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Gall formation by Erwinia species on Douglas-fir 1990

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GALL FORMATION BY EFWINIA SPECIES ON DOUGLAS-FIR BY ROBYN MERRILEE DEYOUNG B.Sc.(Agr.), U n i v e r s i t y of 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 THE FACULTY OF GRADUATE STUDIES (Department of Pl a n t Science) We accept t h i s t h e s i s as conforming t o the req u i r e d standard THE UNIVERSITY OF BRITISH COLUMBIA A p r i l 1990 (k) Robyn M e r r i l e e DeYoung i n In presenting this thesis in partial fulfilment of the requirements for an advanced degree at the University of British Columbia, I agree that the Library shall make it freely available for reference and study. I further agree that permission for extensive copying of this thesis for scholarly purposes may be granted by the head of my department or by his or her representatives. It is understood that copying 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 B a c t e r i a l g a l l s on D o u g l a s - f i r (Pseudotsuga menzeisii [Mirb.] Franco), c o l l e c t e d from the southern t i p of Vancouver I s l a n d , the Greater Vancouver area and the Hope r e g i o n of B r i t i s h Columbia, were g e n e r a l l y globose i n shape w i t h rough, i r r e g u l a r surfaces and measured between 0.5 and 2.0 cm i n diameter. The g a l l s were g e n e r a l l y l o c a t e d on the t i p s of branches or twigs of 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 disease appeared t o a f f e c t few Douglas- f i r t r e e s i n the c o l l e c t i o n areas and b a c t e r i a l g a l l s were not found on any other coniferous species. Furthermore, there have been no r e p o r t s of se 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 disease. Young, greenhouse-grown D o u g l a s - f i r seedlings o c c a s i o n a l l y d i e d i f the t i p of the main stem was a r t i f i c i a l l y i n o c u l a t e d . Often new growing t i p s would be produced a f f e c t i n g the growth form of the s e e d l i n g s . Two types of g a l l - f o r m i n g Erwinia spp. were i s o l a t e d from D o u g l a s - f i r g a l l s . T y p i c a l 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 by f a t t y a c i d a n a l y s i s as Erwinia salicis, produced g a l l s which were rough and i r r e g u l a r i n shape composed of m u l t i p l e outgrowths marked by a s i n g l e or cross-shaped f i s s u r e . The a t y p i c a l 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 by f a t t y a c i d a n a l y s i s as Erwinia herbicola subsp. herbicola, produced g a l l s which were smooth and g e n e r a l l y round i n shape wi t h the surfa c e c r a c k i n g as the g a l l expanded. Colonies of the t y p i c a l i s o l a t e s grown on casein-peptone- glucose 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 i i i somewhat c o n c e n t r i c r i d g i n g observed near the margins of the c o l o n i e s . Three to 4 day o l d c o l o n i e s of the a t y p i c a l i s o l a t e s grown on casein-peptone-glucose media were c h a r a c t e r i s t i c a l l y round and concave while older c o l o n i e s produced an e x t r a c e l l u l a r s l i m e and were more i r r e g u l a r i n shape. In L u r i a Broth, the t y p i c a l i s o l a t e s grew at temperatures of up t o 32°C w h i l e the a t y p i c a l i s o l a t e grew at temperatures of up 34°C. 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 wider range of a n t i b i o t i c s than the a t y p i c a l i s o l a t e . P o l y c l o n a l a n t i s e r a were produced against g l u t a r a l d e h y d e - f i x e d whole c e l l s of both the 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 i s o l a t e s . The p u r i f i e d a n t i s e r a were i s o l a t e s p e c i f i c as t e s t e d by immunodiffusion and an i n d i r e c t ELISA a g a i n s t s e v e r a l d i f f e r e n t phytopathogenic b a c t e r i a i n c l u d i n g Pseudomonas syringae pv. syringae, Erwinia herbicola subsp. herbicola, Agrobacterium tumefaciens, Rhizobium leguminosarum and Erwinia carotovora subsp. carotovora. Plasmid p r o f i l e s of the t y p i c a l Erwinia i s o l a t e s contained one band w h i l e the 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 contained 4 t o 5 bands which appeared to be d i f f e r e n t forms of at l e a s t one plasmid. 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 i s o l a t e s suggested a s i z e of approximately 50 kb w h i l e complex d i g e s t i o n p r o f i l e s were obtained f o r the a t y p i c a l i s o l a t e s because of the 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. From v i s u a l estimates a g a i n s t H i n d l l l - d i g e s t e d lambda DNA, a s i z e of between 10 and 20 kb was suggested f o r the f a s t e s t moving plasmid band of the a t y p i c a l i s o l a t e . No homology was observed between the d i f f e r e n t plasmid types c h a r a c t e r i s t i c of the two i s o l a t e s . The i v r o l e of the plasmid DNA of the a t y p i c a l i s o l a t e i n pathogenesis was not determined because c u r i n g of the plasmid(s) was not s u c c e s s f u l u s i n g high temperature treatments p l u s chemical c u r i n g agents. Heat treatment experiments, i n which the pathogen was s e l e c t i v e l y k i l l e d at various times a f t e r i n o c u l a t i o n , demonstrated t h a t the b a c t e r i a are r e q u i r e d t o be present f o r g a l l i n d u c t i o n and continued development of the g a l l f o r both of the 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 of the 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 Abies, Chamaecyparis, Pinus and Thuja spp. The t y p i c a l i s o l a t e s were weakly pathogenic on Abies, Larix and Picea spp. The a t y p i c a l i s o l a t e was weakly pathogenic on Abies, Chamaecyparis, Larix, Picea and Pinus spp. Due t o the l i m i t e d damage caused on the c o n i f e r s t e s t e d and t o t h e i r infrequent occurrence, these g a l l - forming pathogens do not appear t o be of economic importance t o the f o r e s t r y i n d u s t r y . V TABLE OF CONTENTS Page T i t l e Page i A u t h o r i z a t i o n i A b s t r a c t i i Table of Contents v L i s t of Tables v i i L i s t of Figures i x Acknowledgements x i 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 Detection of the Douglas-Fir Gall-Forming B a c t e r i a I n t r o d u c t i o n 8 M a t e r i a l s and Methods 14 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 14 B. P l a n t m a t e r i a l s 16 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 16 D. P o l y c l o n a l a n t i s e r a production. 17 E. IgG p u r i f i c a t i o n 18 F. A n t i s e r a s e n s i t i v i t y 18 G. S p e c i f i c i t y : immunodiffusion 19 H. S p e c i f i c i t y : i n d i r e c t ELISA 20 I. Host range 20 Re s u l t s 22 A. D e s c r i p t i o n of g a l l s and i s o l a t e s 22 B. A n t i s e r a s e n s i t i v i t y 3 2 C. S p e c i f i c i t y : immunodiffusion 32 D. S p e c i f i c i t y : i n d i r e c t ELISA 3 6 E. Host range 40 D i s c u s s i o n 43 v i TABLE OF CONTENTS (Cont.) Page Chapter 2 The Mechanism of G a l l Formation by Erwinia spp. on Douglas-Fir I n t r o d u c t i o n 52 M a t e r i a l s and Methods 54 A. B a c t e r i a l s t r a i n s and host p l a n t s 54 B. Inoculations/heat treatment 54 C. Growth index 55 D. D i r e c t i s o l a t i o n s / c e l l ELISA 55 E. S t a t i s t i c a l a n a l y s i s 56 R e s u l t s 57 A. T-2789 i s o l a t e 57 B. A-0181 i s o l a t e 61 D i s c u s s i o n 66 Chapter 3 The R e l a t i o n s h i p between the Plasmid Content of the G a l l - f o r m i n g Erwinia I s o l a t e s and Pathogenesis I n t r o d u c t i o n 73 M a t e r i a l s and Methods 7 6 A. B a c t e r i a l s t r a i n s 76 B. T o t a l DNA i s o l a t i o n 7 6 C. Plasmid i s o l a t i o n and p u r i f i c a t i o n 77 D. Southern b l o t s 79 E. Curing p r o t o c o l s 80 F. Colony l i f t s 82 G. 3 2P l a b e l l i n g 82 H. H y b r i d i z a t i o n 83 R e s u l t s 85 A. Plasmid content 85 C. Probe s p e c i f i c i t y / h o m o l o g y 89 B. Curing/colony h y b r i d i z a t i o n 94 Di s c u s s i o n 97 General Discussion 102 References 108 v i i LIST OF TABLES Chapter 1 Page Table 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 species used t o t e s t the s p e c i f i c i t y of p o l y c l o n a l a n t i s e r a 15 Table 1.2 Temperature s e n s i t i v i t y of the g a l l - f o r m i n g Erwinia i s o l a t e s 3 0 Table 1.3 A n t i b i o t i c r e s i s t a n c e s expressed 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 the D o u g l a s - f i r g a l l - f o r m i n g bacterium 31 Table 1.4 Te n t a t i v e i d e n t i f i c a t i o n s of 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 of t h e i r f a t t y a c i d p r o f i l e s and computer- a s s i s t e d comparison wi t h a l i b r a r y of p r o f i l e s from authentic s t r a i n s 33 Table 1.5 S p e c i f i c i t y of whole Erwinia T-2789 antiserum i n an i n d i r e c t ELISA 37 Table 1.6 S p e c i f i c i t y of the p u r i f i e d IgG f r a c t i o n of the Erwinia T-2789 antiserum at a d i l u t i o n of 10"4 i n an i n d i r e c t ELISA 38 Table 1.7 S p e c i f i c i t y of the p u r i f i e d IgG f r a c t i o n of the Erwinia A-0181 antiserum at a d i l u t i o n of 10"3 i n an i n d i r e c t ELISA 39 Table 1.8 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 14 c o n i f e r species 42 Chapter 2 Table 2.1 G a l l formation by and recovery of the pathogen from heat-treated (35°C f o r 18 days) Douglas- f i r s eedlings s t a b - i n o c u l a t e d w i t h the t y p i c a l Erwinia i s o l a t e T-2789 58 Table 2.2 G a l l formation by and recovery of the pathogen from heat-treated (35°C f o r 12 days) Douglas- f i r s eedlings s t a b - i n o c u l a t e d w i t h the t y p i c a l Erwinia i s o l a t e T-2789 59 Table 2.3 G a l l formation by and recovery of the pathogen from heat-treated (3 6°C) D o u g l a s - f i r s e e d l i n g s s t a b - i n o c u l a t e d w i t h the a t y p i c a l Erwinia i s o l a t e A-0181 62 v i i i LIST OF TABLES (Cont.) Page Table 2.4 G a l l formation by and recovery of the pathogen from heat-treated (38°C) D o u g l a s - f i r s e e d l i n g s s t a b - i n o c u l a t e d with the a t y p i c a l Erwinia i s o l a t e A-0181 63 Chapter 3 Table 3.1 Concentrations of chemical c u r i n g agents 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 the g a l l - forming Erwinia i s o l a t e s T-2774 and A-0181 81 i x LIST OF FIGURES Chapter 1 F i g u r e 1.1 Page F i g u r e 1.2 Fig u r e 1.3 Figur e 1.4 Fig u r e 1.5 Figur e 1.6 Fig u r e 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 the a t y p i c a l i s o l a t e A-0181 of the 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 23 Types of 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 the 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 b a c t e r i a 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 g a l l - f o r m i n g b a c t e r i a 24 26 Colonies of the t y p i c a l and the 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 hydrolysate, peptone and glucose medium , 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 against the t y p i c a l T-2789 and A-0181 i s o l a t e s of the 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 against t h e i r r e s p e c t i v e homologous antigens , 28 34 Immunodiffusion patterns 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 the t y p i c a l T-2789 and the 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 Pa 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, Chamaecyparis nootkatensis, Picea engelmannii and Pinus contorta 41 Chapter 2 F i g u r e 2.1 The e f f e c t of a high temperature treatment (35±2°C) , a p p l i e d at various times 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 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 60 X LIST OF FIGURES (Cont.) Chapter 3 F i g u r e 3.1 Figur e 3.2 Figur e 3.3 Page F i g u r e 3.4 Figur e 3.5 Figur e 3.6 Figur e 3.7 Figur e 3.8 Figur e 3.9 Plasmid p r o f i l e s of 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% agarose g e l and s t a i n e d w i t h ethidium bromide Plasmid DNA p r o f i l e of 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 st a i n e d w i t h ethidium bromide Plasmid DNAs from t y p i c a l and a t y p i c a l g a l l - forming Erwinia i s o l a t e s d igested w i t h 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 st a i n e d w i t h ethidium bromide Gel 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 of t o t a l genomic and plasmid DNA of var i o u s phytopathogenic b a c t e r i a probed w i t h plasmid DNA of 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 Gel 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 of plasmid DNA of va r i o u s phytopathogenic b a c t e r i a probed w i t h plasmid DNA of 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 Gel 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 of t o t a l genomic and plasmid DNA of vari o u s phytopathogenic b a c t e r i a probed w i t h plasmid DNA of the 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 Gel 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 of plasmid DNA of va r i o u s phytopathogenic b a c t e r i a probed w i t h plasmid DNA of the 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 Autoradiogram of colony h y b r i d i z a t i o n of plasmid DNA probe of 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 wit h Erwinia spp Autoradiogram of colony h y b r i d i z a t i o n of dAT 3 2P-labelled plasmid DNA of 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 of Erwinia i s o l a t e A-0181 86 87 88 90 91 92 93 95 96 x i ACKNOWLEDGEMENTS I wish t o thank my supervisor, Dr. R.J. Copeman, f o r support and guidance throughout t h i s study. Thanks a l s o are extended t o the other members of my committee namely, Dr. J.C. McPherson, Dr. R.A.J. Warren and Dr. V.C. Runeckles, f o r t h e i r help 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. Ronald f o r p r o v i d i n g s u p p l i e s and lab space f o r r a d i o l a b e l l i n g . Thanks are 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 help 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 of my t h e s i s r e s e a r c h . I would l i k e t o acknowledge Carolyne Jow f o r her t e c h n i c a l a s s i s t a n c e during the p o r t i o n of t h i s study conducted under c o n t r a c t t o the Pl a n t Biotechnology I n s t i t u t e i n Saskatchewan, Len Ward f o r h i s continued help and suggestions and Tom Lowery f o r a s s i s t a n c e w i t h the s t a t i s t i c a l a n a l y s i s and p r e s e n t a t i o n . I would l i k e t o dedicate t h i s t h e s i s t o my parents f o r t h e i r never-ending 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 reaching a c r i t i c a l p e r i o d i n terms of maintaining i t s c o m p e t i t i v e p o s i t i o n i n t e r n a t i o n a l l y . The f o r e s t s of Canada are the n a t i o n ' s most valuable n a t u r a l asset w i t h approximately 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 or i n d i r e c t l y on the f o r e s t i n d u s t r y f o r t h e i r l i v e l i h o o d (Reed, 1989). World consumption of i n d u s t r i a l roundwood i s growing at an average of 2.5% annu a l l y and f o r Canada t o keep i t s share of the market, i t must in c r e a s e the growth and y i e l d of 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 the h i g h q u a l i t y and low cost of the raw m a t e r i a l . Genetic improvement of c o n i f e r s must be c a r r i e d out, i n co n j u n c t i o n w i t h r e f o r e s t a t i o n , t o provide h i g h - q u a l i t y , f a s t e r - growing t r e e s . A study of the p r o d u c t i v i t y of some D o u g l a s - f i r (Pseudotsuga menziesii [Mirb.] Franco) and l o b l o l l y pine (Pinus taeda 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 manipulation has increased 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 to n a t u r a l growth f o r e s t s (Reed, 1989). Research i n t o genetic improvement of p l a n t s i n general has centered on the use of the T i plasmid of Agrobacterium tumefaciens (Smith and Townsend, Conn.), the ca u s a l agent of crown g a l l , as a c l o n i n g vector (Barton and C h i l t o n , 1983; F r a l e y e t a l . , 1983). The st u d i e s t o date have mostly i n v o l v e d dicotyledonous p l a n t s as the r e c i p i e n t s although some monocots and gymnosperms are w i t h i n the wide host range of Agrobacterium (De Cleene and De Ley, 1976). Recently the t r a n s f o r m a t i o n of s e v e r a l c o n i f e r species such as white spruce (Picea glauca 2 (Moench) Voss), Englemann spruce (Picea englemannii Parry ex Engelm.) and S i t k a spruce (Picea sitchensis (Bong.) Carr) as w e l l as l o b l o l l y pine by Agrobacterium has been shown t o occur (Ledig, 1985; Sederoff et al., 1986; Dandekar et a l . , 1987; E l l i s e t al., 1989). D o u g l a s - f i r , n a t i v e t o the coast and i n t e r i o r of B.C., has a l s o been shown t o be s u s c e p t i b l e t o A. tumefaciens (De Cleene and De Ley, 1976). However, A. tumefaciens, i s not commonly found as a n a t u r a l pathogen of D o u g l a s - f i r . Therefore, i t would be of i n t e r e s t t o i n v e s t i g a t e other g a l l - f o r m i n g b a c t e r i a l pathogens of D o u g l a s - f i r with regard t o t h e i r mechanism of g a l l formation and t h e i r p o t e n t i a l f o r harboring a plasmid 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 the 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 , i n s e c t s , 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 . One of the g a l l - f o r m i n g fungal pathogens i s Endocronartium harknessii (J.P. Moore) Y. H i r a t , the causal agent of Western g a l l r u s t on such hosts as lodgepole and ponderosa pines ( S i n c l a i r et al., 1987). Forms of Cronartium quercuum (Berk.) Miy. ex S h i r a i cause Pine-oak g a l l r u s t on d i f f e r e n t pine species such as l o b l o l l y , ponderosa and Scots pine. 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 pests such as the Cooley Spruce g a l l a d e l g i d ( S i n c l a i r et al., 1987). A review of the l i t e r a t u r e on g a l l d i seases on c o n i f e r s i n general, r e v e a l s very few r e p o r t s of b a c t e r i a l i n c i t a n t s . The f i r s t published r e p o r t of a b a c t e r i a l g a l l disease 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 Smith, 1937). The author 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 the twigs, branches and upper stem of Pinus 3 halepensis M i l l . (Aleppo p i n e ) . A s i m i l a r organism was i s o l a t e d from g a l l s on Pinus cembra L. (Swiss stone pine) i n 1911 by von Tubeuf. The f i r s t published r e p o r t of a b a c t e r i a l g a l l disease 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 tumefaciens and A. pseudotsugae (Hansen and Smith 1937) Savulescu 1947 are the only two b a c t e r i a l phytopathogens described as causing g a l l s on c o n i f e r s , more 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; Hansen and Smith, 1937). The work by Hansen and Smith on A. pseudotsugae has not been confirmed and the i s o l a t e d pathogen i s not even l i s t e d i n the most recent v e r s i o n of Bergey's Manual of Determinative B a c t e r i o l o g y (Krieg and H o l t , 1984). In a d d i t i o n t o the s m a l l number of pathogens causing g a l l formation on c o n i f e r s , the frequency of occurrence of these phytopathogens i s very 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 reported on Douglas- f i r from southern Arizona t o B.C. and A l b e r t a ( S i n c l a i r et al., 1987). There have been over 52 r e p o r t s of g a l l s of suspected b a c t e r i a l o r i g i n i n B.C. since 1946, although e x t e n s i v e damage to the hosts was not observed (R.S. Hunt, unpublished). Samples of a g a l l disease on D o u g l a s - f i r have been c o l l e c t e d i n the southwestern corner of B r i t i s h Columbia. The disease was suspected t o be of a b a c t e r i a l o r i g i n due t o the d i f f e r e n t symptoms i n r e l a t i o n t o those associated w i t h the w e l l - documented fungal g a l l s . A s t r a i n of b a c t e r i a was i s o l a t e d from these g a l l s i n the mid 1980's (Muehlchen, 1985). 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 or r e c e i v e d s i n c e t h a t 4 time. One of the f i r s t steps t o be taken when working w i t h a r e l a t i v e l y unknown phytopathogen, such as the D o u g l a s - f i r g a l l - forming bacterium, i s t o choose a method f o r the 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 the organism i n mixed c u l t u r e or i n p l a n t m a t e r i a l . There are many methods of i d e n t i f i c a t i o n or d e t e c t i o n of phytopathogenic 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 assays ( M i l l e r and M a r t i n , 1988), plasmid p r o f i l e s (Morales and Sequeira, 1985), DNArDNA h y b r i d i z a t i o n using e i t h e r chromosome (Thompson e t al., 1989) or plasmid probes ( G i l b e r t s o n et al., 1989) and genome and/or plasmid f i n g e r p r i n t i n g (Cooksey and Graham, 1989). The method used depends upon f a c i l i t i e s and funds a v a i l a b l e , number of samples to be processed, degree or l e v e l of s p e c i f i c i t y d e s i r e d and time 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 of phytopathogenic b a c t e r i a i s widely used as a t o o l i n the researc h l a b o r a t o r y . Serology takes advantage of the r e c o g n i t i o n of a n t i g e n i c determinants on the pathogen t h a t are o f t e n c h a r a c t e r i s t i c of the species. The f i r s t r e p o r t of the use of 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 Jensen working w i t h Agrobacterium tumefaciens ( c i t e d by De Boer, 1987). I n f e c t i o u s agents i n diseased t i s s u e , even when i n low c o n c e n t r a t i o n or i n a l a t e n t phase, can be detected w i t h s e r o l o g i c a l assays ( M i l l e r and M a r t i n , 1988). For the D o u g l a s - f i r g a l l - f o r m i n g bacterium t o be considered as a gene v e c t o r , the pathogen's mechanism of g a l l formation must i n v o l v e a t r a n s f e r of genes t o the host 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 occurs i n the A. tumefaciens system. The A. tumefaciens genes t h a t are i n t e g r a t e d i n t o the host p l a n t genome encode f o r enzymes i n v o l v e d i n the production of phytohormones (Watson et al., 1975; W e i l e r and Spanier, 1981). Once the t r a n s f e r of the b a c t e r i a l genes t o the p l a n t c e l l s occurs, the presence of the pathogen i s no longer r e q u i r e d f o r the continued development of the g a l l s ( C h i l t o n et al., 1977). The b a c t e r i a l genes are t r a n s c r i b e d and t r a n s l a t e d as host DNA and e f f e c t the growth of g a l l t i s s u e . Transformation of host p l a n t c e l l s does not occur i n the Pseudomonas syringae pv. savastanoi ([Smith 1908] Young, Dye & W i l k i e 1978) system. Rather, P. syringae pv. savastanoi, the c a u s a l agent of o l i v e and oleander knot, produces tumors on i t s host p l a n t s through the production of phytohormones by the bacterium i t s e l f . The phytohormones cause hypertrophy, 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 of the host c e l l s surrounding the invading bacterium (Smidt and Kosuge, 1978). Therefore, the presence of the bacterium 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 continued development of the tumor. Although g a l l formation by the two phytopathogens a r i s e s i n the host p l a n t s by d i f f e r e n t mechanisms, the b a s i s of g a l l formation i s s i m i l a r i n both systems. The production of phytohormones, namely auxins and c y t o k i n i n s , i s r e q u i r e d f o r the i n i t i a t i o n and development of the p l a n t tumors (Weiler and Spanier, 1981; Comai et al., 1982). Auxins, such as i n d o l e a c e t i c a c i d (IAA), 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 et al., 1985). Overproduction of these phytohormones i n combination leads t o the unregulated growth and d i v i s i o n of host p l a n t c e l l s and hence to the production of a tumor or g a l l . The a c t u a l s i t e of production of the phytohormones d i s t i n g u i s h e s between the A. tumefaciens and P. syringae pv. savastanoi systems. Whether or not the mechanism of g a l l formation by the D o u g l a s - f i r g a l l - f o r m i n g bacterium i n v o l v e s a t r a n s f e r of genes from a plasmid t o the host p l a n t c e l l s , as occurs i n the crown g a l l system, the r o l e of any plasmid DNA i n pathogenesis remains an i n t e r e s t i n g question. Plasmids are autonomously r e p l i c a t i n g extrachromosomal pieces of DNA. Plasmids c o n t a i n , on average, about 2% of the 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 of the genome (Coplin, 1989). Many phytopathogenic b a c t e r i a , i n c l u d i n g species i n the genera, Agrobacterium, Clavibacter, Erwinia, Pseudomonas, and Xanthomonas, are known to c a r r y one or s e v e r a l plasmids. Plasmids encode f o r many d i f f e r e n t phenotypic c h a r a c t e r i s t i c s which are of t e n n o n e s s e n t i a l yet advantageous t o 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 (Trevors, 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 s t i l l l a b e l l e d as c r y p t i c or have no known f u n c t i o n ( C o p l i n , 1989). However, i n both the A. tumefaciens and P. syringae pv. savastanoi systems, genes involved i n the production of phytohormones, and hence pa t h o g e n i c i t y , have been l o c a t e d on plasmids ( L i u and Kado, 1979; Comai et al., 1982). Furthermore, 7 genes i n v o l v e d i n host s p e c i f i c i t y have been l o c a t e d on plasmids of phytopathogenic b a c t e r i a (Coplin, 1989). The focus of t h i s study on the 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 was not t h e i r r o l e as phytopathogens, but r a t h e r whether or not the b a c t e r i a c a r r i e d plasmids t h a t could serve as p o t e n t i a l gene vecto r s f o r the genetic engineering of c o n i f e r s . 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 formers i s due i n p a r t t o the r e s u l t s of extensive s t u d i e s of the phytopathogenic g a l l formers, A. tumefaciens and P. syringae pv. savastanoi. More s p e c i f i c a l l y , the s u c c e s s f u l use of the T i plasmid of A. tumefaciens as a vector f o r i n t r o d u c i n g r e s i s t a n c e genes i n t o economically important p l a n t s has become an important researc h t o o l (Barton and C h i l t o n , 1983; C h i l t o n , 1983). Therefore the 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 value as an a l t e r n a t e vector t o the T i plasmid of Agrobacterium tumefaciens. When c o n s i d e r i n g the scant 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 formers on D o u g l a s - f i r and the focus of t h i s study, the s p e c i f i c o b j e c t i v e s were the f o l l o w i n g : 1) t o d e s c r i b e the g a l l s produced, i s o l a t e the c a u s a l agent and produce a n t i s e r a against the g a l l - f o r m i n g pathogens; 2) t o determine i f the mechanism of g a l l formation i n v o l v e s the transformation of host p l a n t c e l l s ; 3) t o determine i f there i s a c o r r e l a t i o n between plasmid presence and p a t h o g e n i c i t y . 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 disease, reported by Hansen and Smith (1937), i n some instances suppressed growth and caused dieback 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 areas where the h e a l t h of the host was already compromised such as i n damp areas by streams or ponds. The smooth, globose g a l l s formed on the twigs, branches and upper main stems of 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 c h a r a c t e r i s t i c cross-shaped marking was present across the face of the g a l l s . G a l l s o c c u r r i n g on the main stem of young D o u g l a s - f i r s e e d l i n g s can cause deformation and even death of the s e e d l i n g . The bacterium was not pathogenic on any of the c o n i f e r s t e s t e d , i n c l u d i n g Pinus halepensis M i l l . , P. lambertiana Dougl., Pinus radiata Don. and Tsuga heterophylla (Raf.) Sarg. (Hansen and Smith, 1937), except f o r Pseudotsuga macrocarpa (Vasey) Mayr (Smith, 1940). The bacterium 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 ( G i l l e t t e ) , the Cooley spruce 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 of the bacterium. The s t r u c t u r e of the 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 of g a l l s produced by Pseudomonas syringae pv. savastanoi, i n which the g a l l s are composed of groups of r a p i d l y d i v i d i n g c e l l s . The Hansen and Smith bacterium was l o c a t e d 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 the centers of these groups of c e l l s . The i s o l a t e d g a l l bacterium was determined t o be a Gram- negative, non-motile, 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 using current terminology). I t was named by Hansen and Smith as Bacterium pseudotsugae (1937) and l a t e r renamed t o Agrobacterium pseudotsugae ( S a l v u l e s c u , 1947). The work done on A. pseudotsugae has never been confirmed. The f i r s t step i n the study of any 'new' disease i s the d e s c r i p t i o n of the disease symptoms on the host p l a n t . The 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 the pathogen through biochemical t e s t s , which i s a separate study i n i t s e l f , was not considered a p r i o r i t y i n t h i s study because the major aim was t o i n v e s t i g a t e the mechanism of g a l l formation. C l a s s i f i c a t i o n of the b a c t e r i a l i s o l a t e s using a f a t t y a c i d a n a l y s i s system (Hewlett Packard 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 regarded as s u i t a b l e f o r the context of t h i s research. F a t t y a c i d a n a l y s i s i s touted as being very 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 purposes, due t o the genetic s t a b i l i t y of the f a t t y a c i d s (Anonymous, 1985; Stead, 1988). In t h i s system, comparisons of h i g h r e s o l u t i o n gas chromatographic analyses of the c e l l w a l l f a t t y a c i d s were made wit h a computer l i b r a r y of 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 species and the outcomes were recorded 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 t e n t a t i v e i d e n t i f i c a t i o n by the 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 observations allowed f o r comparison to other g a l l - f o r m i n g 10 phytopathogens. Some of the recognized phytopathogenic b a c t e r i a t h a t cause g a l l s on t h e i r host p l a n t s include Agrobacterium tumefaciens, Pseudomonas syringae pv. savastanoi and Erwinia herbicola f.sp. gypsophilae (Brown) M i l l e r , Quinn and Graham 1981. A. tumefaciens i s the causal 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. G a l l s form on r o o t s and stems e s p e c i a l l y at the crown area or base of the stem ( S i n c l a i r et 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 oleander knot as w e l l as b a c t e r i a l knot on ash. The major symptoms i n c l u d e g a l l i n g or knot formation as w e l l as dieback of twigs and branches ( S i n c l a i r et al., 1987). F i n a l l y , crown and root g a l l s on Gypsophila paniculata, resembling those caused by A. tumefaciens, were f i r s t d e s cribed i n the 1930's. The i s o l a t e d pathogen was o r i g i n a l l y named Bacterium gypsophilae but has since been r e c l a s s i f i e d as Erwinia herbicola f.sp. gypsophilae ( M i l l e r et al., 1981; Cooksey, 1985). E. herbicola i s a l s o reported t o cause g a l l i n g symptoms on roses and c a r n a t i o n s (Maas Geesteranus and Barendsen, 1966). G a l l s are u s u a l l y formed at the cut end of rooted c u t t i n g s or at g r a f t unions when below the 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 of E. herbicola i n r e l a t i o n t o the mechanism of g a l l formation. However, a recent study done on the mechanism of g a l l development by E. herbicola f.sp. gypsophilae, suggests a mechanism s i m i l a r t o P. syringae pv. savastanoi (Clark et al., 1989). T e n t a t i v e i d e n t i f i c a t i o n of the 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 other g a l l - f o r m i n g b a c t e r i a , i n the understanding of the disease c y c l e and i n s o l v i n g the question of the mechanism of g a l l formation. The production 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 i d e n t i f i c a t i o n . Both 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 have been produced agai n s t such b a c t e r i a as Erwinia carotovora, Pseudomonas syringae, Corynebacterium sepedonicum and Xanthomonas spp. (De Boer, 1982; A l l a n and Kelman, 1977; C i v e r o l o and Fan, 1982; Benedict et al., 1989). P o l y c l o n a l or monoclonal a n t i b o d i e s can be produced against the b a c t e r i a l antigens depending upon the f a c i l i t i e s a v a i l a b l e and the s p e c i f i c i t y r e q u i r e d (De Boer, 1987). 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 d e t e c t i o n purposes. As a heterologous mixture of a n t i b o d i e s , p o l y c l o n a l 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 being very s p e c i f i c towards the immunogen. This problem can be a l l e v i a t e d p a r t i a l l y by c a r e f u l p u r i f i c a t i o n and preparation of immunogen. I s o l a t i o n of the immunoglobulin G (IgG) f r a c t i o n a l s o can i n c r e a s e the s p e c i f i c i t y of the antiserum ( M i l l e r and M a r t i n , 1988). The s p e c i f i c i t y of the antiserum must be t e s t e d before r o u t i n e use i n q u a l i t a t i v e and q u a n t i t a t i v e assays. The 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). Ouchterlony double d i f f u s i o n or immunodiffusion was chosen as one of the techniques t o t e s t the s p e c i f i c i t y because i t allows 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 antigens (De Boer et al., 1979). The second technique 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 technique was t o be used i n the experiments as o u t l i n e d i n Chapter 2. Many d i f f e r e n t s e r o l o g i c a l assays have been used t o detect 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 or immunodiffusion, immunofluorescence and immunoelectron microscopy (De Boer, 1982; Briansky e t al., 1982; da Roche et a l . , 1986; Benedict et al., 1989). The enzyme- l i n k e d immunosorbent assay (ELISA) has become a w i d e l y used assay f o r both 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 of phytopathogens i n c l u d i n g v i r u s e s , mycoplasma-like organisms, f u n g i and b a c t e r i a . ELISA was f i r s t introduced as a s e r o l o g i c a l assay i n the area of p l a n t pathology i n 1976 (Clark and Adams, 1977) f o r the d e t e c t i o n of p l a n t v i r u s e s . There are a seemingly i n f i n i t e number of v a r i a t i o n s of the b a s i c ELISA procedure. 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 - support used ( L a z a r o v i t s et al., 1987), the times of i n c u b a t i o n , the number and sources of an t i b o d i e s used i n a s i n g l e assay and the presence or absence of the co a t i n g antibody step. An enrichment step o c c a s i o n a l l y i s used as the i n i t i a l step of the assay t o in c r e a s e the b a c t e r i a l c e l l c o n c e n t r a t i o n i n the t e s t sample. This m o d i f i c a t i o n i s an attempt t o increase the l e v e l of d e t e c t i o n (Beguin et al., 1984). Such a m o d i f i c a t i o n was used f o r the 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 these experiments were t o f i r s t l y d e s c r i b e the 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 l o c a t i o n s i n B.C. The second o b j e c t i v e was t o describe the b a c t e r i a l i n c i t a n t s of these 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 through the use of f a t t y a c i d a n a l y s i s . A t h i r d o b j e c t i v e was the production 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 the g a l l - f o r m i n g 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 i d e n t i f i c a t i o n of i n o c u l a t e d b a c t e r i a i n the host t i s s u e s . 14 MATERIALS AND METHODS B a c t e r i a l Strains and Growth 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 four l o c a t i o n s i n southwestern B r i t i s h Columbia. Two s i t e s were on the southern t i p of Vancouver I s l a n d at the edges of Lorna Lake and Cordova Bay near V i c t o r i a . One s i t e was i n the Dunbar area of g r e a t e r Vancouver and the f i n a l s i t e was i n the Hope reg i o n (Table 1.1). Except f o r the Hope s i t e , i n which D o u g l a s - f i r was the dominant s p e c i e s , these areas were marginal growth areas 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 or i n urban areas. 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 , 0.5% peptone ( D i f c o ) , 0.5% glucose (BDH) (CPG media; Kelman, 1954) pH 7.0 p l a t e s f o r quick i d e n t i f i c a t i o n of the c h a r a c t e r i s t i c growth of the i s o l a t e s . N u t r i e n t agar (Difco) + 1% glucose (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 of 21-2 3°C was used f o r growth of the c u l t u r e s . Broth c u l t u r e s were grown e i t h e r i n L u r i a b roth (LB) (1.0% Tryptone [ D i f c o ] , 0.5% Yeast E x t r a c t [ D i f c o ] , 0.8% NaCl) pH 7.0 (Maniatis et al., 1982) or N u t r i e n t Broth (NB) (Difco) pH 7.0 at room temperature or i n 21°C growth chambers on r o t a r y shakers (150 rpm). A n t i b i o t i c r e s i s t a n c e s were t e s t e d through growth of the i s o l a t e ( s ) on NA/G media c o n t a i n i n g f i l t e r - s t e r i l i z e d a n t i b i o t i c s . 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 sent 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, Inc., Newark, 15 Table 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 species used t o t e s t the s p e c i f i c i t y of p o l y c l o n a l a n t i s e r a B a c t e r i a l I s o l a t e d Geographic Growth species by l o c a t i o n media 8 Family Enterobacteriaceae Erwinia i s o l a t e s LB (ga l l - f o r m i n g ) CPG T y p i c a l i s o l a t e s T-2721 Muehlchen, UBC Cordova Bay T-2722 Muehlchen, UBC Cordova Bay T-2739 Muehlchen, UBC Lorna Lake T-2763 DeYoung, UBC Vancouver T-2774 DeYoung, UBC Vancouver T-2789 DeYoung, UBC Vancouver A t y p i c a l i s o l a t e A-0181 DeYoung, UBC Hope Erwinia carotovora LB subsp. carotovora NA/G (Jones 1901) Bergey et a l . , 1923 s t r a i n E379 Copeman, UBC Creston Erwinia herbicola LB subsp. herbicola NA/G (Lohnis 1911) Dye 1964 NCPPB #2273 s t r a i n 2273 Family Rhizobiaceae Agrobacterium pseudotsugae LB (Hansen and Smith, 1937) CPG S a l v u l e s c u 1947 s t r a i n 180 NCPPB #180 Agrobacterium YDP tumefaciens (Smith and NKS Townsend, 1907) Conn 1942 s t r a i n B - l Copeman, UBC Vancouver s t r a i n CH3 Dion, Laval Quebec Rhizobium leguminosarum YMB (Frank 1879) Frank 1889 H o l l , UBC Vancouver YMA Family Pseudomonadaceae Pseudomonas syringae LB van H a l l 1902 cherry Copeman, UBC Vancouver NA/G s t r a i n 'The media used included: L u r i a broth (LB), c a s e i n h y d r o l y s a t e - peptone-glucose (CPG) (Kelman, 1954), N u t r i e n t agar (Difco) supplemented w i t h 1% glucose (NA/G), yeast-dextrose-peptone (YDP) (Moore, 1977), NKS (Brisbane and Kerr, 1983) and yeast- mannitol broth (YMB) (Vincent, 1970). 16 Delaware, USA, using the Hewlett Packard 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 ( M i l l e r , 1984). A u t h e n t i c c u l t u r e s used i n the a n t i s e r a s p e c i f i c i t y t e s t s are l i s t e d i n Table 1.1. A l l chemicals used were from Sigma Chemical Company unless otherwise 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 obtained as 1-year-old seedlings from year-end grading a t the U n i v e r s i t y of B r i t i s h Columbia F a c u l t y of F o r e s t r y Nursery. The v a r i o u s t r e e species used i n the host range study were obtained from Dr. R.S. Hunt at the P a c i f i c Forest Research Center i n V i c t o r i a , B.C. Seedlings were planted out i n l a r g e styrofoam cups (600 ml) i n a standard s o i l mix wi t h 2400 cm3 peat and 200 cm3 Osmocote, a slow r e l e a s e f e r t i l i z e r (14N-6.0P-11.6K), per 0.16 m3 of s t e r i l i z e d s o i l . F e r t i l i z e r , 20-20-20, was a p p l i e d once every two weeks at a r a t e of 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 through maceration of g a l l t i s s u e i n a small amount of LB broth i n s t e r i l e dimple p l a t e s . No surface s t e r i l i z a t i o n was necessary although surface 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 there was a s u f f i c i e n t amount of t i s s u e remaining t o sample. A f t e r 15-2 0 min, l o o p f u l s of the r e s u l t i n g suspension were streaked 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 through stab i n o c u l a t i o n s using s t r a i g h t p i n s . Branches were i n o c u l a t e d w i t h 5 t o 7-day-old 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 , 3-5 centimeters (cm) below the branch t i p . The branch diameters 17 measured between 2-5 m i l l i m e t e r s (mm) at the p o i n t of i n o c u l a t i o n . The pins were o f t e n unavoidably pushed through the branch and hence through the s t e l e of the branch. P o l y c l o n a l A n t i s e r a Production: Late l o g phase c u l t u r e s grown i n NB were harvested by c e n t r i f u g a t i o n (2100 X g (maximum) f o r 15 min), washed three times w i t h phosphate buffered s a l i n e (PBS) pH 7.3 and adjusted i n PBS t o an o p t i c a l d e n s i t y of 1.0 u n i t at 660 nm. The whole c e l l s were f i x e d w i t h 2.0% glutaraldehyde 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 of PBS (pH 7.3) at 4°C over 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 reparations were then mixed 1:1 w i t h Freund's complete adjuvant ( D i f c o ) . 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 the hind legs of New Zealand white r a b b i t s . A schedule of 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 bleedings 10 days a f t e r the l a s t i n j e c t i o n . Booster shots were administered once a month. Bleedings were done e i t h e r by n i c k i n g the marginal v e i n or by s e d a t i n g the r a b b i t w i t h Innovar Vet and i n s e r t i n g a ca t h e t e r i n t o the a u r i c l e a r t e r y (10-20 ml per b l e e d i n g ) . Whole blood was allowed t o c l o t by p l a c i n g at 37°C f o r 1 h and then at 4°C overn i g h t . The samples were then c e n t r i f u g e d at 2100 X g (maximum) f o r 10 min at 4°C. The supernatants were poured o f f and s t o r e d i n a l i q u o t s at -20°C or at 4°C w i t h 0.02% sodium azide as a p r e s e r v a t i v e . 18 IgG P u r i f i c a t i o n : The IgG f r a c t i o n of the antiserum was p u r i f i e d by a modi f i e d C l a r k and Adams method (1977). One ml of whole antiserum was d i l u t e d w i t h 9 ml of d i s t i l l e d water and mixed w i t h 10 ml of saturated ammonium s u l f a t e at room temperature 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 out at 2100 X g (maximum) at 10°C. The p e l l e t was then resuspended i n 2 ml of 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 then run through 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 monitored at 254 nm wit 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 . P r o t e i n c o n c e n t r a t i o n was measured on a Hewlett Packard spectrophotometer at 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 of 1.4. A n t i s e r a S e n s i t i v i t y : T e s t i n g of the s p e c i f i c i t y the whole a n t i s e r a and the IgG f r a c t i o n s were c a r r i e d out using an i n d i r e c t ELISA ( V o l l e r et al., 1979). This assay involved the in c u b a t i o n of Erwinia c e l l s d i r e c t l y i n the microplate w e l l s (200 j u l / w e l l ) . M i c r o t i t e r 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 E l e c t r i c G25T8 25 W g e r m i c i d a l lamp). The antigens were used a concentration of 5 X 10 8 c e l l s / m l i n PBS pH 7.3 (A 6 6 0 = 0. 14 f o r A-0181 and A 6 6 0 = 0 . 28 f o r T-2789) . A f t e r a 1 h i n c u b a t i o n step at room temperature, the w e l l s were emptied w i t h two, 10-sec r i n s e s of tap water from a homemade p l a t e washer. A 45-min b l o c k i n g step using 200 A i l / w e l l ' B l o t t o ' (0.1% Carnation 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) ( E l l i s , 1988) followed at room temperature. P l a t e s were dumped 19 (not r i n s e d ) and the antiserum 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 ̂ ul/well) was incubated f o r 1 h at room temperature. An antiserum d i l u t i o n s e r i e s (one hundred-fold d i l u t i o n s between 10"1 and 10"12) was r e p l i c a t e d at l e a s t three times. Two, 10-sec washes removed unbound immunoglobulins. Goat a n t i - r a b b i t a l k a l i n e phosphatase enzyme conjugate d i l u t e d 1 i n 1000 w i t h PBS/Tween-2 0/Blotto, 200 ^ u l / w e l l , was incubated at room temperature 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 of the su b s t r a t e , p-nitrophenyl phosphate, d i l u t e d 0.6 mg/ml i n s u b s t r a t e b u f f e r (10% diethanolamine; pH 9.8). Absorbance readings at 405 nm were taken on a T i t e r t e k M u l t i s k a n p l a t e reader, e i t h e r 30 or 45 min, a f t e r s u b s t r a t e a d d i t i o n . S p e c i f i c i t y : Immunodiffusion: Antiserum s p e c i f i c i t y was t e s t e d using Ouchterlony double d i f f u s i o n (3 mm diameter w e l l s were spaced 4 t o 5 mm apart) (De Boer e t a l . , 1979). B a c t e r i a l c e l l s , taken from 1 t o 2-week o l d c u l t u r e s , were suspended i n s t e r i l e d i s t i l l e d water t o form very t u r b i d suspensions. Whole a n t i s e r a or IgG f r a c t i o n s were added u n d i l u t e d t o the middle w e l l s and the r e a c t i o n s were allowed t o proceed f o r 16-24 h at room temperature. Several d i f f e r e n t phytopathogenic 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 : Erwinia carotovora subsp. carotovora, E. herbicola subsp. herbicola s t r a i n 2273, Agrobacterium pseudotsugae s t r a i n 180, A. tumefaciens s t r a i n B - l , Rhizobium leguminosarum (formerly trifolii), Pseudomonas syringae (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 S p e c i f i c i t y : 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 the determination of the a n t i s e r a s e n s i t i v i t y was a l s o used to determine the s p e c i f i c i t y of the a n t i s e r a . The phytopathogenic b a c t e r i a t e s t e d by immunodiffusion were a l s o used i n the i n d i r e c t ELISAs except f o r Agrobacterium pseudotsugae. Tests i n v o l v i n g the whole T-2789 antiserum were c a r r i e d out w i t h a s i n g l e c o n c e n t r a t i o n of c e l l s , 5-7 X 10 8 c e l l s / m l , and 1 0 - f o l d d i l u t i o n s of antiserum ranging from 10"2 t o 10"6 (4 r e p l i c a t i o n s per d i l u t i o n ) . Experiments 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 with v a r y i n g concentrations of c e l l s and a s i n g l e c oncentration of antiserum. Concentrations of 5 X 10 8, 5 X 10 7, 5 X 10 6, 5 X 10 5, 5 X 10 4 c e l l s per ml were used f o r the 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 . The antiserum d i l u t i o n s used were 10"4 f o r T-2789 and 10"3 f o r A-0181. Host Range: A host range study was c a r r i e d out i n v o l v i n g the f o l l o w i n g t r e e s p e c i e s : Abies amabilis Dougl. ex Forbes, A. grandis (Dougl. ex D. Don) L i n d l . , A. lasiocarpa (Hook.) Nutt., Chamaecyparis nootkatensis (D. Don) Spach, Larix o c c i d e n t a l i s Nutt., Picea engelmannii Parry ex. Engelm., P. glauca (Moench) Voss, P. sitchensis (Bong.) Carr, Pinus contorta Dougl. ex Loud., P. monticola Dougl. ex D. Don, P. ponderosa Dougl. ex Laws, Pseudotsuga menziesii (Mirb.) Franco, Thuja p l i c a t a Dpnn ex D. Don 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 plus the 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 the v a r i o u s c o n i f e r s . Two t r e e s (2 branches/tree) were stab i n o c u l a t e d per i s o l a t e . Observations of the i n o c u l a t i o n s i t e s were recorded a f t e r 5 months as e i t h e r p o s i t i v e or negative 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 recorded i f at l e a s t one of the four i n o c u l a t i o n s i t e s d i s p l a y e d v i s i b l e g a l l s . D i r e c t i s o l a t i o n s were made onto CPG media from two s i t e s per t r e e s p e cies per i s o l a t e . R e s u l t s were recorded as percent recovery of c h a r a c t e r i s t i c b a c t e r i a from the two s i t e s . C h a r a c t e r i s t i c 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 using both whole and p u r i f i e d a n t i s e r a . B a c t e r i a i d e n t i f i e d as the i n o c u l a t e d g a l l - forming b a c t e r i a , by immunodiffusion, were back-inoculated i n t o D o u g l a s - f i r t r e e s t o t e s t f o r pa t h o g e n i c i t y . Four i n o c u l a t i o n s i t e s per t r e e species were used per i s o l a t e . R e s u l t s were recorded as the percent of 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 Galls 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 igh numbers w i t h i n l o c a l i z e d marginal growth areas, although a survey of frequency of occurrence was not conducted. The g a l l s were most o f t e n found on the t i p s of branches of mature t r e e s . The g a l l s appeared t o be i n i t i a t e d simply along the length of the new shoot ( F i g . 1.1a) or at the p o i n t where the new year's growth begins ( F i g . 1.1b). Older g a l l s were observed i n the lower dead branches of 10-15 year o l d t r e e s . Most of the n a t u r a l g a l l s were l o c a t e d on branches measuring between 0.2 and 0.8 cm i n diameter w i t h the g a l l s measuring between 0.5 and 2 cm i n diameter ( F i g . 1.1c). The s i n g l e specimen of the a t y p i c a l g a l l c o l l e c t e d from the Hope s i t e was taken from a branch measuring approximately 4.0 cm i n diameter ( F i g . l . l d ) . The g a l l s appeared, i n gen e r a l , t o be i n i t i a t e d at one p o i n t on the branch and t o erupt or expand outward enveloping p a r t of the branch. Two d i f f e r e n t D o u g l a s - f i r g a l l types were c o l l e c t e d . The 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 i n o c u l a t e d g a l l s were observed. The more commonly encountered g a l l s were, i n g e n e r a l , globose i n nature w i t h a rough surface caused by the apparent l o c a l i z e d overgrowth of i s o l a t e d groups of c e l l s ( F i g . 1.2a). The d i s t i n c t i v e n e s s of these outgrowths v a r i e d w i t h i n the t y p i c a l g a l l s both 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 g a l l s ( F i g . 1.2b). The surface of these 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 or r a r e l y a cross-shaped 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 , 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 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 (A ,B,C) and t h e a t y p i c a l i s o l a t e A-0181 (D) 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 ( a c t u a l s i z e ) . 24 F i g . 1.2. Types of 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 t y p e s of t h e 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 months (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 as 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 months (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 as Erwinia herbicola subsp. herbicola. f i s s u r e . As the g a l l matured, t h i s marking was o f t e n l e s s obvious ( F i g . 1.2c). 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 being globose t o oblong i n shape w i t h a smoother, b a r k - l i k e surface t h a t cracked and peeled s l i g h t l y as the 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 t o a f f e c t the s t r u c t u r e of the branch t o a g r e a t e r degree than the t y p i c a l g a l l s . 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 the branch both i n the r e s u l t a n t d i r e c t i o n of growth and i n the s w e l l i n g of the branch below the g a l l . D o u g l a s - f i r g a l l s of both i s o l a t e s 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 they developed and matured, the g a l l s turned brown and o f t e n l o s t the d i s t i n c t i v e markings as noted f o r the younger g a l l s ( F i g 1.2e). The o l d e r 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 the g a l l s from which the i n i t i a l i s o l a t i o n s were made. G a l l s could be a r t i f i c i a l l y - i n d u c e d through stab i n o c u l a t i o n s on most branches of 1-year-old 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 along the length of the main stem ( F i g . 1.3a). Some g a l l s induced on l a t e r a l branches reached a s i z e of 2.0 cm or more i n diameter f o r both of the i s o l a t e types but u s u a l l y g a l l s reached only a s i z e of 0.5 t o 1 cm i n diameter. The r a t i o of g a l l diameter t o branch diameter a f t e r 4 weeks, was g r e a t e r than 3.1 and 2.2 f o r the t y p i c a l and a t y p i c a l i s o l a t e s , r e s p e c t i v e l y . The t y p i c a l i s o l a t e s produced faster-growing g a l l s reaching a diameter of 0.5 cm i n 4 weeks w h i l e the a t y p i c a l g a l l s took 6 weeks to reach a s i m i l a r diameter. The presence of the g a l l i n g symptoms on the host a f f e c t e d the 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 the host t r e e . Death of 26 F i g . 1.3. Galls formed on 1- to 2-year old Douglas-fir trees by stab-inoculation with Douglas-fir gall-forming bacteria. (A) g a l l produced on main stem; (B) g a l l s produced with 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 inoculated branches; (D) insect feeding 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 tissue after insect feeding and (F) apparent spread of g a l l bacterium from inoculation s i t e (arrow) to new s i t e on a Douglas-fir seedling. the i n o c u l a t e d branch or simply the t i p s o f t e n occurred a f t e r a r t i f i c i a l i n o c u l a t i o n . I n o c u l a t i o n of the upper main stem of young D o u g l a s - f i r s e e d l i n g s , l e s s than 6 months o l d , o f t e n r e s u l t e d i n death of the seedlings. W e l l - e s t a b l i s h e d t r e e s (1- y e a r - o l d or older) d i d not appear t o be se v e r e l y a f f e c t e d although the t r e e s sometimes became deformed ( F i g . 1.3b). New branch t i p s were i n i t i a t e d below the g a l l e d t i p o f t e n d i s t o r t i n g the growth p a t t e r n of the t r e e ( F i g . 1.3c). In 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 stop growth a f t e r the f i r s t year. The succulent 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). G a l l s o f t e n continued t o grow a f t e r being eaten by i n s e c t s ( F i g . 1.3e). A cur i o u s observation of the a r t i f i c i a l l y - i n d u c e d g a l l s was t h a t o c c a s i o n a l l y small 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 ( F i g . 1.3f). This would suggest e i t h e r t h a t the pathogen was transported e x t e r n a l l y v i a i n s e c t s or i n t e r n a l l y through the p l a n t ' s v a s c u l a r system. From the two types of g a l l s , two d i s t i n c t s t r a i n s of the 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 larg e numbers of the i n c i t i n g b a c t e r i a . When grown on s o l i d media c o n t a i n i n g c a s e i n h y d r o l y s a t e , peptone and glucose, the common s t r a i n s , t y p i f i e d by the T-2789 i s o l a t e , formed shiny, greyish-white c i r c u l a r 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). The c o l o n i e s have c h a r a c t e r i s t i c surface i n d e n t a t i o n 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 of s i m i l a r c o l o r and tran s l u c e n c e ( F i g . 1.4b), however the shape of the c o l o n i e s d i f f e r e d . Young A-0181 28 F i g . 1 . 4 . Colonies of the t y p i c a l (A) and the a t y p i c a l (B) gall-forming Erwinia i s o l a t e s grown on casein hydrolysate, peptone and glucose medium. 29 c o l o n i e s were convex i n appearance although as the c o l o n i e s matured, depressions were formed i n the center of the c o l o n i e s . E x t r a c e l l u l a r slime was a l s o o f t e n produced as the c o l o n i e s matured causing the coalescence of c l o s e l y spaced c o l o n i e s , g i v i n g the c o l o n i e s an e l l i p t i c a l appearance. Colonies of both g a l l - f o r m i n g i s o l a t e s measured between 1.0 t o 2.0 mm i n diameter a f t e r 5-7 days on s o l i d CPG media. Colonies of the a t y p i c a l i s o l a t e s grew s l i g h t l y f a s t e r than the t y p i c a l i s o l a t e s appearing a f t e r approximately 2 days at 21-2 3°C on s o l i d media. One important c h a r a c t e r i s t i c of the two b a c t e r i a l i s o l a t e s was t h e i r temperature s e n s i t i v i t y (Table 1.2). The t y p i c a l i s o l a t e s were able t o s u r v i v e only at temperatures up t o 3 2±0.5°C i n L u r i a broth. The a t y p i c a l i s o l a t e was able t o s u r v i v e a t s l i g h t l y elevated temperatures of up t o 34±0.5°C i n L u r i a broth. 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 at room temperature on s o l i d CPG media f o r more than 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 or 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 wider range of a n t i b i o t i c s i n c l u d i n g a m p i c i l l i n , erythromycin, kanamycin and t e t r a c y c l i n e . When f a t t y a c i d a n a l y s i s and computer-assisted comparison w i t h a l i b r a r y of authentic 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 than 0.500 placed both of them unequivocally i n the 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 g a l l - forming i s o l a t e A-0181 was p o s i t i v e l y placed i n the E r w i n i a 30 Table 1.2 Temperature s e n s i t i v i t y of 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 Growth i n L u r i a Broth Temperature (+/- 0.5°C) Erwinia T-2789 E r w i n i a A-0181 31 + + 32 + + 33 - + 34 - + 35 - - 31 Table 1.3. A n t i b i o t i c r e s i s t a n c e s expressed 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 the 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 Growth of g a l l - f o r m i n g A n t i b i o t i c Concentration b a c t e r i a l i s o l a t e s (Aig/ml) T y p i c a l A t y p i c a l A m p i c i l l i n 30 + _a 40 + — C a r b e n i c i l l i n 40 + t r a c e 50 + t r a c e Chloramphenicol 10 - - Erythromycin 30 + - F u s i d i c a c i d 31 + + 75 + +a Kanamycin 40 t r a c e — 50 t r a c e — M e t h i c i l l i n 28 + + Novobiocin 75 + + Oleandomycin 25 + t r a c e 3 Streptomycin 25 - - T e t r a c y c l i n e 12 . 5 + - aGrown on NA i n s t e a d of NA/G 32 h e r b i c o l a 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 the E. salicis group (Table 1.4). Antisera S e n s i t i v i t y : The c u t o f f value f o r ELISA values 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 4 0 5) , which was greater than twice the background values (Sutula et al., 1986). The lower l i m i t s of d e t e c t i o n of the 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"6 and 10"3 f o r T-2789 and A-0181, r e s p e c t i v e l y . The p r o t e i n concentrations f o r the 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 of d e t e c t i o n f o r the IgG f r a c t i o n s were 10~4 and 10"3 f o r T-2789 and A-0181, r e s p e c t i v e l y ( F i g . 1.5). S p e c i f i c i t y -Immunodiffusion: The whole T-2789 antiserum 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 antigen and each of the t y p i c a l i s o l a t e s ( F i g . 1.6). One of these l i n e s formed a l i n e of p a r t i a l i d e n t i t y w i t h the s i n g l e l i n e of i d e n t i t y produced i n r e a c t i o n w i t h the A-0181 i s o l a t e . No p r e c i p i t i n l i n e s were observed w i t h Agrobacterium tumefaciens, Agrobacterium pseudotsugae, Erwinia carotovora, Erwinia herbicola subsp. herbicola s t r a i n 2273, Escherichia c o l i , Pseudomonas syringae or Rhizobium leguminosarum. P u r i f i c a t i o n of the T-2789 antiserum removed the cross 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 the whole antiserum ( F i g . 1.6). Both the whole A-0181 antiserum 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 of i d e n t i t y only 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 of the A-0181 antiserum 33 Table 1.4. Tent a t i v e i d e n t i f i c a t i o n of 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 of t h e i r f a t t y a c i d p r o f i l e s and computer-assisted comparison w i t h a l i b r a r y of p r o f i l e s from authentic s t r a i n s S i m i l a r i t y index I s o l a t e P o s s i b l e i d e n t i t i e s w i t h known s t r a i n s T-2739 Hafnia alvei 0.635 Erwinia s a l i c i s 0.593 Erwinia herbicola 0.544 subsp. herbicola T-2763 E r w i n i a salicis 0.767 Hafnia alvei 0.719 Erwinia herbicola 0.597 subsp. herbicola T-2789 E r w i n i a salicis 0.648 Hafnia alvei 0.644 Erwinia herbicola 0.558 subsp. herbicola A-0181 E r w i n i a herbicola 0.840 subsp. herbicola Enterobacter agglomerans 0.545 34 0 . 8 A n t i s e r u m d i l u t i o n F i g . 1.5. S e n s i t i v i t y of the IgG fractions of the antisera r a i s e d against the t y p i c a l T-2789 (•) and A-0181 ( + ) is o l a t e s of the Douglas-fir gall-forming bacteria as determined by an i n d i r e c t ELISA against t h e i r respective homologous antigens. Each point represents the mean of four r e p l i c a t e s . 35 « © o © 0 © ° ©"wo © © © o O © © o © o © © © _ © © o © Q ^ A 0 ° ~ © \ © © ©> © © © © © © e © ~ © © o © © o o © o U ° © © ^ © © ̂  © © o - © o © © © ffi m ° © ° » © © © © o o o © ^ © © © © ^ © © © © © © © © © © © © © ° * ° ° % © © 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 tween 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-2789 and t h e (A) 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 p h y t o p a t h o g e n i c b a c t e r i a . 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-2789, (2) Erwinia A-0181, (3) Erwinia T-2721, (4) Erwinia T-2722, (5) Erwinia T-2739, (6) Erwinia T-2763, (7) Erwinia T-2774, (8) Agrobacterium pseudotsugae, (9) Agrobacterium tumefaciens, (10) Erwinia carotovora s u b s p . carotovora, (11) Erwinia herbicola s t r a i n 2273, (12) Escherichia coli s t r a i n MC1000, (13) Pseudomonas syringae and (14) Rhizobium leguminosarum. 36 r e s u l t e d i n the r e d u c t i o n of the number of p r e c i p i t i n l i n e s , a g a i n s t i t s homologous antigen, from two t o a s i n g l e l i n e . No p r e c i p i t i n l i n e s were observed w i t h any of the other phytopathogenic b a c t e r i a i n c l u d i n g Agrobacterium tumefaciens, Agrobacterium pseudotsugae or even the g a l l - f o r m i n g t y p i c a l i s o l a t e s . S p e c i f i c i t y - I n d i r e c t ELISA: For the whole T-2789 antiserum, at 1.5 h a f t e r s u b s t r a t e a d d i t i o n , a d i l u t i o n of 10"5 detected only the t y p i c a l i s o l a t e s T-2789 and T-2722 (Table 1.5). I s o l a t e A-0181 was detected at antiserum d i l u t i o n s out t o 10"3. E r w i n i a c a r o t o v o r a subsp. c a r o t o v o r a s t r a i n 379 was detected at antiserum d i l u t i o n s out t o 10~4 w h i l e Agrobacterium tumefaciens i s o l a t e CH3 and Rhizobium t r i f o l i i were detected at d i l u t i o n s out t o 10"3. Pseudomonas s y r i n g a e was not detected at any d i l u t i o n a f t e r 1.5 h. Each b a c t e r i a l spp. t e s t e d except f o r A-0181 and A. tumefaciens i s o l a t e CH3 was detected w i t h a 1 0 - f o l d higher d i l u t i o n of antiserum a f t e r 27 h. For the p u r i f i e d T-2789 antiserum, at 2.5 h a f t e r s u b s t r a t e a d d i t i o n , the only b a c t e r i a l species detected was the homologous an t i g e n , down t o a d i l u t i o n of 5 X 10 6 c e l l s / m l (Table 1.6). A T-2789 c e l l d i l u t i o n of 5 X 10 5 c e l l s per ml was detected a f t e r 22 h. A f t e r 22 h the t y p i c a l i s o l a t e T-2722 was a l s o detected a t a c o n c e n t r a t i o n of 5 X 10 8 c e l l s / m l . The A-0181 p u r i f i e d antiserum was a l s o very s p e c i f i c d e t e c t i n g only i t s homologous antigen, at a d i l u t i o n of 5 X 10 7 c e l l s / m l , a f t e r 2.0 h (Table 1.7). A f t e r 27 h an A-0181 37 Table 1.5. S p e c i f i c i t y of whole Erwinia T-2789 antiserum i n an i n d i r e c t ELISA. B a c t e r i a l concentrations of 5-6 X 10 8 c e l l s / m l were used. The A 4 0 5 values f o r each b a c t e r i a l s t r a i n were read at two times a f t e r substrate a d d i t i o n . P o s i t i v e ELISA values were above 0.10 A 4 0 5 at time i n d i c a t e d f o r antiserum d i l u t i o n s B a c t e r i a l Time s t r a i n (h) 10"2 10"3 10"4 10"5 10 Erwinia T-2789 1. 5 a 0. 25 0. 25 0. 33 0. 12 0. 00 (grown i n LB) 27. 5 1. 58 1. 62 1. 87 1. 04 0. 01 Erwinia T-2789 1. 5 0. 28 0. 25 0. 35 0. 23 0. 00 (grown i n NB) 27. 5 1. 72 1. 72 1. 95 1. 63 0. 23 Erwinia T-2722 1. 5 0. 38 0. 39 0. 56 0. 38 0. 00 27. 5 1. 98 1. 98 1. 95 1. 63 0. 23 Erwinia A-0181 1. 5 0. 11 0. 11 0. 00 0. 00 0. 00 27. 5 0. 91 0. 98 0. 00 0. 00 0. 00 Erwinia 1. 5 0. 62 0. 50 0. 12 0. 00 0. 00 carotovora 3 79 27. 5 2. 00 2. 00 1. 87 1. 04 0. 00 Agrobacterium 1. 5 1. 73 0. 32 0. 01 0. 00 0. 00 tumefaciens CH3 27. 5 2 . 00 1. 82 0. 47 0. 01 0. 00 Pseudomonas 1. 5 0. 09 0. 00 0. 00 0. 00 0. 00 syringae 27. 5 0. 81 0. 04 0. 00 0. 00 0. 00 Rhizobium 1. 5 0. 61 0. 21 0. 00 0. 00 0. 00 1eguminosarum 27. 5 2 . 00 1. 53 0. 21 0. 04 0. 00 amean of 4 r e p l i c a t i o n s 38 T a b l e 1 .6 . S p e c i f i c i t y o f the p u r i f i e d IgG f r a c t i o n o f the 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"4 i n an i n d i r e c t E L I S A . The A 4 0 5 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 A 4 0 5 f o r c e l l s / m l B a c t e r i a l Time a t t ime i n d i c a t e d s t r a i n (h) 5 X 10 8 5 X 10 7 5 X 10 6 5 X 10 5 5 X 10 4 Erwinia T-2789 2.5 0 .27 a 0.24 0.18 0.03 0.00 22.0 0.85 0.78 0.61 0.15 0.00 Erwinia T-2722 2.5 0.03 0.05 0.00 0.00 0.00 22.0 0.18 0.04 0.06 0.00 0.00 Erwinia A-0181 2.5 0.00 0.00 0.00 0.00 0.00 22.0 0.05 0.00 0.00 0.00 0.00 Erwinia 2 .5 0.00 0.00 0.00 0.00 0.00 herbicola 2273 22.0 0.04 0.00 0.00 0.00 0.00 Erwinia 2 .5 0.00 0.00 0.00 0.00 0.00 carotovora 379 22.0 0.01 0.00 0.00 0.00 0.00 Agrobacterium 2 .5 0.00 0.00 0.00 0.00 0.00 tumefaciens B - l 22.0 0.00 0.00 0.06 0.00 0.00 Pseudomonas 2 .5 0.00 0.00 0.00 0.00 0.00 syringae 22.0 0.00 0.00 0.00 0.00 0.00 Rhizobium 2 .5 0.00 0.00 0.00 0.00 0.00 leguminosarum 22.0 0.00 0.00 0.00 0.00 0.00 'mean o f 4 r e p l i c a t i o n s 39 Table 1.7. S p e c i f i c i t y of the p u r i f i e d IgG f r a c t i o n of the E r w i n i a A-0181 antiserum at a d i l u t i o n of 10"3 i n an i n d i r e c t ELISA. The A 4 0 5 values f o r each b a c t e r i a l s t r a i n were read at two times a f t e r s ubstrate a d d i t i o n . P o s i t i v e ELISA values were above 0.10 A 4 0 5 f o r c e l l s / m l B a c t e r i a l Time at time i n d i c a t e d s t r a i n (h) 5 X 10° 5 X 10 7 5 X 10 6 5 X 10 5 5 X 10 4 E r w i n i a A-0181 2.0 0.52a 0.25 0.10 0.05 0.05 27.0 1.59 0.78 0.22 0.06 0.05 E r w i n i a T-2789 2.0 0.05 0.05 0.05 0.05 0.05 27.0 0.06 0.05 0.05 0.05 0.04 E r w i n i a T-2722 2.0 0.06 0.04 0.03 0.03 0.03 27.0 0.16 0.05 0.03 0.02 0.02 Erwinia 2.0 0.03 0.01 0.01 0.01 0.01 h e r b i c o l a 2273 27.0 0.07 0.01 0.00 0.00 0.00 Erwinia 2.0 0.01 0.01 0.00 0.00 0.00 carotovora 379 27.0 0.01 0.02 0.00 0.00 0.00 Agrobacterium 2.0 0.07 0.05 0.04 0.05 0.04 tumefaciens B - l 27.0 0.08 0.05 0.04 0.04 0.04 Pseudomonas 2.0 0.06 0.06 0.06 0.06 0.06 syringae 27.0 0.05 0.05 0.06 0.05 0.05 Rhizobium 2.0 0.00 0.00 0.00 0.00 0.00 leguminosarum 27.0 0.00 0.00 0.00 0.00 0.00 a4 r e p l i c a t i o n s per value 40 c e l l d i l u t i o n of 5 X 10 6 c e l l s / m l was detected. The t y p i c a l i s o l a t e T-2722 was a l s o detected a f t e r 27 h at a d i l u t i o n of 5 X 10 8 c e l l s / m l . Host Range: Small growths were observed i n j u s t over h a l f of the c o n i f e r s i n o c u l a t e d w i t h the Erwinia i s o l a t e s ( F i g . 1.7). The l a r g e s t growths and the l a r g e s t numbers of i n o c u l a t e d b a c t e r i a recovered were, however, lo c a t e d on the D o u g l a s - f i r t r e e s . The A-0181 i s o l a t e was recovered from each c o n i f e r species i n o c u l a t e d (Table 1.8). G a l l i n g symptoms produced by the 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, Chamaecyparis, Larix, Picea and Pinus spp. G a l l i n g symptoms produced by the 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 recovered from each i n o c u l a t e d t r e e species. However, when c o n s i d e r i n g the three 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 of t h i s group were recovered 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 g a l l i n g symptoms were not n e c e s s a r i l y those from which the i n o c u l a t e d b a c t e r i a could be s u c c e s s f u l l y recovered. C h a r a c t e r i s t i c b a c t e r i a were recovered from many of the t r e e s t h a t d i d not produce v i s i b l e g a l l i n g symptoms. A l l those s t r a i n s , of both types, i d e n t i f i e d as the 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 back- i 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 of the c u l t u r e s of recovered b a c t e r i a were l o s t and hence were not back-inoculated i n t o D o u g l a s - f i r . 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 branches), wound-only c o n t r o l s (center branches) and a t y p i c a l i s o l a t e A-0181 ( r i g h t branches), r e s p e c t i v e l y . Table 1.8. Path o g e n i c i t y of g a l l - f o r m i n g E r w i n i a f i s o l a t e s on 14 c o n i f e r species. (G) Percentages of observations of g a l l i n g at the i n o c u l a t i o n s i t e are fo l l o w e d by (R) percent recovery of the inoc u l a t e d b a c t e r i a . Percentage of 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 the recovered g a l l - f o r m i n g Erwinia spp. are l i s t e d i n column P Percentages of Erwinia i s o l a t e Dry C o n i f e r T-2774 T-2789 T-2722 A-0181 Stab species G a R b P G R P G R P G R P G R Family Pinaceae Abies amabilis 100 50 100 100 50 100 100 100 100 100 100 100 0 0 Abies grandis 100 50 100 100 50 100 0 0 - 100 100 100 0 0 Abies l a s i o c a r p a 100 100 100 75 0 - 75 50 100 100 50 100 0 0 Larix o c c i d e n t a l i s 100 50 100 75 100 100 100 100 100 100 100 50 0 0 Picea glauca 50 100 100 100 50 100 100 100 75 100 100 100 0 0 Picea engelmannii 100 100 100 100 100 100 0 50 0 100 100 100 0 0 Picea s i t c h e n s i s 75 100 100 50 100 100 100 100 100 100 100 75 0 0 Pinus contorta 50 50 100 75 100 100 100 100 100 100 50 100 0 0 Pinus monticola 0 100 100 0 100 100 50 100 100 75 100 75 0 0 Pinus ponderosa 25 50 100 0 100 50 100 50 50 75 100 100 0 0 Pseudotsuga menzeisii 100 100 50 100 100 100 100 100 100 100 100 100 0 0 Tsuga heterophylla nd d nd nd 25 50 50 nd nd nd 25 100 50 0 0 Family Cupressaceae Chamaecyparis nootkatensis nd nd nd 0 50 nd nd nd nd 100 50 nd 0 0 Thuja p l i c a t a 100 0 — 0 50 75 0 0 — 100 50 50 0 0 a4 r e p l i c a t i o n s b2 r e p l i c a t i o n s c4 r e p l i c a t i o n s dno data 43 DISCUSSION Two d i f f e r e n t types of b a c t e r i a l i s o l a t e s were recovered 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 southwestern B r i t i s h Columbia. A l l of the g a l l s c o l l e c t e d i n t h i s study were s i m i l a r t o those reported by Hansen and Smith i n C a l i f o r n i a i n the 1930's (Hansen and Smith, 1937), e s p e c i a l l y those caused by the a t y p i c a l i s o l a t e . At the disease l e v e l , many s i m i l a r i t i e s were noted between the D o u g l a s - f i r g a l l disease of C a l i f o r n i a , caused by Agrobacterium pseudotsugae, and the B.C. g a l l d i s e a s e , the pathogens of 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 s p e c i e s . Unfortunately, there are no extant c u l t u r e s of the bacterium, 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 described by Hansen and Smith i n 1937. Therefore a d i r e c t comparison between the i n c i t a n t s of the two D o u g l a s - f i r g a l l diseases was not 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 host t r e e , D o u g l a s - f i r . The D o u g l a s - f i r g a l l s i n t h i s study were c o l l e c t e d i n mixed f o r e s t s near lakes or i n urban areas. This s i t e y i e l d e d the s i n g l e source of the second type of b a c t e r i a i s o l a t e d . Hansen and Smith c o l l e c t e d A. pseudotsugae g a l l s i n s i m i l a r areas, a l s o termed as marginal areas where the h e a l t h of the host was already compromised. With both 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 i n o c u l a t e d branches o f t e n d i e . However, due t o the c h a r a c t e r i s t i c s m a l l s i z e of the 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 the n a t u r a l environment, l i t t l e damage was observed. Hansen and Smith (1937) reported that the i s o l a t e d pathogen was 'not of economic importance but of p o t e n t i a l i n t e r e s t t o the lumbering i n d u s t r i e s ' as i t could 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 the pathogen was able t o invade stands of timber dominated by the host, Douglas- f i r , then ' i t might r e a d i l y become an important f a c t o r i n determining the f u t u r e composition of the f o r e s t 1 . F o r t u n a t e l y , t h a t pathogen has not become an important f a c t o r i n the geographical range of D o u g l a s - f i r . Because the g a l l - f o r m i n g 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 death i f i n o c u l a t e d i n the upper main stem of young D o u g l a s - f i r s e e d l i n g s , they may have the 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 study, although the i n i t i a t i o n s i t e of the g a l l s w i t h i n the succulent new year's growth suggests an i n s e c t v e c t o r . Hansen and Smith (1937) proposed t h a t t r a n s m i s s i o n of A. pseudotsugae might be v i a Adelges cooleyi, the Cooley Spruce g a l l a d e l g i d due t o the presence of s i t e s of i n i t i a t i o n of g a l l formation at the s i t e s corresponding t o f e e d i n g punctures formed by the a d e l g i d . S i m i l a r t o g a l l s produced by A. pseudotsugae, g a l l s produced by the E r w i n i a i s o l a t e s only developed when in o c u l a t e d using deep needle stabs. That the succulent g a l l t i s s u e was an a t t r a c t i v e food source f o r i n s e c t s , i n t h i s study, supports the hypothesis t h a t an i n s e c t v e c t o r i s i n v o l v e d . One f u r t h e r s i m i l a r i t y between the 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 was the l o c a t i o n of the g a l l s on the host p l a n t . The g a l l s of both g a l l diseases are found c l o s e t o the t i p s of twigs or branches of D o u g l a s - f i r t r e e s . The D o u g l a s - f i r g a l l s found i n B.C. measured between 0.5 and 2.0 cm i n diameter w h i l e A. pseudotsugae g a l l s were reported t o measure between 1 m i l l i m e t e r and s e v e r a l centimeters (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 the f i e l d a minimum of three months a f t e r i n o c u l a t i o n (Hansen and Smith, 1937). G a l l growth was l i m i t e d t o the a c t i v e growth p e r i o d of the host. Furthermore the pathogen could be present i n the host f o r n e a r l y a year before d e f i n i t e symptoms began to 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 the E r w i n i a i s o l a t e s a f t e r l e s s than 2 weeks i n young succulent t i s s u e . At the microscopic l e v e l , the 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 the diseases caused by the g a l l - f o r m i n g Erwinia and A. pseudotsugae. T h i n - s e c t i o n i n g of the g a l l t i s s u e s formed by the two types of g a l l - f o r m i n g E r w i n i a revealed t h a t both g a l l types were composed of c i r c u l a r groups of 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 one h a l f the s i z e of healthy c o r t i c a l c e l l s (R.J. Copeman, u n p u b l i s h e d ) . The stab i n o c u l a t i o n s penetrated through the s t e l e r e s u l t i n g i n unorganized v a s c u l a r elements extending i n t o the p i t h of the young stems. Both b a c t e r i a l s t r a i n s appeared to be l o c a l i z e d i n the i n t e r c e l l u l a r spaces i n the centers of these groups of d i v i d i n g c e l l s . These f i n d i n g s are s i m i l a r t o those i n the study by Hansen and Smith (1937). D o u g l a s - f i r was i n i t i a l l y thought t o be the only host of the E r w i n i a species as these 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 only from D o u g l a s - f i r g a l l s . A host range study r e v e a l e d t h a t the t y p i c a l and 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 could a l s o form small g a l l s on species w i t h i n the genera Abies, Larix, P i c e a and Pinus. The pathogen stud i e d by Hansen and Smith was 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 , which i n c l u d e d D o u g l a s - f i r , Pinus halepensis (Aleppo p i n e ) , P. lambertiana (sugar p i n e ) , P. radiata (Monterey pine) and Tsuga h e t e r o p h y l l a (coast hemlock). A. pseudotsugae s t r a i n 180 was 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 study. This f i n d i n g confirms the r e s u l t s of a recent study by De Cleene and De Ley (1981) . I t a l s o confirms the comment i n the e i g h t h e d i t i o n of Bergey's Manual (Buchanan and Gibbons, 1974) which s t a t e s t h a t the c u l t u r e s of A. pseudotsugae i n the American 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 observed by Hansen and Smith i n the 1930*s. The g a l l s induced by the Erwinia 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 smaller than those produced on D o u g l a s - f i r g a l l s i n nature and t h e r e f o r e may not be an accurate i n d i c a t i o n of v i r u l e n c e . The 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 pathogenic on some c o n i f e r species other than D o u g l a s - f i r , i n c l u d i n g Abies, Larix, P i c e a and Pinus spp. Some of the g a l l i n g symptoms could have been due t o a wound response, e s p e c i a l l y on Chamaecyparis, Tsuga and Thuja spp. from which e i t h e r low numbers or no b a c t e r i a of the t y p i c a l i s o l a t e s were recovered. In some cases, the i n o c u l a t e d b a c t e r i a were recovered from i n o c u l a t i o n s i t e s that, 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 s i n g l e 47 gene w i t h i n the pathogen can be re s p o n s i b l e f o r determining the v i r u l e n c e of the pathogen on a p a r t i c u l a r host thereby 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 or 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 the other c o n i f e r species t e s t e d were not very s u s c e p t i b l e t o the Erwinia spp., t h i s would a i d i n the ex p l a n a t i o n of the low incidence of t h i s disease. In a d d i t i o n t o d i f f e r i n g i n some respects from A. pseudotsugae, the two g a l l - f o r m i n g Erwinia i s o l a t e types d i f f e r e d from each other i n many respects i n c l u d i n g r e s u l t a n t g a l l morphology. T y p i c a l D o u g l a s - f i r g a l l s c o l l e c t e d i n t h i s study were rough and i r r e g u l a r i n shape w i t h d i s t i n c t i v e f i s s u r e - l i k e markings present on separate, define d outgrowths t h a t c o n s t i t u t e d the g a l l . 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 globose and smooth i n appearance but 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 . The g a l l s produced by A. pseudotsugae were g e n e r a l l y very smooth, l i k e the a t y p i c a l g a l l s , but had a s i n g l e d i s t i n c t i v e cross-shaped marking co v e r i n g the e n t i r e g a l l surface, not observed on any of the 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 other i n colony 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 host range. The c o l o n i e s of both Erwinia i s o l a t e s d i f f e r e d i n morphology from those of A. pseudotsugae s t r a i n 180. Unf o r t u n a t e l y , as mentioned before, the c u l t u r e s of A. pseudotsugae a v a i l a b l e from the N a t i o n a l C o l l e c t i o n of P l a n t Pathogenic B a c t e r i a (NCPPB; H e r t f o r d s h i r e , England) 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 i n the o r i g i n a l d e s c r i p t i o n . Thus, 48 not much could be deduced from such comparisons. With the computer-assisted, f a t t y a c i d a n a l y s i s system (Hewlett Packard) f o r i d e n t i f i c a t i o n of b a c t e r i a , an index of gr e a t e r than 0.500 on a s c a l e of 0.000 and 1.000 i s g e n e r a l l y r e q u i r e d before 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. An index of 0.500 corresponds t o approximately three standard d e v i a t i o n s away from the mean f a t t y a c i d p r o f i l e f o r the l i b r a r y e n t r y (match) i n the computer data base (M. Roy, personal communication). 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 gre a t e r than 0.100 between the f i r s t and second species* 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 the species 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 . F a t t y a c i d a n a l y s i s i s touted as being very 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 purposes, due t o the ge n e t i c s t a b i l i t y of the f a t t y a c i d s (Anonymous, 1985; C l a r k et al., 1988). A p o s i t i v e match, wi t h a s i m i l a r i t y index of 0.840, was made between the p r o f i l e of the a t y p i c a l i s o l a t e , A-0181, and t h a t of E r w i n i a herbicola subsp. h e r b i c o l a . The next c l o s e s t match was at a s i m i l a r i t y index of 0.545, producing a d i f f e r e n c e w e l l above the 0.100 req u i r e d f o r a p o s i t i v e match t o the f i r s t choice of E. herbicola subsp. h e r b i c o l a . The d i f f e r e n c e between the f i r s t choice of E r w i n i a salicis (Day 1924) Chester 1939 and second choice of Hafnia alvei M o l l e r 1954 was l e s s than 0.100 f o r each of the three t y p i c a l i s o l a t e s t e s t e d . However, the f a t t y a c i d p r o f i l e s of species i n the f a m i l y Enterobacteriaceae are h i g h l y s i m i l a r (M. Roy, personal communication) and ofte n 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 between the f i r s t and second choices may not reach 0.100. Hafnia s p e c i e s , u n l i k e 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 , can 49 grow at temperatures up t o 40-42°C and are not reported t o be a s s o c i a t e d w i t h p l a n t s (Krieg and H o l t , 1984). Therefore, the 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 salicis, which i s a known pathogen of Salix sp. (Krieg 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 the t y p i c a l i s o l a t e s , between the f i r s t choice of E. salicis and E. h e r b i c o l a subsp. herbicola (the choice f o r the a t y p i c a l i s o l a t e ) measured c l o s e t o or greater than the 0.100 requirement ( f o r two of the 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 species. Therefore on the b a s i s of f a t t y a c i d p r o f i l e s alone, at l e a s t one of the 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 the a t y p i c a l i s o l a t e . The i d e n t i f i c a t i o n as Erwinia i s supported by other o b s e r v a t i o n s . The g a l l - f o r m i n g Erwinia spp. were shown not t o produce opines i n g a l l e d t i s s u e s nor do they grow on media t h a t c o n t a i n s octopine or nopaline as the s o l e carbon and n i t r o g e n source (R.J. Copeman, unpublished). They a l s o do not grow on any of the common s e l e c t i v e media f o r Agrobacterium and they do not form g a l l s on tomato, raspberry and Kalanchoe daigremontiana, common herbaceous hosts of Agrobacterium tumefaciens, (Muehlchen, 1985). Because the 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 belonging i n the genus Erwinia, comparisons could be made w i t h E. h e r b i c o l a f.sp. gypsophilae, the pathogen of Gypsophila. However, the Erwinia i s o l a t e s were found i n t h i s study not t o form g a l l s on Gypsophila. Furthermore, 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 the 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 s t r a i n 2273. The two g a l l - f o r m i n g Erwinia i s o l a t e types shared t o a degree, 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 detected by immunodiffusion. When the whole t y p i c a l T-2789 antiserum was used, a l i n e of p a r t i a l i d e n t i t y between the t y p i c a l i s o l a t e s and the a t y p i c a l i s o l a t e was formed. This was not observed w i t h any of the other phytopathogenic b a c t e r i a t e s t e d . P u r i f i c a t i o n of the antiserum completely removed the cross r e a c t i o n w i t h the a t y p i c a l i s o l a t e . Whole a t y p i c a l A-0181 antiserum or the A-0181 IgG f r a c t i o n d i d not cross 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 the other phytopathogenic b a c t e r i a when t e s t e d by immunodiffusion or by i n d i r e c t ELISA. A weak r e a c t i o n was observed w i t h the t y p i c a l i s o l a t e T-2722 w i t h both a n t i s e r a . Many s i m i l a r i t i e s were found between the pathogens of the two D o u g l a s - f i r g a l l diseases. As biochemical i d e n t i f i c a t i o n 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 types, 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 could not be made. The i d e n t i t y of Agrobacterium pseudotsugae i s s t i l l i n question (De Cleene and De Ley, 1981) because the o r i g i n a l A. pseudotsugae c u l t u r e s have been l o s t and no g a l l s have been found at the 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. The C a l i f o r n i a bacterium, described 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 placed i n t o the 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 unequivocally i n the genus Erwinia, not i n e i t h e r the Agrobacterium or Pseudomonas genera, which c o n t a i n the most w i d e l y recognized p l a n t g a l l formers. From the observed c h a r a c t e r i s t i c s and the 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 the two g a l l - f o r m i n g i s o l a t e types are two d i f f e r e n t s p e c ies of E r w i n i a . 52 CHAPTER 2 THE MECHANISM OF GALL FORMATION BY ERWINIA SPP. ON DOUGLAS-FIR INTRODUCTION I n i t i a l experiments t o determine the mechanism of g a l l formation by A. tumefaciens focussed on the growth of host tumor t i s s u e s . The growth of healthy p l a n t t i s s u e s in vitro r e q u i r e s an exogenous supply of phytohormones (Parsons et a l . 1986). Crown g a l l t i s s u e s freed of the i n c i t i n g b a c t e r i a were found t o be capable of autonomous growth (Braun 1943). This observation suggested t h a t the pathogen induced the endogenous pro d u c t i o n of phytohormones i n c e r t a i n host c e l l s . To determine the time 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 occur i n the host p l a n t c e l l s , a simple heat treatment was used t o s e l e c t i v e l y k i l l the pathogen but not the host, at s p e c i f i c times a f t e r i n o c u l a t i o n (Braun, 194 3). One t o f i v e days a f t e r i n o c u l a t i o n of A. tumefaciens i n t o p e r i w i n k l e (Vinca rosea L . ) , a high temperature treatment of 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 then returned t o room temperature 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 those produced without the heat treatment, were observed i n those p l a n t s i n i t i a l l y exposed t o the high temperature 4 or more days a f t e r i n o c u l a t i o n (Braun 1943). C e l l u l a r a l t e r a t i o n , or transformation, t h e r e f o r e occurred w i t h i n 4 days of i n o c u l a t i o n a f t e r which time the bacterium's presence was no longer r e q u i r e d f o r g a l l 53 development. No evidence was found t o demonstrate t h a t t r a n s f o r m a t i o n of host p l a n t c e l l s a l s o occurred when i n o c u l a t e d w i t h P. syringae pv. savastanoi (Comai et al., 1982). The continued presence of the 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 has been shown t o produce both IAA and 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 of the phytohormone enzymes w i t h biochemical t e s t s or by assaying f o r the a c t u a l phytohormones w i t h immunoassays (Comai and Kosuge, 1980; Davis et al., 1985; Mertens et al., 1985). A. tumefaciens a l s o produces auxins and c y t o k i n i n s i n c u l t u r e but at a much lower l e v e l than P. syringae pv. savastanoi (Akiyoshi et al., 1987). The production of higher l e v e l s of phytohormones by P. syringae pv. savastanoi may be necessary f o r the continued development of the g a l l s as the host p l a n t c e l l s do not produce phytohormones at a l e v e l r e q u i r e d t o s u s t a i n such unusual growth ( A k i y o s h i et al., 1987). The o b j e c t i v e of these experiments was t o determine i f the continued presence of the 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; of a heat treatment experiment, s i m i l a r t o tha t used f o r the study of A. tumefaciens, was chosen f o r t h i s i n i t i a l study. 54 MATERIALS AND METHODS B a c t e r i a l Strains and Host 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 these experiments were the T-2789 and the A-0181 i s o l a t e s . B a c t e r i a were grown on CPG p l a t e s f o r 5-7 days at 21°C. I s o l a t e d c o l o n i e s were used f o r stab i n o c u l a t i o n s . One t o two year o l d nursery-grown D o u g l a s - f i r t r e e s were p l a n t e d i n l a r g e styrofoam cups (600 ml) i n standard s o i l mix w i t h 2400 cm3 peat and 200 cm3 Osmocote, a slow r e l e a s e f e r t i l i z e r (14N-6.0P-11.6K), per 0.16 m3 of s t e r i l i z e d s o i l . The s e e d l i n g s were p r e v i o u s l y stored at 4°C. Trees were used f o r the heat treatment experiments a minimum of 2 months a f t e r t r a n s p l a n t i n g t o allo w the 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 every two weeks at a r a t e of 0.06 g/L. Inoculations/Heat treatment: Four s e t s of 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 sets and l a b e l l e d . Four branches per t r e e were l a b e l l e d and stab i n o c u l a t e d 2-5 cm below the branch t i p w i t h s t e r i l e p i n s 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 . One b a c t e r i a l colony was i n o c u l a t e d per branch. A f i f t h s e t of t r e e s was stabbed w i t h s t e r i l e pins only t o serve as a c o n t r o l . A l l of the t r e e s were placed i n a growth chamber at 21°C. At i n t e r v a l s of 2, 4, and 7 days a f t e r i n o c u l a t i o n , one set of t r e e s was t r a n s f e r r e d t o a second growth chamber. This chamber was s e t at 35±2°C f o r the T-2789 experiments or 36±2°C f o r the A- 0181 experiments. The temperature f o r the second t r i a l s w i t h 55 the a t y p i c a l i s o l a t e was set 2°C higher than i n the f i r s t t r i a l . Each s e t of t r e e s was l e f t at the elevated temperature f o r a pe r i o d of 8 t o 18 days depending on the experiment. Both growth chambers had a 16 h photoperiod of 80 jaE m"2sec"1 provided by a mixture of c o o l white f l u o r e s c e n t and incandescent l i g h t s . A f t e r the heat treatment, the 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 the 21°C chamber or out i n t o the greenhouse f o r a f u r t h e r 15 t o 18 days f o r the e a r l y t r i a l s or f o r a f u r t h e r 3.5 to 5.5 months f o r the l a t e r t r i a l s . The t r e e s were maintained at 21°C f o r a p e r i o d of time a f t e r the heat treatment t o al l o w f o r growth a t the s i t e of i n o c u l a t i o n t h a t may have been hindered a t the higher temperatures and a l s o t o provide f o r f u r t h e r growth of the c o n t r o l s t o enhance the d i f f e r e n c e i n the treatment responses. The set of wound-only t r e e s and one set of in o c u l a t e d t r e e s were kept at 21°C f o r the e n t i r e experiment t o serve as c o n t r o l s . Growth Index: Diameters of the branches at the i n o c u l a t i o n s i t e s and j u s t below the 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 removal from the high temperature treatment and at the t e r m i n a t i o n of the experiment. Ratios of the two branch measurements were c a l c u l a t e d as an index of growth at the i n o c u l a t i o n s i t e . Direct Isolat ions/Cel l ELISA: The i n o c u l a t i o n s i t e s , three per t r e e , were photographed and then ground up i n 0.5 t o 1.0 ml of LB i n s t e r i l e dimple p l a t e s . The suspensions were l e f t at room temperature f o r 15-20 min t o permit the re l e a s e of the b a c t e r i a from the host 56 t i s s u e . 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 streaked onto CPG p l a t e s and incubated at 21°C f o r 7-10 days. For the 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 Germicidal G25T8 25 W) f o r 15 min. For each i n o c u l a t i o n s i t e , 25 jal of the suspension was added t o each of three w e l l s c o n t a i n i n g 175 _Ail of LB. A l i q u o t s (25 .Ail) of a l a t e l o g phase c u l t u r e were added t o one row of each m i c r o t i t e r p l a t e t o serve as a c o n t r o l on the c e l l ELISA. The samples were covered and allowed t o grow i n the w e l l s at 21°C f o r 90 t o 100 h. CPG p l a t e s were streaked 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 cubation p e r i o d t o t e s t f o r growth and/or contamination. A f t e r the inc 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 Chapter 1 was performed. Whole a n t i s e r a was used f o r the f i r s t t r i a l s of 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 the 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 of 10"5 and 10"3 f o r the 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 , and IgG f r a c t i o n d i l u t i o n s of 10~4 and 10"3 f o r the 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. Absorbance values (405 nm) were read a f t e r 1.5 and 12.5 h f o r the 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 . S t a t i s t i c a l Analysis: R e s u l t s of the heat treatment experiments were analyzed u s i n g the General Li n e a r Model, wi t h d i f f e r e n c e s between treatment means evaluated with Tukey's M u l t i p l e Range Test ( 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 Inc., Cary, NC, USA) . 57 RESULTS T-2789 i s o l a t e : None of the wound-only or heat-treated branches formed g a l l s i n e i t h e r t r i a l w i t h the T-2789 i s o l a t e (Tables 2.1 and 2.2). Even when the heat treatment was delayed 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 of the in o c u l a t e d c o n t r o l t r e e s kept at 21°C throughout ( F i g . 2.1). A minimum of f i v e t r e e s w i t h 3 branches per t r e e were t e s t e d per treatment. 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 w i t h the i n o c u l a t e d c o n t r o l t r e e s producing s i g n i f i c a n t l y higher values than any of the other treatments. In a d d i t i o n t o the s i g n i f i c a n t d i f f e r e n c e s between the c o n t r o l s and the heat treatments i n each of the t r i a l s , the d i f f e r e n c e s between the i n d i v i d u a l t r e e s were a l s o s t a t i s t i c a l l y s i g n i f i c a n t . R a t i o s f o r the wound-only sets and a l l the h e a t - t r e a t e d s e t s 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 each other. R a t i o 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 , of the second t r i a l , i n c r e a s e d 2 8% over the three months a f t e r the heat treatments (Table 2.2). R a t i o means f o r the wound-only and the heat- t r e a t e d s e t s increased only 0% and 8%, r e s p e c t i v e l y , over the same time 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 recovered from any of the wound-only or heat-treated t r e e s by d i r e c t i s o l a t i o n onto CPG 58 Table 2.1. G a l l formation by and recovery of the pathogen from he 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 s t a b - i n o c u l a t e d w i t h 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 indexed f o r the presence of 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 us i n g whole T-2789 antiserum Inoc- u l a t e d w i t h i s o l a t e Heat treatment Days at 21°C 35°C G a l l diameter/ stem diameter Days a f t e r i n o c u l a t i o n 24 d 34 d B a c t e r i a detected i n i n o c u l a t i o n s i t e D i r e c t Absorbance i s o l a t i o n 405nm - 24 0 1.3c1 1. 3b 0/10 0. 02c + 24 0 2. 5a 3 . 2a 20/20 0. 85a + 2 18 1.2c 1.3b 0/20 0.08bc + 4 18 1. 4bc 1.4b 0/20 0.08bc + 6 18 1.4b 1. 5b 0/20 0.10b means i n a column followed by the same l e t t e r are not 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) according t o Tukey's m u l t i p l e range t e s t . 59 Table 2.2. G a l l formation by and recovery of the pathogen from he 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 s t a b - i n o c u l a t e d w i t h 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 indexed f o r the presence of 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 the IgG f r a c t i o n of the T-2789 antiserum Inoc- u l a t e d w i t h i s o l a t e Heat treatment Days at 21°C 35°C G a l l diameter/ stem diameter Days a f t e r i n o c u l a t i o n 26 d 108 d B a c t e r i a detected i n i n o c u l a t i o n s i t e D i r e c t Absorbance i s o l a t i o n 405nm — 19 0 1.2b1 1. l b 0/16 0. 00c + 19 0 1.4a 2. 0a 16/16 0.95a + 2 12 1. l b 1.2b 0/16 0. 00c + 4 12 1.1b 1.3b 0/16 0. 01c + 7 12 1. l b 1.2b 0/16 0. 05b means i n a column followed by the same l e t t e r are not 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) according 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 high temperature treatment (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 w i t h 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) Inoculated c o n t r o l t r e e s kept at 21°C throughout the experiment. Inoculated t r e e s (B,C,D) subjected t o the 18-day heat treatment, 2, 4 and 7 days, r e s p e c t i v e l y , a f t e r i n o c u l a t i o n . 61 p l a t e s (Tables 2.1 and 2.2). Pure c u l t u r e s of the i n o c u l a t e d b a c t e r i a were recovered i n lar g e numbers from each of the 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 confirmed the d i r e c t i s o l a t i o n data t h a t l i v i n g b a c t e r i a were recovered only from the i n o c u l a t e d c o n t r o l t r e e s kept at 21°C throughout (Tables 2.1 and 2.2). R e s u l t s from the two experiments showed ELISA values f o r the c o n t r o l t r e e s t o be s i g n i f i c a n t l y greater than those f o r the wound-only or any of the heat-treated t r e e s . The mean ELISA value f o r p l a n t s r e c e i v i n g the 7-day heat treatment i n the f i r s t t r i a l averaged l e s s than 0.10 when the background wound-only value was sub t r a c t e d . A l l the wound-only and hea t - t r e a t e d t r e e s i n the second t r i a l produced ELISA values l e s s than the c u t o f f absorbance value of 0.10. None of the CPG p l a t e s streaked from the ELISA p l a t e w e l l s of heat-treated branches showed the presence of l i v e Erwinia T-2789 b a c t e r i a i n the m i c r o t i t e r p l a t e w e l l s . A-0181 Isolate: Although a high m o r t a l i t y r a t e was observed i n i n o c u l a t e d t r e e s exposed t o the 3 6±2°C heat treatment of both t r i a l s , no g a l l s were observed at i n o c u l a t i o n s i t e s amongst the s u r v i v o r s (Tables 2.3 and 2.4). A minimum of three t r e e s w i t h f o u r branches per t r e e were t e s t e d per treatment except f o r the 7-day treatment i n the second t r i a l where only two t r e e s s u r v i v e d . R a t i o s of i n o c u l a t i o n s i t e diameter over branch diameter f o r the 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 times g r e a t e r than those f o r a l l of the wound-only and the three h e a t - t r e a t e d s e t s . 62 Table 2.3. G a l l formation by and recovery of the pathogen from heat-treated (36°C) D o u g l a s - f i r s e e d l i n g s s t a b - i n o c u l a t e d w i t h the a t y p i c a l E r w i n i a 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 indexed f o r the presence of 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 using whole A-0181 antiserum Inoc- u l a t e d w i t h i s o l a t e Heat treatment Days at 21°C 36°C G a l l diameter/ stem diameter Days a f t e r i n o c u l a t i o n 17 d 29 d B a c t e r i a detected i n i n o c u l a t i o n s i t e D i r e c t Absorbance i s o l a t i o n 405nm 13 0 + 13 0 + 2 10 + 4 8 + 7 6 1.4b1 1.4b 1.6a 2.2a 1.1c 1.4b 1.2bc 1.4b 1.2bc 1.6b 0/5 0.04c 10/10 0.19a 2/5 0.21a 4/8 0.12b 7/7 0.13b means i n a column followed by the same l e t t e r are not 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) according t o Tukey's m u l t i p l e range t e s t . Table 2.4. G a l l formation by and recovery of the pathogen from heat-treated (38°C) D o u g l a s - f i r s e e d l i n g s s t a b - i n o c u l a t e d with the a t y p i c a l E r w i n i a 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 indexed f o r the presence of 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 using the IgG f r a c t i o n of the A-0181 antiserum Inoc- u l a t e d w i t h i s o l a t e Heat treatment G a l l diameter/ stem diameter B a c t e r i a detected i n i n o c u l a t i o n s i t e Days at 21°C 3 8°C Days a f t e r i n o c u l a t i o n 165 d D i r e c t Absorbance i s o l a t i o n 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 followed by the same l e t t e r are not 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) according 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 the wound-only sets and a l l the h e a t - t r e a t e d s e t s 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 each other at the end of both t r i a l s . S i m i l a r t o the experiments w i t h the 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 the 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 the d i f f e r e n c e s between the c o n t r o l s and the heat treatments, were s i g n i f i c a n t . In both 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 recovered 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 i s o l a t i o n s . In the 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 recovered from each of the heat-treated s e t s but none from the wound-only t r e e s . In the second t r i a l , d i r e c t i s o l a t i o n showed s u r v i v a l and recovery of c h a r a c t e r i s t i c b a c t e r i a from two of the 16 h e a t - t r e a t e d branches of the 2-day treatment. A l s o c h a r a c t e r i s t i c b a c t e r i a were recovered from one of the wound- only t r e e branches, presumably due t o experimental e r r o r . Immunodiffusional a n a l y s i s confirmed the i d e n t i t y of the recovered A-0181 i s o l a t e . The mean ELISA value 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 averaged above the c u t o f f absorbance value of 0.10, even when the background wound-only value was s u b t r a c t e d . Furthermore, the average value f o r the 2-day h e a t - t r e a t e d t r e e s was not s i g n i f i c a n t l y d i f f e r e n t from the average i n o c u l a t e d c o n t r o l value when analyzed w i t h Tukey's m u l t i p l e range t e s t (Table 2.3). Values f o r the other 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 those of the i n o c u l a t e d c o n t r o l s . Mean c e l l ELISA values f o r the i n o c u l a t e d c o n t r o l t r e e s , i n the 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 than f o r any of the other treatments (Table 2.4). The average 65 ELISA values f o r the wound-only and the three h e a t - t r e a t e d sets i n the second t r i a l were a l l l e s s than the c u t o f f value and hence were negative. P l a t e checks on the ELISA assays f o r both t r i a l s showed recovery of b a c t e r i a corresponding t o the d i r e c t i s o l a t i o n data. 66 D I S C U S S I O N These heat treatment experiments demonstrated t h a t f o r both the g a l l - f o r m i n g Erwinia T-2789 and A-0181 i s o l a t e s , the presence of l i v e b a c t e r i a was necessary 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 . G a l l s only formed on those t r e e s not subjected t o the heat treatment and hence only on those t r e e s w i t h l i v e pathogenic b a c t e r i a . No evidence was obtained suggesting t h a t transformation of p l a n t c e l l s had occurred. In order t o demonstrate t h a t a t r a n s f e r of genes t o the 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 t h a t the b a c t e r i a ' s presence was r e q u i r e d f o r g a l l formation, i t was necessary t o k i l l the i n o c u l a t e d b a c t e r i a . The T-2789 and A-0181 i s o l a t e s are able t o grow i n l i q u i d c u l t u r e up t o temperatures of 32°C and 34°C, r e s p e c t i v e l y . The combination of temperature and lengt h of time at t h a t temperature which the host t r e e s could withstand was determined e m p i r i c a l l y . P r e l i m i n a r y experiments i n v o l v i n g heat treatment t o k i l l the g a l l - f o r m i n g E r w i n i a spp. rev e a l e d t h a t the D o u g l a s - f i r seedlings would not r e l i a b l y s u r v i v e heat treatments of 35-38°C l a s t i n g more than about 10 days. Unfor t u n a t e l y each batch of t r e e s and each t r e e w i t h i n each experiment reacted d i f f e r e n t l y t o the heat treatment. The a n t i c i p a t e d high m o r t a l i t y r a t e was the reason f o r the use of 10-12 t r e e s per treatment and four i n o c u l a t i o n s i t e s per t r e e . D i f f e r e n c e s i n temperature and length of heat treatments between experiments were due t o the m o d i f i c a t i o n of these v a r i a b l e s according t o the h e a l t h and s u r v i v a l of the hosts as 67 w e l l as t o the d i f f e r e n t temperatures r e q u i r e d t o k i l l the two i s o l a t e types. A higher temperature was r e q u i r e d t o k i l l the 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 the r e s u l t a n t higher host m o r t a l i t y r a t e s . The t r e e s were incubated a f t e r the heat treatments i n the second t r i a l s t o increase the d i f f e r e n c e s observed between the treatments as w e l l as t o a l l o w f o r the i n c r e a s e i n number and hence ease of d e t e c t i o n of l i v e b a c t e r i a i n the heat 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 or by c e l l ELISA. The i n i t i a l i n c u bation periods at 21°C of 2, 4 and 7 days were decided upon t o encompass the time frame w i t h i n which 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 occur. I n i t i a l l y , the time r e q u i r e d f o r transformation of host p l a n t s by A. tumefaciens was experimentally determined t o occur w i t h i n 4 days (Braun 1943). Further research demonstrated t h a t t r a n s f o r m a t i o n begins approximately 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 Matthysse, 1986). G a l l i n g symptoms caused by the Erwinia spp. are v i s i b l e w i t h i n approximately 2 weeks of s t a b - i n o c u l a t i o n . Thus, 7 days was expected t o be adequate time t o allow t r a n s f o r m a t i o n by the E r w i n i a spp. t o take place i f such an event was i n v o l v e d i n g a l l f ormation. In an attempt t o narrow the time frame, i n the case of a t r a n s f e r of genes to the host p l a n t , i n t e r v a l s of 4 and 2 days were a l s o included. V i s u a l observations and diameter measurements of the 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 techniques aimed at the 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 diameter over branch diameter r e f l e c t e d the 68 l a c k of g a l l formation on the heat-treated and wound-only t r e e s . Some s w e l l i n g or growth occurred at the i n o c u l a t i o n s i t e of h e a t - t r e a t e d or 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 recovery technique. A c e l l ELISA was used as a second t e s t t o determine the presence of l i v e i n o c u l a t e d b a c t e r i a because of 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 of h e a t - t r e a t e d t r e e s demonstrated the b a c t e r i o c i d a l e f f e c t of the heat treatment, e s p e c i a l l y i n the T-2789 t r i a l s . L i v e b a c t e r i a were recovered by d i r e c t i s o l a t i o n from a few of the 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 the A-0181 i s o l a t e . This shows the s u r v i v a b i l i t y of the A-0181 i s o l a t e at temperatures up t o 39- 40°C. One colony 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 corresponding average r a t i o measurements and c e l l ELISA values f o r these few heat 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 the wound-only t r e e s . This would suggest t h a t the small numbers of recovered b a c t e r i a were not enough to produce a v i s i b l e g a l l . From a l l but a couple of i n o c u l a t e d c o n t r o l branches, which u n f o r t u n a t e l y d i e d d u r i n g the course of the experiments, pure c u l t u r e s of the i s o l a t e s were recovered, which corresponds t o every 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 assays are of 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 of the antigen i n e i t h e r low c o n c e n t r a t i o n or i n mixtures of antigens. The degree of s e n s i t i v i t y of the assay depends i n p a r t on the s p e c i f i c i t y and s e n s i t i v i t y of the antiserum used. From Chapter One, i t was demonstrated t h a t the 69 a n t i s e r a towards the two Erwinia i s o l a t e s , e s p e c i a l l y the 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, the d e t e c t i o n l e v e l s of the IgG f r a c t i o n s u sing an i n d i r e c t ELISA were not very 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 the l i m i t of d e t e c t i o n of t h e i r homologous antigens of the 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 . The lower l i m i t f o r the d e t e c t i o n of Erwinia carotovora ssp. carotovora u s i n g a double antibody sandwich ELISA was 10 5 t o 10 6 c e l l s per ml (Caron and Copeman, 1984) while the lower l i m i t of d e t e c t i o n f o r Pseudomonas phaseolicola using the same assay was 10 4 c e l l s per ml ( B a r z i c and T r i g a l e t , 1982) . D i r e c t comparisons t o these values cannot be made as the d e t e c t i o n assay used i n t h i s Chapter was a m o d i f i c a t i o n of the b a s i c double antibody sandwich method. An enrichment step, i n which the t e s t samples are incubated f o r a p e r i o d of time i n L u r i a Broth, was i n c o r p o r a t e d i n t o the assay t o increase the c e l l c o n c e n t r a t i o n i n the sample and hence the 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 the c e l l ELISAs were lower than those i n d i c a t e d i n the 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 of Chapter One. Large numbers of 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 recovered from the i n o c u l a t e d c o n t r o l t r e e s according t o the c e l l ELISA data f o r both the T-2789 and A-0181 i s o l a t e s . Background readings were recorded f o r most of the wound-only and h e a t - t r e a t e d t r e e s . In the e a r l i e r t r i a l s , some of the heat- 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 s u f f e r i n g from the e f f e c t s of the treatment, recorded ELISA values j u s t over the c u t o f f value of 0.10. These r e s u l t s , when combined w i t h the diameter r a t i o s and the d i r e c t i s o l a t i o n data, suggest t h a t the 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 very low con c e n t r a t i o n s of the in 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 heat-treated branches when t e s t e d by ELISA i n the l a t e r t r i a l s , using the p u r i f i e d a n t i s e r a , d i d not produce these low p o s i t i v e values. In each of the t r i a l s , absorbance readings f o r the in 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 eater than those of any of the other treatments i n accordance w i t h the v i s u a l 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 the mechanism of g a l l i n i t i a t i o n and development or t o determine the s u c c e s s f u l t r a n s f o r m a t i o n of p l a n t s by A. tumefaciens. The design of the heat treatment experiment was chosen i n p a r t because i t was a simple technique t h a t was used i n e a r l y experiments w i t h A. tumefaciens (Braun 1943). One of the more recent and d i r e c t approaches t o answer the question of g a l l formation 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 the phytohormone genes, w i t h i n the host p l a n t genome. This was not s e r i o u s l y considered due t o the extensive a n a l y s i s t h a t would be re q u i r e d t o reach a c o n c l u s i v e answer (Sederoff e t al. 1986). Another method, besides the heat treatment, t h a t could answer the question of the mechanism of g a l l formation i s the use of 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 of tumor t i s s u e s and/or d e t e c t i o n of phytohormones were not chosen as primary methods of determination due t o a n t i c i p a t e d problems. Sederoff et al. (1986) found t h a t A. tumefaciens i n f e c t e d pine c a l l u s d i d not 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 to the host p l a n t may- take p l a c e but the expression of the phytohormone genes, i n some cases, i s not s u f f i c i e n t t o allow f o r a u t o t r o p h i c growth in v i t r o . Recently however, Cl a r k et al. (1989) demonstrated, 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.sp. gypsophilae does not t r a n s f e r phytohormone genes t o the host 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 s u f f i c i e n t t o determine the primary producer of phytohormones i n the p l a n t - pathogen i n t e r a c t i o n . A. tumefaciens as w e l l as P. s y r i n g a e pv. savastanoi 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 k i y o s h i et a l . , 1987). Often such i n v e s t i g a t i o n s can lead t o ambiguous r e s u l t s i n r e l a t i o n t o the question of g a l l i n i t i a t i o n and development. To t h i s end, the heat treatments, modelled a f t e r the o r i g i n a l A. tumefaciens experiments, provided a simple yet c o n c l u s i v e answer to the r o l e of the g a l l - f o r m i n g bacterium i n the formation of the g a l l . In the e a r l y heat treatment experiments w i t h A. tumefaciens, g a l l s i n c i t e d w i t h v i r u l e n t b a c t e r i a continued t o develop although l i v e b a c t e r i a were no longer present, four or more days p o s t - i n o c u l a t i o n (Braun, 1943) . This suggested t h a t a t r a n s f o r m a t i o n of the host c e l l s had occurred t o warrant continued unregulated growth and d i v i s i o n . In the present experiments, the 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 both the E r w i n i a T-2789 and the A-0181 i s o l a t e f o r continued g a l l development t o take place. This would i n d i c a t e t h a t no t r a n s f e r of genes t o the host p l a n t genome occurs, at l e a s t not w i t h i n 7 days of i n o c u l a t i o n , as i s the case i n the Agrobacterium tumefaciens system. A mechanism of g a l l formation c l o s e r t o t h a t of the Pseudomonas syringae pv. savastanoi system i s l i k e l y t o be the case w i t h the g a l l - f o r m i n g E r w i n i a spp. Thus, the g a l l - f o r m i n g E r w i n i a spp. are not 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 improvement of 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 Plasmids i n both A. tumefaciens and P. syringae pv. savastanoi c a r r y g e n e t i c determinants t h a t code f o r the sy n t h e s i s of auxins and c y t o k i n i n s , which confer p a t h o g e n i c i t y . Thus, i t was of i n t e r e s t t o determine the plasmid content of the 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 l plasmids range i n s i z e between 1 kb and gr e a t e r than 3 00 kb (Trevors, 1985) . Larger s i z e d plasmids o f t e n are more d i f f i c u l t t o i s o l a t e due t o the shearing of the DNA d u r i n g the e x t r a c t i o n process. Furthermore, each plasmid has 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 c o n d i t i o n s . Plasmids present i n m u l t i p l e c opies, c h a r a c t e r i s t i c a l l y the smaller plasmids, are 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) plasmids are under s t r i n g e n t c o n t r o l and hence are i n a lower copy number (Broda, 1979). Plasmids are u s u a l l y found i n a double-stranded, 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, during the i s o l a t i o n process, one or both strands can be nicked 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 or a l i n e a r form. These d i f f e r e n t forms, along w i t h m u l t i m e r i c forms, can complicate the i n t e r p r e t a t i o n of plasmid p r o f i l e s . P r o f i l e s of plasmid content have been used i n b a c t e r i a l 74 s t r a i n i d e n t i f i c a t i o n (Lazo et 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 at l e a s t e i g h t plasmids t h a t range i n s i z e from 4.1 t o approximately 32 0 kb. A 30 kb plasmid, pEA28, i s found i n a l l s t r a i n s of E. amylovora. Conserved plasmids have a l s o been found i n Clavibacter michiganense subsp. sepedonicum, Curtobacterium flaccumfaciens pv. pointsettae and Rhodococcus f a s c i a n s ( C o p l i n , 1989). Plasmid DNA i s a l s o used f o r probes i n DNA:DNA h y b r i d i z a t i o n s f o r 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 the determination of homology between various DNA fragments ( G i l b e r t s o n et al., 1989) . Plasmid sequences are ofte n i n higher copy number than those on the chromosome producing a stronger h y b r i d i z a t i o n s i g n a l ( C o p l i n , 1989). Conserved plasmid sequences occur between the plasmid DNA of A. tumefaciens and P. s y r i n g a e pv. s a v a s t a n o i . The homology occurs between the genes encoding f o r enzymes i n v o l v e d i n auxin and c y t o k i n i n production (Powell and M o r r i s , 1986; F o l l i n et al., 1985). As i n t h i s case, DNA homology i s o f t e n r e l a t e d to 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 r e l a t i o n t o the g a l l - f o r m i n g E r w i n i a , conserved DNA sequences between the plasmid DNA of the two types could 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 of the o r i g i n a l methods t o determine the f u n c t i o n of plasmid genes i s t o cure the bacterium of the plasmid. Curing a bacterium of i t s plasmid(s) r e s u l t s i n the production of plasm i d - f r e e d e r i v a t i v e s of the wild-type bacterium. Phenotypes of the cured s t r a i n s are then compared w i t h those of the w i l d - type s t r a i n s . Some methods of c u r i n g i n c l u d e the use of a heat treatment such as used w i t h Agrobacterium tumefaciens (Watson et a l . , 1975), E r w i n i a herbicola (Chatterjee and G i b b i n s , 1971) and Xanthomonas campestris pv. v i g n i c o l a (Ulaganathan and Mahadevan, 1988). Chemical c u r i n g agents such as a c r i d i n e orange, sodium dodecyl s u l f a t e (SDS) and ethidium bromide have a l s o been used t o s u c c e s s f u l l y cure b a c t e r i a of one or more of t h e i r plasmids (Sonstein and Baldwin, 1972). A c r i d i n e orange was used f o r c u r i n g the pIAA plasmid 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 these experiments i n c l u d e d the determination of the plasmid content of the 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, experiments aimed at determining the presence of homology between the plasmid content of the two i s o l a t e types were c a r r i e d out. The f i n a l o b j e c t i v e of t h i s s e c t i o n was t o determine the r o l e , i f any, of the plasmid DNA of the g a l l - f o r m i n g Erwinia spp. i n the development of g a l l s . 76 MATERIALS AND METHODS Ba c t e r i a l Strains: The t y p i c a l i s o l a t e , T-2774, and the a t y p i c a l A-0181 i s o l a t e were the main i s o l a t e s used i n these experiments. The other b a c t e r i a l s t r a i n s used and t h e i r sources are as o u t l i n e d i n Table 1.1 i n Chapter 1. The chemicals used were from the Sigma Chemical Company unless otherwise noted. Total DNA I s o l a t i o n : 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 of the app r o p r i a t e broth and a g i t a t e d at 2 00 rpm at room temperature f o r 2-4 days, depending upon the b a c t e r i a l s p e c i e s . B a c t e r i a l c e l l s were harvested by c e n t r i f u g a t i o n at 2100 X g (maximum) f o r 10 min a t 4°C. P e l l e t s were resuspended i n 100 ;ul i c e c o l d Tris-EDTA-Glucose (Maniatis et al., 1982). A l y s i n g s o l u t i o n (200 ,Ail) c o n s i s t i n g of lysozyme (5 mg/ml), SDS (1%) and Pr o t e i n a s e K (2ug/ml) was added and ge n t l y mixed by t i l t i n g the tube. The c e l l suspensions were incubated at 60-65°C f o r 2.5- 3.0 h. The 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 phenol and chloroform. The DNA was p r e c i p i t a t e d w i t h ethanol 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 sheared by passage through 22 gauge needles seven t o e i g h t times. An RNase A treatment (20 jag/ml f o r 10 min at 37°C) was fo l l o w e d by another ethanol p r e c i p i t a t i o n step t o remove the di g e s t e d RNA and the RNase. 77 Plasmid I s o l a t i o n and P u r i f i c a t i o n : 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 attempted f o r the g a l l - f o r m i n g Erwinia i s o l a t e s : l y s i s by b o i l i n g (Holmes and Quigley, 1981 as o u t l i n e d i n M a n i a t i s et al., 1982), l y s i s by SDS (Godson and Vapnek, 1973 as o u t l i n e d i n M a n i a t i s et al., 1982) and l y s i s by a l k a l i (modified Birnboim and Doly, 1979 as o u t l i n e d i n M a n i a t i s et al., 1982; Kado and L i u , 1981; Hardy, 1985). The 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 et al. (1982) was adopted as the best and s i m p l e s t procedure to use. For both t y p i c a l s t r a i n s and A-0181, minipreps 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 of 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 to p u r i f y the plasmid DNA from the two E r w i n i a i s o l a t e s t o determine the method w i t h the h i g h e s t recovery of p u r i f i e d plasmid DNA. E l e c t r o e l u t i o n of the plasmid DNA i n t o d i a l y s i s bags, troughs 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 through Low G e l l i n g Temperature agarose (BioRad) and GeneClean (BioRad) were a l l t e s t e d . For the t y p i c a l i s o l a t e s , DNA obtained from minipreps was run through 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 the plasmid DNA were cut out. The plasmid bands were then 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 us 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 the A-0181 i s o l a t e , large s c a l e plasmid DNA e x t r a c t i o n s were f o l l o w e d by separation on a cesium c h l o r i d e (CsCl) g r a d i e n t . Ethidium bromide was added at a c o n c e n t r a t i o n of 0.5 mg/ml. Samples were c e n t r i f u g e d at 170,000 X g (maximum) f o r 65 78 h on a Type 65 f i x e d angle r o t o r (Beckman). Bands were viewed over 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 Products, Inc. San G a b r i e l , CA) and removed v i a an 18 gauge needle. Ethidium bromide was removed from the s o l u t i o n by e x t r a c t i o n w i t h equal volumes of 1-butanol. This procedure was repeated at l e a s t s i x times or u n t i l the aqueous phase no longer d i s p l a y e d a p i n k i s h c o l o r . The r e s u l t i n g sample was then 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 accomplished by i s o l a t i o n from Low G e l l i n g Temperature agarose or by using GeneClean. DNA con c e n t r a t i o n s were measured at 260 nm on a Hewlett Packard Spectrophotometer (the absorbance values m u l t i p l i e d by 50 t o o b t a i n jaq/ml) or by v i s u a l i z a t i o n on an agarose g e l i f the sample c o n c e n t r a t i o n was too 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 of 20 y U l w i t h up t o 1.0 jag of DNA and at l e a s t two times excess enzyme. Reactions were c a r r i e d out at 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 : 23.13, 9.42, 6.56, 4.36, 2.32, 2.03, 0.56 and 0.13 kb) was used as DNA s i z e markers. Samples were run 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 apparatus (Model GNA-100) i n 0.7% (0.9% f o r d i g e s t s ) agarose at 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 (Maniatis et a l . , 1982) w i t h 0.5 ug/ml ethidium bromide i n the g e l and i n the running 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 of the g e l s were taken using 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 S o u t h e r n B l o t s : DNA samples, digested or undigested, were t r a n s f e r r e d from agarose 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 procedure as o u t l i n e d f o r Nytran nylon membranes ( S c h l e i c h e r & S c h u e l l ) . A f t e r photographs were taken, the g e l s were g e n t l y shaken i n 2 volumes of 0.25M HCl f o r 8-10 min t o f a c i l i t a t e t r a n s f e r of the DNA through fragmentation of the DNA strands. Gentle shaking again accompanied two, 15-min in c u b a t i o n s i n 2 volumes of the b l o t t i n g s o l u t i o n (0.5M NaOH and 1.5M NaCl). The g e l was then placed on a wick made up of three l a y e r s of Whatman 3MM paper, saturated w i t h the b l o t t i n g s o l u t i o n . A piece of Zeta-Probe membrane, soaked f o r 5 min i n the b l o t t i n g s o l u t i o n and nicked on the top r i g h t corner f o r o r i e n t a t i o n , was placed on the g e l and smoothened out t o avoid bubbles underneath. Three l a y e r s of Whatman 3MM, sat u r a t e d w i t h the b l o t t i n g s o l u t i o n , were placed on the membrane, f o l l o w e d by s i x l a y e r s of dry f i l t e r paper. A stack of paper towels, measuring 15-20 cm i n depth, was placed on top. The wick was r e s t i n g on a piece of p l e x i g l a s s with the ends of the wick 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 . The e n t i r e apparatus was wrapped i n Saran Wrap t o avoid excessive evaporation. A piece of g l a s s and a small weight was placed on top of 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 then allowed t o t r a n s f e r t o the membrane v i a c a p i l l a r y a c t i o n f o r 16- 18 h. Once the DNA was t r a n s f e r r e d , the paper towels were taken o f f and then the remaining l a y e r s , down t o the wick, were c a r e f u l l y turned over. A medium b a l l p o i n t pen was used t o mark 80 the lanes of the g e l and t o date the Zeta-probe membrane. To ensure the t r a n s f e r process was complete, the g e l was soaked i n 0.5 ug/ml of ethidium bromide and viewed over a t r a n s i l l u m - i n a t o r . The membrane was soaked i n a small volume of 5X SSC ( s a l i n e sodium c i t r a t e ) (Maniatis et al., 1982) f o r 5 min, p l a c e d between 2 sheets of Whatman 3MM paper, wrapped l o o s e l y i n aluminum f o i l and baked at 60°C f o r 2 h. Before use i n h y b r i d i z a t i o n , b l o t s were stored at room temperature. Curing Protocols: A c t i v e l y growing c u l t u r e s of the t y p i c a l T-2774 or the 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 broth and t r e a t e d w i t h one of the f o l l o w i n g c u r i n g agents: sodium dodecyl s u l f a t e (SDS), ethidium bromide or a c r i d i n e orange at 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 incubated at e l e v a t e d temperatures (28°C f o r T-2774 and 31°C f o r A-0181) f o r 48 h. One or two successive t r a n s f e r s were made w i t h the 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 stored b r i e f l y at 4°C or -80°C before 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 cured 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 plasmid DNA from the a t y p i c a l A-0181 i s o l a t e and the p o t e n t i a l l y cured 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 plasmid DNA from the t y p i c a l T-2774 i s o l a t e . 81 Table 3.1. Concentrations of chemical c u r i n g agents 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 the g a l l - forming E r w i n i a i s o l a t e s T-2774 and A-0181 G a l l - f o r m i n g Sodium A c r i d i n e Ethidium Temp- Erwinia dodecyl orange bromide e r a t u r e i s o l a t e s u l f a t e (%) (yug/ml) (jug/ml) (°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 then the 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 the 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 placed on the agar surface f o r 3-5 min. I d e n t i f y i n g p a t t e r n s were made on the membranes and concomitantly on the p l a t e s w i t h an 18 gauge needle. The membranes were placed, colony s i d e up, on 3-5 l a y e r s of Whatman 3MM paper s a t u r a t e d w i t h 0.5 M NaOH f o r 5 min followed by a b r i e f b l o t t i n g on dry Whatman 3MM paper. This step was repeated once t o ensure l y s i s of the c e l l s . The membranes were then 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 dry on Whatman 3MM, the C/P L i f t membranes were exposed t o UV l i g h t (General E l e c t r i c Germicidal G25T8 25 W) f o r 5-10 min t o ensure covalent l i n k a g e of the DNA t o the nylon membrane. The membranes were st o r e d , i f necessary, at room temperature. 3 2P L a b e l l i n g : Plasmid DNA was l a b e l l e d using the BRL random primers DNA l a b e l l i n g system. An i n i t i a l attempt using n i c k t r a n s l a t i o n (Pharmacia) was not s u c c e s s f u l . The T-2774 probe was prepared from miniprep DNA t h a t had been p u r i f i e d through e l e c t r o e l u t i o n . The A-0181 probe was prepared from C s C l - p u r i f i e d DNA, f u r t h e r p u r i f i e d through GeneClean. Approximately 50 ng of the w i l d - type plasmid DNA was digested w i t h Pst 1 i n a maximum volume of 12 The p r o t o c o l as o u t l i n e d by BRL (Bethesda Research Labs), was fol l o w e d t o l a b e l the DNA (Feinberg and V o g e l s t e i n , 1984). 83 The l a b e l l e d DNA was e t h a n o l - p r e c i p i t a t e d and resuspended i n 300 yul H20. The counts per minute f o r t o t a l and i n c o r p o r a t e d 3 2P-dATP were measured. Twojul samples (1/150th of the t o t a l sample) were spotted onto Whatman GF/C g l a s s - f i b e r d i s c s . To measure t o t a l counts, i n c l u d i n g both incorporated and unincorporated l a b e l , the d i s c was simply placed i n a g l a s s s c i n t i l l a t i o n v i a l , 10 ml of Aquasol was added and counts measured on a Hewlett-Packard S c i n t i l l a t i o n Counter. To measure in c o r p o r a t e d 3 2P-dATP, the d i s c c o n t a i n i n g the sample was r i n s e d 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). A f t e r two, 15- ml r i n s e s w i t h c o l d 95% ethanol, the d i s c was placed i n a g l a s s s c i n t i l l a t i o n v i a l and 10 ml Aquasol added. Counts were measured on the s c i n t i l l a t i o n counter w i t h % i n c o r p o r a t i o n of 3 2P-dATP c a l c u l a t e d from the readings. Hybridization: The C/P L i f t and Zeta-probe membranes were sealed i n p l a s t i c sandwich boxes wi t h approximately 2 0 ml of h y b r i d i z a t i o n b u f f e r (ImM EDTA, 0.5mM NaHP04 (pH 7.2), 7% SDS). An i n c u b a t i o n p e r i o d , i n a shaking water bath set at 65°C, of 5 min f o r C/P L i f t membranes or 2 h f o r Zeta-Probe membranes, preceded h y b r i d i z a t i o n . 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 denaturation p e r i o d i n a b o i l i n g water bath. H y b r i d i z a t i o n was allowed t o proceed f o r 18-24 h at 65°C (shaking) according t o the p r o t o c o l o u t l i n e d f o r the BioRad C/P L i f t Membranes. A f t e r the h y b r i d i z a t i o n p e r i o d , the probe was c a r e f u l l y poured i n t o a 50 ml Falcon tube and stored at -2 0°C. H y b r i d i z a t i o n was followed by 4, 30-min washes at 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 of ImM EDTA, 4 0mM NaHP04 and 5% SDS, wh i l e the f i n a l two washes c o n s i s t e d of ImM EDTA, 4 0mM NaHP04 and 1% SDS. An i n i t i a l r i n s e w i t h the f i r s t washing s o l u t i o n was used t o remove a larg e percentage of unbound l a b e l . The C P / L i f t or Zeta-Probe membranes were wrapped i n Saran Wrap and put up on X-ray f i l m w i t h enhancing screens. The X-ray f i l m was exposed t o the h y b r i d i z e d membranes e i t h e r at room temperature or at -70°C f o r between 3 h and 3 days. 85 RESULTS Plasmid Content: For the 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 plasmid 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 observed (lanes 4-8 i n F i g . 3.1). O c c a s i o n a l l y a d i f f e r e n t form of the plasmid was seen m i g r a t i n g behind the chromosomal band (lane 9 i n F i g . 3.1). The p r o f i l e f o r the 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 of s e v e r a l bands (lane 3 i n F i g . 3.1). Fo l l o w i n g plasmid p u r i f i c a t i o n by CsCl g r a d i e n t c e n t r i f u g a t i o n , the p r o f i l e of the a t y p i c a l A-0181 i s o l a t e contained 4 t o 5 bands which appeared t o c o n s i s t of d i f f e r e n t forms of one or p o s s i b l y two plasmids ( F i g . 3.2). The lower band, D (lane 1), was f u r t h e r p u r i f i e d using LGT agarose, and rerun on an agarose g e l , showing a high degree of p u r i f i c a t i o n (lane 2). S i m i l a r l y the second band, C, was p u r i f i e d as shown i n lane 3. When the top 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 p a t t e r n d i s p l a y e d the four o r i g i n a l bands, suggesting t h a t the lower two bands are modified forms of the upper two bands. R e s t r i c t i o n d i g e s t s f o r plasmids from both the t y p i c a l and the A-0181 i s o l a t e s , are shown i n F i g . 3.3. Digests of the T- 2789 plasmid using Pst 1 and Hind I I I suggested a s i z e of approximately 50 kb (lanes 1 and 2). A l l d i g e s t s of the f a s t e s t moving A-0181 band, Band D, r e s u l t e d i n only p a r t i a l d i g e s t s (lane 4 and 5). When run against Hind I l l - d i g e s t e d lambda DNA, uncut 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 of 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% 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. 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. (1982). Lanes: (1) Erwinia carotovora subsp. carotovora; (2) E. herbicola subsp. herbicola s t r a i n 2273; (3) a t y p i c a l i s o l a t e A-0181; (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-2739; (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 s t a n d a r d s . 87 F i g . 3.2. P l a s m i d DNA p r o f i l e of 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. 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 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 . Lanes: (1) Bands A, B, C, D o f A-0181; (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 Temperature (LGT) agarose 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 agarose 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 at y p i c a l gall-forming Erwinia i s o l a t e s digested with r e s t r i c t i o n endonucleases, fractionated on a 0.9% agarose gel and stained with ethidium bromide. Plasmid DNA was isolated from t y p i c a l i s o l a t e s by a small-scale a l k a l i n e l y s i s procedure (Maniatis et al., 1982) and from the a t y p i c a l i s o l a t e by a large-scale a l k a l i n e l y s i s procedure (Maniatis et a l . , 1982). The at y p i c a l plasmid DNA was further p u r i f i e d through cesium chloride centrifugation and LGT agarose. Lanes: (1) Hindlll-digested t y p i c a l i s o l a t e T-2722; (2) Pst 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) Hindlll-digested Band D from a t y p i c a l i s o l a t e A-0181 (Fig. 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 Specificity/Homology: 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 plasmid probes from g a l l - f o r m i n g Ei-winia T-2774 and A-0181 are shown i n Figures 3.4, 3.5, 3.6 and 3.7. Lane 6 i n Figu r e 3.4 shows stro n g h y b r i d i z a t i o n between the t y p i c a l T-2774 plasmid probe (from a s m a l l - s c a l e plasmid DNA preparation) and the t o t a l DNA p r e p a r a t i o n of t y p i c a l i s o l a t e T-2774. H y b r i d i z a t i o n occurred at a lower degree between the t y p i c a l T-2774 probe and the A-0181 DNA preparations (lanes 5, 8, 9 and 10). Weak h y b r i d i z a t i o n occurred between the t y p i c a l probe and the two Erwinia spp. (E. carotovora and E. herbicola subsp. herbicola s t r a i n 2273) (lanes 3 and 4). No homology was detected between the t y p i c a l T-2774 probe and the E s c h e r i c h i a coli or Agrobacterium tumefaciens DNA preparations (lanes 1 and 2). When t e s t e d against minipreps of other t y p i c a l i s o l a t e s and the a t y p i c a l i s o l a t e A-0181, the T-2774 probe h y b r i d i z e d t o each band of each p r o f i l e of 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 ( F i g . 3.5). No homology was detected between the t y p i c a l T-2774 probe and the DNA preparations of Erwinia carotovora subsp. carotovora, E. h e r b i c o l a subsp. herbicola s t r a i n 2273 and E s c h e r i c h i a coli s t r a i n JM101 (lanes 1, 2 and 10). When the C s C l - p u r i f i e d a t y p i c a l A-0181 plasmid probe was used on s i m i l a r b l o t s , the h y b r i d i z a t i o n only occurred w i t h the homologous samples (lanes 5, 8, 9 and 12 i n F i g . 3.6 and lanes 7 and 13 i n F i g . 3.7). The A-0181 plasmid probe h y b r i d i z e d t o each plasmid band of A-0181. No h y b r i d i z a t i o n occurred 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 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 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, A u t o r a d i o g r a m o f South 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 and 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 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 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) 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 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 BX-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 i s o l a t e A-0181; (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 and (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. 9 1 1 2 3 4 5 6 7 8 9 l O l l F i g . 3.5. Gel 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 of plasmid DNA of various phytopathogenic b a c t e r i a probed w i t h plasmid DNA of 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 using the 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 et al. (1982). A, 0.7% agarose g e l s t a i n e d w i t h ethidium bromide. B, Autoradiogram of Southern b l o t of DNA t r a n s f e r r e d t o Zeta-Probe membrane probed w i t h plasmid DNA of 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 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 ) . 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 DNA/Haelll-digested 5X-174 DNA markers. 92 F i g . 3.6. Gel 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 of t o t a l genomic and plasmid DNA of var i o u s phytopathogenic b a c t e r i a probed w i t h plasmid DNA of the 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 ethidium bromide. B, Autoradiogram of Southern b l o t of DNA t r a n s f e r r e d to Zeta-Probe membrane and probed with plasmid DNA of 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 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). Lanes: (1) Pst 1-digested t o t a l DNA of Escherichia coli JM101; (2) Pst 1-digested t o t a l DNA of Agrobacterium tumefaciens s t r a i n B - l ; (3) Pst 1-digested t o t a l DNA of Erwinia carotovora subsp. carotovora; (4) Pst 1-digested t o t a l DNA of E. herbicola subsp. herbicola s t r a i n 2273; (5) Pst 1-digested t o t a l DNA of a t y p i c a l Erwinia i s o l a t e A-0181; (6) Pst 1-digested t o t a l DNA of 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 DNA/Haelll-digested 5X-174 DNA; (8) Pst 1-digested plasmid Band D (F i g . 3.2) of a t y p i c a l Erwinia i s o l a t e A-0181; (9) uncut plasmid Band D of a t y p i c a l Erwinia i s o l a t e A-0181; (10) Pst 1-digested plasmid DNA of t y p i c a l Erwinia i s o l a t e T-2774; (11) uncut plasmid DNA of t y p i c a l Erwinia i s o l a t e T-2774 and (12) Pst 1-digested plasmid Band D of 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. Gel 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 of plasmid DNA of various phytopathogenic b a c t e r i a probed w i t h plasmid DNA of the 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 . Plasmid DNA was ext r a c t e d using the modified 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 ethidium bromide. B, Auto- radiogram of Southern b l o t of DNA t r a n s f e r r e d t o Zeta-Probe membrane and probed w i t h plasmid DNA of 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 through cesium c h l o r i d e gradient c e n t r i f u g a t i o n and GeneClean). Lanes: (1) Pseudomonas syringae pv. syringae (cherry 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 s t r a i n 2273; (6) H i n d l l l - d i g e s t e d lambda DNA/Haelll-digested JX-174 DNA markers; (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 i s o l a t e 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 plasmid probe and the t y p i c a l i s o l a t e DNA pr e p a r a t i o n s (lanes 6, 10 and 11 i n F i g . 3.6 and lanes 8-12 and 14 i n F i g . 3.7). Curing/Colony Hybridization: H y b r i d i z a t i o n occurred between c o l o n i e s of the t y p i c a l i s o l a t e s and the T-2774 probe and t o a l e s s e r extent between the c o l o n i e s of the a t y p i c a l i s o l a t e and the T-2774 DNA probe. No h y b r i d i z a t i o n occurred between E. carotovora subsp. c a r o t o v o r a and the T-2774 probe ( F i g . 3.8). H y b r i d i z a t i o n occurred w i t h each p o t e n t i a l l y cured colony t e s t e d of both the t y p i c a l and a t y p i c a l i s o l a t e s , i n d i c a t i n g t h a t the plasmids had not been cured from e i t h e r i s o l a t e type ( F i g . 3.9). However, because the 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 res p o n s i b l e f o r the h y b r i d i z a t i o n between the t y p i c a l DNA probe and the t y p i c a l c o l o n i e s . At l e a s t 100 c o l o n i e s per treatment per i s o l a t e were t e s t e d . 95 F i g . 3.8. Autoradiogram of colony h y b r i d i z a t i o n of plasmid DNA probe of 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 the 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 . Colonies of (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 i s o l a t e 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, ly s e d and probed w i t h random- primed dAT 3 2P-label l e d plasmid DNA. 96 • • • • • t • • <<ift • m * • • • -*> • • • * # • • F i g . 3.9. Autoradiogram of colony hybridization of dAT32P- la b e l l e d plasmid DNA of atypical gall-forming Erwinia i s o l a t e A-0181 with p o t e n t i a l l y cured colonies of Erwinia i s o l a t e A-0181. The DNA for the probe was p u r i f i e d through cesium chloride gradient centrifugation and GeneClean. Each colony tested hybridized with the plasmid probe. 97 DISCUSSION Both the 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 the 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 at l e a s t one plasmid. The modified 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 et al. (1982) was adopted as a r a p i d and simple procedure t o use f o r r o u t i n e plasmid i s o l a t i o n . 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 the plasmid p r o f i l e of the A-0181 i s o l a t e c ontains up t o 5 bands which appear to c o n s i s t of the d i f f e r e n t forms of one or p o s s i b l y two plasmids. Large s c a l e plasmid 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 the a t y p i c a l A-0181 i s o l a t e but not w i t h the t y p i c a l T-2 789 i s o l a t e . Problems were encountered when attempting t o i s o l a t e the plasmid DNA from the t y p i c a l i s o l a t e s p o s s i b l y due t o a low copy number and a l a r g e r plasmid s i z e . Furthermore, the T-2789 plasmid u s u a l l y ran w i t h the chromosomal band and r a r e l y i n a d i f f e r e n t form behind the chromosomal DNA hampering attempts t o i s o l a t e and p u r i f y the plasmid DNA. Assessments i n c l u d i n g r e s t r i c t i o n analyses were used t o determine the s i z e of the Erwinia plasmids. The p u t a t i v e l a r g e s i z e of the plasmid of the t y p i c a l Erwinia i s o l a t e s , and the d i f f i c u l t y of p u r i f i c a t i o n of the plasmid, r e s u l t e d i n d i f f i c u l t i e s of p r e c i s e e s t i m a t i o n of s i z e . In the a t y p i c a l i s o l a t e , the complexity of the plasmid content i n t e r f e r e d w i t h d e f i n i n g the s i z e of the p l a s m i d ( s ) . D i f f i c u l t i e s c o uld a l s o have a r i s e n from an i n s u f f i c i e n t amount of DNA f o l l o w i n g g e l p u r i f i c a t i o n and the p o s s i b i l i t y of some contaminants i n h i b i t i n g the r e a c t i o n s . Thus, r e s t r i c t i o n d i g e s t s of the plasmids of both the t y p i c a l and the A-0181 i s o l a t e s d i d not y i e l d 98 c o n c l u s i v e data regarding the s i z e of the plasmids. Both the t y p i c a l plasmid DNA probe (not p u r i f i e d through CsCl g r a d i e n t c e n t r i f u g a t i o n ) and the 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 the g a l l - f o r m i n g Ei-winia i s o l a t e s . No h y b r i d i z a t i o n occurred between e i t h e r probe and t o t a l genomic DNA preparations of Agrobacterium tumefaciens and E s c h e r i c h i a coli. 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 the T-2774 t o t a l DNA p r e p a r a t i o n and t o a l e s s e r extent t o the a t y p i c a l A-0181 DNA p r e p a r a t i o n . These ext 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 the n o n - p u r i f i e d t y p i c a l plasmid probe may contain some h i g h l y r e p e t i t i v e sequences from chromosomal DNA. The presence of chromosomal DNA i n the n o n - p u r i f i e d t y p i c a l T-2774 plasmid DNA probe i s a l s o i n d i c a t e d by the weak h y b r i d i z a t i o n between the n o n - p u r i f i e d t y p i c a l T-2774 probe and the 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 plasmid sequences w i t h i n the va r i o u s 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 when u s i n g DNA:DNA h y b r i d i z a t i o n . 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 plasmid band from the 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 the t y p i c a l T-2774 DNA probe, suggesting 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 of T-2774 occur i n the a t y p i c a l plasmids. The Cs C l - p u r i f i e d plasmid probe from the a t y p i c a l A-0181 i s o l a t e d i d not h y b r i d i z e w i t h any of the 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 the 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 , chromosomally-derived sequences were 99 present i n the A-0181 plasmids, the C s C l - p u r i f i e d plasmid probe would not n e c e s s a r i l y detect the chromosomal analogues i n the t o t a l DNA b l o t of the t y p i c a l i s o l a t e s because of the low co n c e n t r a t i o n of randomly d i s t r i b u t e d sequences. Thus the sequences i n the t o t a l DNA b l o t would be i n a very low copy number as compared t o the plasmid 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, thus 'spread' out over the b l o t . However, i f the r e p e t i t i v e sequences were i n c l u d e d i n the probe they could p i c k up common sequences i n d i s c r e t e plasmid bands, as occurred w i t h the A-0181 probe and i t s homologous b l o t s . These r e s u l t s may i n d i c a t e t h a t there are chromosomal-like r e p e t i t i v e sequences i n the a t y p i c a l g a l l - forming E r w i n i a plasmids, maybe r e s u l t i n g from i n t e g r a t i o n of the plasmids i n t o the chromosome and subsequent e x c i s i o n of some of the chromosomal DNA wi t h the plasmid DNA (Hardman and Gowland, 1985b). Curing of the T i plasmid from A. tumefaciens was o r i g i n a l l y achieved through the use of a heat treatment (Watson e t al., 1975). Curing of the pIAA plasmid from P. syringae pv. savastanoi was achieved using an a c r i d i n e orange treatment (Comai et a l . , 1982). For both of these pathogens, the plasmid- f r e e s t r a i n s were not able t o induce g a l l i n g on the host 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 the p a t h o g e n i c i t y of the pathogen were lo c a t e d on the plasmids. Plasmid-free s t r a i n s were not however produced from 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 u s i n g the c u r i n g p r o t o c o l s . Therefore, no c o r r e l a t i o n between plasmid presence and pa t h o g e n i c i t y could be determined f o r the a t y p i c a l Erwinia i s o l a t e s . 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 of plasmids v i a c u r i n g p r o t o c o l s . Some explanations f o r t h i s d i f f i c u l t y i n c l u d e the f a c t t h a t not a l l plasmids, e s p e c i a l l y l a r g e plasmids, are s u s c e p t i b l e t o the c u r i n g agents (Hardman and Gowland, 1985a). Large plasmids share 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 the chromosome thus a v o i d i n g the e f f e c t s of the c u r i n g agents (Broda, 1979). Other methods t h a t could have provided i n s i g h t i n t o the r o l e ( s ) or f u n c t i o n ( s ) of the g a l l - f o r m i n g E r w i n i a plasmids i n g a l l formation i n c l u d e 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 w i t h or without a helper plasmid (Hamamoto and Murooka, 1987). Escherichia coli or other E r w i n i a spp., such as E. carotovora could serve as r e c i p i e n t s i n the case of g a l l - f o r m i n g Erwinia i s o l a t e s . This 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 or n u t r i t i o n a l markers be used t o screen p o t e n t i a l r e c i p i e n t s . Another method t o determine i f the plasmid genes are i n v o l v e d i n p a t h o g e n i c i t y i n v o l v e s the d i r e c t c l o n i n g of p o r t i o n s of the plasmids ( M a h i l l o n et al., 1988) again i n t o s u i t a b l e hosts such as E. coli or E r w i n i a carotovora. This method would r e q u i r e the express i o n of the E r w i n i a genes w i t h i n the new host organism and a method of determining the expression of v i r u l e n c e genes. The use of transposon mutagenesis to produce mutant d e r i v a t i v e s of the o r i g i n a l pathogen ( M i l l s , 1985; Salch and Shaw, 1988) was considered but not adopted due t o the complex nature of such a study, i e . i n determining the s i t e of mutation and r e l a t i n g i t to a change i n pa t h o g e n i c i t y . The experimental approach of c u r i n g was chosen to r e l a t e plasmid DNA t o p a t h o g e n i c i t y because resources f o r such experiments 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 screening procedures were not found i n 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 . Had cured s t r a i n s been obtained, they 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 the phenotype had been a l t e r e d . Because they were not, the r o l e of the plasmid DNA, i n these i s o l a t e s , i n pathogenesis remains unresolved. 102 GENERAL DISCUSSION The continued 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 from B.C. D o u g l a s - f i r g a l l s , was r e q u i r e d f o r g a l l formation t o occur. This would i n d i c a t e t h a t an interkingdom t r a n s f e r of genes t o the p l a n t host does not occur as i n the 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 to emulate the system of P. syringae pv. savastanoi. A presumptive next step would be to conclude t h a t the g a l l - forming E r w i n i a i s o l a t e s produce and secrete both auxins and c y t o k i n i n s , again s i m i l a r t o P. syringae pv. s a v a s t a n o i . In the P. s y r i n g a e pv. savastanoi system, the host p l a n t c e l l s surrounding the invading bacterium are a f f e c t e d by the exogenous supply of hormones and respond w i t h c e l l d i v i s i o n and c e l l e l o n g a t i o n (Comai et al., 1982). A continued supply of phytohormones from the pathogen induces the unregulated 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 the 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 those produced by A. pseudotsugae, which i n t u r n resembled those produced by P. syringae pv. savastanoi, the presumption t h a t they a l s o produce and secrete phytohormones l i k e P. s y r i n g a e pv. savastanoi i s not without b a s i s . The r a t i o of excess c y t o k i n i n s t o auxins determines the form of the p l a n t g a l l . A high c y t o k i n i n t o auxin r a t i o r e s u l t s i n the development of shoots from the tumor t i s s u e , w h i l e a low c y t o k i n i n t o auxin r a t i o r e s u l t s i n the development of r o o t s t r u c t u r e s . An intermediate r a t i o of the two phytohormones 103 r e s u l t s i n an unorganized tumor. Most of the g a l l s produced by the E r w i n i a spp. f i t i n t o the unorganized category. However, a few of the g a l l s c o n s i s t e d of d i s t i n c t outgrowths resembling the beginnings of teratomatous growths. V a r i a t i o n s i n the c y t o k i n i n t o auxin r a t i o produced by the invading pathogen i t s e l f c o u ld e x p l a i n t h i s phenomenon. The f u n c t i o n of the plasmid genes or simply t h e i r involvement i n pa t h o g e n i c i t y could not be determined. I t was hypothesized t h a t plasmid genes could encode f o r phytohormone enzymes, s i n c e a common denominator of phytohormone production i s l i k e l y f o r a l l of the phytopathogenic g a l l formers. S i m i l a r i t i e s i n regions of DNA involved i n p a t h o g e n i c i t y e x i s t between the two oncogenic phytopathogens, A. tumefaciens and P. s y r i n g a e pv. savastanoi, and could a l s o e x i s t w i t h the g a l l - forming Erwinia herbicola f.sp. gypsophilae and the D o u g l a s - f i r g a l l - f o r m i n g E r w i n i a spp. Therefore, an elegant method t o determine the r o l e of the Erwinia plasmids i n p a t h o g e n i c i t y would be t o probe the plasmids d i r e c t l y w i t h the phytohormone genes of e i t h e r A. tumefaciens or P. syringae pv. savastanoi. This o p t i o n t o d i r e c t l y determine the f u n c t i o n of the plasmid 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 study. 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 r e s p e c t s from A. tumefaciens, Pseudomonas spp. and Erwinia h e r b i c o l a f.sp. gypsophilae, the three most common g a l l - f o r m i n g b a c t e r i a l phytopathogens, as w e l l as from A. pseudotsugae strain 180. The two i s o l a t e s a l s o d i f f e r e d from each other i n a number of c h a r a c t e r i s t i c s , although both i s o l a t e types were c l a s s i f i e d i n the 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 f i n d i n g of two d i s t i n c t i s o l a t e types of the 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 should be conducted on the two i s o l a t e types throughout t h i s t h e s i s . The d i s c o v e r y of two i s o l a t e types 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 of the two i s o l a t e types although i d e n t i f i c a t i o n of the pathogen was not an o b j e c t i v e of t h i s study. The v a r i a t i o n i n c h a r a c t e r i s t i c s of the two i s o l a t e types lead t o s p e c u l a t i o n on the c l a s s i f i c a t i o n of the two i s o l a t e types 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 s l i g h t l y i n colony morphology (convex versus concave s u r f a c e s ) , the upper temperatures l i m i t i n g growth (the a t y p i c a l i s o l a t e was able t o grow a t a higher temperature) and t h e i r r e s i s t a n c e s t o a n t i b i o t i c s (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 wider range 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 reached a s i z e of 1 mm i n diameter on CPG at 2 days and t y p i c a l c o l o n i e s reached a s i m i l a r s i z e at 4 days). The outer appearance of the g a l l s produced by the two Erwinia i s o l a t e types d i f f e r e d s l i g h t l y although the inner c e l l o r g a n i z a t i o n was s i m i l a r (R.J. Copeman, u n p u b l i s h e d ) . Plasmid p r o f i l e s of the two E r w i n i a i s o l a t e types (Chapter 3) suggested t h a t each type contains at l e a s t one plasmid, w i t h the 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 i s o l a t e . The plasmid probe of the 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 the plasmid bands of the a t y p i c a l i s o l a t e but the reverse was not observed i n d i c a t i n g no homology o c c u r r i n g between the plasmid DNA of the two i s o l a t e types. Plasmid p r o f i l e s sometimes are used i n species i d e n t i f i c a t i o n but v a r i a t i o n s occur w i t h i n many species thus making 105 determinations of r e l a t i o n s h i p s d i f f i c u l t v i a t h i s c h a r a c t e r 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 the two i s o l a t e types could be demonstrated by immunodiffusion when usi n g the whole t y p i c a l T-2789 antiserum but not when the p u r i f i e d t y p i c a l T- 2789 antiserum was used. However, as wit h plasmid p r o f i l e s , s e r o l o g i c a l r e l a t i o n s h i p s should not be used alone i n d e s c r i b i n g b a c t e r i a l s t r a i n s . Cross r e a c t i o n s have been observed f o r E r w i n i a spp. w i t h other Enterobacteriaceae when u s i n g p o l y c l o n a l a n t i s e r a 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 serogroups (De Boer, 1987). 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 species w i t h respect t o s e r o l o g i c a l t r a i t s (De Boer et a l . , 1979) which might e x p l a i n the lack of i d e n t i t y between the two i s o l a t e types when u s i n g the a t y p i c a l a n t i s e r a . The host ranges of the 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 E r w i n i a i s o l a t e s were not i d e n t i c a l . The a t y p i c a l i s o l a t e formed g a l l i n g symptoms on more c o n i f e r species than the t y p i c a l i s o l a t e s . I t i s not l i k e l y t h a t the two i s o l a t e types are d i f f e r e n t pathovars of the same species because the i n f r a s u b s p e c i f i c 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 respect t o biochemical 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 but d i f f e r e n t w i t h respect t o p a t h o g e n i c i t y on one or more p l a n t hosts (Krieg and Ho l t , 1984). F a t t y 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 the two E r w i n i a i s o l a t e types. I d e n t i f i c a t i o n of the i s o l a t e s achieved by f a t t y a c i d a n a l y s i s i n d i c a t e d t h a t the two i s o l a t e types were i n f a c t two d i f f e r e n t species. A p o s i t i v e match was made f o r the a t y p i c a l i s o l a t e , while the 'goodness of f i t ' f o r two of the 1 0 6 three t y p i c a l i s o l a t e s analyzed was not indisputable. However, from t h e i r observed and experimentally determined c h a r a c t e r i s t i c s , the two gall-forming i s o l a t e types appear to be two d i f f e r e n t species of Erwinia. Further t e s t i n g should be done to c l e a r l y define the relationship between the two g a l l - forming Erwinia i s o l a t e s . The gall-forming b a c t e r i a l pathogens i s o l a t e d from Douglas- f i r trees i n B.C. do not appear at a high frequency, although as stated before, a survey was not conducted on t h i s aspect of the organism. Furthermore, l i t t l e damage was i n f l i c t e d on the host plant by the gall-forming Erwinia i s o l a t e s . There have been several reports of b a c t e r i a l g a l l s on conifers (R.S. Hunt, personal communication) but i t i s not known i f the i n c i t a n t was i s o l a t e d or the id e n t i t y determined. The host range of the gall-forming Erwinias i s very limited, with Pseudotsuga and some Abies, Larix, Picea and Pinus spp. constituting the majority of susceptible hosts. The potential for spread to surrounding forested areas i s therefore very small, although the presence of an insect vector i s unknown. Due to the infrequently observed natural damage and the li m i t e d host range of the gall-forming erwinias, these pathogens appear to be limited threats to the forest industry i n terms of decreased timber quality. 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