 containing RP4 was then further mated with C1RS (biovarl) or

K128chlr, a spontaneous chloramphenicol-resistant mutant of this naturally Ti-

plasmidless biovar 2 strain. Selection of transconjugants was made using the

antibiotic resistances on RP4 (Km, Cb, Tc) and on the chromosome of the recipients

ClRS (rif, str) and K128 (chl); selection for pTiK309 was made on the basis of its

ability to catablize octopine. All transfers were confirmed by gel electrophoresis and

by pathogenicity testing (see Materials and Methods). Both pTiK309 and RP4 were

transferred and maintained separately in the new background (Figure 4A). RP4 was

maintained separately and a cointegrate was not formed with pTiK309, in contrast to

the repors of others ( Holsters et a1.,1978b; Hooykaas et a1.,1980). Chilton et al.

(1976) reported stable coexistence of RP4 and a Ti-plasmid. However, although RP4

was separately maintained in the strains in this study, it was not stable in the new

background and there was evidence of some breakdown in Aerobacterium (evident in

Figure 4A,lane 1).

b) Transfer of Bior¡ar 2 Ti Plasmid to a Biovar 3 Background

Initial attempts to nansfer the biovar 2 Ti-plasmid,pTi[(Z7 , into a

wild-type biovar 3 background (containing a Ti-plasmid) by RP4 mobilization were

unsuccessful. No wild-type Ti-plasmidless strains of biovar 3 were available so

Figure 44. Agarose gel electrophoresis to confirm transfer

of pTiK309 into biovar 2 strain, K128.

Lanes:

1 to 4: K128 (pTiK309, pRPa)

5: K1286: K3097: K3098: K382 (8. coli carrying pRP4)

a: pTiK309b: cryptic plasmid of K128

c: pRP4

d: chromosomal DNA

Arrows indicate:

ab

+

c-+

d--._

27

attempts were made to cure a \¡/ild-type of its plasmid. Initial attempts using heat

curing (ÉIamilton,lgTl) or ethidium bromide (Lin & Kado,l977) were unsuccessful.

Only partial plasmid deletions were obtained using these methods.

A novel approach was taken,using the knowledge that the Ti-plasmids of the

biovar 1 strain, 46, and the biovar 3 isolate, A9162, are incompatible (Knauf &

Nester,1982). It was thus thought that this may be generally true for biovar 3 Ti-

plasmids. An Aerobacterium strain, NTl, containing both the RP4-derivedplasmid,

pDP35, and a plasmid, pJS400, carrying the Kpnl fragment of A6 which codes for

plasmid incompatibility (Inc) was obtained from Dr. S K. Farand. By transferring

these plasmids into a recombination def,rcient (rec-) mutant of E.coli. strain H8101

rifr, and selecting for the kanamycin resistance caried on pJS400, a cointegrate

between the two plasmids was formed which contained the wide host range capability

of pDP35 as well as the incompatibility function of 46. Cointegrate formation occuls

because pJS400 is unable to replicate in E. coli. The HB101 strain containing the

cointegrate was designated2-4 (Iable 1A) and was then used to eliminate the biovar 3

plasmid, pTiK377, from its wild-type background. In order to eliminate all

Agrobacterium incompatibility functions from the biovar 3 strain, another wide host

range plasmid pPHIJI (Hirsch & Beringer,l984), was used to eliminate the pDP35/

A6 Inc region cointegrate from K377. Both pDP35 and pPHIJI are in the same

plasmid incompatibility group (IncP).In later work,the same scheme forTi-plasmid

elimination was used to eliminate the resident Ti-plasmidof I{252.

A Ti-plasmidless strain of biovar 3 was thus available to be used as a recipient

for the transfer of the biovar 2 Ti-ptasmi d, pTiK27 . Once again, the technique of RP4

mobilization was used to transfer pTiK27 into the K377pTí- background. The ability

of biovar 3 to tolerate 27o NaCI in the growth medium was used to select for the

recipient background; plasmid selection for RP4 was made using its antibiotic

resistances and selection for pTiK27 was made on the basis of its ability to catabolize

nopaline. All transconjugantslilere screened for plasmid transfer by get

electrophoresis (Figure 6A) and for pathogenicity (discussed in the following

Figure 54. Steps involved in transfer of a biovar 2Ti-plasmid,$it<27, into biovar 3 (K377) background.

1. Elimination of pTiK377 by awide host range

plasmid, pDP35, carrying a cloned fragmentof the A grobacterium Ti-plasmid incompatibilityregion (hc A6).

2. Elimination of pDP35 with another IncP plasmid,pPHlJI, containing no Agrobacterium sequences.

3. Inttoduction of pTiK27 into K377 by RP4

mobilization.

rlrc

1

H8101

K R

K377

K377

K377

K377

HBlOl

X

t.

X.1,

pTiRP4

2

X.'t

3

INCA6

pDP35

lnc P

pPtllJl

lncPlncP NC A6

pDP35

pPtllJl

lncP

RP4 pTi

Figure 64. Agarose gel electrophoresis showing

transfer of pTiK27 into biovar 3 strain, K377.

Lanes:

a, b: Í377 (pTiK27,pRPa)c: I(27 (pRP4)

d: K377 pTi- (pPHlJr)

l: pTiK272: pPHIJI3: pRP44: chromosomal DNA

Origin of smaller band in lane d is unknown.

28

secrion). The general scheme for transfer of pTiK27 into K377 is outlined in Figure

54.

Throughout this thesis the transconjugant strains are referred to as

K377(qT1K27) and K128(pTiK309). It should be noted that pRP4 is also present in

these strains but, for simplicity, it is not indicated in the strain designation.

c) Characterization of transconiu gants

i) Patho genicity of transconju gants

when bi."uliit*'tt;,?,oiovar 3 Ti-plasmid i.e. strains+hs

designated K128 (pTiK309) were tested for pathogenicity on tomato stems, it was

observed that the transconjugants were less virulent than wild-type K309 (Figure

7A). When controlled inoculations (5 pl of a 5 x 108 cells/ml bacterial suspension)

were made into tomato stenu with a sterile syringe, mean stem diameters and gall

weighs measured 4 weeks after inoculation differed significantly fum the K309.trurrncur¡tt3cl,,r-8. +l?

control (Table 6A). \Mhen biovar 3 n " õontainingnbiovar 2 Ti-plasmid i.e. strains

d.esignated K377 (pTiK27) were inoculated onto tomato stems, no loss of virulence

of the transconjugants was observed.

Table 7A summarizes the pathogenicity of parental and transconjugant strains

on almonds and grapevines. Both transconjugants had a more limited host range than

wild-type strains. Strain K128 OTiK309) was nonpathogenic on almond while K309

was weakly pathogenic in artifrcial inoculations. Similia¡ly, strain K377 (pTiK27)

retained the pathogenicity of f{27 on almond but was completely nonpathogenic on

grapevine. It was interesting to note that the biovar 2 strain, K27, was pathogenic in

artifrcial inoculations but not in natural infections on grapevine @esults, Section

4.2).

Figure 74. Pathogenicity of biovar 2lbiovar3reciprocal transconjugants on tomato stems.

Photographed 4 weeks after inoculation.

1. AtoD:K128(pTiK309)E: K309 (pRP4)

F: K309

2. A: K377 pTi-B: K377 (pTiK27)C:K27 (pRPa)

1

A

B

c

D

E

F

2

A

B

c

Table 64.

Strain

K309

K128

K128 (pTiK309) #1

K128 (pTiK309) #2

Mean stem diameter 3(mm)

8.33 a

3.67 c

6.00 b

6.83 a,b

t3r.fi a

6b

nJT b

23J7 b

1- method of testing virulence described in Part A Materials and Methods; results

iuL"n afær weeksl Values represent a mean of 6 replicates. One-way analysis ofvariance was performed.

2- plasmid transfer described in Part A Results, Section A'3'

3- values with the same letter not significantþ different

Analysis of Variance: Gall weights

sBetween strains 68.5

Residual 17.5

Totat 86

4- signifrcant at P( 0.01 ( F 5,t8 = 4.25)

df

5

18

23

MS

t3.7

2.t9

Ea

6.26**

Table 74.

Strain

K27K128K309K377

K128 (priK309)K377 (pTiK27)

++¡2

Almond(cv. 'Chellaston')

¡2¡2

+ +

+

1- Method of pathogenicity testing is described in Part A Materials and Methods.

Almonds were inoculated on the young internodes of the stem and at the crown.

Grapevines were inoculated on internodes at the tip of the new season's growth.

2- galls very small.

29

ii) Tartrate utilization by transconjugants

There has been areport in the literature of tarrate catabolic

genes in a biovar 1 strain from grapevine being located not on the chromosome but on

a separate Mkb plasmid, pTAR (Gallie g!-A1., 1984). Biova¡ 3 strains isolated from

grapevine a¡e able to catabolize tartrate (Kerr & Panagopoulos, L977) but biovar 3

isolates from chrysanthemum cannot catabolize tartrate (Bazn & Rosciglione, 1982).

These observations suggest that tartrate catabolism is not a conserved characteristic

among all biovar 3 strains and that the ability to utilize tarEate may be correlated with

colonization of grapevines. Thus, the possibility that tartrate utilization is coded for by

the Ti-plasmid in biovar 3 was investigated.

Biovar 3 strains with and without Ti-plasmids were tested (see Part B,

Materials and Methods) for their ability to catabolize Na-tartrate and results are

presented in Table 84. Typical positive and negative reactions for ta¡trate utilization in

the bromothymol blue indicator medium are depicted in Fig 8A. Results indicate that

tartrate utilization for strains K309, K377 andK252 is chromosomally encoded and

not associated with the Ti-plasmid of biovar 3 @TiK377). No plasmids in the sizet+

range of pTAR (44kb) was observed in any of thenbiovar 3 isolates used in this study.

4.4 Colonization of Almonds and Vines by Constructed Strains

The results discussed in this section were obtained from Almond E;x.2 and 4

and Vine Ex 2 respectively Clable 3A). The purpose of these experiments was to

deterrrine if the Ti-plasmid was important in plant colonization.

i) Almonds: Effect of biovar 2 Ti-plasmid on colonization b]'

biovar 1

In Almond 8x.2, biovar I and2 strains with and without a Ti-plasmid

were compared. Figure 9A shows the pattern of colonization of wild-type and

Table 84. Tarrate Utilization and Biovar 3

Strain

K309r377K252ClRS

C1RS (pTiK309)C1RS (pTi[<z7)K377 pTí-K252pTi-K377 OriK27)

Groupingl

Biovar 3

Biovar 1

Biova¡ 1

Biovar 1

Biovar 3

Biovar 3

Biova¡ 3

Tartrate catabolism

+++

+++

1- biovar grouping of chromosomal background2- construction of transconjugants is described in Part A Results, Section 4.3.

Figure 84. Indicator tubes showing that tartrate utilization

is not Ti-plasmid encoded by biovar 3 strains.

A.B, C.

D, E.

K309 (positive)

clRS #1,2C1RS (pTiK309) #1,2

l¡Jooo

K309- tZ

ClR

S

ClR

S

C1R

S P

Ti K309 2

CIR

S P

T¡K3O

9 1

Figure 94. Colonization of almondroots by srains

K27 (biovar 2), K57 (biovar 1), K57(pTiK27)

and K103 (Ti-plasmidless biovar 2). Data were

obtainedin Almond Ex. 2 and arepresentedin

Appendix C, Table AC-3. Plana were inoculatedin

October.

I

6

4

2

0

ooLô¡E(Jfcto)o

Iø@ø

K27K57K57 pTiI<21K103

Fo NDJmonth of sample

30

transconjugant biovarl and2 strains. Samples were taken over a 5-month period. In

the first 3 months, there were no significant differences betweenK2T, K57 (wild-

type biovar 1) and K57 (pTiK27). However, in January,K2T was significantly more

numerous on the roots @< 0.05) than the two strains with the biovar 1 background

and in February was more numerous than K57 (pTiK27). K57 was chosen for this

experiment because, in a previous study, Shim et al. (1987) had observed poor

colonization of almond roots by the biovar 1 strain, CIRS. The possibility existed

that, because C1RS is an antibiotic-resistant mutant of a Ti-plasmid cured biovar 1

strain ttrat it may be less'ecologically competent' than a wild-type strain. Strains in

this study which had a K57 background exhibited colonization comparable to that of

biovar 2 for much of the experiment.

K27 declined to lower numbers after month 3 (December) in this experiment

than it did in other almond experiments. The reason for this is not clear but could be

related to soil temperatues. Almond Ex. 2 commenced in October and samples for

months 4 and 5 were taken in January and February respectively, the hottest part of

the year. The Ti-plasmidless biovar 2 strain, K103, was not numerous throughout the

experiment. This probably does not reflect any real effect of the Ti-plasmid but is

rather a reflection of the origin of this strain. K103 was not isolated from stonefruit

and although it was identified as biovar 2itmay differ significantly from the biovar 2

stonefruit and rose isolates which comprise a large part of this goup. In subsequent

experiments the wild-type strain from stonefruit, K128, was used to represent Ti-

plasmidless biovar 2 strains.

ii) Almonds: Colonization by biovar 2/biovar 3 reciprocal

transconjugants

In Almond Ex. 4, biovar 2 and 3 wild-type strains were compared with strains

having the same chromosomal background but containing reciprocal Ti-plasmids.

Table 94. Root Populations: Almond Ex. 4 I

Month K27

Treatments: Q-og cfu/ cm2 root)

K309 K377 K128 K377 K128(priK27) (priK309)

4.77 b 4.92b 5.45 a,b 4.90 b 5.66 a,b

4.87 b 5.16b 5.91 a,b 4.95b 5.64a,b

437 b 4.62b 5.91 a 4.74b 5.28 a,b

3.29b 3.99b 6.18 a,b 3.63b 5.02 b

4.47 aþ 3.57 b 5.75 a 4.55 a,b 3.63 b

3.57 b 3.58 b 6.62 à 3.70 b 4.45 a,b

May

June

July

August

Sept.

Oct.

6.03 a

6.41a

5.99 a

634 a

6.17 a

6.98 a

1- Almond Ex. 4 described in Table 34. Data lepresent results of separateinoculations of each strain. Procedures for sampling are described in Part A ,Materialsand Methods.

2- Values with the same letter are not signifrcantly different. Data were analysed byone-way ANOVA and the least squares difference (P< 0.05) between means at eachmonth was determined.

The identity of strain K128 was corfirmed by gel erectrophoresis.

31

Roots: Table 9A indicates the pattern of root colonization over a 6 month period of

witd-type biovar 2 and3 isolates as well as of two transconjugant strains, a biovar 3

strain containing a biovar 2 Ti-plasmid(K377 pTiK27) and a biovar 2 strain

containing a biovar 3 Ti-plasmid (K128 pTiK309).

As in the initial experiments wittr almonds (Section 4.2), the wild-type biovar

2 isolate, K27 , muntained very high (106 cftr/cm2 root or geater) numbers over the

6 month period. Numbers of the Ti-plasmidless biovar 2 strain, K128, did not differ

significantly (P< 0.05) from those of K27 during this period, indicating the

importance of the chromosomal background in determining colonization levels. ('.,"e rt)The wild-type biovar 3 strains, K309 and K377, did not significantly differ

from each othe¡ at any sample time and showed a similia¡ decline in numbers on the

roots. After 3 months, numbers of both strains were1.04 cfulcm2 root or less.

Numbers of both wild-type biovar 3 strains differed significantly (P< 0.05) from K27

at all sample times except in September, when K309 andl(2l were not different. This

confrrms the result observed in the initial almond experiment (Section 4.2), indicating

the marked differences in colonization of almonds by biovars 2 and3.

For the first 4 months of sampling, colonization by K128 (pTiK309), the

biovar 2 strain containing a biovar 3 Ti-plasmid did not differ significantly (P< 0.05)

from strain K128, which has the same chromosomal background but lacks a Ti-

plasmid. It is important to note that in the enumeration procedure for this strain (see

Table 3A), isolation was being made on a medium that was co-selective for the Ti-

plasmid and the RP4 plasmid. In August to October, when media selective for

chromosomal background only was used for isolation of bacteria from the treatrnent

inoculated with K128 (pTiK309), results indicated that total biovar 2 numbers in that

treaünent were about lO-fold higher than populations of K128 (pTiK309) (data not

shown), suggesting that there was some loss of pTiK309 and/or RP4 from the K128

background. Plasmid isolations were made from bacteria isolated on biovar 2medta

and it was observed consistently that 50 to 807o of these isolates had lost the biovar 3

plasmid (demonstrated in Fig 104). Complete loss of the Ti-plasmid and of the RP4

Figure 104. Agarose gel electrophoresis showing plasmid

loss from transconjugant strains in Almond Ex. 4.

Isolations made in month 6 on media selective

for chromosomal background only.

1. Reisolation of K128 (pTiK309)

Lanes:a to c: Isolates recovered on 82 medium

amended with antibiotics selectivefor pRP4.

d to j: Isolates recovered on 82 media.

Loss of pTiK309 observed in lanes a to e.

Arrows indicate:

1. pTiK3092. cryptic plasmid of K1283. pRP44. chromosomal DNA

2. Reisolation of K377 (pTiK27)

Lanes:f: K377 (pTiK27)

a to e: Isolates recovered on RS media.Retention of pTiK27 visible inlanes b,c and d. Poor DNAextraction in lanes a and e.

Arrows indicate:

1. pTiK272. pPHIJI3. pRP44. chromosomal DNA

1

3><1\-2

.+4

+4

I¡¡

trJúdl

32

plasmid was observed; deletions were observed in the RP4 plasmid but there were no

apparent deletions in the Ti-plasmid, as has been reported in a similiar situation in

Rhizobium (tWang et al., 1986).

In contrast, the biovar 3 strain containing the biovar 2 Ti-plasmid, strain K377

(pTiK27),followed the same pattern of root colonization as its wild-type counte{part,

K377.The two strains did not differ signif,rcantþ rtany sample time (P< 0.05). It

should be noted that there were only few and very small galls formed even by strain

K377 OTiK27). In terms of levels of root colonization, the transconjugant strain

displayed the same pattern of colonization as its wild-type background and that there

was no effect of the Ti-plasmid on bacterial numbers on the root.

V/hen plasmid isolations were made for strain K377 (pTiK27) from selective

and non-selective media, Ti-plasmid loss was observed in less than207o of the

isolates tested (demonstrated in Fig 104), indicating ttratpTiK2T was much more

stable in vivo in its new background than was pTiK309.

Stem and lateral roots: The pattern of colonization of all isolates on the stem was

virtually identical to those on the root (Table 104, i). Once again the two wild-type

biova¡ 3 strains were signifrcantly less numerous

(P< 0.05) than K27 at all months except October. Numbers of the transconjugant

strain, K128 (pTiK309), declined in September and October on the stem as they did

on the roots. The actual levels of colonization for all isolates are very similiar to those

on the roots.

In general, levels of all isolates on the lateral roots (fable 104, ü) tended to be

lower than those on stems, although in October (month 6) a general 10-fold increase

was observed for all strains.

iii)

In Vine 8x.2, the same strains were used as in Almond Ex. 4.

Table 104. Sæm and Lateral Root Populations: Almond Ex. 4 I

i) Stem Populations Treatments: 2 (t og cfulcrû stem)

Month

May

June

July

August

Sept.

Oct.

K27 K309

3.95 b

4.47 c

3.89 c

3.55 b

4.27 b

4.t3 aþ

K317 Kl28

4.61 a,b

6.03 a

5.52 a,b

6.18 a

6.51 a

5.58 a

4.94a

5.55 a'b

6.03 a

6.22a

6.55 a

6.12a

4.04 b

4.Cl c

4.19 b,c

3.75b

3.99b

3.53 b

K377 K128(priK27) (priK30e)

4.30 a,b 4.93 a

4.'72b,c 4.68 b,c

4.04c 5.17 a,b,c

3.20b 5.33 a,b

4.50 b 3.49b

3.38 b 4.43 z,b

ii) Lateral Root PopulationsTreatments :2 g-ogcfty'cm2 root)

Month K27 K309 K377 K128 11377 K128C,TiK27) (pTiK309)

May 5.09 a 4.29a 4.66^ 4.51a 4.56a 4.84a

June 6.59 a 5.30 a'b 4.99b 5.40 4b 4.99b 5.04 a,b

July 4.82 a 3.68 a'b 3.89 b 4.49 a,b 3.27 b 3.08 a'b

August 5.07 a 3.38 b 3.42b 4.92^ 3.16 b 3.60 b

Sept. 5.44 a 3.60 b,c a.g6 t,t 5.08 a,b 3.94 a,b,c 3.2g c

oct. 6.01 a 4.68 a,b 4.53a,b 5.52 a,b 4.35 b 5.06 a,b

1- Almond Ex. 4 described in Table 34. Data representresults of separateinoculations of each strain. Procedures for sampling are described in Pa¡t A, Materialsand Methods.

2- Values with the same letter are not sþificantly different Data were analysed byone-way ANOVA and the least squares difference (P< 0.05) between means at eachmonth was determined.

Table 1.14. Root Populations: Vine Ex. 21

Month K27

Treatments: (tog cfu/cm2 root)

K309 K377 K128 K377 K128(priK27) (priK309)

4.22a 4.54a 4.66a 436a 4.56a

4.16 b,c 4.30 b 3.76b,c 4.79 ^ 3.43 c

4.24b 4.26^þ 4.16 b 5.16 a 3.O7 c

3.7ta 4.12a 3.532 3.88 a 2.91b

3.30 a,b 2.65b 3.70a 3.66a 3.06 b

3.00 a 2.92a 3.55 a 4.40a 2.85a

Sept.

Oct.

Nov.

Dec.

Jan.

Feb.

4.56a

3.97 b,c

4.59 ^,b

3.79 a

3.66 a

3.69 a

1- Vine Ex. 2 is described in Table 34. Data represent results of sparate inoculationsof each strain. Procedures fe¡ sampling are described in Part A, Materials andMethods.

2- Values wittr the same letter are not signifrcantly different. Data were analysed byone-way ANOVA and the least squares difference (P< 0.05) at each month betweenmeans was determined.

33

Roots: Table 114 shows the colonization of vine roots over 6 months by the wild-

type and transconjugant biovar 2 and3 strains. There were few signifrcant differences

between trearments. Transconjugant strain, K128 (pTiK309), was significantly (P<

0.05) lower than its wild-type background, K128, in November to January.

Transconjugant strain, K377 (pTiK27), was significantly different from its wild-type

background,K3'l7, and from K27 only in October.

As in the comparable experiment on almonds, isolations from the treatrnents

containing transconjugant strains were made on media selective for only the

chromosomal background and plasmid isolations made from bacteria growing on that

medium. All estimates of plasmid loss are based on observation of isolations made at

3 to 4 times during the sampling period. Figure 114 indicates the pattern of plasmid

loss from these strains. Strain K128 (pTiK309), which was extremely unstable on

almonds, showed a much lower rate of plasmid loss on vines (approximately 50Vo as

compared to90Vo on almonds). Plasmidretention in these strains was confirmed by

checking catabolism of octopine. Total biovar 2 counts for this treatment were not

significantly different from counts made on media which co-selected for pTiK309.

Transconjugant strain, K377 (pTlI{Z7), was more stable than K128

(pTiK309) on vines (Fig 114) as it was on almonds (Fig 104) and plasmid loss was

observed in less than 50Vo of isolates. It seems evident that, in vivo, pTiK27 is

inherently more stable in its reciprocal background than is pTiK309. However,

pTiK309 appears to be less unstable on vines than on almonds which would parallel

the findings of 'Wang et al. (1986), where they found differential plasmid stability

depending on the plant host.In the studies involved in this thesis, no plasmid

instability was observed in either strain in vitro even after multiple subcultures.

Stem and lateral roots: There were no consistent significant differences between

treaünents on either the stem or lateral roots. Numbers on the stem and lateral roots

tended to be more va¡iable but, for all treatrnents, \ilere in the range of 103 to 104

cfu/cmz root for the duration of the experiment (data not shown).

Figure 114. Agarose gel electrophoresis showing plasmid

loss from transconjugant smins in Vine 8x.2.Isolations made in month 5 on media selective

for chromosomal background only.

1. Lanes:h: K309

a to g: Isolates recovered from soilinoculated with K128 (pTiK309)Note loss of cryptic plasmid of K128in all isolates. In lanes d and e the DNAextraction was poor but pTiK309 ispresent in these isolates (visible onnegative) Origin of extra band in lane fis unknown.

Arrows indicate:

1. pTiK3092. pRP4

3. chromosomal DNA

Presence of pTiK309 was confinned by checking octopine

catabolism of isolates.

2. Lanes:

l: K377 (pTiK27)k: K128 (pTiK309)

f to j: Isolates recovered from soil inoculatedwith K377 (pTiK27). Isolatesin lanes f, h and i have lost pTiK27 andisolates in lanes f and i have lost pRP4.

a to e: Isolates recovered from soil inoculatedwith K128 (pTiK309). Isolateshave all retained pTiK309 (confirmedby checking octopine catabolism).

Arrows indicate:

1. pTiK3092. pRP4

3. chromosomal DNA4. pTíK27

<-4

Ø

1+2-3>

34

Systemic infestation: When vascular samples were taken from vines by the vacuum-

flush method (described in Part A, Materials and Methods), only biovar 3 strains

were detected at months 3 to 6, albeit at very low levels (102 cfu/10 cm intemodal

stem section). Biovar 3 suains were isolated only from treatments which had been

inoculated with K309 ,K377 andK377 (pTiK27). It is unlikely that these were

contaminants already present in the vines because they were not detected in any of the

biovar 2 treatrnents.

Bur et al. (1987b) demonstrated that biovar 3 strains can form root lesions

and this may be the first step in systemic infection. However, in this work, no root

lesions were observed in any of the treatments but this was not the primary focus of

the study. Also, no galls were formed in any of the treaünents although all isolates

except K128 are pathogenic to some extent on vines.

The conclusion to be drawn from these experiments is that the Ti-plasmid has

no influence on the colonization of either almond or grapevine by biovar 2 or biovar

3 strains. Any apparent contradictions can be explained by instability of ttre Ti-

plasmids in reciprocal transconj ugants.

4.5 Colonization of Nopaline Galls

The results presented in this section were obtained in Almond Experiment 3

(Table 3A). The purpose of the experiment was to determine if there was a

quantitative difference in the colonization of galls containing nopaline by strains of the

same chromosomal background which differ in their ability to catabolize nopaline.

Galls were formed on almond roots by the biovar 2 strain K27.T}:re colonization of

the galls by two biovar 3 strains, K309 (an octopine süain) andK377 (a nopaline

strain), was compared.

Figure 124 shows the colonization over a S-month sampling period of strains

K27,K3O9 andK377 both on and in nopaline galls induced by K27.Bactenal.

numbers inside and on the surface of the gall followed similiar patterns; levels of l(27

Figure 124. Colonization of: i) nopaline gall surfaces and

ii) inside nopaline galls on almond roots. Galls

were incited by strain K27 anddata were

obtained in Almond Ex. 3. Data are presen.ted

in Appendix C, Table AC-4. Plants were

inoculatedin February.

log

cfu/

cm

'gal

l

oÌu

ÞO

)æ

3 o f J oc-

Ø A) 3 ro õ-

c-

E¡N

Ix^

xo)

(J) r

\)o{

\¡(o

{

iÐ 8

4

2

0

6I K27ø K377El K3oe

:(õo)tE()fC)(')o

AA MJJmonth of sample

35

on the gall surface in all treatrnents remained extremely high (106 cfutcr& gall) and

inside the gall increased rapidly from April to May (up to 107 cful "t¡3 gall). It should

be noted that numbers of K27 on the gall surface were lO-fold more numerous than

those on the root surface (data not shown). Table 124 shows the increase in gall

weight during the initial months of the experiment and it is interesting to speculate on

whether the increase of K27 inside the gall may be due to an increasing amount of

nopaline in the gall environmenl

The two biovar 3 strains, K309 andK377, displayed distinctively different

patterns of colonization both on and in the galls. The nopaline-catabolizing strain,

K377, showed a large increase in May on the gall surface and in May and June inside

the gall. During this period K377 attained numbers comparable to those of K27 but

then dropped to a level comparable to that of the non-nopaline utilizing biovar 3

srrain, K309, which maintained levels of 105 cfu/cm3 gall or less inside the gall for

the entire sampling period. It is tempting to associate this difference in numbers of

biovar 3 strains (signif,rcant in May and June at P< 0.05) with the availability of

nopaline in the galls and with the ability of K377 to catabolize nopaline. This would

then provide preliminary evidence in support of the opine concept.

Table 124. Gall weights: Almond Ex. 3

Month

April

May

June

July

August

Mean fresh weight of galls (g)

Treatments:

Kn K27 + K309 K27 + K377

.04 .r2 .45

0.94 1.70 1.76

4.47 2.45 1.60

0.26 3.54 3.65

t.33 12.78 8.03

1- Almond Ex. 3 is described in Table 34. Galls were formed by inoculation withI{27 andthen plants were dipped in 1:1 suspensions of K27 and the treaûnent strains.Procedures for sampling are described in Part A, Materials and Methods.

2- No significant differences were found between means at each month when datawere analysed by one-way ANOVA.

36

PART A: DISCUSSION

1) Methods and anal]¡sis

The ability of soil-borne plant pathogenic bacteria to colonize the rhizosphere

is essential for their survival and for their ability to cause disease. Therefore it is

important when studying the pathogenic process to find ways of accurately

quantifying bacterial populations on plant root systems. There is no conventional

method for sampling bacterial populations and the approach varies widely, making it

difficult to compare the results of different studies. In some cases entire root systems

constitute the sample unit (Bushby,1981; Mendez-Castro & Alexander, 1983) and in

others the root system is suMivided into segments (Kloepper,1983; Kloepper &

Schroth 1981; Kloepper et al., 1980b; Vy'eller, 1984). In this study, "roots" were

subdivided into three categories- the crown area, the main taproot and lateral roots

without secondary thickening. This was done in order to minimize error due to the

uneven distribution of bacteria on root surfaces which has been noted in electron

microscopy studies (Newman & Bowen,1974; Rovira,1956; Rovira & Campbell,

I974). An effon was made to sample from the same relative locations each time.

Variances obtained in untransformed colonization data were uniformly high

and positively skewed. This indicated that the untransformed data did not satisfy one

of the basic assumptions underlying the analysis of va¡iance test i.e. that variances be

normally distributed. When data were log-transformed, a normal distribution of

variance was obtained. Other workers (Hirano et al., 1982) have found that

populations of epiphytes on a leaf surface were most accurately approximated by a

continuous lognormal distribution. The work of Loper et al. (1984) extended this

finding to rhizosphere bacterial populations. In the latter work, it was commented that

the reason for the appropriateness of a lognormal model was the multiplicative nature

of bacterial growth- any change in the numbers of a bacterial population is

proportional to the bacterial numbers already present. In their f,reld study of

Pseudomonas populations, l-oper et al. (1984) found very large variances and the

need for adequate replication was stressed. Variation in the work presented in this

Because of the level of replication required to carry out this type of experiment,

only a limited number of strains representative of each biovar were tested. 'fhere may be

significant differences between individual strains within a biovar and these were not

observed in this study. Throughout the discussions, conclusions are drawn about

differences between biovars but it should be stressed that these conclusions are based

on data from a limited number of strains.

:

I

i'

I

37

study \ilas not as high, probably because of the more controlled nature of pot

experiments. Generally differences of 1.0 log unit were needed in order to obtain

significant treatment differences with 95Vo confidence.

Variation of bacterial numbers on stem surfaces near the crown tended to be

higher than on the root, possibly due to moisture and temperature fluctuations in this

region. Numbers in the initial samples a¡rd in samples taken towards the end of 12

month experiments also tended to be more variable, the former probably due to

va¡iation in the amount of initial inoculum received by each toot system and the latter

possibly due to an increasing microflora and the increase in nonrandom differences

between pots with respect to temperature, shading effects, fertilizer trea¡nents etc.

accumulated over the 12 month period. The use of non-sterile soil for all experiments

may have increased variation betr¡¿een replicates but it was used in an attempt to

approximate field conditions. Because the almonds were germinated from seed, there

was a certain amount of genetic variation between almond seedlings; vines, on the

other hand, were all of a single clonal type. Differences were observed between

individual experiments in the actual numbers of bacteria achieved though relative

differences benveen treatments were consistenl The differences between separate

experiments \pere probably due to differences in soil microflora and in soil

temperatures in different years. For example, the marked decrease in biova¡ 3

populations on vines observed in months 3 and 4 in Vine Ex. 1 was not noted in Vine

Ex. 2 though populations in the latter were still low and the relative differences

between biovars 2 and 3 were consistent between experiments. The fact that

consistent signifrcant differences were observed in experiments of this type is

testimony to ttre large differences that exist between these bacterial strains in their

colonization of root rr.,.l*ìTÍ?rder to detect more subtle differences between

strains, increased replication would be required and a rigidly controlled sampling

procedure developed so that more uniform root segments were sampled. Factors such

as the number of emerging lateral roots and number of lenticels, wound sites etc. all

must have an impact on colonization levels and would have to be taken into account.

38

Many workers have expressed bacterial populations in tenns of 'cfu/ g root'.

In this study ¡oot surface area was used in preference to root weight because root

weight was felt to be inadequate when comparing different types of roots e.g. fine

lateral roots versus a taproot. Because it is the root surface and not the root as a whole

that is being colonized, estimating root surface area, although somewhat crude,

should give a more realistic assessment of bacterial numbers in the rhizosphere.

The use of selecúve media to quantify bacterial numbers in each sample was

chosen in preference to serology. Serological groups in Agrobacterium a¡e not well-

characterized and serological tests in this study indicated that the biovars are

serologically heterogenous (see Part B). The use of antibiotic resistant mutants was

also avoided due to the possibility of changes in rhizosphere competence by mutants.

This phenomenon has been noted with rifampicin-resistant mutants in Rhizobium

(Lewis eta!, 1986). The use of antibiotic resistance for the construction and recovery

of transconjugants was unavoidable and it is possible that it had some effect on their

recovery. Selective media for biovar 3 were compared and the medium of Roy and

Sasser (1983) was found to be more appropriate. Inaccuracies may arise with the use

of selective media in their relative eff,rciency of colony recovery from soil and growth

of contaminants can be a problem on some selective media. Throughout the

experiments bacteria were randomly selected, rechecked for opine catabolism,

checked on alternate selective media and for plasmid makeup to ensure that the

inoculated isolate was being enumerated.

2) Agrobacterium colonization of almonds and vines

Very little work has been done to date on the colonization of hostplana by

Agrobacterium species. Studies by Schroth et al. (1971) looked largely at disease

incidence in a nursery situation and at ratios of pathogenic and nonpathogenic isolates

in the soil. Dickey (1961) examined the effect of a variety of soil factors on

Agrobacterium survival. Neither study distinguished between different species of

Aerobacterium. Several studies have stated that Agrobacterium is a normal

This difference in numbers cannot be attributed to galls formed by biovar 2 and

not by biovar 3 because the nonpathogenic strain Kl28 also maintained. high

populations on ungalled almoncl roots.

39

rhizosphere inhabitant with an ability to survive for long periods in the soil ( Patel,

L928;Patel,l929; Hildebrand, 1941; Dickey, 1961; Schroth et al., l97I).

In all the experiments performed in ttris study, there was a striking difference

between the relative ability of biovar 2 and 3 strains to colonize the root system of

almonds. Biova¡ 2 consistently colonized almond roots at levels at least 100- fold

higher than biovar 3 and levels of biovar 2 on almond were 10- to 100- fotd higher

than they were on vines. (;"s.nL)

Numbers of biovar 2 on almond roots dropped drastically in month 5 and the

data indicates that this drop could not be attributed to secondary thickening of the

roots but was in fact due to an absolute decline in Aerobacterium numbers. This may

have been at least in part due to the increase in soil temperatures as the decrease

coincided with the onset of the hot South Australian suÍrmer and AÊrobacterium

intolerance of hot conditions has been noted by Dickey (1961). In his work on

nursery populations of biovar 2 on almond, New (L972) also found that there were

no significant differences between bacterial numbers on roots with and without

secondary thickening. The numbers achieved by biovar 2 atthe height of its

colonization of the roots were comparable to those found by New around galled

almond trees in a nusery (106 to 107 biovar 2l gtoot).Both New (1972) and Shim

et al. (1987) suggest that absolute numbers of pathogenic isolates early in the season

may be important for gall formation and data presented in this study lend support to

that suggestion. Certainly the ability of an isolate to form a gall in vitro is not enough

for the formation of a gall in a natural infection, confirmed by the low pathogenicity

on almonds of the biovar 3 transconjugant carrying a biovar 2 Ti-plasmid in this

study. The delivery of the Ti-plasmid to the root by the bacterial chromosome in

sufficient numbers for infection would appear to be as much a determining factor in

pathogenicity as the presence of a Ti-plasmid capable of infecting that host plant. The

low pathogenicity of transconjugants and their restricted host range suggests that the

chromosome may play a part in the transfer of the Ti-plasmid to the plant cell.

40

The association between biovar 2 and stonefruit manifests itself in the high

levels of root colonization achieved by biovar 2. The highly specific interaction

between biovar 3 and grapevine appeals to be due to biovar 3's unique mode of

colonization. Actual levels of biovar 3 on grapevine roots were comparatively low

even in the period shortly after inoculation. Burr et al. (1987b) describe biovar 3 as a

'rhizosphere organism'; it would be diffrcult to completely agree with this statement.

From Burr's work on lesion formation by biovar 3 and from the evidence presented

in this study and those of others (Lehoczky, 1968; Burr & Katz,1983; Bu¡r &Katz,

L984; Tarbah & Goodman, 1986) that biovar 3 is able to colonize grapevine

systemically, it is possible that biovar 3 actually colonizes wound sites on the roots

(and susequently the lesions it forms at these sites) but is not necessarily efficiently

colonizing the vine root system as a whole. Biovar 3 is'rhizosphere-stimulated'to the

extent ttrat it survives around the root system at a higher level than it does in interrow

or fallow soil. Survival in both is very poor (Burr et al., 1987b; Bishop et al.,

unpublished data). Bishop et al. also demonstrated that biovar 3 survival in the oat

rhizosphere was higher than that in fallow soil and that survival in the vine

rhizosphere was higher than either of these. However, results from this study show

that biovar 3's ability to colonize vine roots is no bener than its ability to colonize a

'non-host' such as almond. It is tempting to speculate that because of the poor

saprophytic ability of biovar 3, under certain conditions (as yet undetermined) it

aggressively colonizes wound sites to form the sunken lesions through which there is

circumstantial evidence (Burr et al., 1987a) that it can colonize the vine's vascular

system. The results presented in this study show the systemic movement of a biovar 3

süain containing a biovar 2 Ti-ptasmid, demonstrating that colonization of the vine's

vascula¡ system is not associated with the biovar 3 Ti-plasmid. Burr et al. (1987b)

showed that both pathogenic and nonpathogenic biovar 3 strains were capable of

forming root lesions which indicates that lesion formation is also not Ti-plasmid

coded. It was puzzling that no galls were fomred on vines by proven grapevine-

pathogenic biovar 2 and3 isolates. It may be possible that agrobacteria must colonize

4l

vines systemically in order to form tumours and that not enough biovar 3 were

present in the vines in these experiments for this to occur. The numbers of biovar 3

isolated from stem segments were low in this study but wefe comparable to those

obtained by Tarbah and Goodman (1986). The conditions necessary for systemic

entry are unknown and it is possible that the conditions in these pot experiments were

not conducive to lesion formation and systemic movement. A correlation between

frost damage and crown gall has been observed (Burr, 1978; Lehoc*y,1978) and it

is possible that wounding due to low-temperature injury may facilitate systemic entry

of the bacteria. The ability to catabolize târtrate may be important in the ability of

Agrrobacterium to colonize grapevine, given the lack of ability of chrysanthemum

biovar 3 isolates to utilize taftrate (Bazzi & Rosciglione, 1982) and the ability of

biovar 1 and 3 vine isolates to do so. Ta¡trate catabolism is chromosomally-coded in

the isolates tested in this study but has been shown by others (Gallie et al., 1984) to

be coded for by a unique plasmid, pTAR, in some biovar 1 strains. It is interesting to

speculate on the origin of pTAR; perhaps biovar 1 acquired these genes in order to

extend its host range to grapevine. However, biovar 2 strains a¡e also able to

catabolize tartrate and, although ttrey are capable of colonizing vine roots as efficiently

as biovar 3 and they carry Ti-plasmids which are capable of infecting grapevine in

yitrg, biovar 2 arerarely found in grapevine galls (Peny & Kado,1982, Burr &Katz,

1983, Burr & Katz,1984). Biovar 1 strains can be found at low levels in the

grapevine vascular system (Burr &Kat2,1984) but never biovar 2. In this study,

biovar 2 srains were never isolated from systemic samples. Once again this points to

the essential importance of the ability of the bacteria to become systemic in grapevine

for a successful infection to occur. It is important to note that Burr et al. (1987b)

observed that biovar 3 strains could not form galls in artificial inoculations of vine

roots but could do so on grapevine shoos. Clearly there is a large amount still to be

understood but the data presented in this study and by others indicate that the

chromosomally-coded traits of lesion formation and movement into the vascular

system may be important in the infection process on vines. There is much interesting

42

\4rork to be done on the genetics of the infection process and on the factors leading to

actual plant cell transformation and gall formation once the bacteria have invaded the

vascular system of the vine.

In practical terms, the information available from this work and that of others

suggests that the soil is not the primary source of inoculum for grapevine infections

and that control methods should be targeted at detecting pathogenic biovar 3 in new

planting material. Cïown gall on grapevine has been vfutually unknown in South

Australia until very recently and it seems possible that inoculum is coming from

imported infected vines and not from the soil (unpublished data). The importance of

soil inoculum has not been established but the demonstated poor ability of biovar 3 to

survive in soil suggests that it may be much less important than the systemic bacærial

inoculum. From the work of Bur et al.(1987c), it appears that the bacteria cannot

move into young shoots so green tip propagation appears to be a promising way to

obtain clean planting material, even from infected vines. The poor survival of biovar 3

in soil observed in this study suggests that reinfection from soil inoculum would be

low although this must be shown experimentally. The number of bacte¡ia in the

vascular system required for infection must also be determined. Once detection

methods and levels are determined control of grapevine crown gall may be relatively

easily and inexpensively achieved.

3) Ti-plasmid and chromosome interactions

Because of their conjugative nature (Kerr, 1969; Ken,lg7l) and because all

Ti-plasmids studied to date belong to the same incompatibility group, Rh-1

(Hooykaas et a1.,1980), they would appeil to be completely transferable betr¡veen the

different ch¡omosomal backgrounds of Agrobacterium. Yet at the same time, Ti-

plasmids show tremendous genetic diversity ( Currier & Nester,1976;Perry &

Kado,1982; Sciaky et al., L978; Knauf et a1.,1983; Thomashow et a1.,1981) It is

evident that, although plasmid transfer is relaúvely easily achieved in vitro, in nature

there are specifrc plasmid types associated with each biova¡. Biovar 2 strains virtually

43

always contain nopaline Ti-plasmids and biovar 3 generally carry either wide- or

na¡row-host mnge octopine plasmids. Nopaline Ti-plasmids have been found in

biovar 3 sgains (Knauf et a1.,1983; this study) but are relatively rare and have only

very low homology with their biovar 2 counterparts. Octopine plasmids isolated from

biovars 1 and 3 also have very low (6 to l5%o) homology to each other (fhomashow

et a1.,1981; Knauf et a1.,1983). Therefore, there is a strong correlation between

distinct Ti-plasmid types and their ch¡omosomal backgrounds.

V/ork presented in ttris study suggests that there may be selection at the level

of the bacterial chromosome for particular plasmid types and that there are differences

between ch¡omosomal types in the stringency of that selection. The difference

between plasmid stability of the biovar 2 and 3 reciprocal transconjugants described in

this study in vivo compared with in vitro suggests that there also may be host plant

effects on this selection. The work of V/ang et al. (1986) with Rhizobium suggests

that there is selection by the plant on the plasmid-borne host range genes. That study

also suggested that some host plants were more 'discriminating' in this respect than

others and that selection for particular genes became stronger with successive

passages on the plant. The greater loss of the 'grapevine' plasmid, pTiK309, from the

biovar 2 strain K128 on almonds (807o) than on vines (307o) suggests that there was

selection against pTiK309 on almonds. It is difficult to make too many claims in this

regard but there is, at the least, selection by the biovar 2 chromosome against the

'foreign' biovar 3 plasmid in vivo. The host plant must have some effect on this

process because no plasmid instability was observed in this strain in vitro through

successive subcultures. Biovarl transconjugants which were isolated from grapevine

and found to contain biova¡ 3 Ti-plasmids were unstable in vitro (C.G.

Panagopoulos, personal communication). It could be suggested that the grapevine

host was positively selecting for a'grapevine'plasmid and when this selection was

absent, the Ti-plasmid was rejected. Strain K377 (pTiK27) was relatively stable in

vivo on both vines and almonds in this study. It is possible that some bacterial

44

chromosomal backgrounds or some host plants a¡e less stringent than others in

selecting for aparticular genome.

Knauf et al. (1984) observed that, although pTiA6 and pTiAgl62 are in the

same incompatibility group, the presence of the A9162 incompatibility region on a

cosmid resulted in a rate of establishment in A6 which was 104 lower tha¡r for

cosmids without the region. They termed this phenomenon'one-way

incompatibility'. It is evident that, despite the common incompatibility group of Ti-

plasmids, their acceptance in an Agrobacterium cell may be a more complex process.

Blocking the entry of some plasmids may be a way by which specific Ti-plasmid/

chromosome associations a¡e sustained. Perhaps the more limited host range biova¡ 3

plasmids, such as pTiAg162, are less promiscuous than thei¡ wide host range

counte{parts. The decreased virulence observed in this study of a biova¡ 2 strain

carrying a biovar 3 Ti-plasmid, pTiK309, and its low stability in vivo was

presumably a result of the poor replication and establishment of that plasmid in its

new background.

There was evidence throughout the experiments that plasmids in all

transconjugant strains re-isolated from soil were not completely stable. It would be

interesting to carry out a more controlled set of experiments to study plasmid stability

in vivo and to compare wild-type and transconjugant strains on host and non-host

plants. It is likely that some regions of the plasmid would be highly conserved but

genes involved in host range determination may be quite dynamic in vivo.

The Ti-plasmid has been shown to be the primary determinant of host range in

Agrobacterium (toper & Kado,1979; Thomashow et a1.,1980; Knauf et aI.,1982;

Unger et a1.,1985). The Ti-plasmid of the limited host-range grapevine strain, A9162,

has been mapped (Knauf et a1.,1984) and, in a comparative study of the plasmid of

that strain and a wide host-range plasmid, pTiA6, the involvement of cytokinin

biosynthetic genes in the T-DNA and two of the vir loci, virA and virC, in host range

determination was demonstrated (Yanofsky et al., 1985a;1985b). Yanofsþ et al.

(1985) showed that successful plant cell transformation by the wide host range

45

plasmid pTiA6 required the introduction of both the auxin and cytokinin loci. In

contrast, the timited hostrange plasmid appeafs to be defective at the c¡okinin

biosynthetic locus and it was suggested that plant hosts susceptible to infection by this

strain may have endogenous cytokinin levels high enough to ove¡come the deficiency.

The virA and virC loci also play a role in limiting host range and the role of virC may

be to control the number of T-DNA copies transferred to the plant cell (Yanofsþ et

al., 1985b; Yanofsþ & Nester,1986). Possibly some plant species may require

d.ifferent copy numbers of T-DNA for tumour formation to occur.

Obviously Ti-plasmid genes are essential in the determination of host

specificity. Data presented in this study have also shown chromosomally-encoded

host specificity. If there is indeed a much closer relationship betrreen specific

chromosomal and Ti-plasmid types than has been assumed then this would suggest

that there are 'layers' of host specificity with the host plant selecting for chromosomal

types which in tum are selecting or selected by'host-compatible'Ti-plasmids'

Data from Almond Ex. 4 and from Vine Ex. 2 comparing wild-type and

transconjugant s6ains on non-galled root systems, indicate that the Ti-plasmid has a

negligible effect on initial root colonization. For example, numbers of transconjugant

strain I¡377 (pTiK27) were comparable to those of its wild-type background and it

did not appear to be any more'ecologically competent' on almonds as a result of

carrying a biovar 2 Ti-plasmid. This result is similiar to that obtained in a study of the

effect of the Sym plasmid on the competitive ability of Rhizobium in the legume

rhizosphere (Brewin et al., 1983). In that investigation the plasmid had no effect on a

strain's colonization of the legume root surface. The situation of the biovar 2 strain

carrying a biovar 3 Ti-plasmid was more complex due to the considerable plasmid

loss in this strain. Because of this, strain K128 (pTiK309) as a whole was much less

ecologically competent.

46

4) Role of the opines in nature

The opine concept has been proposed by several authors (Petit et al., 1978a;

Tempe eta1]., 1979; Guyon eta!, 1930). This concept proposes that the main reasons

for the existence of the Ti-plasmid are the opine synthesis and catabolism functions.

By directing the plant to produce opines for its benefit, the Ti-plasmid is ensuring for

itself a selective advantage over non-opine catabolizers and, through the conjugative

functions of the opines, ensuring iS own propogation. To date there has been no

ecological data to support this proposition.

Data presented in this study show initial evidence in support of the opine

concept. In colonization studies on almond galls, two biovar 3 strains, K309 and

K377,which rwere not different from each other in their colonization of almond roots,

were signifrcantly different in their colonization of nopaline galls. The nopaline-

catabolizing strain, K377,was more numerous than K309, an octopine strain, both

inside and on the surface of galls. However, K377 did not maintain the high

population levels and its decline could be attributed either to the competitive effect of

the biovar 2 strain, KZJ, or to the demonstrated inability of biovar 3 strains to survive

at high levels in the rhizosphere.

Data obtained in Part A point to a model where the bacterial chromosome is

completely responsible for the early interaction with the plant root. Chromosomal

genes appear to determine both the level and the mode of colonization of the plant

host. The bacterial chromosome may have a significant amount of control over the

type of Ti-plasmid it caries given the specific associations between Ti-plasmid types

and Agrobacterium biova¡s. The Ti-plasmid is important after bacterial colonization of

the roots when, by inciting gall formation and subsequent opine synthesis, it then

exerts a con¡olling influence on the level of colonization through the expression of its

opine caøbolic functions. Other plant root exudates are likely to be important in the

early stages of colonization. Although acetosyringone has been shown to induce a

chemotactic response from some agrobacteria (Shaw, 1986), this may not tle the case

for all plant hosts or agrobacteria. It is likely that other exudates play a role in plant-

47

bacterial specifrcity. For example, tartrate may play a role in bacterial establishment on

the grapevine. The plant and bacterial factors involved in the formation of root lesions

by biovar 3 are completely unknown at present. Endogenous cytokinin and auxin

levels in the plant host appeü to be at least partially responsible for the host-specific

infection process and the opines produced as a result of infection then play a crucial

role in determining numbers of bacteria on galls.It remains to be shown whether the

conjugative role of the opines provides opine-catabolizing agrobacteria with a

selective advantage. It would be interesting to determine the amount of in vivo Ti-

plasmid transfer and its importance in the survival of the bacteria and on the level of

infection.

In summary, there appears to be a type of symbiotic relationship between the

Ti-plasmid and bacterial chromosome. The Ti-plasmid benefits from the

chromosome's ability to specifically colonize the plant host.In turn, the chromosomal

background benefits from gall formation by the Ti-plasmid because of the opine

substrate that they induce. This is an extension and affÏrmation of the opine concept

and the genetic colonization theory of Schell (1978; Schell et al. ,1979).

Many authors are beginning to view plasmids as discrete organisms (Datta,

1985). It is interesting to speculate on the evolution of the Tiplasmid/ chromosome

relationship in Aerobacterium in this light. Large numbers of nonpathogenic

agrobacteria can be found in natural, undisturbed soils (Bouzar & Moore, 1987).

Perhaps this nonpathogenic, nonspecific state is the'ancestral form'of

Agrobacterium and the Ti-plasmids have in fact'colonized'these nonspecific

chromosomal forms. The plasmid and chromosomal types have then co-evolved, in

the presence of the host and both plasmid and chromosome a¡e host-specific. This

would explain the very low homology between the different Ti-plasmid types and

between the different biova¡s of Aerobacterium and the amount of host specialization

found within the genus.

48

PART B : TAXONOMY OF AGROBACTERIUM ISOLATES FROM RUBUS

AND GRAPEVINE

INTRODUCTTON

Four species of Agrobacterium are described in the 1984 edition of Bergey's

manual- A. tumefaciens, A. radiobacter, A. rhizogenes and A. rubi (Kersters &

Deley,1984). It is recognized by many authors (Del.ey et a1,1966; Keane et

aL.,1970; White,1972; Kersters et a1.,1973; Holmes & Roberts,1981; Kersters &

Deley,1984) that this classification is inadequate. A. tumefaciens, AdfZ@S, and

A. radiobacter differ only in the presence or absence of a tumour-inducing (Ti) or

root-inducing.(Ri) plasmid, which is transferable benveen strains (Van Larebeke et

a1.,1975; Vy'atson et a1.,1975; Albinger & Beiderbeck,1977; Moore et al., 1979).

Therefore, species definitions are presently based solely on a highly unstable

characteristic. Suitable changes to the genus are unfornrnately very diff,rcult to make

because of the rules of bacterial nomenclature (Kersters & Det ey,1984) but it is

agreed that there are at least two well-def,rne{ distinct groups which can be separated

on the basis of chromosomally-coded characteristics, regardless of

phytopathogenicity (Deley et a1.,1966; Keane et a1.,1970; White,t9lZ; Kersters et

a1,L973; Kerr et a1.,1978; Holmes & Roberts,l981; Kersters & De[,ey,1984). These

two groups are considered separate species- A. tumefaciens and A. rhizogenes- by

some (Holmes & Roberts,l981) and different biova¡s or clusters by others (Keane et

a1.,1970; Deley et al.,1973; Kersters et aI..1973).

The position of other Agrobacterium isolates is less clear. The species

Agrobacterium rubi was described by Hildebrand (19a0) and at present only three

strains are included in A.rubi: TR3, TR2 and EU6. These are classified as a separate

species based on thefu low DNA homology with biovars 1 and 2 and their high

homology with each other (Kersters & Deley,1984).Isolates from grapevine have

been charactenzeÃas a separate group, biovar 3, which can be readily distinguished

phenotypically from biovars 1 and 2 (Kerr & Panagopoulos,l9T1; Panagopoulos et

49

a1.,1978; Sule,1978). Isolates from chrysanthemum have also been classified as

biovar 3 (Baz:zr & Rosciglione,1982). The relationship between these biovar 3

isolates and A. rubi is unclear. In a numerical taxonomic study, Holmes and Roberts

(1981) grouped the A. rubi type strain, TR3, with several biovar 3 isolates from

grapevine. However, DuPlessis et al. (1984) found differences between the same

grapevine isolates and the A. rubi strain, TR2, in a study of their protein

electrophoretic patterns.

The purpose of the taxonomic section of this work was to clarify the

relationship of the grapevine and Rubus isolates to the well-defined biovars I andZ.

Relatively recent isolates from both these groups have been used in this study and

compared in their growth characteristics, pathogenicity, opine utilization and their

reaction to a vadety of standard bacteriological tess. Numerical analysis of the

phenotypic data was performed. In addition, isolates have been compared with

respecr to their DNA homology as well as by serological methods. Throughout the

work, isolates conforming to the descriptions of biovars 1,2 and 3 are referred to as

such. The isolates from Scotland (see Table 18) are referred to as the Rubus cane gall

isolates and only the three strains TR2, TR3, and EU6 which presently comprise the

species A. rubi (Kersters & Deley,1984) are referred to as such.

50

PART B: MATERIALS AND METHODS

a) Bacterial strains and culture conditions

All strains used in taxonomic studies and their sources are listed in

Table 18. Bacteria were maintained on nutrient agar (NA) or yeast extract (YE) agar

at 28oC for short-term storage. Isolates in the A. rubi goup were maintained on YE

agar with the growth factors biotin (100 pgl100m1), nicotinic acid (20 pg,/100m1) and

calcium pantothenate (20 pgll00ml). Bacteria were maintained as lyophilized cultures

at 4oC for long-term storage. All culture media are described in Appendix A and

buffers and solutions are described in Appendix B.

b) Pathogenicity tests

The method used for pathogenicity testing is described in Part A,

Materials and Methods. The Rubus isolates were glown on YE agar with added

growth factors (see 'Culture Conditions'). Plants used in the taxonomic paft of this

work were those described in Pa¡t A as well as blackberry ßubus L. cv.

'Silvan')(McGregor & Kroon, 1984), boysenberry (Rubus L. cv.'Boysen') and

raspberry (Mus i¿eaus L.). For pathogenicity testing on boysenberry, inoculations

were made into the woody stems of the plants. Holes were drilled into stems using a

cordless ddll with a 1 mm bit and inoculations were made into the resulting hole using

a Pasteur pipette full of a turbid (109 cells/ml) suspension of the test strain. Results

were recorded afterlO weeks for all inoculations on raspberry, boysenberry and

blackberry.

c) Opine synthesis and catabolism

The presence of opines in gall tissue was confirmed by high voløge

paper electrophoresis GIVPE). Fresh sections of gall tissue (1 cm3) were macerated

in 500 ¡r.l to 1 ml of 70Vo erhanol. Samples were centrifuged for several minutes in an

Eppendorf centrifuge and 10 pl of the resulting supernatant was spotted directly onto

Table 18. Strains used and their origin

Srain CIher Grouping2designations

NCPPB 24373 ICPB TT3ATCC 23308

Biovar I

NCPPB 1OO1

c58Kl87

Isolaæd from Pathogeniol

I $apevlne

sall"gall

this study$uìpel¡rne

Í

Rubus cane

Rubus canegalls

A. rubi Euonymous gall

Additionalinformation

Braun's isolateB6

Romania,1952

A6

South Aust¡alia

South Australia

see Part Aex. T. Burr

Í

yes

yes

yesyes

yesyesfn

yesyesyesyesyesyesyesyesyesyesnoyesnoyesyes

yesyesyesnoyesyes

tnyesyes

yes isolatedBraun, 1

il

ATCC rß254K27K84 NCPPB 2407

scRr 509scRr 5rTscRr 518

NCPPB T856TR3TR3

Biovar

Biovar

A. rubi

Íti

2

3

peachpeach

K305K306K308K309K374r375K376K377K252K253

1(377DTi-cô 49

cG 484CG992K1059

grapeune

As57AES4

lrÍll

lr

sqlsÍltÍ

K864

K870K871K872

TR2NCPPB T8545ATCC 133355

EU6

K868K869

isolated atScottish Crops

Rese¿rch '.Institute

usA,1942

by950

181 - Yes from JJemPé,

ies grouping

3335 were received directly fromthe reçectivetype culture collections.

In some cases, the opine utilization test described by Lippincott et al. (1973) was

used in conjunction with the test for growth on solid medium, if the results on the latter

were not clear.

51

Whatman No. I paper. The apparatus of Tate (1968) was used and alt runs were

performed in 0.75 M formic acidll M acetic acid, pH 1.7 buffer at 3000 V for 15

min. Orange G was used as a reference standa¡d with an arbitrarily assigned relative

mobility (MO.C) of 1.0. The following detection reagants were used: alkaline silver

nitrate dip (frevelyan et al., 1950), modifred phenanthrenequinone reagant (Yamada

& Itano,1966), the Pauly reagant for imidazoles (Ames & Mitchell,1952), and the

xylose-aniline reagant (Smith & Spriestersbach, L954). Table 28 liss the opines

detected in this study, their detection reagants and their M9.6. at pH 1.7.

The minimal medium of Petit et al. (1978b) was used to test opine catabolism.

Filter-sterilized opine solutions (pH 6.0 in double-distilled water) were added to a

final concentr¿tion (w/v) of 0.2Vo when opines used as the sole source of carbon and

nitrogen or to 0.O47o when a carbon source (0.57o mannitol) was included. Fresh

cultures of the test isolates were streaked on the opine-containing media with positive

and negative control strains, incubated at 28oC and results recorded after 4-5days. ( trserà

Growth factors were included for all Rubus strains. Opines tested in this way were

octopine, nopaline, succinamopine and cucumopine. The former three opines were

synthesized by Dr. M.E. Tate in the Dept. of Agricultural Biochemistry. Crude extract

of a cucumber hairy root strain, positive for presence of cucumopine, was used in all

catabolism studies involving cucumopine.

d) Growth on selective media

The selective media used in this study are listed in Appendix A. All

plates were incubated at 28oC and results were recorded after 4to 6 days. Growth

factors (described below) were added for all tests involving Rubus cane gall isolates.

e) Growth factor requirement

The method used was based on that of Keane et al. (1970). Bacteria

from agar slopes were suspended in buffered saline, washed twice with sterile

distilled water and inoculated into broth containing either Petit's (Petit et al., 1978b)

Table 28: Common opines andtheir detection

Common name

Octopine

Structural name

Nopaline ¡2 -1t,3-dicarboxyt propyl)-L-arginine

Structure onlYpartially lnown2

fietection method(s)8

Alkaline silver nitrate4modifred phenanthrenequinone

reagant5

M -(o- t -carUoxyethYl)-L-arginine

1)2)

As above

Mo.c.1

-0.54

-0.45

-0.2rCucumopine

Succinamopine Xylose-aniline reagantT -0.04

1- MO.C. = Relative mobility to Orange G søndard in pH 1'7 buffer:-'

fi.O M acetic acid' 0.75 M formic acid)

2- Ryder, 1984

3- Chilton et aI.,1984a; 1984b

4- Trevelyan et al., 1950

5- Yamada andløno, 1966

6- Ames and Mitchell, 1952

7- Smith & SPriestersbach, 1954

8- detection after high.voltage paper electrophoresis (see Materials and Methods)

Pauly reagantfor imidazoles6

52

or Bergersen's (Bergersen,l961) salts, 17o mannitol and a source of nitrogen, either

with or without growth factors. Cultures \ilere glown for 4 days at 25oC on a rotary

shaker and results were recorded every 24 hours by measurement of optical density at

640 nm. Bacteria were also inoculated onto agil containing Petit's or Bergersen's

salts, 17o mannitol plus either a)O.47o NaNO3, b)0.4Vo NaNO plus 100 pg/100 rrìl

biotin, c) O.ZVo L-gtutamic acid plus 100 pgl100m1 biotin, d) O.2Vo Lglutamic acid

plus 100 ¡rg,/100 ml biotin, 20 ¡rg,/100 ml calcium pantothenate and 20 pglml nicotinic

acid. Plates were evaluated for bacterial $owth after 4 days'incubation at 28oC.

f) B acteriolo gical tests

3 -ketolactose Production

The method of Bernaerts and Deley (1963) was used.

Production of acid from carbon sources

The method of Haywardlg64) was modified to determine acid

production from dulcitol, adonitol, sorbitol, erythritol, melezitose and ethanol. The

indicator medium is described in Appendix A. Tubes were incubated at 25oC for 14

to 21 days.

Production of alkali from L-tarrate

The method of Ayers et al. (1919) was used to test for alkali

production from sodium(+)-tartrate. An alternate medium containing potassium

sod.ium tartrate was also used; both media are described in Appendix A. Tubes were

incubated at 25oC for 7 to 14 days.

Growth on 27o NaCl

Bacteria rwere streaked on nutrient agar with 2Vo (wlv) NaCl

added. Growth was evaluated after 48 h. at 28oC.

Growth at 379e

Bacteria were inoculated into 5 ml cultures of nutrient broth

amended with growth factors and were grown in a stationary water bath at 37oC.

53

Optical density (640 nm) was measured at 2l hour intervals and frnal results were

evaluated after 4 days.

Growth on aniline blue medium

The method of Riker et al. (1930) was used. Bacteria we¡e

streaked onto aniline blue medium (described in Appendix A) and the plates were

incubated at 28oC. Growth was evaluated after 24to 48 hours and reactions were

classified as Type 1 (white mucoid $owth with clearing of the medium) or Type 2

(blue, thin growth with no clearing of the medium).

g) Sensitivitv to agrocin 84

The method of Stonier (1960) was used to test for sensitivity of

isolates to strain K84.

h) Plasmid isolation and visualization

The method used is described in Section A, Materials and Methods.

i) Plasmid incompatibilitv

Strain 2-4 (see TablelA), containing a cloned fragment of the Inc

region of the biovar 1 strain, 46, was used in attempts to eliminate Ti-plasmids from

wild-type strains. This method is described in detail in Part A Results, Section 4.3 .

Transconjugants rwere screened by gel electrophoresis for loss of the Ti-plasmid and

acquisition of plasmid containing the Inc clone. Loss of the Ti-plasmid was further

confinned by checking pathogenicity on a suitable host plant.

j) DNA melting points

The midpoint of the thermal denaturation profile (Tm) was

determined by the method of Mandel and Marmur (1968) for selected strains. All Tm

determinations \ilere made on a Beclcnan DU-8 computing spectrophotometer. DNA

(see below for method of preparation) at a concentration of 50 to 100 pyml in 1 x

54

SSC was loaded into prewarmed (75oC) cuvettes. The temperature was raised in loC

increments to 90oC and in 0.2oC increments to102oC, with an interval time at each

temperarure of 2.2min. A blank containing 1 x SSC and a cuvette containing the type

strain for biovar 1 (NCPPB 2437) were included in each run. All Tm determinations

were made in triplicate. The ratio of absorbance at a given temperature over the initial

absorbance for each sample was plotted versus temperature and the midpoint of the

melting profile was determined graphically. The guanine plus cytosine (GC) content

of the DNA was determined from the equation: GC = (Tm - 69.3) 2.4. (Mandel &

Marmur,1968).

k) Growth rates

Relative growth rates were determined in both nutrient broth and

mannitol-glutamate (MG) b'roth (Appendix A) with and without growth factors (see

'Culture Conditions'). In nutrient broth, bacteria from 48 h cultures were added to

150 ml broth to give initial concentrations of 6 x 106 celVml. Cells were gïown on a

rotary shaker atzsoc and 3 ml of culture were removed and the optical density (640

nm) measured every 2 hours for 42 hours, by which time all cultures hadreached

saturatron.

In mannitol-glutamate broth, bacteria from 48 h cultures were added to 150 ml

broth to give initial cell densities of 1.5 x 105 cetls/rnl. Cultures \ilere grown as above

and optical density measured at th intervals for 5 days, by which time all culn¡res had

reached saturation. All isolates were tested at least in duplicate.

55

l) Serological studies

Preparation of anti gens

The method of Keane et al. (1970) was used.

Preparation of antisera

Antisera to strains K309, K377, and TR3 were prepared using

the method described by Keane et al. (1970). When antiserum titers dropped, 1 ml

booster injections were given intravenously and the rabbits were bled at7 tol4 day

intervals.

Tube agglutination tests

Successive twofold dilutions in buffered saline were made from

an initial serum dilution of'1:50 in buffered saline. Equal volumes (0.5 ml) of each

serum dilution and the bacterial suspension (108 ce[s/ml in buffered saline) were

mixed, incubated 3 h at 37oC and left overnight at 4oC before agglutination endpoints

were determined.

Gel diffusion tests

The method of Ouchterlony (1961) was used. Undiluted

antisera were added to central wells and turbid suspensions (9 x 108 ce[s/rnl) of the

sonicated test isolates were added to peripheral wells.

m) Electron microscopy

Selected Agrobacterium strains were viewed by scanning electron

microscopy (SEM) and transmission electron microscopy (TEM) in order to

determine if flagella were present.

For SEM, two methods of sample preparation were compateÅ- freeze-drying

and critical point drying. For freeze-drying, 48 hour old cultures on YE agar plates

were flooded with SDW and left to sit at room temperature for t hour. The resulting

suspension was gently poured into tubes, centrifuged at 10009 for 15 min. and

resuspended in SDW. Samples were freeze-dried in a Centrifugal Freeze-Drying Unit

56

(Model CDl; Dynavac, Australia). For critical point drying, polycarbonate

membranes (0.22 m; Nuclepore Corp., USA) were floated for t hour on the surface

of plates which had been flooded as above. The membranes were then gently rinsed

with a gfaded series of I\Vo,4OVo,7O7o and 1007o ethanol; each rinse was for 5

minutes except for the two fînal rinses with 1007o ethanol which were for 10 minutes

each. The membranes were critically point dried immediately (CPD 750; Emscope,

UK) and stored in a vacuum dessicator until they were sputter-coated with gold

(Sputter Coater SC500; Emscope, UK) and viewed under a scanning electron

microscope (S tereoScan S E250 ; Cambridge, UK).

For TEM, grids were prepared in the following manner. Bacteria were grown

for 48 hours on Petit's minimal medium amended withD.2%o (NH¿)ZSO4 and 17o L-

glutamate or on YE agar. Formvar ca¡bon-coated grids (400 mesh) were floated for

60 minutes (or ovemight for biovar 3 srains) on the surface of the plates which had

been flooded with buffered saline. Grids were then transferred to LVo

phosphotungstic acid for 30 seconds and rinsed by flotation on sterile double-distilled

\¡/ater for 30 to 60 seconds. Grids were d¡ied on filter paper in petri dishes and

viewed under a transmission electron microscope (EM400; Philips, Australia).

n) Numerical anal)¡sis

The characters listed in Table 78 were used in the numerical

analysis. Results for'growth factor requirement' in both Petit's and Bergersen's salts

were coded separately. Results for all characters except aniline blue reaction and

growth at 37oC were coded as 1 for a positive reaction and 0 for a negative reaction.

Results for growth on aniline blue were coded as Typel or 2 reactions and results for

gtowth at3loc were recorded as final optical densities (640 nm) after 4 days

incubation. Numerical analysis was done on GENSTAT (Rothamsted Experimental

Sration, 1977). Strains were grouped by single linkage cluster analysis. Negative

matches were or were not inctuded as indicated. A minimum spanning tree was

constructed.

57

o) Motilitv

Motility was tested by three methods. Initially, isolates were viewed

under a light microscope in the hanging drop test. Bacteria were grown for 48 hours

on both YE agar and Petit's minimal medium amended tnth I7o Lglutamate and

O.27o (NLL¿)ZSO¿. Plates were flooded with buffered saline as described for TEM

and allowed to sit for t hour. A loopful of the resulting bacterial suspension was then

placed on a cover slip which was then inverted over a well in a glass slide. Slides

were then viewed under 100x magnifrcation.

Bacteria were also tested for motility by stabbing fresh cultures of each isolate

to be tested into a plate or tube of a) ll2} strength YE broth amended with0.3%o agar

or b) 1/5 strength Petit's agarplus LTol-ghutamate and O.2Vo (NH+)ZSO¿ amended

withO.37o agar. Plates and tubes were both assessed visually after 48 hours'

incubation rt25oC and, for plates, the diameter of the swarm was measured.

p) DNA Reassociation Studies

i) DNA preparation

For DNA extractions, bacteria from stock cultures were

inoculated into 5 ml YE broth, grown to saturation and inoculated into 500 ml YEB.

Bacteria were grown to late-loga¡ithmic phase and cells harvested by centrifugation.

DNA \ilas extracted and purified by the method of Marmur (1961), modified

to include O.ZVo Sarkosyl in the initial washing step and a deproteinizalon step using

proteinase K (200 pgrnl at 60oC for 45 minutes) before the addition of sodium

perchlorate. Ribonuclease was used at a concenration of 100 pglrnl.The purified

DNA was dissolved in 0.1 x saline sodium cirate (SSC) to a concentration of l-2

mg/rnl and was stored at 4oC with a few drops of chloroform. DNA was then shea¡ed

to an average fragment size of 400 to 800 base pairs by passing the DNA two to three

times through a French pressure cell at 12,000 to 15,000 psi. Fragment size was

58

confrmed by gel electrophoresis against a lambda Hind Itr marker (Fig lB). DNA

was quantified spectrophotometrically and ttre purity of each preparation ascertained

by measuring the 2601280 nm and 2ffi1230 nm absorbance ratios (Fig 2B). Before

use, fragmented DNA preparations were diluted to a concentration of 150 pgrnl.

Equal volumes of 4 x SSC and DNA were mixed to give final concentrations of 75

tlg/rnl DNA in 2 x SSC.

ii) DNA denaturation and reassociation

The method used was based on that of Deley et al. (1970). A

Beckman DU-8 tlV-visible computing spectrophotometer with a temperature

controlled sample holder and Tm Compuset Module was used for all reassociations.

Four or five cuvettes were filled with a blank containing 2 x SSC, DNA from each of

the strains to be compared and either one or two cuvettes containing a 1:1 mixture of

the two comparison strains. The wavelength was set at 260 nm with a read average of

4, an interval time of 60 sec between readings and a slit width of 1 nm. The cuvettes

were prewarmed to 75oC and the temperature was raised from 75oC to 90oC in

1oQ/min increments and from 90oC to 102oC in 0.2oclmin increments. All samples

were held at 102oC for 5 minutes in order to completely denature the DNA and the

temperature was then dropped to 75oC, the optimal reassociation temperature for

Aerobacterium (Det ey et a1.,1970). The absorbance was recorded every minute for

30 to 40 minutes.

iii) Sources of variation

According to Britten etal. (I974), the parameters which must be

controlled in order for reproducible reassociation values to be obtained are salt

concentration, temperature, DNA fragment size and DNA concentration. In this

study, all comparisons were made in at least duplicate and where there was poor

agreement between the two results up to 5 separate comparisons were performed

Figure 18. Agarose gel electrophoresis of fragmented

DNA preparations used for DNA reassociation

studies.

Lanes:

A. K872B. K871c. K869D. K868E. K309F. Lambda Hind Itr marker DNA(arrows indicate fragment size in bases)

tr-)

- <2027

<564

Figure 2B Typical absorbance profile of DNA used forDNA reassociation studies.

DNA from NCPPB 1854: 1/20 dilution ofDNA in sterile distilled water (SD\Ð. Blank

wasl/2O dilution of 0.1 x SSC in SDV/

EEI T!Ju35 HJ-3l.Jf 'l3r¡L!ï'1

¡B II IIH35 1: ___jttttiI.Ih¡ø' ETE ÜI }.¡Ng' EEE

,aa. Éì 1 ? UEJ L¿ ¡v

EE'e33

8t'¿Ë' T l,lllE'Erf.E

SË}þ'E I.JIJJ'5gEËÊËË'E I,ltJE'gSrE

gEi'eJE

T t¡lJg'g

ELI'E3Ë

EE1BE'BEEÊ-1t:i'EEFOEI'Èggl:J

59

DNA: In all cases, more than one preparation of DNA was used to avoid problems

with contaminants in the preparations which might affect reassociation. In several

cases, the DNA failed to denature properly and these runs were discarded. Ideally the

absorbance of the DNA should increase by 407o for complete denaturation but this is

rarely achieved (N. Scott, personal communication).In this study, absorbance

increase with heating to 102oC was25Vo to357o in all cases and any preparation

showing an increase of less that25Vo was not used.

At all times an effort was made to start each run with a DNA concentration of

75 ¡tglrr,lin all cuvettes. The concentrations were always between 65 and 85 ¡tg/ml

thus falling into the range in which second order kinetics apply (Det ey et al., 1970).

This was important in that it satisfies the assumptions behind the equation used to

calculate degree of binding. Each sample denatured by a slightly different amount and

there were denaturation differences between cuvettes (see below) so it was impossible

to achieve identical absorbance values in each cuvette at the beginning of the

reassociation. This is perceived as the major source of variation in the results.

Fragment size after shearing was checked on an agarose gel against a lamMa

Hind trI marker. In all cases, fragments were consistently in the 400 to 800 bp range

(FigurelB). This is the size range which has been shown to produce the most

accurate results (Del-ey 4!, L97O) so fragment size was not seen as a source of

variability in the results.

DNA purity was assessed spectrophotometrically. In all cases the 26O/280 and

260/230 absorbance ratios were 1.8 or greater. Figure 28 illustrates a t)?ical DNA

spectrum. The purity of all DNA preparations was checked in this way before and

after the shearing step. Protein contamination was thus not seen as an important

source of variation in the results. The same procedure was used for the preparation of

all samples and all DNA pellets were rinsed several times in 7O7o ethanol. However,

there is still some possibility of salt concenEation differences between samples which

may have contributed to denaturation differences.

60

Cuvette differences: Table 38 shows differences between individual

spectrophotometer cuvettes with respect to the amount of DNA denaturation and

renaturation observed when control runs were performed. In these runs, DNA from

the same preparation and dilution was loaded into different cuvettes and denaturation

and renaturation values were recorded. Not only was there variation be¡veen initial

absorbance values but there were differences between cuvettes in the amount of DNA

denaturation and especially renaturation. These differences were observed repeatedly.

Cuvettes were cleaned regularly with a 1/10 strength solution of Trace-Klean

(Beckman Instruments, USA) followed by 8 to 10 rinses with double glass-distilled

water. In order to eliminate any salt residues from cuvettes they were frlled with dilute

HCI (0.01 N) and heated to 40oC for 2 hours. This was followed by numerous rinses

with double distilled water. The latter treatrnent alleviated problems with cuvette

differences to a certain extent but the use of cuvette 4 was discontinued because it

gave consistently lower renaturation rates. Some of the variation between cuvettes

could have been due to differences in ttre temperatues achieved which in turn was

due to impurities in the cuvettes themselves or design problems in the temperature

control.

In summary, the major sources of error in this method were perceived to be

the variation in absorbance readings between cuvettes and the differences in DNA

concentration at the beginning of the renaturation. The latter was likely due to

differences in the starting DNA concenüation which were then compounded by a

certain amount of impurity in the DNA samples andvariation between cuvettes, with

regard to temperatue control. Modifications (described below) were developed in the

calculation of reassociation rates which were designed to take into account DNA

concentration and denaturation differences.

iv) Calculation of the Degree of Binding

Initially, the degree of binding was calculated as suggested by Deley

et al. (1970). The homologous and heterologous reassociations were plotted

Table 38. Differences between cuvettes observed in DNA reassociaúon studiesl

Comparison2

4z

K864Æ(864:denaturation3renaturation4

.341 163

.30.t245

.29.L343

.35.1189

.40.t125

.270508

.321101

.32.rt93

.0597

.35.079r

.32.1400

5

.27.2tt9

.25.0519

K8641K872:denaturationrenaturation

K868Æ(871:denaturationrenaturation

K869Æ(871:denaturationrenatu¡ation

K8691K872denaturationrenaturation

K868A(872denaturationrenaturation

.40

1- The reassociation method and calculation of denaturation andrenasturation rates are

described in Part B, Materials and Methods. Cuvette numbers refer to their position in

a temperature controlled sample holder used with a Beckrnan DU-8 UV-visiblecomputing spectrophotometer. In all cases, cuvette position 1 contained the blank.

2- DNA from the same homologous or heterologous mixture was loaded into different

cuvettes as indicated and run at the same time. DNA preparation for reassociation runs

is described in Part B, Materials and Methods.

3- Denatu¡ation was calculated as the change in absorbance from 75oC to 102oC

divided by the initial absorbance of the sample at 75oC.

4- Renaturation rates were calculated as described in Materials and Methods, Part 8..

61

(absorbance vs. time) graphically and the rates were calculated from the point where

the plots became linear. Figure 38 shows a plot of two closely related strains and

Figure 48 shows the reassociation of two more distantly related strains. Once the

rates were calculàted, the degree of binding (D) between the two strains was

determined from the equation (Deley et al., 1970): D = 4Vmix-VR-Vg/

2(VlVgX100). Vmix is the absorbance change per unit time for the linear portion of

the heterologous reassociation curve. V4 and Vg are the absorbance changes per unit

time for the linear portion of the homologous reassociation curves.

It was perceived that there were sources of va¡iation which may conEibute to

error in the calculated degree of binding so a modified method of calculation was

developed with the help of Dr. Nigel Scott, CSIRO Div. of Horticultural Research,

Adelaide.

In order to eliminate differences in initial DNA concentration values, the

absorbance at 102oC was divide by the initial absorbance at 75oC. This was done to

get an estimate of the amount of denaturation. The renaturation rates were calculated

as previously and divided by the denaturation value (e.g by 0.35 if the absorbance

increase from 75oC to 102oC was35Vo) to obtain adjusted values for V¡¡i¡ , V¡ and

Vg. The denaturation u/¿ìs thus used as an adjustment factor so that the renatu¡ation

rate was not as dependent on initial absorbance reading. Theoretically the adjustrnent

factor provides a more realistic assessment of how much DNA was actually denatured

and thus available for renaturation.

The new Vmix, V4 and Vg values were then used in the equation of DeIæy

et al. (1970) previously described Table 48 presents the recalculated values for D

versus the original values. The results are discussed fully in the next section and

presented in Tables 148 and 158.

In most cases the recalculated results were not very different from the original

results. Recalculated values showed less variation between repeated comparisons of

the same strains, a problem with the original results. Two problems remained. The

first is that there were a certain number of anomalous results; in two cases, very high

Figure 38: $amFle plot of reassociation of homologous

and heterologous mixtures of DNA from twoclosely rolated strains, K864 and K871.Reassociation waSþloned at 75oC over a 50minute period. The calculated degree of bindingof the two strains was757o.

E

L'scqoo(l)oC(ú-oLoU'-o

2.O

2.1

I

1.8

1.7

1

K871

mixture

K864

0 10 20 30 40 50minutes

Figure 48: Sample plot of reassociation of homologous and

heterologous mixtures of DNA from two strains

belonging to different A erobacterium species,

K864 (Rubus isolate) andK377 (biovar3).

Reassociation was plotted at 75oC over a 45

minuteperiod. The calculated degree of bindingof the two strains was 107o.

2.0

Êc0-9Ì\doIo(Jc(ú€o.n-o

2.1

1.9

mixture

K377

K864

0 10 20 30 40 50minutes

Table 48. C-omparison of original and recalculated DNA degrees of bindingl

K864/K871

Replicate

1

2

1

2

1

2

1

2

1

2

1

2

1

2

1

2

1

2

1

2il

957l

9174

9078

t220

8180

293l

511

10081

6262

K8684(872

K8691K872

K869/K870

il

il

il

150

183T

I6968

9656

2736

2733

3448

2425

4540

2828

K868Æ(869

K3O9/NCPPB 2437It

K864/TR3

r<271K309

K8641K377 t311

1- All comparisons and all replicates of each comparison performed in this study are

not included in this table. Results for the DNA reassociation studies are presented inTables 148 and 15B.

62

degrees of binding were obtained for a comparison which had shown low homology

in all other runs. The only possible explanation seems to be that there may have been

salt contamination of those particular runs. The other phenomenon which was

difficult to explain was a low degree of binding between two strains (e.g K8721K868)

which were both highly related to a thi¡d strain, K864 (Table 15B). It was thought

that there were contaminants in the DNA preparations which may have been

responsible for the low degree of binding achieved in these comparisons. These runs

were repeated with new DNA preparations and the same results were obtained so they

must be considered a real result but they are diffrcult to explain in biological terms.

The author feels.that there are several problems with the DNA reassociation

method- It is very diffrcult to obtain identical DNA concentrations in all cuvettes and

denaturation and renaturation values vary with concenftation and, more alarmingly,

with the cuvette used, even after they were extensively cleaned. The problem of

anomalous values and comparisons is difficult to overcome. Ideally in a taxonomic

study a method such as this one should not be the only basis of comparison. Results

should be compared with those obtained from a comparison of phenotypic traits, as in

this study, or with another method such as protein electrophoresis or restriction

fragment length polymorphisms.

63

PART B: RESULTS

B.1 Characteristics of isolates from Rubus and grapevine

a) Pathogenicitv and host range

Table 58 shows the results of pathogenicity tests for biovar 3, Rubus

isolates and A. rubi group strains on a number of hosts. TR2 was completely non-

pathogenic on all host plants tested. Individual Rubus isolates va¡ied in their

pathogenicity on different hosts but were consistently limited in thei¡ host range in

comparison to biovar 3 strains tested and with the published host range of biovars 1

and2 @eCleene & Deley, L976).It was noted that, although only small galls were

formed on the woody stems of boysenberry, galls were not observed when the same

strains were inoculated onto green shoots of boysenberry.

b) Ooine catabolism and synthesis

Table 68 shows the opines catabolized and synthesized in the galls of a

range of biovar 3 and Rubus isolates. There are two opine utilization patterns found in

biovar 3. The presence of octopine and cucumopine (Fig 58) only is by far the most

common situation in vine galls; all samples from South Australian galls observed in

the course of this work contained octopine and cucumopine but not nopaline.

None of the Rubus isolates or EU6 and 181 were able to catabolize octopine;

generally they were able to catabolize either nopaline, succinamopine or both.

Table 5B: Pathogenicity and host range of isolates from Rubus andgraPevinel

Biovar 3

Host: Strain:

K377

+weak *

K309

GrapevineAlmondCarot discTomatoRaspberry

Rubus isolates and others

Host: Strain:

K864 K868 K869 K870 K871 K872 TR2 TR3

Carot discTomatoTobaccoRaspberryBlackberryBoysenberry

1- see Materials and Methods for method and host plant cultivars used forp athogenicity testing.

2- NT = ûot tested

3- Inoculations made into the woody stem of boysenberry (see Materials andMethods)

+

;

K252

+¡12++

NT

+++

+weak+

+++

+

;

++

;NT

+

+NT

+

;

EU6 181

NT NT

NT NT+++NT NT

Table 6B: Opine catabolism and synthesis by isolates from grapevineand Rubus

Opines catobolized: Stain:

K305 K308 K309 K374

I

I

I

I

I

I

Ooines svnthesized:

Cucumopine

Rubus isolates and others:

Ooines catabolized:

OctopineNopalineSuccinamopine

1- Max Tate, personal communication

2- NT = ûot tested

K377 K252

+++

NT

Strain:

TR2 TR3 K864 K868 K869 K870 K871 K872 EU6 181

:+

NT

l+

¡12

+++

:+

++

;+

iNTNT

OctopineNopaline

+

+

+

+

+

+

+

NT

;+

;+

+;++

+;+

+

Figure 58. High voltage paper electrophoresis of grapevine

gall extract showing presence of octopine

in gall tissue. Paper stained for presence ofguanidines and photographed under

LIV (302 nm) illumination.

Lanes: O. octopine standard

1,2. grapevine gall extract

64

c) Plasmids

All biovar 3 isolates tested have single,large (approximately 200 kb)

plasmids. When the ptasmids of K252, K374, K305 andK377 were eliminated by

pDP35 containing the cloned fragment of the incompatibility region of 46,

pathogenicity was lost by all strains. This confrms that the large plasmids were in

fact Ti-plasmids in the Rh-l incompatibility group described by Hooykaas et al.

(1930). In addition,K252 and K305 lost the ability to catabolize octopine but K374

andÍ377 did not (Fig 68), indicating that the genes for octopine catabolism are not

located on the Ti-plasmid in the latter srains.

Figure 78 shows the muttþle plasmid bands visible in Rubus isolates. The

incompatibility group of the Ti-plasmids of the Rubus strains was not established.

There are plasmids in each of the strains of a comparable size to the Ti-plasmid.

d) B iochemical differentiation

Results for all biochemical tess perfomred are listed in Table

78. The differential tests used to separate biovars I and2 have been well-described

previously (De tæy et a1.,1966; Keane et a1.,1970; White,1972; Kersters et a1.,L973;

Holmes & Roberts,1981; Kersters & Deley,1984) and these are confirmed by this

study. Biovar 3 strains can be differentiated from other groups on the basis of growth

on two selective media. They share with the Rubus isolates a negative result for

utilization of many of the carbon sources tested but the groups can be distinguished

by their growth at3Toc,reaction on aniline blue medium and the inability of cane gall

isolates to use Na-tartrate. Two sources of TR3, the type strain for A. rubi, ATCC

13335 and NCPPB 1854, were tested and found to differ from the Rubus isolates in

that they display only a partial requirement for growth factors in the culture medium.

In addition, they did not have the reaction on aniline blue medium typical of the

Scottish Rubus isolates.

Figure 6B: Growth of Ti-plasmidless biova¡ 3 strains on:

A. Minimal media plus0.2%o nopaline

B. Minimal media plus0.2Vo octopine

Strains:

r. K3772. 1<377

3. 1(377

4. Í3745. K374

(wild type)pTi- #1

pTi #2pTi- #1

pTi- #2

Figure 78. Agarose gel electrophoresis showing multipleplasmids in Scottish Rubus isolates.

Lanes:

A. EU6B. K874c. K871D. K870E. K868F. K864G. ATCC 13335 (=TR3)H. K187 (biovar 1)

Arow indicates chromosomat DNA.

Table 78: Biochemical characteristics of strains used in this study

* ú B* Rubus TR3:8 rR2 EU6 181jsoktq2 ATcc NcPPB

Growth on:

B1BZB3RS

Growth on:

Petit's plus:Nal.{O3 aNaNO3 + biotinL-glu + biotinL-glu + yeast extract

Bergersen's plus:NaItIO3NaNO3 + biotinL-glu + biotinL-glu + yeast extractL-glu + growth factors

3-ketolactose prod.

Growth on 27o NaCl

Growth at 37oC

Reaction on aniline blue(Type 1 or 2)

Acid from:

mannitoladonitolerythritoldulcitol

melezitoseethanola¡abitol

Alkali from:

Na-tartrate

++

++

+++

+++

+++

++++

;++

+

+++++

+++++

++

+++

++++

+++

++

++++

+++++

+Ø/q?+(416)5

+++

+

+

+

+

+

I

+

+

I

+

I

+

+

2

+

+

2

++

11TQß)5

+(5/6)a

2

+++

l

+

l++++

++

++

-6rc)6

++

++

;

+

ll+

++

++

-(s/6)7

+ +

1- all methods listed in Part B,lvlaterials and Methods. Composition of all media used clisted in Appendix A.

2-T\e followins sBiovYarBiova¡Biova¡ lA52 (Ae 57)Rubus K872

All strains and their sor¡rces are listed in Table lB.

3- Both K377 andK376 are negative for these characters.

4- K871 is negative for this cha¡acter.

5- NCPPB 2137 gaveaType 2 reaction on anilineblue.

6- K864 positive for this character

7-f376 positive for this characær

8- Two separate sources of TR3 were tested, both received from type cultu¡æollections. ATCC = ATCC13335; NOPPB = NCPPB 1854

65

i) Growth cha¡acteristics in nutrient broth

Table 88 shows the results of three pammeters used to measure

growth rate for selected Agrobacterium strains. Biovar 2 and the Rubus isolates were

very slow-growing in comparison to biovars 1 and 3. Biovar 2 strains did not reach

the same final cell densities as strains from the other groups and took longer to initiate

logarithmic growth.

There was no effect of growth factor addition in nutrient broth for any of the

parameters measured except for the two strains, K868 atdK}7. Both strains showed

approximately 5O7o decreases in mean doubling time as well as increases in final cell

density, indicating a positive response to growth factor addition in nutrient broth.

ii) Growth characteristics in mannitol-glutamate broth

Table 98 shows growth parameters of selected isolates both

with and without addition of growth factors. Strains representing four Agobacterium

groups all showed a decrease in mean doubling time with the addition of growth

factors with the largest decreases shown by the biovar 2 strain ATCC LI325 and

Rubus isolate K868. As in nutrient broth, K868 showed an increase in frnal cell

density when growth factors were added. Lag times appeared to be relatively

unaffected by addition of growth factors. Growth was much slower in mannitol-

glutamate broth but the relative mean doubling times for the four Agrobacterium

groups were the s¿ìme as in nutrient broth. Biovar 2 and Rubus isolates were slower-

growing in both media and showed the largestresponse to the addition of growth

factors.

f) DNA melting points

The midpoins of the thermal denaturation profiles (Tm) of selected

Agobacterium strains are presented in Table 108, along with the corresponding GC

Table 88: Growth characteristics of Aerobacterium strains in nutrient broth

Strain: Grouping: Lag Time(h)4 Mean DoublingTime (min)2

Final Cell Density(cells/ml)3

Kl87c58

K869K868

Biovar 1 66

50.968.8

t72198

45.1

r37r67

2.3 x 1091.8 x 109

1.5 x 1091.5 x 109

2.2x L09

2.3 x 1091.8 x 109

ATCC 11325 Biovar 2K27 'i,

K377 Biovar 3

fl

lt

1010

Rubus isolate

6

86

1- see Materials and Methods; all values represent a mean of 2 replicates when mediadid not include growth factors

2- measured in early log phase of growth

3- cell density where no further growth occurred for 3 consecutive readings

4- time from initiation of experiment to beginning of logarithmic growth

Table 98: Effect of growth factor addiúon on gtowth characteristicsof Aerobacterium in mannitol-glutamate broth

Lag Time (h)4 Mean DoublingTime (min)2

Final CellDensity (cells/d¡3

Strain: Grouping: No GE5 GF No GF GF No GF GF

K187 Biova¡ 1 16 16 r82 167

Biovar 2 16 I 403 355

2.2x lO9

1.7 x 109

1.9 x 109

1.9 x 109ATCCtt325

K377 Biovar 3 40 40 225 208 1.7 x 109

K868 Rubus 24 24 281 218 1.1 x 109isolate

1- experiments done in mannitol-glutamate broth as described in Materials andMethods; average of 2 reps

2- measured in early logarittrmic phase of growth

3- cell density where no further growth occurred for 3 consecutivereadings

4- time from initiation of experiment to beginning of logarithmic growth

5- GF - $owth factors (see'Culture Conditions')

2.3 x t09

1.6 x 109

Table 108. DNA Melting Points of selected Agrobacterium strains

Grouping Tml Ge2

NCPPB 2437 Biovar I 94.3 +/- 0.23 61

K309 Biovar 3 93.6 +/- O.28 59.3

K868 Rubusisolate

94.3 +l- 0.8r 61

1- Tm : midpoint of thermal denaturation profile. Tm determinations weremade as described in Part B, Materials and Methods. Values presented are a mean of3 separate runs plus standard deviation.

2- GC = porcont guanine-plus-cytosine contenl GC content was determinedby the equation: GC = (Tm - 69.3)2.44 (Mandel & Marmur, 1968).

66

contents. The results agree very well with the published GC content of NCPPB 2437

(60.8) (Deley, 1970). The melting points of K309 and K868 are within the range

specifred for Agrobacterium (Kersters & Deley, 1984) with GC contents of 59.3 and

61 respectively.

g) Motilitv

Table 118 shows results of motility tests performed using three

separate methods, with bacteria grown on two different media.

When bacteria were grown on Petit's minimal medium amended with

L-glutamate and (NII+)ZSO+, all biovar 1 and 2 strains were motile but none of the

five biovar 3 strains tested were motile, when tested by the three methods. When YE

was used as the growth medium, biovar 1 and 2 strains were motile as well as 2 out

of the 5 biovar 3 strains tested. Onfy 1 out of the 3 A. rubi strains tested was motile

(K869), and it was motile when it was grown on either of the media. The results

indicate that there is an effect of nutrition on motility of biovar 3 and also that there is

variability between biova¡ 3 and A. rubi strains with respect to motility.'When

preparations were viewed by the hanging drop method, biovar 1 strains appeared to

be more highly motile than either biovar 3 or A. rubi. This was observedrepeatedly.

h) Electron microscop),

Figure 8B(a) shows the Rubus isolate K871 as viewed by

transmission electron microscopy CIEM). The strain is typical of the genus

Agrobacterium in length (1-2 pm), its rod shape and in arrangement of the flagella.

Flagella appear to originate laterally from the bacteria and are 4-5 times the length of

the cell, thus corresponding to the description of the genus (Kersters &DeI-ey,

1984). Figure 8B (b) shows biovar 3 strain K1059 as viewed by TEM. Cells are

typical of the genus in length and shape. Flagella originate laterally from the bacterial

cell but lack the sinuous appearance of functional flagella. A large proportion of

biovar 3 cells were without flagella, in comparison to a reference g¡id of a biovar 1

Table 118. Motility of Agrobacterium isolates

Strain

Biovar 1:

NCPPB2437

ClRS

Biovar 2:

K27

K84

Biovar 3:

K252

CG49

CG484

CG992

K1059

Rubus isolates:

K864

K869

K872

32

YE Minimal YE Minimal

+ + + +

+ +

NT4

NT

+

METHOD & MEDIA: I

Motilit], in tubes: 2

++

16

20

25

20

20

0

20

0

33

35

40

30

NT

NT

+

+

+

+

0

0

0

0

0

+

+

+

+

+

0

15

0

0

15

0

+

NT

NT

NT

++

NT

1- All methods and media described in Paft B, Materials and Methods. All tests weredone in duplicate.

2- Motility scored as positive if 'halo' observed around site of stabinoculation after 48 hours' incubation at25oC.

3- Diameter of swarrn (in mm) after 48 hours' incubation at2soc.Average of 2 plates.

4- NT = rot tested

Figure 88. T¡ansmission electron micrograph

showing peritrichous flagellation of:

A. Rubus cane gall isolate K871(Magnification = 18,000 X)

B. Biovar 3 isolate K1059

@ar = 1pm)

Arrows show points of flagellar attachment to cell.

th

d

tL1

lt.î

,r* ¡3

.tr

1

aa

rla

I

{,ta'pa

I

aA

Jo

67

strain, K188. It was noted repeatedly that when grids were made of biovar 3 strains,

flotation on a flooded culture for 1 to 2 hours resulted in few, if any, cells adhering.

In order to obtain sufficient numbers of biovar 3 cells on the grid, it had to be floated

on the flooded culture for 12 to 18 hours. Possible explanatiorufor this

phenomenonorre that, unlike biovar 1, biovar 3 strains are not attracted to high oxygen

levels or biovar 3 strains have a different surface charge which affects their ability toadhere to the grid.

The preparation of bacteria for scanning electron microscopy (SEM) by freeze-

drying was ineffective. Few flagella were observed and the cells were badly

collapsed. 'When critical-point drying was used for SEM sample preparation, cells

were intact but few flagella were visible and those that were were largely

broken.'When sites of flagellar attachment were visible under SEM for strain K871,

flagella were attached peritrichously to the bacterial cell.

h) Sensitivitv to agrocin 84

All biova¡ 3 strains tested were much less sensitive to strain K84

relative to biovar 2 srains when tested by the method of Stonier (1960). Inhibition

zones of 8-12 mm were produced against the nopaline-type biovar 3 strains K374,

K376 andK377 in comparison with 45 mm zones against K27 , a sensitive biovar 2

strain. K84 produced no inhibition zones at all against any of the Scottish Rubus

isolates tested.

68

8.2 Relatedness among Agrobacterium strains

a) Numerical anal)rsis of phenotypic data/ Differentiation

of eroups

Dendrograms were constructed showing similia¡ities between strains based on

single linkage cluster analysis of the results of 25 biochemical tests. Figure 98 depicts

analysis when zero results were not matched and Figure 10B depicts the analysis

when zero matches were included.

'When zero results were not matched, four major groups, A-D, could be

discerned at the 857o similiarity level. Two of these, A and B, correspond to the

previously described biovars 1 and 2 respectively. A third group, C, comprises

strains isolated from grapevine, all of which have been previously described as biovar

3. The fourth g'oup, D, comprises strains isolated from cane galls on Rubus spp. as

well as strain EU6 which has been previously grouped in A. rubi (Kersters & Deley,

1984). At the 857o similiarity level, the two strains of TR3, ATCC 13335 and

NCPPB 1854, as well as the Rubus isolate K864, do not cluster with the other Rubus

strains but do so at the 77.5Vo level. Strain 181, a succinamopine-catabolizing isolate,

clusters with Group C, the grapevine strains, at the 77.5Vo level. Isolate TR2 is very

isolated phenotypically as it remains unclustered to the 657o similiarity level.

Holmes and Roberts (1981), in a study of Agrobacterium taxonomy based on

cluster analysis of a large number of characters clustered A. rubi strain TR3 with

isolates from grapevine. In that study, zero results were matched. Cluster analysis of

phenotypic data presented in this study was therefore performed with and without

using matched zero values in order to compare the result. Figure 108 depicts the

result when zero matches were included. The same four groups, A to D, were

discerned at the 907o similiarity level as were found in the previous analysis at the

S5Volevel. However, some differences do exist. The two TR3 strains, ATCC 13335

and NCPPB 1854, and K864 all cluster with Group D at the 907o similiarity level,

much higher than in the previous analysis. Strain 181, which was grouped with the

Figure 98. A dendrogram showing relationships between

strains based on single linkage cluster analysis.

Order of strains is as depicted on the minimumspanning tree. Negative matches were not included.

Groups A to D indicate clusters formed atthe&O%o

similarity level.

95 90

Percentage similaritY

85 70 65

ANCPPB 1OO1

c58

NCPPB 2,137

K8A+

NCPPB 1854

ATCÆ 13335

D

K868

KA72

K869

K871

K870

EI,J6

181

K305

K252

K309

K306

K377

K376

KA4

TCC 11325

K27

TR2

c

'þ

Figure 108. A dendrogram showing relationships between

strains based on single linkage cluster analysis.

Negative matches were included. Groups A to Dindicate clusters formed at the 907o similarity level.

Percentage similaritY

95 90 80

A

'l^

K868

K872

K869

K871

K870

EU6

K864

K305

K252

K309

K306

K377

K376

TR2

K84

TCC 11325

K27

NCPPB 1OO1

c58

NCFPB 2¡tÍ17

181

NCPPB 1854

ATCC 13335

D

c

69

grapevine isolates (Group C) previously now clusters with Group D atthe 857o

similiarity level. TR2 remains ungrouped until the 82.57o level. The major effect of

including matched zero results appears to be a 'tightening' of the clusters; all strains

cluster at a higher similiarity level. The actual makeup of each cluster was virtually

identical be¡reen analyses except for the position of strain 181. Its opine utilization

suggests it has more in common with the Rubus isolates, with which it clusters when

zero matches \¡/ere included than wittr the grapevine isolates, with which it clusters

when zero matches were not included.

Table 12B summarizes the differentiating characteristics of the major groups.

The differentiation of biovars 1 and 2 has been well-described previousþ (Deley et

a1.,1966; Keane et al.,L97O; White,1972; Kersters g]!e!., 1973; Holmes and

Roberts,1981; Kersters and Deley,1984). Biovar 3 strains can be differentiated from

other groups on the basis of their growth on selective media. The Rubus isolates can

be differentiated phenotypically from biovar 3 on the basis of their response on aniline

blue medium, their growth at37oc and their inability to use Na-tartrate. A major

distinguishing feature of the Rubus isolates is their requirement for the growth factors

biotin, nicotinic acid and calcium pantothenate in the growth medium.

b) Serological relationships between biovar 3 and Rubus isolates

Table 138 shows the reaction of biovar 3 and Rubus isolates to antisera raised

against K309, K377 and TR3 in both the gel diffusion and tube agglutination assays.

The antisera against biova¡ 3 strains appeared to be quite specific, reacting only with

the closely related strains within the biovar 3 group. The octopine strain, K252,from

Greece did not form precipitin bands with antisera against local octopine or nopaline

strains although these strains are phenotypically similiar (Table 7B). The antigenic

determinant is not Ti-plasmid coded as a pTi- strain of K377 forms precipitin bands

with the K377 antiserum.

None of the isolates tested, including TR3, formed precipitin bands with

antisera to TR3 in the gel diffusion assay. In the tube agglutination assay, the titre

Table 128: Summary: Differcntiation of ¡\¡g9þ4ç1¡9¡i¡¡¡q speciesl

Biovar 1 Biova¡ 2 Biovar 3 A. rubiGrowth on

selective mediæ

Growth factorrequiremenr

a) biotin only

b)^biotin,. calcium pantothenaæ¿k mcoûnrc acrd

3-keolacose production

Growth on 27o NaCl

Growth at 37oC

Aniline blue reaction2

Acidftom:

ethanolarabitol

Alkali fr,om:

Na-târtrate

1- see Table 7B for breakdown of biochemical test results by snain

2- see lvlaærials and Methods, Part B

B1B2B3RS

++

++

+ -l+r

+

+

+

I

;+++

+

+

+

+

2II

ervthrioldúlcitolmelezitose

+

:

++

Table 138: Serological relationships between biovar 3 and Rubus isolates3

Antiserumto:

K309 K377 TR3

AntisencDlT}2@TA@TA¡¡¡3NTNT

NTNTNT

NTNTNT

1- GD = gel diffusion; '+'indicates precipitin band formed; see Materialsand Methods

2-TA= tutle agglutination; number indicates titre; see Materials and Methods

3 - NT = not tested

4 - described in Table 1A

K305K306K308K309K374K375K376K377K252r{253K377P'ri- 4TR3K868K870K871TR2EU6

+++

i1600NT

6400160050NTNT100100NTNT<50NTNTNTNT<50

;+++

+

50NT<50<503200NTNT

1600400NTNT<50NTNTNTNT<50

NTNTNT200NTNTNT<50NTNTNT800<50400<50400200

NT

NTNT

NTNT

70

against TR3 was somewhat low and there was a low level of agglutination with

K870,TR2, and EU6 as well as with K309. None of the other Rubus isolates tested

orK377 showed any reaction with the TR3 antiserum.

c) DNA Homology Studies

i) Relationship of biovar 3 to other groups

Table 148 shows the degree of binding of the biovar 3 strains

to strains from the other Agrobacterium groups and to each other. The biovar 3

isolates tested all displayed a high degree (>70Vo) of binding with each other. K309

had a low degree of binding (40Vo) with the type strain of biovar 1 (NCPPB 2437)

and with two biovar 2 strains, K27 andthe type strain, ATCC 11325. Binding of

K309 andl377 with Rubus isolates was low (<26Vo) and moderately low (45Eo)

with EU6.

ii) Relationships between strains from Rubus and other aerobacteria

Table 15B shows the degree of binding between strains from

Rubus with each other and with isolates from other Agrobacterium groups. Isolates

K864, K868, K869, K870 and K871 all show a high (>8O7o) degree of binding with

each other. K872 has a high (82Vo) degree of binding with K864 but a low degree

(<287o) with both K868 and K869. Two Scottish Rubus isolates, K868 and K871,

were tested for homology to the two sources (ATCC 13335 and NCPPB 1854) of

TR3, the t)?e strain of A. rubi. Both of the Scottish isolates displayed a very high

degree of binding (>82Vo) with ATCC 13335 and a moderately high degree of

binding(>65%o) with NCPPB 1854. It should be noted that the two TR3 isolates

displayed a96Vo degreeof bindingwitheachother. StrainsK868,K869andEU6all

show a low degree of binding with the type strains of biovar 1 (NCPPB 2437) and

biovar 2 (ATCC 1L325) and with biova¡ 3 strains, K309 andK377.

Table 14B. Degree of DNA Bindingl: Grapevine strains and otheragrobacteria

Strain K309 K377

K309K377K374K252

TR33EU63

K864K868K869

A. rubi

Rubusisolates

il

rÍtt

100100_270

1001008396

2945

0226

40

2528

2Til

8

NCPPB 2437 Biovar IATCC 11325 Biovar 2K27 '!,

1- all numbers represent an average of 2-5 separate renaturations

2- '-' indicates comparison not made

3- these strains have been previously grouped in A. rubi (Kersters & Deley,1984)

Table 15B. Degree of DNA Bindinglr Rubus isolates and other agrobacteria

Strain Groupins 8Ét 868 869 &Zt

TR3:3ATCC 13335 A. rubiNCPPB 1854 ',,

9l77

K864 Rubus isolates ioo 100 '.ã3

10081

NCPPB 2437

8826

0

t4

2

28

3

t4

26

8265

ß+

100

il

Ú

tttt

9083

8482

ATCC rr325

K309K377

Biovar 1

Biovar2

Biovar 3Biovar 3 8

1 - all numbers represent an average of 2-5 separate renaturations

2 -'-'indicates comparison not made

3 - TR3 has been previously grouped in A. rubi (Kersters & DeIæy, 1984)

7l

PART B: DISCUSSION

Results of numerical analysis of phenotypic data and DNA binding studies

indicate that there are four distinct groups within the genus Agrobacterium. Analysis

of growth characteristics lend further support to this conclusion. The four groups

identifred in this study, A-D, correspond to biovars 1,2,3 and A. rubi respectively.

Data obtained from biochemical tests, motility tests and melting point detemrinations

confirm that biovar 3 and A. rubi both warant inclusion in the genus Agrobacterium.

The separation of biovars 1. and2 has been well-documented (Keane et al.,

I97O; White, 1972; Kersters et al., 1973; Holmes & Roberts, 1981) and results

obtained in this study conf,rnn this separation. Kersters and Del-ey (1984) have

suggested that biovar 1 should ideally be named A. radiobacter with pathogenic

strains named as pv. tumefaciens and that biovar 2be a separate and as yet unnamed

species. Clearly this is a vast improvement on the existing nomenclature but this

proposal carinot be accepted at present due to the rules of bacterial nomenclature

(Kersters & Del-ey,1984). For the moment, the two groups are designated as biovars

of A. tumefaciens (for pathogenic strains), A. radiobacter (for non-pathogenic strains)

or A. rhizogenes (for rhizogenic strains).

Biovar 3, comprising largely isolates from grapevine, has been described by

several authors (Kerr & Panogopoulous,l9TT; Panogopoulous et a1.,1978;

Sule,1978). The cluster analysis ¡iresented in this study conf,rms the phenotypic

differences be¡ween biovar 3 and strains belonging to biovars I andZ. The DNA

binding studies confirm the sçparation of these groups at the genetic level.In their

numerical taxonomic study, Holmes and Roberts (1981) found that the type strain for

A. rubi, TR3, clustered with grapevine isolates. That finding was not confrrmed by

either numerical analysis or DNA binding studies in this work It is interesting to note

thatHolmes and Roberts included matching of negative results in their single linkage

cluster analysis. It was found in this study that when zero matches were included in

cluster analysis, all strains grouped at a higher similiarity level. Thus, if strains are

negative for a large number of characteristics, they may cluster together falsely. The

72

separation of A. rubi strains, including TR3, from the grapevine isolates is conf,rmed

by resuls obtained in the DNA homology studies.The species A. rubi was originally

described by Hildebrand (1940) as Phytomonas rubi. Ea¡lier authors (Banfield,1930;

Pinckard,1935) had suggested that the disease referred to as'cane gall'on Rubus

spp. ìwas distinct from'crown gall'. Reports indicate that isolations from crown gall

on Rubus yield predominantly biovar 2 strains (Perrf'& Kado,1982; M. Lopez, pers.

communication). Cane galls are characterized not only by being found above soil level

but galls are characteristically smaller and often found in long ridges along the cane-

rarely or never are galls found below soil level in these infections (Hildebrand,1940).

Hildebrand also found fundamental differences between the causal agents of these

diseases. Star (1946) used Hildebrand's original A. rubi isolates, TR2 and TR3

(NCPPB 1856 and 1854 respectively) to demonstrate the auxotrophic nature of A.

rubi; these isolates require L-glutamic acid and the vitamins biotin, calcium

pantothenate and nicotinic acid for gowth. This growth factor requi¡ement of the

strains referred to as the Rubus cane gall isolates was confirmed in this study. The

isolates, all from above-ground galls on Rubus spp., conform to the original

descriptions of A. rubi by Starr and Hildebrand. Based on both cluster analysis and

DNA homology,and emphasized by comparative colony morphology, growth rates,

host range and opine utilization patterns, this study indicates fundamental differences

between A. rubi and biovars 1,2 and 3.

Two sources of the type strain for A. rubi, TR3, were tested and both required

biotin for growth. the TR3 isolate received from the American Type Culture

Collection (ATCC 13335) required both L-glutamate and.biotin for growth. Thus,

neither of the TR3 isolates conformed completely to their original description in terms

of growth requirement but did so in all other respects. In preliminary work, not

included in this study, isolates of TR3 showed low DNA homology with the Scottish

Rubus isolates and did not confonn to the original description in terms of the growth

factor requirement or in terms of the range of carbon sources utilized. It was for this

reason that new source cultures of this strain were obtained. Because of the slow

73

growth of A. rubi and its growth factor requirement, it is likely easily contaminated

and this may be a reason for conflicting reports about the bèhaviour of TR3 in the

pasL When the fresh type cultures of TR3 were used, they showed a high degree of

DNA binding with other Rubus isolates, confrrming that all are the same species.

Strain EU6 and the Rubus strains clustered together in the numerical analysis,

conflicting with the results obtained by Holmes and Roberts (1981). However, in a

DNA binding study, Deley (L974) found high homology between EU6 and TR3.

This anomaly highlights the general problem of correlating phenotypic and genetic

data, a problem occurring in several places in this work. For example, strain K864

shows a high degree of DNA binding with other Rubus isolates but was somewhat

less related to these strains phenotypically, clustering in Group D only at the 77.5Vo

similiarity level, although when negative matches were included in analysis of the

same data, K864 clustered in Group D at9oVo. Conversely,K872 had a higher

degree of DNA binding with K864 than with the phenotlpically more similiar strains,

K868 and K869. Inherent inaccuracies in the methodology used for the measurement

of DNA homology (discussed in Materials and Methods) cannot fully explain the

anomalies which are symptomatic of the general dilemna of relating molecular and

morphological data in taxonomic studies (Mayr,1982).

Despite the problem of correlating phenotypic and genetic data, it is evident

that the Scottish Rubus isolates and TR3 correspond to [Iildebrand's (1940)

description of A. rubi and are a separate group from biovar 3, based on both

phenotypic and genetic data. It is interesting that the panern of opine utilization of

these two $oups is different. Biovar 3 strains are largely (but not always) octopine-

catabolizing (Perrl'& Kado,1982). Selected strains have also been shown to

catablize cucumopine (Table 68) which has been shown to be a conjugative opine

for these strains (4. Petig pers. communication). 'Whether or not this is a general

phenomenon for all biovar 3 strains is unknown. The Rubus isolates to date are all

nopaline and/or succinamopine-catabolizing strains. The conjugative opine(s) for the

Rubus isolates is as yet unknown. Although opine catabolism is generally a Ti- ''

74

plasmid coded trait (Bomhoff et al., 1986; Montoya et al., 1977; Guyon e]!3l.,

1980), it is inæresting that particular plasmid types are consisæntly associated with

particular chromosomal types. Similia¡ly, with respect to another plasmid-coded trait,

pathogenicity, Rubus isolates tested in this study appear to be largely limited in host

range (Iable 5B), confirming the observations of Banfield(l930) and

Hildebrand(1940). Although there are limited host range strains in the biovar 3

grouping (Panagopoulos & Psallidas, L973; Panogopoulous et al., 1978; Loper &

Kado, 1979; Thomashow et a1.,1980; Perry and Kado,1981; Knauf et a1.,1983),

these are limited to grapevine and more commonly biovar 3 srains have a relatively

wide host range (Sule,1978; Burr & Katz,1983).

Serological relationships between agrobacteria are not straighforward and are

not a reliable means for defining groups at the species level though they could be

useful for strain identification. Within biovar 3, there appeff to be at least three

serological subgroups using polyclonal antisera and probably there are more. Only

very similiar isolates were serologrcally related and although South Australian

octopine strains are serologically related, they do not react with phenotypically and

genetically similiar octopine strains from Greece. The serological heterogeneity within

biovars was recently confrrmed by Alarcon et al. (1987) in a comparative study using

several immunological assays as well as protein electrophoresis. The Rubus isolates

and biovar 3 isolates tested are serologically unrelated to TR3, which showed poor

specificity and had low titres even against itself. For this reason,the TR3 antiserum

appears to be unsatisfactory and further studies are required. Recently a monoclonal

antibody to biovar 3 was developed by Bishop et al. ( unpublished results) and has

been shown to react only with biovar 3 and not with biovars I and2 (T. Burr, pers.

communication) or A. rubi (data not shown). The phenomenon of serological

heterogeneity within Agobacterium spp. has been observed in other studies

(Graham,1971; Nesme et a1.,1987). The finding thatK377 andK3TTpTi reacted

serologically also confirms the finding of Hochster and Cole (1967) working with

isolate 86 that antigenic determinants ¿ìre not plasmid coded. Separate species of

75

A8robacterium do appear to be antigenically different from each other; this has been

shown previously by Keane e!-AL, (1970).

In terms of growth characteristics, Agobacterium groups differ in both

gowth rate, as measured by their mean doubling times, and in their response to

gowth factors. Both biovar 2 and Rubus isolates display a marked response to

gowth factor addition in a defined media Clable 9B); biovars 1 and 3 have a limited

response to growth factor addition. The lack of an obvious growth factor response in

nutrient broth as compared to mannitol-glutamate is probably due to the greater

availability of vitamins in the forrrer media.

Motility tess indicated an effect of media nutriúon on the motility of biovar 3

but not of the other Agrobacterium groups. Biovar 3 and A. rubi strains tested were

not all motile and those that were appeff to have a lower percentage of actively

swimming strains than either biovar I or 2 strains. Under TEM, biovar 3 strains

appeared to have a lower proportion of flagellated cells and the flagella look atypical,

in comparison to a biovar 1 isolate. It is possible that, because of the importance for

the grapevine and Rubus strains of 'passive' survival in the host's vascular system as

opposed to su¡vival and dissemination in the soil, that these strains a¡e less motile

than biovars 1 and 2.

Data from phenotypic, genetic and serological tess all indicate that there are at

least four separate groups within Agrobacterium. The low DNA homology between

groups (<257o in most cases) warants these groups being named as separate species.

Although the suggestions for nomenclatural changes to the genus proposed by

Kersters & De Ley(198a) would be ideal, they do not conform to taxonomic rules.

Therefore, it is proposed that Group A (biovar 1) in this study be designated A.

tumefaciens and Group B (biovar 2) be designated A. rhizogenes. Group C,

comprising strains identif,red as biovar 3, has no specific name at present.

Considering the very close association of these strains with grapevine, A. viti seems

appropriate and will be proposed. A. rubi should be retained for isolates conforming

to the original description of this species by Hildebrand (1940), Group D in this

76

study. It should be realized that the specific names forGroup A and B do not describe

pathogenicity which should be indicated by infi:a sub-specific epithets. This proposal

seems to be a less confusing alternative to the present taxonomy and one which

complies with the rules of bacterial nomenclanre.

77

PART C: OTHER OPINE-UTILIZING BACTERIA

INTRODUCTION

The theory of genetic colonization (Schell, 1978; Schell et al., 1979) and the

opine concept (Petit et al., 1978; Tempé et al., 1979; Guyon et al., 1980) botlt

postulate that the opine-related genes are the reason for the existence of the Ti-

plasmid. Since the discovery that the opine synthesis and catabolism genes are on the

Ti-plasmid (Bomhoff et al.,1976; Kerr & Roberts, 1976; Montoya et a1., 1977).,ít

has been theorized that these genes are providing the agrobacteria which carry them

with a competitive advantage in the galls- This theory is discussed and examined more

extensivoly in Part A of this thesis.

The opine concept was formulated at a time when it was presumed that opine

catabolism was exclusive to Ag¡obacterium spp. and that they could then

preferentially colonize galls. Since then there have been reports of Pseudomonas

(Kohn & Beiderbeck, 1982; Beaulieu et a1., 1983; Brisbane & Kerr, 1983; Rossignol

& Dion, 1935) and coryneform bacteria (Tremblay et al., 1987) existing in diverse

environments which can catabolize a number of the opines.

Pafi C of this thesis is divided into two sections. It looks at two separate

genera of bacteria which a¡e both capable of catabolizing opines- fluorescent

Pseudomonas spp. and fermentative bacteria which have not been identified. The

possible use of the former in a biological control strategy is discussed and

speculations are made on the role of these bacteria in the ecology of crown gall.

78

PART C- PSEI.JDOMONAS

MATERIALS AND METFIODS :

1. Strains used and their isolation

Soils were collected from around the base of uninfected, established vines in

three vineyards with no previous history of crown gall. The three sites were the V/aite

Agricultural Research Institute and two commercial vineyards in the Southern Vales

area south of Adelaide- Cambrai and Pirramimma. Ten 1 gram subsamples were taken

from each of the vineyard soil samples and suspended in 10 ml sterile distitled water

(SDV/), diluted and plated on one of the two selective media for biovar 3 and on

King's medium B and incubated at 28oC. Appendix A describes the composition of

all media used.

A second goup of isolates was obtained from galls on K5140 and Salt Creek

rootstocks. Galls were macerated in a mortar and pestle in SDW and five 100 pl

aliquos per gall were plated on nutrient agar and incubated at 28oC.

The final group of strains were isolated from K5140 rootstocks obtained from

the Vine Improvement Committee, Í,oxton, South Australia. These vines were being

checked for the presence of Agrobacterium biovar 3 in the vascular system by the

vacuum-flush method described in Part A, Materials and Methods. Several isolates

which grew on the biovar 3 selective medium of Roy & Sasser (1983) were found to

be positive for fluorescence on King's medium B and were used in this section of the

study. Strain K315 was isolated from grapevine in L977 andhad been found to be a

Pseudomonas spp. Its fluorescence on King's medium B was confirmed.

All isolates, from soil, galls and rootstocks, were purified by streaking for

single colonies on King's medium B and were stored in SDW at 10oC, according to

the method described by Sly (1983). All strains and their origin a¡e listed in Table lC.

Table lC. Str¿ins and their origin

A. Fluorescent isolatesl

Strain designation Isolated from2:

P-2

P-L4P-18

P-20

vineyard soil, Waite Institute octopme

octopine, dMGluoctopine

nopaline

octopine, nopaline

nopaline

octopine, nopaline

octopme

none

I

il

lt

I

il

il

Ir

r

Cambrai 2-6

Cambrai 2-3

Pirr 1-8

Hn l-2

SC4K51 #1

K51#5

v-16Y-2LY-n

K315

R-1

R-6R-16R-20R-25R-30

soil, Cambrai vineyard

soil, Piramimma vineyard

gall, Salt Creek rootstockgall, K5140 rootstock il

il

from vascular system, K5140il

il

from grapevine gall, South Australia octopme

B. Ogine catabolizing isolates from Rubus galls

Strain designation Opine(s) catabolized3

Rubus cv.'Silvan' galls nopalineI

il

r

il

ll

none

nopaline

none

il

1- these isolates are referred to as Pseudomonas in the text All are positive forfluorescein production on the medium of King et al., 1954.

2- see "Materials and Methods", Part C fo¡ details of the isolation of these strains.

3- see "'Materials and Methods", Part C.

79

2. Onine Catabolism

Isolates were checked for catabolism of the opines octopine, nopaline and

dMGlu (mannopinic acid; Dahl et al., 1983) as described in Section B, Materials and

Methods. The galls from which Pseudomonas isolations were made were found to

contain octopine and cucumopine when checked for opines by high-voltage paper

electrophoresis (Part B, Materials and Methods).

3. Biochemical Tests

Standard biochemical tess and tests for gtowth on selective media were

performed as described in Part B, Materials and Methods.-Production of the

fluorescein pigments characteristic of the fluorescent Pseudomonas group was tested

by streaking fresh cultures of each strain on to King's medium B, incubating for 24 to

48 hours at 28oC and checking for fluorescence under W (302 nm) illumination.

Utilization of D-glutamate, thought to be a selective characteristic for Agrobacterium

biovar 3 (Kerr & Brisbane, 1983) was tested by streaking fresh cultures of each strain

on the minimal medium of Petit (described in Appendix A), amended,with0.2%o

mannitol and0.057o D-glutamate. Plates were incubated at 28oC for 2to 4 days and a

biovar 3 strain was used as a positive control.

4.Inhibitory Activit)¡

In vitro: The method of Stonier (1960) was modified to check for inhibitory activity

of Pseudomonas isolates. A 10 pl aliquot of a suspension (108 cels/rnl) of a putative

inhibitory isolate was spotted on agar, allowed to dry and incubated at 28oC for 48

hours. Plates were then exposed to chloroform vapours for 10 minutes, allowed to air

for 15 minutes and a 3 ml agar overlay containing the indicator strain was poured over

the plate. Inhibitory activity was tested on a number of media- nutrient agar,

Pseudomonas minimal medium, King's medium B and King's B amended with either

10 mg/rnl FeEDTA, l0 pM FeCl3 or lmM tri-sodium citrate. The addition of an iron

80

source was rnade to investigate whether inhibitory activity was due to the production

of a siderophore (Kloepper eta!, 1980a) a¡rd the addition of tri-sodium ciuate was

made in order to remove all traces of iron in the medium. All media are described in

Appendix A.

In vivo: Selected Pseudomonas isolates which were inhibitory in in vitro assays were

further tested for inhibition of AÊrobacterium biovar 3 in vivo by several methods.

Coinoculations of Pseudomonas and Agrobacterium were made on tomato

stemsusingthemethodof KerrandHtay (L974). Varyingratios(1:100, L:L0, 1:1,

10:1, 100:1) of turbid (108 cells/ml) suspensions of Pseudomonas: Agrobacterium

were inoculated into tomato stenìs and results recorded after 4 weeks. Gall size

relative to control inoculations of biovar 3 was recorded.

A similiar assay was done on carrot discs (Materials and Methods, Part A)

using 1:1 and 10:1 inoculumratios of Pseudomonas: Agrobacterium. Aliquots (20 ttl)

of the mixed suspensions were inoculated onto the apical side of carrot discs

(described in Materials and Methods, Part A). Discs were inoculated as previously

described and results lilere recorded as gall weighs after 7 weeks. All tests were done

in duplicate.

One isolate (Cambrai 2-6) was chosen for use in an experiment to test in vivo

inhibition of biova¡ 3 isolates K309 andK377 on grapevine. Rooted Cabernet

Sauvignon cüttings were wounded just above the butt of the stem and dipped in a

turbid (9 x108 ce[s/ml in SDW) suspension of isolate Cambrai 2-6for 5 minutes.

They were then planted directly nto25 cmpots conøining a moist, non-sterile 1:1

sand: loam mixture which had been mixed mechanically with 107 cels of K309 or

K377 per litre of soil. Plants were grown under glasshouse conditions. Numben of

both bacteria in soil were recorded af¡er 24 hours and again after 3 and 6 months. Dry

weights of galls were recorded after 3 and 6 months.

81

5. Pathogeniciw tests

Pseudomonas isolates were tested for pathogenicity on tomato stems and

carrot discs using the method described in Part A, Materials and Methods.

82

PART C- PSEI.JDOMONAS

RESULTS:

1. Occurrence and abundance of Pseudomonas

Pseudomonas were isolated from all soil samples; it should be noted that none

of the vineyards from which samples were taken had any history of crown gall.

Numbers of Pseudomonas in soil were 104 to 105 cfu/g soil in all cases. Similiar

isolates were obtained from crown galls and from the vascular washings of

grapevines, indicating that they are widespread and can systemically colonize vines.It

should also be noted that, in sepamte isolations, fluorescent Pseudomonas spp. were

found at levels of 103 to 104 cells/ c-2 root on both Ramsay and K5132 rootstocks

obtained from a Barossa Valley nursery.

2. Opine Utilization

Table lC lists the opines catabolized by representative isolates. A larger

number of isolates from each sample site were tested for opine catabolism and were

found to vary widely in this respect. Isolates from the vine galls, which contained

octopine and cucumopine, were all able to catabolize octopine but not nopaline.

Isolates from soil and vine roots varied in their opine catabolism; isolates from the

snme location could catabolize octopine, nopaline, neither or both. Approximately

807o of rootstock isolates could catabolize at least one of the opines; on the other

hand, only lïVo of the systemically-isolated Pseudomonas were capable of

catabolizing opines. Figure LC shows in vitro octopine catabolism of Pseudomonas

isolates from K5140 rootstocks.

Figure lC. Octopine utilization by pseudomonas isolates.

Growth after 3 days incubation at 28oC onPetit's minimal medium plus0.27o octopine.

K309: positive controlIsolates 1-11: Pseudomonas isolated fromvascular system of Ramsay and K5140dormant cuttings.

83

3. Biochemical tests

Table 2C lists the biochemical characteristics of representative Pseudomonas

isolates. All are positive for fluorescein production on King's medium B. All are able

to grow on the selective media for Agrobacterium, including that of Roy & Sasser in

many cases (1983), but most could be distinguished on the latter from biovar 3 by

their more mucoid colony morphology and lack of red centre. All strains tested were

able to utilize tarftate, a characteristic which is associated with biovar 3 Agrobacterium

strains, with some biovar 1 isolates from grapevine, as well as with biovar 2 strains

(see Part A). Ability to use D-glutamate was tested because this trait was the seleôtive

basis for the biovar 3 medium of Brisbane & Kerr (1983); however, over 507o of

Pseudomonas isolates tested were able to catabolize D-glutamate. There were no

consistent patterns observed in the 5 strains tested for ca¡bon source utilization and no

attempts were made to identify these isolates further.

4. Inhibitorv Activity

In vitro: Approximately 80Vo of fluorescent pseudomonads isolated in this study were

inhibitory to Agrobacterium in varying degrees. Table 3C shows the range of

inhibitory activity of representative Pseudomonas isolates to strains of Agrobacterium

biovars 1,2 and 3. None of the inhibitory Pseudomonas isolated in this study were

active in vitro against biovar 1 but all were inhibitory to the biovar 2 and 3 strains

tested (Figure 2C). Isolates P-2 and P-14 produced very sharply defrned zones of

inhibition against all biovar 2 isolates tested and generally produced larger, more

diffuse zones against biovar 3 isolates @gure 2C). Isolate P-18 differed in the sense

that it produced large, sharply deñned inhibition zones against both biovars 2 and 3.

Isolates V-21 a¡rd V-38 were tested for inhibitory activity against K869, a

representative of the Rubus cane gall group described in Part B. Both V-21 and V-38

Table 2C. Biochemical characteristics of Pseudomonas isolates

Fluorescence on KB

Tartrate utilization

Growth on 81

Growth on 82

Growth on 83

Growth on RS

Acid from:er¡hritolmelezitosedulcitolethanol

Growth on27o NaCl

+

+

+

+

+

P-2 P-14

+

Isolates:

P-18 P-20

++++++++++a2 ¡2

SC4 All othen3

+

+

+

+

+

+l-4

NIINTNTNT

NT

+

+

+

+

+

+

+

+

+

+

+l-2

+ l+

+

+

+

++++

+

+

+

1- not tested

2- colonies grew on RS medium but did not have the red centre characteristic ofbiovar 3 Aerobacterium

3- 36 isolates (not listed in Table lC) from the isolations described inPart C, Materials and Methods

4- 5ß6 strains had the red-centred colonies characteristic of Agrobacterium biovar 3on RS medium

Table 3C. Specificity of in vitro inhibitory activity of Pseudomonas isolatesl

Inhibition of A grobacterium groups:(mm inhibition zone indicated in parenthesis)

Isolate: 814 P+ Y+

P-2P-14P-18

K51 #1 6

K51 #5 6

SC#46

v-38 7

Y-2t7Y-n 7

NTNTNT

8NTNTNT

+ (20-25\2+ (r6-2D2+ (21-40)2

+ (30)

+ (27)+ (32)+ (33)

+ (12-20)3

+ (16-4Ð 3

+ (40-50) 2

+ (32)+ (35)

+ (30)

+ (33)

+ (33)

+ (37)+ (37)

+ (38)

+ (40)

+ (30)

+ (31)

+ (27)+ (25)

1- assay for inhibitory activity described in Part C, Materials and Methods. Tests

performed on King's medium B and results recorded after Vl. hours. Range ofinhibition zone size (mm in diameter) indicated in parenthesis.

2- inhibition zones with clearly defined edges

3- inhibition zones more diffuse; edge of zone not well-defined4- Strains assayed against 10 strains of biovar 1 (B1), 8 strains of biovar 2 (82) and10 strains of biovar 3 (83), except where otherwise indicated.

5- tested against one isolate from each group: B1 (K188), 82 (K108), 83 (K309)6- tested against two 83 isolates (K309, K377)7- tested against one isolate of B 1 (K188), B2 (K108) and two 83 isolates(K309, K377)8- NT = not tested

Figure 2C. Inhibition of Aerrobacterium biovars by

Pseudomonas in plate assay. The method ofStonier (1960) was used.

A. Lack of inhibition of K188 (biova¡ 1) by

isolate Y-21.

B. Inhibition of K869 (A.rubÐ by isolate Y-21.

C. Inhibition of K84 (biovar 2) by isolate P-14.

D. Inhibition of K309 (biovar 3) by isolate P-14.

B

.tù

84

produced large zones of inhibition ( 43 and 41 mm in diameter respectively) against

this isolate (Figure 2C).

Table 4C shows the effect of iron addition to media on the inhibition of biovar

3 strain, K309, by the Pseudomonas isolates. In all experiments, inhibition was not

reversed when 10 pM FeCl3 or l0mg/ml FeEDTA were added to the media. The

complete reversal of inhibition with added iron which has been noted by others

(Kloepper et al., 1980) was not observed with these isolates. In some cases (e.g.

isolate P-2), inhibition decreased on King's B medium when iron was added but

increased when iron was added to the minimal medium.

The effect of iron addition on the inhibition by P-2, P-14 and P-20 of 6 biovar

2 strains was also tested. Inhibition of biovar 2 was observed with and without iron

in all cases and, in some instances (Figure 3C), inhibition zones were larger when the

media was amended with iron. Properly replicated trials would have to be performed

to see if this was a statistically significant effect but once again the results confrm that

the mode of action of these isolates is not via iron-binding siderophores.

Table 5C compares the inhibitory activity of the biological control strain K84

and Pseudomonas isolate P-14. The rate of diffusion of the P-14 inhibitory molecule

suggests that it is of low molecula¡ weighf

In vivo: When tomato plants were coinoculated with Agrobacterium biovar 3 isolates

and Pseudomanas isolates, there were no differences in gall size between control

inoculations with biova¡ 3 only and those plants coinoculated with Pseudomonas.

'When carrot discs were coinoculated, there were reductions in gall size in

comparison with controls at a 10:1 ratio of Pseudomonas: Agrobacterium for three

Pseudomonas isolates, P-14, P-18 a¡rd K315. These differences were not significant

(P< 0.05) because of the large variation between sep¿uate inoculations on carrot discs.

On grapevine, no signifrcant (P< 0.05) differences were noted in gall weights

after 3 and 6 months between inoculations of biovar 3 only and coinoculations of

biovar 3 and Pseudomonas. Galls were formed on all treatrnents at both sample times.

Numbers of biovar 3 on vine roots were approximately 5 x 105 cfr¡/cm2 root after 3

Figure 3C. Effect of addition of iron to growth media oninhibitory activity of Pseudomonas.

Inhibiúon of biovar 2 strain K108 byisolate P-14 on:

A. King's B medium

B. King's B medium amendedwith 10 pM FeCt3

KB t Fc-t{: tot

Table 4C.

Strain:

P-2P-14

P-18

Cambrai 2-3

Cambrai 2-6Ptrrl-2Pirr 1-8

K51#1K51#5

Y-21v-38

bv Pseudomonas

Diameterof inhibition zone (mm) on:

King's B King's B + Fe2 Pl¡tM3

47 24 32

15 24 30

39 25 31

29

74

t420

32

37

32

27

PMM+Fe4

16

35

30

35

424260

NTNTNTNT

NTNT

NTNT

30

¡1sNTNTNT

NTNT

NTNT

33

3l31

31.5

33.5

K315 32 30

L- tested by method of Stonier (1960), as described in PaÍ C, Materials and Methods.

Results presented are means of 2 to 3 separate tests.

2- King's B amended with either 10 pM FeCl3 or 10 mg/ml FeEDTA.

3- PMM = Pseudomonas minimal medium amended with lmM trisodium citrate to

remove all traces of iron from the media.

4- PMM amended with 10 mg/rnl FeEDTA.

5- NT = not tested.

Table 5C. Relative inhibitory activity of snains K84 and P-14 I

Inhibition zones (mm in diameter)oroduced bv:

K84 2 P-r43

2 days

3 days

4 days

5 days

33

47

6l

74

27

37

42

50

1- assayed by method of Stonier (1960), as described in Part C, Materials andMethods. Results presented are a mean of 2 replicates.

2-K84 was overlaid with K198; tested on Stonier's medium

3- P-14 overlaid with K309; tested on King's medium B.

85

months and 5 x 103 cfulctû root after 6 months for both K309 and K377. There

were no signifrcant differences be¡ween numbers of biovar 3 on vines with and

without Pseudomonas. Numbers of the Pseudomanas isolate, Cambrai 2-6, on

sampted roots were comparable to those of biovar 3, i.e. 5 x 105 cfulcrfl root after 3

months and 3 x 104 cfu/cm2 root after 6 months.

86

PART C- PSEUDOMONAS

DISCUSSION

Because biological control of grapevine crown gall cannot be achieved with

strain K84 (Ken & Panogopoulous,l9TT;Ken & Tate, 1984), it would be desirable

either to find or to genetically engineer a biological control agent effective against the

grapevine strains. Fluorescent Pseudomonas spp. isolated in this study appeared to

have some potential for this role from results obtained in in vitro inhibition assays.

The pseudomonads are capable of inhibiting biovar 3 Agrobacterium strains in plate

assays and they appear to have some specificity in that they inhibit biovar 2 and3

strains and the Rubus cane gall isolates but not biovar 1.

The mechanism by which inhibition occurs was not determined in this study

but it is clearly not via the iron-chelating siderophores. The importance of

siderophores in the inhibition of plant pathogens in the rhizosphere is not clear

(Hemming et al., 1982; Lockwood & Schippers, 1984) and the possible role of

antibiotic molecules produced by these strains also has been examined ( Strobel &

Lanier, 1981; Weller& Cook, 1983). Pseudomonads produce a wide variety of

secondary metabolites, including phytotoxins and antibiotics (tæisinger & Margnfl

1979) which are responsible for inhibitory activity in some isolates (Howell &

Stipanovic,1979:1980, V/eller & Cook, 1981; Colyer & Mount, 1984; Xu & Gross,

1986). No further work has been done on the isolates used in this study to determine

the nature of the inhibitory molecule(s) except to ascertain that they a¡e of low

molecular weight. The phenomenon of increased inhibition when iron is added to the

media, observed against biovar 2 isolates in this study, has been noted previously

(Hemming et al., 1982).It is possible thatmore than one inhibitory molecule is being

produced by isolates in this study; inhibition zones produced against biovars 2 and3

were distinctly different and the increased inhibition of biovar 2 but not biovar 3 when

iron was added supports this proposition.

Many of the pseudomonads isolated in this work were able to catabolize

opines and this combined with the fact that they were isolated from the vascular

The high percentage of Pseuclomonas isolates in this snrdy which could catabolize

opines may reflect the media from which they were isolated and may not reflect the

percentage of opine-catabolizing Pseudomonas in the field.

87

(i.ns"rl)system of grapevine, makes them potentially useful in biological control.^The 4't2ih+Y ol p.,:dorron,-

La>

.-okxlta<- +h+ yc..pevc;.re. ' is important given the systemic nature of the biovar 3 pathogens on

vines (discussed in Part A). However, the in vivo inhibition assays performed in this

study were uniformly unsuccessful both in decreasing numbers of Agrobacterium and

in controlling gaü formation. The in vivo assays were crude and limited in the number

of isolates studied. In order to properly determine whether these strains were capable

of controlling biovar 3, an in vivo assay on grapevines would have to be set up to

screen large numbers of isolates. It is probably overly simplistic to apply

Pseudomonas isolates to vine roots and a more interesting possibility for use of these

or other inhibitory isolates might be to introduce them into the vascular system of

'clean' rootstocks or scions as a protectant. The other possibility is to ransfer

antibiotic production from inhibitory strains such as these into nonpathogenic

Agrobacterium biovar 3 isolates; systemic entry into vines does not appeü to be Ti-

plasmid coded ( Bun et al., 1987b) so they would make ideal 'carriers' of antibiotic

genes.

The possible role of these pseudomonads in the ecology of crown gall disease

is discussed in the general discussion at the end of Part C.

88

PART C- FERMENTATTVE BACTERIA

MATERIALS AND METHODS

1. Isolation of strains

All strains used in this section of the study are listed in Table lC. All were

isolated from galls found on the roots or crown of Rubus cv. 'Silvan' (Mc Gregor &

Kroon,1984). Galts were surface-sterilized in one-tenth strength Milton's solution

(l6.5Vo sodium hypochlorite), rinsed with SDV/ and macerated in buffered saline

with a mortar and pestle. Dilutions were made in buffered saline andplated on yeast

extract (YE) agar or yeast mannitol (YM) agar with added gowth factors (Part B,

Materials and Methods). Large, mucoid colonies appeared after24 hours'incubation

at 28oC and were further purified on YM agar (isolates R-1, R-6, R-16, R-20).

Colonies which were slower-growing and less mucoid were isolated after 3 days and

were purified on nutrient agar with added growth factors (isolates R-25, R-30).

2. Opine catabolism and pathogenicity testing of isolates

Isolates were checked for their catabolism of octopine and nopaline, as

described in Part B, Materials and Methods. The galls from which the isolations were

made were found to contain nopaline (Figure 4C) when checked for opine content by

high-voltage papü electrophoresis (Part B, Materials and Methods).

Pathogenicity was tested as described for Agrobacterium isolates (Part A,

Materials and Methods). Indicator hosts used were tomato (L]¡cooersicon esculentum

Mill. va¡. 'Early Dwarf), blackberry ßubus L. cv.'Silvan'), tobacco (NicotianA

glauca Graham.) and broad bean C{icia faba L.). Inoculations were made on roots,

stems and at the crown on all hosts.

3. Biochemical tests

The procedures for all biochemical tests on the Rubus isolates are described in

Part B, Materials and Methods. Fluorescence on King's medium B was tested as

described in Part C (Pseudomonas). Fermentation of glucose was determined by the

Figure 4C. High voltage paper electrophoresis ofRubus cv. 'Silvan' gall extract.

Paper stained with phenanthrenequinone

reagant (Yamada & Itano, 1966) for the

presence of guanidines and photographed

under UV (302 nm) illumination.

Lanes:

1. nopaline standard

2. gall extract

89

merhod described by Fahy and Hayward (1983) using the basal medium of Hayward

(1964) (see Appendix A). An isolate of Erwinia carotovora, K80, was included as a

positive control in the fermentation assay.

4. Growth Rates

Growth rates of selected isolates were compared to those of agrobacteria in

nutrient broth (NB) bottt with and wittrout growth factors, using the method

described in Par:t B, Materials and Methods. Growth was followed by measuring the

optical density of the cultures at 640 nm and cell densities were calculated as

described previously.

5. Microscop]¡

Two isolates, R-1 and R-25, were Gram-stained using the method described

by Fahy and Hayward (1983) and viewed under oil immersion with a light

microscope. Isolate R-25 was also viewed by scanning electron microscopy (SEM).

Preparation for SEM viewing was by critical-point drying as described in Part B,

Materials and Methods.

6. DNA Melting Points

DNA of isolates R-6, R-25 and R-30 was prepared and the midpoint of the

thermal denaturation curves was determined by the methods described in Part B,

Materials and Methods.

7. Plasmid isolation

Plasmid isolations were made from isolates R-1, R-6, R-16, R-25 and

R-30 and visualized by agarose gel electrophoresis as described in Part A, Materials

and Methods. Plasmid DNA isolated from the biovar I strain, NCPPB 2437 (labte

1B), was included as a size marker.

90

PART C- FERMENTATTVE ISOLATES

RESULTS:

1. Biochemical characteristics

Table 6C lists the results of a range of determinative tests performed on the

isolates from Rubus galls. All strains are capable of fermenting glucose and produce

gas very stongly in that test. Their growth on aniline blue medium was different from

the reactions seen with Agrobacterium - bacterial growth was blue andvery mucoid

with no associated clearing of the media- Utilization of the 12 carbon sources tested

was not completely consistent between isolates of this group. Isolates R-16 and R-20,

which do not catabolize nopaline (Iable 6C), were atypical in their ability to utilize

both L-tarrate and ethanol. R-16 was also different in that it has a requirement for

biotin in the growth media and is able to utilize dulcitol. All isolates were able to

utilize the six sugars tested. Four out of the six isolates listed in Table lC were able to

catabolize nopaline; out of a total of 30 strains isolated from Rubus galls, 23 were

able to catabolize nopaline.

2. Growth rates

The growth of strains R-l and R-6 was rapid in comparison with

Agrobacterium strains (Table 8B) in nutrient broth. The mean doubling times for R-l

and R-6 in nutrient broth were 42 and 38 minutes respectively and neither strain was

affected signif,rcantly by the addition of growth factors. The lag phase was very short

for the Rubus isolates (2 hours in both cases), in comparison with lag times of 6 to 10

hours for all of the Agobacterium strains tested. The final cell densities achieved

were comparable to those of AÊrobacterium (109 ce[s/rnl).

3. Pathoeenicity

None of the Rubus strains were pathogenic on any of the host plants tested,

including Rubus cv. 'Silvan', from which they were isolated. In most cases, callus

formation was apparent 2 to 3 weeks after inoculation, giving the appearance of an

Table 6C. Biochemical characteristics of \ubus isolatesl

Isolate:

R-l Bú R-16 R-20 R-25 R-30Growth on selective media

for Aprobacterium:

B1B2RS

3- ketolactose production

Growth factor requirement

Agrocin 84 sensitivity

Growth on27o NaCl

Aniline bluereaction3

Growth at37oC

Fluorescence on KB

Fermentation of glucose

C source utilization:

mannitoldulcitolerythritolarabitolethanolmelezitoseL-tartrateD-galactoseL-arabinoseD-xyloseD-mannoseD-glucose

Opine utilization

nopalineoctopine

¡2

+

3

+

3

+ + +

3 3 3

+ +

aJ

+

++ + +

++

+

I+

;++++

:+

l;+++++

+

:

;+++++

+

l++++++++

;+++++++

;+++++

+++l

1- All tests are dessribed in Pan B, Materials and Methods except fluorescence on KBand fermentation of glucose which a¡e decribed in Part C, Materials and Methods.

2- Isolate R-16 had a requirement for biotin but not nicotinic acid or calciumpantothenate in the growth media. Part B, Materials and Mettrods describes the testfor growth factor requirement.

3- This test is described in Part B, Materials and Methods. Reactions of the isolates inthis table were of neither Typel nor Type 2. A reaction was considered'T¡te 3'when the bacterial growth on aniline blue medium was blue, mucoid and there was noclearing of the surrounding medium.

9T

unhealed wound but no blackening or rotúng of plant ússue was observed nor was

there any tnre gall formation. When the callus tissue was tested for the presence of

opines by high-voltage paper electrophoresis, none were found, indicating that no

plant cell transformation had occured.

4. Microscopy

'When strains R-l and R-25 were Gram-stained, both appeared as Gram-

negative short rods singly or in clusters. Under SEM, isolate R-25 appeared as a

short (1 pm) rod with a large number of protruberances on its surface (Figure 5C).

No flagella were visible in the preparations made by either freeze-drying or critical-

point drying.

5. DNA Melting Points

The midpoint of the thermal denauration profrle (T-) was determined for

DNA isolated from three strains inlx SSC. The melting points (and corresponding

guanine-plus-cytosine contents) of strains R-6, R-30 and R-25 were 94.1 (60.5),

92ß (57.3) and 92.8 (57.3) respectively.

6. Plasmids

Of the 5 strains tested, 2 strains, R-l and R-25 (Figure 6C), contained

plasmids in the size range of the Ti-plasmid of NCPPB 2437 GVIW = 131 x 10-6;

Sciaky et al., 1978). The presence of a plasmid is not correlated with ability to

catabolize nopaline because R-6 and R-30 were able to catabolize nopaline but there

were no visible bands in plasmid preparations of either isolate. The wide host range

mobilization plasmid, RP4 (described in Part A, Materials and Methods), was

introduced into both R-1 and R-25. Attempts were then made to mobilize the plasmids

of R-1 and R-25 into the plasmidless Agrobacterium strain, CIRS. The RP4 plasmid

was successfully transferred and maintained in C1RS but the plasmids from Rubus

isolates were not visible. When the plasmid, pDP35, carrying the cloned fragment of

Figure 5C. Scanning electron micrograph of Rubus

isolate R-25, prepared by critical pointdrying. Bar=2p^. -

Figure 6C. Agarose gel electrophoresis showing plasmids offermentative isolates from Rubus galls.

Lanes:

A. Rubus isolate R-30B. " ' R-25c. " " R-16D.''R-6E.*"R-1F. K188 (biovar 1 Aerobacterium)

Anow indicates chromosomal DNA.

AB CD E F

\r/

- - +..>

92

the AÊrobacteriumplasmid incompatibility (Inc) region (Part A, Materials and

Methods) was introduced into R-l and R-25, it coexisted with the resident plasmids

of those strains. These results indicate that the plasmids resident in the Rubus stains

do not code for nopaline catabolism, are not in the same incompatibility group as Ti-

plasmids nor are they stable in an Agrobacterium background.

93

PART C. DISCUSSION

Initially, because of their opine catabolism, lack of fluorescence on King's

medium B and their DNA melting points, the isolates from Rubus galls were thought

to be agrobacteria. However, thei¡ fermentative nature, carbon source utilization and

appearance under SEM confirms that they are not. Thei¡ genus was not determined

but it is apparent from the results obtained with bottr the Pseudomonas and the

fermentative isolates that there are genera other than Agrobacterium which are capable

of catabolizing opines. This confirms and extends the reports of other workers (Kohn

& Beiderbeckw L982; Beaulieu et al., 1983; Brisbane & Kerr, 1983; Rossignol &

Dion, 1985; Tremblay et al., 1987) The largest number of these reports involved

species of Pseudomonas. It is interesting that opine-catabolizing Pseudomonas were

isolated in this study from vineyards where crown gall had never been observed- It

should then follow that opines had never been present in that environment. This

observation is consistent with the report of Bouzar & Moore (1987) who isolated

large numbers of non-Agrobacterium opine-utilizing bacteria in a natural oak savanna

and tallgrass prairie. Beaulieu et al. (1986) indicated lhat there is very little or no

homology between opine catabolic genes in Pseudomonas and Agrobacterium. This

suggests that Pseudomonas opine catabolic genes have evolved separately, possibly

via a mutation in a previously existing degradative pathway. Because of the size of the

plasmids isolated from the fermentative strains found in the Rubus galls in this study,

it was hypothesized that these could be Ti-plasmids which had been picked up from

pathogenic agrobacteria in the galls. However, the plasmids do not code for nopaJine

catabolism nor are they in the same incompatibility group as Ti-plasmids. Unless

there is a significant amount of plant cell transformation by Agrobacterium which

does not result in gall formation but still results in the production of opines, it is

diff,rcult to see why such diverse opine catabolic systems might have evolved.

Because of the large number of non-Agrobacterium opine-catabolizing bacteria

present in the galls themselves, it is also interesting to speculate on the role of these

bacteria in the ecology of the gall. It is obvious that agrobacteria face much greater

94

competition for available opines than was previously thought. The large number of

Pseudomonas strains inhibitory to Agrobacterium present in vine galls suggests that

pseudomonads aggressively compete with agrobacteria via antibiosis. The

fermentative strains from Rubus were isolated from very old, decayed galls. It is

possible that these very rapidly-growing isolates succeed the more slowly-growing

agrobacteria in the galls. Experiments could be designed to follow the succession of

opine-utilizing bacteria in the galls- it seems possible that pseudomonads and other

opine-utilizing bacteria have a major role in the late stages of the ecology of the gall.

Certainly they appear to exist in much higher numbers in older galls than agrobacteria

themselves.

V/ith their ability to colonize both vines and vine galls, the Pseudomonas

isolates found in ttris study would make interesting candidates for biological control

of presently uncontrolled crown gall of grapevine and Rubus spp. This possibility is

more extensively discussed in the section on the Pseudomonas strains. At the very

least, the presence of non-Agrobacterium opine-catabolizing bacteria, especially in

non-opine environments leads to a quesúoning of the exclusive relationship benreen

opines and Agobacterium and speculation on the reasons for evolution of these

genes.

95

GENERAL DISCUSSION

Host-pathogen specificity and is genetic determinants arc central areas of

research in plant pathology. The advent of molecular biological techniques has led to

increased understanding in this a¡ea and the Agrobacterium system has been one of

the most intensively studied in this respect.

There are a number of stages in plant pathogenesis where specific interactions

can occur. The initial stages of the pathogenic process for a soil-borne pathogen such

as Aerobacterium are the attraction to the plant root (chemotaxis) and subsequent

colonization of the root system. The latter involves attachment, multiplication of

bacteria on the root surface and spread throughout the root system or to the rest of the

plant Once Agobacterium has become established, there a¡e a series of steps leading

to gall formation. These are relatively well-understood- The virulence genes, located

on the Ti-plasmid, are induced by plant exudates. Through a series of steps the T-

DNA is excised, transferred and incorporated into the plant genome where it is then

expressed. Specifrcity may occur at any of these stages- for example, host-specif,rc

bacteria may differ in the number of copies of the T-DNA transferred and in the nanre

of the T-DNA itself, especially with respect to the auxin and cytokinin genes

(Yanofsþ 9]!¿!., 1985a; 1985b). Host specificity in the latter stages of pathogenesis

has been studied more intensively by others (Yanofsky et al. 1985a; 1985b; Yanofsþ

& Nester, 1986). The primary focus of this work was to determine if host specif,rcity

exists in the early stages of the plant-bacterial interaction and, if so, which part of the

bacterial genome codes for this specificity.?ngrJ on ii,æ, lvvtcÈcl nttmþê.( ol s*c,;n9 .|eskd vna,t@-¡ l-he results obtained in Part A indicate that there n specificity which manifests

itself quantitatively in the bacteria s colonization of the host or qualitativety in the

pattern of colonization. The association between biovm 2 and stonefruit is an example

of the former and the association between biovar 3 and grapevine is an example of the

latter. In both cases, colonization appears to be completely determined by the bacterial

ch¡omosome and the Ti-plasmid has no discernible effect at this stage. Understanding

96

the difference between the ¡wo species is of importance when devising new control

strategies- either biological or cultural.

It is interesting that the more host-specific biovar 3 isolates systemically

colonize their hoss as do the Rubus cane gall isolates, (A.rubÐ described in Part B

(Hildebnand, 1940; McKeen, 1954). A. rubi is also very host-specific and has only

been isolated from Rubus spp. Although the phenotypic and genetic data presented in

Part B indicate that biovar 3 and A. rubi are separate species, they have obviously

evolved a similiar strategy in terrns of host colonization. Neither species survives well

in the soil and perhaps loss of saprophytic ability is the tradeoff for increased host

specificity in both cases. Virtually the entire life cycle of these bacteria is spent in

association with the plant host. In both species, motility appeared to be variable

between strains and those strains which were motile appeared to be less so than

strains of biovars 1 and 2. Decreased motility in biovar 3 and A. rubi may be a

reflection of the decreased importance of survival and dissemination in the soil for

these species. Neither of these pathogens are controlled by strain K84 and when

control measures are considered, the systemic nature of A.rubi and biovar 3 will be an

important factor to consider. It would be of great interest to look at the genes for

systemic enbry into the host for both biovar 3 and A. rubi and to determine the amount

of homology both structurally and functionally benveen them. If the genes are highly

homologous in terms of their DNA composition one would expect that they have

evolved from a cornmon ancestor but have different chromosomally-coded

determinants governing which host plant they can colonize.

V/ith respect to the early interaction with plant hostb, the only other studies

using Agrobacterium have been concerned with the process of bacterial-plant cell

attachment. There is evidence (Shaw et al., 1986 ) that agrobacteria are chemotactic to

wound exudates, specifically those that induce the vir genes, but, as yet, no

specificity between host and bacteria has been determined in this interaction. It is

possible that the separate Agrobacterium goups could respond to different levels of

the inducer or that there are separate inducing molecules for different groups. The

97

latter was recently suggested by Leroux et al. (1987) and is supported by evidence

that biova¡ 3 strains sha¡e a unique virA locus (Ma et al., 1987). It is interesting to

note the parallels between the virulence functions of A8robacterium and the

nodulation functions of Rhizobium. In the fast-growing rhizobia, the nodulation

(no{) genes are located on the large symbiotic (Sym) plasmids (Djordjevic eta!,

1986; Evans & Downie, 1986; Kondorosi & Kondorosi, 1986). Flavanoid

compounds present in plant root exudates induce the expression of nod genes @eters

et al., 1986; Redmond et al., 1986). NodD is constituitively expressed and acts as a

positive regulator of the expression of the remaining nod genes (Mulligan & Long,

1985; Rossen e1!3!., 1985). Recent work has shown that the nodD gene products

from rhizobia with different host specificities react with different sensitivity to plant

exudates (Horvath et al., 1987; Spaink g1¡¿!, 1987). Wide host range rhizobia

recognize a broader set of plant exudates than the more limited host range strains. In

this sense, the nodD and virA loci appear to be functionally analogous. If virA plays a

comparable role in determining Agrobacterium host specifrcity, it would be interesting

to examine the range of compounds which induce the vir genes of strains from

grapevine and Rubus spp.

There is some confusion in the literature on the process of attachment.

Agrobacteria attach specifically to wound sites ( Lippincott & Lippincott, 1969;

Glogowski & Galksy, 1978) within minutes of inoculation (Matthyse g]!3!., 1981).

Agrobacteria appeu to cluster at wound sites where they form cellulose frbrils

(Matthyse et al., 1981), which are important in anchoring bacteria to plant cells and to

each other. These fibrils do not appeff to determine attachment as mutants retain

virulence and attachment ability (Matthyse, 1983). There is evidence that a

lipopolysaccharide (LPS) in the bacterial outer membrane (Whatley et al., L976) binds

with pectic fragments in the plant cell wall (Lippincott et al., 1977). Although there is

some evidence that the Ti-plasmid could alter the bacterial LPS (Lippincott et al.,

1978) and binding (Matthyse et al., 1978), most of the evidence suggests that

chromosomal genes a¡e involved in binding between plant and bacteria (Lippincott &

98

Lippincott, t969; \ilhatley e!._al, L976; Ma¡ton et al., L979; Douglas g]!¿!., 1982;

Draper et al., 1983; Douglas et al., 1985). Mutants in chvB, one of the two

chromosomal loci identifred GhvA and ghvB), have been shown to lack flagella

(Douglas et a1., 1985). There has been no evidence for specificity in the attachment

process to date.

This study, however, indicates that there is specifrcity coded for by the

bacterial chromosome in its early colonization of the root. This may be due to plant

root exudates, produced either by the root system as a whole or at a particular site e.g.

a wound or lenticel. The response of the bacteria may be not only to the qualiutive

natue of the exudate but also to the relative quantities produced. Multiplication of

bacteria on the plant root system is almost certainly a response to root exudates and is

likely to be different for different species. Entry into the plant host is important for A.

rubi and biovar 3 strains and appears to be chromosomally coded in the bacteria; on

grapevine systemic enury may be related to the abitity of biovar 3 strains to form root

lesions (Burr e]!3l., 1986b). Plant exudates may play a role in selecting for these host-

specifrc forms but little is known in this area. Lippincott and Lippincon (1969)

showed that addition of growth factors increased tumorigenicity of A. rubi strains

TR2 and TR3 but whether Rubus spp. produce these factors or whether this effect

occurs in vivo is unknown.

Host specificity after artificial inoculation of the plant appears to be largely Ti-

plasmid encoded (Loper & Kado, 1979; Thomashow et al., 1980; Knauf et al. , t982;

Unger et al., 1985), although there may be genes on the bacterial chromosome which

affect host range (Ga¡finkel & Nester, 1980). Tiplasmid encoded host specifrcity

was more extensively discussed both in the General Introduction and Part A

Discussion.

A compounding factor in the association of different Agrobacterium species

with particular host plants is the apparently specific association between Ti-plasmids

and their chromosomal backgrounds. This was observed in vivo in this study @art A)

when some trÍmsconjugant strains lost the introduced Ti-plasmid. In nature, the

99

specificity in early colonization and attachment which is largely chromosomally coded

appears also to be associated with specific Ti-plasmids. The Ti-plasmid carries genes

(cytokinin biosynthesis and vir genes) which enable it to be pathogenic on the specifrc

plant host. This parallels the sitr¡ation in the taxonomically similiar Rhizobium (Detæy

et al., 1966) to a certain extent. Root colonization by rhizobia appears to be

completely under chromosomal control (Brewin g!_ú, 1983) and the large S¡'rn

plasmids present in many Rhizobium spp. (Brewin et al., 1980; Hooykaas et al.,

1981) carry the genes which induce the symbiotic interaction and code for host range.

Wang et al., (1986) also suggest that there is plant host selection for certain plasmids

suggesting that in Rhizobium as well there is a close interrelationship benveen

plasmid, bacterial chromosome and the plant host.fuær) o¡ +V¡e- tr,'nrkd n*ttfu.r^ of eiuains +esþ¿t

'fhe data presented in the gall colonization studies in Part A of this thesis give pret.rnar/arj

evidence that, once tumour formation has occurred, the Ti-plasmid plays the primary

role in determining the level of colonization achieved on the galls. This is at least

preliminary evidence in support of the opine concept. The ability to utilize nopaline in

galls does give nopaline-catabolizing bacteria an advantage in that environment. There

are opines produced in Rhizobium nodules (Tempé & Petit, 1983) and the genes for

their synthesis and catabolism are closely linked and located on the Sym plasmid

(Murphy e][3l., 1987). If these opines also act to give catabolizing rhizobia a selective

advantage then presumably the catabolic genes could be cloned into an 'ecologically

competent'rhizobium enabling it to maintain a higher population after nodule

formation. Unless plant roots are engineered to produce opines it is difficult to see

how the advantage that they provide to agrobacteria could be utilized on apractical

level. It is also important to realize that the initial root colonization and colonization of

the ungalled parts of the root system are still determined by the bacterial chromosome.

Also, opines are not providing a selective advantage to agrobacteria alone but also to

other bacterial genera (Pat C) which can catabolize opines and aggressively colonize

galls and could presumably do the same on opine-containing roots.

100

It is yet to be shown experimentally that the conjugative opines give

agrobacteria an advantage either nutritionally or by promoting Ti-plasmid transfer. In

fact the importance and amount of plasmid transfer in vivo between pathogenic and

plasmidless strains is unknown. There are large numbers of non-pathogenic

agrobacteria in galls and in the soil around galled plants (Kerr, 1969; Schroth et a1.,

L97l; New, 1972) but their role in the infection process and as plasmid recipients has

not been studied.

The study of Bouzar & Moore (1987) indicates that non-pathogenic

agrobacteria ate present in relatively high numbers in soils where there has been no

crown gall and where there are few natural hosts. They are certainly capable of

survival in soil for long periods and are considered'normal rhizosphere inhabitants'

(Schroth e.!¿!., l97l). The Ti-plasmid plays a major role in determining bacterial

levels on the gall (Part A) and so this author agrees with Tempé and others ( Petit et

al.,1978a; Tempé et al., 1979; Guyon et a1.,1980) in the assertion that the opine-

related functions are the'raison d'etre' for the Ti-plasmid- Ti-plasmids certainly seem

to provide no obvious benefit to the bacteria in their survival and multiplication when

galls are not present but they do appear to be c,- determinant of survival onaf lects* tn )¡e((wÐ c>I lVteC alor ti! lo ca)uboLtzÊ. o?ne3.

the galls that they induce¡,In this respect they support the assertion of Sherrat (1982)

that plasmids 'provide non-essential, ephemerally useful functions that allow a

bacterium to occupy a particular ecological niche'. Shenat goes on to suggest that if a

plasmid was coding for traits that were continuously essential for bacterial survival itwould be integrated into the chromosome. So the Ti-plasmids are essential only to

induce gall formation and colonization and not throughout the rest of the bacterial life

cycle.

Once the gall has begutt to decay presumably the chromosomal genes alone are

important in terms of survival. Certainly the different AÊrobacterium species seem to

have evolved quite separately in this respect. Biovars I and2 are both capable of

surviving for long periods in the soil (Patel,1928; 1929; Hildebrand,lg4l; Dickey,

1961; Schroth et al., l97l: New, 1972) but both biovar 3 ( Part A) and A. rubi

(Hildebrand, 1940) do not maintain high populations in soil and systemic movement

into the plant host appears to be of central importance in their survival and

dissemination.

The different chromosomal forms or biovars of Agrobacterium are

taxonomically quite separate with less than25%o DNA homology between them and

they warrant being named as separate species. This and resulting suggestions for

changes to the taxonomy are discussed in Part B. Biovar I (4. tumçfagigns) is not

host-specific and is a very heterogenous group (Kersters & Deley, 1984). Biovar 2 (

A. rhizogenes) is more host-specific in that it is found in association with stonefruit

and rose (New,1972) and appears to be able to colonize its host at an exceptionally

high level (Part A). Biovar 3 and A. rubi are confined virtually exclusively to

grapevine and Rubus respectively. V/ide and na¡row host range Ti-plasmids are also

genetically distinct with only low DNA homology between them (Sciaky et a1., 1978;

Thomashow et al., 1981). Increased host specificity and a closer host association, as

séen with those species which survive systemically in the host, appear to go hand in

hand. Ti-plasmid and chromosome may have co-evolvedin the presence of the host

resulting in greater divergence of the Ti-plasmids and increased speciation.

Further work is needed to pinpoint the exact nature of chromosomally-coded

host specifrcity and much work remains to be done on the role of plant root exudates

in determining both the specificity and level of colonization. It would also be

interesting to look at root colonization in more detail. Bhuvaneswa¡i et al. (1980)

showed that infection sites for Rhizobium are localized to areas just behind the root

tþ. Sites for adherence for Rhizobium and Agrobacterium may be similiar as

suggested by work (Lippincott & Lippincoft,1977) which indicates that R.

leguminosarum competes with Aerobacterium for binding sites on isolated bean leaf

cell walls. Studies could be designed to look at this very localized colonization using

scanning electron microscopy. It would be of particula¡ interest to look atrelative

colonization levels of wound sites and lenticels for biovars 2 and 3 on their respective

hosts. Galls are largely formed on almond seedlings at the point where lateral roots

t02

emerge from the main taproot (Kerr, L972). Colonization patterns in these sites may

be quite different from the rest of the root system. There is also much to be done on

the nature of the specific interactions not only between the Ti-plasmid and the plant

host but also between the Ti-plasmid and the bacterial chromosome. It is probably too

simplistic to think that only one component of the system is of ultimate importance in

deterrnining host specificity. It is more likely that there is a series of messages back

and forth between the host, bacterial chromosome and the Ti-plasmiì and each step in

the pathogenic process involves selection and specifrcity ultimately resulting in the

production of a crown gall tumour.

103

APPENDD( A: CULTURE MEDIA

1. Yeast extract agar @odriguez & Tait, 1983)

nutrientbroth (Difco).yeast exEactsucroseMgSO4.7H2OBacto agardistilled water

2. TY medium (Beringer, 1974\

3. Yeastmannitol agar

KZHPO¿MgSO¿.7HzONaClCaCl2FeCl3yeast exEactmannitolBacto agardistilled water

L3.391.og5.og

0.24g15g

to 1lite

5g3g

1.3e15g

to 1 litre

1.oglog15g

to 1 litre

o.5g

0.01g

104

4. Petit's medium (modified from Petit & Tempé, 1978b)

Agar:

Solution 1:

Solution 2:

purifieddistilled

agarwater

2oe500 ml

1ml1ml

o.2e1ml

to 400 ml

10.5g4-5e

100 ml

CaCI2 (17o soln)FeSO4 (0.57o soln)MgSO4. THzOMnCIZ (0.2Vo soln)distilled water

KZHPO+KH2PO4distilled water

Na2HPO4. L2IJ1OMgSO4. lHzOCaCl2FeCl3thiaminebiotindistilled water

0.45go.1g

0.04g0.02g

1.0 mg0.25 mgto l lire

(pH to 7.0)

For 100 ml: add 40 ml Solution 1 and 10 ml Solution 2to 50ml agar

5. Bergersen's salts (Bergersen, 196l; modified by J. Tempé)

The salt solutions are stored as 5x concentrate. Yeast extract(100 mg,Â) may be used in place of thiamine and biotin.

105

lactoseye¡rst exEactBacto agardistilled water

7. Aniline blue medium (Riker et al., 1930)

L-glutamic acidmannitolMgSO4.7H2OKZFIPO¿NaClCaCDaniline blue*yeast extractBacto agardistilled water

*' - grind in pestle and mortar with 1 to 2 ml water

8. Stonier's medium (Stonier, 1960)

potassium citrateNH¿NO¡L- glutamic acidKZHPO¿NaH2P04MgSO¿.7HZONaClCaSO¿Fe (NO3)3MnCIZZnCI2biotinBacto agardistilled water

log1g

2oeto 1 litre

2g5.ogo.2eo.2eo.2eo.1go.1g1og15g

to 1 litre(pH to 7.0)

log2.7e2.oe

0.88go.3go.2eo.2eo.1g

5.0 mg0.1 mg0.5 mg

o.2e15g

to l lite(pH to 7.0)

106

9. Mannitol- slutamate broth

mannitolLglutamicK2HPO4MgSO4.7H2ONaClbiotindistilled water

L-a¡abitolNH¿NO¡KHzPO¿KzHPO+MgSO4.7H2Osodium taurocholatecrystal violet (0.17o)Bacto agardistilted water

erythritolNH¿NO¡KHZPO¿KZHPO¿MgSO4.'7H2Osodium taurocholateyeast exEact (l%o)malachite green (O.lEo)Bacto agardistilled water

Per 100 ml add:ac¡dione(2Vo)Na2SeO3. 5HZ0 (lVo)

acidlog2g

o.2eo.2e

200 mgto 1 lire

(pH to 7.0)

3.O4g0.1690.54g1.04g0.25g0.29g

2rrd,15g

to 1 lire

3.05g0.1690.54gr.04s0.259O.29g

lmt5ml15g

to 1 litre

1 rnl1 rnl

o.5g

10. Selective media for Agrobacterium biova¡s 1.2 and 3

a) Selective medium for biovar 1 ( Kerr & Brisbane, 1983)

Per 100 ml add:acÞrdtone(2Vo)Na2SeO3. 5}l2O (l7o)

1ml1ml

b) Selective medium for biovar 2 (Ken & Brisbane, 1983)

t07

c) Selective medium for biovar 3 (Kerr & Brisbane, 1983)

A. sodium tarEateD- gluømic acid*NaH2PO4.2H2ONa2FIPO¿NaClMgSO¿. 7It2Osodium taurocholateyeast extract(lVo)Congo Red (1%)distilled water

acndtone (ZVo)Na2SeO3. 5HZO (l%o)

5.75g15 ml6.25g4.2695.8490.25g0.29g

1mt2.5 ml

to 500 ml

* - 47o solution, pH 7.0

B. MnSO4. 4HZOBacto agardistilled water

Autoclave separately. Add 50 rnl (A) to 50 ml (B) and add:

to

2g5gml

1.1I

s00

1ml0.5 ml

d) Selective medium for biovar 3 (RS Medium)(Roy & Sasser, 1983)

MgSO4. TIIZOK2I7PO4KHZPO¿adonitolyeast extractNaClboric acidBacto agar

o.2eo.9go.7e4.og

0.1490.20g

1.og15g

(p}Jto7.2)

108

Add, after dissolving each in 2 ml water:

triphenyl tetrazolium chlorideD- cycloserinetrimethoprim*

* - add 1 drop dilute acid and heat to dissolve

11. Indicator medium (to test carbon source utilization)(modified from Hayward, 1964)

.o8g

.o2e

.o2g

12. Media used to test tartrate utilization

a) Medium 1

5x Bergersen's saltsbromothymol blue*biotin (100 mg/100 ml)nicotinic acid (2 mg/ 100 ml)calcium pantothenate

(2 mel 100 ml)L- glutamic acidBacto agardistilled water

(NII¿)zSO¿KZHPO¿KHZPO+NaClpotassium sodium tartrateyeast extractbromothymol blue (see above)agardistilled water

200 ml40 rnl2rnl

10 ml

10 rnl)a

1.5g750 ml

( pH to 7.0)

* - grind 0.2 g bromothymol blue with 40 rnl 0.01N NaOH in amortil and pestle. Add 60 ml distilled water.

Sterilize carbon sources (107o solutions) and add to a finalconcentrationof LVo in indicator medium.

2go.6go.4g

2g3g

o.1g20 rnl

1.5gto 1 litre

(pH to 7.0)

109

Sterilize sodium tartrate as l07o solution; add to medium 2to a final concentration of l7o in the basal medium.

b) Medium 2 (Ayers g!-al, 1919)NH¿HZPO¿KCIMgSO4. TLrzObromothymol blue (powder)distilted water

13. King's Medium B (King et al.. 1954)

Difco proteose peptone No. 3KZHPO¿MgSO+.7HzOglycerolBacto agardistilled water

14. Pseudomonas minimal medium (PMM)

Nt{¿HZPO¿KCIMgSO4. 7H2OBacto agardistilled water

To 90 ml basal medium, add 10 ml2Vo glucose.

1.ogo.2eo-2e

0.08gto 1 litre

(pH to 7.0)

1.ogo-2eo-2gL2g

to 900 ml( pH to 7.0)

2ogo.2e1.5g

10 ml15g

to l lire( pH to 7.2)

110

APPENDIX B. BUFFERS AND SOLUTIONS

1. Solutions for olasmid isolation and gel electrophoresis

a) TEB buffer: 50 mM Tris-HCl,20 mM EDTA, pH 8.0

e) Electrophoresis buffer: Tris (SigmaT-9)boric aciddisodium EDTAdistilled water

b) Solution 1: 50 mM glucose,25 mM Tris, 10 mM EDTA, pH 8.0.Lysozyme (2mglrnl) added just before use. Kept on ice.

c) Solution 2: 0.2 N NaOH, 17o SDS; made just before use.

d) Sodium acetate (3M): Dissolve 24.6 g sodium acetate in 50 mldistilled twater. Add glacial acetic acid to pH 5.2. Adddistilled water to 100 ml.

10.78 g5.5 g0.93 g

to 1 litre

f¡ Tracking d)¡e:2O7o Ficoll, 0.17o SDS, 0.02Vo bromophenol blue inelectrophoresis buffer.

g) Gel: Agarose (Seakem; 0.7 to 1.0 g) melted in 100 ml electrophoresisbuffer; cooled to 60oC and poured.

2. Solutions for DNA preparation and reassociation (Part B)

a) Saline sodium citrate (SSC) (Maniatis et al., 1982)

t-lre following recipe is for a stock solution of 20 x SSC:

NaCl 175.3 gsodiumcirate 88.2 gdistilled water to 1 lire

(pH to 7.0)

b) Proteinase K: A stock solution of proteinase K (from Tritirachiumalbum Type XI; Sigma Chemical Company, USA) was madein TNE buffer (described below) at a concentration of 5mg/ml, just before use.

c) TNE buffer (Maniatis etal., 7982): 10 mM Tris-HCl, 100 mM NaCl,1 mM EDTA, pH 8.0.

d) RibonucleaseRNase stock solution: RNase (Calbiochem, USA) at a

concentration of 5 mg/rnl was dissolved in 10mM Tris-HCl (pH 7.4),15 mMNaCl, 5Vo glycerol. The solution was heated ina boiling water bath for 20 minutes and allowedto cool before storage at -20oC.

e) Chloroform: a24:l mixture of chloroform: isoamyl alcohol was used.

111

3. Miscellaneous solutions

a) Benedict's reagant (for test of 3- ketolactose production)

Solution A: sodium citrate L7.3 gsodiumcarbonate f0 g

Dissolve in 60 ml distilled water by heating. Make up to 85 ml.

Solution B: CuSO4 1.8 gdistilled water to 15 ml

Slowly add solution B to solution A.

b) Buffered saline

For 200 mll. 0.2 M phosphate buffer (see below), 20 ml;NaCl,l.7 g; distilled \ryater, 180 ml.

0.2 M Phosphate buffer pH 7.3 (10 x conc.):

0.2 M Na2HPO40.2 M NaHZPO¿

81 ml19 rnl

tt2

APPENDD( C

The tables presented in this appendix represent data which has been

presented graphically in Part A of this thesis. The graph to which an

individual table refers is indicated.

Table AC-1. Root Populations: Almond Ex. 1 I

Month K27ß\2

Log cfu/ cm2 root

Treatments:3

K309(s) 2 K27(m\2

3.81 c 6.19 a

4.95b 5.57 a

3.80 c 6.42a

4.60 b 6.t4a

4.85 b 5.94a

3.68 b 5.02a

4.00 a 4.L6a

3.07 b 4.43 a

2.59 a 2.gg a

2.65 c 4.43 a

2.57 b 3.59 a

z.fib 3.54 a

June

July

August

September

October

November

December

fanuary

February

March

April

May

6.58 a

5.84 a

6.2L a

634a

6.23 a

5.14a

3.89 b

3.84a

3.07 a

4.09 aþ

3.86 a

3.50 a

K309(m) 2

5.57 b

5.00 b

5.02b

4.18 b

4.10 b

4.95 a

4.00 a

2.95b

2.9I a

3.20b,c

3.00 b

2.88 b

1- Almond Ex. 1 is described in Table 34. Procedures for sampling are described inPart A, Materials and Methods.

2- (Ð = single inoculations; data presented graphically in Fig. 2A(m) = mixed inoculations; data not presented elsewhere

3- Values with the same_letter are not significantly different. Data were analysed byone-way ANOVA and the least squares difference was used to determine diiferencesbetween means at each month.

Table AC-z. Root Populations: Vine Ex. 1l

I-og cfu/ cm2 root

Month

September

October

November

December

January

February

March

April

May

June

July

August

K27(.s)2

5.73 a

4.96 ^

4.94a

5.08 a

4.90 a

4.97 a

4.2I a

3.82a

3.56 a

2.91a

3.06 a

3.52a

Treatments: 3

K309(s) 2

4.49b

4.25 b

3.70 b

2.9L c

4.70 a

4.48 a

4.07 a

4.46 a

332a

3.00 a

3.61a

3.O7 a

K27în) 2

5.44 a

4.4r b

4.78 a

4.50 b

3.92 a

4.79 a

4.79 à

4.18 a

3-81 a

2.gg a

2.gl a

3.43 a

K309(m) 2

4.50 b

4.2tb

3.7tb

3.11 c

4.26a

4.73 a

434 a

4.00 a

3.22a

2.gg a

334 a

2.52 a

1- Vine Ex. 1 is described in Table 34. Procedures for sampling are described in PartA, Materials and Methods.

2- dataforK2T and K309 in single inoculations arc presonted graphically inFigure 34.

3- Values with the same letter are not significantly different. Data were analysed byone-rway ANOVA and the least squares difference was used to determine differencesbetween means at each month.

Table AC-3. Root Populations: Almond Ex. 2 I

Month K57

Log cfu/ cm2 rcg!

Treatments:2

K27 K57(pTiK27)

October

November

December

January.,

February

6.26a

5.99 ^

5.66 a

3.63 b

4.40 ^

5.53 a

5.67 a

5.50 a

4.51a

4.19 a

6.11 a

6.24î

5.40 a

3.45b

3.70b

K103

4.00 b

4.93b

4.03 b

2.90b

3.04 b

1- Almond Ex.2 is described in Table 3,A'. Procedures for sampling a¡e described inPart A, Materials and Methods. Data are presented graphically in Figure 94.

2- Values with the same letter Íre not significantly different. Data were analysed withone-way ANOVA and the least squares difference was used to determine differencesbetween means at each month.

Table AC-4. Gall Populations: Almond Ex. 3 1

Month

April

May

June

July

August

ii) Inside Galls:

Month

Ap.il

May

June

July

August

K27 3

5.48 a

6.96a

7.08 a

6.29 a

6.59 a

Iog cfu/cm2 g4![

Treaûnents:2

K377

5.86 a

735a

4.90 b

4.23b

4.05 b

K309

6.12a

4.69b

4.48b

4.7tb

436b

K27 3

4.54a

6.55 a

7.43 ^

7.I5 a

7.29 a

K309

2.00 b

4.79b

5.55 b

5.77 b

4.56b

Log cfu/ cm3 41K377

4.90a

6.28a

6.71a'b

5.45 b

3.88 b

1- Almond Ex. 3 is described in Table 34. Data are presented graphically inFigure 124.

2- Values with the same letter are not significantly different. Data were analysed byone-way ANOVA and the least squares fference was used to detemrine differencesbetween means at each month.

3- There were no signifrcant differences between populations of K27 in treaments A,B and C so only data f¡om treatnent A is shown.

113

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