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Etiology and epidemiology of bacterial blight of red raspberry in British Columbia Sinnott, Nancy Marie 1979

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ETIOLOGY AND EPIDEMIOLOGY OF BACTERIAL BLIGHT OF RED RASPBERRY IN BRITISH COLUMBIA by NANCY MARIE SINNOTT B.Sc. St. Edward's University, 1975 A THESIS SUBMITTED IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF MASTER OF SCIENCE in THE FACULTY OF GRADUATE STUDIES Department of Plant Science We accept this thesis as conforming to the required standard THE UNIVERSITY OF A p r i l , (c) Nancy Marie BRITISH COLUMBIA 1979 Sinnott, 1979 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 representatives. It is understood that copying or publication of this thesis for financial gain shall not be allowed without my written permission. Department O f Plant Sciences The University of British Columbia 2075 Wesbrook Place Vancouver, Canada V 6 T 1W5 D a t e Apr i l , 6 , 1979 A b s t r a c t Pseudomonas syringae van H a l l was recovered from 31 of 32 samples of red raspberry t i s s u e showing t y p i c a l symptoms of b a c t e r i a l b l i g h t . Of the 99 i s o l a t e s recovered, 85 were p h y s i o l o g i c a l l y - t y p i c a l y P . syringae i s o l a t e s , three d i d not produce the fl u o r e s c e n t pigment, s i x d i d not u t i l i z e l a c t a t e and f i v e d i d not produce t o x i n as determined by the Geotrichum candidum bioassay. When a suspension of 10^ CFU/ml was sprayed on the leaves of 6-week-old raspberry p l a n t s , 42 of 48 i s o l a t e s caused necrosis w i t h i n 4 days. No other bacterium recovered from the diseased raspberry t i s s u e was pathogenic to raspberry i n greenhouse t e s t s . P. syringae i s o l a t e s remained v i a b l e and re t a i n e d t h e i r toxin-producing a b i l i t y when stored for one year on n u t r i e n t g l y c e r o l agar at 5°C. o I d e n t i c a l i s o l a t e s stored on n u t r i e n t agar at 5 C or i n s t e r i l e d i s t i l l e d water at room temperature e i t h e r d i d not s u r v i v e or l o s t t h e i r toxin-producing a b i l i t y . There seemed to be a r e l a t i o n s h i p between toxin-producing a b i l i t y and an i s o l a t e ' s v i r u l e n c e as about 50% of toxin-producing i s o l a t e s were rated pathogenic and 35% weakly pathogenic while none of the non-toxin-producing i s o l a t e s were rated pathogenic and 60% were rated weakly pathogenic. A scheme was devised for r a p i d i d e n t i f i c a t i o n of P_^  syringae from raspberry t i s s u e . An i s o l a t e was determined to be P. syringae i f i t produced a d i s t i n c t i v e r a i s e d mucoid colony on n u t r i e n t sucrose agar, produced a fl u o r e s c e n t pigment, was oxidase negative and reacted i n drop a g g l u t i n a t i o n - i i i -t e s t s w i t h an antiserum prepared against syringae. These t e s t s could be done w i t h i n three days. This scheme was used to study the ov e r w i n t e r i n g s i t e of P_^  syringae on raspberry. P^ syringae was found to n a t u r a l l y populate 4 25-75% of raspberry buds during the winter months. Populations of 10 CFU/six bud sample were most common i n the buds that contained P_^  s yringae. During the s p r i n g and summer months, P^ syringae survived as an epiphyte on raspberry leaves both i n the f i e l d and i n the greenhouse t r i a l s . P. syringae was also shown to cause brownish-red spots surrounded by yellow halos on the leaves of raspberry during the summer months. About 90% of the P^ syringae i s o l a t e s from raspberry were i c e n u c l e a t i o n a c t i v e . Raspberry plants that had been sprayed w i t h a suspension of 10^ CFU/ml and then held at -2°C for 4 hours developed symptoms s i m i l a r to those of b a c t e r i a l b l i g h t w i t h i n 12 hours of the freeze treatment. In p r e l i m i n a r y t e s t s , raspberry c u l t i v a r s showed v a r y i n g degrees of r e s i s t a n c e to P^ syringae i n f e c t i o n . Raspberry c u l t i v a r C h i l c o t i n showed greatest r e s i s t a n c e and c u l t i v a r M a i l i n g Leo showed greatest s u s c e p t i b i l i t y to Pseudomonas syringae i n f e c t i o n . Three d i f f e r e n t species of b a c t e r i a were found i n the normal m i c r o f l o r a of the raspberry that were a n t a g o n i s t i c to P.  syringae i n v i t r o . - i v -Table of Contents Page i i ABSTRACT vi LIST OF TABLES v i i LIST OF FIGURES ix ACKNOWLEDGEMENTS INTRODUCTION 1 PART I: ETIOLOGY OF BACTERIAL BLIGHT OF RED RASPBERRY IN B.C. 4 LITERATURE REVIEW 5 MATERIALS AND METHODS 17 Source and Storage of Cultures 17 S e l e c t i v e Media E v a l u a t i o n 17 I s o l a t i o n and Grouping of B a c t e r i a Recovered from Diseased 19 raspberry P a t h o g e n i c i t y Tests 20 I d e n t i f i c a t i o n of I s o l a t e s i n Pathogenic Groups 21 Survey of B a c t e r i a l B l i g h t i n Commercial F i e l d s 21 Storage of B a c t e r i a 21 RESULTS 23 S e l e c t i v e Media E v a l u a t i o n 23 Grouping of B a c t e r i a Recovered from Diseased Raspberry Tissue 28 Pa t h o g e n i c i t y Tests 28 I d e n t i f i c a t i o n of Pathogenic I s o l a t e s 33 Survey of B a c t e r i a l B l i g h t i n Commercial F i e l d s 33 Storage of B a c t e r i a 35 DISCUSSION 39 PART I I : EPIDEMIOLOGY OF BACTERIAL BLIGHT OF RED RASPBERRY IN B.C. 42 LITERATURE REVIEW 43 MATERIALS AND METHODS 56 Culture Maintenance 56 Antiserum Production and Te s t i n g of S e r o l o g i c a l Methods 56 Monitoring Overwintering Populations of P^ syringae i n Raspberry 58 Buds Summer S u r v i v a l of P_^  syringae 59 Ice N u c l e a t i o n A b i l i t y 61 Survey for B a c t e r i a A n t a g o n i s t i c to P^ syringae 62 C u l t i v a r S u s c e p t i b i l i t y T r i a l s 63 RESULTS 63 Antiserum Production and T e s t i n g 64 Overwintering Populations of P_^  syringae i n Raspberry Buds 69 Summer S u r v i v a l of P. syringae 69 Ice Nucleation A c t i v i t y 71 B a c t e r i a A n t a g o n i s t i c to P^ _ syringae 78 C u l t i v a r S u s c e p t i b i l i t y T r i a l s 78 DISCUSSION 79 THESIS SUMMARY 92 LITERATURE CITED 94 - v i -L i s t of Tables Page Table 1. Comparison of estimated versus a c t u a l y i e l d s of raspberry 3 f r u i t from 1966-1973 Table 2. T y p i c a l r e a c t i o n s of Pseudomonas syringae and E r w i n i a 13 amylovora to the t e s t s normally used f o r i d e n t i f i c a t i o n of I \ syringae Table 3. Authentic c u l t u r e s received from other sources 18 Table 4. Recovery rates for Pseudomonas syringae and E r w i n i a amylovora 24 on s e l e c t i v e media Table 5. Types of b a c t e r i a i s o l a t e d from b l i g h t e d raspberry 29 Table 6. P a t h o g e n i c i t y to raspberry of d i f f e r e n t types of b a c t e r i a 31 i s o l a t e d from b l i g h t e d raspberry Table 7. C h a r a c t e r i s t i c s of Types I , I I IV and authentic Pseudomonas 34 syringae i s o l a t e s Table 8. E f f e c t of the method of storage on the toxin-producing 36 a b i l i t y of Pseudomonas syringae i s o l a t e d from raspberry Table 9. Toxin-producing a b i l i t y of Pseudomonas syringae s i n g l e - c e l l 38 i s o l a t e s of Ps-346 and Ps-505 a f t e r 2 and 3 months i n water storage Table 10. Reactions of v a r i o u s b a c t e r i a l i s o l a t e s w i t h cross-absorbed 67 Pseudomonas syringae antiserum Table 11. Number of nights during which temperatures of -1.8°C or 77 lower were recorded i n the years since b a c t e r i a l b l i g h t was d iscovered Table 12. Ratings of raspberry c u l t i v a r s f or s u s c e p t i b i l i t y to 80 Pseudomonas syringae i n f e c t i o n - v i i - -L i s t of Figures Page F i g . 1. Comparison of Pseudomonas syringae colony to Erwinia 25 amylovora colony on NSA F i g . 2. Comparison of Pseudomonas syringae colony to Erwinia 26 amylovora colony both grown on CVSA F i g . 3. Recovery of Pseudomonas syringae colonies on CVSA and NSA 27 from ground raspberry bud suspensions. Saprophytes were commonly present i n these i s o l a t i o n s while pure cultures of P. syringae on CVSA were r a r e l y seen F i g . 4. Raspberry l a t e r a l showing t y p i c a l symptoms of b a c t e r i a l b l i g h t . 30 Type I bacteria were iso l a t e d from this tissue F i g . 5. a. Blackening of petioles following stem prick inoculation 32 with Pseudomonas syringae b. Necrosis surrounded by a yellow halo on le a f following l e a f spray inoculation with a 10^ CFU/ml suspension F i g . 6. E f f e c t of storage method on toxin-producing a b i l i t y of 37 Pseudomonas syringae, Ps-248 as demonstrated by the Geotrichum candidum bioassay. F i g . 7. Agglutination reaction of Pseudomonas syringae, Ps-346 a n t i - 65 serum and normal antiserum with homologous culture Ps-346 and P. marginalis F i g . 8. Agglutination reaction of Pseudomonas syringae, Ps-346 and P_^  66 phaseolicola, Pp-1 with Ps-346 antiserum and Ps-346 antiserum that has been cross-absorbed with P^ phaseolicola F i g . 9. B a c t e r i a l c e l l s stained with FITC-conjugated Pseudomonas 68 syringae antiserum that had been cross-absorbed with P.  phaseolicola F i g . 10. Populations of Pseudomonas syringae recovered from rasp- 70 berry buds throughout the winter of 1976-1977 F i g . 11. Raspberry l e a f from a n a t u r a l l y - i n f e c t e d plant from the 72 Clearbrook Substation showing t y p i c a l l e a f spots F i g . 12. Leaf spots from n a t u r a l l y - i n f e c t e d raspberry plants at the 73 Clearbrook Substation from which Pseudomonas syringae was is o l a t e d and l e a f spots on a six-week-old raspberry plant from spray inoculation with P^ syringae under greenhouse cond i t i o n s - v i i i -Page F i g . 13. Ice n u c l e a t i o n t e s t showed Pseudomonas syringae to be i c e 74 n u c l e a t i o n a c t i v e and E r w i n i a carotovora to be i n a c t i v e F i g . 14. E f f e c t of c u l t u r e medium on i c e n u c l e a t i o n a c t i v i t y 74 F i g . 15. E f f e c t of c u l t u r e age on ice n u c l e a t i o n a c t i v i t y of Pseudomonas 76 syringae, Ps-346 c e l l s grown on NGA f o r 2 days and 4 days F i g . 16. D i f f e r i n g i c e n u c l e a t i o n of suspensions of two d i f f e r e n t 76 i s o l a t e s of Pseudomonas syringae, Ps-205 and Ps-346 F i g . 17. I n h i b i t i o n of growth of Pseudomonas syringae, Ps-346 by an 79 u n i d e n t i f i e d bacterium from raspberry F i g . 18. a. Symptoms on raspberry c u l t i v a r M a i l i n g Leo caused by spray 81 i n o c u l a t i o n s with Pseudomonas syringae and water, b. Symptoms on raspberry c u l t i v a r Willamette caused by spray i n o c u l a t i o n with water and P^ syringae F i g . 19. Symptoms and corresponding disease r a t i n g s for raspberry 82 c u l t i v a r s F i g . 20. Scheme for r a p i d d e t e c t i o n of Pseudomonas syringae from 85 raspberry F i g . 21. Proposed disease c y c l e of b a c t e r i a l b l i g h t of raspberry 88 caused by Pseudomonas syringae Acknowledgements I wish to thank Dr. R.J. Copeman for suggesting t h i s p r o j e c t and p a r t i c u l a r l y f o r h i s help throughout the course of t h i s study. I a l s o wish to thank Dr. H.S. Pepin and Dr. H.A. Daubeny of A g r i c u l t u r e Canada f o r t h e i r guidance and f o r p r o v i d i n g plant m a t e r i a l and f i e l d research area. I appreciate the t e c h n i c a l a s s i s t a n c e of Dr. N.S. Wright, Dr. Fred McElroy, Mr. Ray Palmer, Mrs. Dana Stary and Mrs. Connie Hyams, a l l of A g r i c u l t u r e Canada. I would e s p e c i a l l y l i k e to thank Dr. S.H. De Boer for h i s suggestions and o encouragement so v i t a l to the completion of t h i s t h e s i s . I acknowledge with greatest a p p r e c i a t i o n the e x c e l l e n t typing job done by Sandra J . Sturgeon. And, f i n a l l y , my most sincere thanks to Timothy S i n n o t t fo r h i s i n s p i r a t i o n . This study was supported by a grant from the F a c u l t y of A g r i c u l t u r a l Sciences Research and Development Grant Fund. I am a l s o g r a t e f u l f o r the UBC Graduate Fellowship I r e c e i v e d i n 1976-1977. - 1 -INTRODUCTION B a c t e r i a l b l i g h t of red raspberry, Rubus idaeus L. ( h e r e a f t e r r e f e r r e d to as raspberry) was f i r s t observed i n B r i t i s h Columbia (B.C.) i n the s p r i n g of 1964 i n a commercial f i e l d i n the Lower Mainland area (79). The symptoms of the disease were a sudden w i l t i n g and blackening of new shoots, cane t i p s and young l a t e r a l s . In 1967, Pepin ej: a l . (79) reported i s o l a t i o n of a bacterium from diseased t i s s u e that would produce s i m i l a r symptoms on raspberry under greenhouse c o n d i t i o n s . The bacterium was i d e n t i f i e d as Pseudomonas syringae van H a l l although i t d i d not produce a f l u o r e s c e n t pigment or a c i d from glucose or sucrose, t y p i c a l c h a r a c t e r i s t i c s of P. syringae (32). By A p r i l of 1972, "...some b l i g h t was found i n every one-year-old f i e l d examined i n Matsqui, Abbotsford and Yarrow areas w i t h serious damage evident i n two f i e l d s " (68). The symptoms of the disease were most n o t i c e a b l e during A p r i l and May but were not evident during the summer months. They sometimes reappeared i n the f a l l w i t h the r e t u r n of cool temperatures and increased r a i n f a l l . Disease occurence was e r r a t i c . I t was reported to be widespread i n 1968, 1970, 1972 and 1973 (12,14,68,70) but no damage was a t t r i b u t e d to the disease i n the other years since i t s discovery. There seemed to be a reduced amount of l a t e r a l bud emergence i n raspberry p l a n t i n g s where b l i g h t had been seen. While a causal r e l a t i o n s h i p has not been e s t a b l i s h e d , economic losses were a t t r i b u t e d to the dead bud problem i n 1968, 1972 and 1973 (13,15,17) the same years when b l i g h t was a problem. U n f o r t u n a t e l y , b a c t e r i a l i s o l a t i o n s were not done i n the years since 1967 and a l l subsequent reports were based only on observation of symptoms. Mone-ta r y losses due s o l e l y to b a c t e r i a l b l i g h t are therefore d i f f i c u l t to estimate - 2 -because the disease was never the only problem in any one growing season. However, in the 4 years when bacterial blight was reported to be widespread actual yields were well below estimated yields while actual yields approximated estimated yields in years when blight was not a problem (Table 1 ) . The objectives of this study, therefore, were: 1. to establish the causal organism of bacterial bl ight , 2. to investigate the sites inhabited by the bacterium during the winter and summer and establish a disease cycle, 3. to determine the relationship of bacterial blight to the dead bud syndrome, and 4. to study possible controls for the disease. This thesis is divided into two parts. The etiology of the disease is examined in Part I and the epidemiology in Part II . - 3 -Table 1. Comparison of estimated versus a c t u a l y i e l d s of raspberry f r u i t from 1966-1973* Year Y i e l d i n kilograms Estimated A c t u a l 1966 7.3 X io 6 7,556,244 1967 7.8 X 6 10 7,685,463 + 1968 5.9 X io 6 5,595,446 1969 6.4 X io 6 6,171,800 + 1970 7.3 X 6 10 4,820,100 1971 4.3 X io 6 4,636,364 +1972 6.4 X 6 10 4,970,455 + 1973 6.4 X io 6 5,000,000 i n f o r m a t i o n obtained from H o r t i c u l t u r e Newsletters, B r i t i s h Columbia Department of A g r i c u l t u r e , V i c t o r i a , B.C. +Years when b a c t e r i a l b l i g h t was reported to be widespread - 4 -PART I E t i o l o g y of Raspberry i n B.C. L i t e r a t u r e Review A b a c t e r i a l b l i g h t of raspberry was f i r s t reported by Detmers i n Ohio i n 1891 (29). Laterals and flowers were infected by an u n i d e n t i f i e d bacterium. Detmers reported that he sent a sample to T.J. B u r r i l l who i d e n t i f i e d the disease as "pear b l i g h t " noting that raspberry i n I l l i n o i s was also infected by the disease. Pear b l i g h t , now commonly known as f i r e b l i g h t , was l a t e r found to be caused by Erwinia amylovora ( B u r r i l l ) Winslow £t a l . (60). In 1932, Lehman (59) reported that E. amylovora was responsible for necro-sis of raspberry l a t e r a l s i n North Carolina. Infected leaves had dead margins bordered by brown, water-soaked tissue s . White droplets of b a c t e r i a l ooze were observed on necrotic tissues. B a c t e r i a l i s o l a t e s recovered from these diseased plants were not pathogenic to apple, which i s commonly infected by E.  amylovora. Apple i s o l a t e s of E^ amylovora did not i n f e c t raspberry. The i n a b i l i t y to c r o s s - i n f e c t suggests that strains of E^ amylovora ex i s t which have d i f f e r e n t pathogenic c a p a b i l i t i e s on d i f f e r e n t hosts. Starr et^ a l . (94) studied an outbreak of f i r e b l i g h t i n plantings of the raspberry c u l t i v a r (cv) Latham i n Maine. The i n f e c t i o n started i n the flowers or leaves and spread downward into the stem or started i n the stem and moved upward into the leaves or growing t i p s . Infected flowers and growing t i p s turned purple then became curved and n e c r o t i c . P e t i o l e s and veins of leaves turned purplish-black and adjacent l e a f tissue became brown. Drops of bac-t e r i a l ooze exuded from infected t i s s u e . B l i s t e r s f u l l of b a c t e r i a developed i n the young bark. Bacteria i s o l a t e d from infected tissue were i d e n t i f i e d as E. amylovora. As i n the previous example, raspberry i s o l a t e s of E^ amylovora did not i n f e c t apple and apple i s o l a t e s did not i n f e c t raspberry. Raspberry - 6 -plants, cv Newburg, and wild raspberry plants (Rubus strigosus Michx.) growing near the diseased Latham planting were not infected by this bacterium. E . amylovora was implicated as the cause of f ireblight on several cu l -tivars of thornless blackberry (Rubus sp. hybrid) in I l l ino i s in 1977 (82). The symptoms observed on blackberry were similar to those described for rasp-berry except that the blackberry fruits were also infected. Infected fruits became dry, brown, hard and remained attached to the pedicel. Different cu l -tivars showed varying degrees of susceptibi l i ty to the disease. Neither apple nor pear plants could be infected with these blackberry strains. Apple and pear isolates of E_^  amylovora did not infect raspberry or blackberry plants. Another bacterial blight of raspberry was reported from B.C. in 1964 (79). Within a few years the disease had become fa ir ly widespread in the Lower Main-land area (68). Typical symptoms included brown water-soaked spots on leaves, petioles, internodes of young shoots and developing laterals . Spots enlarged and blackened. Brown streaks extended from the blackened tips into the vas-cular tissues. Entire laterals often blackened and died. Occasionally new growth was k i l l e d to ground level . In fields where blight was a problem an unusually high percentage of buds failed to open in the spring. The causal organism was identif ied as Pseudomonas syringae although the isolates recovered did not produce a fluorescent pigment and did not produce acid from glucose and sucrose, which are common characteristics of P_^  syringae. Another report of a raspberry disease caused by P^ syringae appeared in 1977 (80). In Yugoslavia, this bacterium was isolated from raspberry plants with halo-spotting disease. Typical symptoms were reported to be o i l y -appearing spots at the ends of veins which enlarged and became necrotic surrounded by yellow halos. Bacterial ooze was noted on the underside of - 7 -leaves below the spots. The morphological and biochemical c h a r a c t e r i s t i c s reported were s i m i l a r to those expected for P. syringae (32). Except f o r the Yugoslavian r e p o r t , the published d e s c r i p t i o n s of the symptoms of f i r e b l i g h t and b a c t e r i a l b l i g h t of raspberry were very s i m i l a r . Symptoms of both i n i t i a l l y appeared as water-soaked l e s i o n s which became nec-r o t i c as the t i s s u e s d r i e d out. C u r l i n g of i n f e c t e d shoot t i p s and l a t e r a l s was c h a r a c t e r i s t i c of both diseases. However, n e i t h e r the i n f e c t i o n of flowers nor the production of b a c t e r i a l ooze was reported for b a c t e r i a l b l i g h t . The s i m i l a r i t y of symptoms caused i n many hosts by P^ syringae and E.  amylovora has led to m i s i d e n t i f i c a t i o n of the i n c i t a n t of a disease i n the past. In England, B i l l i n g et^ al_. (9) found both P^ syringae and E^ _ amylovora on pear trees as i n c i t a n t s of pear b l a s t and f i r e b l i g h t , r e s p e c t i v e l y . These two diseases could not be d i s t i n g u i s h e d on the b a s i s of symptoms. I s o l a t i o n s were necessary to determine which organism was r e s p o n s i b l e for the symptoms observed. An e p i p h y t o t i c of blossom and twig b l i g h t assumed to be f i r e b l i g h t on the basis of symptoms occurred i n the Saanich P e n i n s u l a of Vancouver I s l a n d (64). Attempts to i s o l a t e E^ amylovora from diseased trees f a i l e d and P.  syringae was found i n s t e a d . S i m i l a r l y , f i r e b l i g h t had been reported as a common disease i n C h i l e (19) but, when i s o l a t i o n s were attempted, only P.  syringae could be recovered from i n f e c t e d t i s s u e s . Subsequent surveys f a i l e d to detect E^ amylovora anywhere i n the country. In A l b e r t a , both E_^  amylovora and P^ syringae were recovered from b l i g h t e d raspberry plants near Edmonton (44). P. syringae alone was i s o l a t e d from diseased raspberry near Wainwright. P a t h o g e n i c i t y t e s t s were not reported. The r e l a t i o n s h i p of the two b a c t e r i a to the disease i s c u r r e n t l y being i n v e s t i g a t e d . - 8 -The i dea l i s o l a t i o n medium for determining the causal agent of b a c t e r i a l b l i gh t of raspberry should allow for the growth of P_^  syringae and amylovora but i n h i b i t the saprophytic m i c r o f l o r a . Morphology of the co lonies of these two genera should be s u f f i c i e n t l y d i f f e ren t so that each can be d i s t ingu i shed from the other. K ing 's Medium B (KMB) has been used for the i s o l a t i o n of P.  syringae (50). This medium enhances the production of the f luorescent pigment normally synthesized by th i s bacterium. Colonies of amylovora or a non-f luorescent i s o l a te of P^ syringae would not be d i s t i n c t i v e on th i s medium. A l so , i t is d i f f i c u l t to d i s t i n gu i sh f luorescent P^ syringae colonies from the other f luorescent pseudomonads, commonly found as saprophytes on plant t i s sues (56). Consequently King 's Medium B would not be a su i tab le s e l e c t i v e medium in th is study. Moustafa et^  a l . (67) described a p a r t i a l l y s e l ec t i ve medium for P.  syr ingae. The medium contained p ro l i ne which enhances f luorescence, and manganese su l fa te which is tox ic to non-pathogenic bac te r i a associated with in fec ted t i s sue. Recovery rates for P. syringae on th is medium were not reported nor was the growth of Erwinia species tes ted. M i l l e r and Schroth (66) developed a complex medium which was s e l e c t i v e for Erwinia species but which permitted only slow growth of 12 species of Pseudomonas. Subsequently Cuppels and Kelman (28) showed that the medium became increas ing ly tox ic to a l l bac te r i a as i t aged. Because the medium is time-consuming to prepare, has to be used immediately and does not allow for growth of Pseudomonas species i t would not be a des i rab le medium to use i n th i s case. _ 9 -Kado and Heskett (47) proposed s e v e r a l media f o r the i s o l a t i o n of p l a n t pathogenic b a c t e r i a . A recovery rate of 77% for amylovora was a t t a i n e d on t h e i r D3 medium compared to recovery rates on a standard medium. No pseudo-monads grew on t h i s medium. Their D4 medium was reported to be a b s o l u t e l y s e l e c t i v e f o r syringae but only a 6% recovery rate was a t t a i n e d . A l l other b a c t e r i a were i n h i b i t e d on t h i s medium. Otta (73) found that i n l i q u i d form the D4 medium could be used as an enrichment medium. Tissue to be sampled was homogenized, put i n the medium, and incubated f or 24 hours. P. syringae, i f present, would m u l t i p l y and was detected by s t r e a k i n g some of the broth c u l -ture onto KMB. This medium would be u s e f u l i f the n a t u r a l populations of P.  syringae were so low compared to the saprophytic p o p u l a t i o n that enrichment would be required p r i o r to p l a t i n g . Neither D3 nor D4 medium allowed the growth of both P^ syringae and amylovora. N u t r i e n t sucrose agar (NSA) has been used by Garre t t et _al. (41) f o r the i s o l a t i o n of P^ syringae from f r u i t t r e e s . The medium contained 5% sucrose which i s u t i l i z e d by both P^ syringae and E^ amylovora to produce levan. Levan production r e s u l t s i n the formation of d i s t i n c t i v e r a i s e d mucoid c o l o n i e s by both b a c t e r i a . The a d d i t i o n of c r y s t a l v i o l e t (CVSA) made the medium more s e l e c t i v e as gram-positive p l a n t saprophytes are i n h i b i t e d (25). This medium has been used by s e v e r a l workers for i s o l a t i o n of syringae (36,42). Crosse and Goodman (27) found that by i n c r e a s i n g the sucrose c o n c e n t r a t i o n to 40% a greater percentage of the background m i c r o f l o r a was reduced and a f t e r 60 hours of i n c u b a t i o n , p i t s formed on the surface of the E^ amylovora c o l o -n i e s . They d i d not report the c h a r a c t e r i s t i c s of P_^  syringae on the medium. - 10 -The best medium f o r i s o l a t i o n of b a c t e r i a from raspberry t i s s u e s would probably be NSA. Both amylovora and syringae form d i s t i n c t i v e c o l o n i e s on t h i s medium. The a d d i t i o n of more sucrose, manganese s u l f a t e or c r y s t a l v i o l e t might a l s o prove b e n e f i c i a l as a l l have been reported to reduce numbers of other b a c t e r i a without i n t e r f e r i n g w i t h the growth of the suspected i n c i t a n t s . Once a levan-forming colony has been i s o l a t e d , i t s i d e n t i f i c a t i o n r e q u i r e s se v e r a l t e s t s . To i d e n t i f y an i s o l a t e as E. amylovora the f o l l o w i n g t e s t s are u s u a l l y done: check for formation of d i s t i n c t i v e colony type on M i l l e r - S c h r o t h s o r b i t o l medium (MSS) ( 6 6 ) , presence of p e r i t r i c h o u s f l a g e l l a ( 6 5 ) , absence of polypectate g e l p i t t i n g ( 4 5 ) , production of a h y p e r s e n s i t i v e r e a c t i o n i n tobacco ( 5 1 ) . I d e n t i f i c a t i o n of a b a c t e r i a l i s o l a t e as syringae i s complicated by d i f f e r i n g concepts of what c o n s t i t u t e s the species. The dilemma i n v o l v i n g the separation of the f l u o r e s c e n t pseudomonads i n t o d i s t i n c t species was summar-i z e d by L e l l i o t e_t a l . ( 6 1 ) : "The f l u o r e s c e n t pseudomonads are d i v i d e d i n t o some 70 species whose p a t h o g e n i c i t y on d i f f e r e n t hosts appears to be the only grounds f o r ma i n t a i n i n g them as separate species. The method of c l a s s i f i c a t i o n g e n e r a l l y used has l i t t l e p r e d i c t i v e merit taxonomically, i t i s cumbersome and of t e n i m p r a c t i c a b l e i n d i a g n o s t i c work." These workers proposed a determin-a t i v e scheme to d i v i d e the phytopathogenic f l u o r e s c e n t pseudomonads i n t o f i v e groups based on the i s o l a t e ' s oxidase r e a c t i o n , a b i l i t y to r o t potato, presence of a r g i n i n e d i h y d r o l a s e , levan formation from sucrose and production of a h y p e r s e n s i t i v e r e a c t i o n i n tobacco. 'P_L syringae gave negative r e a c t i o n s f o r the f i r s t three t e s t s and p o s i t i v e r e s u l t s for the l a s t two. Sands, Schroth and Hildebrand (87) d i v i d e d the phytopathogenic pseudo-monads i n t o four groups with Groups I and I I c o n t a i n i n g the f l u o r e s c e n t species. Group I included pseudomonads that do not possess a r g i n i n e dihydro-l a s e , do produce a h y p e r s e n s i t i v e r e a c t i o n i n tobacco and do produce a f l u o r -escent pigment. Of 62 nomenspecies that f e l l i n t h i s category the authors proposed that the group be d i v i d e d i n t o two species, oxidase p o s i t i v e s t r a i n s designated as Pseudomonas c i c h o r i i (Swingle) Stapp and oxidase negative s t r a i n s as P. syringae, and that d i f f e r e n t s t r a i n s from d i f f e r e n t hosts be designated as pathotypes u n t i l t h e i r status could be determined. Hildebrand and Schroth (45) l a t e r r e v i s e d t h e i r o r i g i n a l scheme and recognized 11 d i f f e r e n t species of oxidase negative phytopathogenic, f l u o r e s c e n t pseudomonads. An i s o l a t e i s considered P^ syringae i f i t i s oxidase negative, does not p i t polypectate gel medium and does u t i l i z e l a c t a t e . Doudoroff and P a l l e r o n i (60) e s t a b l i s h f i v e c r i t e r i a f o r i d e n t i f i c a t i o n . To be P_^  syringae an i s o l a t e must n e i t h e r accumulate poly-8-hydroxybutyrate as an i n t r a c e l l u l a r carbon reserve nor possess a r g i n i n e d i h y d r o l a s e . I t must produce a f l u o r e s c e n t pigment, be pathogenic, and be oxidase negative. In t h i s treatment, 42 nomenspecies have been " . . . p r o v i s i o n a l l y included i n t h i s species [P. syringae] which may be synonyms, bi o t y p e s , pathotypes, or v a r i e t i e s and some of which may even deserve independent s p e c i f i c rank". Dye et^ a l . (35), however, found t h i s treatment "inadequate from a p l a n t p a t h o l o g i s t ' s viewpoint because i t does not provide a nomenclature that ex-presses the phytopathogenic i n d i v i d u a l i t y of these b a c t e r i a " . They proposed that 18 of 42 nomenspecies lumped i n t o P\_ syringae by Doudoroff and P a l l e r o n i be r e t a i n e d as species pending f u r t h e r research and the remaining 24 - 12 -nomenspecies be discarded because the species names are " i l l e g i t i m a t e , synonyms of an accepted species, represent a c u l t u r e that has obviously been m i s i d e n t i f i e d or represents a c u l t u r e that i s not found i n a recognized c u l t u r e c o l l e c t i o n " . In order to s a t i s f y a l l of the schemes, 10 d i f f e r e n t t e s t s must be done to a b a c t e r i a l c u l t u r e to i d e n t i f y i t as P. syringae (Table 2). This i s f u r t h e r complicated by the v a r i a b i l i t y of r e s u l t s for some t e s t s . I s o l a t e s that do not produce a f l u o r e s c e n t pigment have been reported (79, J.E. DeVay, personal communication). Sands et a l . (87) found only 8 of 12 P. syringae i s o l a t e s that they examined u t i l i z e d l a c t a t e . About 10% of P. syringae i s o l a t e s t e s t e d by Doudoroff and P a l l e r o n i (60) d i d not produce levan when grown on NSA. A l s o , the s i m i l a r i t y of P_^  syringae to E^ amylovora i n these t e s t s (Table 2) emphasizes the importance of having a s e l e c t i v e medium which permits d i s t i n -guishing these species. Another c o n s i d e r a t i o n of any e t i o l o g i c a l study i s the development of a r e l i a b l e method f o r t e s t i n g p a t h o g e n i c i t y of an i s o l a t e . B a c t e r i a must be introduced i n t o plant t i s s u e s i n some way to cause symptoms. K i r a l y et a l . (51) pointed out that concentrations used are c r i t i c a l . I f too few b a c t e r i a are i n o c u l a t e d i n t o the p l a n t , no symptoms r e s u l t even i f the b a c t e r i a are pathogenic. I f too many b a c t e r i a are used, h y p e r s e n s i t i v e r e a c t i o n s o f t e n occur which are e a s i l y confused w i t h disease symptoms. To induce shoot dieback they suggested p r i c k i n g stems of p l a n t s w i t h a needle and rubbing the wound with a suspension of 5 x 10^ b a c t e r i a l c e l l s / m l or i n j e c t i o n of a s i m i l a r suspension i n t o shoots. To simulate l e a f spot diseases they suggested spraying the undersurface of leaves w i t h e i t h e r a - 13 -Table 2. Typ i c a l reactions of Pseudomonas syringae and Erwinia amylovora to the biochemical tests normally used for i d e n t i f i c a t i o n of P_. syringae TESTS Pseudomonas syringae Erwinia amylovora I n t r a c e l l u l a r accumulation of poly-8-hydroxybutyrate Oxidase reaction Tobacco h y p e r s e n s i t i v i t y Levan production from sucrose Fluorescent pigment production A b i l i t y to p i t polypectate gels Arginine dihydrolase Lactate u t i l i z a t i o n A b i l i t y to rot potato Pathogenic to raspberry not reported v a r i a b l e suspension of greater than 5 x 10 cel ls/ml from a low pressure sprayer or a suspension of less than 5 x 10^ cells/ml from an atomizer providing a 2 pressure of 1.5 kg/cm . The ab i l i t y of P^ syringae to cause disease has been associated with the production of the antibiotic syringomycin by this organism. Backman and DeVay (3) isolated syringomycin from syringae grown on potato dextrose broth (PDB) containing 4% casamino acids and found that i t caused damage to peach leaves similar to the symptoms produced when l ive bacterial suspensions of P_^  syringae were sprayed on peach leaves. Gross and DeVay (42) found that the toxin could reproduce the symptoms of holcus spot disease of maize caused by P_^  syringae. A simple bioassay was developed to check for toxin production (30). An isolate was spotted in the center of a plate of PDA with 4% casamino acids, allowed to incubate for 6 days and then sprayed with a suspension of Geotrichum  candidum Pers. ex Links. A zone of inhibit ion surrounding the bacteria i n d i -cated toxin production. DeVay e £ a l . (30) consistently found P^ syringae isolates to produce this toxin. They used this test as one of their c r i t e r i a for identif icat ion of an isolate as P^ syringae. However, isolates that do not produce toxin but are identical to P^ syringae in a l l other biochemical tests and are pathogenic to peach seedlings have been reported (4,74) so this test may not be absolutely rel iable for determination of pathogenicity. If pathogenic B.C. isolates produce this toxin and i f this toxin can be shown to be responsible for symptom production i t may be unnecessary to conduct green-house pathogenicity tests. The simple bioassay only may be required to deter-mine an isolate's pathogenic capabil i t ies . - 15 -Maintenance of a b a c t e r i a l i s o l a t e i n a s t a b l e , pathogenic c o n d i t i o n i s e s s e n t i a l to any long term study. P_^  syringae c u l t u r e s have been suspected of l o s i n g t h e i r v i r u l e n c e i n storage by s e v e r a l workers (80, H.S. Pepin, personal communication). Various storage methods have been employed. P. syringae has commonly been stored on n u t r i e n t agar (NA) (72,79) or n u t r i e n t g l y c e r o l agar (NGA) (36,41). L y o p h i l i z a t i o n has been used f o r long term storage (31). No data on v i r u l e n c e of i s o l a t e s stored i n these ways was reported. DeVay and Schnathorst (31) reported s u c c e s s f u l storage of IP^ syringae i n o s t e r i l e glass d i s t i l l e d water at 10 C. A f t e r 20 months i n storage no l o s s of v i a b i l i t y or p a t h o g e n i c i t y to peach was noted. Storage of i s o l a t e s on KMB was discouraged by Otta (72) because he found that " w i l d type" smooth colony c u l t u r e s converted more r a p i d l y to a t y p i c a l rough colony forms on t h i s medium than on NA or NA w i t h 1% dextrose. The storage of amylovora has not posed the same problem. V i a b l e , v i r u l e n t c u l t u r e s have been maintained on modified Emerson's medium f o r over 2 years (81). In summary, the i n c i t a n t of b a c t e r i a l b l i g h t of raspberry i n B.C. has not been a b s o l u t e l y e s t a b l i s h e d . The symptoms of the disease are very s i m i l a r to those described for f i r e b l i g h t of raspberry caused by E^ amylovora. The pathogenic bacterium i s o l a t e d from diseased raspberry plants i n B.C. was i d e n t i f i e d as P^ syringae although i t d i f f e r e d from t y p i c a l s t r a i n s of t h i s species i n s e v e r a l important aspects. The organism gave r e s u l t s to b i o -chemical t e s t s s i m i l a r to those expected for E^ amylovora. The purpose of t h i s study was, t h e r e f o r e , to e s t a b l i s h what the i n c i t a n t of b a c t e r i a l b l i g h t - 16 -of raspberry i n B.C. i s . Thus the objectives were: 1. to find an i s o l a t i o n medium suitable for this study, 2. to e s t a b l i s h c r i t e r i a for i d e n t i f i c a t i o n of the i n c i t a n t to species, 3. to develop a r e l i a b l e technique for testing pathogenicity of a suspected pathogen, and 4. to determine a method for maintaining a b a c t e r i a l culture i n a pathogenic condition for long periods of time. - 17 -MATERIALS AND METHODS SOURCE AND STORAGE OF CULTURES A l l c u l t u r e s used i n t h i s study that were received from other sources are l i s t e d i n Table 3. Cultures were r o u t i n e l y stored on n u t r i e n t g l y c e r o l agar NGA (41) at 4°C. • SELECTIVE MEDIA EVALUATION P. syringae i s o l a t e Ps-1 was in o c u l a t e d onto KMB p l a t e s . A f t e r 48 hours c e l l s were taken up i n s t e r i l e d i s t i l l e d water (SDW) and the c o n c e n t r a t i o n of 9 b a c t e r i a adjusted to 5 x 10 colony forming u n i t s (CFU)/ml. A l i q u o t s of s e r i a l d i l u t i o n s of t h i s suspension were p l a t e d on each of the f o l l o w i n g : (1) NSA (41), (2) NSA c o n t a i n i n g 6% c r y s t a l v i o l e t (CVSA) (25), (3) CVSA w i t h .25% manganese s u l f a t e (MCVSA) as prepared by E r c o l a n i et a_l. (36), (4) M i l l e r and Schroth's s o r b i t o l E r w i n i a medium (MSS) (66), and (5) Crosse and Goodman's 40% sucrose medium (CGSA) (27). Numbers and c h a r a c t e r i s t i c s of c o l o n i e s growing on each of the f i v e media were noted a f t e r 3 days. This experiment was repeated on three separate occasions. A suspension of E^ amylovora i s o l a t e , Ea-1, grown on NA, was s i m i l a r l y prepared and d i l u t i o n - p l a t e d on NSA, CVSA, CGSA and MSS. This experiment was repeated twice. To determine the s e l e c t i v i t y of the media, s i x dormant raspberry buds that g had been sprayed with a suspension of 10 CFU/ml of P. syringae were ground i n 3 ml of SDW using a s t e r i l e mortar and p e s t l e . The r e s u l t i n g s o l u t i o n was d i l u t i o n p l a t e d on NSA, CVSA and CGSM. Types and numbers of each type of b a c t e r i a growing on each medium were recorded. - 18 -Table 3. Authentic cultures received from other sources Numb er Species Host Source Source No. Ps-1 P. syringae Li lac D . J . Hagedorn, Madison, Wisconsin N.G.* Ps-2 P^ syringae Forsythia R . J . Copeman, Vancouver, B.C. N.G. Ps-3 P^ syringae Forsythia R . J . Copeman, Vancouver, B.C. N.G. Ps-4 syringae Forsythia R . J . Copeman, Vancouver, B.C. N.G. Ps-5 P^ syringae Lima bean D . J . Hagedorn, Madison, Wisconsin N.G. Ps-6 P. syringae Pear M.P. Starr, Davis, Cal i fornia ICPB PS-269 Ps-7 syringae Bean D . J . Hagedorn, Madison, Wisconsin N.G. Ps-8 I\ syringae Almond J . E . DeVay, Davis, Cal i fornia B-15+ Ps-9 I\ syringae Pear J . E . DeVay, Davis, Cal i fornia B-301 Ps-10 syringae Peach J . E . DeVay, Davis, Cal i fornia B-3A Ps-11 I\ syringae Unknown J . E . DeVay, Davis, Cal i fornia P-359 Ps-12 I\ syringae Almond J . E . DeVay, Davis, Cal i fornia B-15 Ps-13 I\ syringae Peach J . E . DeVay, Davis, Cal i fornia B-3A Ea-1 E^ amylovora Pear W.G. Bonn, Harrow, Ontario E2017P Ec-1 E . carotovora var carotovora Potato R . J . Copeman, Vancouver, B.C. 755 Pp-1 I\ phaseolicola Bean D . J . Hagedorn, Madison, Wisconsin N.G. Pm-1 P^ marginalis Unknown A. Kelman, Madison , Wisconsin PM6 *Source number not given - 1 9 -Otta's m o d i f i c a t i o n (73) of Kado and Heskett's D4 medium was tested as an enrichment medium. One ml of a 10 CFU/ml suspension of i s o l a t e Ps-1 or the ground i n o c u l a t e d raspberry bud suspension was dispensed i n t o 9 ml of l i q u i d D4 medium, incubated f or 24 hours and d i l u t i o n - p l a t e d on NSA. The raspberry bud suspension was a l s o d i l u t i o n - p l a t e d immediately on NSA. A f t e r 2 days on NSA, numbers of each d i f f e r e n t colony type were recorded. ISOLATION AND GROUPING OF BACTERIA RECOVERED FROM DISEASED RASPBERRY In the s p r i n g and summer of 1976, raspberry plants showing symptoms assoc-i a t e d w i t h b a c t e r i a l b l i g h t from the p l o t s at the Vancouver Research S t a t i o n ' s Substation at Clearbrook ( h e r e a f t e r r e f e r r e d to as the Clearbrook Substation) were sampled. Tissues from 31 d i f f e r e n t c u l t i v a r s or s e l e c t i o n s were c o l -l e c t e d from May 7 to J u l y 18 on 11 d i f f e r e n t sampling dates. Approximately 0.5 g of t i s s u e from the h e a l t h y / n e c r o t i c i n t e r f a c e was crushed i n 3 ml SDW using a s t e r i l e mortar and p e s t l e . This suspension was then s e r i a l l y d i l u t e d and p l a t e d on NSA. A f t e r 48 hours r e p r e s e n t a t i v e s of every colony type present on the pl a t e s were restreaked on NSA to insure p u r i t y . A f t e r 48 hours i n c u b a t i o n , colony morphology was noted and i s o l a t e s were streaked onto KMB. The oxidase r e a c t i o n (45) and f l u o r e s c e n t pigment production checked under u l t r a v i o l e t l i g h t were done a f t e r 24 hours. A l l i s o l a t e s were a l s o tested f o r t o x i n production using the Geotrichum candidum bioassay (30). C e l l s of the bacterium to be tested were t r a n s f e r r e d to the center of a p e t r i d i s h of potato dextrose agar (PDA) c o n t a i n i n g 4% casamino a c i d s . A f t e r 6 days i n c u b a t i o n , G. candidum was sprayed onto the p l a t e . Twenty-four hours l a t e r the c l e a r zone of i n h i b i t i o n from the edge of the b a c t e r i a l growth to the - 20 -s t a r t of the fungal growth was measured. I s o l a t e s were put i n t o groups based on t h e i r colony type and r e a c t i o n s to these t e s t s . I s o l a t e s having c h a r a c t e r i s t i c s s i m i l a r to amylovora (non-fluorescent on KMB, mucoid co l o n i e s on NSA, and oxidase negative) were streaked onto CGSA. Colonies were checked a f t e r 48 and 64 hours f o r the d i s t i n c t i v e p i t s which form on the surface of amylovora and P^ syringae i s o l a t e s . PATHOGENICITY TESTS Raspberry plants of the cv Willamette were grown from root c u t t i n g s i n the greenhouse, f e r t i l i z e d every ten days with 20-20-20, and used f o r p a t h o g e n i c i t y t e s t s when the plants were 6 to 10 weeks o l d . Representative i s o l a t e s from each of the groups were tested f o r p a t h o g e n i c i t y . The i s o l a t e s to be t e s t e d were grown for 48 hours on KMB. For stem p r i c k i n o c u l a t i o n s the stem of a raspberry p l a n t was p r i c k e d w i t h a s t e r i l e needle near the growing t i p . Some of the b a c t e r i a from the KMB p l a t e were smeared i n the wound. Inoculated p l a n t s were placed i n a mist chamber i n the greenhouse at 16-23°C. The p l a n t s were checked a f t e r 10 and 14 days for systemic n e c r o s i s spreading from the point of i n o c u l a t i o n . For l e a f spray i n o c u l a t i o n s , b a c t e r i a from a 48-hour KMB p l a t e were suspended i n SDW and adjusted s p e c t r o p h o t o m e t r i c a l l y to 10^ CFU/ml. The suspension was sprayed from a hand sprayer onto the underside of the leaves. S t e r i l e water was sprayed on s i m i l a r p lants as c o n t r o l s . The t i s s u e appeared water-soaked a f t e r spraying. The pl a n t s were placed i n a mist chamber as described for the stem p r i c k i n o c u l a t i o n s and checked for n e c r o s i s spreading from the sprayed areas a f t e r 4 and 7 days. Diseased t i s s u e s from s e v e r a l plants showing symptoms were ground i n SDW and d i l u t i o n - p l a t e d on NSA - 21 -i n an attempt to r e i s o l a t e the bacterium that had been i n o c u l a t e d i n t o the p l a n t s . IDENTIFICATION OF ISOLATES IN PATHOGENIC GROUPS I s o l a t e s i n the pathogenic groups were tested f o r the f o l l o w i n g c h a r a c t e r i s t i c s : Gram r e a c t i o n as recommended by Bradbury (10), production of h y p e r s e n s i t i v e r e a c t i o n i n tobacco, N i c o t i a n a tabacum L. (51), u t i l i z a t i o n of l a c t a t e (85), a b i l i t y to p i t polypectate g e l medium (45), presence of a r g i n i n e dihydrolase tested using Thornley's 2A medium (95). These t e s t s were a l s o done on the known c u l t u r e s l i s t e d i n Table 3. SURVEY OF BACTERIAL BLIGHT IN COMMERCIAL FIELDS In the summer of 1976, raspberry t i s s u e s showing t y p i c a l b l i g h t symptoms were taken from the Dyck and Hoogie farms, two commercial raspberry p l a n t i n g s near Clearbrook, B.C. N e c r o t i c t i s s u e s from raspberry plants showing a t y p i c a l symptoms were a l s o taken from the Maddocks farm near Richmond, B.C. and the Reynolds farm on Westham I s l a n d , B.C.. The t i s s u e s were ground and p l a t e d on NSA as described. Raised, mucoid co l o n i e s were s e l e c t e d and tested to see i f they were P^ syringae. STORAGE OF BACTERIA The b a c t e r i a i s o l a t e d from diseased raspberry and i d e n t i f i e d as P^ syringae were stored i n each of the f o l l o w i n g ways: (1) on NA at 5 C, (2) on NGA at 5°C, and (3) as a t u r b i d suspension i n SDW at room temperature ( 2 3 - 3 0 ° c ) . A f t e r 1 year, i s o l a t e s were streaked onto NSA to check for s u r v i v a l . Each - 22 -i s o l a t e was a l s o tested f o r t o x i n production using the G_^  candidum bioassay. Cultures of r e p r e s e n t a t i v e i s o l a t e s Ps-205 and Ps-346, stored 1 year on NGA and i n water, were tested f o r p a t h o g e n i c i t y using the l e a f spray i n o c u l a t i o n method p r e v i o u s l y described. Two raspberry i s o l a t e s , Ps-346 and Ps-248, stored on NGA f o r 1 year were put i n t o water storage. A f t e r 2 and 3 months storage, 0.1 ml of these water stocks was d i l u t i o n - p l a t e d on NSA. Twelve s i n g l e c o l o n i e s of each c u l t u r e were s e l e c t e d and tested i n the Gj_ candidum bioassay to determine what per-centage of the b a c t e r i a i n water r e t a i n e d the a b i l i t y to produce t o x i n . S i x of these s i n g l e colony c u l t u r e s that had l o s t t h e i r toxin-producing a b i l i t y a f t e r 3 months storage i n water were t r a n s f e r r e d to an NGA s l a n t and tested a f t e r 1 month to determine i f r e v e r s i o n to a toxin-producing form would occur. - 23 -RESULTS SELECTIVE MEDIA EVALUATION Colony d e s c r i p t i o n s and the average number of b a c t e r i a recovered from a 5 9 x 10 suspension of b a c t e r i a on each medium were recorded (Table 4). Using recovery of b a c t e r i a on NSA as a standard, recovery rates were found to be highest on CVSA and CGSA for both species. On NSA E^ amylovora c o l o n i e s appeared slimy and had c l e a r margins while P^ syringae c o l o n i e s appeared dry and the margins were not c l e a r ( F i g . 1). The c o l o n i e s are d i f f i c u l t to d i s t i n g u i s h i n these photographs but are more e a s i l y d i f f e r e n t i a t e d on the p l a t e s . On CVSA the two species are i n d i s t i n g u i s h a b l e ( F i g . 2). The a d d i t i o n of 0.25% manganese s u l f a t e to CVSA caused a p r e c i p i t a t e to form even i f the manganese s u l f a t e was s t e r i l i z e d s e p a r a tely and then added to the agar component j u s t before pouring i n t o p l a t e s . For t h i s reason, growth of E. amylovora on t h i s medium was not te s t e d . No b a c t e r i a grew on the MSS medium. E. amylovora c o l o n i e s were smaller than those of P_^  syringae on CGSA. A l s o p i t s formed on the surface of Ej_ amylovora c o l o n i e s a f t e r 48 hours on t h i s medium. P i t s formed on the surface of P_^  syringae c o l o n i e s but not u n t i l a f t e r 60 hours of in c u b a t i o n . Several m o r p h o l o g i c a l l y d i f f e r e n t types of b a c t e r i a were recovered from the d i l u t i o n s of the ground bud suspensions on both CVSA and NSA ( F i g . 3 ) . The growth of a s l i m y , yellow-pigmented bacterium, commonly recovered from raspberry t i s s u e , was not i n h i b i t e d on any of the media t e s t e d . P. syringae was e a s i l y recovered on NSA when i t was f i r s t grown alone i n l i q u i d D4 medium. However, when l i q u i d D4 med ium was i n o c u l a t e d w i t h the - 24 -Table 4. Recovery rates f o r Pseudomonas syringae and E r w i n i a amylovora on s e l e c t i v e media a f t e r 72 hrs incubation Media Colony D e s c r i p t i o n Number of B a c t e r i a Recovered* Recovery Rate P• syringae NSA (standard) MVSA CVSA CGSA MSS E. amylovora NSA (standard) CVSA r a i s e d , greenish-white 50 opaque, mucoid, dry, 4 mm r a i s e d , p u r p le, opaque, 8 mucoid, 4 mm r a i s e d , p u r p le, opaque, 20 mucoid, 4 mm r a i s e d , l i g h t purple, 20 c l e a r , mucoid, p i t s on surface a f t e r 60 h r s . no growth 0 r a i s e d , white, opaque 50 center w i t h c l e a r margin, mucoid, sli m y , 5 mm r a i s e d , p u r p le, mucoid, 30 5 mm 100% 16% 40% 40% 0% 100% 60% CGSA r a i s e d , l i g h t purple, mucoid, c l e a r 1-2 mm, p i t s on surface a f t e r 48 h r s . 30 60% MSS no growth 0% *Number of b a c t e r i a recovered from a 10 -^ d i l u t i o n of a 5 x 10^ suspension of the bacterium being t e s t e d . Each number represents the average on three r e p l i c a t e p l a t e s i n three experiments for P^ syringae and i n two experiments f o r E^ amylovora. - 25 -F i g . 1 Comparison of Pseudomonas syringae colony (a) to Erwinia amylovora colony (b) on NSA (6 x magnification) - 26 -a F i g . 2 Comparison of Pseudomonas syringae colony (a) with E r w i n i a amylovora colony (b) both grown on CVSA ( 6 x m a g n i f i c a t i o n ) . - 27 -a b F i g . 3 Recovery of Pseudomonas syringae c o l o n i e s (arrows) on CVSA ( l e f t ) and NSA ( r i g h t ) from ground raspberry bud suspensions. Saprophytes were commonly present i n these i s o l a t i o n s (a) while pure c u l t u r e s of P.  syringae on CVSA were r a r e l y seen (b). - 28 -ground raspberry bud suspension known to contain P^ syringae, incubated f o r 24 hours and 1 ml of t h i s medium d i l u t i o n - p l a t e d on NSA, no syringae was recovered because the slimy, yellow-pigmented bacterium completely overgrew any other b a c t e r i a on the p l a t e s . GROUPING OF BACTERIA RECOVERED FROM DISEASED RASPBERRY TISSUE From the diseased raspberry samples, 174 b a c t e r i a were i s o l a t e d . These were d i v i d e d i n t o e i g h t types based on colony morphology on NSA, oxidase r e a c t i o n , f l u o r e s c e n t pigment production and G^ candidum i n h i b i t i o n (Table 5). Most of the b a c t e r i a recovered f e l l i n t o Type I. An example of i n f e c t e d t i s s u e from which Type I i s o l a t e s were recovered i s shown i n F i g . 4. This sample shows symptoms t y p i c a l of those a s s o c i a t e d w i t h b a c t e r i a l b l i g h t . B a c t e r i a i n Types IV and V had c h a r a c t e r i s t i c s s i m i l a r to E_^  amylovora. When these i s o l a t e s were streaked onto CGSM, Type V i s o l a t e s d i d not grow. Type IV i s o l a t e s grew on t h i s medium and p i t s formed on the surface of the c o l o n i e s when they were 64 hours o l d . PATHOGENICITY TESTS Most i s o l a t e s i n Type I were pathogenic to raspberry i n greenhouse t e s t s (Table 6 ) . Stem-prick i n o c u l a t i o n s w i t h these i s o l a t e s r e s u l t e d i n v a s c u l a r blackening w i t h i n 10 days that spread away from the point of i n o c u l a t i o n ( F i g . 5). Leaf spray i n o c u l a t i o n s caused necrosis of t i s s u e d i r e c t l y h i t by the spray and adjacent t i s s u e . Often a yellow halo would form around the n e c r o t i c area ( F i g . 5). These symptoms u s u a l l y developed w i t h i n 4 days. I s o l a t e s of Type I I were e i t h e r weakly or non-pathogenic. No other bacterium i n the other types was pathogenic to raspberry. B a c t e r i a , which formed r a i s e d , mucoid - 29 -Table 5 . Types of bacteria isolated from blighted raspberry Type Colony Description Fluorescent Pig- Oxidase Geotrichum Number on NSA ment Production Reaction candidum isolated inhibit ion in type I raised, mucoid, greenish II raised, mucoid, greenish III raised, mucoid, greenish, very large IV raised, mucoid, white V raised, white, clear, slimy VI f la t , white VII f lat , reddish VIII f la t , yellow or white 91 3 4 4 7 5 5 - 30 -F i g . 4 Raspberry l a t e r a l showing t y p i c a l symptoms of b a c t e r i a l b l i g h t . Type I b a c t e r i a were i s o l a t e d from t h i s t i s s u e . - 31 -Table 6. The pa t h o g e n i c i t y to raspberry of d i f f e r e n t types of b a c t e r i a i s o l a t e d from b l i g h t e d r a s p b e r r i e s Type T o t a l Number of Number that were  I s o l a t e s Tested Pathogenic Weakly Pathogenic Non-Pathogenic I 48 25 17 6 I I 5 0 3 2 I I I 5 0 0 5 IV 3 0 0 3 V 3 0 0 3 VI 3 0 0 3 V I I 3 0 0 3 V I I I 10 0 0 10 - 32 F i g . 5 a. Blackening of p e t i o l e s f o l l o w i n g stem p r i c k i n o c u l a t i o n w i t h Pseudomonas syringae b. Necrosis surrounded by yellow halo on l e a f f o l l o w i n g l e a f spray i n o c u l a t i o n with a 10^ CFU/ml suspension of P.  syringae. - 33 -colonies on NSA, were oxidase negative and produced a fluorescent pigment, were recovered from each raspberry plant showing symptoms in these patho-genicity tests. IDENTIFICATION OF PATHOGENIC ISOLATES Further characterization of the pathogenic types I and II indicated at least 85 of 91 isolates of Type I and four of five isolates of Type II were P.  syringae (Table 7). Type IV isolates were non-fluorescent P. syringae and not E . amylovora• A l l biochemical tests done on the P^ syringae cultures received from other sources (Table 2) gave expected results with the following exceptions. Isolates Ps-1 and Ps-8 did not u t i l i z e lactate. Ps-2 and Ps-4 did not produce levan from sucrose. No toxin was produced by isolates Ps-1, Ps-2, Ps-3, Ps-4, Ps-5, Ps-7, Ps-11, Ps-12 or Ps-13. None of the authentic cultures were pathogenic to raspberry (Table 7). SURVEY OF BACTERIAL BLIGHT IN COMMERCIAL FIELDS Three of four samples of blighted plant material taken from the Dyck farm near Clearbrook, B.C. contained Type I P. syringae isolates. Both samples from the Hoogie farm in the same area contained Type I P_^  syringae. Blight was evident but not widespread in these f ie lds . Only five of 17 samples of necrotic tissue from the Maddock farm in Richmond, B.C. contained P. syringae. No P^ syringae was isolated from the three samples from the Reynolds farm on Westham Island, B.C. Table 7 C h a r a c t e r i s t i c s of Types I , I I , IV and authentic Pseudomonas syringae i s o l a t e s Type Gram Reaction H y p e r s e n s i t i v i t y Lactate Polypectate Presence of P a t h o g e n i c i t y U t i l i z a t i o n Gel P i t t i n g A r g i n i n e to Raspberry Dihydrolase 0/91* 91/91 85/91 0/91 0/91 42/48 I I 0/5 4/5 4/5 0/5 0/5 3/5 IV 0/3 3/3 3/3 0/3 0/3 0/3 Authentic 0/3 P. syringae 13/13 11/13 0/13 0/13 0/13 * f r a c t i o n s represent r a t i o of i s o l a t e s g i v i n g p o s i t i v e reactions to t o t a l number of i s o l a t e s tested I - 35 -STORAGE OF BACTERIA I s o l a t e s survived 1 year stored on NGA at 5°C or i n water at room temperature. Five of seven i s o l a t e s survived when stored on NA at 5°C (Table 8 ) . I s o l a t e s that survived on NGA and NA r e t a i n e d t h e i r a b i l i t y to produce t o x i n as demonstrated i n the G_^  candidum bioassay while the same i s o l a t e s stored i n water could not produce t o x i n ( F i g . 6). Ps-205 and Ps-248, stored on NGA at 5°C for 1 year, were s t i l l pathogenic to raspberry. The i d e n t i c a l i s o l a t e s stored i n water were not pathogenic a f t e r 1 year i n storage. A f t e r toxin-producing i s o l a t e s Ps-346 and Ps-505 had been stored i n water fo r 2 months, some s i n g l e colony c u l t u r e s of each had l o s t t h e i r toxin-producing a b i l i t y (Table 9). A f t e r 3 months i n water storage, none of the s i n g l e colony c u l t u r e s produced t o x i n . A f t e r 1 month on NGA, none of s i x non-toxin-producing s i n g l e colony c u l t u r e s of Ps-346 had reverted to a toxin-producing form. - 36 -Table 8. The e f f e c t of the method of storage on the toxin-producing a b i l i t y of Pseudomonas syringae i s o l a t e d from raspberry I s o l a t e Number Zone of I n h i b i t i o n of G. candidum i n mm. w i t h i n 2 weeks one year a f t e r i s o l a t i o n  of i s o l a t i o n stored on NA stored on NGA stored i n water at 5°C at 5°C at room temp. 205 5* no growth 4 0 223 0 0 0 0 231 8 no growth 6 0 248 3 4 5 0 327 8 4 5 0 368 3 3 4 0 505 5 3 4 0 *average of the of the zone of distance from i n h i b i t i o n i n the margin of the three r e p l i c a t i o n s b a c t e r i a l growth to the edge - 37 -F i g . 6 E f f e c t of storage method on toxin-producing a b i l i t y of Pseudomonas  syringae, Ps-248 as demonstrated by the Geotrichum candidum bioassay. a) I s o l a t e stored for one year on NGA at 5°c and b) i d e n t i c a l i s o l a t e stored i n water at room temperature. - 38 -Table 9. Toxin-producing a b i l i t y of Pseudomonas syringae s i n g l e - c e l l i s o l a t e s , Ps-346 and Ps-505 a f t e r 2 and 3 months i n water storage O r i g i n a l t o x i n - A f t e r 2 months A f t e r 3 months producing a b i l i t y * water storage water storage Ps-346 4 mm 10/12+ 1/12 Ps-505 5 mm 6/12 0/12 *zone of i n h i b i t i o n as determined i n the Geotrichum candidum bioassay done immediately a f t e r i s o l a t i o n from raspberry + f r a c t i o n of i s o l a t e s r e t a i n i n g toxin-producing a b i l i t y - 39 -DISCUSSION E. amylovora and syringae both grew w e l l on NSA forming d i s t i n c t i v e r a i s e d c o l o n i e s on t h i s medium that could be d i s t i n g u i s h e d from one another and from other b a c t e r i a l components of the raspberry m i c r o f l o r a . A sapro-p h y t i c pseudomonad was f r e q u e n t l y recovered which formed co l o n i e s s i m i l a r to P. syringae on NSA but t h i s pseudomonad was oxidase p o s i t i v e and could be e a s i l y d i s t i n g u i s h e d from P^ syringae by t h i s biochemical t e s t . A d d i t i o n s to NSA d i d not improve i t s performance. When c r y s t a l v i o l e t was added to the medium, P_^  syringae c o l o n i e s could not be d i s t i n g u i s h e d from those of E.  amylovora. The a d d i t i o n of c r y s t a l v i o l e t also reduced the number of other b a c t e r i a recovered but because there was some question about the i d e n t i t y of the i n c i t a n t i t was f e l t that i t should not be added so that a more represen-t a t i v e m i c r o f l o r a could be i s o l a t e d and tested for p a t h o g e n i c i t y . This a d d i t i v e a l s o reduced the numbers of P^ syringae. The a d d i t i o n of manganese s u l f a t e to CVSA caused a p r e c i p i t a t e to form. I t also reduced the numbers of P^ syringae that could be recovered. For these reasons i t was not tested f u r t h e r as a s e l e c t i v e medium. Kado and Heskett's l i q u i d D4 medium could not be used as an enrichment medium i n t h i s study because other b a c t e r i a i n the raspberry m i c r o f l o r a were not i n h i b i t e d and overgrew the P^ syringae present. Of the 174 b a c t e r i a l i s o l a t e s recovered from b l i g h t e d raspberry t i s s u e , 91 belonged i n Type I . E i g h t y - f i v e of these i s o l a t e s could be i d e n t i f i e d as P.  syringae by any of the i d e n t i f i c a t i o n schemes c u r r e n t l y i n use. S i x of the i s o l a t e s d i d not u t i l i z e l a c t a t e . They gave r e s u l t s s i m i l a r to P^ syringae i n a l l other biochemical t e s t s and were pathogenic to raspberry i n greenhouse - 40 -tests. Because only the identif icat ion scheme proposed by Hildebrand and Schroth (35) considers lactate u t i l i za t ion necessary for identif icat ion as P.  syringae and because two authentic P_^  syringae cultures were also negative for lactate u t i l i za t ion , these six isolates were considered to be P^ syringae. Four of the five Type II isolates were also positively identif ied as P.  syringae in spite of the fact that the isolates did not produce toxin as determined by the candidum bioassay. This concurs with the finding of non-toxin-producing pathogenic isolates of P^ syringae by other workers (4,74). The f i f th isolate of this group caused no hypersensitivity reaction in tobacco. Because most of the P. syringae identif ication schemes considered this ab i l i ty essential, this f i f th isolate was not considered to be P.  syringae. Non-fluorescent isolates of P^ syringae were recovered in this survey confirming the i n i t i a l report of Pepin et a l . (79). However the isolates in the present survey differed from those found by Pepin et_ al_. in that they were not pathogenic to raspberry in greenhouse tests. The leaf spray inoculation was preferred to the stem prick method for pathogenicity tests because fewer bacteria were required so the spray more closely approximated populations that would be expected in nature (51). Also symptoms developed sooner with the leaf spray technique. Pathogenic P^ syringae isolates were recovered from 22 of 31 cultivars or selections of raspberry showing blight symptoms in 31 out of 40 isolation attempts. Eight of the nine failures to recover P. syringae occurred during a 2-week period of dry weather in June, 1976. The samples taken were dry, a condition not conducive to isolation of bacteria. The recovery of P^ syringae from most of the diseased raspberry samples indicated this organism was probably responsible for the blight seen on the - 41 -Dyck and Hoogie farms. B l i g h t was not widespread i n these f i e l d s and probably had l i t t l e e f f e c t on y i e l d s i n these p l a n t i n g s . The low recovery r a t e of P.  syringae from the Maddocks farm and i t s absence from the Reynolds farm i n d i c a t e d b a c t e r i a l b l i g h t was probably not the problem. Checks f o r P.  syringae were only made i n these few f i e l d s because b a c t e r i a l b l i g h t was not a problem i n the Lower Mainland Area i n 1976 or 1977, the p e r i o d of t h i s study. The best method of storage of P_^  syringae was on NGA at 5°C as both v i a b i l i t y and toxin-producing a b i l i t y were maintained f o r at l e a s t 1 year without t r a n s f e r s . On NA, the i s o l a t e s d i d not remain v i a b l e for 1 year and i n water the i s o l a t e s l o s t t h e i r toxin-producing a b i l i t y w i t h i n 3 months. Whether t o x i n production i s e s s e n t i a l f o r p a t h o g e n i c i t y could not be c l e a r l y e s t a b l i s h e d . Only h a l f of the toxin-producing Type I i s o l a t e s were s t r o n g l y pathogenic i n greenhouse t e s t s . The remainder were weakly pathogenic or non-pathogenic even though they produced t o x i n . About 50% of the non-toxin-producing i s o l a t e s i n Type I I were weakly pathogenic. An i s o l a t e ' s a b i l i t y to produce t o x i n seems to improve i t s pathogenic c a p a b i l i t i e s but i s not e s s e n t i a l f o r p a t h o g e n i c i t y . The e f f e c t of the p u r i f i e d t o x i n on raspberry p l a n t s would be required to e s t a b l i s h a r o l e f o r the t o x i n i n pathogenesis. No amylovora was i s o l a t e d from raspberry during t h i s i n v e s t i g a t i o n and no other bacterium i s o l a t e d from b l i g h t e d p l a n t s caused symptoms on raspberry i n p a t h o g e n i c i t y t e s t s . T y p i c a l , f l u o r e s c e n t i s o l a t e s of P_^  syringae are therefore confirmed as the causal agent of b a c t e r i a l b l i g h t of red raspberry i n B.C. Now that the i n c i t a n t of b a c t e r i a l b l i g h t has been e s t a b l i s h e d and techniques f o r i t s i s o l a t i o n , i d e n t i f i c a t i o n and storage have been determined, a study of the epidemiology of the disease can be undertaken. - 42 -PART II EPIDEMIOLOGY OF BACTERIAL BLIGHT OF RED RASPBERRY IN B.C. - 43 -LITERATURE REVIEW Confirmation of Pseudomonas syringae as the i n c i t a n t of b a c t e r i a l b l i g h t of raspberry i n B.C. made i t p o s s i b l e to begin an i n v e s t i g a t i o n of the epidemiology of the disease. Very l i t t l e was known about the disease c y c l e at the beginning of t h i s study. Neither the source of the primary inoculum nor the oversummering s i t e for the causal agent were known. The set of con-d i t i o n s r e q u i r e d f o r the i n i t i a t i o n of the disease had not been e s t a b l i s h e d . I t was f e l t that the study of these f a c t o r s might e x p l a i n why the disease was widespread and damaging some years and absent i n others. I t was a l s o thought that the connection between b a c t e r i a l b l i g h t and the dead bud syndrome could be determined. Moreover i t was f e l t that a more e f f e c t i v e c o n t r o l measure might be found once the disease c y c l e was understood. An i n v e s t i g a t i o n of the disease c y c l e would be g r e a t l y f a c i l i t a t e d by the development of a quick, r e l i a b l e technique for d e t e c t i n g P^ syringae. Tech-niques i n v o l v i n g the use of s e l e c t i v e media, bacteriophages or s e r o l o g i c a l methods are c u r r e n t l y used for r a p i d d e t e c t i o n of phytopathogenic b a c t e r i a . S e l e c t i v e media have been s u c c e s s f u l l y developed f o r many plant pathogenic b a c t e r i a (28,66,83), but no r e l i a b l e medium has been developed for P. syringae as was discussed i n P a r t I of t h i s t h e s i s . Another method of r a p i d d e t e c t i o n i s based on the a b i l i t y of bacteriophages to s e l e c t i v e l y l y s e a l l i s o l a t e s of one species of b a c t e r i a . Baldwin and Goodman (6) used phage to detect E r w i n i a  amylovora i n apple buds. Klement (53) discovered phages i n Corynebacterium  flaccumfaciens (Hedges) Dowson and Xanthomonas p h a s e o l i (Erw. Smith) Dowson that would only l y s e c e l l s of the species from which they were found. He l a t e r reported a phage of Xanthomonas v e s i c a t o r i a (Doidge) Dowson that was - 44 -s p e c i f i c f o r i s o l a t e s recovered from tomato (54). A d i f f e r e n t phage l y s e d c e l l s of X. v e s i c a t o r i a i s o l a t e s from pepper. Klement (52) found phage of P.  syringae from bean but t h i s phage a l s o lysed i s o l a t e s of other species of Pseudomonas. Crosse and Garret (26) al s o found n o n - s p e c i f i c phage which attacked P^ syringae from cherry and plum. B i l l i n g (8) found phage u s e f u l f o r i d e n t i f i c a t i o n of P. syringae from s e v e r a l d i f f e r e n t hosts but only when used with other biochemical t e s t s . The most promising method for quick d e t e c t i o n of plant pathogenic b a c t e r i a i n v o l v e s the use of s e r o l o g i c a l techniques. In studies of the r e l a t i o n s h i p s among s e v e r a l Pseudomonas species, Friedman (40) found that an antiserum prepared against u n f i x e d , unheated P^ syringae c e l l s reacted i n immuno-d i f f u s i o n t e s t s w i t h three of four i s o l a t e s of P. syringae and w i t h three i s o l a t e s of P^ fluorescens Migula. This e a r l y work i n d i c a t e d t h i s technique might not be s p e c i e s - s p e c i f i c . Otta and E n g l i s h (74) s u c c e s s f u l l y prepared an antiserum, against sonicated c e l l s that was u s e f u l f o r P^ syringae d e t e c t i o n . They d i f f e r e n t i a t e d 10 serotypes of P^ syringae from i s o l a t e s from 20 d i f -f erent hosts. Two s t r a i n s tested had been i s o l a t e d from raspberry and both belonged to the same serotype although host of o r i g i n d i d not u s u a l l y cor-r e l a t e w i t h serotype. A r a b b i t was i n j e c t e d w i t h a mixture of seven d i f f e r e n t serotypes and the antiserum obtained reacted w i t h any s t r a i n of P^ syringae from any host. The antiserum a l s o reacted i n double d i f f u s i o n t e s t s w i t h other species of Pseudomonas, a l l of which are now considered to be P.  syringae by Doudorff and P a l l e r o n i (32). The f l u o r e s c e n t antibody s t a i n (FAS) technique was a f u r t h e r development of the s e r o l o g i c a l d e t e c t i o n method. Paton (78) used t h i s technique to detect - 45 -P. syringae on t u r n i p s . Antiserum against P^ syringae was conjugated w i t h f l u o r e s c e i n i s o t h i o c y a n a t e (FITC), a dye that f l u o r e s c e s under high i n t e n s i t y l i g h t . This method, which allows f o r the v i s u a l i z a t i o n of s i n g l e c e l l s under the microscope, i s more s e n s i t i v e than the other s e r o l o g i c a l techniques which depend on macroscopic observation of the p r e c i p i t a t e formed when b a c t e r i a l c e l l s react w i t h the antiserum. Once a quick, r e l i a b l e method for d e t e c t i o n of Pj_ syringae has been pe r f e c t e d , the e p i d e m i o l o g i c a l study can be s t a r t e d . The f i r s t c o n s i d e r a t i o n of t h i s study i s the source of the primary inoculum. I s o l a t e s of P^ syringae which cause b a c t e r i a l speck of tomato were i s o l a t e d from rhizospheres of weed species near the tomato f i e l d s (89). However, Schuster and Coyne (91) s t a t e d that P_^  syringae was incapable of p e r s i s t i n g i n a f r e e s t a t e i n the s o i l . H o i t i n k et^ a l . (46) i n Wisconsin found that P^ syringae, cause of brown spot of bean, could be detected i n the s o i l from August to March, but they could not recover the b a c t e r i a from the s o i l i n l a t e s p r i n g or e a r l y summer when the beans were i n f e c t e d w i t h the pathogen. L a t e r , E r c o l a n i et a l . (36) found that t h i s bean pathogen overwintered as an epiphyte on the leaves of h a i r y vetch, a common weed along bean f i e l d borders. The b a c t e r i a from the vetch were spashed by r a i n onto the bean crop i n the s p r i n g . The primary inoculum f o r some diseases caused by P_^  syringae was found on the host p l a n t s . P. syringae was found to cause cankers i n which i t over-wintered on peach, plum, a p r i c o t , sweet and sour cherry, almond, pear and n e c t a r i n e (18,22). Cameron (18) found v i a b l e P. syringae i n the bark, buds and vascular t i s s u e of cherry t r e e s . Crosse (24) reported l i v e P. syringae overwintered i n the dead buds of cherry and plum t r e e s . Leben et a l . (58) - 46 -found that sudden outbreaks of b l a s t on c i t r u s , almond and pear i n c i t e d by P.  syringae followed a night of f r o s t or a h a i l storm i n the s p r i n g i n d i c a t i n g that the primary inoculum was present on the host before environmental con-d i t i o n s occurred which predisposed the plants to i n f e c t i o n . In B.C. overwintering of P^ syringae has not been i n v e s t i g a t e d . In the f i r s t report of b a c t e r i a l b l i g h t of raspberry i n B.C., the i n f e c t e d rasp-b e r r i e s were found to be i n c l o s e p r o x i m i t y to a l i l a c i n f e c t e d w i t h P.  syringae. I t seems p o s s i b l e that the bacterium might have overwintered on the l i l a c and spread i n t o the raspberry crop i n the s p r i n g . The presence of P.  syringae i n the s o i l s of raspberry f i e l d s i n B.C. has never been i n v e s t i g a t e d . Cankers have not been observed on raspberry p l a n t s w i t h b a c t e r i a l b l i g h t . In a d d i t i o n , no check for P. syringae i n the buds, bark, or v a s c u l a r t i s s u e of raspberry p l a n t s has been undertaken. The most l i k e l y s t r a t e g y f o r over-w i n t e r i n g would be for the bacterium to e s t a b l i s h i t s e l f on the host p l a n t . A raspberry produces b i e n n i a l f r u i t i n g canes from p e r e n n i a l r o o t s . Because new shoots are sent up from the roots every s p r i n g , new canes and one-year-old canes are together throughout the growing season. I f the b a c t e r i a pass from the o l d canes and become e s t a b l i s h e d on the young canes, the organism could e a s i l y overwinter on these canes and be r e a d i l y a v a i l a b l e for i n f e c t i o n of new canes i n the s p r i n g . During the d r i e r summer months i n B.C., b a c t e r i a l b l i g h t was not evident. P. syringae s u r v i v e d as a non-symptom-causing r e s i d e n t on other p l a n t s on which i t i s pathogenic (56). E n g l i s h and Davis (37) i s o l a t e d P. syringae from healthy leaves, f r u i t s , and twigs of peach and almond during the summer months. Crosse (25) found populations of P. mors-prunonum Wormald (considered - 47 -to be syringae by Doudoroff and P a l l e r o n i (32)) as high as 10^ b a c t e r i a / 2 cm on healthy cherry leaves. Leben et^ a l . (58) found P_^  syringae to co l o n i z e healthy buds of bean p l a n t s from which i t spread onto u n f u r l i n g leaves. No check has been made for P^ syringae on healthy raspberry p l a n t s . Many p l a n t s i n f e c t e d by P_;_ syringae develop l e a f spots as part of the disease syndrome. On the leaves of f r u i t trees P_^  syringae caused two d i f -f e rent kinds of i n f e c t i o n s (18). On peach, plum, cherry and a p r i c o t , l e a f spots s t a r t e d as dark green water-soaked angular spots about 1-2 mm i n diameter. The spots turned red or brown and were u s u a l l y surrounded by a yellow or reddish-brown h a l o . The spots became n e c r o t i c and i n the case of peach, plum and cherry, the n e c r o t i c t i s s u e became dry, b r i t t l e and e v e n t u a l l y dropped out producing a shot-hole e f f e c t . The second type of l e a f i n f e c t i o n , common on pear, began as a d i s t i n c t n e c r o t i c spot but spread r a p i d l y over the e n t i r e l e a f area. P^ syringae was e a s i l y i s o l a t e d from both of these types of spots during the sp r i n g but as the l e a f matured and the n e c r o t i c t i s s u e d r i e d i s o l a t i o n s became i n c r e a s i n g l y d i f f i c u l t . P_^  syringae a l s o caused n e c r o t i c spots surrounded by yellow halos on corn, bean, wheat and w i l d grass species (49,71,77). In 1973, Rancovic and S u t i c (80) reported that raspberry plants were i n f e c t e d w i t h h a l o - s p o t t i n g disease. Water-soaked spots appeared at the ends of l e a f v e i n s , enlarged, became n e c r o t i c and were surrounded by yellow h a l o s . P. syringae was shown to be the cause of these symptoms. Leaf spots have not been reported on raspberry i n B.C. B a c t e r i a l b l i g h t was a problem i n 1968, 1970, 1972 and 1973 but has not been a problem i n the Lower Mainland Area of B.C. since then. To e x p l a i n t h i s - 48 -e r r a t i c incidence i t i s e s s e n t i a l to consider the three components which i n t e r a c t i n pathogenesis: the i n c i t a n t , the host plant and the environment. Changes that have occurred i n any one of these f a c t o r s over the years may account for the e r r a t i c disease incidence. There are two p o s s i b l e changes that may have occurred to the i n c i t a n t . The f i r s t i s that P^ syringae has disappeared from raspberry f i e l d s i n B.C. as suddenly as i t appeared. This s u s p i c i o n i s encouraged by the absence of b l i g h t symptoms i n recent years. A population study should be done to confirm whether or not P. syringae i s c u r r e n t l y present i n the raspberry f i e l d s . Secondly, a change i n the genetic makeup of the pathogen may have occurred. Such changes are not uncommon i n b a c t e r i a . A l l i s o l a t e s of E r w i n i a amylovora recovered from an apple orchard were found to be non-pathogenic (6). I t was p o s t u l a t e d that the i s o l a t e s had changed g e n e t i c a l l y i n some way as outbreaks of f i r e b l i g h t caused by E^ amylovora had been common i n t h i s orchard i n years p r i o r to that study. S t r e p t o m y c i n - r e s i s t a n t mutants of P^ syringae have been found i n a p r i c o t orchards (98). Both p a t h o g e n i c i t y and the a b i l i t y to r e s i s t a n t i b i o t i c s have been a t t r i b u t e d to the i n c o r p o r a t i o n of an epichromosomal f a c t o r , known as a plasmid, i n t o the genetic m a t e r i a l of the bacterium. The p a t h o g e n i c i t y of some human b a c t e r i a l pathogens has been shown to be deter-mined by plasmids (84,93). Panagopolous et_ aL. (76) speculated that plasmids were res p o n s i b l e f o r p a t h o g e n i c i t y of p l a n t pathogens a l s o . G a l l - i n d u c i n g a b i l i t y was shown to be determined by large plasmids i n Agrobacterium  tumefaciens (Smith and Townsend) Conn (96). L a i et^ a l . (55) showed that plasmids, r e s p o n s i b l e f o r a n t i b i o t i c r e s i s t a n c e , were l o s t spontaneously from the p l a n t pathogenic b a c t e r i a Xanthomonas v e s i c a t o r i a i n storage. I f plasmids - 49 -are r e s p o n s i b l e f o r the p a t h o g e n i c i t y of syringae and i f these plasmids are r e l a t i v e l y e a s i l y acquired and l o s t to the n a t u r a l population of P^ syringae, v a r i a b i l i t y i n the incidence of disease would be expected. The s u s c e p t i b i l i t y of a host p l a n t to i n f e c t i o n of P^ syringae i s depen-dent on s e v e r a l f a c t o r s most important of which i s the genetic makeup of the host p l a n t . D i f f e r e n t raspberry c u l t i v a r s d i s p l a y e d v a r y i n g s u s c e p t i b i l i t y to i n f e c t i o n by amylovora (94) and syringae (79). In the Fraser V a l l e y i n B.C. no change has occurred that could account for the e r r a t i c incidence of the disease. Willamette was the most commonly grown c u l t i v a r i n the years when b l i g h t was a problem and i t remains the most commonly grown c u l t i v a r today (H.A. Daubeny, personal communication). The rat e of f e r t i l i z e r a p p l i c a t i o n i s another f a c t o r that a f f e c t s host s u s c e p t i b i l i t y . K a r l e n et. a l . (48) found that the incidence of chocolate spot of corn caused by P^ syringae was most severe when the crop was not f e r t i -l i z e d . A d d i t i o n s of 160 lb / a c r e of n i t r o g e n r e s u l t e d i n a s l i g h t decrease i n i n f e c t i o n w h i le a d d i t i o n of 40 lb/acre of potassium almost e l i m i n a t e d the disease. E n g l i s h et^ al. (39) found that b a c t e r i a l canker caused by P^ syringae k i l l e d 85% of the peach trees grown i n s o i l s w i t h low l e v e l s of n i t r o g e n . When the s o i l was adequately f e r t i l i z e d by adding n i t r o g e n , potassium and phos-phorus, only 13% of the trees died. I f manure and moderate amounts of n i t r o g e n were added 39% of the trees were k i l l e d . I t was concluded that some f e r t i l i z -a t i o n decreased the damage due to P^ syringae i n f e c t i o n but high l e v e l s of n i t r o g e n were a c t u a l l y detrimental to the tr e e s . In B.C., b a c t e r i a l b l i g h t was most severe i n f i e l d s that had been h e a v i l y f e r t i l i z e d w i t h chicken manure (16). Since t h i s observation was made, the i n d i s c r i m i n a t e use of chicken - 50 -manure to increase n i t r o g e n content i n the s o i l s of raspberry f i e l d s has been discouraged (69). The ease with which plant t i s s u e s can be penetrated by the causal organism i s another f a c t o r a f f e c t i n g the host p l a n t ' s s u s c e p t i b i l i t y . A l l p l a n t pathogenic b a c t e r i a enter host t i s s u e through n a t u r a l openings or wounds (51). The n a t u r a l p l a n t openings u t i l i z e d by b a c t e r i a i n c l u d e l e n t i c e l s , hydathodes and stomates. Wounds due to c u l t u r a l p r a c t i c e s or n a t u r a l causes more commonly provide entrance f o r b a c t e r i a . Common raspberry c u l t i v a t i o n p r a c t i c e s include t y i n g canes to wires strung along the plant rows to support the canes. When i t i s windy, the canes rub against the wires and the bark i s removed at these p o i n t s . N a t u r a l wounding occurs when wind-blown dust scratches the surface of leaves. Wind-blown r a i n causes water-soaking of leaves. Xanthomonas malvacearum, Pseudomonas t a b a c i (Wolf and F o s t e r ) Stevens and P^ mors-prunorum are known to enter host t i s s u e through water-soaked areas (23). Water-soaked l e a f t i s s u e was commonly seen i n the raspberry f i e l d s i n B.C. during periods of wet, windy weather. Leaf scars r e s u l t i n g from n a t u r a l l e a f f a l l i n autumn provided entry for P^ syringae i n t o cherry trees which r e s u l t e d i n canker formation (21). No changes have occurred i n B.C. to any of these mechanisms that would discourage the chances of P^ syringae e n t e r i n g pl a n t t i s s u e and account f o r the d e c l i n e of the disease. V a r i a t i o n s i n environmental c o n d i t i o n s from year to year may account f o r the e r r a t i c incidence of the disease. Several attempts have been made to determine the environmental c o n d i t i o n s required by P^ syringae for disease i n i t i a t i o n . E n g l i s h and Davis (38) found peach tree i n f e c t i o n by P_^  syringae more l i k e l y to occur at 12°C than at 28°C. Cameron (18) noted i n f e c t i o n - 51 -of sweet cherry was most common during c o o l , wet periods i n the s p r i n g i n Oregon. In C a l i f o r n i a l e a f spot and canker of c i t r u s was much more common i n wet years (70). Schmidle and Z e l l e r (88) reported temperatures of 15-25°C accompanied by a 12-hour period of 100% r e l a t i v e humidity were most conducive to i n i t i a t i o n of sour cherry b l a s t . Crosse (23) found that windy, wet periods were responsible f o r e p i p h y t o t i c s of cherry l e a f spot and spur w i l t caused by P. syringae. In B.C., such wet, c o o l , windy periods are recorded almost every s p r i n g and f a l l . There i s a p o s s i b i l i t y that i n i t i a t i o n of b a c t e r i a l b l i g h t of raspberry depends on f r e e z i n g temperatures. Sudden outbreaks of diseases caused by P. syringae have of t e n followed a night of f r o s t . Wormald (99) reported an outbreak of blossom b l i g h t of pear a f t e r a l a t e f r o s t i n May. Baker (5) and Leben et a l . (58) observed a s i m i l a r phenomenon. Panagopoulos and Crosse (75) reported that P. syringae was u n i v e r s a l l y d i s t r i b u t e d on pear trees as the dominant component of surface m i c r o f l o r a . They speculated that freeze i n j u r y r e s u l t e d i n a " l i f t i n g of the s k i n of the receptacles and p e d i c e l s due to separation of the hypoderm from u n d e r l y i n g c o r t i c a l c e l l s " . This i n j u r y allowed the entry of the b a c t e r i a i n t o p l a n t t i s s u e followed by the i n i t i a t i o n of disease. Recent i n v e s t i g a t i o n s have i m p l i c a t e d P^ syringae as the cause of freeze i n j u r y and not merely i t s b e n e f i c i a r y . In 1974, Maki et al_. (63) found that drops of s t e r i l e water or suspensions of most b a c t e r i a f r o z e between -9 and o -17 C wh i l e drops of suspensions of P^ syringae i s o l a t e d from alder leaves f r o z e at temperatures of -1.8 to -3.8°c. Concentrations of at l e a s t 10 4  P. syringae c e l l s per drop were required f o r t h i s e f f e c t to occur. They - 52 -proposed that one bacterium i n 10,000 was able to seed i c e c r y s t a l formation. Amy £t a l . (2) found that the a b i l i t y of P_^  syringae to cause i c e n u c l e a t i o n a l s o depended on the medium on which the c e l l s had been grown. When corn p l a n t s , sprayed w i t h e i t h e r P^ syringae or E r w i n i a h e r b i c o l a (Lohnis) Dye, were placed i n a mist chamber f o r 24 hours and then placed i n a freeze chamber, freeze i n j u r y occurred at -3 to -4°C. No damage was observed on c o n t r o l p l a n t s sprayed w i t h s t e r i l e d i s t i l l e d water. They speculated that e i t h e r bacterium, when present on plant t i s s u e s , could act as a nucleus f or i c e c r y s t a l formation. Because water expands on f r e e z i n g , p l a n t c e l l s ruptured and damage occurred. Attempts were made to determine i f P. syringae c e l l s had to be a l i v e i n order to be i c e n u c l e a t i o n a c t i v e . C e l l s were trea t e d w i t h dyes, a n t i b i o t i c s , chemicals or were h e a t - k i l l e d or sonicated (63). Only treatment with the a n t i b i o t i c s polymyxin B and streptomycin s u l f a t e e f f e c t i v e l y reduced the numbers of v i a b l e b a c t e r i a i n a suspension without destroying the i c e n u c l e a t i o n a b i l i t y . Weaver (98) has r e c e n t l y shown that b a c t e r i a l canker on peach i s a r e s u l t of the i n t e r a c t i o n of Pj_ syringae and freeze i n j u r y . Cankers developed on excised peach twigs that were o ino c u l a t e d w i t h P_^  syringae, h e l d at -10 C for 36 hours and then kept at o 15 C for 10 days. Neither i n o c u l a t i o n w i t h P. syringae or the freeze treatment alone caused canker formation. I f the i s o l a t e s of P_^  syringae i n B.C. possess t h i s i ce n u c l e a t i o n a b i l i t y i t f o l l o w s that raspberry p l a n t s i n f e s t e d w i t h P^ syringae may be more s u s c e p t i b l e to freeze i n j u r y than raspberry plants free of P^ syringae. I t may even be that freeze i n j u r y to the p l a n t , f a c i l i t a t e d by the presence of P.  syringae i s r e s p o n s i b l e for the symptoms associated w i t h b a c t e r i a l b l i g h t . A l s o t h i s i c e n u c l e a t i o n a b i l i t y may provide an exp l a n a t i o n f o r the dead bud - 5 3 -syndrome which i s most l i k e l y to occur i n f i e l d s where b a c t e r i a l b l i g h t has been a problem. Present recommendations for c o n t r o l of b a c t e r i a l b l i g h t of raspberry i n B.C. are not e f f e c t i v e . The B r i t i s h Columbia Department of A g r i c u l t u r e recommends that f i x e d copper or Bordeaux mixture sprays be a p p l i e d at bud-burst followed by three more sprays at 14-day i n t e r v a l s f o r c o n t r o l of b a c t e r i a l b l i g h t of raspberry (3). S a t i s f a c t o r y c o n t r o l of the disease was not achieved using t h i s schedule (17,70). The development of a b e t t e r c o n t r o l method for b a c t e r i a l b l i g h t would therefore be very d e s i r a b l e . Other diseases caused by Pj_ syringae have not been e f f e c t i v e l y c o n t r o l l e d w i t h Bordeaux or f i x e d copper sprays (18,22,34). B e t t e r r e s u l t s have been r e a l i z e d w i t h streptomycin s u l f a t e sprays. Crosse (24) reported c o n t r o l of the l e a f spot but not the canker phase of b a c t e r i a l canker of cherry w i t h t h i s spray. Dye (34) found streptomycin s u l f a t e sprays provided good c o n t r o l f o r stone f r u i t b l a s t caused by P_^  syringae. Bethel et a l . (7) found streptomycin-f i x e d copper sprays to be very e f f e c t i v e f o r c o n t r o l of pear blossom b l a s t a l s o caused by P^ syringae. There are disadvantages for the use of t h i s a n t i b i o t i c f o r c o n t r o l . I t i s expensive and b a c t e r i a have been known to develop r e s i s t a n c e to i t . Streptomycin had been s u c c e s s f u l l y used to c o n t r o l f i r e b l i g h t of pears i n C a l i f o r n i a u n t i l an e p i p h y t o t i c broke out i n 1970 which was found to be caused by s t r e p t o m y c i n - r e s i s t a n t s t r a i n s (66). Pathogenic, s t r e p t o m y c i n - r e s i s t a n t P^ syringae s t r a i n s were recovered from a p r i c o t f r u i t l e s i o n s and l e a f surfaces i n New Zealand (100). Sands and Mclntyre (86) have reported good c o n t r o l of Pj_ syringae i n f e c t i o n of pear wit h aqueous sprays of sodium t a r t r a t e . The advantages of using t h i s organic s a l t f o r c o n t r o l were that i t was biodegradable, inexpensive, and was not phytotoxic to pear. - 54 -Attempts have been made to c o n t r o l P_^  syringae or c l o s e l y r e l a t e d species b i o l o g i c a l l y w i t h a n t a g o n i s t i c b a c t e r i a . Crosse (25) i s o l a t e d a bacterium w i t h c h a r a c t e r i s t i c s s i m i l a r to E r w i n i a species from cherry t r e e s . This bacterium reduced the incidence and s e v e r i t y of l e a f scar i n f e c t i o n s caused by P. mors-prunorum when i t was sprayed on the tree p r i o r to i n o c u l a t i o n s w i t h t h i s pathogen. The population of the antagonist on the f r u i t trees was increased by spraying b a c t e r i a l suspensions on the trees but the po p u l a t i o n dropped o f f r a p i d l y and n a t u r a l e q u i l i b r i u m was r e e s t a b l i s h e d w i t h i n a few days. Crosse proposed that competition f o r space and n u t r i e n t s was the b a s i s of t h i s antagonism. Dowler (33) reported i n h i b i t i o n of P^ syringae i s o l a t e d from peach trees by a f l u o r e s c e n t , non-pathogenic, gram-negative b a c t e r i a the same s i z e as P^ syringae. In mixed c u l t u r e s at 25°C the saprophyte markedly i n h i b i t e d the pathogen. When equal numbers of both antagonist and pathogen were i n o c u l a t e d i n t o peach trees at 25°C the saprophyte was recovered i n greater numbers than the pathogen a f t e r 48 hours. A l s o i n f e c t i o n was not as severe when peach seedlings were in o c u l a t e d w i t h both antagonist and pathogen as when the seedlings were i n o c u l a t e d with the pathogen alone. Leben (57) i s o l a t e d three b a c t e r i a l s t r a i n s from the seed coats of soybeans that produced t o x i c a n t s that d i f f u s e d i n agar and i n h i b i t e d P^ _ g l y c i n e a Coerper (now con-sidered P_^  syringae by Doudoroff and P a l l e r o n i (32)) which i n f e c t e d soybean see d l i n g s . Of seedlings developing from seeds n a t u r a l l y i n f e c t e d w i t h P_^  : g l y c i n e a , between 7 and 17% were i n f e c t e d when the seeds were tre a t e d w i t h the antagonist while 46-98% of the seedlings were i n f e c t e d i f the seeds were not t r e a t e d . - 55 -There is also hope that genetic resistance against bacterial blight may provide control. Crosse (22) reported that cherry cv Napoleon was more sus-ceptible to bacterial canker than cherry cv Frogmore. Plums cv Victor ia was also found to be susceptible to P_^  syringae infection while cv Purple Egg was highly resistant. Different wheat cultivars also showed varying resistance to P. syringae (103). Glenlea was highly resistant to infection while cv Era was moderately susceptible. Pepin et a l . (79) found that cv Latham was susceptible while Newburgh and Viking cultivars were most resistant to bacterial blight of red raspberry in B.C. Starr (94) also observed cv Latham to be susceptible and cv Newburgh to be resistant to f ireblight infection of red raspberry. This s imilarity in reaction might indicate some raspberry cultivars are generally resistant or susceptible to bacterial infections. Because new cultivars of raspberry are being developed in B.C. in a breeding program, a method of screening the new cultivars for resistance to bacterial blight is needed. In summary, bacterial blight of raspberry is an errat ic , uncontrolled disease which appears in the spring and/or f a l l of some years. How and where P. syringae survives the rest of the year is not known. Environmental conditions may prove to be very important for disease in i t ia t ion because of the unique ice nucleation ab i l i ty of strains of this bacterium found elsewhere. Therefore the objectives of this study were: 1. to develop a technique for rapid detection of P_^  syringae on red raspberry so that the overwintering and oversummering sites for this bacterium can be located, 2. to investigate the ice nucleation ab i l i t y of local P. syringae isolates and determine the effect of this ab i l i ty on the disease syndrome, and 3. to evaluate some potential control measures for this disease. - 56 -MATERIALS AND METHODS CULTURE MAINTENANCE Authentic c u l t u r e s used i n t h i s study are l i s t e d i n Table 3 (Part I ) . Cultures were stored on n u t r i e n t g l y c e r o l agar (NGA) at 5°C. Unless otherwise s t a t e d , P^ syringae c u l t u r e s were grown on King's Medium B (KMB) f o r 48 hours at 27°C before use i n t e s t s . ANTISERUM PRODUCTION AND TESTING OF SEROLOGICAL METHODS P. syringae c u l t u r e Ps-346, recovered from a b l i g h t e d raspberry from the Clearbrook Substation of the Vancouver Research S t a t i o n of A g r i c u l t u r e Canada, was grown on n u t r i e n t agar (NA) for 48 hours. The c e l l s were glutaraldehyde-9 f i x e d by the procedure of A l l a n and Kelman (20) and then adjusted to 10 c e l l s / m l . One ml of t h i s suspension was mixed w i t h 1 ml of Freud's incomplete adjuvant ( D i f c o , D e t r o i t , Michigan) and 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 a 2 kg New Zealand r a b b i t . The r a b b i t was t h e r e a f t e r i n j e c t e d w i t h 1 ml of the same suspension at weekly i n t e r v a l s f o r 4 weeks. P r i o r to the f i r s t i n j e c t i o n and 2 weeks a f t e r the l a s t weekly i n j e c t i o n , 20 ml of blood was c o l l e c t e d by bleeding from the ear v e i n of the r a b b i t (105). The blood was allowed to c l o t o overnight at 5 C, the serum c o l l e c t e d and ce n t r i f u g e d at 12,000 g f o r 20 minutes i n an I n t e r n a t i o n a l Equipment Co. c e n t r i f u g e , Model HT. The t i t r e of the antiserum was determined by drop a g g l u t i n a t i o n . A g suspension of 10 colony-forming u n i t s (CFU)/ml of the homologous c u l t u r e Ps-346 was prepared i n 0.01M buffered s a l i n e (PBS), pH 7.2. The antiserum was d i l u t e d 1:10 and then s e r i a l l y - d i l u t e d 1:2 ten times with PBS. One drop of the b a c t e r i a l suspension was mixed with one drop of each conc e n t r a t i o n of antiserum i n a p l a s t i c p e t r i p l a t e . For c o n t r o l s , one drop of PBS was tested - 57 -with each concentration of antiserum and w i t h the b a c t e r i a l suspension. The p l a t e was placed i n a humid chamber at room temperature and checked for a g g l u t i n a t i o n under 6 times m a g n i f i c a t i o n a f t e r 2, 4 and 8 hours. The denom-i n a t o r of the highest d i l u t i o n that s t i l l reacted w i t h the b a c t e r i a l suspension was taken as the t i t r e of the antiserum. Because the t i t r e was high enough for r o u t i n e t e s t i n g a f t e r the four weekly i n j e c t i o n s the r a b b i t was t h e r e a f t e r b l e d once every month and i n j e c t e d two weeks a f t e r each b l e e d i n g . To determine the s p e c i f i c i t y of the antiserum PBS suspensions of 10^ and g 10 CFU/ml of each of Ps-346, Ps-205, P. p h a s e o l i c o l a (Burkholder) Dowson (Pp-1) and P^ m a r g i n a l i s (Brown) Stevens (Pm-l) which had been grown on NGA were made. One drop of each concentration of b a c t e r i a was mixed w i t h one drop of a 1:80 d i l u t i o n of each of the normal and anti-Ps-346 serum i n PBS as above and checked a f t e r 4 hours for a g g l u t i n a t i o n . Because the antiserum reacted w i t h i s o l a t e Pp-1, the antiserum was cross-absorbed. Ten NA p l a t e s were in o c u l a t e d w i t h Pp-1 and incubated f o r 48 hours. C e l l s were taken up i n 100 ml of PBS and c e n t r i f u g e d at 12,000 g f o r 20 minutes. The supernatant was discarded and the c e l l s were taken up i n 8 ml of PBS. Of the r e s u l t i n g suspension 0.7 ml were added to each of four small o t e s t tubes c o n t a i n i n g 0.1 ml of antiserum each. The tubes were h e l d at 50 C i n a waterbath f o r 2 hours, c e n t r i f u g e d and the supernatant t r a n s f e r r e d to a clean t e s t tube (97). The supernatant was cross-absorbed two more times using 0.6 ml of b a c t e r i a l suspension each time. The supernatants were pooled g i v i n g about 8 ml of a 1:20 d i l u t i o n of cross-absorbed antiserum. The s p e c i f i c i t y of t h i s cross-absorbed antiserum was determined by t e s t i n g the f o l l o w i n g a u t h e n t i c i s o l a t e s w i t h the antiserum i n drop a g g l u t i n a t i o n : - 58 -Ps-1, Ps-2, Ps-3, Ps-4, Ps-5, Ps-6, Ps-7, Ps-8, Ps-9, Ps-10, Ps-11, Ps-12, Ps-13, Pm-1, Pp-1, E r w i n i a amylovora ( E a - l ) , and E r w i n i a carotovora var carotovora Dye (Ec-1). A l s o tested were 51 i s o l a t e s b i o c h e m i c a l l y i d e n t i f i e d as P. syringae, 5 u n i d e n t i f i e d pseudomonads, and 41 other b a c t e r i a , a l l i s o l a t e d from diseased raspberry p l a n t s from the Clearbrook Substation i n 1976. S i x P_^  syringae i s o l a t e s from diseased raspberry plants from Richmond were a l s o t e s t e d . F l u o r e s c e i n i s o t h i o c y a n a t e (FITC) was conjugated to the cross-absorbed antiserum as described by A l l a n and Kelman (104). Suspensions of b a c t e r i a l i s o l a t e s Ps-346, Pp-1, and Pm-1 and 10 non-pseudomonad b a c t e r i a , commonly i s o l a t e d from diseased raspberry from the Clearbrook S u b s t a t i o n , were smeared i n 5 mm diameter c i r c l e s etched on glass microscope s l i d e s . The s l i d e s were a i r - d r y e d , h e a t - f i x e d , and stained w i t h a 1:1 d i l u t i o n of the congugated antiserum i n PBS for 30 minutes. The s l i d e s were r i n s e d i n PBS and viewed with a Z e i s s U n i v e r s a l microscope equipped f o r i n c i d e n t f l u o r e s c e n t microscopy using a Neofluar 40 o b j e c t i v e , e x c i t a t i o n f i l t e r BG-12, and b a r r i e r f i l t e r 50. Smears c o n t a i n i n g f l u o r e s c i n g b a c t e r i a l c e l l s were noted. MONITORING OVERWINTERING POPULATIONS OF P^ SYRINGAE IN RASPBERRY BUDS Populations of P_^  syringae on raspberry buds from the Clearbrook Substation were monitored throughout the winter of 1976-1977. The sample area c o n s i s t e d of two 17-m rows, 2 m apart. Each row was d i v i d e d i n t o s i x 2-m s e c t i o n s of raspberry plants with 1-m spaces between s e c t i o n s . Each s e c t i o n contained s i x raspberry p l a n t s cv Willamette w i t h e i g h t canes per p l a n t . Because no b a c t e r i a l b l i g h t had been seen i n t h i s p l o t during the 1976 growing - 59 -season, dormant canes i n s i x randomly s e l e c t e d sections were sprayed to r u n o f f g w i t h a suspension of 5 x 10 CFU/ml of Ps-346 from a hand sprayer on November 19. P l a n t s i n the other s e c t i o n s were sprayed with water. Before and immediately a f t e r spraying, and at monthly i n t e r v a l s t h e r e a f t e r u n t i l March, two buds were s e l e c t e d at random from each pla n t and the 12 buds from each s e c t i o n were grouped. These 12 buds were d i v i d e d i n t o two six-bud samples and each sample was ground i n 3 ml of s t e r i l e d i s t i l l e d water (SDW) using a s t e r i l e mortar and p e s t l e . Two ml of the r e s u l t i n g s o l u t i o n were r> s e r i a l l y d i l u t e d and p l a t e d on n u t r i e n t sucrose agar (NSA). A f t e r 2 days r a i s e d , mucoid c o l o n i e s were counted. Representative c o l o n i e s were streaked on NSA to ensure p u r i t y and then t r a n s f e r r e d to KMB. A f t e r 24 hr i n c u b a t i o n the oxidase r e a c t i o n and production of a f l u o r e s c e n t pigment were checked. Each i s o l a t e was then t r a n s f e r r e d to a NGA s l a n t . A f t e r 48 hours i n c u b a t i o n , a loop of the c u l t u r e was t r a n s f e r r e d to 2 ml of PBS and tested i n drop a g g l u t i n a t i o n with a 1:80 d i l u t i o n of the cross-absorbed antiserum prepared against Ps-346. SUMMER SURVIVAL OF I \ SYRINGAE Healthy leaves were obtained from raspberry plants cv Willamette from the Clearbrook S u b s t a t i o n on four sampling dates during the summer of 1977. Seven two-leaf samples (each approximately 1 g) were ground i n SDW and d i l u t i o n - p l a t e d on NSA. A f t e r 48 hours, r a i s e d , mucoid c o l o n i e s were s e l e c t e d and c h a r a c t e r i z e d as described above. The p a t h o g e n i c i t y of four of these i s o -g l a t e s was tested by spraying a b a c t e r i a l suspension of 10 CFU/ml on the leaves of 6-week-old rapsberry p l a n t s cv Willamette. A f t e r spraying, the p l a n t - 60 -t i s s u e appeared water-soaked. The p l a n t s were then placed i n a mist chamber i n the greenhouse and checked for symptoms a f t e r 7 days. To determine how long P^ syringae could s u r v i v e on the raspberry leaves g under greenhouse c o n d i t i o n s a suspension of 10 CFU/ml of i s o l a t e Ps-346 was sprayed to runoff on fo u r , 6-week-old raspberry p l a n t s cv Willamette. P l a n t t i s s u e s d i d not appear water-soaked a f t e r spraying. A f t e r 4 and 6 weeks, two leaves from each pl a n t were ground and p l a t e d as was done w i t h the healthy l e a f samples. A f t e r incubation r a i s e d , mucoid c o l o n i e s were counted. In the s p r i n g and summer of 1977, l e a f spots were observed on red raspberry plants at the Clearbrook Substation. To determine i f these spots were caused by P. syringae, samples of leaves w i t h spots were c o l l e c t e d . A s t e r i l e 5 mm diameter cork-borer was used to cut d i s c s of t i s s u e that included a l e a f spot and some surrounding healthy t i s s u e . Eighteen^20-disc samples were ground and d i l u t i o n - p l a t e d on NSA. A f t e r 48 hours i n c u b a t i o n , c o l o n i e s suspected to be P^ syringae were s e l e c t e d and c h a r a c t e r i z e d as described 8 above. A suspension of 2 x 10 CFU/ml of each of three c u l t u r e s c h a r a c t e r i z e d as P^ syringae recovered from the l e a f spot samples were each sprayed on two 6-week-old raspberry plants cv Willamette. The t i s s u e s d i d not appear water-soaked a f t e r spraying. The pl a n t s were placed i n a humid chamber and checked for l e a f spots 10 days l a t e r . Discs of t i s s u e surrounding the spots that d i d develop were cut from the pl a n t s and i s o l a t i o n s were made as described above. The col o n i e s obtained were compared to those i n o c u l a t e d i n t o the p l a n t to f u l f i l l Koch's p o s t u l a t e s . - 61 -ICE NUCLEATION ABILITY Two P. syringae c u l t u r e s , Ps-282 and Ps-346, i s o l a t e d from diseased raspberry p l a n t s i n the summer of 1976, and one E r w i n i a carotovora c u l t u r e , Ec-1, from potato were e x t e n s i v e l y tested for i c e n u c l a t i o n a b i l i t y (INA). The i s o l a t e s were grown on KMB and NGA f o r 1, 2, 4 and 7 days. C e l l s were 9 suspended i n SDW and adjusted to 10 CFU/ml. D i l u t i o n s were made to obt a i n suspensions of 10^, 10^, and 10^ CFU/ml i n SDW. A piece of aluminum f o i l was coated w i t h a 1% s o l u t i o n of p a r a f f i n i n xylene (62). The xylene was allowed to evaporate i n a dry i n g oven at 60°C. The coated aluminum f o i l was f l o a t e d i n a waterbath c o n t a i n i n g ethylene g l y c o l (1:2 Prestone a n t i f r e e z e to water) h e l d at -5°C. A 10 y l droplet of each concentration from each medium for the three b a c t e r i a l i s o l a t e s and a s t e r i l e water c o n t r o l were placed on the f o i l . A f t e r 1 minute the drops were checked f o r f r e e z i n g . This was repeated using the same suspension to confirm r e s u l t s . Subsequently, 48 other P_^  syringae c u l t u r e s i s o l a t e d i n the s p r i n g and summer of 1976 from the diseased r a s p b e r r i e s , 28 P. syringae c u l t u r e s i s o l a t e d during the winter of 1976-1977 from dormant buds, 5 pseudomonads from raspberry and 10 other species of b a c t e r i a were grown on NGA f o r 4 days. Suspensions of 10^ and 10^ CFU/ml were prepared and test e d as above. Ps-346 was grown on NGA f o r 4 days, suspended i n SDW and adjusted to 10^ CFU/ml. This suspension was tested f o r INA before and a f t e r heat t r e a t i n g i t o fo r 20 minutes at 121 C. A 4-day-old NGA c u l t u r e of Ps-346 was exposed to fo r m a l i n vapors f o r 20 minutes. (To confirm that a l l b a c t e r i a were k i l l e d , a loop of c e l l s was streaked on a f r e s h NSA p l a t e and no growth was noted a f t e r 48 hours.) The k i l l e d b a c t e r i a from the p l a t e were suspended i n SDW and adjusted to 10^ c e l l s / m l . The INA of the suspension was then t e s t e d . - 62 -Four 6-week-old raspberry p l a n t s cv Willamette were sprayed w i t h a suspension of 10^ CFU/ml of Ps-346 or SDW. The p l a n t s were put i n t o a o growth chamber at -2 C for 4 hours and then returned to the greenhouse. A f t e r 24 hours, i n j u r y to the plants was recorded. In an attempt to c o r r e l a t e frequency of f r o s t s w i t h disease i n c i d e n c e , the o numbers of nights during which temperatures of -1.8 C or lower were recorded at the Abbotsford A i r p o r t , 2 km from the Clearbrook S u b s t a t i o n , between March 25 and May 31 for the years 1973-1978 were obtained. The period between March 25 and May 31 was chosen because the raspberry buds have u s u a l l y broken dormancy by March 25 and the disease u s u a l l y was n o t i c e a b l e i n A p r i l and May. SURVEY FOR BACTERIA ANTAGONISTIC TO I\_ SYRINGAE Several d i f f e r e n t b a c t e r i a representing the normal m i c r o f l o r a on raspberry plants i s o l a t e d from commercial raspberry p l a n t i n g s i n the Clearbrook-Abbotsford area i n the summer of 1975 were obtained from Dr. R.J. Copeman. These b a c t e r i a along w i t h P^ syringae i s o l a t e s from F o r s y t h i a , were tested to determine i f they could i n h i b i t P^ syringae from raspberry. B a c t e r i a l c e l l s from a 24-hour NA c u l t u r e of Ps-346 were suspended i n 2 ml SDW. From t h i s a suspension of 10~* CFU/ml was prepared and 0.1 ml was spread on a NA p l a t e and allowed to dry. One s t e r i l e 6-mm f i l t e r paper d i s c was saturated w i t h a suspension c o n t a i n i n g 10^ CFU/ml of each of the suspected antagonists and then placed immediately on the lawn of Ps-346. F i v e d i s c s were placed on each p l a t e . A f t e r the p l a t e s were incubated for 3 days at 27°C, the zone of i n h i b i t i o n from the edge of the d i s c to the beginning of the b a c t e r i a l growth . was measured. - 63 -CULTIVAR SUSCEPTIBILITY TRIALS Several c u l t i v a r s of raspberry were tested for t h e i r s u s c e p t i b i l i t y to P.  syringae i n f e c t i o n i n greenhouse t e s t s . Root c u t t i n g s of c u l t i v a r s Haida, M a i l i n g Promise, M a i l i n g Leo, Meeker, and Willamette and the r e c e n t l y developed c u l t i v a r s C h i l c o t i n , Nootka and Skeena were obtained from the A g r i c u l t u r e Canada Red Raspberry Breeding Program i n B.C. Three leaves on each of three 6-week-old p l a n t s were sprayed w i t h a d i r e c t stream of a suspension of 10^ CFU/ml of Ps-346 from a hand sprayer. One plant of each c u l t i v a r was s i m i l a r l y sprayed w i t h s t e r i l e water. Leaves appeared water-soaked where they had been sprayed. Inoculated plants were placed i n a mist chamber i n the greenhouse f o r 7 days at which time they were rated f o r symptoms. Ratings were based on the number of sprayed leaves that showed symptoms and the s e v e r i t y of these symptoms expressed according to the f o l l o w i n g scheme: 0 - no leaves i n f e c t e d 1 - one l e a f s l i g h t l y i n f e c t e d 2 - one or two leaves i n f e c t e d with necrosis at areas d i r e c t l y sprayed 3 - at l e a s t two leaves i n f e c t e d w i t h n e c r o s i s spreading from the area d i r e c t l y sprayed 4 - a l l sprayed leaves i n f e c t e d w i t h necrosis spreading from the area d i r e c t l y sprayed. These t r i a l s were repeated at l e a s t once for each c u l t i v a r . - 64 -RESULTS ANTISERUM PRODUCTION AND TESTING The antiserum obtained a f t e r four weekly i n j e c t i o n s had a t i t r e of 5,120 as determined i n drop a g g l u t i n a t i o n t e s t s . Subsequent bleedings f o r 3 months had an i d e n t i c a l t i t r e but a f t e r that time the t i t r e dropped to 2,560 and the r a b b i t was s a c r i f i c e d . No a g g l u t i n a t i o n occurred when e i t h e r the b a c t e r i a l suspension or the antiserum alone was mixed with PBS. A g g l u t i n a t i o n d i d occur w i t h both P^ syringae i s o l a t e s and the P_^  p h a s e o l i c o l a i s o l a t e but not w i t h the P^ m a r g i n a l i s i s o l a t e . The normal r a b b i t serum d i d not react w i t h any of these four i s o l a t e s ( F i g . 7). A f t e r c r o s s - a b s o r p t i o n w i t h P. p h a s e o l i c o l a , the Ps-346 antiserum no longer reacted w i t h P_^  p h a s e o l i c o l a but s t i l l reacted w i t h Ps-346 ( F i g . 8 ) . I t reacted w i t h a l l 51 P^ syringae i s o l a t e s recovered from diseased rasp-b e r r i e s from the Clearbrook s u b s t a t i o n (Table 10). Three of these I s o l a t e s d i d not produce a f l u o r e s c e n t pigment when grown on KMB. Of the s i x Richmond P. syringae i s o l a t e s t e s t e d , four reacted s t r o n g l y , one reacted weakly and one d i d not react w i t h the antiserum. Authentic P^ syringae i s o l a t e s from other hosts from C a l i f o r n i a and Wisconsin reacted s t r o n g l y w i t h the antiserum. None of the other 41 b a c t e r i a tested reacted w i t h the antiserum. B a c t e r i a l c e l l s s t ained s t r o n g l y w i t h FITC—conjugated antiserum i n the smears of Ps-346, and weakly with P^ p h a s e o l i c o l a and one of the 10 other b a c t e r i a commonly found on diseased raspberry i n the summer of 1976 ( F i g . 9). The photomicrographs i n F i g . 9 were a l l taken w i t h i d e n t i c a l exposure times and m a g n i f i c a t i o n s . C e l l s of P_^  m a r g i n a l i s and the other nine b a c t e r i a were not s t a i n e d . The other bacterium that stained was rod-shaped w i t h s l i g h t l y - 65 -F i g . 7 A g g l u t i n a t i o n r e a c t i o n of Pseudomonas syringae antiserum ( l e f t ) and normal antiserum ( r i g h t ) w i t h homologous c u l t u r e Ps-346 (a) and Pseudomonas m a r g i n a l i s (b). 6 x m a g n i f i c a t i o n . - 66 -a b F i g . 8 A g g l u t i n a t i o n r e a c t i o n of Pseudomonas syringae, Ps-346 ( l e f t ) and Pseudomonas p h a s e o l i c o l a , Pp-1 ( r i g h t ) w i t h Ps-346 antiserum (a) and Ps-346 antiserum that had been cross-absorbed wit h P^ p h a s e o l i c o l a (b). 6 x m a g n i f i c a t i o n . - 67 -Table 10. Reactions of various b a c t e r i a l i s o l a t e s w i t h cross-absorbed Pseudomonas syringae, Ps-346 antiserum I s o l a t e tested Number of I s o l a t e s Producing Each Reaction Type ^ _ Strong Weak No Reaction P. syr ingae (raspberry, Clearbrook, B.C.) 51 0 0 P. syringae (raspberry, Richmond, B.C.) 4 1 1 Ps-1 L i l a c , Wisconsin 1 0 0 Ps-2 F o r s y t h i a , B.C. 0 1 0 Ps-3 F o r s y t h i a , B.C. 0 0 1 Ps-4 F o r s y t h i a , B.C. 0 0 1 Ps-5 Lima bean, Wisconsin 1 0 0 Ps-6 Pear, Wisconsin 1 0 0 Ps-7 Bean, Wisconsin 1 0 0 Ps-8 Almond, C a l i f o r n i a 1 0 0 Ps-9 Pear, C a l i f o r n i a 1 0 0 Ps-10 Peach, C a l i f o r n i a 1 0 0 Other pseudomonads 0 0 5 (raspberry, Clearbrook, B.C.) Other b a c t e r i a (raspberry, Clearbrook, B.C.) 0 0 41 E r w i n i a carotovora, Ec-1, potato, B.C. 0 0 1 E r w i n i a amylovora, Ea-1, pear, Ontario 0 0 1 - 68 -c F i g . 9 Bacterial cells stained with FITC-conjugated, Ps-346 antiserum that had been cross-absorbed with Pseudomonas phaseolicola. a) Pseudomonas syringae, Ps-346 (b) P_^  phaseolicola (c) unidentified bacterium commonly isolated from raspberry (400 x magnification, 2 sec exposures). - 69 -larger cells than syringae. When grown on NSA, this bacterium produced large, mucoid colonies that were almost clear. This bacterium did not react with the cross-absorbed Ps-346 antiserum in drop agglutination tests. OVERWINTERING POPULATIONS OF I\ SYRINGAE IN RASPBERRY BUDS P. syringae was recovered from about 30% of the six-bud samples from the dormant raspberry canes before they were sprayed with the bacterial suspension (Fig. 10). In the samples that did contain P^ syringae, the mode (the number 4 of bacteria that was most commonly recovered from the buds) was 10 . In the sections that were sprayed with bacteria, 92% of the bud samples contained P. 6 syringae with a mode of 10 CFU per sample immediately after spray. By 4 December 17, one month later the mode had returned to 10 CFU per sample and remained at this level for the rest of the winter. The natural populations of P. syringae in the water-sprayed buds were similar to the populations in the buds sprayed with the bacterial suspension from January 14 to March 18. Of 196 P_^  syringae isolates recovered from the bud samples, 190 were oxidase negative, produced a fluorescent pigment and did react in drop agglutination tests with the Ps-346 antiserum. The remaining six isolates were oxidase negative, produced a fluorescent pigment but reacted weakly or not at a l l with the antiserum. These isolates, when subsequently subjected to additional biochemical tests, were identified as P. syringae. SUMMER SURVIVAL OF I\ SYRINGAE P. syringae survived as an epiphyte on raspberry leaves in greenhouse 3 tests. Approximately 10 CFU/leaf could be recovered from healthy raspberry - 70 -£:£: sprayed w i t h j J s p r a y e d w i t h a iiiiiiiil w a t e r j j bac te r ia l suspension before a f t e r spray spray F i g . 10 Populations of Pseudomonas syringae recovered from raspberry buds throughout the winter of 1976-1977. Top graph gives percentage of six-bud samples that contained P. syringae on each sampling date. Bottom graph shows the l o g of the most common number of b a c t e r i a (the mode) recovered per six-bud sample on each sampling date. - 71 -leaves 4 and 6 weeks a f t e r these plants had been sprayed with a suspension of g 10 CFU/ml. S i m i l a r l y , P. syringae was recovered from four of seven healthy 4 l e a f samples from raspberry plants i n the f i e l d . An average of 10 CFU/leaf were recovered from those samples c o n t a i n i n g P_^  syringae. Four r e p r e s e n t a t i v e i s o l a t e s caused symptoms on raspberry plants i n greenhouse p a t h o g e n i c i t y t e s t s . P. syringae was recovered from 16 of 18 l e a f spot samples. Leaf spots from which P^ syringae was i s o l a t e d ( F i g . 11) were t y p i c a l l y 2 to 5 mm i n diameter with brownish-red centers surrounded by yellow halos. These spots would enlarge, turn brown and often coalesce w i t h nearby spots. I s o l a t e s recovered from f i e l d l e a f samples when i n o c u l a t e d onto young raspberry p l a n t s produced symptoms s i m i l a r to those on the n a t u r a l m a t e r i a l ( F i g . 12) except that c u r l i n g and savoying of the young expanding leaves i n the greenhouse t e s t s were also noted. T y p i c a l P. syringae i s o l a t e s were recovered from these i n o c u l a t e d leaves. ICE NUCLEATION ACTIVITY The a b i l i t y of a P^ syringae i s o l a t e to cause i c e n u c l e a t i o n depended on se v e r a l f a c t o r s but E r w i n i a carotovora ( i n c l u d e d as a c o n t r o l ) was never found to be i c e n u c l e a t i o n a c t i v e ( F i g . 13). Culture medium e f f e c t e d i c e n u c l e a t i o n a b i l i t y (INA) as a suspension of 10^ CFU/ml of Ps-346 grown on NGA was 9 n u c l e a t i o n a c t i v e w h i l e a concentration of 10 CFU/ml was r e q u i r e d f o r INA i f the b a c t e r i a were grown on KMB ( F i g . 14). The age of the c u l t u r e was a l s o important. A suspension of 10 7 CFU/ml of a 4-day-old c u l t u r e grown on NGA 9 was n u c l e a t i o n a c t i v e w h i l e a suspension of 10 CFU/ml was required i f the - 72 -F i g . 11 Raspberry l e a f from a n a t u r a l l y - i n f e c t e d plant from the Clearbrook Substation showing t y p i c a l l e a f spots (a) and l e a f spots magnified 30 x (b). - 73 -F i g . 12 Leaf spots from n a t u r a l l y - i n f e c t e d raspberry plants at the Clearbrook Substation from which Pseudomonas syringae was isolated (a. and b.) and l e a f spots on a six-week-old raspberry plant four days af t e r spray inoculation with P^ syringae under greenhouse conditions ( c ) . - 74 -F i g . 13 Ice n u c l e a t i o n a c t i v i t y t e s t showed Pseudomonas syringae, Ps-346 (top) to be ice n u c l e a t i o n a c t i v e and E r w i n i a carotovora to be i n a c t i v e (bottom). Concentrations of bacteria/ml were (from l e f t to 9 7 5 r i g h t ) 10 ^ 10 , 10 and SDW. F i g . 14 E f f e c t of c u l t u r e medium on i c e n u c l e a t i o n a c t i v i t y . Four-day-old c u l t u r e grown on NGA (top) and on KMB (bottom). Concentrations of 9 7 5 bacteria/ml were (from l e f t to r i g h t ) 10 , 10 , 10 and SDW. c u l t u r e was 2 days o l d ( F i g . 15). INA a l s o v a r i e d among c u l t u r e s . Ps-205 and Ps-346 were s i m i l a r i n t h e i r response to a l l f a c t o r s except that 10 times fewer Ps-205 b a c t e r i a were required to induce i c e n u c l e a t i o n as Ps-346 ( F i g . 16). Of 48 P^ syringae c u l t u r e s recovered from b l i g h t e d red raspberry p l a n t s i n the s p r i n g and summer of 1976, 43 were i c e n u c l e a t i o n a c t i v e . Of 28 P.  syringae c u l t u r e s recovered from buds i n the winter of 1976-1977, 22 were i c e n u c l e a t i o n a c t i v e . No other b a c t e r i a from red raspberry were i c e n u c l e a t i o n a c t i v e i n these t e s t s . A suspension of 10^ CFU/ml of P^ syringae was i c e n u c l e a t i o n a c t i v e before but not a f t e r heat-treatment. The formaldehyde-killed b a c t e r i a l suspension was also i c e n u c l e a t i o n i n a c t i v e . When raspberry plants were sprayed w i t h Ps-346, placed at -2°C for 4 hours and then l e f t i n the greenhouse, necrosis was evident on the p l a n t s by 12 hours a f t e r i n o c u l a t i o n . Stems and leaves turned brown and e v e n t u a l l y became blackened. The symptoms were s i m i l a r to those t y p i c a l of b a c t e r i a l b l i g h t except the t i s s u e s became very dry. P l a n t s that were sprayed w i t h water and then given the 4-hour freeze treatment d i d not develop these symptoms. Temperatures of -1.8°C or lower were recorded two or more times i n the years when b l i g h t was a problem (Table 11). In the years when b l i g h t was not seen one or no f r o s t s were recorded except i n 1965 and 1975 when s i x and f i v e such f r e e z i n g periods were recorded r e s p e c t i v e l y . Temperatures of -3.5°C or lower were only noted once i n t h i s 16 year p e r i o d , i n 1975. - 76 -F i g . 15 E f f e c t of culture age on ice nucleation a c t i v i t y of Pseudomonas syringae, Ps-346 c e l l s grown on NGA for two days (top) and four days (bottom). Concentrations of bacteria/ml were (from l e f t to r i g h t ) 10 9, 10 7, 10 5 and SDW. F i g . 16 D i f f e r i n g ice nucleation a b i l i t y of suspensions of two d i f f e r e n t Pseudomonas syringae s t r a i n s , Ps-205 (top) and Ps-346 (bottom) both grown on NGA for four days. Concentrations of bacteria/ml were (from l e f t to r i g h t ) 1Q9, 1Q7, 1Q6, 1Q5 and SDW. - 77 -Table 11. Numbers of nights during which temperatures of -1.8°C or lower were recorded i n the years since b a c t e r i a l b l i g h t of raspberry was f i r s t seen Year Number of nights temperatures of -1.8°C were recorded* 1963 1 + 1964 3 1965 6 1966 0 1967 1 +1968 3 1969 0 +1970 6 1971 1 *1972 2 +1973 2 1974 1 1975 5 1976 0 1977 1 1978 0 *Data from Abbotsford A i r p o r t taken from Monthly Records of Canada M e t e r o l o g i c a l Information, F i s h e r i e s and Environment Canada + f i r s t year b a c t e r i a l b l i g h t was seen i n the Lower Fraser R i v e r V a l l e y +years when b a c t e r i a l b l i g h t was reported to be widespread - 78 -BACTERIA ANTAGONISTIC TO P_;_ SYRINGAE Three b a c t e r i a from the normal f l o r a of the raspberry were found to be a n t a g o n i s t i c to Ps-346. An 8-mm zone of i n h i b i t i o n surrounded i s o l a t e UN-1 on a lawn of Ps-346 ( F i g . 17). I s o l a t e s UN-2 and Ps-2 caused 3-mm zones of i n h i b i t i o n on a Ps-346 lawn. CULTIVAR SUSCEPTIBILITY TRIALS Raspberry c u l t i v a r s seemed to d i f f e r i n t h e i r s u s c e p t i b i l i t y to P. syringae i n f e c t i o n (Table 12). Although d e f i n i t e d i f f e r e n c e s could not be e s t a b l i s h e d i n t h i s p r e l i m i n a r y study there was a continuum i n s u s c e p t i b i l i t y from M a i l i n g Leo which was rated most s u s c e p t i b l e to C h i l c o t i n which was most r e s i s t a n t ( F i g s . 18 and 19). - 19 -F i g . 17 Zone of i n h i b i t i o n of growth of Pseudomonas syringae, Ps-346 (arrow) by an u n i d e n t i f i e d bacterium from raspberry. - 80 -Table 12 Ratings of raspberry cultivars for susceptibi l i ty to Pseudomonas  syringae infection Cultivar T r i a l Date Nov, . 17 Feb. 11 Feb. 17 March 2 March 13 Mean Haida 1.5* 1.3 1.4 Mailing Promise 1.8 1.8 1.8 Mailing Leo 3.0 2.3 2.3 2.5 Meeker 2. 2 2.0 2.0 2.1 Willamette 1. .7 1.5 2.3 1.8 Chilcotin 1.3 .6 1.0 Nootka 1.3 2.0 1.7 Skeena 1. 3 2.0 1.5 2.6 1.9 ratings vary from 0-resistant to 4-highly susceptible - 81 -F i g . 18 a. Symptoms on raspberry c u l t i v a r M a i l i n g Leo caused by spray i n o c u l a t i o n s w i t h Pseudomonas syringae ( l e f t ) and water ( r i g h t ) , b. Symptoms on raspberry c u l t i v a r Willamette caused by spray i n o c u l a t i o n w i t h water ( l e f t ) and P. syringae ( r i g h t ) . - 82 -a b F i g . 19 Symptoms and corresponding disease r a t i n g s f o r raspberry c u l t i v a r s . a. Meeker ( l e f t ) r a t i n g - 3 and Willamette ( r i g h t ) r a t i n g - 2. b. Skeena ( l e f t ) r a t i n g - 2 and C h i l c o t i n ( r i g h t ) r a t i n g - 1. - 83 -DISCUSSION The cross-absorbed antiserum developed against i s o l a t e Ps-346 proved to be a val u a b l e t o o l f or the d e t e c t i o n of P^ _ syringae from diseased raspberry t i s s u e s from the Clearbrook area. A l l of the i s o l a t e s recovered i n the summer of 1976 and i d e n t i f i e d as P^ syringae i n biochemical t e s t s reacted i n drop a g g l u t i n a t i o n t e s t s w i t h the antiserum. Of the P^ syringae i s o l a t e s recovered from raspberry buds during the winter of 1976-1977, 97% reacted with the antiserum. The usefulness of t h i s antiserum f or d e t e c t i o n of P^ syringae from other hosts or from other areas i s not c e r t a i n . The antiserum reacted w i t h a l l a u thentic c u l t u r e s from C a l i f o r n i a and Wisconsin which had been i s o l a t e d from a v a r i e t y of hosts. However, t h i s antiserum reacted s t r o n g l y w i t h only four of s i x raspberry i s o l a t e s from Richmond, B.C. and d i d not react s t r o n g l y w i t h any of the three F o r s y t h i a i s o l a t e s from B.C. These data i n d i c a t e that there may be s e r o l o g i c a l l y d i f f e r e n t s t r a i n s of P^ _ syringae present i n B.C. This i s not unexpected since Otta and E n g l i s h (74) found 10 d i f f e r e n t serotypes of P^ syringae from v a r i o u s hosts. Immunodiffusional a n a l y s i s w i t h antiserum prepared against other i s o l a t e s of P^ syringae would be required to confirm t h i s s u s p i c i o n . Because a l l of the P_^  syringae i s o l a t e s that d i d not react w i t h the antiserum produced a fl u o r e s c e n t pigment and were oxidase negative, the scheme given i n F i g . 20 can be used i n future work for quick d e t e c t i o n of P.  syringae. With t h i s scheme, d e t e c t i o n of the t y p i c a l P. syringae i s o l a t e s , which accounted for greater than 90% of the i s o l a t e s recovered, can be com-pl e t e d i n 2 days and the remaining a t y p i c a l i s o l a t e s can be i d e n t i f i e d i n - 84 -an add i t i ona l 2 days. Before the antiserum was employed, 6 to 10 days were required to i d e n t i f y an i s o l a te as P. syringae. One reservat ion remains, however, due to the f ind ing by Pepin et _a_l. (79) of pathogenic P_^  syringae i so l a te s that d id not produce a f luorescent pigment. In th is current study such i so l a te s were found and a l l of them reacted with the antiserum so they would be detected by the scheme in F i g . 20. Problems would a r i se i f i s o l a te s are found in the future that do not react with the antiserum and do not produce f luorescent pigment. The FAS technique could not be rou t ine l y used for the detect ion of P.  syringae from raspberry. Ce l l s of an un iden t i f i ed saprophytic bacterium commonly present in the m ic ro f l o ra of raspberry were n o n - s p e c i f i c a l l y s ta ined. Although the unknown bacterium had c e l l s larger than those of P.  syringae and did not s t a in as b r i g h t l y i t was s t i l l d i f f i c u l t to d i f f e r e n t i a t e these c e l l s from,P. syringae c e l l s . Cross-absorpt ion of the antiserum with the unknown was not attempted because P_^  phaseo l i co la c e l l s s t i l l s ta ined f a i n t l y with the FITC-conjugated antiserum cross-absorbed with P.  phaseo l i co l a . The increased s e n s i t i v i t y of the FAS technique proved to be i t s downfall here. Even s l i gh t react ions between antiserum and b a c t e r i a l c e l l give pos i t i ve r e s u l t s . In the drop agg lut inat ion method, however, greater react ion of antiserum with b a c t e r i a l c e l l s must occur for the formation of the large p r e c i p i t a t e needed for v i s u a l i z a t i o n at 6 x magn i f i ca t ion. The drop agg lut inat ion technique was therefore pre fer red in th i s ep idemio log ica l study. The recovery of P_^  syringae from the buds of dormant raspberry canes before they were sprayed with a b a c t e r i a l suspension ind icated P^ syringae n a t u r a l l y inhabits the buds of raspberry p lants . The high populations of P_^  - 85 -RAISED, MUCOID COLONY ON NSA AGGLUTINATION WITH PSEUDOMONAS SYRINGAE ANTISERUM PSEUDOMONAS SYRINGAE NO AGGLUTINATION WITH PSEUDOMONAS SYRINGAE ANTISERUM OXIDASE-NEGATIVE AND PRODUCES A FLUORESCENT PIGMENT OXIDASE POSITIVE OR DOES NOT PRODUCE FLUOR-ESCENT PIGMENT PSEUDOMONAS SYRINGAE NOT PSEUDOMONAS SYRINGAE F i g . 20 Scheme f o r r a p i d d e t e c t i o n of Pseudomonas syringae from raspberry. - 86 -syringae i n the buds a f t e r spraying with the b a c t e r i a l suspension qu ick ly dropped o f f to a l e v e l equal to the natura l populations i n the water-sprayed buds. This would suggest that there is an optimum number of P_^  syringae which can be na tu ra l l y supported i n the buds. Cameron (18) and Crosse (24) a lso found P. syringae to overwinter in the buds of Rosaceous hosts. It was sur -p r i s i n g to f ind P_^  syringae overwintering i n the raspberry buds here because very l i t t l e b a c t e r i a l b l i gh t was seen in the f i e l d s i n the two years p r i o r to monitoring the buds and i t had been suspected that no P^ syringae would be found na tura l l y i n the f i e l d s . The i n c i t a n t of b a c t e r i a l b l i gh t therefore i s not dependent on the presence of an a l ternate host for overwinter ing. This f ind ing does not preclude the p o s s i b i l i t y that the in fec ted l i l a c was the o r i g i n a l source of inoculum. From the raspberry buds the bac te r i a are r e a d i l y ava i l ab le for i n f e c t i o n of the l a t e r a l s that develop from the buds. The i s o l a t i o n of pathogenic P^ syringae from healthy leaves throughout the summer ind icated the pathogen is present on the raspberry during th i s time although no symptoms are evident. This concurs with the f ind ing of P. syringae e x i s t i n g as an epiphyte on the leaves of peach and almond (37). That popula-3 t ions of 10 CFU/leaf of syringae survived on raspberry leaves for at leas t 6 weeks i n greenhouse tests is further evidence that a stable populat ion can be maintained on apparently healthy leaves. The l ea f spot symptom f i r s t observed on raspberry in the summer of 1976 was shown to be caused by P^ syringae. While these spots could be found throughout the summer, recovery of bac te r i a from them proved to be more d i f f i c u l t a f ter a dry per iod. The 2 of 18 attempts to i s o l a te P_^  syringae from lea f spots that f a i l e d were done on leaves c o l l e c t e d during a dry per iod - 87 -i n J u l y . Crosse (22) a l s o found i s o l a t i o n s from l e a f spots to be more d i f f i -c u l t when the l e a f became mature and the n e c r o t i c t i s s u e became dry. This l e a f spot phase i s an important part of the disease c y c l e because i t provides a means for the bacterium to survive the dry, hot summer i n a place where i t i s r e a d i l y a v a i l a b l e to cause i n f e c t i o n i n the f a l l when more s u i t a b l e environmental c o n d i t i o n s r e t u r n . The b a c t e r i a can also be r a i n - s p l a s h e d from the leaves onto the developing buds of the f i r s t year canes and the disease c y c l e i s complete ( F i g . 21). While i t i s p o s s i b l e to propose a disease c y c l e , the reasons why the disease i s so e r r a t i c s t i l l remain a mystery. Very l i t t l e change has occurred i n the host p l a n t . The same c u l t i v a r of raspberry i s grown now as was grown i n the years when the disease was a problem. No c u l t u r a l p r a c t i c e s or n a t u r a l processes that might have changed the degree of wounding of the host p l a n t have occurred during these years. Chicken mature i s no longer a p p l i e d to raspberry f i e l d s i n large q u a n t i t i e s which may somewhat reduce the sus-c e p t i b i l i t y of the host to disease but t h i s alone cannot account f o r the disappearance of b a c t e r i a l b l i g h t . No marked change i n weather patterns has occurred over the years. Wet, cool periods are recorded every s p r i n g and f a l l i n B.C. There i s i n d i c a t i o n that f r e e z i n g temperatures do have an e f f e c t on the occurrence of b l i g h t . Amy et a l . (2) found that damage to corn p l a n t s sprayed w i t h syringae occurred at temperatures of -3.5 to -4°C. Temperatures i n t h i s range were recorded only one night i n B.C. during the period of March 25 to May 30 i n the years 1963 to 1978. A temperature of , o -4 C was recorded i n 1975, a year when b l i g h t was not a problem. Maki e_t a l . (63) found suspensions of J?^ syringae would freeze from -1.8 to -3°C i n - 88 -WINTER Organism overwinters i n buds on dormant canes Dead bud may occur over winter FALL Blight symptoms may recur on succulent growth SPRING Bl i g h t of young l a t e r a l s and shoots SUMMER Organism survives as epiphyte on leaves May cause l e a f spots F i g . 21 Proposed disease cycle of b a c t e r i a l b l i g h t of raspberry caused by Pseudomonas syringae. - 89 -l a b o r a t o r y t e s t s and i n a p r e l i m i n a r y t e s t i n t h i s current study damage to young raspberry plants sprayed w i t h syringae was recorded a f t e r the p l a n t s o had been exposed to -2 C for 4 hours. In years when b l i g h t was a problem (1968, 1970, 1972, 1973) temperatures of -2°C or lower were recorded two or more times between March 25 and May 31. One or no such f r o s t s occurred i n the other years when b a c t e r i a l b l i g h t was not n o t i c e d except i n 1965 and 1975 when temperatures below -2°C were recorded s i x and f i v e times r e s p e c t i v e l y . So except f o r these 2 years a higher number of f r o s t s were recorded i n years when b l i g h t was a problem than i n years when i t was not seen. Further experimen-t a t i o n must be done to f i r s t e s t a b l i s h a b s o l u t e l y the r o l e of freeze i n j u r y caused by P^ syringae i n b a c t e r i a l b l i g h t and second, the exact c o n d i t i o n s required f o r i c e n u c l e a t i o n ( c o n c e n t r a t i o n of b a c t e r i a needed, the exact temp-erature and time of exposure to t h i s temperature). There i s one other p o s s i b l e e x p l a nation f o r the change i n the incidence of b a c t e r i a l b l i g h t . The i n c i t a n t may have changed g e n e t i c a l l y over the years to a form that i s not h i g h l y v i r u l e n t . This s u p p o s i t i o n i s based on the f a c t that i t was d i f f i c u l t to produce symptoms on plants when conducting patho-g e n i c i t y t e s t s i n t h i s study even with c u l t u r e s f r e s h l y i s o l a t e d from diseased raspberry p l a n t s . In the o r i g i n a l work done on t h i s disease, Pepin et a l . (79) d i d not have problems reproducing symptoms i n greenhouse t e s t s when f r e s h l y i s o l a t e d c u l t u r e s of P^ syringae were used. Also i n the Geotrichum candidum bioassay raspberry s t r a i n s of P^ syringae from B.C. u s u a l l y caused zones of i n h i b i t i o n ranging up to 10-mm wide w h i l e i s o l a t e s from various hosts tes t e d by Gross (41) i n C a l i f o r n i a produced i n h i b i t i o n zones ranging up to 20 mm. I t - 90 -may be that highly virulent isolates of syringae were on the infected l i l a c found in the f i r s t f ie ld infected with bacterial bl ight. If i ts virulence was due to a plasmid, as has been suggested for other pathogenic bacteria (55) , i t would have been passed to the natural population of P_^  syringae on the raspberry. The plasmid may have been lost from the isolates in the f ie ld as they have a tendency to be lost from the bacteria while in storage ( 76 ) . The role of P_^  syringae in the dead bud syndrome could not be exactly established. The ice nucleation ab i l i ty of syringae provides an explanation for the syndrome but further experimentation in the laboratory must be done to confirm that the presence of syringae in buds exposed to freezing temperatures increases the chance of bud death. Since the popula-tions of P^ syringae in the raspberry buds were monitored over the winter of 1976-1977, i t was hoped that a relationship between bacterial population levels and the number of dead buds in the spring could be found. However, spur bl ight , caused by Didymella aplanata (Niessl) Sacc. was also prevalent and could also have been responsible for bud deaths. It was therefore not possible to ascertain the cause of the bud deaths seen. Only under controlled environment conditions using raspberry canes free of spur blight can the relationship of dead bud and the ice nucleation ab i l i ty of syringae be established. No chemical control methods were attempted during this study because there was no natural outbreak of the disease in the f ie ld and because of the d i f f i cu l ty of infecting plants in the greenhouse. If a natural outbreak does occur i t would be interesting to attempt to control the disease with sodium tartrate as suggested by Sands and Mclntyre (86) . The b a c t e r i a found i n t h i s study that were a n t a g o n i s t i c to P_^  syringae have p o t e n t i a l i n c o n t r o l of b a c t e r i a l b l i g h t . Because they were a n a t u r a l component of the raspberry m i c r o f l o r a , attempts to increase the populations of the antagonists by spraying suspensions onto host plants pose no threat to the he a l t h of the host p l a n t . Even i f the n a t u r a l populations of the antagonists cannot be increased f or long periods of time as Crosse (25) found, there i s s t i l l hope f o r c o n t r o l because the antagonists produce substances that d i f -fused out i n t o the agar medium. I f these substances are produced i n l i q u i d c u l t u r e s i t may be p o s s i b l e that the substances could be sprayed on the p l a n t s . Further experimentation must be done to tes t these p o s s i b i l i t i e s . Some c u l t i v a r s were found to be more r e s i s t a n t to P^ syringae i n f e c t i o n than others. This confirms the report by Pepin et: al. (79). C h i l c o t i n showed l e s s s u s c e p t i b i l i t y to i n f e c t i o n that most c u l t i v a r s . I t s Newburgh parent was also found to be r e s i s t a n t i n t e s t s done by Pepin et a l . (79). Nootka was found to d i s p l a y a s u s c e p t i b i l i t y s i m i l a r to i t s Willamette parent. The r e s u l t s obtained i n these c u l t i v a r t r i a l s must be accepted w i t h c a u t i o n because of the d i f f i c u l t y of o b t a i n i n g i n f e c t i o n i n greenhouse t r i a l s and the small number of times these t r i a l s were r e p l i c a t e d . Observations on i n f e c t i o n of the c u l t i v a r s during n a t u r a l i n f e c t i o n i n the f i e l d must be made before meaningful conclusions about t h e i r r e l a t i v e r e s i s t a n c e s can be made. Many questions remain unanswered but they w i l l remain so u n t i l an epiphy-t o t i c of b a c t e r i a l b l i g h t i s again seen i n the f i e l d s , an event e n l i g h t e n i n g to the academic but d i s a s t r o u s i n the short term to the farmer. - 92 -THESIS SUMMARY 1. T y p i c a l P. syringae i s o l a t e s were confirmed as the causal agent of b a c t e r i a l b l i g h t of raspberry i n B.C. 2. N u t r i e n t sucrose agar was found to be the best medium for i s o l a t i o n of P. .syringae from raspberry t i s s u e s . 3. The biochemical t e s t s that are most u s e f u l for i d e n t i f i c a t i o n of an i s o l a t e as P. syringae are gram r e a c t i o n , oxidase t e s t , a r g i n i n e d i h y d r o l a s e t e s t , polypectate g e l p i t t i n g a b i l i t y , f l u o r e s c e n t pigment production, tobacco h y p e r s e n s i t i v i t y , t o x i n production bioassay, l a c t a t e u t i l i z a t i o n and production of levan when grown on sucrose medium. P^ syringae i s o l a t e s t y p i c a l l y gave negative r e s u l t s f o r the f i r s t four t e s t s and p o s i t i v e r e s u l t s for the l a s t f i v e although some raspberry i s o l a t e s were found that d i d not produce a f l u o r e s c e n t pigment, could not u t i l i z e l a c t a t e or d i d not produce a t o x i n . 4. For p a t h o g e n i c i t y t e s t i n g , the l e a f - s p r a y i n o c u l a t i o n technique was pre-f e r r e d to the stem-prick i n o c u l a t i o n because symptoms developed sooner and fewer b a c t e r i a were required to i n i t i a t e disease. 5. The a b i l i t y to produce t o x i n improved an i s o l a t e ' s pathogenic c a p a b i l i t i e s but t h i s a b i l i t y was not e s s e n t i a l for p a t h o g e n i c i t y . 6. The best method f o r long-term storage of P^ syringae i s o l a t e s was on n u t r i e n t g l y c e r o l agar s l a n t s at 5°C. I s o l a t e s remained v i a b l e and pathogenic f o r 1 year when stored i n t h i s manner. 7. An antiserum prepared against P. syringae was u s e f u l for quick d e t e c t i o n of t h i s organism from raspberry e s p e c i a l l y when used i n conjunction w i t h - 93 -t e s t s f o r levan production on a sucrose medium, f l u o r e s c e n t pigment production and oxidase r e a c t i o n . Three days were required for completion of these t e s t s . 8. 'P_L syringae was found to n a t u r a l l y populate 25-75% of raspberry buds 4 during the winter months. Populations of about 10 CFU/six bud sample are most common i n buds that do contain b a c t e r i a . 9. During the s p r i n g and summer months P^ syringae was found to s u r v i v e as an epiphyte on leaves of raspberry p l a n t s i n the f i e l d . In greenhouse t e s t s , P. syringae survived on healthy raspberry leaves for at l e a s t 6 weeks. P.  syringae was a l s o shown to cause brown spots surrounded by yellow halos on the leaves of raspberry during t h i s time. 10. About 90% of P^ syringae i s o l a t e s from raspberry were i c e n u c l e a t i o n a c t i v e . Raspberry p l a n t s that had been sprayed w i t h a P. syringae suspension and then h e l d at -2°C for 4 hours developed symptoms s i m i l a r to those of b a c t e r i a l b l i g h t w i t h i n 12 hours of the freeze treatment. 11. B a c t e r i a were found i n the normal m i c r o f l o r a of the raspberry that were a n t a g o n i s t i c to P^ syringae i n v i t r o and may have p o t e n t i a l f o r c o n t r o l of b a c t e r i a l b l i g h t . 12. In p r e l i m i n a r y t e s t s , raspberry c u l t i v a r s showed va r y i n g degrees of s u s c e p t i b i l i t y to P_^  syringae i n f e c t i o n i n d i c a t i n g some c o n t r o l of b a c t e r i a l b l i g h t might be achieved by growing r e s i s t a n t v a r i e t i e s . - 94 -L i t e r a t u r e C i t e d 1. A l l a n , E. and A. Kelman. 1977. Immunofluorescent s t a i n procedures for de t e c t i o n and i d e n t i f i c a t i o n of E r w i n i a carotovora var a t r o s e p t i c a . Phytopathology 67:1305-1312. 2. Amy, D.C, S.E. Lindow and CD. Upper. 1976. Frost s e n s i t i v i t y of Zea  mays increased by a p p l i c a t i o n of Pseudomonas syringae. Nature 262:282-284. 3. Backman, P.A. and J.E. De Vay. 1971. 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