UBC Theses and Dissertations

UBC Theses Logo

UBC Theses and Dissertations

Gall formation by Erwinia species on Douglas-fir DeYoung, Robyn Merrilee 1990-12-31

You don't seem to have a PDF reader installed, try download the pdf

Item Metadata

Download

Media
UBC_1990_A6_7 D49.pdf [ 11.8MB ]
[if-you-see-this-DO-NOT-CLICK]
Metadata
JSON: 1.0098122.json
JSON-LD: 1.0098122+ld.json
RDF/XML (Pretty): 1.0098122.xml
RDF/JSON: 1.0098122+rdf.json
Turtle: 1.0098122+rdf-turtle.txt
N-Triples: 1.0098122+rdf-ntriples.txt
Original Record: 1.0098122 +original-record.json
Full Text
1.0098122.txt
Citation
1.0098122.ris

Full Text

GALL FORMATION BY EFWINIA SPECIES ON DOUGLAS-FIR  BY ROBYN MERRILEE DEYOUNG B . S c . ( A g r . ) , U n i v e r s i t y o f B r i t i s h Columbia, 1985  A THESIS SUBMITTED IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF MASTER OF SCIENCE  in  THE FACULTY OF GRADUATE STUDIES (Department o f P l a n t S c i e n c e )  We a c c e p t t h i s t h e s i s as c o n f o r m i n g to the required standard  THE UNIVERSITY OF BRITISH COLUMBIA A p r i l 1990 (k) Robyn M e r r i l e e DeYoung  In  presenting  this  degree at the  thesis  in  University of  partial  fulfilment  of  of  department  this or  thesis for by  his  or  requirements  British Columbia, I agree that the  freely available for reference and study. I further copying  the  representatives.  an advanced  Library shall make it  agree that permission for extensive  scholarly purposes may be her  for  It  is  granted  by the  understood  that  head of copying  my or  publication of this thesis for financial gain shall not be allowed without my written permission.  Department  of  if*((tH £ Sc >&yr CZ,  The University of British Columbia Vancouver, Canada  DE-6 (2/88)  ABSTRACT  Bacterial  g a l l s on D o u g l a s - f i r  [Mirb.] Franco),  (Pseudotsuga  menzeisii  c o l l e c t e d from t h e s o u t h e r n t i p o f Vancouver  I s l a n d , the Greater  Vancouver a r e a and t h e Hope r e g i o n o f  B r i t i s h Columbia, were g e n e r a l l y g l o b o s e i n shape w i t h rough, irregular diameter.  s u r f a c e s and measured between 0.5 and 2.0 cm i n The g a l l s were g e n e r a l l y l o c a t e d on t h e t i p s o f  b r a n c h e s o r t w i g s o f 10- t o 20-year o l d D o u g l a s - f i r t r e e s . The  b a c t e r i a l g a l l d i s e a s e appeared t o a f f e c t  few Douglas-  f i r t r e e s i n t h e c o l l e c t i o n a r e a s and b a c t e r i a l g a l l s were n o t found on any o t h e r c o n i f e r o u s s p e c i e s .  F u r t h e r m o r e , t h e r e have  been no r e p o r t s o f s e r i o u s damage t o n a t u r a l f o r e s t s i n B r i t i s h Columbia due t o b a c t e r i a l g a l l d i s e a s e .  Young, greenhouse-grown  D o u g l a s - f i r s e e d l i n g s o c c a s i o n a l l y d i e d i f t h e t i p o f t h e main stem was a r t i f i c i a l l y  inoculated.  O f t e n new g r o w i n g t i p s would  be produced a f f e c t i n g  t h e growth form o f t h e s e e d l i n g s .  Two t y p e s o f g a l l - f o r m i n g Erwinia Douglas-fir galls.  spp. were i s o l a t e d  Typical isolates, tentatively  f a t t y a c i d a n a l y s i s as Erwinia were rough and i r r e g u l a r  salicis,  i d e n t i f i e d by  produced g a l l s w h i c h  i n shape composed o f m u l t i p l e  o u t g r o w t h s marked by a s i n g l e o r c r o s s - s h a p e d f i s s u r e . atypical as Erwinia  isolate, tentatively herbicola  from  The  i d e n t i f i e d by f a t t y a c i d a n a l y s i s  subsp. herbicola,  produced g a l l s w h i c h were  smooth and g e n e r a l l y round i n shape w i t h t h e s u r f a c e c r a c k i n g as t h e g a l l expanded. Colonies  o f t h e t y p i c a l i s o l a t e s grown on c a s e i n - p e p t o n e -  g l u c o s e media were c h a r a c t e r i s t i c a l l y round, s l i g h t l y domed w i t h  iii somewhat c o n c e n t r i c r i d g i n g observed near t h e m a r g i n s o f t h e colonies.  Three t o 4 day o l d c o l o n i e s of t h e a t y p i c a l  isolates  grown on c a s e i n - p e p t o n e - g l u c o s e media were c h a r a c t e r i s t i c a l l y round and concave w h i l e o l d e r c o l o n i e s produced s l i m e and were more i r r e g u l a r i n shape.  an  extracellular  In L u r i a Broth, the  t y p i c a l i s o l a t e s grew a t temperatures  of up t o 32°C w h i l e t h e  a t y p i c a l i s o l a t e grew a t temperatures  of up 34°C.  i s o l a t e was  The  typical  r e s i s t a n t t o a w i d e r range o f a n t i b i o t i c s t h a n t h e  atypical isolate. Polyclonal  a n t i s e r a were produced  against glutaraldehyde-  f i x e d whole c e l l s o f b o t h t h e t y p i c a l T-2789 and a t y p i c a l A-0181 g a l l - f o r m i n g Erwinia  isolates.  The p u r i f i e d a n t i s e r a  i s o l a t e s p e c i f i c as t e s t e d by i m m u n o d i f f u s i o n and an ELISA a g a i n s t s e v e r a l d i f f e r e n t p h y t o p a t h o g e n i c including  Pseudomonas syringae  subsp. herbicola, leguminosarum  Agrobacterium  and Erwinia  pv. syringae,  indirect  bacteria  Erwinia  tumefaciens,  carotovora  were  herbicola  Rhizobium  subsp.  P l a s m i d p r o f i l e s o f t h e t y p i c a l Erwinia  carotovora. i s o l a t e s contained  one band w h i l e t h e a t y p i c a l i s o l a t e c h a r a c t e r i s t i c a l l y c o n t a i n e d 4 t o 5 bands which appeared t o be d i f f e r e n t forms of a t l e a s t one p l a s m i d . suggested  R e s t r i c t i o n d i g e s t s of the t y p i c a l  isolates  a s i z e o f a p p r o x i m a t e l y 50 kb w h i l e complex  digestion  p r o f i l e s were o b t a i n e d f o r t h e a t y p i c a l i s o l a t e s because o f t h e d i f f i c u l t y i n i s o l a t i n g i n d i v i d u a l plasmid types. estimates against Hindlll-digested 10 and 20 kb was  lambda DNA,  From v i s u a l  a s i z e o f between  suggested f o r t h e f a s t e s t moving p l a s m i d band  of t h e a t y p i c a l i s o l a t e .  No homology was  observed between t h e  d i f f e r e n t p l a s m i d t y p e s c h a r a c t e r i s t i c o f t h e two i s o l a t e s .  The  iv r o l e o f t h e p l a s m i d DNA  of t h e a t y p i c a l i s o l a t e i n p a t h o g e n e s i s  was n o t d e t e r m i n e d because c u r i n g of t h e p l a s m i d ( s ) was  not  s u c c e s s f u l u s i n g h i g h temperature t r e a t m e n t s p l u s c h e m i c a l c u r i n g agents. Heat t r e a t m e n t e x p e r i m e n t s , i n which t h e pathogen s e l e c t i v e l y k i l l e d at v a r i o u s times a f t e r demonstrated  was  inoculation,  t h a t t h e b a c t e r i a a r e r e q u i r e d t o be p r e s e n t f o r  g a l l i n d u c t i o n and c o n t i n u e d development o f t h e g a l l f o r b o t h of t h e g a l l - f o r m i n g Erwinia  i s o l a t e types.  P a t h o g e n i c i t y o f t h e i s o l a t e d b a c t e r i a was t e s t e d on 14 c o n i f e r s p e c i e s , other than D o u g l a s - f i r , i n c l u d i n g Chamaecyparis,  Pinus  and Thuja spp.  w e a k l y p a t h o g e n i c on Abies,  Larix  The t y p i c a l i s o l a t e s were  and Picea  i s o l a t e was weakly p a t h o g e n i c on Abies, Picea  and Pinus  spp.  Abies,  spp.  The  Chamaecyparis,  atypical Larix,  Due t o t h e l i m i t e d damage caused on t h e  c o n i f e r s t e s t e d and t o t h e i r i n f r e q u e n t o c c u r r e n c e , t h e s e g a l l f o r m i n g pathogens the f o r e s t r y  do not appear t o be of economic i m p o r t a n c e t o  industry.  V  TABLE OF CONTENTS Page  T i t l e Page  i  Authorization  i  Abstract  i i  Table o f Contents L i s t of Tables  v v i i  L i s t of Figures  ix  Acknowledgements  xi  General Introduction  1  Chapter l  I s o l a t i o n , D e s c r i p t i o n and S e r o l o g i c a l D e t e c t i o n of t h e Douglas-Fir Gall-Forming B a c t e r i a Introduction  8  M a t e r i a l s and Methods A. B a c t e r i a l s t r a i n s and growth c o n d i t i o n s B. P l a n t m a t e r i a l s C. D i r e c t i s o l a t i o n s / i n o c u l a t i o n s D. P o l y c l o n a l a n t i s e r a p r o d u c t i o n . E. IgG p u r i f i c a t i o n F. A n t i s e r a s e n s i t i v i t y G. S p e c i f i c i t y : immunodiffusion H. S p e c i f i c i t y : i n d i r e c t ELISA I . Host range  14 14 16 16 17 18 18 19 20 20  Results A. B. C. D. E.  22 22 32 32 36 40  D e s c r i p t i o n o f g a l l s and i s o l a t e s Antisera sensitivity Specificity: immunodiffusion Specificity: i n d i r e c t ELISA Host range  Discussion  43  vi TABLE OF CONTENTS (Cont.) Page Chapter 2  The Mechanism o f G a l l Formation by Erwinia on D o u g l a s - F i r  spp.  Introduction  52  M a t e r i a l s and Methods A. B a c t e r i a l s t r a i n s and h o s t p l a n t s B. Inoculations/heat treatment C. Growth i n d e x D. D i r e c t i s o l a t i o n s / c e l l ELISA E. S t a t i s t i c a l analysis  54 54 54 55 55 56  Results A. B.  57 57 61  T-2789 i s o l a t e A-0181 i s o l a t e  Discussion  66  Chapter 3  The R e l a t i o n s h i p between t h e P l a s m i d Content o f t h e G a l l - f o r m i n g Erwinia I s o l a t e s and P a t h o g e n e s i s Introduction  73  M a t e r i a l s and Methods A. B a c t e r i a l s t r a i n s B. T o t a l DNA i s o l a t i o n C. P l a s m i d i s o l a t i o n and p u r i f i c a t i o n D. S o u t h e r n b l o t s E. Curing protocols F. Colony l i f t s G. P labelling H. H y b r i d i z a t i o n  76 76 76 77 79 80 82 82 83  Results A. C. B.  85 85 89 94  32  Plasmid content Probe s p e c i f i c i t y / h o m o l o g y Curing/colony h y b r i d i z a t i o n  Discussion  97  General D i s c u s s i o n  102  References  108  vii LIST OF TABLES Chapter 1  T a b l e 1.1  T a b l e 1.2 T a b l e 1.3  T a b l e 1.4  T a b l e 1.5 T a b l e 1.6  Page  G a l l - f o r m i n g Erwinia i s o l a t e s and b a c t e r i a l s p e c i e s used t o t e s t t h e s p e c i f i c i t y o f polyclonal antisera  15  Temperature s e n s i t i v i t y o f t h e g a l l - f o r m i n g Erwinia isolates  30  A n t i b i o t i c r e s i s t a n c e s e x p r e s s e d by r e p r e s e n t a t i v e t y p i c a l and a t y p i c a l i s o l a t e s of t h e D o u g l a s - f i r g a l l - f o r m i n g b a c t e r i u m  31  Tentative i d e n t i f i c a t i o n s of Douglas-fir g a l l - f o r m i n g b a c t e r i a l i s o l a t e s by a n a l y s i s o f t h e i r f a t t y a c i d p r o f i l e s and computera s s i s t e d comparison w i t h a l i b r a r y o f p r o f i l e s from a u t h e n t i c s t r a i n s  33  S p e c i f i c i t y o f whole Erwinia i n an i n d i r e c t ELISA  37  T-2789 a n t i s e r u m  S p e c i f i c i t y o f t h e p u r i f i e d IgG f r a c t i o n o f t h e Erwinia T-2789 a n t i s e r u m a t a d i l u t i o n o f 10" i n an i n d i r e c t ELISA  38  S p e c i f i c i t y o f t h e p u r i f i e d IgG f r a c t i o n o f t h e Erwinia A-0181 a n t i s e r u m a t a d i l u t i o n o f 10" i n an i n d i r e c t ELISA  39  P a t h o g e n i c i t y o f g a l l - f o r m i n g Erwinia on 14 c o n i f e r s p e c i e s  42  4  T a b l e 1.7  3  T a b l e 1.8  isolates  Chapter 2  T a b l e 2.1  T a b l e 2.2  T a b l e 2.3  G a l l f o r m a t i o n by and r e c o v e r y o f t h e pathogen from h e a t - t r e a t e d (35°C f o r 18 days) D o u g l a s f i r seedlings stab-inoculated with the t y p i c a l Erwinia i s o l a t e T-2789  58  G a l l f o r m a t i o n by and r e c o v e r y o f t h e pathogen from h e a t - t r e a t e d (35°C f o r 12 days) D o u g l a s f i r seedlings stab-inoculated with the t y p i c a l Erwinia i s o l a t e T-2789  59  G a l l f o r m a t i o n by and r e c o v e r y o f t h e pathogen from h e a t - t r e a t e d (3 6°C) D o u g l a s - f i r s e e d l i n g s stab-inoculated with the a t y p i c a l Erwinia i s o l a t e A-0181  62  viii LIST OF TABLES  (Cont.) Page  T a b l e 2.4  G a l l f o r m a t i o n by and r e c o v e r y o f t h e pathogen from h e a t - t r e a t e d (38°C) D o u g l a s - f i r s e e d l i n g s stab-inoculated with the a t y p i c a l Erwinia i s o l a t e A-0181  63  C o n c e n t r a t i o n s o f c h e m i c a l c u r i n g a g e n t s added t o LB i n c u r i n g p r o t o c o l s used w i t h t h e g a l l f o r m i n g Erwinia i s o l a t e s T-2774 and A-0181  81  Chapter 3  T a b l e 3.1  ix LIST OF FIGURES Page  Chapter 1  Figure  Figure  Figure  Figure  Figure  Figure  Figure  1.1  1.2  1.3  1.4  1.5  1.6  1.7  The c h a r a c t e r i s t i c l o c a t i o n , appearance and s i z e of n a t u r a l l y - o c c u r r i n g b a c t e r i a l g a l l s of D o u g l a s - f i r produced by t y p i c a l i s o l a t e s and t h e a t y p i c a l i s o l a t e A-0181 o f t h e Douglas-fir gall-forming bacteria  23  Types o f g a l l s formed on D o u g l a s - f i r t r e e s a r t i f i c i a l l y i n o c u l a t e d w i t h t h e two d i f f e r e n t i s o l a t e types of the D o u g l a s - f i r g a l l bacteria  24  G a l l s formed on 1- t o 2-year o l d D o u g l a s - f i r t r e e s by s t a b - i n o c u l a t i o n w i t h D o u g l a s - f i r gall-forming bacteria  26  C o l o n i e s o f t h e t y p i c a l and t h e a t y p i c a l g a l l - f o r m i n g Erwinia i s o l a t e s grown on c a s e i n h y d r o l y s a t e , peptone and g l u c o s e medium  ,  28  S e n s i t i v i t y o f t h e IgG f r a c t i o n s o f t h e a n t i s e r a r a i s e d a g a i n s t t h e t y p i c a l T-2789 and A-0181 i s o l a t e s of t h e D o u g l a s - f i r g a l l - f o r m i n g b a c t e r i a as d e t e r m i n e d by an i n d i r e c t ELISA a g a i n s t t h e i r r e s p e c t i v e homologous a n t i g e n s ,  34  Immunodiffusion p a t t e r n s produced by r e a c t i o n s between a n t i s e r a e l i c i t e d a g a i n s t t h e t y p i c a l T-2789 and t h e a t y p i c a l A-0181 g a l l - f o r m i n g Erwinia i s o l a t e s and s e v e r a l phytopathogenic b a c t e r i a  35  Pathogenicity of g a l l - f o r m i n g Erwinia i s o l a t e s on Abies amabilis, Chamaecyparis nootkatensis, Picea engelmannii and Pinus contorta  41  The e f f e c t o f a h i g h t e m p e r a t u r e t r e a t m e n t (35±2°C) , a p p l i e d a t v a r i o u s t i m e s a f t e r i n o c u l a t i o n w i t h g a l l - f o r m i n g Erwinia isolate T-2789, on t h e development o f g a l l i n g symptoms on D o u g l a s - f i r  60  Chapter 2  Figure  2.1  X  LIST OF FIGURES (Cont.) Page  Chapter 3  F i g u r e 3.1  F i g u r e 3.2  F i g u r e 3.3  F i g u r e 3.4  F i g u r e 3.5  F i g u r e 3.6  F i g u r e 3.7  F i g u r e 3.8  F i g u r e 3.9  P l a s m i d p r o f i l e s o f t y p i c a l and a t y p i c a l g a l l - f o r m i n g Erwinia isolates fractionated on a 0.7% agarose g e l and s t a i n e d w i t h e t h i d i u m bromide  86  P l a s m i d DNA p r o f i l e o f Erwinia i s o l a t e A-0181 f r a c t i o n a t e d on a 0.7% agarose g e l and s t a i n e d w i t h e t h i d i u m bromide  87  P l a s m i d DNAs from t y p i c a l and a t y p i c a l g a l l f o r m i n g Erwinia isolates digested with r e s t r i c t i o n endonucleases, f r a c t i o n a t e d on a 0.9% agarose g e l and s t a i n e d w i t h e t h i d i u m bromide  88  G e l e l e c t r o p h o r e s i s p r o f i l e and S o u t h e r n a n a l y s i s o f t o t a l genomic and p l a s m i d DNA o f v a r i o u s p h y t o p a t h o g e n i c b a c t e r i a probed w i t h p l a s m i d DNA o f a t y p i c a l g a l l - f o r m i n g Erwinia isolate  90  G e l e l e c t r o p h o r e s i s p r o f i l e and S o u t h e r n a n a l y s i s o f p l a s m i d DNA o f v a r i o u s p h y t o p a t h o g e n i c b a c t e r i a probed w i t h p l a s m i d DNA o f a t y p i c a l g a l l - f o r m i n g Erwinia isolate  91  G e l e l e c t r o p h o r e s i s p r o f i l e and S o u t h e r n a n a l y s i s o f t o t a l genomic and p l a s m i d DNA o f v a r i o u s p h y t o p a t h o g e n i c b a c t e r i a probed w i t h p l a s m i d DNA o f t h e a t y p i c a l g a l l - f o r m i n g Erwinia isolate  92  G e l e l e c t r o p h o r e s i s p r o f i l e and S o u t h e r n a n a l y s i s o f p l a s m i d DNA o f v a r i o u s p h y t o p a t h o g e n i c b a c t e r i a probed w i t h p l a s m i d DNA o f t h e a t y p i c a l g a l l - f o r m i n g Erwinia isolate  93  Autoradiogram of colony h y b r i d i z a t i o n o f p l a s m i d DNA probe o f t y p i c a l g a l l - f o r m i n g Erwinia i s o l a t e T-2774 w i t h Erwinia spp  95  Autoradiogram of colony h y b r i d i z a t i o n o f d A T P - l a b e l l e d p l a s m i d DNA o f a t y p i c a l g a l l - f o r m i n g Erwinia i s o l a t e A-0181 w i t h p o t e n t i a l l y cured c o l o n i e s o f Erwinia i s o l a t e A-0181  96  32  xi ACKNOWLEDGEMENTS  I w i s h t o thank my s u p e r v i s o r , Dr. R . J . Copeman, f o r s u p p o r t and g u i d a n c e throughout t h i s study.  Thanks a l s o a r e  extended t o t h e o t h e r members o f my committee namely, Dr. J.C. McPherson, Dr. R.A.J. Warren and Dr. V.C. R u n e c k l e s , f o r t h e i r h e l p and c r i t i c a l  readings.  I would a l s o l i k e t o thank Dr. M. Shaw and Mr. B. R o n a l d f o r p r o v i d i n g s u p p l i e s and l a b space f o r r a d i o l a b e l l i n g .  Thanks  a r e e s p e c i a l l y extended t o Dr. P. E l l i s f o r h i s h e l p and a l l o w i n g me t o work i n h i s l a b o r a t o r y f o r a p o r t i o n o f my t h e s i s research.  I would l i k e t o acknowledge C a r o l y n e Jow f o r h e r  t e c h n i c a l a s s i s t a n c e during t h e p o r t i o n of t h i s study under c o n t r a c t t o t h e P l a n t B i o t e c h n o l o g y Saskatchewan, Len Ward f o r h i s c o n t i n u e d  conducted  Institute i n h e l p and s u g g e s t i o n s  and Tom Lowery f o r a s s i s t a n c e w i t h t h e s t a t i s t i c a l a n a l y s i s and presentation. I would l i k e t o d e d i c a t e t h i s t h e s i s t o my p a r e n t s f o r t h e i r n e v e r - e n d i n g f a i t h and support.  1 GENERAL INTRODUCTION  The  f o r e s t i n d u s t r y i n B.C., and Canada as a whole, i s now  r e a c h i n g a c r i t i c a l p e r i o d i n terms o f m a i n t a i n i n g i t s competitive position internationally.  The f o r e s t s o f Canada a r e  t h e n a t i o n ' s most v a l u a b l e n a t u r a l a s s e t w i t h a p p r o x i m a t e l y one m i l l i o n f a m i l i e s depending d i r e c t l y o r i n d i r e c t l y on t h e f o r e s t industry for their livelihood  (Reed, 1989).  o f i n d u s t r i a l roundwood i s growing  World consumption  a t an average o f 2.5%  a n n u a l l y and f o r Canada t o keep i t s share o f t h e market, i t must i n c r e a s e t h e growth and y i e l d o f i t s f o r e s t s w h i l e m a i n t a i n i n g t h e h i g h q u a l i t y and low c o s t o f t h e raw m a t e r i a l . G e n e t i c improvement o f c o n i f e r s must be c a r r i e d o u t , i n conjunction with reforestation, t o provide h i g h - q u a l i t y , f a s t e r growing  trees.  (Pseudotsuga taeda  A study o f t h e p r o d u c t i v i t y o f some D o u g l a s - f i r  menziesii  [Mirb.] Franco) and l o b l o l l y p i n e  (Pinus  L.) p l a n t a t i o n s showed t h a t s i l v i c u l t u r a l and g e n e t i c  m a n i p u l a t i o n has i n c r e a s e d t h e i r p r o d u c t i v i t y 70 and 300%, r e s p e c t i v e l y , i n comparison t o n a t u r a l growth f o r e s t s 1989).  Research  i n t o g e n e t i c improvement o f p l a n t s i n g e n e r a l  has c e n t e r e d on t h e use o f t h e T i p l a s m i d o f tumefaciens  (Reed,  Agrobacterium  (Smith and Townsend, Conn.), t h e c a u s a l agent o f  crown g a l l , as a c l o n i n g v e c t o r (Barton and C h i l t o n , 1983; F r a l e y e t a l . , 1983).  The s t u d i e s t o date have m o s t l y i n v o l v e d  d i c o t y l e d o n o u s p l a n t s as t h e r e c i p i e n t s a l t h o u g h some monocots and gymnosperms a r e w i t h i n t h e wide h o s t range o f (De Cleene and De Ley, 1976).  Agrobacterium  Recently the transformation of  s e v e r a l c o n i f e r s p e c i e s such as w h i t e spruce  (Picea  glauca  2  (Moench) V o s s ) , Englemann s p r u c e (Picea englemannii Engelm.) and S i t k a s p r u c e (Picea sitchensis w e l l as l o b l o l l y p i n e by Agrobacterium ( L e d i g , 1985; S e d e r o f f e t al., E l l i s e t al.,  P a r r y ex  (Bong.) C a r r ) as  has been shown t o o c c u r  1986; Dandekar e t a l . ,  1987;  1989).  D o u g l a s - f i r , n a t i v e t o t h e c o a s t and i n t e r i o r o f B.C., a l s o been shown t o be s u s c e p t i b l e t o A. tumefaciens and De Ley, 1976).  However, A. tumefaciens,  found as a n a t u r a l pathogen of D o u g l a s - f i r .  has  (De Cleene  i s n o t commonly T h e r e f o r e , i t would  be o f i n t e r e s t t o i n v e s t i g a t e o t h e r g a l l - f o r m i n g b a c t e r i a l pathogens o f D o u g l a s - f i r w i t h r e g a r d t o t h e i r mechanism o f g a l l f o r m a t i o n and t h e i r p o t e n t i a l f o r h a r b o r i n g a p l a s m i d w i t h c a p a b i l i t i e s s i m i l a r t o t h e Agrobacterium  T i plasmid.  G a l l s on c o n i f e r s can be caused by e i t h e r f u n g i , b a c t e r i a , m i s t l e t o e s or environment-related s t r e s s e s . the  g a l l - f o r m i n g f u n g a l pathogens i s Endocronartium  (J.P.  insects, One  of  harknessii  Moore) Y. H i r a t , t h e c a u s a l agent of Western g a l l r u s t on  such h o s t s as l o d g e p o l e and ponderosa p i n e s ( S i n c l a i r e t al., 1987).  Forms o f Cronartium  quercuum  (Berk.) M i y . ex S h i r a i  cause P i n e - o a k g a l l r u s t on d i f f e r e n t p i n e s p e c i e s such as l o b l o l l y , ponderosa and S c o t s p i n e .  C o n i f e r g a l l s can a l s o be  caused by i n s e c t p e s t s such as t h e Cooley Spruce g a l l ( S i n c l a i r e t al.,  1987).  adelgid  A r e v i e w of t h e l i t e r a t u r e on g a l l  d i s e a s e s on c o n i f e r s i n g e n e r a l , r e v e a l s v e r y few r e p o r t s o f bacterial incitants.  The f i r s t p u b l i s h e d r e p o r t o f a b a c t e r i a l  g a l l d i s e a s e on c o n i f e r s was i n 1888 by V u i l l e m i n ( c i t e d by Hansen and S m i t h , 1937).  The a u t h o r i s o l a t e d b a c t e r i a from  g a l l s l o c a t e d on t h e t w i g s , branches and upper stem of  Pinus  3 halepensis  Mill.  (Aleppo p i n e ) .  from g a l l s on Pinus Tubeuf.  A s i m i l a r organism was  isolated  cembra L. (Swiss s t o n e p i n e ) i n 1911 by von  The f i r s t p u b l i s h e d r e p o r t of a b a c t e r i a l g a l l d i s e a s e  on D o u g l a s - f i r was  i n 1933 by Hansen and Smith i n C a l i f o r n i a  (Hansen and Smith,  1933).  Agrobacterium Smith 1937)  tumefaciens  and A. pseudotsugae  S a v u l e s c u 1947 a r e t h e o n l y two  phytopathogens  (Hansen and  bacterial  d e s c r i b e d as c a u s i n g g a l l s on c o n i f e r s ,  s p e c i f i c a l l y , D o u g l a s - f i r (De Cleene and De Ley, 1976; and S m i t h , 1937). pseudotsugae  more Hansen  The work by Hansen and Smith on A.  has not been c o n f i r m e d and t h e i s o l a t e d pathogen i s  n o t even l i s t e d i n t h e most r e c e n t v e r s i o n of Bergey's Manual o f D e t e r m i n a t i v e B a c t e r i o l o g y ( K r i e g and H o l t , 1984). t o t h e s m a l l number of pathogens conifers,  In addition  c a u s i n g g a l l f o r m a t i o n on  t h e f r e q u e n c y o f o c c u r r e n c e of t h e s e phytopathogens  v e r y r e s t r i c t e d (R.S. Hunt,  unpublished).  G a l l s a t t r i b u t e d t o b a c t e r i a have been r e p o r t e d on f i r from s o u t h e r n A r i z o n a t o B.C. 1987).  is  Douglas-  and A l b e r t a ( S i n c l a i r e t  al.,  There have been over 52 r e p o r t s of g a l l s o f s u s p e c t e d  b a c t e r i a l o r i g i n i n B.C.  s i n c e 1946, a l t h o u g h e x t e n s i v e damage  t o t h e h o s t s was not observed (R.S. Hunt, unpublished).  Samples  of a g a l l d i s e a s e on D o u g l a s - f i r have been c o l l e c t e d i n t h e s o u t h w e s t e r n c o r n e r o f B r i t i s h Columbia.  The d i s e a s e was  s u s p e c t e d t o be o f a b a c t e r i a l o r i g i n due t o t h e d i f f e r e n t symptoms i n r e l a t i o n t o t h o s e a s s o c i a t e d w i t h t h e w e l l documented f u n g a l g a l l s .  A s t r a i n of b a c t e r i a was  t h e s e g a l l s i n t h e mid 1980's (Muehlchen,  1985).  isolated  from  Several  D o u g l a s - f i r g a l l specimens were c o l l e c t e d o r r e c e i v e d s i n c e t h a t  4  time. One o f t h e f i r s t s t e p s t o be t a k e n when w o r k i n g w i t h a r e l a t i v e l y unknown phytopathogen, such as t h e D o u g l a s - f i r  gall-  f o r m i n g b a c t e r i u m , i s t o choose a method f o r t h e d e t e c t i o n and/or i d e n t i f i c a t i o n of t h e organism i n mixed c u l t u r e o r i n plant material.  There a r e many methods o f i d e n t i f i c a t i o n o r  d e t e c t i o n o f p h y t o p a t h o g e n i c agents such as s e l e c t i v e media (Meneley and S t a n g h e l l i n i , 1976), s e r o l o g i c a l a s s a y s ( M i l l e r and M a r t i n , 1988), p l a s m i d p r o f i l e s (Morales and S e q u e i r a , 1985), DNArDNA h y b r i d i z a t i o n u s i n g e i t h e r chromosome (Thompson e t al., 1989) o r p l a s m i d probes ( G i l b e r t s o n e t al., and/or p l a s m i d f i n g e r p r i n t i n g  (Cooksey and Graham, 1989).  method used depends upon f a c i l i t i e s of  1989) and genome The  and funds a v a i l a b l e , number  samples t o be p r o c e s s e d , degree or l e v e l o f s p e c i f i c i t y  d e s i r e d and t i m e a v a i l a b l e . The s e r o l o g i c a l d e t e c t i o n and i d e n t i f i c a t i o n o f p h y t o p a t h o g e n i c b a c t e r i a i s w i d e l y used as a t o o l i n t h e research laboratory.  S e r o l o g y t a k e s advantage o f t h e  r e c o g n i t i o n o f a n t i g e n i c d e t e r m i n a n t s on t h e pathogen t h a t a r e o f t e n c h a r a c t e r i s t i c of t h e s p e c i e s .  The f i r s t r e p o r t o f t h e  use o f a s e r o l o g i c a l t e s t t o i d e n t i f y a p l a n t pathogen was i n 1918 by J e n s e n w o r k i n g w i t h Agrobacterium De Boer, 1987).  tumefaciens  ( c i t e d by  I n f e c t i o u s agents i n d i s e a s e d t i s s u e , even when  i n low c o n c e n t r a t i o n o r i n a l a t e n t phase, can be d e t e c t e d w i t h s e r o l o g i c a l a s s a y s ( M i l l e r and M a r t i n , 1988). For  t h e D o u g l a s - f i r g a l l - f o r m i n g b a c t e r i u m t o be c o n s i d e r e d  as a gene v e c t o r , t h e pathogen's mechanism o f g a l l f o r m a t i o n must i n v o l v e a t r a n s f e r of genes t o t h e h o s t p l a n t genome, a  5 t r a n s f o r m a t i o n event, such as t h a t which o c c u r s i n t h e A. tumefaciens  system.  The A. tumefaciens  genes t h a t a r e  i n t e g r a t e d i n t o t h e h o s t p l a n t genome encode f o r enzymes i n v o l v e d i n t h e p r o d u c t i o n of phytohormones (Watson e t 1975; W e i l e r and S p a n i e r , 1981).  al.,  Once t h e t r a n s f e r o f t h e  b a c t e r i a l genes t o t h e p l a n t c e l l s o c c u r s , t h e p r e s e n c e o f t h e pathogen i s no l o n g e r r e q u i r e d f o r t h e c o n t i n u e d development o f the g a l l s  ( C h i l t o n e t al.,  1977).  The b a c t e r i a l genes a r e  t r a n s c r i b e d and t r a n s l a t e d as h o s t DNA gall  and e f f e c t t h e growth of  tissue. T r a n s f o r m a t i o n o f h o s t p l a n t c e l l s does not o c c u r i n t h e  Pseudomonas W i l k i e 1978)  syringae  pv. savastanoi  system.  ( [ S m i t h 1908]  Rather, P. syringae  Young, Dye  pv. savastanoi,  &  the  c a u s a l agent o f o l i v e and o l e a n d e r k n o t , produces tumors on i t s h o s t p l a n t s t h r o u g h t h e p r o d u c t i o n of phytohormones by t h e bacterium i t s e l f .  The phytohormones cause h y p e r t r o p h y ,  h y p e r p l a s i a and v a s c u l a r d i f f e r e n t i a t i o n o f t h e h o s t surrounding the invading bacterium  (Smidt and Kosuge,  cells 1978).  T h e r e f o r e , t h e presence of t h e b a c t e r i u m i s r e q u i r e d f o r i n i t i a t i o n as w e l l as c o n t i n u e d development o f t h e tumor. A l t h o u g h g a l l f o r m a t i o n by t h e two phytopathogens a r i s e s i n t h e h o s t p l a n t s by d i f f e r e n t mechanisms, t h e b a s i s o f g a l l f o r m a t i o n i s s i m i l a r i n both systems.  The p r o d u c t i o n o f  phytohormones, namely a u x i n s and c y t o k i n i n s , i s r e q u i r e d f o r t h e i n i t i a t i o n and development of t h e p l a n t tumors ( W e i l e r and S p a n i e r , 1981; Comai e t al., acetic acid  1982).  A u x i n s , such as i n d o l e  ( I A A ) , r e g u l a t e c e l l expansion and e l o n g a t i o n w h i l e  c y t o k i n i n s , such as t r a n s - z e a t i n , r e g u l a t e c e l l d i v i s i o n  (Davis  6  e t al.,  1985).  O v e r p r o d u c t i o n of t h e s e phytohormones i n  c o m b i n a t i o n l e a d s t o t h e u n r e g u l a t e d growth and d i v i s i o n o f h o s t p l a n t c e l l s and hence t o t h e p r o d u c t i o n o f a tumor o r g a l l .  The  a c t u a l s i t e o f p r o d u c t i o n of t h e phytohormones d i s t i n g u i s h e s between t h e A. tumefaciens  and P. syringae  pv.  savastanoi  systems. Whether o r not t h e mechanism of g a l l f o r m a t i o n by t h e D o u g l a s - f i r g a l l - f o r m i n g b a c t e r i u m i n v o l v e s a t r a n s f e r o f genes from a p l a s m i d t o t h e h o s t p l a n t c e l l s , as o c c u r s i n t h e crown g a l l system, t h e r o l e of any p l a s m i d DNA an i n t e r e s t i n g q u e s t i o n . extrachromosomal  i n pathogenesis  P l a s m i d s a r e autonomously  p i e c e s o f DNA.  remains  replicating  P l a s m i d s c o n t a i n , on  average,  about 2% o f t h e b a c t e r i a l chromosome but can c o n s t i t u t e up t o a t h i r d o f t h e genome ( C o p l i n , 1989).  Many p h y t o p a t h o g e n i c  b a c t e r i a , i n c l u d i n g s p e c i e s i n t h e genera, Clavibacter,  Erwinia,  Agrobacterium,  Pseudomonas, and Xanthomonas,  a r e known t o  c a r r y one o r s e v e r a l p l a s m i d s . P l a s m i d s encode f o r many d i f f e r e n t p h e n o t y p i c c h a r a c t e r i s t i c s which a r e o f t e n n o n e s s e n t i a l y e t advantageous  to  the bacterium, i n c l u d i n g a n t i b i o t i c r e s i s t a n c e , metal degradation, n i t r o g e n f i x a t i o n , conjugation, the p r o d u c t i o n of b a c t e r i o c i n s and r e s t r i c t i o n enzymes ( T r e v o r s , 1985).  Most of  the plasmids i d e n t i f i e d i n p l a n t pathogenic b a c t e r i a are l a b e l l e d as c r y p t i c o r have no known f u n c t i o n ( C o p l i n , However, i n b o t h t h e A. tumefaciens savastanoi  and P. syringae  still  1989).  pv.  systems, genes i n v o l v e d i n t h e p r o d u c t i o n o f  phytohormones, and hence p a t h o g e n i c i t y , have been l o c a t e d on p l a s m i d s ( L i u and Kado, 1979; Comai e t al.,  1982).  Furthermore,  7  genes i n v o l v e d i n h o s t s p e c i f i c i t y have been l o c a t e d on p l a s m i d s o f p h y t o p a t h o g e n i c b a c t e r i a ( C o p l i n , 1989). The  f o c u s of t h i s s t u d y on the D o u g l a s - f i r  b a c t e r i a was  gall-forming  not t h e i r r o l e as phytopathogens, but  rather  whether o r not t h e b a c t e r i a c a r r i e d p l a s m i d s t h a t c o u l d s e r v e as p o t e n t i a l gene v e c t o r s f o r the g e n e t i c e n g i n e e r i n g The  s h i f t i n research  i n t e r e s t of b a c t e r i a l g a l l f o r m e r s i s due  i n p a r t t o t h e r e s u l t s of e x t e n s i v e s t u d i e s of p h y t o p a t h o g e n i c g a l l f o r m e r s , A. tumefaciens savastanoi. plasmid  the  and P. syringae  pv.  More s p e c i f i c a l l y , t h e s u c c e s s f u l use of t h e T i  of A. tumefaciens  genes i n t o e c o n o m i c a l l y research  of c o n i f e r s .  as a v e c t o r f o r i n t r o d u c i n g r e s i s t a n c e i m p o r t a n t p l a n t s has become an  t o o l (Barton and C h i l t o n , 1983;  important  C h i l t o n , 1983).  T h e r e f o r e t h e i s o l a t i o n of a r e l a t i v e l y unknown b a c t e r i a l g a l l former prompted i n v e s t i g a t i o n i n t o i t s p o t e n t i a l economic v a l u e as an a l t e r n a t e v e c t o r t o the T i p l a s m i d  of  Agrobacterium  tumefaciens. When c o n s i d e r i n g t h e s c a n t amount of i n f o r m a t i o n  on  b a c t e r i a l g a l l f o r m e r s on D o u g l a s - f i r and t h e f o c u s of  this  s t u d y , t h e s p e c i f i c o b j e c t i v e s were t h e f o l l o w i n g : 1) t o d e s c r i b e t h e g a l l s produced, i s o l a t e t h e agent and produce a n t i s e r a a g a i n s t t h e  causal  gall-forming  pathogens; 2) t o d e t e r m i n e i f the mechanism of g a l l i n v o l v e s the transformation  formation  of h o s t p l a n t  cells;  3) t o d e t e r m i n e i f t h e r e i s a c o r r e l a t i o n between p r e s e n c e and  pathogenicity.  plasmid  8  CHAPTER 1  ISOLATION, DESCRIPTION AND SEROLOGICAL DETECTION OP THE DOUGLAS-FIR GALL-FORMING BACTERIA  INTRODUCTION  The D o u g l a s - f i r g a l l d i s e a s e , r e p o r t e d by Hansen and Smith (1937), i n some i n s t a n c e s suppressed growth and caused d i e b a c k of the host t r e e .  The a f f e c t e d D o u g l a s - f i r t r e e s were l o c a t e d  i n a r e a s where t h e h e a l t h o f t h e h o s t was a l r e a d y such a s i n damp a r e a s by streams o r ponds.  compromised  The smooth, g l o b o s e  g a l l s formed on t h e t w i g s , branches and upper main stems o f young D o u g l a s - f i r t r e e s .  The g a l l s measured between one  m i l l i m e t e r t o s e v e r a l centimeters i n diameter.  A characteristic  c r o s s - s h a p e d marking was p r e s e n t a c r o s s t h e f a c e o f t h e  galls.  G a l l s o c c u r r i n g on t h e main stem o f young D o u g l a s - f i r  seedlings  can cause d e f o r m a t i o n and even death o f t h e s e e d l i n g .  The  b a c t e r i u m was not p a t h o g e n i c on any o f t h e c o n i f e r s t e s t e d , i n c l u d i n g Pinus radiata  Don.  halepensis  M i l l . , P. lambertiana  and Tsuga heterophylla  S m i t h , 1937), e x c e p t f o r Pseudotsuga ( S m i t h , 1940).  Dougl.,  Pinus  (Raf.) S a r g . (Hansen and macrocarpa  (Vasey) Mayr  The b a c t e r i u m was determined t o be h i g h l y  s p e c i f i c t o Pseudotsuga  spp.  Adelges  cooleyi  (Gillette), the  C o o l e y s p r u c e g a l l a d e l g i d , was suggested as an i n s e c t v e c t o r o f the bacterium. The s t r u c t u r e o f t h e D o u g l a s - f i r g a l l was l i k e n e d t o t h a t  9 o f g a l l s produced by Pseudomonas syringae  pv. savastanoi,  w h i c h t h e g a l l s a r e composed o f groups o f r a p i d l y cells.  The Hansen and Smith b a c t e r i u m was  in  dividing  located i n r e l a t i v e l y  l a r g e i n t e r c e l l u l a r spaces i n t h e c e n t e r s o f t h e s e groups o f cells.  The  i s o l a t e d g a l l b a c t e r i u m was  negative, non-motile,  d e t e r m i n e d t o be a Gram-  f a c u l t a t i v e aerobe (more a p p r o p r i a t e l y  termed f a c u l t a t i v e anaerobe u s i n g c u r r e n t t e r m i n o l o g y ) . named by Hansen and Smith as Bacterium l a t e r renamed t o Agrobacterium The work done on A. pseudotsugae The  pseudotsugae  pseudotsugae  (1937) and  ( S a l v u l e s c u , 1947).  has never been  f i r s t s t e p i n t h e study of any  I t was  'new'  confirmed.  disease i s the  d e s c r i p t i o n o f t h e d i s e a s e symptoms on t h e h o s t p l a n t . i d e n t i f i c a t i o n and d e s c r i p t i o n of t h e pathogen  The  through  b i o c h e m i c a l t e s t s , which i s a s e p a r a t e study i n i t s e l f , was  not  c o n s i d e r e d a p r i o r i t y i n t h i s study because t h e major aim was i n v e s t i g a t e t h e mechanism of g a l l f o r m a t i o n .  to  C l a s s i f i c a t i o n of  t h e b a c t e r i a l i s o l a t e s u s i n g a f a t t y a c i d a n a l y s i s system ( H e w l e t t P a c k a r d M i c r o b i a l I d e n t i f i c a t i o n System) was as s u i t a b l e f o r t h e c o n t e x t of t h i s r e s e a r c h .  regarded  Fatty acid  a n a l y s i s i s t o u t e d as b e i n g v e r y r e l i a b l e f o r i d e n t i f i c a t i o n p u r p o s e s , due t o t h e g e n e t i c s t a b i l i t y of t h e f a t t y a c i d s (Anonymous, 1985;  Stead, 1988).  I n t h i s system, comparisons of  h i g h r e s o l u t i o n gas chromatographic a n a l y s e s o f t h e c e l l w a l l f a t t y a c i d s were made w i t h a computer l i b r a r y o f p r o f i l e s from v a r i o u s known b a c t e r i a l s p e c i e s and t h e outcomes were r e c o r d e d as s i m i l a r i t y i n d i c e s ( M i l l e r , 1984;  Stead, 1988).  A tentative  i d e n t i f i c a t i o n by t h e f a t t y a c i d a n a l y s i s system i n a d d i t i o n t o v i s u a l o b s e r v a t i o n s a l l o w e d f o r comparison t o o t h e r g a l l - f o r m i n g  10 phytopathogens. Some o f t h e r e c o g n i z e d p h y t o p a t h o g e n i c b a c t e r i a t h a t g a l l s on t h e i r h o s t p l a n t s i n c l u d e Agrobacterium pv. savastanoi  cause  tumefaciens,  Pseudomonas  syringae  gypsophilae  (Brown) M i l l e r , Quinn and Graham 1981.  tumefaciens  i s t h e c a u s a l agent of crown g a l l which a f f e c t s many  d i c o t s , monocots and gymnosperms.  and Erwinia  herbicola  f.sp.  A.  G a l l s form on r o o t s and stems  e s p e c i a l l y a t t h e crown a r e a o r base o f t h e stem ( S i n c l a i r e t al.,  1987).  Pseudomonas syringae  pv. savastanoi  i s the  pathogen  t h a t i s r e s p o n s i b l e f o r o l i v e and o l e a n d e r k n o t as w e l l as b a c t e r i a l k n o t on ash.  The major symptoms i n c l u d e g a l l i n g o r  k n o t f o r m a t i o n as w e l l as d i e b a c k of t w i g s and ( S i n c l a i r e t al.,  branches  1987).  F i n a l l y , crown and r o o t g a l l s on Gypsophila r e s e m b l i n g t h o s e caused by A. tumefaciens, i n t h e 1930's.  were f i r s t d e s c r i b e d  The i s o l a t e d pathogen was o r i g i n a l l y named  Bacterium  gypsophilae  herbicola  f . s p . gypsophilae  1985).  paniculata,  E. herbicola  but has s i n c e been r e c l a s s i f i e d as ( M i l l e r e t al.,  Erwinia  1981; Cooksey,  i s a l s o r e p o r t e d t o cause g a l l i n g symptoms  on r o s e s and c a r n a t i o n s (Maas Geesteranus and Barendsen,  1966).  G a l l s a r e u s u a l l y formed a t t h e c u t end of r o o t e d c u t t i n g s o r a t g r a f t u n i o n s when below t h e s o i l l i n e .  L i t t l e work however has  been done on t h i s g a l l - f o r m i n g s t r a i n o f E. herbicola r e l a t i o n t o t h e mechanism of g a l l f o r m a t i o n .  in  However, a r e c e n t  s t u d y done on t h e mechanism of g a l l development by E.  herbicola  f . s p . gypsophilae,  syringae  pv. savastanoi  s u g g e s t s a mechanism s i m i l a r t o P.  ( C l a r k e t al.,  1989).  Tentative i d e n t i f i c a t i o n  o f t h e D o u g l a s - f i r g a l l - f o r m i n g b a c t e r i a can a i d , by  comparisons  11 w i t h o t h e r g a l l - f o r m i n g b a c t e r i a , i n the u n d e r s t a n d i n g of d i s e a s e c y c l e and gall  the  i n s o l v i n g the q u e s t i o n of t h e mechanism of  formation. The  p r o d u c t i o n of p o l y c l o n a l a n t i s e r a a g a i n s t b a c t e r i a l  pathogens has  been used f o r t h e i r d e t e c t i o n and  identification.  B o t h p o l y c l o n a l a n t i s e r a and monoclonal a n t i b o d i e s produced a g a i n s t  such b a c t e r i a as Erwinia  Pseudomonas  syringae,  Xanthomonas  spp.  C i v e r o l o and  Corynebacterium  (De Boer, 1982;  Fan,  1982;  monoclonal antibodies  have been  carotovora,  sepedonicum  and  A l l a n and Kelman,  B e n e d i c t e t al.,  1989).  can be produced a g a i n s t  1977;  Polyclonal  the b a c t e r i a l  a n t i g e n s depending upon the f a c i l i t i e s a v a i l a b l e and s p e c i f i c i t y required  (De Boer, 1987).  or  the  S e r o l o g i c a l assays  i n v o l v i n g p o l y c l o n a l a n t i s e r a are f a i r l y s p e c i f i c f o r most detection  purposes.  As a h e t e r o l o g o u s m i x t u r e of a n t i b o d i e s ,  polyclonal  a n t i s e r a o f t e n i s c r i t i c i z e d as not b e i n g v e r y s p e c i f i c towards the  immunogen.  T h i s problem can be a l l e v i a t e d p a r t i a l l y  c a r e f u l p u r i f i c a t i o n and p r e p a r a t i o n t h e i m m u n o g l o b u l i n G (IgG)  of immunogen.  f r a c t i o n a l s o can  I s o l a t i o n of  increase  s p e c i f i c i t y of t h e a n t i s e r u m ( M i l l e r and M a r t i n ,  by  the  1988).  The  s p e c i f i c i t y of the a n t i s e r u m must be t e s t e d b e f o r e r o u t i n e i n q u a l i t a t i v e and The  use  q u a n t i t a t i v e assays.  measurement of s p e c i f i c i t y of a n t i s e r a depends upon the  s e r o l o g i c a l method employed and the number of organisms t e s t e d (De Boer, 1982).  O u c h t e r l o n y double d i f f u s i o n o r  i m m u n o d i f f u s i o n was  chosen as one  of the t e c h n i q u e s t o t e s t  the  s p e c i f i c i t y because i t a l l o w s the v i s u a l i z a t i o n of r e l a t i o n s h i p s  12 between a n t i g e n s (De Boer e t al.,  1979).  The second t e c h n i q u e  chosen was an i n d i r e c t ELISA as a m o d i f i c a t i o n of t h i s t e c h n i q u e was t o be used i n t h e experiments as o u t l i n e d i n C h a p t e r 2. Many d i f f e r e n t s e r o l o g i c a l a s s a y s have been used t o d e t e c t b a c t e r i a i n c l u d i n g l a t e x a g g l u t i n a t i o n , Ouchterlony double d i f f u s i o n o r i m m u n o d i f f u s i o n , immunofluorescence  and  immunoelectron m i c r o s c o p y (De Boer, 1982; B r i a n s k y e t al., da Roche e t a l . , 1986; B e n e d i c t e t al.,  1989).  1982;  The enzyme-  l i n k e d immunosorbent assay (ELISA) has become a w i d e l y  used  a s s a y f o r b o t h q u a l i t a t i v e and q u a n t i t a t i v e d e t e c t i o n o f phytopathogens i n c l u d i n g v i r u s e s , m y c o p l a s m a - l i k e o r g a n i s m s , f u n g i and b a c t e r i a .  ELISA was f i r s t i n t r o d u c e d  a s s a y i n t h e a r e a of p l a n t p a t h o l o g y i n 1976 1977)  as a s e r o l o g i c a l  ( C l a r k and Adams,  f o r t h e d e t e c t i o n of p l a n t v i r u s e s . There a r e a seemingly i n f i n i t e number of v a r i a t i o n s o f t h e  b a s i c ELISA p r o c e d u r e .  V a r i a t i o n s a r i s e i n the type of s o l i d -  s u p p o r t used ( L a z a r o v i t s e t al.,  1987), t h e t i m e s o f i n c u b a t i o n ,  t h e number and s o u r c e s of a n t i b o d i e s used i n a s i n g l e a s s a y and t h e p r e s e n c e o r absence of t h e c o a t i n g a n t i b o d y s t e p .  An  e n r i c h m e n t s t e p o c c a s i o n a l l y i s used as t h e i n i t i a l s t e p o f t h e assay t o increase the b a c t e r i a l c e l l concentration sample.  i n the t e s t  T h i s m o d i f i c a t i o n i s an attempt t o i n c r e a s e t h e l e v e l  of d e t e c t i o n  (Beguin e t al.,  1984).  Such a m o d i f i c a t i o n  was  used f o r t h e d e t e c t i o n of l i v e b a c t e r i a i n h e a t - t r e a t e d , i n o c u l a t e d branches as o u t l i n e d i n Chapter 2. The o b j e c t i v e s of t h e s e experiments were t o  firstly  d e s c r i b e t h e D o u g l a s - f i r g a l l s c o l l e c t e d from v a r i o u s i n B.C.  locations  The second o b j e c t i v e was t o d e s c r i b e t h e b a c t e r i a l  incitants  o f t h e s e g a l l s and t o o b t a i n a t e n t a t i v e  i d e n t i f i c a t i o n t h r o u g h the use of f a t t y a c i d a n a l y s i s . o b j e c t i v e was gall-forming  A  the p r o d u c t i o n of p o l y c l o n a l a n t i s e r a a g a i n s t b a c t e r i a t o f a c i l i t a t e d e t e c t i o n and  o f i n o c u l a t e d b a c t e r i a i n the h o s t t i s s u e s .  third the  identification  14 MATERIALS AND  B a c t e r i a l S t r a i n s and  Growth  METHODS  Conditions:  The g a l l - f o r m i n g b a c t e r i a l i s o l a t e s a l l o r i g i n a t e d from D o u g l a s - f i r g a l l s c o l l e c t e d from f o u r l o c a t i o n s i n s o u t h w e s t e r n B r i t i s h Columbia.  Two s i t e s were on t h e s o u t h e r n t i p o f  Vancouver I s l a n d a t t h e edges of Lorna Lake and Cordova Bay near Victoria.  One s i t e was i n t h e Dunbar a r e a o f g r e a t e r  and t h e f i n a l s i t e was i n t h e Hope r e g i o n  Vancouver  ( T a b l e 1.1).  Except  f o r t h e Hope s i t e , i n which D o u g l a s - f i r was t h e dominant s p e c i e s , t h e s e a r e a s were m a r g i n a l growth a r e a s f o r D o u g l a s - f i r , e i t h e r i n mixed f o r e s t s o r i n urban a r e a s . The b a c t e r i a were grown on 0.01% c a s e i n h y d r o l y s a t e , peptone  ( D i f c o ) , 0.5% g l u c o s e (BDH)  (CPG media;  0.5%  Kelman,  1954)  pH 7.0 p l a t e s f o r q u i c k i d e n t i f i c a t i o n of t h e c h a r a c t e r i s t i c growth o f t h e i s o l a t e s .  N u t r i e n t agar ( D i f c o ) + 1% g l u c o s e  (NA/G media) pH 7.0 p l a t e s were used o c c a s i o n a l l y i f a s h o r t e r growth p e r i o d was r e q u i r e d .  A temperature o f 21-2 3°C was used  f o r growth o f t h e c u l t u r e s .  B r o t h c u l t u r e s were grown e i t h e r i n  L u r i a broth  (LB) (1.0% Tryptone [ D i f c o ] , 0.5% Y e a s t E x t r a c t  [ D i f c o ] , 0.8% NaCl) pH 7.0 B r o t h (NB)  ( M a n i a t i s e t al.,  1982) o r N u t r i e n t  ( D i f c o ) pH 7.0 a t room temperature o r i n 21°C  chambers on r o t a r y s h a k e r s (150 rpm).  Antibiotic  resistances  were t e s t e d t h r o u g h growth of t h e i s o l a t e ( s ) on NA/G containing f i l t e r - s t e r i l i z e d  growth  media  antibiotics.  B a c t e r i a l i s o l a t e s , A-0181, T-2739, T-2763 and T-2789, were s e n t away t o be i d e n t i f i e d by M i c r o b i a l ID, I n c . , Newark,  15 T a b l e 1.1. G a l l - f o r m i n g Erwinia i s o l a t e s and b a c t e r i a l used t o t e s t t h e s p e c i f i c i t y o f p o l y c l o n a l a n t i s e r a Bacterial species Family Enterobacteriaceae Erwinia isolates (gall-forming) Typical isolates T-2721 T-2722 T-2739 T-2763 T-2774 T-2789 Atypical isolate A-0181 Erwinia carotovora subsp. carotovora (Jones 1901) Bergey e t a l . , 1923 s t r a i n E379 Erwinia herbicola subsp. herbicola ( L o h n i s 1911) Dye 1964 s t r a i n 2273 Family Rhizobiaceae Agrobacterium pseudotsugae (Hansen and Smith, 1937) S a l v u l e s c u 1947 s t r a i n 180  Isolated by  Geographic location  Growth media 8  LB CPG Muehlchen, UBC Muehlchen, UBC Muehlchen, UBC DeYoung, UBC DeYoung, UBC DeYoung, UBC  Cordova Bay Cordova Bay Lorna Lake Vancouver Vancouver Vancouver  DeYoung, UBC  Hope LB NA/G  Copeman,  UBC  Creston LB NA/G  NCPPB #2273  LB CPG  NCPPB #180  Agrobacterium tumefaciens (Smith and Townsend, 1907) Conn 1942 s t r a i n B - l s t r a i n CH3  Copeman, UBC Dion, L a v a l  Vancouver Quebec  Rhizobium leguminosarum (Frank 1879) Frank 1889  Holl,  Vancouver  F a m i l y Pseudomonadaceae Pseudomonas syringae van H a l l 1902 c h e r r y strain  species  YDP NKS  UBC  Copeman,  UBC  Vancouver  YMB YMA LB NA/G  'The media used i n c l u d e d : L u r i a b r o t h ( L B ) , c a s e i n h y d r o l y s a t e p e p t o n e - g l u c o s e (CPG) (Kelman, 1954), N u t r i e n t agar ( D i f c o ) supplemented w i t h 1% g l u c o s e (NA/G), y e a s t - d e x t r o s e - p e p t o n e (YDP) (Moore, 1977), NKS ( B r i s b a n e and K e r r , 1983) and y e a s t m a n n i t o l b r o t h (YMB) ( V i n c e n t , 1970).  16 Delaware, USA, u s i n g t h e H e w l e t t P a c k a r d 5898A M i c r o b i a l I d e n t i f i c a t i o n System  (Miller,  1984).  A u t h e n t i c c u l t u r e s used i n t h e a n t i s e r a s p e c i f i c i t y are  l i s t e d i n T a b l e 1.1.  tests  A l l c h e m i c a l s used were from Sigma  C h e m i c a l Company u n l e s s o t h e r w i s e noted. Plant  materials:  D o u g l a s - f i r t r e e s used f o r p a t h o g e n i c i t y t e s t s were o b t a i n e d as 1 - y e a r - o l d s e e d l i n g s from year-end g r a d i n g a t t h e U n i v e r s i t y o f B r i t i s h Columbia F a c u l t y o f F o r e s t r y N u r s e r y .  The  v a r i o u s t r e e s p e c i e s used i n t h e h o s t range s t u d y were o b t a i n e d from Dr. R.S. Hunt a t t h e P a c i f i c F o r e s t R e s e a r c h C e n t e r i n V i c t o r i a , B.C. cups cm  3  S e e d l i n g s were p l a n t e d o u t i n l a r g e s t y r o f o a m  (600 ml) i n a s t a n d a r d s o i l mix w i t h 2400 cm  Osmocote, a slow r e l e a s e f e r t i l i z e r  0.16 m  3  of s t e r i l i z e d s o i l .  3  p e a t and 200  (14N-6.0P-11.6K), p e r  F e r t i l i z e r , 20-20-20, was a p p l i e d  once e v e r y two weeks a t a r a t e o f 0.06 g/L. Direct  Isolations/Inoculations:  D i r e c t i s o l a t i o n s were c a r r i e d out t h r o u g h m a c e r a t i o n o f g a l l t i s s u e i n a s m a l l amount o f LB b r o t h i n s t e r i l e d i m p l e plates.  No s u r f a c e s t e r i l i z a t i o n was n e c e s s a r y a l t h o u g h s u r f a c e  t i s s u e was removed w i t h s t e r i l e t o o l s i f t h e r e was a s u f f i c i e n t amount o f t i s s u e r e m a i n i n g t o sample. of  A f t e r 15-2 0 min, l o o p f u l s  t h e r e s u l t i n g s u s p e n s i o n were s t r e a k e d onto CPG p l a t e s .  C o l o n i e s appeared a f t e r 4 t o 5 days. A r t i f i c i a l l y - i n d u c e d g a l l s were produced t h r o u g h s t a b inoculations using straight pins.  Branches were i n o c u l a t e d w i t h  5 t o 7 - d a y - o l d b a c t e r i a l c o l o n i e s from CPG p l a t e s , c e n t i m e t e r s (cm) below t h e branch t i p .  3-5  The b r a n c h d i a m e t e r s  17 measured between 2-5 m i l l i m e t e r s (mm) a t t h e p o i n t o f inoculation.  The p i n s were o f t e n u n a v o i d a b l y  pushed t h r o u g h t h e  b r a n c h and hence t h r o u g h t h e s t e l e o f t h e b r a n c h . P o l y c l o n a l Antisera Production: L a t e l o g phase c u l t u r e s grown i n NB were h a r v e s t e d by c e n t r i f u g a t i o n (2100 X g (maximum) f o r 15 m i n ) , washed t h r e e t i m e s w i t h phosphate b u f f e r e d s a l i n e (PBS) pH 7.3 and a d j u s t e d i n PBS t o an o p t i c a l d e n s i t y o f 1.0 u n i t a t 660 nm.  The whole  c e l l s were f i x e d w i t h 2.0% g l u t a r a l d e h y d e by d i a l y s i s f o r 2 h and washed w i t h s e v e r a l changes o f PBS (pH 7.3) a t 4°C o v e r a 20 h p e r i o d ( A l l a n and Kelman, 1977).  The g l u t a r a l d e h y d e - f i x e d  whole c e l l p r e p a r a t i o n s were t h e n mixed 1:1 w i t h Freund's complete a d j u v a n t  (Difco).  Two m i l l i l i t e r  (ml) a l i q u o t s were  i n j e c t e d i n t r a m u s c u l a r l y i n t o t h e h i n d l e g s o f New Zealand rabbits.  A schedule  white  o f 5 weekly i n j e c t i o n s was f o l l o w e d by t e s t  b l e e d i n g s 10 days a f t e r t h e l a s t i n j e c t i o n .  Booster  s h o t s were  a d m i n i s t e r e d once a month. B l e e d i n g s were done e i t h e r by n i c k i n g t h e m a r g i n a l v e i n o r by s e d a t i n g t h e r a b b i t w i t h Innovar V e t and i n s e r t i n g a c a t h e t e r i n t o t h e a u r i c l e a r t e r y (10-20 ml p e r b l e e d i n g ) .  Whole b l o o d  was a l l o w e d t o c l o t by p l a c i n g a t 37°C f o r 1 h and t h e n a t 4°C overnight.  The samples were t h e n c e n t r i f u g e d a t 2100 X g  (maximum) f o r 10 min a t 4°C.  The s u p e r n a t a n t s  were poured o f f  and s t o r e d i n a l i q u o t s a t -20°C o r a t 4°C w i t h 0.02% sodium a z i d e as a p r e s e r v a t i v e .  18 IgG  Purification: The  IgG f r a c t i o n o f t h e a n t i s e r u m was p u r i f i e d by a  modified  C l a r k and Adams method (1977).  One ml o f whole  a n t i s e r u m was d i l u t e d w i t h 9 ml o f d i s t i l l e d w a t e r and mixed w i t h 10 ml o f s a t u r a t e d ammonium s u l f a t e a t room t e m p e r a t u r e f o r 30 min. The p r e c i p i t a t e was c e n t r i f u g e d o u t a t 2100 X g (maximum) a t 10°C.  The p e l l e t was t h e n resuspended i n 2 ml o f  h a l f - s t r e n g t h PBS.  The r e s u l t i n g sample was t h e n r u n t h r o u g h a  DEAE-cellulose  column p r e - e q u i l i b r a t e d w i t h h a l f - s t r e n g t h PBS  and m o n i t o r e d a t 254 nm w i t h an ISCO Model UA-4 Absorbance Monitor.  The f i r s t major peak was c o l l e c t e d .  Protein  c o n c e n t r a t i o n was measured on a H e w l e t t P a c k a r d s p e c t r o p h o t o m e t e r a t 2 54 nm assuming an e x t i n c t i o n c o e f f i c i e n t o f 1.4. Antisera  Sensitivity:  T e s t i n g o f t h e s p e c i f i c i t y t h e whole a n t i s e r a and t h e IgG f r a c t i o n s were c a r r i e d o u t u s i n g an i n d i r e c t ELISA ( V o l l e r e t al.,  1979).  T h i s assay i n v o l v e d t h e i n c u b a t i o n o f Erwinia  d i r e c t l y i n t h e m i c r o p l a t e w e l l s (200 j u l / w e l l ) .  cells  Microtiter  p l a t e s ( T i t e r t e k ) were p r e v i o u s l y s t e r i l i z e d f o r 15 min under a UV l i g h t  (General  antigens  were used a c o n c e n t r a t i o n  o f 5 X 10 c e l l s / m l i n PBS pH  7.3 ( A  = 0. 14 f o r A-0181 and A  = 0 . 28 f o r T-2789) .  660  E l e c t r i c G25T8 25 W g e r m i c i d a l lamp).  660  The  8  After a  1 h i n c u b a t i o n s t e p a t room temperature, t h e w e l l s were emptied w i t h two, 10-sec r i n s e s o f t a p water from a homemade p l a t e washer.  A 45-min b l o c k i n g s t e p u s i n g 200 A i l / w e l l  Carnation (Ellis,  'Blotto'  (0.1%  I n s t a n t Skim M i l k Powder d i l u t e d i n PBS pH 7.3)  1988) f o l l o w e d a t room temperature.  P l a t e s were dumped  19 (not r i n s e d ) and t h e a n t i s e r u m d i l u t e d i n PBS p l u s 0.05% Tween 20 and 0.1% B l o t t o (200 ^ u l / w e l l ) was i n c u b a t e d f o r 1 h a t room temperature.  An a n t i s e r u m d i l u t i o n s e r i e s (one h u n d r e d - f o l d  d i l u t i o n s between 10" and 10" ) was r e p l i c a t e d a t l e a s t t h r e e 1  times.  12  Two, 10-sec washes removed unbound  Goat a n t i - r a b b i t a l k a l i n e phosphatase  immunoglobulins.  enzyme c o n j u g a t e d i l u t e d 1  i n 1000 w i t h PBS/Tween-2 0 / B l o t t o , 200 ^ u l / w e l l , was i n c u b a t e d a t room t e m p e r a t u r e f o r 1 h.  Two, 10-sec washes were f o l l o w e d by  a d d i t i o n o f t h e s u b s t r a t e , p - n i t r o p h e n y l phosphate,  diluted  0.6  mg/ml i n s u b s t r a t e b u f f e r (10% d i e t h a n o l a m i n e ; pH 9.8). Absorbance  r e a d i n g s a t 405 nm were t a k e n on a T i t e r t e k M u l t i s k a n  p l a t e r e a d e r , e i t h e r 30 o r 45 min, a f t e r s u b s t r a t e a d d i t i o n . Specificity:  Immunodiffusion:  A n t i s e r u m s p e c i f i c i t y was t e s t e d u s i n g O u c h t e r l o n y d o u b l e diffusion  (3 mm d i a m e t e r w e l l s were spaced 4 t o 5 mm a p a r t ) (De  Boer e t a l . , 1979).  B a c t e r i a l c e l l s , t a k e n from 1 t o 2-week o l d  c u l t u r e s , were suspended t u r b i d suspensions.  i n s t e r i l e d i s t i l l e d water t o form v e r y  Whole a n t i s e r a o r IgG f r a c t i o n s were added  u n d i l u t e d t o t h e m i d d l e w e l l s and t h e r e a c t i o n s were a l l o w e d t o p r o c e e d f o r 16-24 h a t room temperature. S e v e r a l d i f f e r e n t p h y t o p a t h o g e n i c b a c t e r i a were a l s o t e s t e d t o determine the s p e c i f i c i t y of the a n t i s e r a : carotovora  subsp. carotovora,  s t r a i n 2273, Agrobacterium tumefaciens trifolii),  E. herbicola  pseudotsugae  s t r a i n B - l , Rhizobium Pseudomonas  syringae  Erwinia  subsp.  herbicola  s t r a i n 180, A.  leguminosarum  (formerly  (cherry s t r a i n ) , along w i t h the  a t y p i c a l i s o l a t e A-0181 and two t y p i c a l i s o l a t e s , T-2789 and T-2722.  20 Specificity:  I n d i r e c t ELISA:  An i n d i r e c t ELISA as o u t l i n e d above f o r t h e d e t e r m i n a t i o n o f t h e a n t i s e r a s e n s i t i v i t y was s p e c i f i c i t y of t h e a n t i s e r a . t e s t e d by i m m u n o d i f f u s i o n  a l s o used t o d e t e r m i n e  the  The p h y t o p a t h o g e n i c b a c t e r i a  were a l s o used i n t h e i n d i r e c t ELISAs  e x c e p t f o r Agrobacterium  pseudotsugae.  T e s t s i n v o l v i n g t h e whole T-2789 a n t i s e r u m were c a r r i e d w i t h a s i n g l e c o n c e n t r a t i o n o f c e l l s , 5-7  X 10  1 0 - f o l d d i l u t i o n s of a n t i s e r u m r a n g i n g from 10" r e p l i c a t i o n s per d i l u t i o n ) .  Experiments  cells/ml,  8  2  t o 10"  out  and  (4  6  w i t h the p u r i f i e d  IgG  f r a c t i o n s were c a r r i e d out w i t h v a r y i n g c o n c e n t r a t i o n s o f c e l l s and a s i n g l e c o n c e n t r a t i o n of a n t i s e r u m . 10 , 5 X 10 , 5 X 10 , 5 X 10 , 5 X 10 8  7  6  5  4  Concentrations of 5 X  c e l l s per ml were used f o r  t h e i n d i r e c t ELISAs w i t h 4 r e p l i c a t i o n s per c e l l c o n c e n t r a t i o n per b a c t e r i a l s t r a i n . T-2789 and 10"  3  The a n t i s e r u m d i l u t i o n s used were 10"  4  for  f o r A-0181.  Host Range: A h o s t range study was tree species:  Abies  (Dougl. ex D. Don) Chamaecyparis N u t t . , Picea  c a r r i e d out i n v o l v i n g t h e  amabilis  Dougl. ex Forbes, A.  L i n d l . , A. lasiocarpa  nootkatensis engelmannii  (D. Don)  Spach, Larix  Loud., P. monticola  Dougl. ex D. Don,  ex D. Don  occidentalis  P a r r y ex. Engelm., P. glauca  (Bong.) C a r r , Pinus  menziesii  grandis  (Hook.) N u t t . ,  V o s s , P. sitchensis  Laws, Pseudotsuga  following  contorta  (Moench)  Dougl.  P. ponderosa  ex  Dougl.  ex  (Mirb.) Franco, Thuja p l i c a t a Dpnn  and Tsuga heterophylla  (Raf.) Sarg.  Three t y p i c a l  i s o l a t e s T-2789, T-2722 and T-2774 p l u s t h e a t y p i c a l A-0181 i s o l a t e were t e s t e d f o r p a t h o g e n i c i t y on t h e v a r i o u s c o n i f e r s .  Two t r e e s isolate.  (2 b r a n c h e s / t r e e ) were s t a b i n o c u l a t e d p e r  O b s e r v a t i o n s o f t h e i n o c u l a t i o n s i t e s were r e c o r d e d  a f t e r 5 months as e i t h e r p o s i t i v e o r n e g a t i v e f o r g a l l i n g .  A  p o s i t i v e r e s u l t was r e c o r d e d i f a t l e a s t one o f t h e f o u r inoculation s i t e s displayed  visible galls.  Direct isolations  were made onto CPG media from two s i t e s p e r t r e e s p e c i e s p e r isolate.  R e s u l t s were r e c o r d e d as p e r c e n t r e c o v e r y o f  c h a r a c t e r i s t i c b a c t e r i a from t h e two s i t e s .  Characteristic  b a c t e r i a were t e s t e d by double d i f f u s i o n u s i n g b o t h whole and purified antisera.  Bacteria  i d e n t i f i e d as t h e i n o c u l a t e d  gall-  f o r m i n g b a c t e r i a , by i m m u n o d i f f u s i o n , were b a c k - i n o c u l a t e d i n t o Douglas-fir  trees to t e s t f o r pathogenicity.  s i t e s p e r t r e e s p e c i e s were used p e r i s o l a t e .  Four  inoculation  R e s u l t s were  r e c o r d e d as t h e p e r c e n t o f s i t e s which produced v i s i b l e g a l l i n g .  22 RESULTS  Description  of G a l l s and  Isolates:  The D o u g l a s - f i r g a l l s were i n f a i r l y  h i g h numbers w i t h i n  l o c a l i z e d m a r g i n a l growth a r e a s , a l t h o u g h a s u r v e y o f f r e q u e n c y of o c c u r r e n c e was not conducted.  The g a l l s were most o f t e n  found on t h e t i p s of branches of mature t r e e s .  The  galls  appeared t o be i n i t i a t e d s i m p l y a l o n g t h e l e n g t h o f t h e s h o o t ( F i g . 1.1a)  new  o r a t t h e p o i n t where t h e new y e a r ' s growth  b e g i n s ( F i g . 1.1b).  O l d e r g a l l s were observed i n t h e l o w e r dead  b r a n c h e s o f 10-15 y e a r o l d t r e e s .  Most o f t h e n a t u r a l  were l o c a t e d on branches measuring between 0.2  and 0.8  galls cm i n  d i a m e t e r w i t h t h e g a l l s measuring between 0.5 and 2 cm i n d i a m e t e r ( F i g . 1.1c).  The s i n g l e specimen o f t h e a t y p i c a l g a l l  c o l l e c t e d from t h e Hope s i t e was t a k e n from a b r a n c h a p p r o x i m a t e l y 4.0 cm i n d i a m e t e r ( F i g . l . l d ) .  The  measuring  galls  appeared, i n g e n e r a l , t o be i n i t i a t e d a t one p o i n t on t h e branch and t o e r u p t o r expand outward e n v e l o p i n g p a r t o f t h e b r a n c h . Two  d i f f e r e n t D o u g l a s - f i r g a l l t y p e s were c o l l e c t e d .  d i f f e r e n c e s were more apparent when a r t i f i c i a l l y g a l l s were observed.  The  inoculated  The more commonly e n c o u n t e r e d g a l l s were,  i n g e n e r a l , g l o b o s e i n n a t u r e w i t h a rough s u r f a c e caused by t h e a p p a r e n t l o c a l i z e d overgrowth of i s o l a t e d groups o f c e l l s ( F i g . 1.2a). the  The d i s t i n c t i v e n e s s of t h e s e outgrowths v a r i e d w i t h i n  t y p i c a l g a l l s b o t h i n n a t u r a l and a r t i f i c i a l l y - i n o c u l a t e d  galls  ( F i g . 1.2b).  The s u r f a c e of t h e s e outgrowths were  c h a r a c t e r i s t i c a l l y marked by a s i n g l e o r r a r e l y a c r o s s - s h a p e d  23  F i g . 1.1. The c h a r a c t e r i s t i c l o c a t i o n , a p p e a r a n c e a n d s i z e o f naturally-occurring g a l l s o f D o u g l a s - f i r p r o d u c e d by t y p i c a l i s o l a t e s ( A , B , C ) a n d t h e a t y p i c a l i s o l a t e A-0181 (D) o f t h e Douglas-fir gall-forming bacteria (actual s i z e ) .  24  F i g . 1.2. T y p e s o f g a l l s f o r m e d on D o u g l a s - f i r t r e e s a r t i f i c i a l l y i n o c u l a t e d w i t h t h e two d i f f e r e n t i s o l a t e t y p e s o f the D o u g l a s - f i r g a l l b a c t e r i a . T y p i c a l g a l l s formed, a f t e r 2 m o n t h s (A and B) and a f t e r 2 y e a r s ( C ) , by i s o l a t e s t e n t a t i v e l y i d e n t i f i e d a s Erwinia s a l i c i s ; a t y p i c a l g a l l s formed, a f t e r 2 m o n t h s (D) and a f t e r 2 y e a r s ( E ) , by an i s o l a t e t e n t a t i v e l y i d e n t i f i e d a s Erwinia herbicola subsp. herbicola.  fissure.  As t h e g a l l matured,  o b v i o u s ( F i g . 1.2c).  t h i s marking was o f t e n l e s s  A t y p i c a l D o u g l a s - f i r g a l l s were  c h a r a c t e r i z e d by b e i n g g l o b o s e t o oblong i n shape w i t h a smoother,  b a r k - l i k e s u r f a c e t h a t c r a c k e d and p e e l e d s l i g h t l y  t h e g a l l expanded ( F i g . 1.2d).  The a t y p i c a l g a l l s appeared  as to  a f f e c t t h e s t r u c t u r e of t h e branch t o a g r e a t e r degree t h a n t h e typical galls.  The a t y p i c a l g a l l s o f t e n d i s t o r t e d t h e b r a n c h  both i n the r e s u l t a n t d i r e c t i o n t h e b r a n c h below t h e g a l l .  of growth and i n t h e s w e l l i n g o f  D o u g l a s - f i r g a l l s of both  isolates  were i n i t i a l l y l i g h t green i n c o l o r but as t h e y d e v e l o p e d matured,  and  t h e g a l l s t u r n e d brown and o f t e n l o s t t h e d i s t i n c t i v e  markings as noted f o r t h e younger g a l l s  ( F i g 1.2e).  The  older  a r t i f i c i a l l y i n o c u l a t e d g a l l s resembled t h e g a l l s from which t h e i n i t i a l i s o l a t i o n s were made. G a l l s c o u l d be a r t i f i c i a l l y - i n d u c e d t h r o u g h s t a b i n o c u l a t i o n s on most branches of 1 - y e a r - o l d D o u g l a s - f i r s e e d l i n g s as w e l l as a l o n g t h e l e n g t h of t h e main stem ( F i g . 1.3a). 2.0  Some g a l l s induced on l a t e r a l branches r e a c h e d a s i z e of  cm o r more i n d i a m e t e r f o r both of t h e i s o l a t e t y p e s but  u s u a l l y g a l l s r e a c h e d o n l y a s i z e of 0.5 t o 1 cm i n d i a m e t e r . The r a t i o o f g a l l d i a m e t e r t o branch d i a m e t e r a f t e r 4 weeks, was g r e a t e r t h a n 3.1 and 2.2 respectively.  f o r t h e t y p i c a l and a t y p i c a l  isolates,  The t y p i c a l i s o l a t e s produced f a s t e r - g r o w i n g  g a l l s r e a c h i n g a d i a m e t e r of 0.5 cm i n 4 weeks w h i l e t h e atypical  g a l l s took 6 weeks t o r e a c h a s i m i l a r  diameter.  The p r e s e n c e of t h e g a l l i n g symptoms on t h e h o s t a f f e c t e d t h e h e a l t h and s t r u c t u r a l  i n t e g r i t y of t h e h o s t t r e e .  Death of  26  F i g . 1.3. G a l l s formed on 1- t o 2-year o l d D o u g l a s - f i r t r e e s by s t a b - i n o c u l a t i o n with D o u g l a s - f i r g a l l - f o r m i n g b a c t e r i a . (A) g a l l produced on main stem; (B) g a l l s produced w i t h the a t y p i c a l and (C) t y p i c a l i s o l a t e s causing d i s t o r t i o n of i n o c u l a t e d branches; (D) i n s e c t f e e d i n g a c t i v i t y on an a t y p i c a l g a l l ; (E) regrowth of g a l l t i s s u e a f t e r i n s e c t f e e d i n g and (F) apparent spread of g a l l bacterium from i n o c u l a t i o n s i t e (arrow) t o new s i t e on a D o u g l a s - f i r s e e d l i n g .  t h e i n o c u l a t e d branch o r s i m p l y t h e t i p s o f t e n o c c u r r e d artificial  inoculation.  after  I n o c u l a t i o n o f t h e upper main stem o f  young D o u g l a s - f i r s e e d l i n g s , l e s s t h a n 6 months o l d , o f t e n r e s u l t e d i n death of the seedlings.  Well-established trees (1-  y e a r - o l d o r o l d e r ) d i d n o t appear t o be s e v e r e l y a f f e c t e d a l t h o u g h t h e t r e e s sometimes became deformed ( F i g . 1.3b). b r a n c h t i p s were i n i t i a t e d below t h e g a l l e d t i p o f t e n  New  distorting  t h e growth p a t t e r n o f t h e t r e e ( F i g . 1.3c). I n a r t i f i c i a l l y - i n o c u l a t e d , greenhouse-grown t r e e s , g a l l s appeared t o s t o p growth a f t e r t h e f i r s t y e a r .  The s u c c u l e n t  g a l l t i s s u e was an a t t r a c t a n t t o i n s e c t s ( F i g . 1.3d). often continued 1.3e).  Galls  t o grow a f t e r b e i n g e a t e n by i n s e c t s ( F i g .  A curious observation  of the a r t i f i c i a l l y - i n d u c e d  galls  was t h a t o c c a s i o n a l l y s m a l l g a l l s were produced a t l o c a t i o n s other than the i n o c u l a t i o n s i t e  (Fig. 1.3f).  T h i s would s u g g e s t  e i t h e r t h a t t h e pathogen was t r a n s p o r t e d e x t e r n a l l y v i a i n s e c t s o r i n t e r n a l l y t h r o u g h t h e p l a n t ' s v a s c u l a r system. From t h e two t y p e s o f g a l l s , two d i s t i n c t s t r a i n s o f t h e g a l l - f o r m i n g b a c t e r i a were i s o l a t e d .  Simple d i r e c t i s o l a t i o n  from young g a l l t i s s u e r e s u l t e d i n l a r g e numbers o f t h e i n c i t i n g bacteria.  When grown on s o l i d media c o n t a i n i n g  hydrolysate,  peptone and g l u c o s e ,  casein  t h e common s t r a i n s , t y p i f i e d  by t h e T-2789 i s o l a t e , formed s h i n y , g r e y i s h - w h i t e convex c o l o n i e s w i t h e n t i r e margins ( F i g . 1.4a). have c h a r a c t e r i s t i c s u r f a c e i n d e n t a t i o n s .  circular The c o l o n i e s  The a t y p i c a l A-0181  i s o l a t e formed c o l o n i e s o f s i m i l a r c o l o r and t r a n s l u c e n c e ( F i g . 1.4b), however t h e shape o f t h e c o l o n i e s d i f f e r e d .  Young A-0181  28  Fig. 1.4. C o l o n i e s of the t y p i c a l (A) and the a t y p i c a l (B) g a l l - f o r m i n g Erwinia i s o l a t e s grown on c a s e i n h y d r o l y s a t e , peptone and g l u c o s e medium.  29 c o l o n i e s were convex i n appearance a l t h o u g h as t h e c o l o n i e s matured, d e p r e s s i o n s were formed i n t h e c e n t e r o f t h e c o l o n i e s . E x t r a c e l l u l a r s l i m e was a l s o o f t e n produced  as t h e c o l o n i e s  matured c a u s i n g t h e c o a l e s c e n c e of c l o s e l y spaced g i v i n g t h e c o l o n i e s an e l l i p t i c a l appearance. g a l l - f o r m i n g i s o l a t e s measured between 1.0 a f t e r 5-7  days on s o l i d CPG media.  colonies,  C o l o n i e s of both  t o 2.0 mm  Colonies of the  i n diameter atypical  i s o l a t e s grew s l i g h t l y f a s t e r t h a n t h e t y p i c a l i s o l a t e s a p p e a r i n g a f t e r a p p r o x i m a t e l y 2 days a t 21-2 3°C on s o l i d media. One was  i m p o r t a n t c h a r a c t e r i s t i c o f t h e two b a c t e r i a l  t h e i r temperature  s e n s i t i v i t y (Table 1.2).  The  isolates  typical  i s o l a t e s were a b l e t o s u r v i v e o n l y a t t e m p e r a t u r e s  up t o  3 2±0.5°C i n L u r i a b r o t h .  able to  The a t y p i c a l i s o l a t e was  s u r v i v e a t s l i g h t l y e l e v a t e d temperatures Luria broth.  o f up t o 34±0.5°C i n  The a t y p i c a l i s o l a t e , however, d i d not s u r v i v e a t  room t e m p e r a t u r e  on s o l i d CPG media f o r more t h a n 10 days.  The  t y p i c a l i s o l a t e s s u r v i v e d under s i m i l a r c o n d i t i o n s f o r 2 weeks o r more.  The two i s o l a t e s d i f f e r e d i n t h e i r a n t i b i o t i c  r e s i s t a n c e s (Table 1.3).  The t y p i c a l i s o l a t e s were r e s i s t a n t t o  a w i d e r range o f a n t i b i o t i c s i n c l u d i n g kanamycin and  a m p i c i l l i n , erythromycin,  tetracycline.  When f a t t y a c i d a n a l y s i s and c o m p u t e r - a s s i s t e d  comparison  w i t h a l i b r a r y o f a u t h e n t i c s t r a i n s was used t o t e n t a t i v e l y i d e n t i f y r e p r e s e n t a t i v e i s o l a t e s , s i m i l a r i t y i n d i c e s of g r e a t e r t h a n 0.500 p l a c e d b o t h of them u n e q u i v o c a l l y i n t h e genus E r w i n i a i n the f a m i l y Enterobacteriaceae.  The a t y p i c a l  gall-  f o r m i n g i s o l a t e A-0181 was p o s i t i v e l y p l a c e d i n t h e E r w i n i a  30  T a b l e 1.2 Temperature s e n s i t i v i t y o f t h e g a l l - f o r m i n g Erwinia isolates Temperature (+/- 0.5°C) 31 32 33 34 35  Growth i n L u r i a B r o t h Erwinia  T-2789 + + -  E r w i n i a A-0181 + + + + -  31  T a b l e 1.3. A n t i b i o t i c r e s i s t a n c e s e x p r e s s e d byr e p r e s e n t a t i v e t y p i c a l and a t y p i c a l i s o l a t e s o f t h e Douglas-fir gall-forming bacteria Antibiotic  Concentration (Aig/ml)  Typical  Atypical  30 40  + +  _a  Carbenicillin  40 50  + +  trace trace  Chloramphenicol  10  -  Erythromycin  30  +  -  Fusidic acid  31 75  + +  + +  Kanamycin  40 50  trace trace  —  Methicillin  28  +  +  Novobiocin  75  +  +  Oleandomycin  25  +  trace  Streptomycin  25  -  -  Tetracycline  12 . 5  +  -  Ampicillin  a  Growth o f g a l l - f o r m i n g bacterial isolates  Grown on NA i n s t e a d o f  NA/G  —  a  —  3  32 herbicola  subsp. herbicola  group.  The t y p i c a l i s o l a t e s were  matched most c l o s e l y w i t h t h e E. salicis Antisera  group (Table 1.4).  Sensitivity:  The c u t o f f v a l u e f o r ELISA v a l u e s was chosen t o be 0.1 o p t i c a l d e n s i t y u n i t (A ) , which was g r e a t e r t h a n t w i c e t h e 405  background v a l u e s ( S u t u l a e t al., 1986).  The lower l i m i t s o f  d e t e c t i o n o f t h e whole a n t i s e r a , as determined  by an i n d i r e c t  c e l l ELISA were 10" and 10" f o r T-2789 and A-0181, r e s p e c t i v e l y . 6  3  The p r o t e i n c o n c e n t r a t i o n s f o r t h e p u r i f i e d IgG f r a c t i o n were 1.86  mg/ml f o r A-0181 and 1.0 mg/ml f o r T-2789.  The lower  l i m i t s o f d e t e c t i o n f o r t h e IgG f r a c t i o n s were 10~ and 10" f o r 4  3  T-2789 and A-0181, r e s p e c t i v e l y ( F i g . 1.5). Specificity  -Immunodiffusion:  The whole T-2789 a n t i s e r u m produced two major p r e c i p i t i n l i n e s w i t h i t s homologous a n t i g e n and each o f t h e t y p i c a l isolates  ( F i g . 1.6). One o f t h e s e l i n e s formed a l i n e o f  p a r t i a l i d e n t i t y w i t h t h e s i n g l e l i n e o f i d e n t i t y produced i n r e a c t i o n w i t h t h e A-0181 i s o l a t e . o b s e r v e d w i t h Agrobacterium pseudotsugae,  Erwinia  No p r e c i p i t i n l i n e s were  tumefaciens,  carotovora,  herbicola  s t r a i n 2273, Escherichia  Rhizobium  leguminosarum.  Erwinia coli,  Agrobacterium herbicola Pseudomonas  subsp. syringae  or  P u r i f i c a t i o n o f t h e T-2789 a n t i s e r u m  removed t h e c r o s s r e a c t i o n w i t h i s o l a t e A-0181 observed w i t h t h e whole a n t i s e r u m  ( F i g . 1.6).  Both t h e whole A-0181 a n t i s e r u m and p u r i f i e d A-0181 IgG f r a c t i o n formed l i n e s o f i d e n t i t y o n l y w i t h t h e i r homologous antigens  ( F i g . 1.6). P u r i f i c a t i o n o f t h e A-0181 a n t i s e r u m  33  T a b l e 1.4. T e n t a t i v e i d e n t i f i c a t i o n o f D o u g l a s - f i r g a l l - f o r m i n g b a c t e r i a l i s o l a t e s by a n a l y s i s o f t h e i r f a t t y a c i d p r o f i l e s and c o m p u t e r - a s s i s t e d comparison w i t h a l i b r a r y o f p r o f i l e s from a u t h e n t i c s t r a i n s Isolate  T-2739  Possible  Hafnia  Erwinia Erwinia  identities  alvei  salicis herbicola  subsp.  T-2763  Erwinia  Hafnia Erwinia  Erwinia Hafnia Erwinia  salicis  herbicola  salicis  alvei herbicola  subsp.  A-0181  herbicola  alvei herbicola  subsp.  T-2789  S i m i l a r i t y index w i t h known strains  herbicola  E r w i n i a herbicola subsp. herbicola  Enterobacter  agglomerans  0.635  0.593 0.544  0.767  0.719 0.597  0.648  0.644 0.558  0.840 0.545  34  0.8  Antiserum  dilution  F i g . 1.5. S e n s i t i v i t y of the IgG f r a c t i o n s of the a n t i s e r a r a i s e d a g a i n s t the t y p i c a l T-2789 (•) and A-0181 ( + ) i s o l a t e s of t h e D o u g l a s - f i r g a l l - f o r m i n g b a c t e r i a as determined by an i n d i r e c t ELISA a g a i n s t t h e i r r e s p e c t i v e homologous a n t i g e n s . Each p o i n t r e p r e s e n t s the mean of f o u r r e p l i c a t e s .  35  «  ©  o  A ° ~ © © © ° ^\ © ©"wo © © ©> © ©© © © o © © O © © e © © o ©© o © ~ ©© o ©_©© o © © o o © o 0  ©  U °  0  Q  ©  o  o  ©^©© © © ^ © ©^ ©© ^ © © © © o © © - © o © © © ©© © © © © ffi © © * ° ° ©% ° »© © © ° © © o m  °  ©  F i g . 1.6. I m m u n o d i f f u s i o n p a t t e r n s p r o d u c e d by r e a c t i o n s b e t w e e n a n t i s e r a e l i c i t e d a g a i n s t t h e (T) t y p i c a l T - 2 7 8 9 a n d t h e (A) a t y p i c a l A - 0 1 8 1 g a l l - f o r m i n g Erwinia i s o l a t e s and s e v e r a l phytopathogenic bacteria. Crude (c) and p u r i f i e d (p) a n t i s e r a were t e s t e d a g a i n s t (1) Erwinia T - 2 7 8 9 , (2) Erwinia A - 0 1 8 1 , (3) Erwinia T - 2 7 2 1 , (4) Erwinia T - 2 7 2 2 , (5) Erwinia T - 2 7 3 9 , (6) Erwinia T - 2 7 6 3 , (7) Erwinia T - 2 7 7 4 , (8) Agrobacterium  pseudotsugae, (9) Agrobacterium tumefaciens, (10) Erwinia carotovora s u b s p . carotovora, ( 1 1 ) Erwinia herbicola strain 2 2 7 3 , ( 1 2 ) Escherichia coli s t r a i n M C 1 0 0 0 , ( 1 3 ) Pseudomonas syringae a n d ( 1 4 ) Rhizobium leguminosarum.  36 r e s u l t e d i n t h e r e d u c t i o n of t h e number of p r e c i p i t i n  lines,  a g a i n s t i t s homologous a n t i g e n , from two t o a s i n g l e l i n e . p r e c i p i t i n l i n e s were observed w i t h any of t h e  other  p h y t o p a t h o g e n i c b a c t e r i a i n c l u d i n g Agrobacterium Agrobacterium  pseudotsugae  No  tumefaciens,  or even t h e g a l l - f o r m i n g t y p i c a l  isolates. S p e c i f i c i t y - I n d i r e c t ELISA: For t h e whole T-2789 a n t i s e r u m , a d d i t i o n , a d i l u t i o n of 10"  10~  4  I s o l a t e A-0181 was  d i l u t i o n s out t o 10" .  w h i l e Agrobacterium  tumefaciens  i s o l a t e CH3  and  b a c t e r i a l spp. t e s t e d e x c e p t f o r A-0181 and A.  antiserum  h.  Each  tumefaciens  d e t e c t e d w i t h a 1 0 - f o l d h i g h e r d i l u t i o n of  a f t e r 27  h.  For t h e p u r i f i e d T-2789 a n t i s e r u m ,  a t 2.5  h after  a d d i t i o n , t h e o n l y b a c t e r i a l s p e c i e s d e t e c t e d was a n t i g e n , down t o a d i l u t i o n of 5 X 10 T-2789 c e l l d i l u t i o n of 5 X 10 22 h.  Rhizobium  Pseudomonas  not d e t e c t e d a t any d i l u t i o n a f t e r 1.5  was  at  d e t e c t e d a t a n t i s e r u m d i l u t i o n s out t o  3  i s o l a t e CH3  isolates  detected  t r i f o l i i were d e t e c t e d a t d i l u t i o n s out t o 10" . s y r i n g a e was  substrate  E r w i n i a c a r o t o v o r a subsp.  3  c a r o t o v o r a s t r a i n 379 was  h after  detected only the t y p i c a l  5  T-2789 and T-2722 (Table 1.5). antiserum  a t 1.5  5  6  cells/ml  c e l l s p e r ml was  A f t e r 22 h t h e t y p i c a l i s o l a t e T-2722 was  a t a c o n c e n t r a t i o n of 5 X 10  8  substrate  t h e homologous  (Table 1.6). detected  after  also detected  cells/ml.  The A-0181 p u r i f i e d a n t i s e r u m was  a l s o very  specific  d e t e c t i n g o n l y i t s homologous a n t i g e n , a t a d i l u t i o n of 5 X c e l l s / m l , a f t e r 2.0  A  h (Table 1.7).  A f t e r 27 h an A-0181  10  7  37  T a b l e 1.5. S p e c i f i c i t y o f whole Erwinia T-2789 a n t i s e r u m i n an i n d i r e c t ELISA. B a c t e r i a l c o n c e n t r a t i o n s o f 5-6 X 1 0 c e l l s / m l were used. The A v a l u e s f o r each b a c t e r i a l s t r a i n were r e a d a t two t i m e s a f t e r s u b s t r a t e a d d i t i o n . P o s i t i v e ELISA v a l u e s were above 0.10 8  405  A a t time i n d i c a t e d f o r antiserum d i l u t i o n s 405  Bacterial strain  Time (h)  T-2789 (grown i n LB)  1. 5 27. 5  0.25 1. 58  0.25 1. 62  0. 33 1. 87  0. 12 1. 04  0. 00 0. 01  T-2789 (grown i n NB)  1. 5 27. 5  0.28 1. 72  0.25 1. 72  0. 35 1. 95  0. 23 1. 63  0. 00 0.23  Erwinia  T-2722  1. 5 27. 5  0. 38 1. 98  0. 39 1. 98  0. 56 1. 95  0. 38 1. 63  0. 00 0. 23  Erwinia  A-0181  1. 5 27. 5  0. 11 0.91  0.11 0.98  0.00 0. 00  0. 00 0. 00  0. 00 0. 00  1. 5 27. 5  0. 62 2. 00  0.50 2. 00  0. 12 1. 87  0. 00 1. 04  0. 00 0. 00  1. 5 27. 5  1. 73 2 .00  0. 32 1. 82  0. 01 0.47  0. 00 0. 01  0. 00 0. 00  Pseudomonas syringae  1. 5 27. 5  0. 09 0.81  0.00 0. 04  0. 00 0. 00  0. 00 0. 00  0. 00 0. 00  Rhizobium 1eguminosarum  1. 5 27. 5  0.61 2 .00  0.21 1. 53  0. 00 0. 21  0. 00 0. 04  0. 00 0. 00  Erwinia Erwinia  Erwinia carotovora Agrobacterium tumefaciens  a  3 79 CH3  10" a  mean o f 4 r e p l i c a t i o n s  2  10"  3  10"  4  10"  5  10  38  Table 1.6. S p e c i f i c i t y o f t h e p u r i f i e d IgG f r a c t i o n o f t h e Erwinia T - 2 7 8 9 a n t i s e r u m a t a d i l u t i o n o f 10" i n an i n d i r e c t ELISA. The A v a l u e s f o r e a c h b a c t e r i a l s t r a i n were r e a d a t two t i m e s a f t e r s u b s t r a t e a d d i t i o n . P o s i t i v e E L I S A v a l u e s were above 0.10 4  4 0 5  A for c e l l s / m l at time i n d i c a t e d 4 0 5  Bacterial strain  Time (h) 5 X 10  Erwinia  T-2789  2.5 22.0  0.27 0.85  Erwinia  T-2722  2.5 22.0  Erwinia  A-0181  8  5 X 10  7  5 X 10  6  5 X 10  5  5 X 10  0.24 0.78  0.18 0.61  0.03 0.15  0.00 0.00  0.03 0.18  0.05 0.04  0.00 0.06  0.00 0.00  0.00 0.00  2.5 22.0  0.00 0.05  0.00 0.00  0.00 0.00  0.00 0.00  0.00 0.00  2273  2.5 22.0  0.00 0.04  0.00 0.00  0.00 0.00  0.00 0.00  0.00 0.00  379  2.5 22.0  0.00 0.01  0.00 0.00  0.00 0.00  0.00 0.00  0.00 0.00  2.5 22.0  0.00 0.00  0.00 0.00  0.00 0.06  0.00 0.00  0.00 0.00  Pseudomonas syringae  2.5 22.0  0.00 0.00  0.00 0.00  0.00 0.00  0.00 0.00  0.00 0.00  Rhizobium leguminosarum  2.5 22.0  0.00 0.00  0.00 0.00  0.00 0.00  0.00 0.00  0.00 0.00  Erwinia herbicola Erwinia carotovora Agrobacterium tumefaciens  'mean o f  4  B-l  replications  a  4  39  T a b l e 1.7. S p e c i f i c i t y o f t h e p u r i f i e d IgG f r a c t i o n o f t h e E r w i n i a A-0181 a n t i s e r u m a t a d i l u t i o n o f 10" i n an i n d i r e c t ELISA. The A v a l u e s f o r each b a c t e r i a l s t r a i n were r e a d a t two t i m e s a f t e r s u b s t r a t e a d d i t i o n . P o s i t i v e ELISA v a l u e s were above 0.10 3  405  Bacterial strain  Time (h)  A for cells/ml a t time i n d i c a t e d 405  5 X 10° 5 X 1 0 5 X 10 7  E r w i n i a A-0181  2.0 27.0  0.52 1.59  E r w i n i a T-2789  2.0 27.0  E r w i n i a T-2722  2.0 27.0  Erwinia  5 X 10  5  5 X 10  0.25 0.78  0.10 0.22  0.05 0.06  0.05 0.05  0.05 0.06  0.05 0.05  0.05 0.05  0.05 0.05  0.05 0.04  0.06 0.16  0.04 0.05  0.03 0.03  0.03 0.02  0.03 0.02  0.07  0.01  0.01  0.01  0.01  0.00  0.00  2.0 27.0  0.01 0.01  0.01 0.02  0.00 0.00  0.00 0.00  0.00 0.00  2.0 27.0  0.07 0.08  0.05 0.05  0.04 0.04  0.05 0.04  0.04 0.04  Pseudomonas syringae  2.0 27.0  0.06 0.05  0.06 0.05  0.06 0.06  0.06 0.05  0.06 0.05  Rhizobium leguminosarum  2.0 27.0  0.00 0.00  0.00 0.00  0.00 0.00  0.00 0.00  0.00 0.00  Erwinia carotovora Agrobacterium tumefaciens  a  379 B-l  4 r e p l i c a t i o n s per value  0.03  a  27.0  h e r b i c o l a 2273  2.0  6  0.00  0.01  4  40 c e l l d i l u t i o n o f 5 X 10 c e l l s / m l was d e t e c t e d . 6  i s o l a t e T-2722 was a l s o d e t e c t e d  The t y p i c a l  a f t e r 27 h a t a d i l u t i o n o f 5 X  10 c e l l s / m l . 8  Host Range:  S m a l l growths were observed i n j u s t over h a l f o f t h e c o n i f e r s i n o c u l a t e d w i t h t h e Erwinia  i s o l a t e s ( F i g . 1.7).  The  l a r g e s t growths and t h e l a r g e s t numbers o f i n o c u l a t e d b a c t e r i a r e c o v e r e d were, however, l o c a t e d on t h e D o u g l a s - f i r t r e e s . A-0181 i s o l a t e was r e c o v e r e d from each c o n i f e r inoculated  (Table 1.8).  Picea  and Pinus  species  G a l l i n g symptoms produced by t h e  a t y p i c a l i s o l a t e , A-0181, were v i s i b l e on Abies, Larix,  The  Chamaecyparis,  spp.  G a l l i n g symptoms produced by t h e t y p i c a l i s o l a t e s were most v i s i b l e on Abies,  Larix  and Picea  spp.  Each t y p i c a l i s o l a t e was  not r e c o v e r e d from each i n o c u l a t e d t r e e s p e c i e s .  However, when  c o n s i d e r i n g t h e t h r e e t y p i c a l i s o l a t e s as one group, b a c t e r i a c h a r a c t e r i s t i c o f t h i s group were r e c o v e r e d from a t l e a s t one i n o c u l a t e d t r e e per species.  Those t r e e s t h a t d i s p l a y e d  galling  symptoms were n o t n e c e s s a r i l y t h o s e from which t h e i n o c u l a t e d b a c t e r i a c o u l d be s u c c e s s f u l l y r e c o v e r e d .  Characteristic  b a c t e r i a were r e c o v e r e d from many o f t h e t r e e s t h a t d i d n o t produce v i s i b l e g a l l i n g symptoms. A l l t h o s e s t r a i n s , o f both t y p e s ,  i d e n t i f i e d as t h e  i n o c u l a t e d g a l l - f o r m i n g b a c t e r i a caused g a l l s when backi n o c u l a t e d i n t o D o u g l a s - f i r , except f o r i s o l a t e T-2722 on P i c e a engelmannii.  Two o f t h e c u l t u r e s o f r e c o v e r e d b a c t e r i a were  l o s t and hence were n o t b a c k - i n o c u l a t e d  into Douglas-fir.  41  F i g . 1.7. P a t h o g e n i c i t y of g a l l - f o r m i n g Erwinia i s o l a t e s on Abies amabilis ( A ) , Chamaecyparis nootkatensis ( B ) , Picea engelmannii (C) and Pinus contorta (D). S i t e s i n o c u l a t e d w i t h : t y p i c a l i s o l a t e T-2789 ( l e f t b r a n c h e s ) , wound-only c o n t r o l s ( c e n t e r branches) and a t y p i c a l i s o l a t e A-0181 ( r i g h t b r a n c h e s ) , respectively.  T a b l e 1.8. P a t h o g e n i c i t y o f g a l l - f o r m i n g E r w i n i a i s o l a t e s on 14 c o n i f e r s p e c i e s . (G) P e r c e n t a g e s o f o b s e r v a t i o n s o f g a l l i n g a t t h e i n o c u l a t i o n s i t e a r e f o l l o w e d by (R) p e r c e n t r e c o v e r y o f t h e i n o c u l a t e d b a c t e r i a . Percentage o f g a l l e d s i t e s on D o u g l a s - f i r i n o c u l a t e d w i t h t h e r e c o v e r e d g a l l - f o r m i n g Erwinia spp. a r e l i s t e d i n column P f  P e r c e n t a g e s o f Erwinia Conifer species Family Pinaceae Abies amabilis Abies grandis Abies lasiocarpa Larix o c c i d e n t a l i s Picea glauca Picea engelmannii Picea sitchensis Pinus contorta Pinus monticola Pinus ponderosa Pseudotsuga menzeisii Tsuga heterophylla F a m i l y Cupressaceae Chamaecyparis nootkatensis Thuja plicata a b c d  4 replications 2 replications 4 replications no d a t a  T-2774 G  a  R  b  T-2789 P  G  R  T-2722  P  G  100 100 100 100 50 100 75 50 0 25 100 nd  50 50 100 50 100 100 100 50 100 50 100 nd  100 100 100 100 100 100 100 100 100 100 50 nd  100 100 75 75 100 100 50 75 0 0 100 25  50 50 0 100 50 100 100 100 100 100 100 50  100 100  nd 100  nd 0  nd  0 0  50 50  d  —  isolate  R  Dry Stab  A-0181  P  G  R  P  G R  100 100 100 100 100 100 50 100 50  100 0 75 100 100 0 100 100 50 100 100 nd  100 0 50 100 100 50 100 100 100 50 100 nd  100 100 100 75 0 100 100 100 50 100 nd  100 100 100 100 100 100 100 100 75 75 100 25  100 100 50 100 100 100 100 50 100 100 100 100  100 100 100 50 100 100 75 100 75 100 100 50  0 0 0 0 0 0 0 0 0 0 0 0  0 0 0 0 0 0 0 0 0 0 0 0  nd 75  nd 0  nd 0  nd  100 100  50 50  nd 50  0 0  0 0  -  —  43 DISCUSSION  Two d i f f e r e n t t y p e s o f b a c t e r i a l i s o l a t e s were r e c o v e r e d from D o u g l a s - f i r g a l l s c o l l e c t e d from s o u t h w e s t e r n Columbia. to  British  A l l o f t h e g a l l s c o l l e c t e d i n t h i s s t u d y were s i m i l a r  t h o s e r e p o r t e d by Hansen and Smith i n C a l i f o r n i a i n t h e  1930's (Hansen and Smith, 1937), e s p e c i a l l y t h o s e caused by t h e atypical isolate.  A t t h e d i s e a s e l e v e l , many s i m i l a r i t i e s were  n o t e d between t h e D o u g l a s - f i r g a l l d i s e a s e o f C a l i f o r n i a , by Agrobacterium  pseudotsugae,  caused  and t h e B.C. g a l l d i s e a s e , t h e  pathogens o f which were t e n t a t i v e l y i d e n t i f i e d as E r w i n i a species.  U n f o r t u n a t e l y , t h e r e a r e no e x t a n t c u l t u r e s o f t h e  b a c t e r i u m , o r i g i n a l l y named A. pseudotsugae,  possessing the  c h a r a c t e r i s t i c s f i r s t d e s c r i b e d by Hansen and Smith i n 1937. T h e r e f o r e a d i r e c t comparison between t h e i n c i t a n t s o f t h e two D o u g l a s - f i r g a l l d i s e a s e s was n o t p o s s i b l e . N e i t h e r A. pseudotsugae  nor the g a l l - f o r m i n g E r w i n i a  i s o l a t e s caused s i g n i f i c a n t economic damage t o t h e i r h o s t t r e e , Douglas-fir.  The D o u g l a s - f i r g a l l s i n t h i s s t u d y were c o l l e c t e d  i n mixed f o r e s t s near l a k e s o r i n urban a r e a s .  This s i t e  y i e l d e d t h e s i n g l e s o u r c e o f t h e second t y p e o f b a c t e r i a isolated.  Hansen and Smith c o l l e c t e d A. pseudotsugae  galls i n  s i m i l a r a r e a s , a l s o termed as m a r g i n a l a r e a s where t h e h e a l t h o f t h e h o s t was a l r e a d y compromised.  With b o t h g a l l - f o r m i n g  E r w i n i a i s o l a t e s , the t i p s of a r t i f i c i a l l y often die.  inoculated  branches  However, due t o t h e c h a r a c t e r i s t i c s m a l l s i z e o f t h e  g a l l s i n r e l a t i o n t o the s i z e of the host t r e e s observed i n t h e n a t u r a l environment,  l i t t l e damage was observed.  Hansen and  S m i t h (1937) r e p o r t e d  that  t h e i s o l a t e d pathogen was  'not  economic importance but of p o t e n t i a l i n t e r e s t t o t h e  of  lumbering  i n d u s t r i e s ' as i t c o u l d deform and even g i r d l e young t r e e s l e a d i n g t o t h e i r death.  They suggested t h a t i f t h e pathogen  a b l e t o i n v a d e s t a n d s of t i m b e r dominated by t h e h o s t , fir,  was  Douglas-  t h e n ' i t might r e a d i l y become an i m p o r t a n t f a c t o r i n  determining  t h e f u t u r e c o m p o s i t i o n of t h e f o r e s t . 1  Fortunately,  t h a t pathogen has not become an i m p o r t a n t f a c t o r i n t h e geographical  range of D o u g l a s - f i r .  Because t h e  gall-forming  e r w i n i a s can s i m i l a r l y cause g i r d l i n g and d e a t h i f i n o c u l a t e d i n t h e upper main stem of young D o u g l a s - f i r s e e d l i n g s , t h e y  may  have t h e p o t e n t i a l t o become a problem i n t r e e n u r s e r i e s . Methods of t r a n s m i s s i o n of the g a l l - f o r m i n g e r w i n i a s were not i n v e s t i g a t e d i n t h i s s t u d y , a l t h o u g h t h e i n i t i a t i o n s i t e t h e g a l l s w i t h i n t h e s u c c u l e n t new insect vector.  of  y e a r ' s growth s u g g e s t s an  Hansen and Smith (1937) proposed t h a t  t r a n s m i s s i o n o f A.  pseudotsugae  might be  v i a Adelges  cooleyi,  t h e C o o l e y Spruce g a l l a d e l g i d due t o t h e p r e s e n c e of s i t e s of i n i t i a t i o n of g a l l formation  a t the s i t e s c o r r e s p o n d i n g t o  f e e d i n g p u n c t u r e s formed by t h e a d e l g i d . produced by A. pseudotsugae,  Similar to g a l l s  g a l l s produced by t h e E r w i n i a  i s o l a t e s o n l y developed when i n o c u l a t e d u s i n g deep n e e d l e s t a b s . T h a t t h e s u c c u l e n t g a l l t i s s u e was  an a t t r a c t i v e f o o d s o u r c e f o r  i n s e c t s , i n t h i s s t u d y , s u p p o r t s the h y p o t h e s i s  t h a t an  insect  vector i s involved. One  f u r t h e r s i m i l a r i t y between t h e two g a l l - f o r m i n g E r w i n i a  i s o l a t e s and A. pseudotsugae the host p l a n t .  The  was  t h e l o c a t i o n of t h e g a l l s  g a l l s of both g a l l d i s e a s e s  on  a r e found c l o s e  to  t h e t i p s o f t w i g s o r branches  D o u g l a s - f i r g a l l s found i n B.C.  of D o u g l a s - f i r t r e e s . measured between 0.5  i n d i a m e t e r w h i l e A. pseudotsugae  The  and 2.0  cm  g a l l s were r e p o r t e d t o measure  between 1 m i l l i m e t e r and s e v e r a l c e n t i m e t e r s (Hansen and  Smith,  1937) . A. pseudotsugae  formed g a l l s on a r t i f i c i a l l y i n o c u l a t e d  D o u g l a s - f i r i n t h e f i e l d a minimum of t h r e e months a f t e r i n o c u l a t i o n (Hansen and Smith, 1937). to  G a l l growth was  t h e a c t i v e growth p e r i o d of t h e h o s t .  Furthermore  limited the  pathogen c o u l d be p r e s e n t i n t h e h o s t f o r n e a r l y a y e a r b e f o r e d e f i n i t e symptoms began t o appear.  In the present study only  greenhouse i n o c u l a t i o n s were c a r r i e d out.  However, g a l l s were  formed by t h e E r w i n i a i s o l a t e s a f t e r l e s s t h a n 2 weeks i n young succulent tissue. A t t h e m i c r o s c o p i c l e v e l , t h e g a l l t i s s u e o r g a n i z a t i o n and make-up was  found t o be s i m i l a r between t h e d i s e a s e s caused  t h e g a l l - f o r m i n g Erwinia of  and A. pseudotsugae.  by  Thin-sectioning  t h e g a l l t i s s u e s formed by t h e two t y p e s o f g a l l - f o r m i n g  E r w i n i a r e v e a l e d t h a t b o t h g a l l t y p e s were composed o f c i r c u l a r groups o f r a p i d l y d i v i d i n g c e l l s .  These c e l l s were about  h a l f the s i z e of healthy c o r t i c a l c e l l s unpublished).  ( R . J . Copeman,  The s t a b i n o c u l a t i o n s p e n e t r a t e d t h r o u g h  s t e l e r e s u l t i n g i n u n o r g a n i z e d v a s c u l a r elements t h e p i t h o f t h e young stems.  one  the  extending  into  Both b a c t e r i a l s t r a i n s appeared t o  be l o c a l i z e d i n t h e i n t e r c e l l u l a r spaces i n t h e c e n t e r s o f t h e s e groups o f d i v i d i n g c e l l s .  These f i n d i n g s a r e s i m i l a r t o t h o s e  i n t h e s t u d y by Hansen and Smith D o u g l a s - f i r was  (1937).  i n i t i a l l y thought t o be t h e o n l y h o s t of  t h e E r w i n i a s p e c i e s as t h e s e b a c t e r i a were s u c c e s s f u l l y i s o l a t e d o n l y from D o u g l a s - f i r g a l l s . t h e t y p i c a l and  A h o s t range s t u d y r e v e a l e d  that  the a t y p i c a l g a l l - f o r m i n g E r w i n i a i s o l a t e s c o u l d  a l s o form s m a l l g a l l s on s p e c i e s w i t h i n the genera A b i e s ,  Larix,  P i c e a and Pinus.  was  The  pathogen s t u d i e d by Hansen and  Smith  found t o be nonpathogenic on a l l of the c o n i f e r s t e s t e d , w h i c h i n c l u d e d D o u g l a s - f i r , Pinus lambertiana  halepensis  (sugar p i n e ) , P. radiata  heterophylla  ( c o a s t hemlock).  (Aleppo p i n e ) ,  (Monterey p i n e ) and  A. pseudotsugae  f i n d i n g c o n f i r m s the r e s u l t s of a r e c e n t (1981) .  Tsuga  s t r a i n 180  found t o be a v i r u l e n t on D o u g l a s - f i r i n t h i s s t u d y .  De Ley  P.  was  This  s t u d y by De C l e e n e  I t a l s o c o n f i r m s the comment i n t h e  eighth  e d i t i o n of Bergey's Manual (Buchanan and Gibbons, 1974) s t a t e s t h a t t h e c u l t u r e s of A. pseudotsugae  and  which  i n t h e A m e r i c a n Type  C u l t u r e C o l l e c t i o n do not d i s p l a y the c h a r a c t e r i s t i c s as o b s e r v e d by Hansen and The  Smith i n the  g a l l s induced by the Erwinia  1930*s. i s o l a t e s through  a r t i f i c i a l i n o c u l a t i o n were s m a l l e r t h a n t h o s e produced  on  D o u g l a s - f i r g a l l s i n n a t u r e and t h e r e f o r e may  accurate  i n d i c a t i o n of v i r u l e n c e .  The  not be an  r e s u l t i n g symptoms, however,  i n d i c a t e d t h a t the i s o l a t e s were weakly p a t h o g e n i c on some c o n i f e r species  o t h e r t h a n D o u g l a s - f i r , i n c l u d i n g Abies,  P i c e a and Pinus  spp.  been due and  Thuja  Larix,  Some of the g a l l i n g symptoms c o u l d have  t o a wound r e s p o n s e , e s p e c i a l l y on Chamaecyparis, spp.  Tsuga  from which e i t h e r low numbers o r no b a c t e r i a of  t h e t y p i c a l i s o l a t e s were r e c o v e r e d .  I n some c a s e s ,  the  i n o c u l a t e d b a c t e r i a were r e c o v e r e d from i n o c u l a t i o n s i t e s t h a t , were not g a l l e d , i n d i c a t i n g an e p i p h y t i c r e l a t i o n s h i p .  A single  47  gene w i t h i n t h e pathogen can be r e s p o n s i b l e  for determining the  v i r u l e n c e o f t h e pathogen on a p a r t i c u l a r h o s t t h e r e b y r e s u l t i n g i n e i t h e r a s u c c e s s f u l i n f e c t i o n o r an e p i p h y t i c r e l a t i o n s h i p ( S u r i c o e t al., 1985).  As t h e o t h e r c o n i f e r s p e c i e s t e s t e d were  not v e r y s u s c e p t i b l e t o t h e Erwinia explanation  spp.,  o f t h e low i n c i d e n c e o f t h i s  t h i s would a i d i n t h e disease.  I n a d d i t i o n t o d i f f e r i n g i n some r e s p e c t s pseudotsugae,  t h e two g a l l - f o r m i n g Erwinia  d i f f e r e d from each o t h e r i n many r e s p e c t s g a l l morphology.  from A.  i s o l a t e types including resultant  Typical Douglas-fir g a l l s collected i n t h i s  s t u d y were rough and i r r e g u l a r i n shape w i t h  distinctive  f i s s u r e - l i k e markings p r e s e n t on s e p a r a t e , d e f i n e d that constituted the g a l l .  outgrowths  The a r t i f i c i a l l y - i n d u c e d a t y p i c a l  D o u g l a s - f i r g a l l s were g l o b o s e and smooth i n appearance b u t d i d not have any d i s t i n c t markings on t h e i r s u r f a c e . produced by A. pseudotsugae  The g a l l s  were g e n e r a l l y v e r y smooth, l i k e t h e  a t y p i c a l g a l l s , b u t had a s i n g l e d i s t i n c t i v e c r o s s - s h a p e d marking covering  t h e e n t i r e g a l l s u r f a c e , n o t o b s e r v e d on any o f  t h e B.C. g a l l s . The  two g a l l - f o r m i n g Erwinia  i s o l a t e s d i f f e r e d from each  o t h e r i n c o l o n y morphology, temperature s e n s i t i v i t y , growth r a t e , a n t i b i o t i c r e s i s t a n c e s , s e r o l o g i c a l p r o p e r t i e s and h o s t range.  The c o l o n i e s o f both Erwinia  isolates differed i n  morphology from t h o s e o f A. pseudotsugae Unfortunately, pseudotsugae  as mentioned b e f o r e ,  s t r a i n 180.  t h e c u l t u r e s o f A.  a v a i l a b l e from t h e N a t i o n a l C o l l e c t i o n o f P l a n t  Pathogenic Bacteria  (NCPPB;  H e r t f o r d s h i r e , England) do n o t  display the c h a r a c t e r i s t i c s i n the o r i g i n a l description.  Thus,  48 not much c o u l d be deduced from such comparisons. W i t h t h e c o m p u t e r - a s s i s t e d , f a t t y a c i d a n a l y s i s system ( H e w l e t t Packard) f o r i d e n t i f i c a t i o n o f b a c t e r i a , an i n d e x o f g r e a t e r t h a n 0.500 on a s c a l e o f 0.000 and 1.000 i s g e n e r a l l y r e q u i r e d b e f o r e a p o s i t i v e i d e n t i f i c a t i o n can be made. of 0.500 c o r r e s p o n d s t o a p p r o x i m a t e l y t h r e e s t a n d a r d  deviations  away from t h e mean f a t t y a c i d p r o f i l e f o r t h e l i b r a r y (match) i n t h e computer d a t a base (M. Roy, communication).  An i n d e x  entry  personal  A d i f f e r e n c e i n s i m i l a r i t y i n d i c e s of greater  t h a n 0.100 between t h e f i r s t and second s p e c i e s *  choices i s  u s u a l l y r e q u i r e d f o r d i s t i n g u i s h i n g between t h e s p e c i e s and hence a p o s i t i v e i d e n t i f i c a t i o n .  Fatty a c i d a n a l y s i s i s touted  as b e i n g v e r y r e l i a b l e f o r i d e n t i f i c a t i o n p u r p o s e s , due t o t h e genetic al.,  s t a b i l i t y of the f a t t y acids  1988).  (Anonymous, 1985; C l a r k e t  A p o s i t i v e match, w i t h a s i m i l a r i t y i n d e x o f 0.840,  was made between t h e p r o f i l e o f t h e a t y p i c a l i s o l a t e , A-0181, and  t h a t o f E r w i n i a herbicola  subsp. h e r b i c o l a .  The n e x t  c l o s e s t match was a t a s i m i l a r i t y i n d e x o f 0.545, p r o d u c i n g a d i f f e r e n c e w e l l above t h e 0.100 r e q u i r e d f o r a p o s i t i v e match t o t h e f i r s t c h o i c e o f E. herbicola The  subsp. h e r b i c o l a .  d i f f e r e n c e between t h e f i r s t c h o i c e o f E r w i n i a  salicis  (Day  1924) C h e s t e r 1939 and second c h o i c e o f Hafnia  1954  was l e s s t h a n 0.100 f o r each o f t h e t h r e e t y p i c a l i s o l a t e s  tested.  However, t h e f a t t y a c i d p r o f i l e s o f s p e c i e s  family Enterobacteriaceae communication)  alvei  Moller  i n the  a r e h i g h l y s i m i l a r (M. Roy,  personal  and o f t e n t h e d i f f e r e n c e s i n s i m i l a r i t y  indices  between t h e f i r s t and second c h o i c e s may n o t r e a c h 0.100. Hafnia  s p e c i e s , u n l i k e t h e g a l l - f o r m i n g E r w i n i a i s o l a t e s , can  49 grow a t t e m p e r a t u r e s up t o 40-42°C and a r e n o t r e p o r t e d t o be associated with plants  ( K r i e g and H o l t , 1984). T h e r e f o r e , t h e  t y p i c a l i s o l a t e s were t e n t a t i v e l y i d e n t i f i e d as E r w i n i a w h i c h i s a known pathogen o f Salix  salicis,  sp. ( K r i e g and H o l t , 1984).  The d i f f e r e n c e s i n s i m i l a r i t y i n d i c e s , f o r t h e t y p i c a l i s o l a t e s , between t h e f i r s t c h o i c e o f E. salicis h e r b i c o l a subsp. herbicola  and E.  (the c h o i c e f o r t h e a t y p i c a l i s o l a t e )  measured c l o s e t o o r g r e a t e r than t h e 0.100 r e q u i r e m e n t ( f o r two of t h e three t y p i c a l i s o l a t e s analyzed:  0.170, 0.091, and  0.049) f o r a d i s t i n c t i o n between s p e c i e s .  T h e r e f o r e on t h e  b a s i s o f f a t t y a c i d p r o f i l e s a l o n e , a t l e a s t one o f t h e t y p i c a l i s o l a t e s i s indeed a d i f f e r e n t species than t h e a t y p i c a l isolate. The i d e n t i f i c a t i o n as Erwinia observations.  i s s u p p o r t e d by o t h e r  The g a l l - f o r m i n g Erwinia  spp. were shown n o t t o  produce o p i n e s i n g a l l e d t i s s u e s n o r do t h e y grow on media t h a t contains octopine  o r n o p a l i n e as t h e s o l e c a r b o n and n i t r o g e n  s o u r c e ( R . J . Copeman, unpublished). any  They a l s o do n o t grow on  o f t h e common s e l e c t i v e media f o r Agrobacterium  not form g a l l s on tomato, r a s p b e r r y and daigremontiana, tumefaciens,  and t h e y do  Kalanchoe  common herbaceous h o s t s o f  Agrobacterium  (Muehlchen, 1985).  Because t h e D o u g l a s - f i r g a l l - f o r m i n g b a c t e r i a were i d e n t i f i e d as b e l o n g i n g  i n t h e genus Erwinia,  be made w i t h E. h e r b i c o l a f . s p . gypsophilae, Gypsophila.  However, t h e Erwinia  comparisons c o u l d t h e pathogen o f  i s o l a t e s were found i n t h i s  s t u d y n o t t o form g a l l s on Gypsophila.  F u r t h e r m o r e , no  s e r o l o g i c a l r e l a t i o n s h i p s were observed between t h e g a l l - f o r m i n g  50 E r w i n i a and E. h e r b i c o l a subsp. herbicola The  two g a l l - f o r m i n g Erwinia  s t r a i n 2273.  i s o l a t e types shared t o a  d e g r e e , a s e r o l o g i c a l r e l a t i o n s h i p as d e t e c t e d immunodiffusion.  by  When t h e whole t y p i c a l T-2789 a n t i s e r u m  u s e d , a l i n e of p a r t i a l i d e n t i t y between t h e t y p i c a l and t h e a t y p i c a l i s o l a t e was  formed.  T h i s was  isolates  not o b s e r v e d w i t h  any of t h e o t h e r p h y t o p a t h o g e n i c b a c t e r i a t e s t e d . of t h e a n t i s e r u m  was  Purification  c o m p l e t e l y removed t h e c r o s s r e a c t i o n w i t h  atypical isolate.  Whole a t y p i c a l A-0181 a n t i s e r u m  the  o r t h e A-0181  IgG f r a c t i o n d i d not c r o s s r e a c t w i t h the t y p i c a l T-2789 i s o l a t e nor w i t h any of t h e o t h e r p h y t o p a t h o g e n i c b a c t e r i a when t e s t e d by i m m u n o d i f f u s i o n  or by i n d i r e c t ELISA.  A weak r e a c t i o n  was  o b s e r v e d w i t h t h e t y p i c a l i s o l a t e T-2722 w i t h b o t h a n t i s e r a . Many s i m i l a r i t i e s were found between t h e pathogens of two D o u g l a s - f i r g a l l d i s e a s e s .  As b i o c h e m i c a l  the  identification  t e s t s were not c a r r i e d out on the g a l l - f o r m i n g E r w i n i a i s o l a t e t y p e s , a d i r e c t comparison and hence a taxonomic r e l a t i o n s h i p t o A. pseudotsugae  c o u l d not be made.  i d e n t i t y of  Agrobacterium  pseudotsugae  De Ley,  because t h e o r i g i n a l A. pseudotsugae  1981)  is still  The  i n question  (De C l e e n e  and  c u l t u r e s have  been l o s t and no g a l l s have been found a t t h e o r i g i n a l s i t e i n C a l i f o r n i a t o enable f u r t h e r research. bacterium,  The  California  d e s c r i b e d as a Gram-negative, p l a n t  pathogenic  f a c u l t a t i v e anaerobe, would l i k e l y be p l a c e d i n t o t h e genus E r w i n i a today. I d e n t i f i c a t i o n of the B.C.  i s o l a t e s by f a t t y a c i d a n a l y s i s  p l a c e d them u n e q u i v o c a l l y i n t h e genus Erwinia, t h e Agrobacterium  or Pseudomonas  not i n e i t h e r  genera, which c o n t a i n t h e most  widely recognized  p l a n t g a l l formers.  From t h e o b s e r v e d  c h a r a c t e r i s t i c s and t h e r e s u l t s of f a t t y a c i d p r o f i l e s , i t would appear t h a t t h e two g a l l - f o r m i n g i s o l a t e t y p e s a r e two s p e c i e s of  Erwinia.  different  52 CHAPTER 2  THE MECHANISM OF GALL FORMATION BY ERWINIA  SPP. ON DOUGLAS-FIR  INTRODUCTION  I n i t i a l e x p e r i m e n t s t o determine t h e mechanism o f g a l l f o r m a t i o n by A. tumefaciens tissues.  f o c u s s e d on t h e growth o f h o s t tumor  The growth o f h e a l t h y p l a n t t i s s u e s in vitro  requires  an exogenous s u p p l y o f phytohormones (Parsons e t a l . 1986). Crown g a l l t i s s u e s f r e e d o f t h e i n c i t i n g b a c t e r i a were found t o be c a p a b l e o f autonomous growth s u g g e s t e d t h a t t h e pathogen  (Braun 1943).  This observation  induced t h e endogenous p r o d u c t i o n o f  phytohormones i n c e r t a i n h o s t c e l l s .  To d e t e r m i n e t h e t i m e  frame i n v o l v e d f o r t h i s b a c t e r i a l - i n d u c e d a l t e r a t i o n t o o c c u r i n t h e h o s t p l a n t c e l l s , a s i m p l e heat t r e a t m e n t was used t o s e l e c t i v e l y k i l l t h e pathogen but not t h e h o s t , a t s p e c i f i c t i m e s a f t e r i n o c u l a t i o n (Braun, 194 3). i n o c u l a t i o n o f A. tumefaciens  One t o f i v e days a f t e r  i n t o p e r i w i n k l e (Vinca  rosea  L.),  a h i g h t e m p e r a t u r e t r e a t m e n t o f 4 6-47°C was a p p l i e d f o r 5 days. Host p l a n t s were t h e n r e t u r n e d t o room t e m p e r a t u r e f o r a f u r t h e r 3 months.  G a l l s , s i m i l a r i n s i z e t o t h o s e produced w i t h o u t t h e  h e a t t r e a t m e n t , were observed i n t h o s e p l a n t s i n i t i a l l y to  exposed  t h e h i g h temperature 4 o r more days a f t e r i n o c u l a t i o n  1943).  (Braun  C e l l u l a r a l t e r a t i o n , or transformation, therefore  o c c u r r e d w i t h i n 4 days o f i n o c u l a t i o n a f t e r which t i m e t h e b a c t e r i u m ' s p r e s e n c e was no l o n g e r r e q u i r e d f o r g a l l  53 development. No e v i d e n c e was found t o demonstrate t h a t t r a n s f o r m a t i o n o f h o s t p l a n t c e l l s a l s o o c c u r r e d when i n o c u l a t e d w i t h P. pv.  savastanoi  (Comai e t al., 1982).  The c o n t i n u e d  syringae  presence of  t h e b a c t e r i a i s r e q u i r e d f o r g a l l i n i t i a t i o n and development. P. s y r i n g a e pv. savastanoi and  has been shown t o produce b o t h IAA  c y t o k i n i n s i n c u l t u r e by d e t e c t i o n o f t h e phytohormone  enzymes w i t h b i o c h e m i c a l  t e s t s o r by a s s a y i n g  for the actual  phytohormones w i t h immunoassays (Comai and Kosuge, 1980; D a v i s e t al., 1985; Mertens e t al., 1985).  A. tumefaciens  also  p r o d u c e s a u x i n s and c y t o k i n i n s i n c u l t u r e b u t a t a much l o w e r l e v e l t h a n P. syringae  pv. savastanoi  ( A k i y o s h i e t al., 1987).  The  production  o f h i g h e r l e v e l s o f phytohormones by P.  pv.  savastanoi  may be n e c e s s a r y f o r t h e c o n t i n u e d  syringae  development o f  t h e g a l l s as t h e h o s t p l a n t c e l l s do n o t produce phytohormones a t a l e v e l r e q u i r e d t o s u s t a i n such u n u s u a l growth ( A k i y o s h i e t al.,  1987). The  continued  o b j e c t i v e o f t h e s e experiments was t o d e t e r m i n e i f t h e p r e s e n c e o f t h e g a l l - f o r m i n g E r w i n i a spp. was r e q u i r e d  f o r g a l l formation  t o occur.  The design; o f a heat t r e a t m e n t  e x p e r i m e n t , s i m i l a r t o t h a t used f o r t h e s t u d y o f A. tumefaciens,  was chosen f o r t h i s i n i t i a l  study.  54 MATERIALS AND  B a c t e r i a l S t r a i n s and Host  METHODS  Plants:  The two g a l l - f o r m i n g Erwinia  i s o l a t e s used i n t h e s e  e x p e r i m e n t s were t h e T-2789 and t h e A-0181 i s o l a t e s . were grown on CPG p l a t e s f o r 5-7 days a t 21°C.  Bacteria  Isolated  c o l o n i e s were used f o r s t a b i n o c u l a t i o n s . One t o two y e a r o l d nursery-grown D o u g l a s - f i r t r e e s were planted  i n l a r g e s t y r o f o a m cups (600 ml) i n s t a n d a r d s o i l mix  w i t h 2400 cm fertilizer  3  p e a t and 200 cm  (14N-6.0P-11.6K),  3  Osmocote, a s l o w r e l e a s e p e r 0.16 m  3  of s t e r i l i z e d  The s e e d l i n g s were p r e v i o u s l y s t o r e d a t 4°C.  soil.  T r e e s were used  f o r t h e h e a t t r e a t m e n t experiments a minimum o f 2 months a f t e r t r a n s p l a n t i n g t o a l l o w t h e t r e e s t o become e s t a b l i s h e d under greenhouse c o n d i t i o n s .  Trees were f e r t i l i z e d w i t h 2 0-2 0-2 0 once  e v e r y two weeks a t a r a t e o f 0.06 g/L. Inoculations/Heat  treatment:  Four s e t s o f 10 t o 12 D o u g l a s - f i r t r e e s were s e l e c t e d f o r t h e i r u n i f o r m i t y between s e t s and l a b e l l e d .  Four branches p e r  t r e e were l a b e l l e d and s t a b i n o c u l a t e d 2-5 cm below t h e b r a n c h t i p with s t e r i l e pins c a r r y i n g the b a c t e r i a l i s o l a t e . b a c t e r i a l c o l o n y was i n o c u l a t e d p e r branch.  One  A f i f t h s e t of  t r e e s was s t a b b e d w i t h s t e r i l e p i n s o n l y t o s e r v e as a c o n t r o l . A l l o f t h e t r e e s were p l a c e d i n a growth chamber a t 21°C.  At  i n t e r v a l s o f 2, 4, and 7 days a f t e r i n o c u l a t i o n , one s e t o f t r e e s was t r a n s f e r r e d t o a second growth chamber.  T h i s chamber  was s e t a t 35±2°C f o r t h e T-2789 e x p e r i m e n t s o r 36±2°C f o r t h e A0181 e x p e r i m e n t s .  The temperature f o r t h e second t r i a l s w i t h  55 t h e a t y p i c a l i s o l a t e was s e t 2°C h i g h e r t h a n i n t h e f i r s t Each s e t o f t r e e s was l e f t a t t h e e l e v a t e d period  trial.  temperature f o r a  o f 8 t o 18 days depending on t h e e x p e r i m e n t .  B o t h growth  chambers had a 16 h p h o t o p e r i o d o f 80 jaE m" sec" p r o v i d e d by a 2  mixture of cool white fluorescent  1  and i n c a n d e s c e n t l i g h t s .  A f t e r t h e h e a t t r e a t m e n t , t h e t r e e s were t r a n s f e r r e d  back  t o e i t h e r t h e 21°C chamber o r out i n t o t h e greenhouse f o r a f u r t h e r 15 t o 18 days f o r t h e e a r l y t r i a l s o r f o r a f u r t h e r 3.5 t o 5.5 months f o r t h e l a t e r t r i a l s .  The t r e e s were m a i n t a i n e d  a t 21°C f o r a p e r i o d o f t i m e a f t e r t h e heat t r e a t m e n t t o a l l o w f o r growth a t t h e s i t e o f i n o c u l a t i o n t h a t may have been h i n d e r e d a t t h e h i g h e r temperatures and a l s o t o p r o v i d e f o r f u r t h e r growth o f t h e c o n t r o l s t o enhance t h e d i f f e r e n c e treatment responses. inoculated  i n the  The s e t o f wound-only t r e e s and one s e t o f  t r e e s were k e p t a t 21°C f o r t h e e n t i r e e x p e r i m e n t t o  serve as c o n t r o l s . Growth Index: D i a m e t e r s o f t h e branches a t t h e i n o c u l a t i o n s i t e s and j u s t below t h e i n o c u l a t i o n s i t e s were measured, w i t h c a l i p e r s , upon r e m o v a l from t h e h i g h temperature t r e a t m e n t and a t t h e termination  o f t h e experiment.  R a t i o s o f t h e two b r a n c h  measurements were c a l c u l a t e d as an i n d e x o f growth a t t h e inoculation  site.  Direct I s o l a t i o n s / C e l l ELISA: The i n o c u l a t i o n s i t e s , t h r e e per t r e e , were photographed and t h e n ground up i n 0.5 t o 1.0 ml o f LB i n s t e r i l e d i m p l e plates.  The s u s p e n s i o n s were l e f t a t room t e m p e r a t u r e f o r  15-20 min t o p e r m i t t h e r e l e a s e  o f t h e b a c t e r i a from t h e h o s t  56 tissue.  For d i r e c t i s o l a t i o n s , l o o p f u l s of these suspensions  were s t r e a k e d  onto CPG p l a t e s and i n c u b a t e d  a t 21°C f o r 7-10  days. For t h e c e l l ELISA, m i c r o t i t e r p l a t e s were s t e r i l i z e d 15 cm below a UV l i g h t  (General  E l e c t r i c G e r m i c i d a l G25T8 25 W) f o r 15  F o r each i n o c u l a t i o n s i t e , 25 jal  min.  o f t h e s u s p e n s i o n was  added t o each o f t h r e e w e l l s c o n t a i n i n g 175 _Ail o f LB.  Aliquots  (25 .Ail) o f a l a t e l o g phase c u l t u r e were added t o one row o f each m i c r o t i t e r p l a t e t o s e r v e as a c o n t r o l on t h e c e l l  ELISA.  The samples were c o v e r e d and a l l o w e d t o grow i n t h e w e l l s a t 21°C f o r 90 t o 100 h.  CPG p l a t e s were s t r e a k e d w i t h l o o p f u l s  from s e l e c t e d w e l l s a f t e r t h i s i n c u b a t i o n p e r i o d t o t e s t f o r growth and/or  contamination.  A f t e r t h e i n c u b a t i o n p e r i o d , an i n d i r e c t ELISA as o u t l i n e d i n C h a p t e r 1 was performed. first trials  Whole a n t i s e r a was used f o r t h e  o f t h i s experiment f o r both i s o l a t e s w h i l e IgG  f r a c t i o n s were used f o r t h e second t r i a l s .  Whole a n t i s e r a  d i l u t i o n s o f 10" and 10" f o r t h e T-2789 and A-0181 5  3  isolates,  r e s p e c t i v e l y , and IgG f r a c t i o n d i l u t i o n s o f 10~ and 10" f o r t h e 4  T-2789 and A-0181 i s o l a t e s , r e s p e c t i v e l y , were used. values  3  Absorbance  (405 nm) were r e a d a f t e r 1.5 and 12.5 h f o r t h e f i r s t and  second t r i a l s , r e s p e c t i v e l y . Statistical  Analysis:  R e s u l t s o f t h e heat t r e a t m e n t e x p e r i m e n t s were  analyzed  u s i n g t h e G e n e r a l L i n e a r Model, w i t h d i f f e r e n c e s between t r e a t m e n t means e v a l u a t e d  w i t h Tukey's M u l t i p l e Range T e s t  ( S t a t i s t i c a l A n a l y s i s Systems, SAS I n s t i t u t e I n c . , Cary, NC, USA) .  57 RESULTS  T-2789 i s o l a t e : None o f t h e wound-only o r h e a t - t r e a t e d  branches formed  g a l l s i n e i t h e r t r i a l w i t h t h e T-2789 i s o l a t e 2.2).  (Tables  2.1 and  Even when t h e heat t r e a t m e n t was d e l a y e d u n t i l 1 week  a f t e r i n o c u l a t i o n , no g a l l s were formed.  G a l l s had formed on  each i n o c u l a t e d branch o f t h e i n o c u l a t e d c o n t r o l t r e e s k e p t a t 21°C t h r o u g h o u t ( F i g . 2.1). A minimum o f f i v e t r e e s w i t h 3 b r a n c h e s p e r t r e e were t e s t e d p e r t r e a t m e n t . In both t r i a l s , the r a t i o s of i n o c u l a t i o n s i t e diameter/branch diameter numerically r e f l e c t e d these observations  with the inoculated control trees producing  s i g n i f i c a n t l y h i g h e r v a l u e s t h a n any o f t h e o t h e r  treatments.  I n a d d i t i o n t o t h e s i g n i f i c a n t d i f f e r e n c e s between t h e c o n t r o l s and t h e h e a t t r e a t m e n t s i n each o f t h e t r i a l s , t h e d i f f e r e n c e s between t h e i n d i v i d u a l t r e e s were a l s o  statistically  significant. R a t i o s f o r t h e wound-only s e t s and a l l t h e h e a t - t r e a t e d s e t s were n o t s i g n i f i c a n t l y d i f f e r e n t from each o t h e r . means f o r i n o c u l a t e d c o n t r o l t r e e s , o f t h e second  Ratio  trial,  i n c r e a s e d 2 8% over t h e t h r e e months a f t e r t h e h e a t t r e a t m e n t s (Table 2.2).  R a t i o means f o r t h e wound-only and t h e h e a t -  t r e a t e d s e t s i n c r e a s e d o n l y 0% and 8%, r e s p e c t i v e l y , o v e r t h e same t i m e p e r i o d . No c h a r a c t e r i s t i c b a c t e r i a were r e c o v e r e d from any o f t h e wound-only o r h e a t - t r e a t e d  t r e e s by d i r e c t i s o l a t i o n onto CPG  58  T a b l e 2.1. G a l l f o r m a t i o n by and r e c o v e r y o f t h e pathogen from h e a t - t r e a t e d (35°C f o r 18 days) Douglas-fir seedlings stab-inoculated with the t y p i c a l E r w i n i a i s o l a t e T-2789. I n o c u l a t i o n s i t e s were i n d e x e d f o r t h e p r e s e n c e o f l i v e b a c t e r i a by d i r e c t i s o l a t i o n onto CPG media and by a c e l l ELISA u s i n g whole T-2789 a n t i s e r u m Inoculated with isolate  Heat treatment Days a t 21°C 35°C  -  +  24 24  0 0  + + +  2 4 6  18 18 18  G a l l diameter/ stem d i a m e t e r Days a f t e r inoculation 24 d 34 d 1.3c 2. 5a  1. 3b 3 . 2a  1.2c 1. 4bc 1.4b  1.3b 1.4b 1. 5b  1  Bacteria detected i n inoculation site D i r e c t Absorbance i s o l a t i o n 405nm 0/10 20/20  0. 02c 0. 85a  0/20 0/20 0/20  0.08bc 0.08bc 0.10b  means i n a column f o l l o w e d by t h e same l e t t e r a r e n o t s i g n i f i c a n t l y d i f f e r e n t (P=0.05) a c c o r d i n g t o Tukey's m u l t i p l e range t e s t .  59  T a b l e 2.2. G a l l f o r m a t i o n by and r e c o v e r y o f t h e pathogen from h e a t - t r e a t e d (35°C f o r 12 days) Douglas-fir seedlings stab-inoculated with the t y p i c a l E r w i n i a i s o l a t e T-2789. I n o c u l a t i o n s i t e s were i n d e x e d f o r t h e presence o f l i v e b a c t e r i a by d i r e c t i s o l a t i o n onto CPG media and by a c e l l ELISA u s i n g t h e IgG f r a c t i o n o f t h e T-2789 a n t i s e r u m Inoculated with isolate  —  + + + +  Heat treatment Days a t 21°C 35°C 19 19 2 4 7  0 0 12 12 12  G a l l diameter/ stem d i a m e t e r Days a f t e r inoculation 26 d 108 d 1.2b 1.4a  1  1. l b 1.1b 1. l b  Bacteria detected i n inoculation site D i r e c t Absorbance i s o l a t i o n 405nm  1. l b 2. 0a  0/16 16/16  0. 00c 0.95a  1.2b 1.3b 1.2b  0/16 0/16 0/16  0. 00c 0. 01c 0. 05b  means i n a column f o l l o w e d by t h e same l e t t e r a r e n o t s i g n i f i c a n t l y d i f f e r e n t (P=0.05) a c c o r d i n g t o Tukey's m u l t i p l e range t e s t .  6 0  F i g . 2.1. The e f f e c t of a h i g h temperature t r e a t m e n t (35±2°C), a p p l i e d at v a r i o u s times a f t e r i n o c u l a t i o n with g a l l - f o r m i n g Erwinia i s o l a t e T-2789, on the development of g a l l i n g symptoms on D o u g l a s - f i r . (A) I n o c u l a t e d c o n t r o l t r e e s k e p t a t 21°C t h r o u g h o u t t h e experiment. I n o c u l a t e d t r e e s (B,C,D) s u b j e c t e d t o t h e 18-day h e a t t r e a t m e n t , 2, 4 and 7 days, r e s p e c t i v e l y , after inoculation.  61 p l a t e s (Tables  2.1 and 2.2).  Pure c u l t u r e s o f t h e i n o c u l a t e d  b a c t e r i a were r e c o v e r e d i n l a r g e numbers from each o f t h e i n o c u l a t e d c o n t r o l t r e e branches. C e l l ELISA r e s u l t s c o n f i r m e d t h e d i r e c t i s o l a t i o n d a t a t h a t l i v i n g b a c t e r i a were r e c o v e r e d o n l y from t h e i n o c u l a t e d c o n t r o l t r e e s k e p t a t 21°C t h r o u g h o u t (Tables 2.1 and 2.2). from t h e two e x p e r i m e n t s showed ELISA v a l u e s  Results  f o r the control  t r e e s t o be s i g n i f i c a n t l y g r e a t e r t h a n t h o s e f o r t h e wound-only o r any o f t h e h e a t - t r e a t e d  trees.  The mean ELISA v a l u e f o r  p l a n t s r e c e i v i n g t h e 7-day heat t r e a t m e n t i n t h e f i r s t  trial  a v e r a g e d l e s s t h a n 0.10 when t h e background wound-only v a l u e subtracted.  A l l t h e wound-only and h e a t - t r e a t e d  second t r i a l produced ELISA v a l u e s absorbance v a l u e o f 0.10.  trees i n the  l e s s than the c u t o f f  None o f t h e CPG p l a t e s s t r e a k e d  t h e ELISA p l a t e w e l l s o f h e a t - t r e a t e d p r e s e n c e o f l i v e Erwinia  was  from  branches showed t h e  T-2789 b a c t e r i a i n t h e m i c r o t i t e r p l a t e  wells. A-0181  Isolate:  A l t h o u g h a h i g h m o r t a l i t y r a t e was o b s e r v e d i n i n o c u l a t e d t r e e s exposed t o t h e 3 6±2°C heat t r e a t m e n t o f b o t h t r i a l s ,  no  g a l l s were o b s e r v e d a t i n o c u l a t i o n s i t e s amongst t h e s u r v i v o r s (Tables  2.3 and 2.4).  A minimum o f t h r e e t r e e s w i t h  four  b r a n c h e s p e r t r e e were t e s t e d p e r t r e a t m e n t e x c e p t f o r t h e 7-day t r e a t m e n t i n t h e second t r i a l where o n l y two t r e e s  survived.  R a t i o s o f i n o c u l a t i o n s i t e d i a m e t e r over b r a n c h d i a m e t e r f o r t h e i n o c u l a t e d c o n t r o l t r e e s were over 1 . 5 t i m e s g r e a t e r t h a n t h o s e f o r a l l o f t h e wound-only and t h e t h r e e h e a t - t r e a t e d  sets.  62  T a b l e 2.3. G a l l f o r m a t i o n by and r e c o v e r y o f t h e pathogen from h e a t - t r e a t e d (36°C) D o u g l a s - f i r seedlings stab-inoculated with the a t y p i c a l Erwinia i s o l a t e A-0181. I n o c u l a t i o n s i t e s were i n d e x e d f o r t h e p r e s e n c e o f l i v e b a c t e r i a by d i r e c t i s o l a t i o n o n t o CPG media and by a c e l l ELISA u s i n g whole A-0181 antiserum Inoculated with isolate  Heat treatment Days a t 21°C 36°C  G a l l diameter/ stem diameter Days a f t e r inoculation 17 d 29 d  +  13 13  0 0  1.4b 1.6a  + + +  2 4 7  10 8 6  1.1c 1.2bc 1.2bc  1  Bacteria detected i n inoculation site D i r e c t Absorbance i s o l a t i o n 405nm  1.4b 2.2a  0/5 10/10  0.04c 0.19a  1.4b 1.4b 1.6b  2/5 4/8 7/7  0.21a 0.12b 0.13b  means i n a column f o l l o w e d by t h e same l e t t e r a r e n o t s i g n i f i c a n t l y d i f f e r e n t (P=0.05) a c c o r d i n g t o Tukey's m u l t i p l e range t e s t .  T a b l e 2.4. G a l l f o r m a t i o n by and r e c o v e r y o f t h e pathogen from h e a t - t r e a t e d (38°C) D o u g l a s - f i r seedlings stab-inoculated with the a t y p i c a l Erwinia i s o l a t e A-0181. I n o c u l a t i o n s i t e s were i n d e x e d f o r t h e p r e s e n c e o f l i v e b a c t e r i a by d i r e c t i s o l a t i o n onto CPG media and by a c e l l ELISA u s i n g t h e IgG f r a c t i o n o f t h e A-0181 a n t i s e r u m Inoculated with isolate  +  + + +  Heat treatment Days a t 21°C 3 8°C  G a l l diameter/ stem diameter Days a f t e r inoculation 165 d  Bacteria detected i n inoculation site D i r e c t Absorbance isolation 405nm  18 18  0 0  1. l b 2. 2a  1/16 10/12  0.03b 0.71a  2 4 7  11 11 11  1. l b 1.2b 1.2b  2/16 0/12 0/4  0. 07b 0. 05b 0. 03b  means i n a column f o l l o w e d by t h e same l e t t e r a r e n o t s i g n i f i c a n t l y d i f f e r e n t (P=0.05) a c c o r d i n g t o Tukey's m u l t i p l e range t e s t .  64 R a t i o s f o r t h e wound-only s e t s and a l l t h e h e a t - t r e a t e d s e t s were n o t s i g n i f i c a n t l y d i f f e r e n t from each o t h e r a t t h e end both t r i a l s .  S i m i l a r t o t h e experiments  of  w i t h t h e t y p i c a l T-2789  i s o l a t e , d i f f e r e n c e s between t h e i n d i v i d u a l t r e e s , i n a d d i t i o n to  t h e d i f f e r e n c e s between the c o n t r o l s and t h e h e a t  treatments,  were s i g n i f i c a n t . In b o t h t r i a l s w i t h A-0181, l i v e b a c t e r i a were r e c o v e r e d from each i n o c u l a t e d c o n t r o l branch t e s t e d u s i n g d i r e c t isolations.  I n t h e f i r s t t r i a l , however, l i v e b a c t e r i a were  a l s o r e c o v e r e d from each of t h e h e a t - t r e a t e d s e t s but none from t h e wound-only t r e e s .  I n the second t r i a l ,  direct  isolation  showed s u r v i v a l and r e c o v e r y of c h a r a c t e r i s t i c b a c t e r i a from o f t h e 16 h e a t - t r e a t e d branches of t h e 2-day t r e a t m e n t .  two  Also  c h a r a c t e r i s t i c b a c t e r i a were r e c o v e r e d from one o f t h e woundo n l y t r e e branches, Immunodiffusional  presumably due t o e x p e r i m e n t a l  error.  a n a l y s i s confirmed the i d e n t i t y of the  r e c o v e r e d A-0181 i s o l a t e . The mean ELISA v a l u e f o r the 2-day h e a t - t r e a t e d t r e e s i n the f i r s t t r i a l 0.10,  even when the background wound-only v a l u e was  Furthermore, was  averaged above the c u t o f f absorbance v a l u e o f subtracted.  t h e average v a l u e f o r t h e 2-day h e a t - t r e a t e d t r e e s  n o t s i g n i f i c a n t l y d i f f e r e n t from t h e average i n o c u l a t e d  c o n t r o l v a l u e when a n a l y z e d w i t h Tukey's m u l t i p l e range t e s t ( T a b l e 2.3).  V a l u e s f o r t h e o t h e r h e a t - t r e a t e d s e t s and  the  wound-only s e t were s i g n i f i c a n t l y d i f f e r e n t from t h o s e o f t h e inoculated controls.  Mean c e l l ELISA v a l u e s f o r t h e i n o c u l a t e d  c o n t r o l t r e e s , i n t h e second t r i a l , were s i g n i f i c a n t l y g r e a t e r t h a n f o r any o f t h e o t h e r t r e a t m e n t s  (Table 2.4).  The  average  65  ELISA v a l u e s f o r t h e wound-only and t h e t h r e e h e a t - t r e a t e d s e t s i n t h e second t r i a l were a l l l e s s than t h e c u t o f f v a l u e hence were n e g a t i v e .  and  P l a t e checks on t h e ELISA a s s a y s f o r b o t h  t r i a l s showed r e c o v e r y of b a c t e r i a c o r r e s p o n d i n g t o t h e d i r e c t i s o l a t i o n data.  66 DISCUSSION  These h e a t t r e a t m e n t experiments demonstrated t h e g a l l - f o r m i n g Erwinia  t h a t f o r both  T-2789 and A-0181 i s o l a t e s , t h e  p r e s e n c e o f l i v e b a c t e r i a was n e c e s s a r y f o r g a l l i n i t i a t i o n and development on D o u g l a s - f i r s e e d l i n g s . those t r e e s not subjected  G a l l s o n l y formed on  t o t h e heat t r e a t m e n t and hence o n l y  on t h o s e t r e e s w i t h l i v e p a t h o g e n i c b a c t e r i a . obtained  suggesting that transformation  No e v i d e n c e was  o f p l a n t c e l l s had  occurred. I n o r d e r t o demonstrate t h a t a t r a n s f e r o f genes t o t h e host p l a n t d i d take place or a l t e r n a t i v e l y that the b a c t e r i a ' s p r e s e n c e was r e q u i r e d f o r g a l l f o r m a t i o n , k i l l the inoculated bacteria.  i t was n e c e s s a r y t o  The T-2789 and A-0181 i s o l a t e s  a r e a b l e t o grow i n l i q u i d c u l t u r e up t o t e m p e r a t u r e s o f 32°C and 34°C, r e s p e c t i v e l y .  The c o m b i n a t i o n o f t e m p e r a t u r e and  l e n g t h o f t i m e a t t h a t temperature which t h e h o s t t r e e s withstand  was determined e m p i r i c a l l y .  Preliminary  could  experiments  i n v o l v i n g h e a t t r e a t m e n t t o k i l l t h e g a l l - f o r m i n g E r w i n i a spp. r e v e a l e d t h a t t h e D o u g l a s - f i r s e e d l i n g s would n o t r e l i a b l y s u r v i v e h e a t t r e a t m e n t s o f 35-38°C l a s t i n g more t h a n about 10 days.  Unfortunately  each b a t c h o f t r e e s and each t r e e w i t h i n  each e x p e r i m e n t r e a c t e d d i f f e r e n t l y t o t h e heat t r e a t m e n t .  The  a n t i c i p a t e d h i g h m o r t a l i t y r a t e was t h e r e a s o n f o r t h e use o f 10-12 t r e e s p e r t r e a t m e n t and f o u r i n o c u l a t i o n s i t e s p e r t r e e . D i f f e r e n c e s i n temperature and l e n g t h o f h e a t t r e a t m e n t s between e x p e r i m e n t s were due t o t h e m o d i f i c a t i o n o f t h e s e v a r i a b l e s according  t o t h e h e a l t h and s u r v i v a l o f t h e h o s t s as  67 w e l l as t o t h e d i f f e r e n t temperatures r e q u i r e d t o k i l l t h e two i s o l a t e types.  A h i g h e r temperature was r e q u i r e d t o k i l l t h e A-  0181 i s o l a t e and t h i s i s r e f l e c t e d i n t h e r e s u l t a n t h i g h e r h o s t mortality rates.  The t r e e s were i n c u b a t e d a f t e r t h e h e a t  t r e a t m e n t s i n t h e second t r i a l s t o i n c r e a s e t h e d i f f e r e n c e s o b s e r v e d between t h e t r e a t m e n t s as w e l l as t o a l l o w f o r t h e i n c r e a s e i n number and hence ease o f d e t e c t i o n o f l i v e in  bacteria  t h e h e a t t r e a t e d t r e e s by e i t h e r d i r e c t i s o l a t i o n o r by  cell  ELISA. The i n i t i a l i n c u b a t i o n p e r i o d s a t 21°C o f 2, 4 and 7 days were d e c i d e d upon t o encompass t h e t i m e frame w i t h i n w h i c h p o t e n t i a l t r a n s f o r m a t i o n was expected t o o c c u r .  Initially,  the  t i m e r e q u i r e d f o r t r a n s f o r m a t i o n of h o s t p l a n t s by A. tumefaciens  was e x p e r i m e n t a l l y determined t o o c c u r w i t h i n 4 days  (Braun 1943).  F u r t h e r r e s e a r c h demonstrated t h a t t r a n s f o r m a t i o n  b e g i n s a p p r o x i m a t e l y 4 h a f t e r i n o c u l a t i o n and i s completed by 8 h a f t e r i n o c u l a t i o n (Sykes and M a t t h y s s e , 1986). symptoms caused by t h e Erwinia  Galling  spp. a r e v i s i b l e w i t h i n  a p p r o x i m a t e l y 2 weeks o f s t a b - i n o c u l a t i o n .  Thus, 7 days  was  e x p e c t e d t o be adequate time t o a l l o w t r a n s f o r m a t i o n by t h e E r w i n i a spp. t o t a k e p l a c e i f such an event was formation. of  involved i n g a l l  I n an attempt t o narrow t h e t i m e frame, i n t h e case  a t r a n s f e r o f genes t o t h e h o s t p l a n t , i n t e r v a l s o f 4 and 2  days were a l s o i n c l u d e d . V i s u a l o b s e r v a t i o n s and d i a m e t e r measurements o f t h e i n o c u l a t i o n s i t e s were c o r r e l a t e d w i t h t e c h n i q u e s aimed a t t h e d e t e c t i o n of l i v e b a c t e r i a at those s i t e s .  R a t i o s of  i n o c u l a t i o n s i t e d i a m e t e r over branch d i a m e t e r r e f l e c t e d t h e  68 l a c k o f g a l l f o r m a t i o n on t h e h e a t - t r e a t e d and wound-only t r e e s . Some s w e l l i n g o r growth o c c u r r e d a t t h e i n o c u l a t i o n s i t e o f h e a t - t r e a t e d o r wound-only t r e e s p o s s i b l y due t o wound h e a l i n g . D i r e c t i s o l a t i o n was used as a b a s i c s e l e c t i v e r e c o v e r y technique.  A c e l l ELISA was used as a second t e s t t o d e t e r m i n e  t h e p r e s e n c e o f l i v e i n o c u l a t e d b a c t e r i a because o f i t s s e n s i t i v i t y and i d e n t i f i c a t i o n p r o p e r t i e s as a s e r o l o g i c a l assay.  D i r e c t i s o l a t i o n from i n o c u l a t i o n s i t e s o f h e a t - t r e a t e d  t r e e s demonstrated t h e b a c t e r i o c i d a l e f f e c t of t h e h e a t t r e a t m e n t , e s p e c i a l l y i n t h e T-2789 t r i a l s .  L i v e b a c t e r i a were  r e c o v e r e d by d i r e c t i s o l a t i o n from a few of t h e h e a t - t r e a t e d branches i n o c u l a t e d w i t h t h e A-0181 i s o l a t e .  T h i s shows t h e  s u r v i v a b i l i t y o f t h e A-0181 i s o l a t e a t t e m p e r a t u r e s up t o 3940°C.  One c o l o n y was s u f f i c i e n t t o produce a p o s i t i v e r e s u l t i n  the d i r e c t i s o l a t i o n s .  The c o r r e s p o n d i n g average  ratio  measurements and c e l l ELISA v a l u e s f o r t h e s e few h e a t t r e a t e d branches were not s i g n i f i c a n t l y d i f f e r e n t from t h e wound-only trees.  T h i s would suggest t h a t t h e s m a l l numbers o f r e c o v e r e d  b a c t e r i a were n o t enough t o produce a v i s i b l e g a l l .  From a l l  b u t a c o u p l e o f i n o c u l a t e d c o n t r o l branches, w h i c h u n f o r t u n a t e l y d i e d d u r i n g t h e c o u r s e of t h e e x p e r i m e n t s , pure c u l t u r e s o f t h e i s o l a t e s were r e c o v e r e d , which c o r r e s p o n d s t o e v e r y i n o c u l a t i o n s i t e t h a t formed a g a l l . S e r o l o g i c a l a s s a y s a r e o f t e n noted f o r t h e i r s e n s i t i v i t y i n d e t e c t i o n o f t h e a n t i g e n i n e i t h e r low c o n c e n t r a t i o n o r i n mixtures of antigens.  The degree of s e n s i t i v i t y o f t h e a s s a y  depends i n p a r t on t h e s p e c i f i c i t y and s e n s i t i v i t y o f t h e a n t i s e r u m used.  From Chapter One,  i t was demonstrated t h a t t h e  69 a n t i s e r a towards t h e two Erwinia  i s o l a t e s , e s p e c i a l l y t h e IgG  f r a c t i o n s , were e s s e n t i a l l y i s o l a t e s p e c i f i c .  However, t h e  d e t e c t i o n l e v e l s o f t h e IgG f r a c t i o n s u s i n g an i n d i r e c t ELISA were n o t v e r y s e n s i t i v e w i t h 5 X 10  6  and 5 X 10  7  c e l l s / m l being  t h e l i m i t o f d e t e c t i o n of t h e i r homologous a n t i g e n s o f t h e p u r i f i e d T-2789 and A-0181 a n t i s e r a , r e s p e c t i v e l y . l i m i t f o r t h e d e t e c t i o n of Erwinia  carotovora  u s i n g a d o u b l e a n t i b o d y sandwich ELISA was ml for  The  ssp.  10  5  t o 10  lower  carotovora 6  c e l l s per  (Caron and Copeman, 1984) w h i l e t h e lower l i m i t o f d e t e c t i o n Pseudomonas  p e r ml  phaseolicola  u s i n g t h e same assay was  ( B a r z i c and T r i g a l e t , 1982) .  10  4  cells  D i r e c t comparisons t o t h e s e  v a l u e s cannot be made as t h e d e t e c t i o n assay used i n t h i s C h a p t e r was a m o d i f i c a t i o n of t h e b a s i c double a n t i b o d y sandwich method.  An enrichment s t e p , i n which t h e t e s t samples a r e  i n c u b a t e d f o r a p e r i o d of t i m e i n L u r i a B r o t h , was i n c o r p o r a t e d i n t o t h e a s s a y t o i n c r e a s e t h e c e l l c o n c e n t r a t i o n i n t h e sample and hence t h e p o s s i b i l i t i e s of d e t e c t i o n .  Therefore, the  o v e r a l l d e t e c t i o n l e v e l s of t h e c e l l ELISAs were l o w e r t h a n t h o s e i n d i c a t e d i n t h e i n d i r e c t ELISA s p e c i f i c i t y t e s t s o f Chapter  One.  L a r g e numbers o f l i v e g a l l - f o r m i n g E r w i n i a b a c t e r i a were r e c o v e r e d from t h e i n o c u l a t e d c o n t r o l t r e e s a c c o r d i n g t o t h e c e l l ELISA d a t a f o r both t h e T-2789 and A-0181 i s o l a t e s . Background  r e a d i n g s were r e c o r d e d f o r most o f t h e wound-only and  heat-treated trees.  I n t h e e a r l i e r t r i a l s , some o f t h e h e a t -  t r e a t e d i n o c u l a t i o n s i t e s , a few of which were v i s i b l y  suffering  from t h e e f f e c t s of t h e t r e a t m e n t , r e c o r d e d ELISA v a l u e s j u s t o v e r t h e c u t o f f v a l u e of 0.10.  These r e s u l t s , when combined  w i t h t h e d i a m e t e r r a t i o s and t h e d i r e c t i s o l a t i o n d a t a ,  suggest  t h a t t h e s e r o l o g i c a l assay was d e t e c t i n g e i t h e r v e r y low c o n c e n t r a t i o n s o f t h e i n o c u l a t e d b a c t e r i a and/or dead b a c t e r i a l c e l l s i n the host t i s s u e .  The h e a t - t r e a t e d branches when t e s t e d  by ELISA i n t h e l a t e r t r i a l s , u s i n g t h e p u r i f i e d a n t i s e r a , d i d not produce t h e s e low p o s i t i v e v a l u e s .  I n each o f t h e t r i a l s ,  absorbance r e a d i n g s f o r t h e i n o c u l a t e d c o n t r o l t r e e s were s i g n i f i c a n t l y g r e a t e r t h a n those o f any o f t h e o t h e r i n accordance w i t h the v i s u a l  treatments  observations.  S e v e r a l methods have been used t o e i t h e r i n v e s t i g a t e t h e mechanism o f g a l l i n i t i a t i o n and development o r t o d e t e r m i n e t h e s u c c e s s f u l t r a n s f o r m a t i o n o f p l a n t s by A. tumefaciens. d e s i g n o f t h e heat t r e a t m e n t  The  experiment was chosen i n p a r t  because i t was a s i m p l e t e c h n i q u e t h a t was used i n e a r l y e x p e r i m e n t s w i t h A. tumefaciens  (Braun 1943).  One o f t h e more  r e c e n t and d i r e c t approaches t o answer t h e q u e s t i o n o f g a l l f o r m a t i o n i s t o probe f o r t r a n s f e r r e d b a c t e r i a l genes, s p e c i f i c a l l y t h e phytohormone genes, w i t h i n t h e h o s t p l a n t genome.  T h i s was n o t s e r i o u s l y c o n s i d e r e d due t o t h e e x t e n s i v e  a n a l y s i s t h a t would be r e q u i r e d t o r e a c h a c o n c l u s i v e answer ( S e d e r o f f e t al. 1986). A n o t h e r method, b e s i d e s t h e heat t r e a t m e n t ,  that could  answer t h e q u e s t i o n o f t h e mechanism o f g a l l f o r m a t i o n i s t h e use o f an analogous treatment  i n a t i s s u e c u l t u r e system.  C u l t u r e o f tumor t i s s u e s and/or d e t e c t i o n o f phytohormones were not chosen as p r i m a r y methods o f d e t e r m i n a t i o n due t o a n t i c i p a t e d problems. tumefaciens  S e d e r o f f e t al. (1986) found t h a t A.  i n f e c t e d p i n e c a l l u s d i d n o t grow on hormone-free  71 media.  The  t r a n s f e r of b a c t e r i a l genes t o t h e h o s t p l a n t  may-  t a k e p l a c e but t h e e x p r e s s i o n of the phytohormone genes, i n some cases,  i s not s u f f i c i e n t t o a l l o w f o r a u t o t r o p h i c growth in  vitro.  R e c e n t l y however, C l a r k e t al.  (1989) d e m o n s t r a t e d ,  u s i n g a t i s s u e c u l t u r e system, t h a t E. h e r b i c o l a f . s p . gypsophilae  does not t r a n s f e r phytohormone genes t o t h e h o s t  p l a n t ' s genome. The  d e t e c t i o n of phytohormones i n c u l t u r e i s not  sufficient  t o d e t e r m i n e t h e p r i m a r y producer of phytohormones i n t h e p l a n t pathogen i n t e r a c t i o n . A. tumefaciens savastanoi  as w e l l as P. s y r i n g a e pv.  produces phytohormones i n c u l t u r e a l b e i t i n a  c o n s i d e r a b l y lower l e v e l (Smidt and Kosuge, 1978; a l . , 1987).  Akiyoshi et  O f t e n such i n v e s t i g a t i o n s can l e a d t o ambiguous  r e s u l t s i n r e l a t i o n t o t h e q u e s t i o n of g a l l i n i t i a t i o n development.  To t h i s end,  t h e o r i g i n a l A. tumefaciens  t h e heat t r e a t m e n t s ,  and  modelled a f t e r  experiments, provided a simple  yet  c o n c l u s i v e answer t o t h e r o l e of t h e g a l l - f o r m i n g b a c t e r i u m the formation  of t h e  gall.  I n t h e e a r l y heat t r e a t m e n t e x p e r i m e n t s w i t h tumefaciens,  A.  g a l l s i n c i t e d with v i r u l e n t b a c t e r i a continued  develop although  in  l i v e b a c t e r i a were no l o n g e r p r e s e n t ,  more days p o s t - i n o c u l a t i o n (Braun, 1943) .  to  f o u r or  This suggested t h a t a  t r a n s f o r m a t i o n of t h e h o s t c e l l s had o c c u r r e d t o w a r r a n t continued  unregulated  growth and d i v i s i o n .  In the  present  e x p e r i m e n t s , t h e presence of l i v e b a c t e r i a i s r e q u i r e d w i t h b o t h t h e E r w i n i a T-2789 and the A-0181 i s o l a t e f o r c o n t i n u e d development t o t a k e p l a c e .  gall  T h i s would i n d i c a t e t h a t no t r a n s f e r  o f genes t o t h e h o s t p l a n t genome o c c u r s , a t l e a s t not w i t h i n 7  days o f i n o c u l a t i o n , as i s t h e case i n t h e tumefaciens  system.  Agrobacterium  A mechanism o f g a l l f o r m a t i o n  t h a t o f t h e Pseudomonas  syringae  pv. savastanoi  closer to  system i s l i k e l y  t o be t h e c a s e w i t h t h e g a l l - f o r m i n g E r w i n i a spp.  Thus, t h e  g a l l - f o r m i n g E r w i n i a spp. a r e not p o t e n t i a l gene v e c t o r s f o r t h e g e n e t i c improvement o f D o u g l a s - f i r .  73 CHAPTER 3  THE RELATIONSHIP BETWEEN THE PLASMID CONTENT OF THE GALL-FORMING ERWINIA  ISOLATES AND PATHOGENESIS  INTRODUCTION  P l a s m i d s i n b o t h A. tumefaciens savastanoi  and P. syringae  pv.  c a r r y g e n e t i c d e t e r m i n a n t s t h a t code f o r t h e  s y n t h e s i s o f a u x i n s and c y t o k i n i n s , which c o n f e r p a t h o g e n i c i t y . Thus, i t was o f i n t e r e s t t o determine t h e p l a s m i d c o n t e n t o f t h e gall-forming Erwinia.  B a c t e r i a l p l a s m i d s range i n s i z e between  1 k b and g r e a t e r t h a n 3 00 kb ( T r e v o r s , 1985) .  Larger s i z e d  p l a s m i d s o f t e n a r e more d i f f i c u l t t o i s o l a t e due t o t h e s h e a r i n g o f t h e DNA d u r i n g t h e e x t r a c t i o n p r o c e s s .  Furthermore,  each  p l a s m i d h a s a c h a r a c t e r i s t i c copy number under d e f i n e d conditions.  Plasmids present i n m u l t i p l e copies,  c h a r a c t e r i s t i c a l l y t h e s m a l l e r p l a s m i d s , a r e under r e l a x e d c o n t r o l w h i l e l a r g e (>50 kb) p l a s m i d s a r e under s t r i n g e n t c o n t r o l and hence a r e i n a lower copy number (Broda,  1979).  P l a s m i d s a r e u s u a l l y found i n a d o u b l e - s t r a n d e d , c o v a l e n t l y c l o s e d c i r c u l a r form.  However, d u r i n g t h e i s o l a t i o n p r o c e s s ,  one o r b o t h s t r a n d s c a n be n i c k e d r e s u l t i n g i n e i t h e r an open c i r c u l a r o r a l i n e a r form.  These d i f f e r e n t forms, a l o n g w i t h  m u l t i m e r i c forms, c a n c o m p l i c a t e t h e i n t e r p r e t a t i o n o f p l a s m i d profiles. P r o f i l e s o f p l a s m i d c o n t e n t have been used i n b a c t e r i a l  74 strain identification  (Lazo e t al., 1987).  V i r u l e n t s t r a i n s of  E. s t e w a r t i i c o n t a i n a t l e a s t e i g h t p l a s m i d s t h a t range i n s i z e from 4.1 t o a p p r o x i m a t e l y 32 0 kb. A 30 kb p l a s m i d , pEA28, i s found i n a l l s t r a i n s o f E. amylovora. a l s o been found i n Clavibacter Curtobacterium  fascians  flaccumfaciens  Conserved p l a s m i d s have  michiganense  subsp.  pv. pointsettae  and  sepedonicum, Rhodococcus  ( C o p l i n , 1989).  P l a s m i d DNA i s a l s o used f o r probes i n DNA:DNA hybridizations for the i d e n t i f i c a t i o n or d i f f e r e n t i a t i o n of phytopathogenic  s t r a i n s , as w e l l as f o r t h e d e t e r m i n a t i o n o f  homology between v a r i o u s DNA fragments ( G i l b e r t s o n e t al., 1989) .  P l a s m i d sequences a r e o f t e n i n h i g h e r copy number t h a n  t h o s e on t h e chromosome p r o d u c i n g a s t r o n g e r h y b r i d i z a t i o n signal  ( C o p l i n , 1989).  Conserved p l a s m i d sequences o c c u r  between t h e p l a s m i d DNA o f A. tumefaciens savastanoi.  and P. s y r i n g a e pv.  The homology o c c u r s between t h e genes e n c o d i n g  for  enzymes i n v o l v e d i n a u x i n and c y t o k i n i n p r o d u c t i o n ( P o w e l l and M o r r i s , 1986; F o l l i n e t al., 1985).  As i n t h i s c a s e , DNA  homology i s o f t e n r e l a t e d t o common b i o l o g i c a l f u n c t i o n (Von Bodman and Shaw, 1987).  In relation to the gall-forming  E r w i n i a , c o n s e r v e d DNA sequences between t h e p l a s m i d DNA o f t h e two t y p e s c o u l d f u r t h e r e x p l a i n t h e i r r e l a t i o n s h i p . One  o f t h e o r i g i n a l methods t o determine  the function of  p l a s m i d genes i s t o c u r e t h e b a c t e r i u m o f t h e p l a s m i d .  Curing a  bacterium of i t s plasmid(s) r e s u l t s i n the production of plasmid-free d e r i v a t i v e s of the wild-type bacterium.  Phenotypes  o f t h e c u r e d s t r a i n s a r e t h e n compared w i t h t h o s e o f t h e w i l d type s t r a i n s .  Some methods o f c u r i n g i n c l u d e t h e u s e o f a heat  t r e a t m e n t such as used w i t h Agrobacterium al.,  1975), E r w i n i a herbicola  Xanthomonas  1988).  campestris  pv.  tumefaciens  (Watson e t  ( C h a t t e r j e e and G i b b i n s , 1971) vignicola  (Ulaganathan and  and  Mahadevan,  C h e m i c a l c u r i n g agents such as a c r i d i n e orange,  sodium  d o d e c y l s u l f a t e (SDS) and e t h i d i u m bromide have a l s o been used t o s u c c e s s f u l l y c u r e b a c t e r i a of one o r more o f t h e i r p l a s m i d s ( S o n s t e i n and B a l d w i n , 1972).  A c r i d i n e orange was used f o r  c u r i n g t h e pIAA p l a s m i d from P. syringae  pv.  savastanoi  (Watanabe and Fukasawa, 1961; Comai and Kosuge, 1980). The o b j e c t i v e s of t h e s e experiments i n c l u d e d t h e d e t e r m i n a t i o n o f t h e p l a s m i d c o n t e n t of t h e two g a l l - f o r m i n g E r w i n i a i s o l a t e types.  Secondly, e x p e r i m e n t s aimed a t  d e t e r m i n i n g t h e presence of homology between t h e p l a s m i d c o n t e n t o f t h e two i s o l a t e t y p e s were c a r r i e d o u t .  The f i n a l  objective  o f t h i s s e c t i o n was t o determine t h e r o l e , i f any, o f t h e p l a s m i d DNA of g a l l s .  of t h e g a l l - f o r m i n g Erwinia  spp. i n t h e  development  76 MATERIALS AND METHODS  Bacterial  Strains:  The t y p i c a l i s o l a t e , T-2774, and t h e a t y p i c a l A-0181 i s o l a t e were t h e main i s o l a t e s used i n t h e s e e x p e r i m e n t s .  The  o t h e r b a c t e r i a l s t r a i n s used and t h e i r s o u r c e s a r e as o u t l i n e d i n T a b l e 1.1 i n Chapter 1.  The c h e m i c a l s used were from t h e  Sigma C h e m i c a l Company u n l e s s o t h e r w i s e n o t e d . T o t a l DNA  Isolation:  S i n g l e b a c t e r i a l c o l o n i e s were i n o c u l a t e d i n t o 5 ml o f t h e a p p r o p r i a t e b r o t h and a g i t a t e d a t 2 00 rpm a t room t e m p e r a t u r e for  2-4 days, depending upon t h e b a c t e r i a l s p e c i e s .  Bacterial  c e l l s were h a r v e s t e d by c e n t r i f u g a t i o n a t 2100 X g (maximum) f o r 10 min a t 4°C.  P e l l e t s were resuspended  Tris-EDTA-Glucose  i n 100 ;ul i c e c o l d  ( M a n i a t i s e t al., 1982).  (200 ,Ail) c o n s i s t i n g o f lysozyme  A lysing  solution  (5 mg/ml), SDS (1%) and  P r o t e i n a s e K (2ug/ml) was added and g e n t l y mixed by t i l t i n g t h e tube. 3.0 h.  The c e l l s u s p e n s i o n s were i n c u b a t e d a t 60-65°C f o r 2.5The r e s u l t i n g c e l l l y s a t e s were e x t r a c t e d w i t h p h e n o l  and c h l o r o f o r m .  The DNA was p r e c i p i t a t e d w i t h e t h a n o l and  c o l l e c t e d by c e n t r i f u g a t i o n .  Resuspended DNA samples were  s h e a r e d by passage t h r o u g h 22 gauge n e e d l e s seven t o e i g h t times.  An RNase A t r e a t m e n t (20 jag/ml  f o r 10 min a t 37°C) was  f o l l o w e d by a n o t h e r e t h a n o l p r e c i p i t a t i o n s t e p t o remove t h e d i g e s t e d RNA and t h e RNase.  77 Plasmid I s o l a t i o n and  Purification:  Several d i f f e r e n t plasmid  e x t r a c t i o n p r o t o c o l s were  a t t e m p t e d f o r t h e g a l l - f o r m i n g Erwinia boiling al.,  (Holmes and Q u i g l e y ,  1982), l y s i s by SDS  M a n i a t i s e t al., and Liu,  D o l y , 1979 1981;  1982)  1981  isolates:  lysis  as o u t l i n e d i n M a n i a t i s  (Godson and Vapnek, 1973  and  l y s i s by a l k a l i  The  et  as o u t l i n e d i n  (modified  as o u t l i n e d i n M a n i a t i s e t al.,  Hardy, 1985).  by  1982;  Birnboim Kado  and  alkaline extraction protocol  o u t l i n e d i n M a n i a t i s e t al.  (1982) was  adopted as t h e b e s t  s i m p l e s t p r o c e d u r e t o use.  For both t y p i c a l s t r a i n s and  as and  A-0181,  m i n i p r e p s were c a r r i e d out on 5 ml l a t e l o g phase LB c u l t u r e s . For l a r g e s c a l e a l k a l i n e l y s i s of A-0181, an i n i t i a l volume of 1 l i t e r o f l a t e l o g phase LB c u l t u r e was  harvested.  D i f f e r e n t methods were used t o p u r i f y t h e p l a s m i d t h e two E r w i n i a i s o l a t e s t o determine t h e method w i t h highest recovery plasmid  DNA  of p u r i f i e d p l a s m i d  DNA.  DNA  from  the  E l e c t r o e l u t i o n of  the  i n t o d i a l y s i s bags, t r o u g h s and onto d i a l y s i s  membranes as w e l l as p u r i f i c a t i o n t h r o u g h Low  Gelling  Temperature agarose (BioRad) and GeneClean (BioRad) were a l l tested. was  For t h e t y p i c a l i s o l a t e s , DNA  obtained  from m i n i p r e p s  r u n t h r o u g h agarose g e l s and g e l s l i c e s c o n t a i n i n g  plasmid  DNA  were c u t out.  The  plasmid  the  bands were t h e n  e l e c t r o e l u t e d i n t o d i a l y s i s tubes or onto d i a l y s i s membranes u s i n g a B i o r a d E l e c t r o - e l u t o r Model  422.  For t h e A-0181 i s o l a t e , l a r g e s c a l e p l a s m i d were f o l l o w e d by s e p a r a t i o n on a cesium c h l o r i d e gradient. mg/ml.  Ethidium  bromide was  DNA  extractions  (CsCl)  added a t a c o n c e n t r a t i o n  of  0.5  Samples were c e n t r i f u g e d a t 170,000 X g (maximum) f o r 65  78 h on a Type 65 f i x e d a n g l e r o t o r (Beckman).  Bands were viewed  o v e r a mid-wave U.V. t r a n s i l l u m i n a t o r (Model TM36 U l t r a v i o l e t P r o d u c t s , I n c . San G a b r i e l , CA) and removed v i a an 18 gauge needle.  E t h i d i u m bromide was removed from t h e s o l u t i o n by  e x t r a c t i o n w i t h e q u a l volumes o f 1 - b u t a n o l .  T h i s p r o c e d u r e was  r e p e a t e d a t l e a s t s i x t i m e s o r u n t i l t h e aqueous phase no l o n g e r displayed a pinkish color.  The r e s u l t i n g sample was t h e n  concentrated v i a ethanol p r e c i p i t a t i o n .  Further p u r i f i c a t i o n of  i n d i v i d u a l A-0181 bands was a c c o m p l i s h e d by i s o l a t i o n from Low G e l l i n g Temperature agarose o r by u s i n g GeneClean.  DNA  c o n c e n t r a t i o n s were measured a t 260 nm on a H e w l e t t P a c k a r d Spectrophotometer o b t a i n jaq/ml)  (the absorbance v a l u e s m u l t i p l i e d by 50 t o  o r by v i s u a l i z a t i o n on an agarose g e l i f t h e  sample c o n c e n t r a t i o n was t o o low. R e s t r i c t i o n enzyme d i g e s t s were performed i n a r e a c t i o n volume o f 20 y U l w i t h up t o 1.0 jag o f DNA and a t l e a s t two t i m e s e x c e s s enzyme.  R e a c t i o n s were c a r r i e d o u t a t 37°C f o r 2 h.  H i n d l l l - d i g e s t e d lambda DNA (fragment s i z e s : 4.36,  23.13, 9.42, 6.56,  2.32, 2.03, 0.56 and 0.13 kb) was used as DNA s i z e  markers. Samples were r u n on a Pharmacia h o r i z o n t a l g e l e l e c t r o p h o r e s i s a p p a r a t u s (Model GNA-100) i n 0.7% (0.9% f o r d i g e s t s ) agarose a t 60-90 mA f o r 2 t o 2.5 h i n IX T r i s - a c e t a t e EDTA (TAE) pH 8.0 ( M a n i a t i s e t a l . , 1982) w i t h 0.5 ug/ml e t h i d i u m bromide i n t h e g e l and i n t h e r u n n i n g b u f f e r .  Gels  were viewed over a mid-wave u l t r a v i o l e t t r a n s i l l u m i n a t o r . Photographs  o f t h e g e l s were t a k e n u s i n g a 605 mu narrow band  pass f i l t e r and P o l a r o i d Type 57 sheet f i l m .  79 Southern  Blots:  DNA samples, d i g e s t e d o r u n d i g e s t e d , were t r a n s f e r r e d from a g a r o s e g e l s t o Zeta-Probe (BioRad) membranes by an a l k a l i n e b l o t t i n g p r o c e d u r e as o u t l i n e d f o r N y t r a n n y l o n membranes (Schleicher & Schuell).  A f t e r photographs were t a k e n , t h e g e l s  were g e n t l y shaken i n 2 volumes o f 0.25M H C l f o r 8-10 m i n t o f a c i l i t a t e t r a n s f e r o f t h e DNA through strands.  fragmentation  o f t h e DNA  G e n t l e s h a k i n g a g a i n accompanied two, 15-min  i n c u b a t i o n s i n 2 volumes o f t h e b l o t t i n g s o l u t i o n (0.5M NaOH and 1.5M N a C l ) .  The g e l was then p l a c e d on a w i c k made up o f t h r e e  l a y e r s o f Whatman 3MM paper, s a t u r a t e d w i t h t h e b l o t t i n g solution.  A p i e c e o f Zeta-Probe membrane, soaked f o r 5 m i n i n  t h e b l o t t i n g s o l u t i o n and n i c k e d on t h e t o p r i g h t c o r n e r f o r o r i e n t a t i o n , was p l a c e d on t h e g e l and smoothened o u t t o a v o i d b u b b l e s underneath.  Three l a y e r s o f Whatman 3MM, s a t u r a t e d w i t h  t h e b l o t t i n g s o l u t i o n , were p l a c e d on t h e membrane, f o l l o w e d by six  layers of dry f i l t e r  paper.  A s t a c k o f paper t o w e l s ,  m e a s u r i n g 15-20 cm i n depth, was p l a c e d on t o p .  The w i c k was  r e s t i n g on a p i e c e o f p l e x i g l a s s w i t h t h e ends o f t h e w i c k soaking i n a container f u l l of the b l o t t i n g s o l u t i o n . e n t i r e apparatus evaporation.  The  was wrapped i n Saran Wrap t o a v o i d e x c e s s i v e  A p i e c e o f g l a s s and a s m a l l w e i g h t was p l a c e d on  top o f the p i l e t o l e v e l the s t r u c t u r e .  The DNA was t h e n  a l l o w e d t o t r a n s f e r t o t h e membrane v i a c a p i l l a r y a c t i o n f o r 1618 h. Once t h e DNA was t r a n s f e r r e d , t h e paper t o w e l s were t a k e n off  and t h e n t h e r e m a i n i n g  c a r e f u l l y t u r n e d over.  l a y e r s , down t o t h e w i c k , were  A medium b a l l p o i n t pen was used t o mark  80 t h e l a n e s o f t h e g e l and t o date t h e Zeta-probe membrane.  To  e n s u r e t h e t r a n s f e r p r o c e s s was complete, t h e g e l was soaked i n 0.5 ug/ml o f e t h i d i u m bromide and viewed o v e r a t r a n s i l l u m inator.  The membrane was soaked i n a s m a l l volume o f 5X  ( s a l i n e sodium c i t r a t e )  ( M a n i a t i s e t al.,  1982)  SSC  f o r 5 min,  p l a c e d between 2 s h e e t s of Whatman 3MM paper, wrapped l o o s e l y i n aluminum f o i l and baked a t 60°C f o r 2 h.  B e f o r e use i n  h y b r i d i z a t i o n , b l o t s were s t o r e d a t room t e m p e r a t u r e . Curing  Protocols:  A c t i v e l y growing c u l t u r e s of t h e t y p i c a l T-2774 o r t h e a t y p i c a l A-0181 i s o l a t e were d i l u t e d 1/1000 i n t o L u r i a b r o t h and t r e a t e d w i t h one of t h e f o l l o w i n g c u r i n g a g e n t s : sulfate  sodium d o d e c y l  (SDS), e t h i d i u m bromide o r a c r i d i n e orange a t v a r y i n g  c o n c e n t r a t i o n s (Table 3.1).  The shake c u l t u r e s were i n c u b a t e d  a t e l e v a t e d t e m p e r a t u r e s (28°C f o r T-2774 and 31°C f o r A-0181) f o r 48 h.  One o r two s u c c e s s i v e t r a n s f e r s were made w i t h t h e  c u r i n g agents.  R e s u l t i n g c u l t u r e s were s t o r e d b r i e f l y a t 4°C o r  -80°C b e f o r e d i l u t i o n p l a t i n g . The p o t e n t i a l l y c u r e d a t y p i c a l c o l o n i e s were probed w i t h 3 2  P - l a b e l l e d p l a s m i d DNA  from t h e a t y p i c a l A-0181 i s o l a t e and t h e  p o t e n t i a l l y c u r e d t y p i c a l c o l o n i e s were probed w i t h p l a s m i d DNA  from t h e t y p i c a l T-2774 i s o l a t e .  3 2  P-labelled  81  T a b l e 3.1. C o n c e n t r a t i o n s o f c h e m i c a l c u r i n g a g e n t s added t o LB i n c u r i n g p r o t o c o l s used w i t h t h e g a l l f o r m i n g E r w i n i a i s o l a t e s T-2774 and A-0181 Gall-forming Erwinia  isolate  Sodium Acridine dodecyl orange s u l f a t e (%) (yug/ml)  Ethidium bromide (jug/ml)  Temperature (°C)  T-2774  0.0175  60  15  28  A-0181  0. 0300  70  40  31  82 Colony L i f t s : G r i d p l a t e s or d i l u t i o n p l a t e s of the c o l o n i e s of i n t e r e s t were grown up f o r 2 days on n u t r i e n t agar (NA) p l a t e s and t h e n t h e c o l o n i e s were t r a n s f e r r e d t o BioRad C P / L i f t membranes according  t o t h e accompanying i n s t r u c t i o n s .  The membranes were  c a r e f u l l y p l a c e d on t h e agar s u r f a c e f o r 3-5 min.  Identifying  p a t t e r n s were made on t h e membranes and c o n c o m i t a n t l y p l a t e s w i t h an 18 gauge n e e d l e . colony  on t h e  The membranes were p l a c e d ,  s i d e up, on 3-5 l a y e r s o f Whatman 3MM paper  saturated  w i t h 0.5 M NaOH f o r 5 min f o l l o w e d by a b r i e f b l o t t i n g on d r y Whatman 3MM paper. of t h e c e l l s .  T h i s s t e p was r e p e a t e d once t o e n s u r e l y s i s  The membranes were t h e n r i n s e d i n 2X SSC and 0.2%  SDS b r i e f l y t o remove c e l l d e b r i s .  A f t e r b l o t t i n g d r y on  Whatman 3MM, t h e C/P L i f t membranes were exposed t o UV  light  (General  E l e c t r i c G e r m i c i d a l G25T8 25 W) f o r 5-10 min t o ensure  covalent  l i n k a g e o f t h e DNA t o t h e n y l o n membrane.  The  membranes were s t o r e d , i f n e c e s s a r y , a t room t e m p e r a t u r e . 32  P Labelling: Plasmid  DNA was l a b e l l e d u s i n g t h e BRL random p r i m e r s DNA  l a b e l l i n g system.  An i n i t i a l attempt u s i n g n i c k t r a n s l a t i o n  (Pharmacia) was n o t s u c c e s s f u l .  The T-2774 probe was p r e p a r e d  from m i n i p r e p DNA t h a t had been p u r i f i e d t h r o u g h e l e c t r o e l u t i o n . The A-0181 probe was p r e p a r e d from C s C l - p u r i f i e d DNA, p u r i f i e d t h r o u g h GeneClean. type plasmid 12  A p p r o x i m a t e l y 50 ng o f t h e w i l d -  DNA was d i g e s t e d w i t h P s t 1 i n a maximum volume o f  The p r o t o c o l as o u t l i n e d by BRL (Bethesda R e s e a r c h  L a b s ) , was f o l l o w e d t o l a b e l t h e DNA 1984).  further  ( F e i n b e r g and V o g e l s t e i n ,  83 The  l a b e l l e d DNA  i n 300 yul H 0.  The  2  32  was  e t h a n o l - p r e c i p i t a t e d and  c o u n t s per minute f o r t o t a l and  P-dATP were measured.  measure t o t a l c o u n t s ,  32  simply placed i n a g l a s s  10 ml of A q u a s o l was  added and  counts To measure  P-dATP, t h e d i s c c o n t a i n i n g t h e sample was  w i t h 15 ml c o l d 10% t r i c h l o r o a c e t i c a c i d (TCA). ml r i n s e s w i t h c o l d 95% e t h a n o l , t h e d i s c was s c i n t i l l a t i o n v i a l and  To  and  measured on a H e w l e t t - P a c k a r d S c i n t i l l a t i o n C o u n t e r . incorporated  total  glass-fiber discs.  i n c l u d i n g both incorporated  l a b e l , t h e d i s c was  scintillation vial,  incorporated  Twojul samples (1/150th o f t h e  sample) were s p o t t e d onto Whatman GF/C  unincorporated  resuspended  10 ml A q u a s o l added.  rinsed  A f t e r two,  15-  placed i n a glass Counts were  measured on t h e s c i n t i l l a t i o n c o u n t e r w i t h % i n c o r p o r a t i o n o f 32  P-dATP c a l c u l a t e d from t h e  readings.  Hybridization:  The  C/P  L i f t and  Zeta-probe membranes were s e a l e d i n  p l a s t i c sandwich boxes w i t h a p p r o x i m a t e l y buffer  (ImM  EDTA, 0.5mM NaHP0  4  (pH 7.2),  2 0 ml of h y b r i d i z a t i o n 7% SDS).  An  p e r i o d , i n a s h a k i n g water b a t h s e t a t 65°C, o f 5 min  incubation for  C/P  L i f t membranes o r 2 h f o r Zeta-Probe membranes, p r e c e d e d hybridization.  The  l a b e l l e d probe was  added t o  the  h y b r i d i z a t i o n b u f f e r a f t e r a 5 min d e n a t u r a t i o n p e r i o d i n a b o i l i n g water bath. 18-24  H y b r i d i z a t i o n was  h a t 65°C (shaking)  t h e B i o R a d C/P  allowed t o proceed f o r  according t o the p r o t o c o l o u t l i n e d f o r  L i f t Membranes.  A f t e r t h e h y b r i d i z a t i o n p e r i o d , t h e probe was  carefully  p o u r e d i n t o a 50 ml F a l c o n tube and s t o r e d a t -2 0°C. H y b r i d i z a t i o n was  f o l l o w e d by 4, 30-min washes a t 65°C.  The  84 f i r s t two washing s o l u t i o n s c o n s i s t e d o f ImM EDTA, 4 0mM NaHP0  4  and 5% SDS, w h i l e t h e f i n a l two washes c o n s i s t e d o f ImM EDTA, 4 0mM NaHP0 and 1% SDS. 4  An i n i t i a l r i n s e w i t h t h e f i r s t  s o l u t i o n was used t o remove a l a r g e p e r c e n t a g e o f unbound  washing label.  The C P / L i f t o r Zeta-Probe membranes were wrapped i n Saran Wrap and p u t up on X-ray f i l m w i t h enhancing s c r e e n s .  The X-ray  was exposed t o t h e h y b r i d i z e d membranes e i t h e r a t room t e m p e r a t u r e o r a t -70°C f o r between 3 h and 3 days.  film  85 RESULTS  Plasmid Content:  For t h e t y p i c a l E r w i n i a i s o l a t e s , a s i n g l e p l a s m i d  band  m i g r a t i n g w i t h any i s o l a t e d chromosomal DNA was o b s e r v e d 4-8 i n F i g . 3.1). was  Occasionally a different  (lanes  form o f t h e p l a s m i d  seen m i g r a t i n g b e h i n d t h e chromosomal band ( l a n e 9 i n F i g .  3.1).  The p r o f i l e f o r t h e a t y p i c a l A-0181 i s o l a t e was more  complex, c o n s i s t i n g  o f s e v e r a l bands ( l a n e 3 i n F i g . 3 . 1 ) .  F o l l o w i n g p l a s m i d p u r i f i c a t i o n by C s C l centrifugation, contained  gradient  t h e p r o f i l e o f t h e a t y p i c a l A-0181 i s o l a t e  4 t o 5 bands which appeared t o c o n s i s t o f d i f f e r e n t  forms o f one o r p o s s i b l y two p l a s m i d s ( F i g . 3.2).  The lower  band, D ( l a n e 1 ) , was f u r t h e r p u r i f i e d u s i n g LGT a g a r o s e , and r e r u n on an agarose g e l , showing a h i g h degree o f p u r i f i c a t i o n (lane 2).  S i m i l a r l y t h e second band, C, was p u r i f i e d as shown  i n l a n e 3.  When t h e t o p two bands, A and B, were e x c i s e d and  p u r i f i e d together the r e s u l t i n g pattern displayed the four o r i g i n a l bands, s u g g e s t i n g  t h a t t h e lower two bands a r e m o d i f i e d  forms o f t h e upper two bands. Restriction  d i g e s t s f o r p l a s m i d s from b o t h t h e t y p i c a l and  t h e A-0181 i s o l a t e s , a r e shown i n F i g . 3.3. D i g e s t s o f t h e T2789 p l a s m i d u s i n g P s t 1 and Hind I I I suggested a s i z e o f approximately  50 kb ( l a n e s 1 and 2 ) .  moving A-0181 band, Band D, r e s u l t e d ( l a n e 4 and 5 ) .  A l l digests of the fastest i n only p a r t i a l  When r u n a g a i n s t Hind I l l - d i g e s t e d  digests lambda DNA,  u n c u t Band D appeared t o be between 10 and 2 0 kb i n s i z e  (lanes  86  1 2 3 4 5 6 7 8 9  lO 11  F i g . 3.1. P l a s m i d DNA p r o f i l e s o f t y p i c a l and a t y p i c a l g a l l f o r m i n g Erwinia i s o l a t e s f r a c t i o n a t e d on a 0.7% a g a r o s e g e l and s t a i n e d w i t h e t h i d i u m bromide. P l a s m i d DNA was e x t r a c t e d u s i n g t h e s m a l l - s c a l e a l k a l i n e l y s i s p r o t o c o l as o u t l i n e d i n M a n i a t i s e t al. ( 1 9 8 2 ) . Lanes: (1) Erwinia carotovora subsp. carotovora; (2) E. herbicola s u b s p . herbicola s t r a i n 2 2 7 3 ; (3) a t y p i c a l i s o l a t e A - 0 1 8 1 ; (4) t y p i c a l i s o l a t e T-2774; (5) t y p i c a l i s o l a t e T-2789; (6) t y p i c a l i s o l a t e T-2721; (7) t y p i c a l i s o l a t e T - 2 7 3 9 ; (8) t y p i c a l i s o l a t e T-2763; (9) t y p i c a l i s o l a t e T-2722; (10) Escherichia coli s t r a i n JM101 and (11) H i n d l l l - d i g e s t e d lambda D N A / H a e l l l - d i g e s t e d BX-174 DNA p r o v i d i n g DNA s i z e standards.  87  F i g . 3.2. P l a s m i d DNA p r o f i l e o f Erwinia i s o l a t e A-0181 f r a c t i o n a t e d on a 0.7% a g a r o s e g e l and s t a i n e d w i t h e t h i d i u m bromide. P l a s m i d DNA was i s o l a t e d u s i n g t h e l a r g e - s c a l e a l k a l i n e l y s i s p r o t o c o l as o u t l i n e d i n M a n i a t i s e t al. (1982) and p u r i f i e d t h r o u g h c e s i u m c h l o r i d e g r a d i e n t centrifugation. Lanes: (1) B a n d s A, B, C, D o f A - 0 1 8 1 ; (2) Band D p u r i f i e d t h r o u g h Low G e l l i n g T e m p e r a t u r e (LGT) a g a r o s e f o l l o w e d by p h e n o l / c h l o r o f o r m e x t r a c t i o n and e t h a n o l p r e c i p i t a t i o n ; (3) Band C p u r i f i e d t h r o u g h LGT a g a r o s e ; (4) Bands A and B p u r i f i e d t h r o u g h LGT a g a r o s e and (5) H i n d l l l - d i g e s t e d lambda DNA marker.  88  F i g . 3.3. Plasmid DNAs from t y p i c a l and a t y p i c a l g a l l - f o r m i n g Erwinia i s o l a t e s d i g e s t e d with r e s t r i c t i o n endonucleases, f r a c t i o n a t e d on a 0.9% agarose g e l and s t a i n e d w i t h e t h i d i u m bromide. Plasmid DNA was i s o l a t e d from t y p i c a l i s o l a t e s by a s m a l l - s c a l e a l k a l i n e l y s i s procedure (Maniatis e t al., 1982) and from the a t y p i c a l i s o l a t e by a l a r g e - s c a l e a l k a l i n e l y s i s procedure (Maniatis et a l . , 1982). The a t y p i c a l p l a s m i d DNA was f u r t h e r p u r i f i e d through cesium c h l o r i d e c e n t r i f u g a t i o n and LGT agarose. Lanes: (1) H i n d l l l - d i g e s t e d t y p i c a l i s o l a t e T-2722; (2) P s t 1-digested t y p i c a l i s o l a t e T-2722; (3) H i n d l l l - d i g e s t e d lambda DNA marker; (4) H i n d l l l - d i g e s t e d Band D from a t y p i c a l i s o l a t e A-0181 ( F i g . 3.2) and (5) Pst 1-digested Band D from a t y p i c a l i s o l a t e A-0181.  89 1-4 i n F i g . 3.1). Probe S p e c i f i c i t y / H o m o l o g y :  Southern analyses of d i f f e r e n t b a c t e r i a l s t r a i n s u s i n g p l a s m i d p r o b e s from g a l l - f o r m i n g Ei-winia  T-2774 and A-0181 a r e  shown i n F i g u r e s 3.4, 3.5, 3.6 and 3.7.  Lane 6 i n F i g u r e 3.4  shows s t r o n g h y b r i d i z a t i o n between t h e t y p i c a l T-2774 p l a s m i d probe (from a s m a l l - s c a l e p l a s m i d DNA p r e p a r a t i o n ) and t h e t o t a l DNA p r e p a r a t i o n o f t y p i c a l i s o l a t e T-2774.  Hybridization  o c c u r r e d a t a lower degree between t h e t y p i c a l T-2774 probe and t h e A-0181 DNA p r e p a r a t i o n s ( l a n e s 5, 8, 9 and 1 0 ) .  Weak  h y b r i d i z a t i o n o c c u r r e d between t h e t y p i c a l probe and t h e two Erwinia  spp. (E. carotovora  and E. herbicola  s t r a i n 2273) ( l a n e s 3 and 4 ) .  subsp.  No homology was d e t e c t e d  t h e t y p i c a l T-2774 probe and t h e E s c h e r i c h i a coli Agrobacterium  tumefaciens  herbicola  between  or  DNA p r e p a r a t i o n s ( l a n e s 1 and 2 ) .  When t e s t e d a g a i n s t m i n i p r e p s o f o t h e r t y p i c a l i s o l a t e s and t h e a t y p i c a l i s o l a t e A-0181, t h e T-2774 probe h y b r i d i z e d t o each band o f each p r o f i l e o f t h e g a l l - f o r m i n g E r w i n i a i s o l a t e s ( F i g . 3.5).  No homology was d e t e c t e d between t h e t y p i c a l T-2774 probe  and t h e DNA p r e p a r a t i o n s o f Erwinia carotovora,  carotovora  E. h e r b i c o l a subsp. herbicola  E s c h e r i c h i a coli  subsp.  s t r a i n 2273 and  s t r a i n JM101 ( l a n e s 1, 2 and 1 0 ) .  When t h e C s C l - p u r i f i e d a t y p i c a l A-0181 p l a s m i d probe was used on s i m i l a r b l o t s , t h e h y b r i d i z a t i o n o n l y o c c u r r e d w i t h t h e homologous  samples ( l a n e s 5, 8, 9 and 12 i n F i g . 3.6 and l a n e s 7  and 13 i n F i g . 3.7).  The A-0181 p l a s m i d probe h y b r i d i z e d t o  each p l a s m i d band o f A-0181.  No h y b r i d i z a t i o n o c c u r r e d  between  90  1  2 3 4 5 6 7 8 9  10  F i g . 3.4. G e l e l e c t r o p h o r e s i s p r o f i l e and Southern a n a l y s i s o f t o t a l g e n o m i c a n d p l a s m i d DNA o f v a r i o u s p h y t o p a t h o g e n i c b a c t e r i a p r o b e d w i t h p l a s m i d DNA o f a t y p i c a l g a l l - f o r m i n g Erwinia isolate. A, 0.9% a g a r o s e g e l s t a i n e d w i t h e t h i d i u m bromide. B, A u t o r a d i o g r a m o f S o u t h e r n b l o t o f DNA t r a n s f e r r e d t o Z e t a - P r o b e membrane a n d p r o b e d w i t h p l a s m i d DNA o f t y p i c a l i s o l a t e T-2774 ( i s o l a t e d u s i n g t h e s m a l l - s c a l e a l k a l i n e l y s i s p r o c e d u r e a s o u t l i n e d i n M a n i a t i s e t al., 1982 a n d p u r i f i e d through e l e c t r o e l u t i o n ) . Lanes: (1) P s t 1 - d i g e s t e d t o t a l DNA o f Escherichia coli s t r a i n J M 1 0 1 ; (2) P s t 1 - d i g e s t e d t o t a l DNA o f Agrobacterium tumefaciens s t r a i n B - l ; (3) P s t 1 - d i g e s t e d t o t a l DNA o f Erwinia carotovora s u b s p . carotovora; (4) P s t 1d i g e s t e d t o t a l DNA o f E. herbicola s u b s p . herbicola s t r a i n 2273 (5) P s t 1 - d i g e s t e d t o t a l DNA o f a t y p i c a l Erwinia i s o l a t e A-0181 (6) P s t 1 - d i g e s t e d t o t a l DNA o f t y p i c a l Erwinia i s o l a t e T-2774; (7) H i n d l l l - d i g e s t e d l a m b d a D N A / H a e l l l - d i g e s t e d BX-174 DNA; (8) P s t 1 - d i g e s t e d p l a s m i d B a n d D ( F i g . 3.2) o f a t y p i c a l Erwinia i s o l a t e A - 0 1 8 1 ; (9) P s t 1 - d i g e s t e d Band C ( F i g . 3.2) o f a t y p i c a Erwinia i s o l a t e A-0181 a n d (10) P s t 1 - d i g e s t e d t o t a l DNA o f a t y p i c a l Erwinia i s o l a t e A-0181.  91  1 2 3 4 5 6  7 8 9 lOll  F i g . 3.5. G e l e l e c t r o p h o r e s i s p r o f i l e and Southern a n a l y s i s o f p l a s m i d DNA o f v a r i o u s p h y t o p a t h o g e n i c b a c t e r i a probed w i t h p l a s m i d DNA o f a t y p i c a l g a l l - f o r m i n g E r w i n i a i s o l a t e . Plasmid DNA was e x t r a c t e d u s i n g t h e s m a l l - s c a l e a l k a l i n e l y s i s p r o t o c o l as o u t l i n e d i n M a n i a t i s e t al. (1982). A, 0.7% agarose g e l s t a i n e d w i t h e t h i d i u m bromide. B, A u t o r a d i o g r a m o f Southern b l o t o f DNA t r a n s f e r r e d t o Zeta-Probe membrane probed w i t h p l a s m i d DNA o f t y p i c a l i s o l a t e T-2774 ( i s o l a t e d by a s m a l l - s c a l e a l k a l i n e l y s i s p r o c e d u r e as o u t l i n e d i n M a n i a t i s e t al., 1982 and p u r i f i e d t h r o u g h e l e c t r o e l u t i o n ) . Lanes: (1) E r w i n i a carotovora  subsp. carotovora;  (2) E. herbicola  subsp.  herbicola  s t r a i n 2273; (3) a t y p i c a l Erwinia i s o l a t e A-0181; (4) t y p i c a l E r w i n i a i s o l a t e T-2774; (5) t y p i c a l Erwinia i s o l a t e T-2789; (6) t y p i c a l E r w i n i a i s o l a t e T-2721; (7) t y p i c a l E r w i n i a i s o l a t e T-2739; (8) t y p i c a l Erwinia i s o l a t e T-2763; (9) t y p i c a l E r w i n i a i s o l a t e T-2722; (10) Escherichia coli s t r a i n JM101 and (11) H i n d l l l - d i g e s t e d lambda D N A / H a e l l l - d i g e s t e d 5X-174 DNA markers.  92  F i g . 3.6. G e l e l e c t r o p h o r e s i s p r o f i l e and Southern a n a l y s i s o f t o t a l genomic and p l a s m i d DNA o f v a r i o u s p h y t o p a t h o g e n i c b a c t e r i a probed w i t h p l a s m i d DNA o f t h e a t y p i c a l g a l l - f o r m i n g Erwinia i s o l a t e . A, 0.9% agarose g e l s t a i n e d w i t h e t h i d i u m bromide. B, Autoradiogram o f Southern b l o t o f DNA t r a n s f e r r e d t o Zeta-Probe membrane and probed w i t h p l a s m i d DNA o f a t y p i c a l i s o l a t e A-0181 ( p u r i f i e d t h r o u g h cesium c h l o r i d e g r a d i e n t c e n t r i f u g a t i o n and GeneClean). Lanes: (1) P s t 1 - d i g e s t e d t o t a l DNA o f Escherichia coli JM101; (2) P s t 1 - d i g e s t e d t o t a l DNA o f Agrobacterium tumefaciens s t r a i n B - l ; (3) P s t 1 - d i g e s t e d t o t a l DNA o f Erwinia carotovora subsp. carotovora; (4) P s t 1 - d i g e s t e d t o t a l DNA o f E. herbicola subsp. herbicola s t r a i n 2273; (5) P s t 1 - d i g e s t e d t o t a l DNA o f a t y p i c a l Erwinia i s o l a t e A-0181; (6) P s t 1 - d i g e s t e d t o t a l DNA o f t y p i c a l Erwinia i s o l a t e T-2774; (7) H i n d l l l - d i g e s t e d lambda D N A / H a e l l l - d i g e s t e d 5X-174 DNA; (8) P s t 1 - d i g e s t e d p l a s m i d Band D ( F i g . 3.2) o f a t y p i c a l Erwinia isolate A-0181; (9) u n c u t p l a s m i d Band D o f a t y p i c a l Erwinia isolate A-0181; (10) P s t 1 - d i g e s t e d p l a s m i d DNA o f t y p i c a l Erwinia i s o l a t e T-2774; (11) uncut p l a s m i d DNA o f t y p i c a l Erwinia i s o l a t e T-2774 and (12) P s t 1 - d i g e s t e d p l a s m i d Band D o f a t y p i c a l Erwinia i s o l a t e A-0181.  93  1 2 3 4 5 6 7 8 91011121314  4  •  F i g . 3.7. G e l e l e c t r o p h o r e s i s p r o f i l e and Southern a n a l y s i s o f p l a s m i d DNA o f v a r i o u s p h y t o p a t h o g e n i c b a c t e r i a probed w i t h p l a s m i d DNA o f t h e a t y p i c a l g a l l - f o r m i n g Erwinia isolate. P l a s m i d DNA was e x t r a c t e d u s i n g t h e m o d i f i e d s m a l l - s c a l e a l k a l i n e l y s i s p r o t o c o l as o u t l i n e d i n M a n i a t i s e t al. (1982). A, 0.7% agarose g e l s t a i n e d w i t h e t h i d i u m bromide. B, A u t o r a d i o g r a m o f Southern b l o t o f DNA t r a n s f e r r e d t o Zeta-Probe membrane and probed w i t h p l a s m i d DNA o f a t y p i c a l i s o l a t e A-0181 ( p u r i f i e d t h r o u g h cesium c h l o r i d e g r a d i e n t c e n t r i f u g a t i o n and GeneClean). Lanes: (1) Pseudomonas syringae pv. syringae ( c h e r r y s t r a i n ) ; (2) Escherichia coli s t r a i n MC1000; (3) Agrobacterium pseudotsugae s t r a i n 180; (4) Erwinia carotovora subsp. carotovora; (5) E. herbicola subsp. herbicola strain 2273; (6) H i n d l l l - d i g e s t e d lambda D N A / H a e l l l - d i g e s t e d JX-174 DNA m a r k e r s ; (7) a t y p i c a l Erwinia i s o l a t e A-0181; (8) t y p i c a l Erwinia i s o l a t e T-2739; (9) t y p i c a l Erwinia i s o l a t e T-2721; (10) t y p i c a l Erwinia i s o l a t e T-2789; (11) t y p i c a l Erwinia isolate T-2722; (12) t y p i c a l Erwinia i s o l a t e T-2774; (13) a t y p i c a l Erwinia i s o l a t e A-0181 and (14) t y p i c a l Erwinia i s o l a t e T-2763.  the  a t y p i c a l p l a s m i d probe and t h e t y p i c a l i s o l a t e  DNA  p r e p a r a t i o n s ( l a n e s 6, 10 and 11 i n F i g . 3.6 and l a n e s 8-12 and 14 i n F i g . 3.7). Curing/Colony H y b r i d i z a t i o n :  H y b r i d i z a t i o n o c c u r r e d between c o l o n i e s o f t h e t y p i c a l i s o l a t e s and t h e T-2774 probe and t o a l e s s e r colonies of the a t y p i c a l hybridization and  e x t e n t between t h e  i s o l a t e and t h e T-2774 DNA probe.  o c c u r r e d between E. carotovora  No  subsp. c a r o t o v o r a  t h e T-2774 probe ( F i g . 3.8). H y b r i d i z a t i o n o c c u r r e d w i t h each p o t e n t i a l l y  t e s t e d o f b o t h t h e t y p i c a l and a t y p i c a l  isolates,  t h a t t h e p l a s m i d s had n o t been c u r e d from e i t h e r  cured colony indicating i s o l a t e type  ( F i g . 3.9). However, because t h e t y p i c a l T-2774 DNA probe appeared t o c o n t a i n chromosomal DNA, i t i s p o s s i b l e t h a t t h i s chromosomal DNA was r e s p o n s i b l e f o r t h e h y b r i d i z a t i o n the  t y p i c a l DNA probe and t h e t y p i c a l c o l o n i e s .  c o l o n i e s p e r t r e a t m e n t p e r i s o l a t e were t e s t e d .  between  A t l e a s t 100  95  F i g . 3.8. Autoradiogram o f c o l o n y h y b r i d i z a t i o n o f p l a s m i d DNA probe o f t y p i c a l g a l l - f o r m i n g Erwinia i s o l a t e T-2774 w i t h Erwinia spp. The DNA f o r t h e probe was i s o l a t e d by a s m a l l s c a l e a l k a l i n e l y s i s procedure as o u t l i n e d i n M a n i a t i s e t al., 1982 and p u r i f i e d through e l e c t r o e l u t i o n . C o l o n i e s o f (1) t y p i c a l Erwinia i s o l a t e T-2789 (2) t y p i c a l Erwinia isolate T-2774 (3) a t y p i c a l Erwinia i s o l a t e A-0181 (4) t y p i c a l Erwinia i s o l a t e T-2763 (5) t y p i c a l Erwinia i s o l a t e T-2722 (6) t y p i c a l Erwinia i s o l a t e T-2721 (7) t y p i c a l Erwinia i s o l a t e T-2763 (8) t y p i c a l Erwinia i s o l a t e T-2739 (9) E. carotovora subsp. carotovora and (10) t y p i c a l Erwinia i s o l a t e T-2774 were t r a n s f e r r e d t o C/P L i f t membranes, l y s e d and probed w i t h randomprimed d A T P - l a b e l l e d p l a s m i d DNA. 32  96  •• • • • • • •• • • t  <<ift  m  *  -*>  •*  •  #  •  •  •  F i g . 3.9. Autoradiogram of colony h y b r i d i z a t i o n of dAT Pl a b e l l e d plasmid DNA of a t y p i c a l g a l l - f o r m i n g Erwinia isolate A-0181 with p o t e n t i a l l y cured c o l o n i e s of Erwinia isolate A-0181. The DNA f o r the probe was p u r i f i e d through cesium c h l o r i d e g r a d i e n t c e n t r i f u g a t i o n and GeneClean. Each colony t e s t e d h y b r i d i z e d with the plasmid probe. 32  97 DISCUSSION  Both t h e t y p i c a l and a t y p i c a l i s o l a t e s of t h e g a l l - f o r m i n g E r w i n i a c a r r i e d a t l e a s t one p l a s m i d .  The m o d i f i e d a l k a l i n e  e x t r a c t i o n p r o t o c o l as o u t l i n e d i n M a n i a t i s e t al. adopted as a r a p i d and s i m p l e procedure plasmid i s o l a t i o n .  (1982)  was  t o use f o r r o u t i n e  The t y p i c a l i s o l a t e s c o n t a i n one  plasmid  band w h i l e t h e p l a s m i d p r o f i l e of t h e A-0181 i s o l a t e c o n t a i n s up t o 5 bands w h i c h appear t o c o n s i s t of t h e d i f f e r e n t forms o f o r p o s s i b l y two p l a s m i d s .  one  Large s c a l e p l a s m i d DNA e x t r a c t i o n s  were s u c c e s s f u l w i t h t h e a t y p i c a l A-0181 i s o l a t e but n o t w i t h t h e t y p i c a l T-2 789  isolate.  Problems were encountered  a t t e m p t i n g t o i s o l a t e t h e p l a s m i d DNA  from t h e t y p i c a l  p o s s i b l y due t o a low copy number and a l a r g e r p l a s m i d Furthermore,  when isolates size.  t h e T-2789 p l a s m i d u s u a l l y r a n w i t h t h e chromosomal  band and r a r e l y i n a d i f f e r e n t form behind t h e chromosomal hampering a t t e m p t s t o i s o l a t e and p u r i f y t h e p l a s m i d  DNA  DNA.  Assessments i n c l u d i n g r e s t r i c t i o n a n a l y s e s were used t o determine  t h e s i z e of the Erwinia  plasmids.  s i z e o f t h e p l a s m i d of t h e t y p i c a l Erwinia  The p u t a t i v e l a r g e i s o l a t e s , and  the  d i f f i c u l t y o f p u r i f i c a t i o n of the p l a s m i d , r e s u l t e d i n d i f f i c u l t i e s o f p r e c i s e e s t i m a t i o n of s i z e .  In the  atypical  i s o l a t e , t h e c o m p l e x i t y of the p l a s m i d c o n t e n t i n t e r f e r e d w i t h d e f i n i n g t h e s i z e of t h e p l a s m i d ( s ) .  D i f f i c u l t i e s could also  have a r i s e n from an i n s u f f i c i e n t amount of DNA  following gel  p u r i f i c a t i o n and t h e p o s s i b i l i t y of some contaminants the r e a c t i o n s .  inhibiting  Thus, r e s t r i c t i o n d i g e s t s of t h e p l a s m i d s  b o t h t h e t y p i c a l and t h e A-0181 i s o l a t e s d i d not  yield  of  98 conclusive data regarding the s i z e of the plasmids. Both t h e t y p i c a l plasmid  DNA probe (not p u r i f i e d t h r o u g h  C s C l g r a d i e n t c e n t r i f u g a t i o n ) and t h e C s C l - p u r i f i e d a t y p i c a l plasmid  DNA probe were s p e c i f i c t o t h e g a l l - f o r m i n g  isolates.  No h y b r i d i z a t i o n o c c u r r e d  t o t a l genomic DNA p r e p a r a t i o n s E s c h e r i c h i a coli.  Ei-winia  between e i t h e r probe and  o f Agrobacterium  tumefaciens  and  The n o n - p u r i f i e d t y p i c a l T-2774 probe  h y b r i d i z e d s t r o n g l y t o t h e T-2774 t o t a l DNA p r e p a r a t i o n l e s s e r e x t e n t t o t h e a t y p i c a l A-0181 DNA p r e p a r a t i o n .  and t o a These  e x t e n s i v e h y b r i d i z a t i o n p r o f i l e s suggest t h a t t h e n o n - p u r i f i e d t y p i c a l plasmid  probe may c o n t a i n some h i g h l y r e p e t i t i v e  sequences from chromosomal DNA.  The p r e s e n c e o f chromosomal DNA  i n t h e n o n - p u r i f i e d t y p i c a l T-2774 p l a s m i d  DNA probe i s a l s o  i n d i c a t e d by t h e weak h y b r i d i z a t i o n between t h e n o n - p u r i f i e d t y p i c a l T-2774 probe and t h e Erwinia  carotovora  and E. h e r b i c o l a  subsp. h e r b i c o l a s t r a i n 2273 t o t a l genomic DNA p r e p a r a t i o n s . There appeared t o be conserved p l a s m i d v a r i o u s t y p i c a l g a l l - f o r m i n g Erwinia hybridization.  sequences w i t h i n t h e  i s o l a t e s when u s i n g DNA:DNA  Furthermore DNA:DNA h y b r i d i z a t i o n p r o f i l e s  showed each p l a s m i d  band from t h e a t y p i c a l i s o l a t e h y b r i d i z e d  w i t h t h e t y p i c a l T-2774 DNA probe, s u g g e s t i n g  the p o s s i b i l i t y  t h a t h i g h l y r e p e t i t i v e sequences homologous t o chromosomal DNA sequences o f T-2774 o c c u r i n t h e a t y p i c a l p l a s m i d s . p u r i f i e d plasmid  The C s C l -  probe from t h e a t y p i c a l A-0181 i s o l a t e d i d n o t  h y b r i d i z e w i t h any o f t h e t y p i c a l i s o l a t e p r o f i l e s , i n d i c a t i n g t h a t t h e r e was no sequence homology between t h e t y p i c a l and a t y p i c a l plasmid  types.  I f h i g h l y r e p e t i t i v e , c h r o m o s o m a l l y - d e r i v e d sequences were  99 p r e s e n t i n t h e A-0181 p l a s m i d s , t h e C s C l - p u r i f i e d p l a s m i d probe would n o t n e c e s s a r i l y d e t e c t t h e chromosomal analogues i n t h e t o t a l DNA b l o t o f t h e t y p i c a l i s o l a t e s because o f t h e low c o n c e n t r a t i o n o f randomly d i s t r i b u t e d sequences.  Thus t h e  sequences i n t h e t o t a l DNA b l o t would be i n a v e r y low copy number as compared t o t h e p l a s m i d bands and a l s o may be w i t h i n d i f f e r e n t s i z e d r e s t r i c t i o n fragments, the b l o t .  thus  'spread' o u t over  However, i f t h e r e p e t i t i v e sequences were i n c l u d e d i n  t h e probe t h e y c o u l d p i c k up common sequences i n d i s c r e t e p l a s m i d bands, as o c c u r r e d w i t h t h e A-0181 probe and i t s homologous b l o t s . chromosomal-like  These r e s u l t s may i n d i c a t e t h a t t h e r e a r e r e p e t i t i v e sequences i n t h e a t y p i c a l  gall-  f o r m i n g E r w i n i a p l a s m i d s , maybe r e s u l t i n g from i n t e g r a t i o n o f t h e p l a s m i d s i n t o t h e chromosome and subsequent e x c i s i o n o f some o f t h e chromosomal DNA w i t h t h e p l a s m i d DNA (Hardman and Gowland, 1985b). C u r i n g o f t h e T i p l a s m i d from A. tumefaciens a c h i e v e d t h r o u g h t h e use o f a heat t r e a t m e n t 1975). savastanoi  was o r i g i n a l l y  (Watson e t al.,  C u r i n g o f t h e pIAA p l a s m i d from P. syringae was a c h i e v e d u s i n g an a c r i d i n e orange  (Comai e t a l . ,  1982).  pv. treatment  F o r both o f t h e s e pathogens, t h e p l a s m i d -  f r e e s t r a i n s were n o t a b l e t o induce g a l l i n g on t h e h o s t p l a n t s i n d i c a t i n g t h a t genes e s s e n t i a l t o t h e p a t h o g e n i c i t y o f t h e pathogen were l o c a t e d on t h e p l a s m i d s .  Plasmid-free strains  were n o t however produced from t h e a t y p i c a l g a l l - f o r m i n g E r w i n i a i s o l a t e s using the curing protocols.  T h e r e f o r e , no c o r r e l a t i o n  between p l a s m i d presence  and p a t h o g e n i c i t y c o u l d be  f o r t h e a t y p i c a l Erwinia  isolates.  determined  100 I t i s o f t e n d i f f i c u l t t o r i d b a c t e r i a l pathogens o f plasmids v i a curing protocols.  Some e x p l a n a t i o n s  d i f f i c u l t y include the f a c t that not a l l plasmids, large plasmids,  for this especially  a r e s u s c e p t i b l e t o t h e c u r i n g a g e n t s (Hardman  and Gowland, 1985a).  Large p l a s m i d s s h a r e many chromosomal  r e p l i c a t i o n c h a r a c t e r i s t i c s and a l s o may i n t e g r a t e i n t o t h e chromosome t h u s a v o i d i n g t h e e f f e c t s o f t h e c u r i n g a g e n t s (Broda, 1979). Other methods t h a t c o u l d have p r o v i d e d  insight into the  r o l e ( s ) or function(s) of the gall-forming E r w i n i a plasmids i n g a l l formation  include the t r a n s f e r of the E r w i n i a plasmids i n t o  s u i t a b l e hosts v i a conjugation  with or without a helper  (Hamamoto and Murooka, 1987).  Escherichia  spp.,  such as E. carotovora  coli  plasmid  or other E r w i n i a  c o u l d s e r v e as r e c i p i e n t s i n t h e  c a s e o f g a l l - f o r m i n g Erwinia  isolates.  T h i s method would  r e q u i r e t h a t e i t h e r c h a r a c t e r i s t i c a n t i b i o t i c markers o r n u t r i t i o n a l markers be used t o s c r e e n p o t e n t i a l r e c i p i e n t s . A n o t h e r method t o determine i f t h e p l a s m i d pathogenicity  genes a r e i n v o l v e d i n  involves the d i r e c t cloning of portions of the  p l a s m i d s ( M a h i l l o n e t al., 1988) a g a i n i n t o s u i t a b l e h o s t s such as E. coli  o r E r w i n i a carotovora.  expression  o f t h e E r w i n i a genes w i t h i n t h e new h o s t organism and  a method o f d e t e r m i n i n g  T h i s method would r e q u i r e t h e  the expression  o f v i r u l e n c e genes.  The  use o f t r a n s p o s o n mutagenesis t o produce mutant d e r i v a t i v e s o f t h e o r i g i n a l pathogen ( M i l l s , 1985; S a l c h and Shaw, 1988) was considered  b u t n o t adopted due t o t h e complex n a t u r e o f such a  study, i e . i n determining  t h e s i t e o f m u t a t i o n and r e l a t i n g i t  t o a change i n p a t h o g e n i c i t y .  The e x p e r i m e n t a l approach p l a s m i d DNA  of c u r i n g was chosen t o r e l a t e  t o p a t h o g e n i c i t y because r e s o u r c e s f o r such  e x p e r i m e n t s were a v a i l a b l e and u s e f u l a n t i b i o t i c markers f o r s c r e e n i n g p r o c e d u r e s were not found i n t h e g a l l - f o r m i n g E r w i n i a isolates.  Had c u r e d s t r a i n s been o b t a i n e d , t h e y would have been  i n o c u l a t e d back i n t o D o u g l a s - f i r t o see i f t h e phenotype had been a l t e r e d . DNA,  Because they were n o t , t h e r o l e o f t h e p l a s m i d  i n t h e s e i s o l a t e s , i n p a t h o g e n e s i s remains u n r e s o l v e d .  102 GENERAL DISCUSSION  The  continued  from B.C. occur.  presence of g a l l - f o r m i n g b a c t e r i a , i s o l a t e d  D o u g l a s - f i r g a l l s , was  required f o r g a l l formation  to  T h i s would i n d i c a t e t h a t an i n t e r k i n g d o m t r a n s f e r of  genes t o t h e p l a n t h o s t does not o c c u r as i n t h e crown g a l l system.  Rather, the g a l l - f o r m i n g b a c t e r i a , i d e n t i f i e d  as  E r w i n i a s p e c i e s , were found t o emulate t h e system of P. pv.  syringae  savastanoi.  A p r e s u m p t i v e n e x t s t e p would be t o c o n c l u d e t h a t t h e  gall-  f o r m i n g E r w i n i a i s o l a t e s produce and s e c r e t e b o t h a u x i n s  and  c y t o k i n i n s , a g a i n s i m i l a r t o P. syringae  In  P. s y r i n g a e pv. savastanoi surrounding supply  pv. s a v a s t a n o i .  system, t h e h o s t p l a n t  cells  t h e i n v a d i n g b a c t e r i u m are a f f e c t e d by t h e exogenous  of hormones and respond w i t h c e l l d i v i s i o n and  elongation  the  (Comai e t al.,  1982).  A continued  supply  cell of  phytohormones from t h e pathogen i n d u c e s t h e u n r e g u l a t e d  growth  and d i v i s i o n of p l a n t c e l l s c h a r a c t e r i s t i c of tumor t i s s u e s .  As  t h e g a l l - f o r m i n g E r w i n i a spp. produce g a l l s s i m i l a r i n s t r u c t u r e t o t h o s e produced by A. pseudotsugae, t h o s e produced by P. syringae t h a t t h e y a l s o produce and s y r i n g a e pv. The  savastanoi  which i n t u r n resembled  pv. savastanoi,  the presumption  s e c r e t e phytohormones l i k e  P.  i s not w i t h o u t b a s i s .  r a t i o of e x c e s s c y t o k i n i n s t o a u x i n s d e t e r m i n e s t h e  form of t h e p l a n t g a l l .  A high c y t o k i n i n to auxin r a t i o r e s u l t s  i n t h e development of s h o o t s from t h e tumor t i s s u e , w h i l e a c y t o k i n i n t o a u x i n r a t i o r e s u l t s i n the development o f structures.  An i n t e r m e d i a t e  r a t i o of t h e two  root  phytohormones  low  103 r e s u l t s i n an u n o r g a n i z e d tumor.  Most o f t h e g a l l s produced by  t h e E r w i n i a spp. f i t i n t o t h e u n o r g a n i z e d c a t e g o r y . few o f t h e g a l l s c o n s i s t e d o f d i s t i n c t outgrowths b e g i n n i n g s o f teratomatous growths. t o a u x i n r a t i o produced  However, a  resembling the  Variations i nthe cytokinin  by t h e i n v a d i n g pathogen i t s e l f c o u l d  e x p l a i n t h i s phenomenon. The f u n c t i o n o f t h e p l a s m i d genes o r s i m p l y t h e i r involvement  i n p a t h o g e n i c i t y c o u l d n o t be d e t e r m i n e d .  I t was  h y p o t h e s i z e d t h a t p l a s m i d genes c o u l d encode f o r phytohormone enzymes, s i n c e a common denominator o f phytohormone p r o d u c t i o n i s l i k e l y f o r a l l of t h e phytopathogenic  gall  formers.  S i m i l a r i t i e s i n r e g i o n s o f DNA i n v o l v e d i n p a t h o g e n i c i t y e x i s t between t h e two oncogenic phytopathogens, s y r i n g a e pv. savastanoi, f o r m i n g Erwinia  A. tumefaciens  and P.  and c o u l d a l s o e x i s t w i t h t h e g a l l -  herbicola  g a l l - f o r m i n g E r w i n i a spp.  f . s p . gypsophilae  and t h e D o u g l a s - f i r  T h e r e f o r e , an e l e g a n t method t o  d e t e r m i n e t h e r o l e o f t h e Erwinia  plasmids i n p a t h o g e n i c i t y  would be t o probe t h e p l a s m i d s d i r e c t l y w i t h t h e phytohormone genes o f e i t h e r A. tumefaciens  o r P. syringae  pv.  savastanoi.  T h i s o p t i o n t o d i r e c t l y determine t h e f u n c t i o n o f t h e p l a s m i d genes would be i n t e r e s t i n g t o i n v e s t i g a t e i n a f u t u r e s t u d y . The two g a l l - f o r m i n g D o u g l a s - f i r i s o l a t e s d i f f e r e d i n many respects  from A. tumefaciens,  h e r b i c o l a f . s p . gypsophilae, b a c t e r i a l phytopathogens, 180.  Pseudomonas  spp. and  Erwinia  t h e t h r e e most common g a l l - f o r m i n g as w e l l as from A. pseudotsugae  strain  The two i s o l a t e s a l s o d i f f e r e d from each o t h e r i n a number  o f c h a r a c t e r i s t i c s , a l t h o u g h both i s o l a t e t y p e s were c l a s s i f i e d i n t h e genus E r w i n i a by f a t t y a c i d a n a l y s i s .  The unexpected  104 finding  o f two d i s t i n c t i s o l a t e t y p e s of t h e g a l l - f o r m i n g  b a c t e r i a d i c t a t e d t h a t p a r a l l e l s t u d i e s s h o u l d be conducted t h e two i s o l a t e t y p e s throughout t h i s t h e s i s .  on  The d i s c o v e r y  of  two i s o l a t e t y p e s a l s o r e q u i r e d d e s c r i p t i o n and t e n t a t i v e i d e n t i f i c a t i o n o f t h e two i s o l a t e t y p e s a l t h o u g h i d e n t i f i c a t i o n of t h e pathogen was not an o b j e c t i v e o f t h i s s t u d y . variation  i n characteristics  The  of t h e two i s o l a t e t y p e s l e a d t o  s p e c u l a t i o n on t h e c l a s s i f i c a t i o n of t h e two i s o l a t e t y p e s as two d i f f e r e n t  species.  The two g a l l - f o r m i n g Erwinia  i s o l a t e types v a r i e d  slightly  i n c o l o n y morphology (convex v e r s u s concave s u r f a c e s ) , t h e upper temperatures  l i m i t i n g growth  grow a t a h i g h e r temperature) antibiotics  (the a t y p i c a l  i s o l a t e was  able to  and t h e i r r e s i s t a n c e s t o  (the t y p i c a l i s o l a t e was r e s i s t a n t t o a w i d e r  of a n t i b i o t i c s ) , growth r a t e ( a t y p i c a l c o l o n i e s r e a c h e d a of 1 mm  i n d i a m e t e r on CPG a t 2 days and t y p i c a l  reached a s i m i l a r s i z e at 4 days). g a l l s produced  by t h e two Erwinia  size  colonies  The o u t e r appearance o f t h e i s o l a t e types  s l i g h t l y a l t h o u g h t h e i n n e r c e l l o r g a n i z a t i o n was Copeman, u n p u b l i s h e d ) .  range  differed similar  (R.J.  P l a s m i d p r o f i l e s of t h e two E r w i n i a  i s o l a t e t y p e s (Chapter 3) suggested t h a t each t y p e c o n t a i n s a t l e a s t one p l a s m i d , w i t h t h e t y p i c a l i s o l a t e s c o n t a i n i n g a l a r g e r plasmid than the a t y p i c a l  isolate.  The p l a s m i d probe o f t h e  t y p i c a l i s o l a t e h y b r i d i z e d t o t h e p l a s m i d bands o f t h e i s o l a t e but t h e r e v e r s e was not observed i n d i c a t i n g o c c u r r i n g between t h e p l a s m i d DNA  atypical  no homology  o f t h e two i s o l a t e t y p e s .  P l a s m i d p r o f i l e s sometimes a r e used i n s p e c i e s i d e n t i f i c a t i o n b u t v a r i a t i o n s o c c u r w i t h i n many s p e c i e s t h u s making  105 determinations of relationships  d i f f i c u l t v i a t h i s character  alone. A s e r o l o g i c a l r e l a t i o n s h i p between t h e two i s o l a t e t y p e s c o u l d be demonstrated by i m m u n o d i f f u s i o n when u s i n g t h e whole t y p i c a l T-2789 a n t i s e r u m b u t n o t when t h e p u r i f i e d t y p i c a l T2789 a n t i s e r u m was used.  However, as w i t h p l a s m i d p r o f i l e s ,  serological relationships  s h o u l d n o t be used a l o n e i n d e s c r i b i n g  bacterial strains. Erwinia  spp. w i t h o t h e r E n t e r o b a c t e r i a c e a e when u s i n g  antisera 1987).  C r o s s r e a c t i o n s have been o b s e r v e d f o r  t o s o r t b a c t e r i a l s t r a i n s i n t o s e r o g r o u p s (De Boer, D i f f e r e n c e s have a l s o been observed w i t h i n a s p e c i e s  with respect t o serological t r a i t s might e x p l a i n  The  (De Boer e t a l . , 1979) w h i c h  t h e l a c k o f i d e n t i t y between t h e two i s o l a t e t y p e s  when u s i n g t h e a t y p i c a l  Erwinia  polyclonal  antisera.  h o s t ranges o f t h e t y p i c a l and a t y p i c a l i s o l a t e s were n o t i d e n t i c a l .  gall-forming  The a t y p i c a l  isolate  formed g a l l i n g symptoms on more c o n i f e r s p e c i e s t h a n t h e t y p i c a l isolates.  I t i s n o t l i k e l y t h a t t h e two i s o l a t e t y p e s a r e  d i f f e r e n t p a t h o v a r s o f t h e same s p e c i e s because t h e infrasubspecific  ranking of pathovar r e f e r s t o s t r a i n s t h a t are  s i m i l a r w i t h r e s p e c t t o b i o c h e m i c a l and b a c t e r i o l o g i c a l c h a r a c t e r i s t i c s b u t d i f f e r e n t w i t h r e s p e c t t o p a t h o g e n i c i t y on one  o r more p l a n t h o s t s ( K r i e g and H o l t , Fatty  a c i d p r o f i l e s a l s o d i f f e r e d f o r t h e two E r w i n i a  i s o l a t e types. acid analysis two  1984).  I d e n t i f i c a t i o n o f t h e i s o l a t e s a c h i e v e d by f a t t y indicated  d i f f e r e n t species.  t h a t t h e two i s o l a t e t y p e s were i n f a c t A p o s i t i v e match was made f o r t h e  a t y p i c a l i s o l a t e , w h i l e t h e 'goodness o f f i t '  f o r two o f t h e  106  t h r e e t y p i c a l i s o l a t e s analyzed was not i n d i s p u t a b l e . from t h e i r observed  and e x p e r i m e n t a l l y  However,  determined  c h a r a c t e r i s t i c s , the two g a l l - f o r m i n g i s o l a t e types appear t o be two  d i f f e r e n t s p e c i e s of Erwinia.  F u r t h e r t e s t i n g s h o u l d be  done t o c l e a r l y d e f i n e the r e l a t i o n s h i p between t h e two g a l l forming Erwinia  isolates.  The g a l l - f o r m i n g b a c t e r i a l pathogens i s o l a t e d from Douglasf i r t r e e s i n B.C. do not appear a t a h i g h frequency, s t a t e d b e f o r e , a survey was not conducted organism.  on t h i s aspect o f t h e  Furthermore, l i t t l e damage was i n f l i c t e d on t h e host  p l a n t by t h e g a l l - f o r m i n g Erwinia  isolates.  There have been  s e v e r a l r e p o r t s of b a c t e r i a l g a l l s on c o n i f e r s personal  although as  communication)  (R.S. Hunt,  but i t i s not known i f t h e i n c i t a n t was  i s o l a t e d o r t h e i d e n t i t y determined.  The host range o f t h e  g a l l - f o r m i n g E r w i n i a s i s very l i m i t e d , w i t h Pseudotsuga Abies,  Larix,  Picea  susceptible hosts.  and Pinus  and some  spp. c o n s t i t u t i n g t h e m a j o r i t y o f  The p o t e n t i a l f o r spread t o s u r r o u n d i n g  f o r e s t e d areas i s t h e r e f o r e very s m a l l , although t h e presence o f an i n s e c t v e c t o r i s unknown. Due t o t h e i n f r e q u e n t l y observed  n a t u r a l damage and t h e  l i m i t e d h o s t range of the g a l l - f o r m i n g e r w i n i a s , these pathogens appear t o be l i m i t e d t h r e a t s t o the f o r e s t i n d u s t r y i n terms of decreased Erwinia  timber q u a l i t y .  Thus, although t h e g a l l - f o r m i n g  i s o l a t e s p r o v i d e no s c i e n t i f i c  advantage i n terms of  p r o v i d i n g a p o t e n t i a l gene v e c t o r , they, on t h e o t h e r hand, a r e not an economic t h r e a t t o the t r e e s i n terms o f d i s e a s e . g a l l - f o r m i n g Erwinia  The  i s o l a t e s c o u l d serve very w e l l as  a d d i t i o n a l models both i n the study of phytohormone p r o d u c t i o n  107 by b a c t e r i a  i n plant-pathogen i n t e r a c t i o n s  phytohormone genes i n b a c t e r i a l  and i n t h e o r i g i n o f  phytopathogens.  108 REFERENCES A k i y o s h i , D.E., production  R e g i e r , D.A.  by Agrobacterium  169: 4242-4248.  and Gordon, M.P.  and Pseudomonas  1987.  spp.  J.  Cytokinin  Bacteriol.  A l l a n , E. and Kelman, A. 1977. Immunofluorescent s t a i n p r o c e d u r e s f o r d e t e c t i o n and i d e n t i f i c a t i o n o f E r w i n i a carotovora  v a r . atroseptica.  Phytopathology  67:  1305-1312.  Anonymous. 1985. H e w l e t t P a c k a r d Gas Chromatography Data Sheet, June, 1985. P u b l i c a t i o n No. 43-5953-1857. 4 pp. B a r t o n , K.A. and C h i l t o n , M.-D. 1983. Agrobacterium p l a s m i d s as v e c t o r s f o r p l a n t g e n e t i c e n g i n e e r i n g . Enzymol. 101: 527-539. B a r z i c , M.-R. phaseolicola 2: 389-397.  Ti Methods  and T r i g a l e t , A. 1982. D e t e c t i o n de (Burkh.) Dowson p a r l a t e c h n i q u e ELISA.  Pseudomonas Agronomie  B e g u i n , N., G u g e r l i , P. and C a z e l l e s . 0. 1984. Detection d i n f e c t i o n s l a t e n t e s de jambe n o i r e dans l e s t u b e r c u l e s de pomme de t e r r e p a r l a dosage immuno-enzymatique ELISA. Revue 1  Suisse  Agric.  16:  321-324.  B e n e d i c t , A.A., A l v a r e z , A.M., B e r e s t e c k y , J . , Imanaka, W., Mizumoto, C.Y., P o l l a r d , L.W., Mew, T.W. and G o n z a l e z , C.F. 1989. P a t h o v a r - s p e c i f i c monoclonal a n t i b o d i e s f o r Xanthomonas campestris pv. oryzae and oryzicola. Phytopathology  f o r Xanthomonas 79: 322-328.  campestris  pv.  Braun, A.C. 1943. S t u d i e s on tumor i n c e p t i o n i n t h e crown d i s e a s e . Am. J. Bot. 30: 674-677.  gall  B r i a n s k y , R.H., Lee, R.F., Timmer, L.W., P u r c i f u l l , D.E. and R a j u , B.C. 1982. Immunofluorescent d e t e c t i o n o f x y l e m - l i m i t e d b a c t e r i a i n s i t u . Phytopathology 72: 1444-1448. B r i s b a n e , P.G.  and K e r r , A.  b i o v a r s o f Agrobacterium.  B r o d a , P. 1979. Plasmids. Fransisco. 197 pp.  1983.  J. Appl.  W.H.  S e l e c t i v e media f o r t h r e e  B a c t e r i o l . 54:  Freeman and Co.,  Buchanan, R.E. and Gibbons, N.E. (Eds.) of D e t e r m i n a t i v e Bacteriology. 8 t h ed. Co., B a l t i m o r e , U.S.A. 1246 pp.  425-431.  San  1974. Bergey's Manual W i l l i a m s and W i l k i n s  Caron, M. and Copeman, R.J. 1984. E f f e c t of t h e p l a t e washing p r o c e d u r e on t h e d e t e c t i o n of Erwinia carotovora subsp. a t r o s e p t i c a by an enzyme immunoassay ( E I A ) . P h y t o p r o t e c t i o n 65: 17-25.  109 C h a t t e r j e e , A.K. and G i b b i n s , L.N. 1971. Induction of nonpigmented v a r i a n t s of Erwinia h e r b i c o l a by i n c u b a t i o n s u p r a o p t i m a l t e m p e r a t u r e s . J. Bacteriol. 105: 107-112.  at  C h i l t o n , M.-D., Drummond, M.H., M e r i o , D.J., S c i a k y , D., Montoya, A.L., Gordon, M.P. and N e s t e r , E.W. 1977. Stable i n c o r p o r a t i o n of p l a s m i d DNA i n t o h i g h e r p l a n t c e l l s : the m o l e c u l a r b a s i s of crown g a l l t u m o r i g e n e s i s . Cell 11: 263-271. C h i l t o n , M.-D. 1983. p l a n t s . S c i . Am. 248: C i v e r o l o , E.L.  and  A v e c t o r f o r i n t r o d u c i n g new 50-59.  Fan,  F.  1982.  Xanthomonas  genes i n t o  campestris  pv.  c i t r i d e t e c t i o n and i d e n t i f i c a t i o n by e n z y m e - l i n k e d immunosorbent assay. Plant Dis. 66: 231-236.  C l a r k , E., Vigodsky-Hass, H. and G a f n i , Y. 1989. C h a r a c t e r i s t i c s i n t i s s u e c u l t u r e of h y p e r p l a s i a s i n d u c e d  Erwinia  Pathol.  herbicola  35:  p a t h o v a r gypsophilae.  Physiol.  383-390.  Mol.  Pi.  by  C l a r k , M.F. and Adams, A.N. 1977. C h a r a c t e r i s t i c s of t h e m i c r o p l a t e method of enzyme-linked immunosorbent a s s a y f o r t h e d e t e c t i o n of p l a n t v i r u s e s . J. Gen. V i r o l . 34: 475-483. Comai, L. and Kosuge, T. 1980. Involvement of p l a s m i d deoxyribonucleic acid i n indoleacetic acid synthesis i n  Pseudomonas  savastanoi.  J.  Bacteriol.  143:  950-957.  Comai, L., S u r i c o , G. and Kosuge, T. 1982. R e l a t i o n of p l a s m i d DNA t o i n d o l e a c e t i c a c i d p r o d u c t i o n i n d i f f e r e n t s t r a i n s of  Pseudomonas  2157-2163.  Cooksey, D.A.  Erwinia  syringae  herbicola.  1985.  pv.  savastanoi.  J.  G a l l s of Gypsophila  Plant  Dis.  70:  Gen.  Microbiol.  128:  p a n i c u l a t a caused by  464-468.  Cooksey, D.A. and Graham J.H. 1989. Genomic f i n g e r p r i n t i n g of two p a t h o v a r s of p h y t o p a t h o g e n i c b a c t e r i a by r a r e - c u t t i n g r e s t r i c t i o n enzymes and f i e l d i n v e r s i o n g e l e l e c t r o p h o r e s i s . Phytopathology 79: 745-750. C o p l i n , D.L. 1989. P l a s m i d s and t h e i r r o l e i n t h e e v o l u t i o n of p l a n t p a t h o g e n i c b a c t e r i a . Annu. Rev. Phytopathol. 27: 187-212. Dandekar, A.M., Gupta, P.K., Durzan, D.J. and Knauf, V. 1987. T r a n s f o r m a t i o n and f o r e i g n gene e x p r e s s i o n i n m i c r o p r o p a g a t e d Douglas-fir  (Pseudotsuga  menziesii).  Bio/Technology  5:  587-590.  D a v i s , G.C, H e i n , M.B., N e e l y , B.C., Sharp, C R . and Carnes, M.G. 1985. S t r a t e g i e s f o r t h e d e t e r m i n a t i o n of p l a n t hormones. A n a l . Chem. 57: 638-646.  110 De Boer, S.H. 1987. Use o f monoclonal a n t i b o d i e s t o i d e n t i f y and d e t e c t p l a n t p a t h o g e n i c b a c t e r i a . Can. J. P l a n t Pathol. 9: 182-187. De Boer, S.H., Copeman, R.J. and V r u g g i n k , H. 1979. Serogroups of E r w i n i a carotovora potato s t r a i n s determined w i t h d i f f u s i b l e s o m a t i c a n t i g e n s . Phytopathology 69: 316-319. De C l e e n e , M. and De Ley, J . 1976. The h o s t range o f crown g a l l , Agrobacterium tumefaciens. Bot. Rev. 42: 389-466. De C l e e n e , M. and De Ley, J . 1981. The r e e v a l u a t i o n o f  p a t h o g e n i c i t y o f "Agrobacterium  101: 185-188.  pseudotsugae".  Phytopathol.  Z.  E l l i s , P . J . 1988. A s u r v e y o f common weeds o f B r i t i s h Columbia f o r b e e t w e s t e r n y e l l o w s v i r u s and p o t a t o l e a f r o l l v i r u s u s i n g m o n o c l o n a l a n t i b o d i e s . Can. J. Plant Pathol. 10: 363 ( A b s t r . ) . E l l i s , D., R o b e r t s , D., S u t t o n , B., L a z a r o f f , W., Webb, D. and F l i n n , B. 1989. T r a n s f o r m a t i o n o f w h i t e s p r u c e and o t h e r  c o n i f e r s p e c i e s by Agrobacterium  8: 16-20.  tumefaciens.  Plant  Cell  Repts.  F e i n b e r g , A.P. and V o g e l s t e i n , B. 1983. A t e c h n i q u e f o r r a d i o l a b e l i n g DNA r e s t r i c t i o n endonuclease fragments t o h i g h s p e c i f i c a c t i v i t y . Anal. Biochem. 132: 6-13. F r a l e y , R.T., Rogers, S.G., Horsch, R.B., Sanders, P.R., F l i c k , J.S., Adams, S.P., B i t t n e r , M.L., Brand. L.A., F i n k , C.L., F r y , J.S., G a l l u p p i , G.R., G o l d b e r g , S.B., Hoffmann, N.L. and Woo, S.C. 1983. E x p r e s s i o n o f b a c t e r i a l genes i n p l a n t c e l l s .  Proc.  Natl.  Acad.  Sci. USA 80: 4830-4807.  F o l l i n , A., I n z e , D., Budar, F., G e n e t e l l o , C., Van Montagu, M. and S c h e l l , J . 1985. G e n e t i c e v i d e n c e t h a t t h e t r y p t o p h a n 2mono-oxygenase gene o f Pseudomonas savastanoi i s functionally e q u i v a l e n t t o one o f t h e T-DNA genes i n v o l v e d i n p l a n t tumour formation  178-185.  by Agrobacterium  tumefaciens.  Mol. Gen. Genet.  201:  G i l b e r t s o n , R.L., M a x w e l l , D.P., Hagedorn, D.J. and Leong, S.A. 1989. Development and a p p l i c a t i o n o f a p l a s m i d DNA probe f o r d e t e c t i o n o f b a c t e r i a c a u s i n g common b a c t e r i a l b l i g h t o f bean. Phytopathology 79: 518-525. Hamamoto, A. and Murooka, Y. 1987. P l a s m i d v e c t o r s used i n v a r i o u s s t r a i n s o f Erwinia s p e c i e s . Appl. M i c r o b i o l . B i o t e c h n o l . 26: 242-247. Hansen, H.N. and Smith, R.E. 1933. A b a c t e r i a l g a l l d i s e a s e o f the Douglas f i r . Science 77: 628. Hansen, H.N. and Smith, R.E. Douglas f i r ,  Pseudotsuga  1937. A b a c t e r i a l g a l l d i s e a s e o f  taxifolia.  Hilgardia  10: 569-577.  Ill Hardman, D.J, and Gowland, P.C. 1985a. L a r g e p l a s m i d s i n b a c t e r i a . P a r t 1. A survey of a s s o c i a t e d phenotypes. Microbiol. S c i . 2: 90-94. Hardman, D.J, and Gowland, P.C. 1985b. L a r g e p l a s m i d s i n b a c t e r i a . P a r t 2. G e n e t i c s and e v o l u t i o n . M i c r o b i o l . S c i . 2: 184-190. Hardy, K.G. (Ed.).  Oxford.  DNA  1985.  245  Cloning:  Bacillus  pp.  c l o n i n g methods.  a P r a c t i c a l Approach.  I n D.M.  Vol.  2.  Glover  IRL  Press,  Kado, C.I. and L i u , S.-T. 1981. Rapid procedure f o r d e t e c t i o n and i s o l a t i o n of l a r g e and s m a l l p l a s m i d s . J. B a c t e r i o l . 145: 1365-1373. K r i e g , N.R. and H o l t , J.G. D e t e r m i n a t i v e Bacteriology. B a l t i m o r e , MD, U.S.A. 964  (Eds.) 1984. Bergey's Manual of Volume 1. W i l l i a m s and W i l k i n s , pp.  L a z a r o v i t s , G., Z u t r a , D., and Bar-Joseph, M. 1987. Enzymel i n k e d immunosorbent assay on n i t r o c e l l u l o s e membranes ( d o t ELISA) i n t h e s e r o d i a g n o s i s of p l a n t p a t h o g e n i c b a c t e r i a . Can. J. M i c r o b i o l . 33: 98-103. L a z o , G.R. and G a b r i e l , D.W. 1987. C o n s e r v a t i o n of p l a s m i d DNA sequences and p a t h o v a r i d e n t i f i c a t i o n of s t r a i n s of Xanthomonas campestris.  L e d i g , F.T. For. Chron. L i u , S.-T.  Phytopathology  77:  448-453.  1985. Genetic transformation i n f o r e s t t r e e s . 61: 454-465. and Kado, C.I.  1979.  a p l a s m i d f u n c t i o n of Agrobacterium Biophys. Res. Commun. 90: 171-178.  Maas G e e s t e r a n u s , H.P.  Agrobacterium  gypsophilae.  Indoleacetic acid production: tumefaciens  and Barendsen, H. Neth.  J.  Pi.  1966.  Path.  C58.  Biochem.  Hostplants  72:  231-232.  M a h i l l o n , J . , H e s p e l , F., P i e r s s e n s , A.-M. and D e l c o u r , J . 1988. C l o n i n g and p a r t i a l c h a r a c t e r i z a t i o n of t h r e e s m a l l c r y p t i c p l a s m i d s from Bacillus thuringiensis. Plasmid 19: 173. M a n i a t i s , T., F r i t s c h , E.F. and Sambrook, J . 1982. C l o n i n g . C o l d S p r i n g Harbor L a b o r a t o r y , New York.  of  169-  Molecular 545 pp.  Meneley, J.C. and S t a n g h e l l i n i , M.E. 1976. I s o l a t i o n of s o f t r o t E r w i n i a spp. from a g r i c u l t u r a l s o i l s u s i n g an e n r i c h m e n t technique. Phytopathology 66: 367-370. M e r t e n s , R., E b e r l e , J . , A r n s c h e i d t , A., Ledebur, A. and W e i l e r , E.W. 1985. M o n o c l o n a l a n t i b o d i e s t o p l a n t growth r e g u l a t o r s . II. I n d o l e - 3 - a c e t i c a c i d . Planta 166: 389-393.  112 Muehlchen, A.M. 1985. B a c t e r i a l g a l l s of D o u g l a s - f i r . B.Sc.(Agr.) T h e s i s . U n i v e r s i t y of B r i t i s h Columbia. 25 M i l l e r , S.A. and M a r t i n , R.R. 1988. p l a n t d i s e a s e . Ann. .Rev. Phytopathol.  pp.  M o l e c u l a r d i a g n o s i s of 26: 409-432.  M i l l e r , L.T. 1984. G a s - l i q u i d chromatography of c e l l u l a r a c i d s as a b a c t e r i a l i d e n t i f i c a t i o n a i d . Hewlett-Packard Application  Note  228-37.  M i l l e r , H.J.,  Quinn, C.E.  PI.  167-172.  Erwinia  Path.  herbicola  87:  fatty  and Graham, D.C.  1981.  p a t h o g e n i c on Gypsophila  paniculata.  A s t r a i n of Neth.  J.  M i l l s , D. 1985. Transposon mutagenesis and i t s p o t e n t i a l f o r s t u d y i n g v i r u l e n c e genes i n p l a n t pathogens. Ann. Rev. Phytopathol. 23: 297-320. Moore, L.D. 1977. P r e v e n t i o n of crown g a l l on Prunus b a c t e r i a l a n t a g o n i s t s . Phytopathology 67: 139-144. Morales,  V.M.  Pseudomonas  and S e q u e i r a , L.  solanacearum.  1985.  Phytopathology  r o o t s by  Indigenous plasmids 75:  767-771.  in  P a r s o n s , T.J., S i n k a r , V.P., S t e t t l e r , R.F., N e s t e r , E.W. and Gordon, M.P. 1986. T r a n s f o r m a t i o n of p o p l a r by Agrobacterium tumefaciens.  Bio/Technology  4:  533-536.  P o w e l l , G.K. and M o r r i s , R.O. 1986. N u c l e o t i d e sequence and e x p r e s s i o n of a Pseudomonas savastanoi cytokinin biosynthetic gene: homology w i t h Agrobacterium tumefaciens tmr and tzs l o c i .  Nucl.  Acids  Res.  14:  2555-2565.  Reed, F.L.C. 1989. The p o t e n t i a l economic impact of b i o t e c h n o l o g y and r e l a t e d r e s e a r c h on t h e f o r e s t s e c t o r . Chron. 65: 185-189. S a l c h , Y.P. and Shaw, P.D. 1988. I s o l a t i o n and c h a r a c t e r i z a t i o n of p a t h o g e n i c i t y genes of Pseudomonas  pv.  tabaci.  J.  B a c t e r i o l . 170:  2584-2591.  For.  syringae  S e d e r o f f , R. , Stomp, A.-M., C h i l t o n , W.S. and Moore, L.W. 1986. Gene t r a n s f e r i n t o l o b l o l l y p i n e by Agrobacterium tumefaciens. Bio/Technology 4: 647-649. S i n c l a i r , W.A., Lyon, H.H. and Johnson, W.T. 1987. Diseases of Trees and Shrubs. C o r n e l l U n i v e r s i t y P r e s s , New York. 574 pp. Smidt, M. and Kosuge, T. 1978. The r o l e of i n d o l e - 3 - a c e t i c a c i d a c c u m u l a t i o n by a l p h a methyl t r y p t o p h a n - r e s i s t a n t mutants of Pseudomonas savastanoi i n g a l l f o r m a t i o n on o l e a n d e r s .  Physiol.  Plant  Smith, CO.  Bacterium  Pathol.  1940.  13:  203-214.  G a l l s on Pseudotsuga  pseudotsugae.  Phytopathology  macrocarpa  30:  624.  induced  by  113 S o n s t e i n , S.A. and B a l d w i n , J.N. 1972. Loss o f t h e p e n i c i l l i n a s e p l a s m i d a f t e r t r e a t m e n t o f Staphylococcus aureus w i t h sodium d o d e c y l s u l f a t e . J. B a c t e r i o l . 109: 262-265. S t e a d , D.E. 1988. I d e n t i f i c a t i o n o f b a c t e r i a by computera s s i s t e d f a t t y a c i d p r o f i l i n g . Acta. Hort. 225: 39-46. S u r i c o , G., I a c o b e l l i s , N.S. and S i s t o , A. 1985. S t u d i e s on t h e r o l e o f i n d o l e - 3 - a c e t i c a c i d and c y t o k i n i n s i n t h e f o r m a t i o n o f k n o t s on o l i v e and o l e a n d e r p l a n t s by Pseudomonas syringae  pv. savastanoi.  Physiol.  Plant  Pathol.  26:  309-320.  S u t u l a , C.L., G i l l e t t , J.M., M o r r i s s e y , S.M. and R a m s d e l l , D.C. 1986. I n t e r p r e t i n g ELISA d a t a and e s t a b l i s h i n g t h e p o s i t i v e n e g a t i v e t h r e s h o l d . Plant Dis. 70: 722-726. Sykes, L.C. and M a t t h y s s e , A.G. i n d u c t i o n by Agrobacterium  M i c r o b i o l . 52: 597-598. T r e v o r s , J.T. purification. Ulaganathan,  K. and Mahadevan, A.  92-96.  Vincent,  J.M.  Time r e q u i r e d f o r tumor Appl.  Environ.  1985. B a c t e r i a l p l a s m i d i s o l a t i o n and J. Microbiol. Methods 3: 259-271.  from Xanthomonas  Root-nodule 164 pp.  1986.  tumefaciens.  campestris  1970.  Bacteria.  1988.  pv. v i g n i c o l a .  A Manual  for  I s o l a t i o n of a plasmid J. Phytopathol.  the P r a c t i c a l Study  1979.  S l a t e r s Lane, A l e x a n d r i a , V i r g i n i a 22314.  128 pp.  immunosorbent  the  IBP Handbook No. 15. B l a c k w e l l , O x f o r d .  V o l l e r , A., B i d w e l l , D.E. and A. B a r t l e t t linked  of  123:  assay  (ELISA).  The  enzyme  Dynatech L a b o r a t o r i e s ,  900  Von Bodman, S.B. and Shaw, P.D. 1987. C o n s e r v a t i o n o f p l a s m i d s among p l a n t - p a t h o g e n i c Pseudomonas syringae i s o l a t e s of diverse o r i g i n s . Plasmid 17: 240-247. Watanabe, T. and Fukasawa, T. 1961. Episome-mediated t r a n s f e r of drug r e s i s t a n c e i n Enterobacteriaceae I I . E l i m i n a t i o n of r e s i s t a n c e f a c t o r s w i t h a c r i d i n e dyes. Biochem. Biophys. Res. Commun. 8: 679-683. Watson, B., C u r r i e r , T.C. Gordon, M.P., C h i l t o n , M.-D. and N e s t e r , E.W. 1975. P l a s m i d r e q u i r e d f o r v i r u l e n c e o f Agrobacterium  tumefaciens.  J. B a c t e r i o l .  12 3: 255-264.  W e i l e r , E.W. and S p a n i e r , K. 1981. Phytohormones i n t h e f o r m a t i o n o f crown g a l l tumors. Planta 153: 326-337.  

Cite

Citation Scheme:

        

Citations by CSL (citeproc-js)

Usage Statistics

Country Views Downloads
United States 14 0
Egypt 6 0
China 5 4
New Zealand 1 0
Turkey 1 0
Canada 1 0
Italy 1 0
City Views Downloads
Unknown 8 5
Ashburn 5 0
Des Moines 4 0
Shenzhen 3 4
Seattle 2 0
Beijing 2 0
Redmond 1 0
Victoria 1 0
Fort Worth 1 0
Civitanova Marche 1 0
Ataturk 1 0

{[{ mDataHeader[type] }]} {[{ month[type] }]} {[{ tData[type] }]}
Download Stats

Share

Embed

Customize your widget with the following options, then copy and paste the code below into the HTML of your page to embed this item in your website.
                        
                            <div id="ubcOpenCollectionsWidgetDisplay">
                            <script id="ubcOpenCollectionsWidget"
                            src="{[{embed.src}]}"
                            data-item="{[{embed.item}]}"
                            data-collection="{[{embed.collection}]}"
                            data-metadata="{[{embed.showMetadata}]}"
                            data-width="{[{embed.width}]}"
                            async >
                            </script>
                            </div>
                        
                    
IIIF logo Our image viewer uses the IIIF 2.0 standard. To load this item in other compatible viewers, use this url:
http://iiif.library.ubc.ca/presentation/dsp.831.1-0098122/manifest

Comment

Related Items