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Incidence and survival of Erwinia Carotovora in B.C. Schneider, Frank Frithjof 1977

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THE INCIDENCE AND SURVIVAL OF ERWINIA CAROTOVORA IN B.C. by FRANK FRITHJOF SCHNEIDER B.Sc. (Agr.), U n i v e r s i t y of B r i t i s h Columbia, 1972 A THESIS SUBMITTED IN PARTIAL FULFILMENT OF THE REQUIREMENTS FOR THE DEGREE OF MASTER OF SCIENCE i n the Department of Plant Science i n the Faculty of Agri c u l t u r e We accept t h i s thesis as conforming to the required standard THE UNIVERSITY OF BRITISH COLUMBIA October, 1976 (o) Frank Frithjof Schneider, 1976 In p r e s e n t i n g t h i s t h e s i s i n p a r t i a l f u l f i l m e n t o f the r e q u i r e m e n t s f o r an advanced degree at the U n i v e r s i t y o f B r i t i s h C o lumbia, I agree t h a t the L i b r a r y s h a l l make i t f r e e l y a v a i l a b l e f o r r e f e r e n c e and s t u d y . I f u r t h e r agree t h a t p e r m i s s i o n f o r e x t e n s i v e c o p y i n g o f t h i s t h e s i s f o r s c h o l a r l y p u rposes may be g r a n t e d by the Head o f my Department o r by h i s r e p r e s e n t a t i v e s . I t i s u n d e r s t o o d t h a t c o p y i n g o r p u b l i c a t i o n o f t h i s t h e s i s f o r f i n a n c i a l g a i n s h a l l not be a l l o w e d w i t h o u t my w r i t t e n p e r m i s s i o n . Department o f ~P I. ft-K^ Scte^^/ The U n i v e r s i t y o f B r i t i s h Columbia 2075 Wesbrook Place Vancouver, Canada V6T 1W5 D a t e N r»u 2 . 19 i ABSTRACT The extent of tuber contamination with Erwinia carotovora in B.C. grown seed potatoes was determined by incubating tubers in a moist, anaerobic atmosphere. Only 2.1% and 11.3% of the tubers indexed in 1972 and 1975, respectively, were found to be latently-infected with 15. carotovora. Recoveries within individual lots ranged from 0 to a high of 27.7%. Both var. carotovora and var. atroseptica were recovered from tubers with equal frequency. No correlation was found between the field incidence of blackleg and the degree of latent tuber infection. Representative isolates of both varietal forms were able to infect faba beans, cucumbers and peppers while none was able to infect cereals, forage legumes or common weeds like black nightshade, horsetail, and lamb's quarters. Approximately 50% of the var. carotovora isolates recovered in the surveys incited blackleg symptoms in stem-prick inoculated Erwinia-tested potato stem cuttings grown at 18° C. Pathogenic carotovora isolates were also found to have a much wider host range than the nonpatho-genic carotovora isolates. The additional hosts expressing symptoms were onions, peas, petunias, tobacco, tomato, lady's thumb and Chenopodium  quinoa L. The majority of the pathogenic isolates (73%) reacted with an antiserum prepared against a pathogenic var. carotovora isolate. The atroseptica varietal form was found not to survive in artificially infested fallow field soils beyond 8 weeks under winter conditions and 4 weeks under summer conditions. By contrast var. carotovora was recovered from similarly infested soil at the conclusion of the over-wintering experiments (139 and 225 days). The carotovora form also survived i i 29 days longer in soils in the oversummering experiment. The atroseptica form could not be detected around inoculated seed pieces after 4 weeks. However, var. carotovora was s t i l l recovered from soil in 2 out of 5 replicates at the end of the experiment (70 days). Both pathogenic and nonpathogenic isolates of the carotovora form were easily recovered from the rhizosphere of horsetail, lady's thumb, lamb's quarters, nightshade and redroot pigweed during the f a l l but rarely in similar surveys during the spring. The atroseptica form was recovered only once, from the rhizosphere of a horsetail plant during the September 1974 survey. The atroseptica form could s t i l l be recovered from artificially infested rhizospheres of lamb's quarters at the end of the experiment (140 days) but was not detected in the rhizospheres of lady's thumb after 42 days or in the rhizospheres of horsetail after 112 days. Parallel experiments with var. carotovora indicated that while this varietal form could survive in soils, weed rhizospheres help to maintain a high inoculum potential. Survival on tubers was found to be the only way in which var. atroseptica could survive winter in numbers which were detectable the following spring. Recovery f rom tubers buried 10 cm deep was greater than from those on the surface after 225 days. Surface-borne tubers inoculated with the atroseptica form tended to decay more rapidly which accounted for the lower recovery rate. The survival of var. carotovora was less dependent upon burial of tubers but the recovery rates were generally higher i f tubers were buried. i i i TABLE OF CONTENTS Page ABSTRACT . i TABLE OF CONTENTS ; i i i LIST OF TABLES . , • • • v LIST OF FIGURES v i i ACKNOWLEDGEMENT i x INTRODUCTION . . . . . . . . 1 LITERATURE REVIEW 3 (1) Importance of The Disease . 3 (2) Symptomatology 5 (3) Taxonomy and Etiology 6 (4) Detection, I s o l a t i o n and I d e n t i f i c a t i o n of Erwinia  carotovora 9 (5) Epidemiology 11 (a) The H i s t o r i c a l Approach 11 (b) The Modern Concept of Overwintering 12 (c) Factors Influencing Incidence 14 (d) Re-evaluation of S o i l Survival 17 (e) Host Range 18 (f) Insect Transmission 19 (6) Control 21 (7) Summary and Objectives 24, MATERIALS AND METHODS 26 (1) Incidence of E_. carotovora i n Seed Tubers 26 (a) Source of Tubers 26 (b) Detection Methods 26 (c) Isolate Characterization > 27 Continued.. . i v TABLE OF CONTENTS (Contd.) Page (2) Incidence of E_. carotovora i n Weeds 29 (3) Survival of Erwinia carotovora 32 (a) General Techniques 32 (i) Inoculum Preparation and Inoculation Techniques . 32 ( i i ) Detection and I d e n t i f i c a t i o n Techniques . . . . . . 35 (b) Overwintering Experiment UBC 1974-1975 36 (c) Oversummering Experiment Pemberton 1975 38 (d) Overwintering Experiment Pemberton 1975-1976 40 (e) Overwintering Experiments UBC 1975-1976 . . . . . . . 43 (f) Host Range Studies 44 RESULTS 48 (1) Survey of.Seed Potato Stocks . 48 (.2) Survey of Volunteer Tubers 55 (3) Survey of Weed Rhizospheres 55 (4) Surv i v a l i n S o i l , Tubers and Weed Rhizospheres . 59 (a) Overwintering i n S o i l 59 (b) Overwintering i n Tubers . 67 (c) Overwintering i n Weed Rhizospheres 70 (d) Oversummering i n S o i l at Pemberton i n 1975 75 (5) Host Range Studies 79 DISCUSSION 83 LITERATURE CITED 93 V LIST OF TABLES Table Page I The t e s t s used f o r the d i f f e r e n t i a t i o n of the v a r i e t a l forms of Erwinia carotovora. 30 II An a l p h a b e t i c a l l i s t i n g of the plant species included i n the host range experiment 46 II I A l i s t i n g of the various carotovora and atroseptica i s o l a t e s used i n the alternate host experiment 47 IV Levels of l a t e n t tuber i n f e c t i o n with Erwinia carotovora i n B.C. seed potato stocks harvested i n 1972 49 V Levels of l a t e n t contamination with Erwinia carotovora v a r i e t a l forms i n f r e s h l y harvested seed potato stocks grown i n 1975 . 52 VI Latent Erwinia i n f e c t i o n of seed potato tubers produced i n the Creston V a l l e y i n 1974 53 VII Pathogenicity and s e r o l o g i c a l reactions of Erwinia i s o l a t e s recovered from two d i f f e r e n t Creston seed tuber l o t s 54 VIII The recovery of Erwinia carotovora from overwintering volunteer tubers i n the Pemberton Valley 56 IX The recovery of Erwinia carotovora v a r i e t a l forms from the rhizospheres of common weed species i n the Pemberton Seed Control Area 57 X The s u r v i v a l of Erwinia carotovora i n a r t i f i c i a l l y -i n f e s t e d f i e l d s o i l on the UBC campus 61 XI The overwintering of Erwinia carotovora i n a r t i f i c i a l l y - I n f e s t e d f i e l d s o i l at UBC 1975-1976 . . . 63 XII Survival of Erwinia carotovora i n a r t i f i c i a l l y -i n f e sted s o i l at Pemberton 66 XIII The recovery of Erwinia carotovora v a r i e t a l forms from a r t i f i c i a l l y - i n o c u l a t e d tubers buried i n fallow s o i l on the UBC campus i n 1974 , 68 XIV The s u r v i v a l of Erwinia carotovora i n inoculated potato tubers l e f t on the s o i l surface or buried at 10 cm on the UBC campus i n 1975 69 Continued. v i LIST OF TABLES (Contd.) Table Page XV The recovery of Erwinia carotovora from inoculated tubers overwintering on the surface and buried i n f i e l d s o i l at Pemberton 71 XVI The recovery of Erwinia carotovora from infested h o r s e t a i l rhizospheres under natural f i e l d conditions i n Pemberton 72 XVII The recovery of Erwinia carotovora from rhizospheres of common weed species on the UBC campus 74 XVIII The recovery of s o f t - r o t t i n g Erwinia forms from carotovora- and atroseptica-inoculated potato tubers buried at 10 cm 76 XIX Host reactions at 18° C when inoculated with representative i s o l a t e s of the atroseptica and carotovora v a r i e t a l forms . . . . . . . . . 80 XX Host reactions at 18° C when inoculated with representative i s o l a t e s of Erwinia carotovora v a r i e t a l forms 81 v i i LIST OF FIGURES Figure Page 1 Appearance of Erwinia carotovora. on the selective crystal violet pectate (CVP) medium. (a) Typical pitting after 48 hours growth. (b) Characteristic cross-hatched colony morphology illuminated with oblique light 28 2 The stem-prick method of inoculating potato cuttings, (a) A sterilized toothpick with bacteria at the tip inserted into the axil of the 3rd leaf. (b) Inocu-lation site covered with moistened s t e r i l e cotton batting to prevent desiccation 31 3 The inoculation of tubers for survival studies. (a) Tubers were slashed with a pocket knife. (b) Tubers were soaked in a bacterial suspension. Cc) Tubers were buried at a depth of 10 cm 33 4 The preparation of soilscells for survival studies, (a) Glass ring (9 cm dia) with cheesecloth covering 1 side. (b) Glass ring f i l l e d with a r t i f i c i a l l y infested s o i l . (c) Loaded s o i l c e l l ready for burial. (d) Soil c e l l buried at 10 cm . . . . 34 5 Longitudinal section through the paper cup showing how ste r i l i z e d tuber slices were used to bait s o i l for Erwinia carotovora 37 6 The replicator board used to randomly sample s o i l cores, taken 10 cm away from the decaying seed piece . . 41 7 A replicated treatment consisted of 3 horsetail plants transplanted 34 cm apart 42 8 Symptoms produced in potato cuttings at 18° C, 1 week after stem-prick inoculation with Erwinia  carotovora. (a) Typical blackleg symptoms incited by the atroseptica varietal form. (b) Symptoms similar to blackleg caused by pathogenic isolates of the carotovora form. (c) Localized stem rotting symptoms caused by nonpathogenic isolates of the carotovora form 50 9 Survival of Erwinia carotovora in a r t i f i c i a l l y - i n f e s t e d , nutrient-amended fallow soils at U.B.C. during the winter of 1974-1975. (a) Survival of the atroseptica and the carotovora forms in plastic bags and s o i l cells Continued. LIST OF FIGURES (Contd.) Figure Page with time. (b) S o i l temperature at 10 cm during the study period. (c) A i r temperature during the study period. (d) To t a l p r e c i p i t a t i o n during the experimental period . . . 60 10 Survival of Erwinia carotovora i n a r t i f i c i a l l y -i n f ested fallow s o i l at U.B.C. during the winter of 1975-1976. (a) Survival of the atroseptica and carotovora forms i n s o i l c e l l s with time. (b) S o i l temperature at 10 cm during the study period. (c) A i r temperature during the study period. (d) T o t a l p r e c i p i t a t i o n during the experimental period . . 62 11 Survival of Erwinia carotovora i n a r t i f i c i a l l y -i n f e s t e d fallow s o i l at Pemberton i n the winter of 1975-1976. (a) Survival of the atroseptica and carotovora forms i n s o i l c e l l s with time. (b) A i r temperature during the experimental period. (c) T o t a l p r e c i p i t a t i o n during the study period. (d) Accumulated snowpack during the study period . . . 65 12 Survival of Erwinia carotovora i n a r t i f i c i a l l y -i n f e s t e d fallow s o i l and around inoculated tubers i n Pemberton during the summer of 1975. •(?•',: (a) S u r v i v a l of the atroseptica and carotovora forms i n s o i l c e l l s and around decaying seed tubers buried i n s o i l with time. (b) A i r temperature during the study period. (c) Total p r e c i p i t a t i o n during the experimental period*., . 77 13 Erwinia carotovora i n f e c t i o n s i n stem-prick inoculated faba beans. Isolates of both atroseptica and carotovora v a r i e t a l forms were pathogenic to faba beans 82 ACKNOWLEDGEMENT S The author wishes to express h i s appreciation to Dr. R.J. Copeman, who o r i g i n a l l y suggested the project and who provided the guidance and d i r e c t i o n necessary f o r the successful completion of t h i s t h e s i s . His constructive comments i n i t s preparation and h i s photographic contributions are g r a t e f u l l y acknowledged. Many thanks also to the other members of my committee, Dr. V.C. Runeckles and Dr. N.S. Wright, f o r t h e i r support and encourage-ment. Special thanks go to Dr. N.S. Wright, whose e f f o r t s resulted i n the co-operation of Pemberton seed potato growers. The co-operation of the B.C. seed potato growers was greatly appreciated. The author wishes to express his gratitude to Mr. C l i f f Ronayne and Mr. Tony van Loon who kindly donated t h e i r f i e l d s f o r the Pemberton studies. Thanks also to Dr. R.G. Wilson of the Environment and Land Use Committee Secretariat f o r supplying the c l i m a t o l o g i c a l data f o r the Pemberton Meadows. The author would also l i k e to express h i s appreciation to Mr. Ben Lawson, Mr. Ted Cole and Mr. Paul Froese of the Plant Protection D i v i s i o n (CDA) f o r securing the seed potato samples used i n the survey work. I wish to express my appreciation to Dr. G.W. Eaton, who helped me with the s t a t i s t i c a l analysis of the data. Many thanks, also, go to Dr. N.S. Wright and Mrs. Connie Hyams, X who aided i n the preparation of the antisera used i n t h i s study. The services of Mr. Ilmars Derics i n the preparation of the s o i l c e l l s and i n the photographic work i s g r a t e f u l l y acknowledged. Heartiest thanks should also be extended to the rest of the Plant Science Department for bearing with me during those aromatically unpleasant seed potato surveys. Special thanks go to Mrs. Nancy Sinnott for her moral support throughout the w r i t i n g of t h i s t h e s i s . Acknowledged, also, i s the f i n a n c i a l a i d granted to Dr. R.J. Copeman i n the form of an A g r i c u l t u r e Canada Operating Grant No. 3037. F i n a l l y , I wish to express my gratitude to the B.C. Department of Ag r i c u l t u r e (Plant Pathology Branch) for t h e i r support through Careers 73 and Careers 74 funding. 1 INTRODUCTION Seed potatoes are commonly contaminated by the phytopathogenic b a c t e r i a l species, Erwinia carotovora. The two v a r i e t a l forms of t h i s species inducing losses i n B r i t i s h Columbia seed potato stocks are Erwinia carotovora var. carotovora (Jones) Dye and Erwinia carotovora var. atroseptica (Van Hall) Dye. The former or true soft rot form has h i s t o r i c a l l y been l a r g e l y credited with storage losses while the atroseptica v a r i e t a l type has been known to i n c i t e both storage soft rot and blackleg i n the f i e l d . Recent findings i n d i c a t e that the carotovora form may also be implicated i n the blackleg syndrome. Blackleg of potato i s an economically important disease wherever the crop i s c u l t i v a t e d . Losses may not only be incurred at the storage l e v e l or i n the post-emergent stage but may also r e s u l t from seed piece decay i n the f i e l d . Losses a t t r i b u t e d to t h i s disease and to soft rot vary considerably from one year to the next, reaching epidemic proportions under favorable conditions. The sev e r i t y of such losses i s la r g e l y governed by such factors as the l e v e l s of latent i n f e c t i o n of the seed material, the degree of sanitary precaution exercised at planting time, storage environments, f i e l d conditions and the extent of fecontamination of clean stocks i n the f i e l d . Early attempts to control the blackleg disease by chemical treatments or through the manipulation of c u l t u r a l p ractices have proven f u t i l e . The f a c t that the disease has recently been shown to be tuber-borne has generated new i n t e r e s t i n c o n t r o l . With the advent of the 2 S c o t t i s h Seed Potato C e r t i f i c a t i o n Scheme and, i n p a r t i c u l a r , the v i r u s tested stem cutting (VTSC) program, a new dimension was added to the control arsenal. The use of only "clean" stem cutting material provided the cornerstone for t h i s scheme. A s i m i l a r system has been employed i n B r i t i s h Columbia to produce v i r u s - f r e e potato stocks. More recently, t e s t i n g cuttings f o r Erwinia has become part of t h i s program. I n i t i a l success i n Scotland i n reducing the l e v e l s of f i e l d incidence of blackleg have now been overshadowed by the continuing problem of recontamination of Erwinia-tested stocks i n potato f i e l d s . Surveys of seed potato stocks i n B.C. since the inception of the stem cutting program have r e f l e c t e d no major reduction i n blackleg f i e l d incidence. The f e a s i b i l i t y of producing Erwinia-tested seed potatoes i s dependent upon the i d e n t i f i c a t i o n and elimination of the sources of recontamination for "clean" stocks. I t i s hoped that by eliminating such sources, the l e v e l of iLatent i n f e c t i o n can be reduced. 3 LITERATURE REVIEW (1) Importance of The Disease Blackleg of potatoes i s endemic i n a l l potato growing regions of the world but generally i s more prevalent i n cool, moist l o c a l i t i e s (5,9,10,55,64,80,89,95). F i e l d losses may range from 10-20% i n commercial stocks but generally they seldom exceed 5% of the crop i n seed potato stocks (5^54,61,80,94). However, losses i n seed potato f i e l d s ranging from 30 to 50% have been reported i n Scotland (38) . Probably the most r e l i a b l e records of blackleg have been kept i n the Scottish c e r t i f i c a t i o n program, which since 1962 allows a maximum f i e l d incidence of 2%. The t o t a l acreage rejected f o r blackleg was w e l l under 2% (46). Nevertheless, s t r i c t e r tolerance l e v e l s against blackleg have accounted for one-third of the r e j e c t i o n s or down-grading of Sc o t t i s h seed potato crops (114). I t i s i n t e r e s t i n g to note that where such apparently clean seed has been exported abroad to warmer countries such as I s r a e l or Egypt.for l o c a l production purposes, losses due to the blackleg organism have been as high as 50 to 60% i n the f i e l d (28,50). In addition to causing downgrading of seed, blackleg causes y i e l d losses which vary from year to year and from one f i e l d to the next (81,138). Moreover, there i s no c o r r e l a t i o n between the blackleg f i e l d incidence and the l e v e l of tuber contamination. Perombelon (114) has shown that most seed potato stocks i n Scotland are contaminated with Erwinia carotovora, i r r e s p e c t i v e of whether they were derived from blackleg-infected or blackleg-free crops. Webb and Wood (151) demonstrated that, while the blackleg pathogen was present i n 40% of the tested seed tubers, only 0.3% of the crop developed blackleg. L a t e n t l y - i n f e c t e d seed tubers, however, have been shown to contaminate the progeny tubers upon decaying (116). Such contaminated tubers may then rot i n storage. Losses i n storage a t t r i b u t e d to Erwinia carotovora are d i f f i c u l t to assess. Lazar and Bucur (78) c i t e the work of Savuluscu et a l . from 1930-1949 and 1951-1963 who estimated storage losses to be 60%. In Germany losses ranging from 5-90% have been recorded (138). Other p e c t o l y t i c b a c t e r i a l species, including those belonging to the genera Pseudomonas, B a c i l l u s , Clostridium, Aerobacter and Flavobacterium, may be involved (87, 129,130). Interactions with c e r t a i n Fusarium species are also known to e x i s t (52) . Estimations of storage losses due to E_. carotovora often r e f l e c t damage caused by the large numbers of secondary saprophytic fungi and b a c t e r i a invading injured t i s s u e . Moreover, i f conditions are favorable i n storage, there may be no c o r r e l a t i o n between the f i e l d incidence and the extent of decay i n storage. Seed piece decay i s an important problem c l o s e l y associated with the control of blackleg. Such losses are commonly att r i b u t e d to Erwinia carotovora or a Fusarium spp.-E. carotovora complex (92,97). Planting contaminated seed may r e s u l t i n reduction i n stands due to misses i n the row, fewer stems per plant, weak or small plants and loss i n y i e l d . Seed piece decay has accounted f o r a 28 to 30% reduction i n stand i n some instances (10). A subsequent decrease i n y i e l d i s often associated with a decline i n stand due to seed piece decay (132). Blackleg has been c i t e d as one of the p r i n c i p a l causes for the r e j e c t i o n of seed potato f i e l d s i n B.C. (19). However, recent compilations 5 by the Plant Protection D i v i s i o n (PPD) do not bear t h i s out. Only 7 seed potato f i e l d s ( t o t a l l i n g 31.5 acres) were rejected f or having a blackleg incidence greater than 2% through the years 1970-1975 i n c l u s i v e (B.M. Lawson, PPD, personal communication). Although blackleg was apparent i n a much higher percentage of the seed potato f i e l d s , the majority of these f i e l d s had only a trace (up to 0.1%). Estimations of the crop loss due to blackleg i n the Lower Fraser Valley amounted to 2% and 1% for the years 1965 and 1966, r e s p e c t i v e l y (145,146). Blackleg incidence i s more frequently recorded i n the Kootenays than i n the Lower Fraser V a l l e y or the Cariboo regions of B.C. (19). S t a t i s t i c s for losses due to soft rot i n storage f a c i l i t i e s and seed piece decay are v i r t u a l l y non-existent for B.C. The d i f f i c u l t i e s associated with i d e n t i f y i n g the causal f a c t o r ( s ) and quantifying those losses a t t r i b u t e d to Erwinia carotovora have p r a c t i c a l l y negated such assessments. Nevertheless, losses are common i n a l l potato growing regions of the province. In 1965 and 1966, f o r example, storage losses caused by 15. carotovora accounted for a 10% y i e l d reduction i n the Lower Fraser V a l l e y (145). Seed piece decay i n the f i e l d , on the other hand, has been reported to exceed 50% i n some cases (20). (2) Symptomatology Blackleg i s characterized by the development of a slimy, moist, black l e s i o n on the potato haulm, usually near the s o i l l i n e but occasionally extending r i g h t into stem regions (5,38,81). The i n t e n s i t y of t h i s black d i s c o l o r a t i o n i s not as c h a r a c t e r i s t i c as f i r s t suggested. The decaying s t a l k may take on a l i g h t brown or greenish-brown color, depending on the environmental conditions and on the potato c u l t i v a r (81). Frequently only one of the haulms expresses such symptoms while others a r i s i n g from the same seed piece look quite healthy. In the early stages of disease development, plants may appear c h l o r o t i c , stunted and may express l e a f - r o l l symptoms. Foliage tends to r o l l upwards and inwards, assuming a m e t a l l i c l u s t r e . In dry weather, diseased plants may begin to w i l t and s h r i v e l i n response to the associated vascular d i s r u p t i o n . In cases of early blackleg, encountered when the plants are barely 5 to 10 cm high, daughter tubers f a i l to develpp. Seed piece decay may frequently precede blackleg. I t i s p a r t i c u l a r l y prevalent i n North America where cut seed i s extensively planted. Growers and f i e l d inspectors tended to blame misses i n rows on malfunctioning seeding machinery. Seed piece decay may r e s u l t from an i n f e c t i o n by e i t h e r var. carotovora or var. atroseptica or may be due to an i n t e r a c t i o n e f f e c t between these s o f t rot coliforms and species of Fusarium (10,38,52,132) or Phoma tuberosa (10). The s t r u c t u r a l i n t e g r i t y of the i n f e c t e d tuber i s soon l o s t , as the parenchyma c e l l s separate from each other to form a wet r o t . Secondary saprophytes enter and convert such tubers into dark, foul-smelling pulp. (3) Taxonomy and E t i o l o g y In 1901, L.R. Jones described the p e r i t r i c h o u s l y - f l a g e l l a t e d bacterium responsible for the breakdown of carrot tissue i n storage. He named the causal organism B a c i l l u s carotovorus (65). Concurrent studies i n Holland and i n Germany by Van H a l l and Appel, r e s p e c t i v e l y , i n the following year l e d to the discovery that potato blackleg was i n c i t e d by a c l o s e l y - r e l a t e d bacterium. The i n c i t a n t was named B a c i l l u s atrosepticus by 7 Van Hall and 13. phytophthorus by Appel (4,26). While these findings prompted researchers elsewhere to examine the bacterial flora associated with soft rot diseases in these and other hosts, i t is generally conceded today that these subsequent isolation attempts resulted in the recovery of one or the other of these previously described coliforms (14,26,34, 40,139). A great deal of disagreement exists today among taxonomists of soft rot coliforms. Graham (40) concluded that "speciation in the soft rot coliform group is in a state of utter taxonomic confusion". The taxonomy of the soft-rotting Erwinia spp. has been extensively reviewed and thus, warrants l i t t l e further attention (26,40,142,150). However, it should be noted that three schools of thought have existed concerning this important group of phytopathogenic bacteria. Waldee (150) contended that the inclusion of the soft rot coliforms in the genus Erwinia constitutes an ar t i f i c i a l system. Instead, he established a new genus, Pectobacterium, to include a l l those gram-negative, peritrichously- ...„; flagellated coliforms associated with soft rot diseases, thus distinguishing them from those bacterial pathogens associated with necrosis and dry wilts typified by Erwinia amylovora. A second school, represented by Burkholder and Smith (14) was prepared to accept the generic classification but insisted on retaining the species differentiation between E_. carotovora and E_. atro- septica prevalent in the older literature. The proponents of the final school of soft rot taxonomy did not agree on the generic classification but a l l agree that there should be only one species? E. carotovora (26,40,42,58,59,128). A l l other forms then become variants of this species. This was the approach taken by Buchanan 8 et al. in Bergey's 8th Edition of the Manual of Determinative Bacteriology (12). Dye (26) recognized five variants: namely, var. carotovora, var. atroseptica, var. chrysanthemi, var. rhapontici, and var. cypredii. I shall follow this nomenclatural regime and will refer to var. carotovora and var. atroseptica in this text. A great many morphological and physiological aimilarities exist between the soft rot organism discovered by Jones, var. carotovora, and the blackleg organism, var. atroseptica (26,41,142). The organisms are physically alike in being peritrichously flagellated rods and biochemically similar in being gram-negative, facultatively anaerobic and in fermenting many carbohydrates to acids. Graham (41) has demonstrated that only seven of 90 tests may be used with any consistency for distinguishing var. carotovora from var. atroseptica or var. chrysanthemi. The diagnostic responses induced by var. atroseptica and var. carotovora in potato stem tissue have been cited by some workers as occurring with enough consistency to warrant a differentiation of the two organisms taxonomically when used in conjunction with biochemical data (14,77,133). The atroseptica varietal form has generally been credited with causing the typical blackleg symptom in the field (44,78,86,102,114). While i t was known that this varietal form could cause soft rot of healthy vegetable tissue, recent studies indicate that in some instances i t may be the primary cause of potato soft rot in storage and transit (52,114). The carotovora form has long been known as the cause of soft rot. The role of the latter organism in blackleg symptom expression has also been examined. Rudd Jones (128), for example, reported that several of his carotovora isolates could incite a blackleg infection on 9 experimental inoculation into young potato stalks. This experiment was repeated by Hellmers and Dowson (59) and their findings indicated that blackleg symptoms could be induced by a wide range of strains so long as the inoculum was introduced directly into the vascular bundle of the stem. Noble and Marshall (102), however, were unable to duplicate these results. This inconsistency prompted others to examine the pathogenic capacity of both varietal forms in relation to temperature (43). All the isolates tested produced typical blackleg lesions at temperatures exceeding 24.5°C, while several could also produce symptoms at temperatures below 19°C. The latter corresponded to the atroseptica varietal form while the high temperature group consisted of the carotovora, chrysanthemi and aroideae forms. Graham and Dowson (43) cautioned against using pathogenicity trials at one arbitrarily selected temperature. Stanghellini and Menely (137) recently reported that var. carotovora was the primary cause of a blackleg outbreak in Arizona involving atypical symptoms. Moreover, their isolates were pathogenic to potato stems at 18.3°C. Evidence incriminating the carotovora variant as causing some cases of blackleg exists also in British Columbia (R.J. Copeman, personal  communication). (4) Detection, Isolation and Identification of Erwinia carotovora A distinctive feature of E_. carotovora is rapid pectate gel liquefaction. This has been used extensively as a criterion in selective isolation media (6,21,25,82,112,144). While pectolytic pseudomonad species are common contaminants in tubers, these are readily distinguished from soft rot Erwinia spp. by the rather shallow pits that they produce on such media. 10 Several biochemical tests, based primarily on their utilization of certain carbon sources, are now used in standard identification procedures, specifically to differentiate between the atroseptica, carotovora and chrysanthemi forms (26,40,41,114). Being facultatively anaerobic, these varietal forms are able to metabolize numerous carbon compounds fermentatively. It should be noted, thoughj' ~-that only 7 of 90 tests examined by Graham (41) showed sufficient consistency to be used for such purposes. The use of serology in the detection and identification of soft rot coliforms is gaining increasing acceptance (78,149). Early studies, using agglutination tests for the rapid diagnosis of infected potato stems, indicated that the antisera prepared against unfixed whole bacterial cells lacked sufficient specificity to be considered reliable for routine identification procedures (39,1.03),' These early problems have largely been overcome by fixing the cells with gluteraldehyde (Allan and Kelman, personal communication). Attempts to increase the sensitivity of the atroseptica antiserum have also led to the development of a fluorescent antibody staining technique (FAS) (3). Allan and Kelman (3) found that 74 of 77 atroseptica isolates reacted positively with the FAS while 77 carotovora and 10 chrysanthemi isolates failed to do so. Moreover, atroseptica cells could be detected on slides prepared from suspensions g containing 500 cells/ml in a total population of 10 cells/ml of var. carotovora. Jones and Paton (66) have used FAS to detect intracellular L-phase forms of var. atroseptica. Vruggink and Maas Gesteranus (149) have successfully used micro-agglutination tests and agar double diffusion tests for rapid diagnosis 11 of blackleg in the field. Their experiments to determine the degree of latent infection of stored seed potatoes, however, indicated that rotted tissue could not be tested directly using the micro-agglutination tests. A more reliable method was to streak tissue sampled on selective medium and serologically identify Erwinia colonies which grew out. Studies by Elrod (30) indicated that the common antigenic components of the soft rot Erwinia group were localized in the flagella. Mushin e_t al. (96) not only found that somatic antigens were primarily type-specific but also that different isolates displayedwast: antigenic variability and cross-agglutinations. Moreover, Graham (AO) cites the work of Goto and Okabe in Japan in suggesting the presence of group-specific and strain^specific somatic antigens in a l l isolates. Similar conclusions were formulated by Tanii et al. (143) according to Vruggink and Maas Gesteranus (149). Thermolabile capsular (K) antigens have also been implicated in cross-agglutination experiments (40). To date, a l l evidence points to antigenic specificity among the atroseptica isolates (40). (5) Epidemiology (a) The Historical Approach The controversy ^'surrounding the viability of Erwinia carotovora in soil over the winter has raged through much of this century. The studies of Rosenbaum and Ramsay (127) and Ramsay (125) suggested that the blackleg organism did not survive in soils. This supported the then prevailing viewpoint of Morse (95), that infected seed was the primary source of inoculum. However, Patel (104) and Leach (80) demonstrated that soft rotting. Erwinia varietal forms could survive in soil for a comparatively 12 long time. The survival of E_. carotovora in soils more easily explained why the same seed lot, when planted in different fields, expressed different levels of blackleg. Leach's work was readily accepted without further question for this reason. While Bonde (10) experienced l i t t l e difficulty in securing apparent Erwinia isolates from fallow fields in South Carolina, concurrent studies in Maine by the same researcher negated the importance of such overwintering under this state's cool climatic conditions. According to Eddins et al. (27) , E_. carotovora was present in most Florida soils, playing a significant role in the contamination of potato seed pieces in the field. However, i t should be pointed out that Florida soils are intensively cropped, unlike conditions elsewhere where potatoes are grown as part of a rotation. Subsequent studies by Kerr in Scotland (68) and van den Boom in Germany (147) inferred that many soils contain the black-leg causal agent and suggested that soil was the main source of infection. Graham (37), however, disputed Kerr's findings, noting that only isolates of the genus Pseudomonas were recovered from soils in Scotland. The prevailing viewpoint today is that pectolytic coliforms are soil invaders rather than soil inhabitants in temperate potato growing areas (35,37,38,44,78,84,109,114, 148). This concept may apply to tropical areas as well (126). (b) The Modern Concept of Overwintering The search for a plausible overwintering mechanism has recently prompted a shift in emphasis from soil survival to the role of seed tubers in the transmission of these soft rot coliforms from one crop to the next. Early work done by Perombelon (10.8,109,114,121) indicated that nearly a l l 13 tubers from a l l seed stocks were contaminated with Erwinia carotovora. Both varietal forms could survive on tubers for 6 to 7 months but the recovery ratio of 4:1 suggested that the atroseptica form was more prevalent in Scotland. Similar studies by Harrison (53) and DeBoer and Kelman (24) in the USA revealed levels of contamination in seed stocks varying from 0-100%. These bacteria contaminated the surface layers of seed tubers and remained latent until changes occurring within the tuber allowed these coliforms to enter through the lenticels (151). Most of these soft rot coliforms were localized in the lenticels, a few in the periderm layer and none in the vascular ring (108,115). These findings corroborated the suggestion of Harper e_t al. (51) that the periderm offers a barrier to bacterial entry. Lenticel infection of tuber initials was observed by Davidson (22), in much the same way as i t was described by Smith and Ramsay (135) for mature tubers. Laboratory studies showed that i f lenticels were subjected to relative humidities of below 80%, they were blocked by the deposition of secondary periderm (33). Storing tubers at 100% relative humidity, on the other hand'/ji promoted lenticel proliferation, thus encouraging the subsequent invasion by soft rot coliforms (7,100). The development of lenticels from tuber stomata and their modification, as mediated by soil moisture, has also been investigated (1). Lenticel proliferation may vary with tuber age, with mature tubers becoming more resistant to bacterial invasion. Adams (2) postulated that the most severe lenticel infection is likely to result when there is an early breakdown of seed tubers. 14 (c) Factors Influencing Incidence It is well established that prolonged cool, wet environmental conditions during the growing season favour blackleg symptom expression (46,131). Field incidence in Romania was correlated with high average rainfall during the growing season (250-300 mm) and average summer temperatures of 17-20°C. The disease was uncommon in areas with a rainfall of 150-200 mm and an average temperature of 22-23°C (78). Similarly, in Australia, heavy rainfall early in the season was considered conducive to blackleg incidence in susceptible varieties (52). Graham and Harper (46) contend that the blackleg epidemic of 1966 in Scotland early in the growing season reflected the poor sprout development in the coldcsoil and the subsequent wet weather. This concurs with Davidson's (22) demonstration that waterlogged soil conditions favoured blackleg. Moreover, studies in Scotland indicated that lenticel and soil contamination levels over the growing season tended to be low during dry periods when the soil was warm but rose after heavy rain when the soil was cool (118). Irrigation procedures currently are coming under close scrutiny as they may assist the spread of bacteria from h i l l to h i l l and may favour tuber infection through enlarged lenticels (47). Handling and cleansing procedures are important in the contamination of stored tubers. Storage and transit losses may be large when solutions used to move potatoes hydraulically are not regularly changed or disinfectants added (R.J. Copeman, personal communication). Jet pressure washing may force soft rot coliforms further into lenticels and damaged tissues (52). Improperly used washing procedures only ensure mass inoculation of tubers prior to storage or transit. 15 The widespread use of cut seed in North America favours both disease development and spread. Problems arise particularly i f the seed pieces are stored for any length of time after cutting (10,80). The healing process is adversely influenced by the locally anaerobic conditions which may exist in storage facilities. These conditions may cause the inhibition of wound cork formation (80), thus allowing contaminating Erwinia forms, spread by cutting knives or mechanical seed cutters (11,134) to proliferate in the susceptible tissue. Blackleg development is enhanced i f such cut seed is planted in cold, wet and poorly aerated soils (123). Tubers previously infected by other phytopathogenic organisms or those damaged by frost injury, are more prone to produce blackleg-infected plants than sound tubers (10). Graham and Harper (46) indicated that large tubers are more likely to produce blackleg-infected plants than smaller tubers. This may be related to the increased cracking and mechanical damage associated with larger tubers (51). Growth crack formation (and thus soft rot) was enhanced by weather conditions and high rates of fertilizer application (51,63). An increase in nitrogen alone resulted in a decrease in the proportion of stems affected by potato blackleg (45). The role of seed piece decay on thexsubsequent contamination of its progeny tubers and those of neighbouring plants has been extensively documented (108,109,110,111,114,116,118,119,121). Graham and Harper (46) have indicated that tubers removed from apparently health plants could produce just as many infected plants as tubers taken from plants affected by blackleg. This finding appeared to negate the importance of systemic infections. Obviously, tubers on "healthy" plants were becoming contaminated in some other way. It became apparent that the infected tubers and stems of blackleg plants in the crop could serve as focal points of inoculum and that bacteria released into the soil could be spread to adjacent healthy plants. The organism could be recovered from up to four feet away from such infected plants (46). As previously noted, most Scottish seed potato stocks are latently infected with soft rot Erwinia forms. The breakdown of such seed may result in extensive contamination of daughter tuber lenticels. This occurs only after the seed piece has decomposed and has liberated the pathogen into the soil (109,116). The timing of the harvest greatly influences such contamination, with stocks lifted after mid-September being more heavily contaminated than those harvested earlier (108). Erwinia carotovora populations fluctuate in the soil, with levels tapering off only after the seed piece has totally decayed (23,110,118). Soil temperatures and water movement played an equally important role in bacterial population fluctuations (118,122). Numerous.environmental factors are involved in the storage breakdown as incited by E_. carotovora. These include such conditions as poor ventilation (91,124), heating (62), high humidities (67,86), oxygen depletion (88,99,124) and increased carbon dioxide levels (60,88,98,99,152 154). Under moist, anaerobic conditions, water is absorbed forcing the lenticels to swell and open. The reduced oxygen levels disrupt the cell membrane integrity, resulting in the leakage of water and solutes from the turgid cells (124). Intruding pectolytic bacteria establish themselves in the deeper lenticel regions and proliferate rapidly. Soft rot lesions are thus established. Bacteria may be carried onto adjacent healthy tubers when the gas pressure inside the tuber is vented. Should aerobic conditions prevail, however, a dry, hard rot may result (83). Tuber suberization depends on the temperature and the relative humidity in the storage bins, with high temperatures favouring bacterial multiplication and humidities below saturation delineating the advancing bacterial edge (129). Perombelon and others (24,113), in their isolation techniques, have been able to transform tubers into semi-selective enrichment media for E_. carotovora by providing adequate moisture and anaerobic conditions. (d) Re-evaluation of Soil Survival Ficke et_ al. (32) have demonstrated that var. atroseptica can survive in soils for long periods of time (up to 16 weeks in unfertilized soils). Soil temperatures were found to reduce bacterial numbers more significantly than soil moisture levels. Moreover, long frosty periods and lasting snow covers prolonged the organism's presence in soil. Others have observed that its viability is much greater in sterile soils maintained at low temperatures than in field soils at higher temperatures (78,119). Scottish studies demonstrated that Erwinia populations survive in field soils for only one to two weeks i f temperatures rose above 16°C. Much of the work discrediting survival in soil is based on the inability to recover the pathogens on semi-selective media (21,82,84,116). Recently, however, a more sensitive enrichment medium has been developed that can detect E_. carotovora levels as low as 2 to 7 cells per gram dry weight soil (90). The extremely sensitive FAS technique may also be used to resolve the survival question (3,69,105). 18 (e) Host Range The ubiquitous nature of the true s o f t rot pathogen E. c. var. carotovora i s w e l l documented (29,42,65,106,126,139). The accounts of natural and laboratory i n f e c t i o n s by t h i s v a r i e t a l form are beyond the scope of t h i s review but s u f f i c e i t to say that numerous a g r i c u l t u r a l l y important plant species are affected. Vegetables, f r u i t s and other fleshy tissues are e s p e c i a l l y susceptible to such soft r o t . Soft rot i n the b o t a n i c a l family Gramineae i s not common but Goto (36) has, nevertheless, been able to i s o l a t e the carotovora form from a sheath rot of r i c e i n Indonesia. I t has since been recorded as a l e a f pathogen of r i c e by Hashioka (56). Also noteworthy i s i t s a b i l i t y to survive on the seeds of c e r t a i n agronomic plant species. Korol'ova (74) reported i t s seedborne nature i n lupine species. Both s o f t r o t v a r i e t a l forms have subsequently been detected i n stored clover seed as w e l l (101). Host range studies were i n i t i a t e d by-Appel (5) and Stapp (138). The former reported blackleg f i e l d symptoms i n broad beans ( V i c i a faba), Campanula rapunculus and Symphytum o f f i c i n a l e and i d e n t i f i e d the causal agent as B a c i l l u s phytophthorus (= E. c^ . var. a t r o s e p t i c a ) . Cucumbers developed soft r o t symptoms but d i d not express blackening symptoms. The pathogen could occasionally induce blackleg i n lupines but more often was responsible for seed germination f a i l u r e . Beans, carrots, turnips, vegetable marrows, beets and marigolds were a l l considered susceptible. E l l i o t t . (29), however, l i s t s only green peppers, tomatoes, tobacco, Solanum melongena and potatoes as hosts of the a t r o s e p t i c a v a r i e t a l form. Stapp (139), on the other hand, demonstrated that tomatoes were susceptible to s o f t r o t damage only when the r e l a t i v e humidity exceeded 95% and the temperature rose to 22°C. The same 19 applied to tobacco, Nicotiana rustica, broad beans, carrots and onions but not to Pelargonium zbnale or; lamb's quarters. Delphiniums and Taraxacum kok-saghyz were listed as hosts by Stapp (139) as well. Susceptibility to blackleg varied greatly among solanaceous hosts (140, 141). Blackleg of broad beans is one of the most important diseases of this crop in the Soviet Union (8,15). It has also affected bush beans in the field %13). Erwinia carotovora forms are able.to survive in the rhizosphere of certain plant species without the host plant expressing diagnostic symptoms. Butler and Jones (16) suggested that cruciferous weeds may play a part in "carrying over" populations of these pathogens from one crop to the next in this manner. Krivets (76) and Klingner (72) have documented the close association of E_. _c. var. carotovora with the root systems of lupine and cotton plants, respectively. Furthermore, E_. aroideae has been isolated from the rhizasphere of Chinese cabbage, cucumber and Pharbitis  n i l thirty-two days following the ar t i f i c i a l inoculation of soil (70). Using immunofluorescent staining techniques these workers could detect the pathogen in the rhizosphere of radish, wheat, oat, red bean, tomato and sponge gourd. Finally, Erwinia aroideae was recovered in the rhizosphere of such non-inoculated plants as cruciferous crops, teosinte (Euchlaena spp.), Chinese chive, tomato, wild lettuce (Sonchus oleraceus), lamb's quarters (Chenopodium album) and Commelina communis after 67 and 82 days. (f) Insect Transmission Insects have long been incriminated in the transmission of blackleg. Leach (79,81) was the first to report that the larva of the seed-corn maggot (Hylemyia cilicrura Rond.) was an important agent of dissemination of var. atroseptica. Maggots were contaminated on emergence from their egg shells, from the soil or from the surface of contaminated seed. The feeding maggots were shown to be very effective agents of inoculation, introducing these soft-rotting bacteria deep into the seed piece. Infected seed pieces decayed quickly and the bacteria invaded stems of the plants thereby producing typical blackleg symptoms. Pupation was usually found to be well under way by this time. Leach (79) also demonstrated that the organism passed through the intestinal tract of the feeding larva, could survive in the puparia and was found on the emerging adult fly in the early spring. This meant that the adult could act as a vector in nature. Leach (81) demonstrated that there were two broods of the seed-corn maggot in Minnesota. Maggots of the second brood were frequently found in the stems of blackleg plants while the maggots of the first were chiefly confined to the seed pieces. The insect is also a pest of corn, peas, turnips, onions, beets, tomatoes and many other cultivated crops. Bonde (10) isolated the atroseptica form from the pupae of potato maggot (H. trichodactylo Rond.) that had overwintered in Maine soils as well as from H. brassica feeding on cruciferous crops. While numerous other insects are known to feed on decaying vegetable matter, few have been examined to ascertain their role in the recontamination of blackleg-free stocks. Molina et al. (93) have reported that the blackleg-inciting organism may be transmitted by the common fruit fly, Drosophila melanogaster. Further studies in Colorado indicated that at least five genera of dipterous insects, Allotrichoma, Drosophila^Hylemyia  Leptocera and Scatopse, were contaminated with both soft-rotting Erwinia 21 varietal forms (73). The percentage of atroseptica-contaminated insects declined with time whereas the percentage of carotovora-contaminated insects increased from £a 25% in June to 100% in August. Moreover, the common f r u i t f l y , D. melanogaster, was found to transmit both organisms to injured potato plants. Graham et a l . (49) reported that dipterous insects caught in an adjacent dump of decomposing organic matter were contaminated with the carotovora varietal form several weeks before infection was found on potato stalks. Serological studies of the recovered isolates from insects and from infected plants suggested a very close serotypic similarity. This would imply that the dump was a reservoir of inoculum. (6) Control Blackleg control measures in commercial crops have been "ineffective. Chemical disinfection of tubers with sodium hypochlorite or mercurial compounds hase shown l i t t l e promise in the control of seed piece decay (10,50,132). Antibacterial preparations have, likewise, not reduced the incidence of blackleg in the f i e l d (50). Moreover, no potato varieties have proven to be resistant to either blackleg or soft rot; i t appears that some varieties may be more susceptible than others (38). Graham and Harper (46) suggested that successful control measures be based on such sound agronomic practices as roguing out infected plants, preventing the entry of these pathogens into tubers at harvest, grading and during storage and through the use of healthy sound seed tubers. One way to control blackleg i s to break the disease cycle. The simplest approach may be to prevent the seed piece from contaminating the progeny tubers. As the seed piece is responsible for liberating the soft rot coliform bacteria which w i l l , i n turn, contaminate the developing progeny tubers, i t i s e s s e n t i a l that t h i s l i n k i n the disease cycle be broken. This may be r e a l i z e d e i t h e r through the use of tested stem cuttings or by using only whole seed and harvesting the progeny tubers before the seed piece has a chance to break down. Moreover, a long r o t a t i o n and i s o l a t i o n may be valuable as co n t r o l measures. The S c o t t i s h Seed Potato C e r t i f i c a t i o n Scheme was devised to reduce y i e l d losses a t t r i b u t e d to v i r a l i n f e c t i o n s . The basic premise of t h i s program i s to refuse the c e r t i f i c a t i o n of growing seed crops showing more than a s p e c i f i e d l e v e l of i n f e c t i o n . This program i s based upon the use of v i r u s - t e s t e d stem cuttings (VTSC) ( 4 4 ) . The p o s s i b i l i t y of producing blackleg-free seed material within the framework of the program became f e a s i b l e when i t was apparent that the*causal organism was seed-borne, not soil-borne as previously suspected. As mother tubers constituted the major source of contamination of daughter tubers, i t followed that i f stem cuttings, removed before the pathogens spread to them, were used to propagate a new generation of seed tubers, these would be free from i n f e c t i o n . Cuttings were tested to ensure that they were, indeed, uninfected ( 4 4 ) . The method of m u l t i p l i c a t i o n of v i r u s - t e s t e d stocks has been described by Perombelon (116). As tubers may become re-contaminated with Erwinia carotovora, they are constantly replaced by material derived from new stem cuttings. Propagation of potato stocks from tested cuttings has produced seed material almost e n t i r e l y free from i n f e c t i o n with Erwinia carotovora ( 4 9 ) . A s i m i l a r " f l u s h out" program has been introduced i n B.C. using 23 virus-free nuclear stock propagated by stem cuttings (136,155). Since this scheme employed cuttings a lowered incidence of blackleg might have been expected due to the break in the tuber to tuber cycle. However, this anticipated reduction was not observed. With the testing of stock mother plants for soft rot coliforms, an Erwinia-tested program similar to that in Scotland was instituted in 1972 (R.J. Copeman, personal  communication). Seed stocks derived from stem cuttings have shown great promise in Scotland but the maintenance of such "clean" seed has s proven a real challenge. Though levels of recontamination may be relatively low, they remain a major concern anywhere this blackleg-free program has been instituted (17,123). In Scotland only 2 of some 20,000 'mother plants'used fo'r.-«ul5lSfin?g8-.»vwef.ei£n found infected with soft-rotting Erwinia species (49). The origin of these infections with Erwinia carotovora remains ^unknown. Graham e_t _al. (49) speculated that these infections were established by insects associated with a dump of vegetable matter nearby. Harvesting and grading equipment has come under close scrutiny in recent years as well (31,156). Graham and Harper (46) emphasized the need for disinfection of riddles and spool graders. The blackleg-inciting pathogen can overwinter on tuber tissue and soil adhering to graders (44). Contaminated cutting knives can readily recontaminate seed stocks as well. Volunteer tubers remaining in soils following harvest may play a significant role in the re-infection of stem cutting-derived materials. Lumkes and Beukema (85) demonstrated that i f the winter temperatures are sufficiently low, such tubers break down before planting time the following spring, especially i f the tubers are located near the soil surface. Studies 24 in Scotland, on the other hand, indicate that as many as 124,000 tubers per hectare (most contaminated by Erwinia carotovora) remained in a field after harvest (117). Only one fifth were on the surface, the rest being buried as deep as 20 cm. Moreover, these may survive through five years of cereal monoculture. Both .varietal forms of E. carotovora have been recovered from turnips growing in a field where potato volunteers had been carried over through two successive barley crops (119). Blackleg incidence levels do not provide realistic approximations of the severity of such contamination. Yet, to date, certification of seed potato fields has been based exclusively on the visual aspects of the disease. This is rather unfortunate as there is a definite lack of correlation between field and tuber infections. Wood et al. (153) have found that, in one instance, where 25-50% of the mother tubers were known to be latently infected in a crop of 8500 plants, only 25 developed diagnostic symptoms. Sometimes, on planting seed that was derived from a field expressing blackleg the previous season, blackleg symptoms will f a i l to appear in the successive crop. Yet, planting out the progeny seed material will result in a crop with a moderate to high field incidence. (7) Summary and Objectives Scottish studies indicate that nearly a l l seed potato stocks are infected by Erwinia carotovora. Moreover, decomposing seed pieces are primarily responsible for the subsequent contamination of progeny tubers. These bacteria gain entry into the tubers through the proliferating . lenticels and remain latent in the lenticels until favourable environmental conditions allow the bacterial cells to multiply and initiate soft rot. 25 Blackleg may be.incited by var. atroseptica or less commonly, var. carotovora. Host range studies demonstrate that the latter is '. „' ubiquitous but, also, that both forms can remain viable in the rhizosphere of certain plant species for comparatively long periods of time. Further-more, these soft rot coliforms'may, under suitable meteorological conditions, overwinter in the soil. While the VTSC program has experienced success, the feasibility of maintaining a "blackleg-free" stem-cutting program depends largely on preventing recontamination. A virus-tested seed potato program using stem cuttings has existed in B.C. for several years. This provided the framework for an Erwinia tested program, as the seed was virtually free of Erwinia from the start. Yet, even with the introduction of seed derived from Erwinia-tested stock, the blackleg incidence has not decreased. The recurrence of soft rot coliform bacterial infections in this program has prompted the search for sources of recontamination. The objectives of this study were: 1) To survey the extent of latent contamination of tubers derived from the virus-free program. 2) To evaluate soil, tubers and alternate hosts including weeds as possible reservoirs of inoculum permitting recontamination. 26 MATERIALS AND METHODS (1) Incidence of E. .carotovora i n Seed Tubers (a) Source of Tubers Seed tubers were harvested by co-operating growers i n the Lower Fraser V a l l e y , the Kootenays and i n the Pemberton Seed Control Area. In the 1972 survey, samples c o n s i s t i n g of 40 tubers from E l i t e I I I , Foundation and C e r t i f i e d seed grades were c o l l e c t e d i n the spring at grading. Where convenient, both apparently healthy tubers and c u l l tubers were tested. C u l l tubers were those which were obviously diseased (dry rot , sof t r o t , scab, etc.) or those which were too small to be of any value as seed tubers. A s i m i l a r survey was conducted i n 1975, except that the tubers were indexed within s i x weeks a f t e r harvest. They were stored at 5.0° C u n t i l used. Samples from two stocks-of Creston seed were tested to compare the l e v e l s of Erwinia i n f e c t i o n i n seed derived from a common nuclear stock but m u l t i p l i e d out i n d i f f e r e n t areas p r i o r to being grown i n Creston. Some of the tubers had begun to deteriorate during storage and t r a n s i t p r i o r to being received. Only apparently f i r m tubers (100-150) were selected f o r t e s t i n g . (b) Detection Methods' The detection of E.. carotovora i n potato stocks was f a c i l i t a t e d by using Perombelon's tuber rot induction technique (113). In the 1972 survey no attempt was made to inj u r e the l e n t i c e l s . I n d i v i d u a l l y wrapped tubers were placed i n a p o l y v i n y l c h l o r i d e (PVC) chamber (33 cm x 33 cm x 46 cm) which was flushed Iwith nitrogen gas for 20 minutes every 4 to 6 hours. 27 Tubers were held under anaerobic conditions f o r 10 days. Rotting ti s s u e was then plated d i r e c t l y onto Perombelon's modified Stewart's medium and incubated f o r 48 hours at 27° C (112). In subsequent surveys of seed stocks and volunteer tubers, the tubers were injured p r i o r to wrapping by puncturing ten l e n t i c e l s per tuber with si n g l e s t e r i l i z e d toothpicks (24). The incubation period was reduced to 5 days at 20° C. Isolations were then made by removing the toothpicks with any adhering pieces of the soft rot l e s i o n , mixing t h i s material i n 0.5 ml s t e r i l e , d i s t i l l e d water blanks and p l a t i n g the suspension on c r y s t a l v i o l e t pectate medium (CVP) (21). Colonies formed a f t e r 2-3 days d i s p l a y i n g the deep cup-like depressions and the cross-hatched morphology under oblique l i g h t (Figs, l a and lb) pec u l i a r to p e c t o l y t i c Erwinia spp. were transferred to nutrient agar slants for storage. In most cases only one t y p i c a l E r w i n i a - l i k e colony per plate was saved f o r i d e n t i f i c a t i o n . (c) Isolate Characterization Suspected s o f t - r o t coliform i s o l a t e s from the 1972 survey were f i r s t confirmed as belonging to the genus Erwinia by standard p h y s i o l o g i c a l tests (26,41). The c r i t e r i a used were: the a b i l i t y to rot potato s l i c e s (48), absence of f l u o r e s c e i n production on King's Medium B (71), no oxidation of gluconate (18) and oxidase negative (75). Isolates meeting these c r i t e r i a were further d i f f e r e n t i a t e d by acid production from lactose and maltose (26), the production of reducing substances from sucrose (114) and the a b i l i t y to produce indole (48). A l l tests were run i n t r i p l i c a t e and were repeated at le a s t once. With the use of CVP and the d i s t i n c t i v e colony morphology of 2 8 F i g . 1 Appearance of Erwinia carotovora on the s e l e c t i v e c r y s t a l v i o l e t pectate (CVP) medium. (a) Typ i c a l p i t t i n g a f t e r 48 hours growth, (b) C h a r a c t e r i s t i c cross-hatched colony morphology illuminated with oblique l i g h t . v 29. E_. carotovora on this medium the need to confirm^genus identification became unnecessary in later work. Varietal form differentiation was accomplished using an expanded series of tests (Table I). Isolates were also serologically identified. Agglutination tests were carried out using antisera prepared against a pathogenic atroseptica isolate, a pathogenic carotovora isolate and a nonpathogenic carotovora isolate. The procedure described by Allan and Kelman (A. Kelman, personal communication) was used to prepare the antisera. The stem-prick method of Graham and Dowson (1960) was used to test pathogenicity. Sterilized toothpicks were used to inoculate 21 day old potato cuttings with bacteria (Fig. 2a) grown for 48 hours on King's Medium B. Moistened cotton plugs were then taped to the wounding;site to maintain a high humidity (Fig. 2b). Five replicate plants were used per isolate. Inoculated plants were placed in a growth cabinet at 18° C and with a 12 hour photoperiod. Symptoms were recorded after 7 days. (2) Incidence of j E . carotovora in Weeds Collection was restricted to the Pemberton Seed Control Area. Weeds selected for examination were chosen at random. In the case of perennial species with rhizomatous root stocks, such as horsetail and couch-grass, i t was difficult to assess what constituted an individual plant unit. Vegetative stalks or culms at intervals of 7 to 8 feet were considered separate plants. Samples were placed in plastic bags with soil to prevent desiccation and stored at 5° C until indexed (3r-5 days). For the first two sampling dates,^weedsv'Were treated similarly to the tubers; they were wrapped in moist tissue paper and anaerobically incubated. In subsequent Table I The tests used for the differentiation of the varietal forms of Erwinia carotovora Tests var. carotovora Erwinia carotovora var. atroseptica var. chrysanthemi Acid from lactose Acid from maltose Acid from e(-methyl glucoside Growth at 37° C Reducing substances from sucrose + + + + + - or d d = delayed reaction F i g . 2 The stem-prick method of inoculating potato cuttings. (a) A s t e r i l i z e d toothpick with b a c t e r i a at the t i p inserted into the a x i l of the 3rd l e a f , (b) Inoculation s i t e covered with moistened s t e r i l e cotton batting to prevent desiccation. surveys, however, the weed roots were shaken to remove any loose soil and cut into 5-7 cm pieces with a sterilized scalpel. No attempt was made to standardize the amount of tissue used. These pieces were deposited in a 250 ml Erlenmeyer flask containing 100 ml sterile, distilled water. These flasks were then vigorously shaken and dilution series of the original suspension were plated on CVP. These plates were examined for Erwinia  carotovora isolates after 48 hours incubation at 24° C. (3) Survival of Erwinia carotovora (a) General Techniques (I) Inoculum Preparation and Inoculation Techniques Nutrient broth shake cultures (40 ml/250 ml Erlenmeyer flask) of the carotovora and atroseptica varietal forms were grown for 36 hours at 24° C. These suspensions were centrifuged at 150 x G for 8 minutes on a Sorvall Superspeed RC 2B centrifuge (with an SS-34 head type). The pellets were then resuspended in 250 ml distilled water. Tubers were slashed and allowed to soak in a liter of a 1/10 aqueous dilution of the appropriate original stock culture for 10 to 15 minutes (Figs. 3a and 3b). These bacterial suspensions ranged from 1 x 10* 9 to 3 x 10 CFU/ml. Two treatments were tested: some tubers were left on the soil surface while others were buried at a 10 cm depth (Fig. 3c). Uninoculated tubers were included as control treatments. Soil cells consisted of 4 cm sections of clear Pyrex glass tubing (9 cm diameter) which, when f u l l , were sealed at both ends with double-layered cheesecloth and secured with an.elastic band (Fig. 4a). Fig. 3 The inoculation of tubers for survival studies. (a) Tubers were slashed with a pocket knife. (b) Tubers were soaked in a bacterial suspension. (c) Tubers were buried at a depth of 10 cm. 34 Fig. 4 The preparation of s o i l c e l l s f o r s u r v i v a l studies, (a) Glass r i n g (9 cm dia) with cheesecloth covering 1 side. (b) Glass r i n g f i l l e d with a r t i f i c i a l l y infested s o i l , (c) Loaded s o i l c e l l ready f o r b u r i a l . (d) S o i l c e l l buried at 10 cm. 35 Field soils were inoculated with a 1/10 aqueous dilution of the appropriate original stock culture. This suspension was sprayed on to the soil with a mist hand sprayer and thoroughly mixed-in. Soil cells were subsequently loaded and sealed (Figs. 4b and ,4c). One liter of field soil was sufficient for f i l l i n g two soil cells. These soil cells were then buried perpendicularly to the soil surface at a 10 cm depth (Fig. 4d). Weed rhizospheres were inoculated by pouring a 15 ml aliquot of a 1/10 dilution of the appropriate original stock solution around the base of each plant. Control plants were treated with water. The detection of Erwinia carotovora in weed rhizospheres was facilitated by using the serial dilution technique previously described for the weed study. (ii) Detection and Identification Techniques Upon recovery, the inoculated soil was poured into a polythene bag and shaken vigorously. A 10 gram wet weight sample was removed and added to a 250 ml Erlenmeyer flask containing 100 ml sterile distilled water. This was well shaken and a standard soil dilution series was prepared. A 0.1 ml aliquot of each dilution was then spread over the surface of duplicate CVP plates. These plates were then incubated at 24° C for 48 hours and the number of carotovora colonies per plate was recorded. Finally, a 10 gram sample of the mixed soil was taken from each replicate and allowed to dry at 105° C for 24 hours. The Erwinia population could thus be expressed on a gram oven-dry weight basis. Baiting of soils was necessitated when the levels of these pathogens decreased below the detection capacity of the dilution plating technique. Erwinia-tested tubers (cultivar Red La Soda) were surface-36 sterilized in a 5% hypochlorite solution for 20 minutes. The epidermal layers were peeled away using a sterilized knife and tubers were dipped into 95% ethanol and flamed. Five millimeter wedges were cut from these tubers and resterilized. Soil from glass cells or from around inoculated tubers was poured into paper cups with drainage holes. The sterile tuber slices were then placed in these cups and covered with soil (Fig. 5). These cups were set into Petri plates containing sterile distilled water. The tissue slices were allowed to remain in soil for five days then transferred to 2 ml sterile distilled water blanks. A sample of the resulting bacterial suspension was streaked on CVP. A few of the isolates recovered at every harvest date were retained for identification. One isolate out of every 10 was tested for varietal differentiation, using the biochemical tests previously outlined. Al l isolates saved were tested against the antisera previously mentioned. (b) Overwintering Experiment UBC 1974-1975 The ability of Erwinia carotovora varietal forms to overwinter in field soil and on tubers in field plots on the UBC campus was examined from Dec. 14, 1974 to Feb. 1, 1975. Survival in soil containers of two different designs was compared. Wisconsin isolates of the atroseptica (SR8) and the carotovora (SR40, a nonpathogenic form) varietal forms were used for this experiment. Shake cultures were grown in nutrient broth at 21° C for 80 hours. These cultures were not centrifuged; rather, 35 ml of the appropriate bacterial suspension was sprayed on to one kilogram of field soil (1 kg of 37 Fig. 5 Longitudinal section through the paper cup showing how sterilized tuber slices were used to bait s o i l for Erwinia carotovora. 38 f i e l d s o i l = 3 s o i l c e l l s ) and mixed-in w e l l . S o i l c e l l s and non-perforated polythene bags were then f i l l e d . Population l e v e l s at b u r i a l were determined to be 6.8 x 10^ and 8.2 x 10^ colony forming units (CFU) per gram oven-dry s o i l for the at r o s e p t i c a and carotovora forms, r e s p e c t i v e l y . Tubers ( c u l t i v a r Netted Gem) were inoculated i n the manner previously described. The inoculum concentration f o r both forms was determined, by s e r i a l d i l u t i o n g techniques, to approximate 2.0 x 10 CFU/ml. Half of these tubers were buried at a 17-22 cm depth while the rest were l e f t on the s o i l surface. Treatments, set out 1 m apart, were arranged i n a completely randomized design. The dimensions of the experimental area approximated 6 m x 35 m. The s o i l c e l l s and p l a s t i c bags were buried adjacent to stakes to f a c i l i t a t e t h e i r recovery. C l i m a t o l o g i c a l data f o r the UBC campus was obtained from the Uni v e r s i t y of B r i t i s h Columbia Meteorological Station. Five r e p l i c a t e s per treatment were examined at each sampling date. D i l u t i o n p l a t i n g techniques were used to recover E_. carotovora from inoculated s o i l s . No s o i l b a i t i n g , however, was attempted. Tubers recovered were cut i n h a l f , with only one hal f of each tuber used f o r indexing purposes. The other h a l f was kept at room temperature f o r 48 hours before being grown out i n 10 cm p l a s t i c pots i n the greenhouse. Symptoms were recorded a f t e r one month. (c) Oversummering Experiment Pemberton 1975 The l e v e l s of Erwinia carotovora around; a r t i f i c i a l l y - i n o c u l a t e d seed pieces and i n fallow s o i l were monitored from June 3, 1975 to Aug. 10, 1975 i n Pemberton. Two experimental areas, designated the "at r o s e p t i c a p l o t s " 39 and the "carotovora plots", were laid out 25-30 meters apart in an oat field to minimize the risk of cross-contamination. The appropriate inoculum was used in these areas to inoculate tubers and soil cells. Within each of these experimental areas there were separate control tuber plots, inoculated tuber plots and soil cell plots. To reduce the chance of cross-contamination, 45 m fallow guard strips were left between the respective plots. Soil cells were randomized equidistantly (1.5 meter) within a 5 x 5 Latin square design. The ten inoculated tubers were buried 1.5 meters apart in their appropriate plots. A similar arrangement was used for the control tuber plots. All treatments were fertilized with 8-20-20 at 600 lbs/A. . Two isolates, originally recovered from latent tuber infections by Dr. R.J. Copeman, were selected as the test bacteria. The atroseptica isolate (# 32-2) was recovered from a Kootenay seed potato sample, whereas the carotovora isolate (# 755-2) was a pathogenic form isolated from a Pemberton seed sample. Nutrient broth shake cultures of these organisms were grown for 72 hours at 21° C. Cells were then centrifuged and washed to remove any nutrients. The concentrations of the aqueous stock suspensions 9 9 were determined to be 1.3 x 10 and 1.8 x 10 CFU/ml for the atroseptica and carotovora forms, respectively. Soil and tubers were inoculated with a 1/10 aqueous dilution of these stock suspensions. Tubers were derived from stem cuttings and were assumed to be free of blackleg. Five soil cells were recovered at each harvest date. Soil dilution plates were used for detection purposes until baiting was necessitated. A non-destructive sampling technique was devised for testing the soil around seed pieces. A replicator board (a square sheet of 1.8 cm 40 plywood with a 22 cm diameter circle cut out in the centre), marked every 60°, was used to randomly select a sampling site for each date (Fig. 6). Soil samples 10 cm away from the seed piece were removed with a 1.8 cm diameter soil auger. Soil cores were carefully transferred to plastic bags which were stored at 5° C until samples were removed for dilution plating on CVP. (d) Overwintering Experiment Pemberton 1975-1976 The survival of Erwinia carotovora varietal forms in soil, on tubers and in the rhizospheres of horsetail plants was monitored under field conditions in Pemberton from Sept. 16, 1975 to April 22, 1976. The same carotovora isolate (# 755) was used in this study that was used in the previous experiment but a different atroseptica isolate (# 744), recovered from a latently-infected tuber obtained from Pemberton, was selected.. The inoculum was prepared in the standard manner. The concen-tration of the atroseptica arid the carotovora forms were determined by 9 10 dilution plating to be 7.0 x 10 and 3.0 x 10 CFU/ml, respectively. Soil cells were again used in this study. Soil, horsetail rhizospheres arid tubers were inoculated with 150 ml, 15 ml and 1000 ml, respectively, of a 1/10 dilution of the appropriate aqueous stock suspensions. The seed material (cultivar Netted Gem)wwasggrownooutoovertthe summer months in the appropriate plots as were the horsetail plants. Three replicates of each treatment were buried equidistantly as illustrated in Figure 7. Tubers left on the surface were covered with potato debris to discourage rodent damage. Stakes were used to distinguish test horsetail plants from other plants growing in the experimental plot. Fig. 6 The replicator board used to randomly sample soil cores, taken 10 cm away from the decaying seed piece. F i g . 7 A r e p l i c a t e d treatment consisted of 3 h o r s e t a i l plants transplanted 34 cm apart. A3 These p l o t s were located adjacent to the experimental p l o t s of the same v a r i e t a l form i n the oversummering experiment. The treatments were l a i d out 1.5 m apart i n a 5 x 7 randomized block design. There were 3 r e p l i c a t e s per treatment i n each block. Only three harvest dates (Sept. 16, 1975, Oct. 20, 1975j A p r i l 22, 1976) were f e a s i b l e due to the early snowfall i n Pemberton. Isolates were saved at each sampling date for s e r o l o g i c a l i d e n t i f i c a t i o n . Weather information was obtained (when available) from the Gingerbread Creek Cl i m a t o l o g i c a l Station. (e) Overwintering Experiments UBC 1975-1976 Population l e v e l s of both v a r i e t a l forms of E_. carotovora were monitored i n a r t i f i c i a l l y - i n o c u l a t e d lamb's quarters, lady's thumb and h o r s e t a i l rhizospheres from Oct. 27, 1975 u n t i l March 15, 1976. Surv i v a l on inoculated tubers was checked f o r comparative purposes. The same Erwinia i s o l a t e s were selected f o r t h i s study as were used i n the over-wintering studies i n Pemberton. Aqueous suspensions of the carotovora 9 9 and atroseptica forms contained 1.2 x 10 and 4.1 x 10 CFU/ml, res p e c t i v e l y . A 15 ml aliqu o t of a ten-fold d i l u t i o n of the appropriate stock suspension was poured around the base of test plants. These plants had been c o l l e c t e d outside the experimental area and were transplanted into t h e i r respective s i t e s two weeks p r i o r to rhizosphere i n f e s t a t i o n . Erwinia-tested tubers ( c u l t i v a r Warba) were inoculated by the standard procedure described previously but a l l tubers were buried to a depth of 10 cm. A 3 x 3 randomized block design covering a 22.5 m x 38.0 m area was used. Blocks were separated from one another by a 5.8 m fallow s t r i p and the treatments within each block were 1.5 m apart. Six r e p l i c a t e s were grown i n each p l o t , each separated from the next by 150 cm. One replicate per plot was randomly selected for indexing at each date. A few E_. carotovora isolates were saved for serological identification. No attempt was made to standardize the amount of root tissue used for detection purposes. The tubers recovered were indexed in the usual manner. The same carotovora and atroseptica isolates were used to study their overwintering ability in field soils on the UBC campus compared to the survival on tubers either left on the soil surface or buried (10 cm). Stock bacterial suspensions containing equal concentrations 9 (1.5 x 10 CFU/ml) of these pathogens were used. Ten-fold dilutions of these suspensions were made for inoculation purposes. The soil and tuber inoculation techniques outlined previously were used. Tubers (cultivar Pontiac) were derived from Erwinia-tested stocks. Only soil cells were used for soil survival studies. The plots were set up in a field seeded to field peas and oats. A 3 x 3 randomized block design was used. The dimensions of the plot approximated 32.5 m x 38.0 m and the blocks were separated by 6 meter guard strips. At each harvest date dilution plating and baiting techniques were used to detect these Erwinia populations in soil. The standard tuber indexing method was used to check survival in tubers. Some of the isolates recovered were saved.for identification purposes. (f) Host Range Studies An experiment was set up to establish which, i f any, of a pre-determined l i s t of plant species could express diagnostic symptoms upon art i f i c i a l inoculation. Plant species were selected on the basis of three main criteria: (1) previous documentation of Erwinia-infection or the ability of Erwinia carotovora to survive in their rhizospheres; (2) importance as a rotation crop or weed in potato fields; (3) member of the Solanaceae. A listing of the plant species included in this study was made (Table II). A representative collection of carotovora and atroseptica isolates was.selected for this study (Table III). The stem-prick inoculation method was used for most of the test species. Such plants as white clover, red clover, alfalfa and horsetail were grown in flats for 2-3 months before being root-inoculated prior to transplanting. Five replicates were used per treatment and the inoculated plants were kept on a temperature-controlled (18° C) bench under lights (12 hour photoperiod). The symptoms were recorded after one week. Faba beans, peas and lady's thumb were also tested with these isolates at 25° C. 46 Table II An alphabetical l i s t i n g of the plant species included i n the host range experiment Common Name Botanical Name Cu l t i v a r a l f a l f a Medicago s a t i v a L. Rhizoma black nightshade Solanum nigrum L. -? Chenopodium quinoa L. corn Zea mays L. Golden Bantam cucumber Cucumis sativus L. Challenger faba bean V i c i a faba L. Broad Windsor h o r s e t a i l Equisetum arvense L. -lady's thumb Polygonum p e r s i c a r i a L. -lamb's quarters Chenopodium album L. -oat Avena s a t i v a L. Pendek onion Allium cepa L. Autumn Spice pea Pisum sativum L. Super Sweetpod pepper Capsicum annuum L. Liberty B e l l petunia Petunia hybrida Vilm. Single M u l t i f l o r a potato Solanum tuberosum L. Netted Gem red clover T r i f o l i u m pratense L. Delta tobacco Nicotiana tabacum L. Xanthi-nc tomato Lycopersicum esculentum M i l l . Bonny Best wheat Triticum aestivum L. Marquis white clover T r i f o l i u m repens L. 47 Table I II A l i s t i n g of the various carotovora and atroseptica i s o l a t e s used i n the alternate host experiment Isolate Number V a r i e t a l Characterization Host Location 755 var. carotovora Potato-tuber Pemberton WR 19 it II Potato-tuber II SR 40 II II Potato-tuber Wisconsin PRO 5 II it C a r r o t - f i e l d LFV SM 2 II it Lady's thumb- Pemberton rhizosphere CH 4 it it Chrys anthemum-s t em LFV WA 10-2 ti ii Potato-tuber Pemberton MNP 14 II II Potato-tuber Creston 744 var. atroseptica Potato-tuber Pemberton 32-2 II II Potato-tuber Invermere SR 8 II II Potato-tuber Wisconsin S 13 II II Potato-blackleg •Pemberton stem S 31 II j II Potato-blackleg II 400-2 stem II ti Potato-tuber Invermere 316-2 it it Potato-tuber II LFV = Lower Fraser V a l l e y , B.C. RESULTS (1) Survey of Seed Potato Stocks The levels of latent contamination which Erwinia carotovora of B.C. seed potato stocks grown in 1972 were determined to be relatively low (Table IV). While 73 pectolytic isolates were recovered, only 8 were characterized as E_. carotovora. Four of the seven lots surveyed appeared to be Erwinia-free. These lots corresponded to the stocks with the lowest field incidence. It's noteworthy that apparently healthy tubers and culls from -the seed lots with the highest field incidence (Lot E) displayed an equally high level of latent infection. The contamination levels of foundation and certified stocks did not appear to differ greatly. Both varietal forms of the pathogen were isolated from Lots A and E. Pathogenicity tests carried out at 18° C on the two week old Epicure plants demonstrated that the carotovora forms could be further subdivided into pathogenic and nonpathogenic isolates (Fig. 8). The former induced typical blackleg lesions, undistinguishable from those expressed by atroseptica forms. With the nonpathogenic form only a localized soft rot occurred at the inoculation site which the plants outgrew. Blackleg symptoms were induced within 72 hours i f the relative humidity was sufficiently high. Serologically, atroseptica forms reacted with only the atroseptica antiserum (AS 744). Many of the nonpathogenic carotovora isolates failed to react with antisera prepared against the atroseptica, the pathogenic or the nonpathogenic carotovora isolates. Table IV Levels of latent tuber i n f e c t i o n with Erwinia carotovora i n B.C. seed potato stocks harvested i n 1972 Lot C u l t i v a r Number of Tubers== Tested Seed Grade Blackleg F i e l d Incidence % * E. carotovora ;Varietal Form Recovered S e r o l o g i c a l Identifitr c a t i o n * * • .Pathogenicity at 18°C *** A Kennebec 1) Healthy 2) Culls 40 40 Foundation .25,.1 1 at r o s e p t i c a 1 carotovora A/S A no reaction + + (w) B Norgold Russet 1) Healthy 2) Culls 40 40 Foundation .25,.1 1 at r o s e p t i c a 1 A/S A + C Kennebec 1) Healthy 40 C e r t i f i e d tr.,.1 - — — D Pontiac 1) Cutseed 2) Cu l l s 40 40 E l i t e I I I 0,0,tr. - - -E Netted Gem, 1) Healthy 18 Foundation C e r t i f i e d Fl.0,.25, l' -!?525,. C.5,.8,.8 ,- 2 carotovora ••' 3 at r o s e p t i c a 2 no react. 3 A/S A 1-1+ (w) 3+ F Netted Gem 1) Cu l l s 40 Foundation . l , t r . , 0 - — — G Netted Gem 1) Healthy 40 Virus-free plants 0,0,0 - -* t r . = les s than 0.1% blackleg incidence. * A/S A = atroseptica, A/S PC = pathogenic carotovora, A/S NPC = nonpathogenic carotovora. * + (w) = only 1 or 2 of 5 r e p l i c a t e plants k i l l e d . 50 Fig. 8 Symptoms produced in potato cuttings at 18 C, 1 week after stem-prick inoculation with Erwinia carotovora. (a) Typical blackleg symptoms incited by the atroseptica varietal form, (b) Symptoms similar to blackleg caused by pathogenic isolates of the carotovora form, (c) Localized stem rotting symptoms caused by nonpathogenic isolates of the carotovora form. There was an o v e r a l l increased l e v e l of contamination i n a si m i l a r study c a r r i e d out i n the f a l l of 1975 using f r e s h l y harvested tubers. Again, no d i r e c t c o r r e l a t i o n was found between the l e v e l s of latent i n f e c t i o n and the recorded f i e l d incidence for the crops. Both v a r i e t a l forms were recovered from some l o t s . The r a t i o of the carotovora: atro s e p t i c a forms recovered from 300 tubers was almost 1:1. Once again both pathogenic and nonpathogenic carotovora types were recovered and i n a few cases sin g l e tubers were found to be infected with both types. The p o s s i b i l i t y of d i s t i n c t carotovora serotypes i s indicated i n Table V. The majority of the nonpathogenic i s o l a t e s reacted with none of the a v a i l a b l e a n t i s e r a (riot even with an antiserum prepared against a nonpathogenic carotoyora i s o l a t e ) . Pathogenic forms i s o l a t e d from Lots B, C and D reacted with the antiserum prepared against a pathogenic carotovora i s o l a t e . A few pathogenic carotovora i s o l a t e s recovered from one of the tubers i n Lot E reacted with the antiserum prepared against the atroseptica i s o l a t e . S e r o l o g i c a l s p e c i f i c i t y was observed among atroseptica i s o l a t e s . Tubers from f i e l d s i n the Creston Va l l e y having widely d i f f e r i n g f i e l d incidence of blackleg'were found to be extensively contaminated with Erwinia carotovora (Table VI). The carotovora form was pr i m a r i l y recovered from both l o t s . Pathogenicity tests and s e r o l o g i c a l studies indicated that there were d i s t i n c t forms of var. carotovora (Table VII). Pathogenic carotovora forms were i s o l a t e d from both l o t s , but i s o l a t e s from Lot A were s e r o l o g i c a l l y s i m i l a r to the pathogenic carotovora i s o l a t e used i n antiserum preparation. Only 3 of 13 i s o l a t e s recovered from Lot B, however, reacted with t h i s antiserum. Table V Levels of latent contamination with Erwinia carotovora varietal forms in freshly harvested seed potato stocks grown in 1975 Lot Cultivar Number of Tubers Tested Seed Grade Blackleg Field Incidence /° E. carotovora Varietal Form Recovered Serological Identifi-cation ** Pathogenicity at 18°C *** A Netted Gem 50 ^Foundation • 1 3 atroseptica . 3 A/S A 3+ B Kennebec 50 Foundation 0,0 3 carotovora 8 .atroseptica 1 A/S PC 2 A/S A 8 A/S A 1+ 2-8+ C Red, weo Pontiac 50 Foundation .1,0 4 carotovora 3 A/S PC 1 A/S A 3+ 1+ (w) D Norgold Russet 50 Certified 00,0 6 carotovora 5 A/S PC 1 no react. 5+ 1-E Kennebec 50 Certified tr.,tr. 4 carotovora 2 atroseptica 1 " (?) KA/SA+A/SPC) 2 no react. 1 A/S PC 3 A/S A + & -2-1+ 3+ F Netted Gem 50 Foundation ? 2 atroseptica 1 carotovora 2 A/S A 1 no react. 2+ 1-* tr. = less than 0.1% blackleg incidence. ** A/S A = atroseptica, A/S PC = pathogenic carotovora, A/S NPC = nonpathogenic carotovora. *** + (w) = only 1 or 2 of 5 replicate plants killed. 53 Table VI Latent Erwinia infection of seed potato tubers produced in the Creston Valley in 1974 Seed Lot Sample Size Blackleg Field E. carotovora Incidence Varietal Forms Recovered A 120 low or no 14 carotovora field incidence 2 atroseptica B 100 "high" incidence 33 carotovora 2 atroseptica Table VII Pathogenicity and serological reactions of Erwinia isolates recovered from two different Creston seed tuber lots Seed Lot Number of Pathogenicity Serological Varietal Forms at 18°C * Identification Isolated (P) (P+NP) (NP) 14 carotovora 2 atroseptica 33 carotovora '- at»"-septics 2 atroseptica 8+ 2± 4- All NP = no reaction 8 P = A/S PC 2 P± = A/S A 2+ 0 0 2 P = A/S A 13+ 0 20- 20 NP = no reaction 3 P = A/S PC 10 P = no reaction 2+ 0 0 2 P = A/S A * (P) = number of pathogenic isolates recovered; (NP) = number of nonpathogenic isolates recovered; (P+NP) = number of tubers latently infected with both pathogenic and nonpathogenic forms. ** Multiple isolate recoveryd from single tuber. *** Only three of the carotovora isolates proved to be virulent pathogens. 55 (2) Survey of Volunteer Tubers Volunteer tubers, both on the soil surface and buried in fallow fields following lifting of the crop, persisted well into the succeeding spring and summer in Pemberton. The majority of these volunteers appeared quite healthy, bearing only harvest scars. Several had developed into plants 7 to 15 cm high and no blackleg symptoms were observed. As noted in Table VIII, the level of Erwinia carotovora recovery from volunteer tubers was quite low. The only latently-infected tubers sampled were collected in a field rejected for seed purposes due to its high blackleg incidence. Pathogenic carotovora forms were primarily recovered. The atroseptica varietal form could not be detected. Yet, serologically these pathogenic carotovora isolates were found to be most similar to the atroseptica form on the basis of agglutination tests-More recent tests using the FAS technique did not confirm this close relationship. (3) Survey of Weed Rhizospheres The predominant species of weeds associated with seed potato fields in Pemberton were horsetail, lady's thumb, lamb's quarters and redroot pigweed. Erwinia carotovora varietal forms were detected in weed rhizospheres early in the summer only once (Table IX). By contrast they were readily isolated in the f a l l from weeds growing in fields seeded to potatoes. The carotovora form was primarily recovered in these studies and was detected9in the rhizospheres of lady's thumb, horsetail, redroot pigweed, nightshade and lamb's quarters but not those of couchgrass or yellow cress. A single atroseptica isolate was recovered from one horsetail 56 Table VIII The recovery of Erwinia carotovora from overwintering volunteer tubers in the Pemberton Valley Seed Sampling Sample Tubers Varietal Serological Pathogenicity Lot Date Size Contam- Character- Indentifi- at 18°C inated ization * cation A June 40 tubers 1974 18 potato - — _ plants B June 68 tubers 2(5)** carotovora 4 A/S A 4+ 1975 1 no reaction" .1-C June 50 tubers 1975 A/S A = atroseptica, A/S PC = pathogenic carotovora, A/S NPC = non-pathogenic carotovora. • Multiple isolate recovery. Two isolates were recovered from 1 tuber and 3 from another. 57 Table IX The recovery of Erwinia carotovora varietal forms from the rhizospheres of common weed species in the Pemberton Seed Control Area Sampling Weed Number of Varietal Serological Patho-Date Species Plants Character- Identifi- genicity Tested ization cation* at 18°C of Isolates Recovered July 1973 horsetail 31 0 0 0 lady's thumb , 0 0 0 lamb's ..... ^ quarters 5 0 0 0 turnips 3 0 0. 0 May.1974 horsetail** 37 0 0 0 horsetail*** 46 0 0 0 June 1974 horsetail + 20 0 0 0 lady's thumb 15 3 carotovora 3 no react. 3-redroot pigweed 4 0 0 0 SSept 1974 couchgrass 6 0 0 0 horsetail 43 5 carotovora 1 no react. 2+ 4 A/S A 3-1 atroseptica ?++ 1 lady's thumb 22 2 carotovora 2 A/S A 1 lamb"s quarters 19 3 carotovora 3 A/S A 3+ nightshade 9 2 carotovora 2 A/S A ? redroot pigweed 7 1 ? ? ? yellow cress 3 0 0 0 June 1975 horsetail 64 0 0 0 * A/S A = atroseptica, A/S PC = pathogenic carotovora, A/S NPC = non= pathogenic carotovora. ** F i e l d seeded to potatoes. *** F i e l d seeded to oats. +3?" F i e l d seeded to oats. ++ Isolate(s) l o s t i n cul t u r e . 58 rhizosphere but the isolate was lost before pathogenicity and serological identification could be carried out. No symptoms were observed in spite of rhizosphere contamination. While horsetail constituted the major weed problem in the Pemberton seed potato fields, Erwinia carotovora was infrequently isolated from the rhizospheres of horsetail plants. E, carotovora was detected in redroot pigweed and nightshade rhizospheres in spite of the small sample size. Unfortunately, these isolates were lost in culture and pathogenicity tests could not be carried out. Several isolates biochemically identified as var. carotovora, which were recovered from the root zones of lady's thumb and lamb's quarters in the September, 1974 survey, reacted serologically with the atroseptica antiserum. These f a l l lamb's quarters isolates were also pathogenic, compared to the nonpathogenic isolates recovered from earlier surveys of lady's thumb rhizospheres. Moderate levels of pectolytic pseudomonad species were recovered on CVP in these surveys. Their abundance proved a nuisance as these bacterial species interfered with the recovery of Erwinia forms. Erwinia-type pits on this pectate medium were often observed, particularly around horsetail tissue, yet no E_. carotovora varietal forms could be isolated. However, pectolytic pseudomonad species were consistently recovered from these pits. An antagonistic, fluorescent pseudomonad isolate recovered from the rhizospheres of weed species was especially troublesome. This unidentified species was responsible for the loss of several cultures in storage. Its amorphous growth habit and bluish hue made i t difficult to detect on CVP medium. 59 (4) Su r v i v a l i n S o i l , Tubers and Weed Rhizospheres (a) Overwintering i n S o i l Both E_. carotovora v a r i e t a l forms were able to survive winter conditions at UBC f o r at le a s t 8 weeks beginning Dec. 17, 1974 i n a r t i -f i c i a l l y i n f ested s o i l amended with nutrient broth (Fig. 9a). The atroseptica form could not be detected by d i l u t i o n p l a t i n g i n e i t h e r s o i l c e l l s or p l a s t i c bags beyond t h i s time. The carotovora v a r i e t a l form by contrast could be recovered from two of f i v e s o i l c e l l s a f t e r 4 months without b a i t i n g . The population of the carotovora v a r i e t a l form was -s i g n i f i c a n t l y higher (P = 0.01) u n t i l the Feb. 11 sampling date (Table X). The longevity of E.ecarotovora populations was greater i n s o i l c e l l s than i n p l a s t i c bags. Winter temperatures were r e l a t i v e l y mild (Figs. 9b and 9c). S o i l temperatures did not drop below 0° C although freezing ambient ,air temperatures were recorded. Presumably the l i m i t e d snowfall during these periods prevented the s o i l from freezing. Ample p r e c i p i t a t i o n ( F i g . 9d) was recorded so that desiccation was not a factor i n s u r v i v a l . The experiment was repeated i n 1975-1976 i n unamended s o i l at UBC. Under these conditions both v a r i e t a l forms could be detected by d i l u t i o n p l a t i n g at 28 days (Fig. 10a). Only the carotovora v a r i e t a l form could beo recovered by d i l u t i o n p l a t i n g at subsequent sampling dates although the l e v e l s had dropped o f f s i g n i f i c a n t l y (Table XI). The atroseptica v a r i e t a l form could not be recovered even by b a i t i n g at l a t e r sampling dates. S e r o l o g i c a l i d e n t i f i c a t i o n of the i s o l a t e s recovered revealed that 85% of 60 Fig. 9 Survival of Erwinia carotovora in artificially-infested, nutrient-amended fallow soils at U.B.C. during the winter of 1974-1975. (a?);. Survival of the atroseptica and the carotovora forms in plastic bags and soil cells with time. (b) Soil temperature at 10 cm during the study period, (c) Air temperature during the study period, (d) Total precipitation during the experimental period. Table X The survival of Erwinia carotovora in artificially-infested field soil on the UBC E. carotovora Time Interval (Days) Varietal Forms 0 7 14 21 28 Dec.17 Dec.24 Dec.31 Jan.7 Jan.14 1974 1975 var. atroseptica (.1) soil cells 4.6 (2) plastic bags 5.0 Averages 4.8 var. carotovora (1) soil cells 2.9 (2) plastic bags 3.0 Average 3.0 Statistical Significance** Effects 0 7 14 21 28 42 56 112 Bacterial 0.01 Container 0.05 Bacteria x Container 0.05 0.01 ns ns 0.01 0.01 0.05 0.01 0.01 ns 0.01 0.01 ns 0.01 0.01 0.01 ns ns ns ns ns ns * All values are expressed as data values. log reciprocal counts. An arbitrary value of 0.5 was added to a l l Values listed indicate significance at the respective probability level, ns = P Z 0.05 0.05 = P "C 0.05 0.01 = P ^  0.01 42 56 112 Jan.28 Feb.11 April 14 1975 5.7 6.1 5.9 7.9 7.9 8.9 8.4 10.7 13.1 11.9 14.0 13.9 14.0 14.5 14.5 14.5 3.2 3.3 3.3 7.0 7.3 7.1 10.4 11.0 10.7 14.0 14.5 14.3 14.5 14.5 14.5 62 Fig. 10 Survival of Erwinia carotovora in artificially-infested fallow soil at U.B.C. during the winter of 1975-1976. (a) Survival of the atroseptica and carotovora forms in soil cells with time. (b) Soil temperature at 10 cm during the study period, (c) Air temperature during the study period. (d) Total precipitation during the experimental period. 63 Table XI The overwintering of Erwinia carotovora in artificially-infested field soil at UBC 1975-1976* Sampling Date Time CFU/;gram oven-dry Soil of Interval Erwinia carotovora** (Days) var. carotovora var. atroseptica Nov. 4, 1975 0 4.0 x 106 ( a ) 1.5 x 106 (b) Nov. 17, 1975 13 5.3 x 105 ( C ) 2.2 x 104 (d) Dec. 2, 1975 28 1.8 x 104 ( d e ) 2.7 x 103 (e) Dec. 18, 1975 44 5.0 x 102 ( f ) 0 ( f ) Feb. 24, 1976 112 4.5 x 10 ( f ) 0 <f> Mar. 22, 1976 139 2.3 x 10 ( f ) 0 (f> Each value is a mean of 3 observations. Means followed by the same letter are not significantly different at P = 0.05. 64 the isolates from carotovora and 93% of the isolates from the atroseptica plots were serologically similar to the species started with. Cross-contamination accounted for the 7% discrepancy in the atroseptica plots, while some isolates with a different serotype were recovered from the carotovora plot. Isolates from two of the four control plots yielding var. carotovora were of the same serotype as that used as inoculum. Weather conditions during the 1975 study period (Figs. 10b and 10c) were generally similar to those of the previous year. Increased precipitation (Fig. lOd) and poor drainage resulted in flooding, occasionally being a problem in some portions of the plot. Moderate levels of both varietal forms were detected after 35 days in a parallel overwintering study of infested soil under natural field conditions in Pemberton in 1975-76 (Fig. 11a). No significant difference was noted in the ability of the varietal forms to survive under these conditions (Table XII). While the average ambient temperatures dropped rapidly, the snowpack protected the soil from freezing (Figs, lib and 11c). The carotovora form could be recovered from only one of 5 soil cells by baiting in the spring after 225 days. The isolate recovered was serologi-cally identical to the isolate used for infestation. Serological identi-fication of the representative isolates showed that 88% of the carotovora isolates and 85% of the atroseptica isolates were of the same serotype as the inoculum. The difference was due to cross-contamination in the atroseptica plots and presence in the carotovora plots of a carotovora of a differing serotype. 65 z o L i . >-z o _l o u o o o >-Q < tt o z tt »— < z o S: E <-» E LU tt a. O « z to 7y 6-5-4-3-2-• var. atroseptica var. carotovora maximum P r-0. 0-' - -minimum l5 lo~ 20 24 TIME (WEEKS) APR. 22 Fig. 11 Survival of Erwinia carotovora, in artificially-infested fallow soil at Pemberton in the winter of 1975-1976. (a) Survival of the atroseptica and carotovora forms in soil cells with time, (b) Air temperature during the experimental period. (c) Total precipitation during the study period. (d) Accumulated snowpack during the study period. '66 Table XII Survival of Erwinia carotovora in artificially^infested soil at Pemberton Sampling Date Time Interval Number of Colony Forming Units/Gram Oven-Dry Soil E. carotovora var. carotovora var. atroseptica Sept. 16, 1975 Oct. 20, 1975 April 22, 1976 0 35 days 225 days 2.2 x 10 6* (a) 5.5 x 10 + 1/5 bait 3 (b) 1.4 x 10 3.9 x 10 0 6 (a) 3 (b) * Each value represents a mean of 5 observations. Means followed by the same letter did not differ significantly (P = 0.05). 67 (b) Overwintering in Tubers Both varietal forms could be recovered from at least some inoculated volunteer, tubers exposed to 16 weeks of 1974 winter weather conditions at UBC (.Table XIII). Tubers inoculated with either varietal form buried at 10 cm were found to be more decayed than those buried at 20 cm. The latter tubers were frequently sound when dug. Tubers inoculated with var. atroseptica were generally more decayed than those inoculated with var. carotovora. The recovery rate of both varietal forms from the more deeplybburied tubers was lower (Table XIII). Of the 80 tubers inoculated with var. atroseptica, 26 produced apparently healthy plants, 5 produced blackleg-infected plants and 49 did not sprout due to seed piece decay. The tubers inoculated with var. carotovora produced 59 apparently healthy plants and 21 did not sprout due to seed piece decay. When the experiment was repeated (with modifications) in 1975-1976, var. atroseptica was found not to survive as well as var. carotovora (Table XIV). Similar rates of recovery from buried tubers (~25%) were recorded for atroseptica-inoculated tubers at 44 days and carotovora-inoculated tubers at 139 days. Survival of both forms was better at 10 cm than when the tubers were left on the surface. Tubers left on the surface were found to decompose much more readily than those buried, particularly those inoculated with var. atroseptica. At the later sampling dates often only traces of skin remained. In contrast, those buried at 10 cm were found to be sound and unrotted. Cross-contamination was not a problem as the isolates recovered were serologically similar to that used as inoculum. However, two of three Table XIII The recovery of Erwinia carotovora varietal forms from artificially-inoculated tubers buried in fallow soil on the UBC campus in 1974 Sampling Date Time Interval (Days) Number of Tubers Yielding Erwinia it carotovora var. i 10 cm carotovora 20 cm var. 10 cm atroseptica 20 cm Dec. 17, 1974 0 +4** +3 +4 +3 Dec. 24, 1974 7 +3 +4 +4 42 Dec. 31, 1974 14 +3 +2 +2 +3 Jan. 7, 1975 21 +5 +2 +3 +1 Jan. 14, 1975 28 +4 +1 +4 +1 Jan. 28, 1975 42 +3 +1 +5 +2 Feb. 11, 1975 56 +5 +5 +3 +1 April 14, 1975 112 +2 +2 +2 +1 The carotovora and atroseptica isolates used in this study were the Wisconsin isolates SR40 and SR8,rrespectively. Five replicate tubers per treatment were tested at any given sampling date. 69 Table XIV The s u r v i v a l of Erwinia carotovora i n inoculated potato tubers l e f t on the s o i l surface or buried at 10 cm on the UBC campus i n 1975 Sampling Date Time Inte r v a l (Days) Number of Tubers Y i e l d i n g Erwinia carotovora* var. carotovora var. atroseptica surface buried surface buried Nov. 4, 1975 0 +6 +6 +6 +6 Nov. 17, 1975 13 +5 +5 +4 +4 Dec. 2, 1975 28 +6 +5 +5 +6 Dec. 18, 1975 44 +5 +4 +2 +3 Feb. 24, 1976 112 +1 +4 +Q +2 Mar. 22, 1976 139 +1 +3 +0 +0 * Six r e p l i c a t e s per treatment were sampled at each date. 70 control control tubers were found to be contaminated with var. carotovora of the same serotype as the inoculum. Both varietal forms also overwintered on inoculated tubers under natural field conditions in Pemberton (Table XV). As in the trials at UBC, the percentage recovery from tubers left on the soil surface was low by the end of the experimentaX225 days). The tubers were found in varying stages of decay. At least 90% of the recovered isolates corresponded to the serotypes used for inoculation. However, there was a high degree of control tuber contamination. When a sample of the tubers used in this experiment were indexed for latent infection, approximately 16% were contaminated with var. carotov.ora. Some isolates had the same serotype as the inoculum while others did not. (c) Overwintering in Weed Rhizospheres Populations of both varietal forms could be detected in arti-f i c i a l l y infested horsetail rhizospheres at 35 days under natural field conditions at Pemberton. Neither form, however, could be recovered either by dilution plating or baiting after 225 days. As the root tissue weight could not easily be standardized, a rating system was devised to relate the ease of recovery to the population levels (Table XVI). Serological identification procedures indicated that the recovery percentage of the appropriate carotovora and atroseptica isolates from inoculated rhizospheres totalled 100% for both forms. However, the control horsetail rhizospheres in the carotovora plots were contaminated with var. carotovora. Isolates recovered from 4/5 and 1/5 control plots on Sept. 16 and Oct. 20, respectively, were serologically identical to the inoculum used. However, the isolate recovered on April 22, 1976 was not of the same serotype. 71 Table XV The recovery of Erwinia carotovora from inoculated tubers overwintering on the surface and buried in field soil at Pemberton* (A) Erwinia carotovora var. carotovora Sampling Date Control bSuffaee Control Buried Inoculated Surface Inoculated Buried Sept. 16, 1975 77/10 6/10 8/10 10/10 Oct. 20, 1975 0/10 2/10 10/10 10/10 April 22, 1976 0/10 0/10 4/10 6/10 (B) Erwinia carotovora var. atroseptica Sampling Date Control Surface Control Buried Inoculated Surface Inoculated Buried Sept. 16, 1975 5/10 5/10 4/10 7/10 Oct. Z0, 1975 5/10 1/10 10/10 10/10 April 22, 1976 0/10 0/10 3/10 6/10 * Ten replicates per treatment were sampled at each. date.. 72 Table XVI The recovery 'of Erwinia carotovora from infested horsetail rhizospheres under natural field conditions in Pemberton Sampling Date Time Interval (Days) Erwinia carotovora var. carotovora var. atroseptica Control Inoculated Control Inoculated Horsetail Horsetail Horsetail Horsetail Sept. 16, 1975 0 Oct. 20, 1975 35 April 22, 1976 225 +5/5*(+1)** +5/5(+5) 0/5(0) +5/5(+5) +1/5(+1) +5/5(+5) 0/5(0) +5/5(+5) +1/5(+1) 0/5(0) 0/5(0) 0/5(0) * Five replicates of each treatment were tested at each sampling date. ** Rating the ease of isolation (0) = none (+1) = recovery by baiting (+5) = recovery by dilution plating. 73 Similarly, populations of both varietal forms were detected at 4 weeks in the artificially inoculated rhizospheres of common weeds grown at UBC (Table XVII). There was, however, a rapid drop in the population during the first few weeks. After 112 days, the atroseptica form could be recovered from only one horsetail replicate whereas the carotovora form was s t i l l detectable in the rhizospheres of a l l three species. Baiting had to be used for lady's thumb and horsetail. By 140 days the atroseptica form was recovered from only 1 replicate of lamb's quarters by baiting. The soft rot form, similarly, was baited from one replicate of lady's thumb and horsetail. The identification of representative isolates recovered revealed that at least 80% were serologically similar to those used as inoculum. There was some cross-contamination in the rhizospheres of control plants. Carotovora isolates similar to the inoculum were recovered from 3/18 lady's thumb and 2/18 lambrs quarters plants. Contaminants detected in control horsetail rhizospheres were identified as an:atroseptica and a serologically different carotovora isolate. Cross-contamination was not surprising considering the heavy rain (Fig. lOd) and surface water movement during the experiment. The tops of the horsetail and lady's thumb plants were dead by Nov. 24, 1975, coinciding with the decrease in ambient temperatures. Root systems of lamb's quarters and lady's thumb were s t i l l intact in spite of the dead tops. Horsetail plants had overwintered successfully and new shoots appeared the following spring. No diagnostic blackleg symptoms were observed at any time. Table XVII The recovery of Erwinia carotovora from rhizospheres of common weed species on the UBC campus (A) Erwinia carotovora var. atroseptica Weed Species Time Interval (Days) 0 Oct.27,1975 14 Nov.10 28 Nov.24 42 Dec. 8 112 Feb.16 140 Mar.15 1976 (1) lamb's quarters (2) lady's thumb (3) h o r s e t a i l 3(++)* 3(4+) 3 C++) 3 C++) 3(++) 3(++) O(-) K++) 2(4+) K++) i:(+» 3(++) O(-) O(-) K++) K+) O(-) 'O(-) (B) Erwinia carotovora var. carotovora Weed Species Time Interval (Days) 0 14 28 42 112 140 (1) lamb's quarters (2) lady's thumb (3) h o r s e t a i l 3(++) 3(++) 3 C++) 3(++) 3 C++) 3(++) 3(++) 3(++) 3(++) 2 (++) 3 (++) 3 C++) K++) 2(+) 2(+) O(-) K+) K+) * Number out of three r e p l i c a t e s per treatment sampled at each date from which Erwinia carotovora was recovered. The ease of i s o l a t i o n i s indicated i n the following manner: (-) = no recovery (+) = recovery by s o i l b a i t i n g (++) = recovery by d i l u t i o n p l a t i n g . 75 The survival in weed rhizospheres at UBC was compared to survival in tubers under similar plot conditions. Once again tubers inoculated with the atroseptica varietal form tended to decay more readily than those treated with the carotovora form. A l l of the tubers inoculated with the former were rotten after 112 days. Buried tubers in the carotovora plots, on the other hand, were not as extensively decayed. Some were even beginning to sprout. A l l 30 tubers yielded soft-rotting Erwinia forms in the caroppvora plot and 15 out of 30 did in the atroseptica plots (Table XVIII). Virtually a l l of the isolates identified were of the inoculum type. However, several tubers (5) in the control plots were infected with the carotovora form. Four of these tubers harboured isolates identical to the serotype of the carotovora inoculum used. (d) Oversummering in Soil at Pemberton in 1975 A marked decline in the Erwinia population in infested soils was observed after four weeks (Fig. 12a). This reduction corresponded to a drop 7 3 7 3 from 1.1 x 10 to 1.9 x 10 and from 2.3 x 10 to 2.4 x 10 CFU per gram oven-dry soil for var. carotovora and var. atroseptica, respectively. Neither organism was detected after six weeks using serial dilution plating on CVP. However, var. carotovora was reisolated from 3 out of 5 soil cellf', replicates by baiting at 58 days. No atroseptica isolates could be recovered by baiting at this time. The carotovora isolates recovered were serologically similar to the serotype of the inoculum used to infest the soil. By mid-August neither varietal form could be recovered using dilution plating or by baiting. The recovery of the appropriate carotovora and atroseptica isolates from these plots was 80% ana 87%, respectively. 76 Table XVIII The recovery of soft-rotting Erwinia.forms from carotovora- and atroseptica-inoculated potato tubers buried at 10 cm Sampling Date Time Interval (Days) Number of Erwinia var. carotovora Tubers Yielding carotovora * var. atroseptica Oct. 27, 1975 0 +3 +3 Nov. 10, 1975 14 +3 +2 Nov. 24, 1975 28 +3 +3 Dec. 8, 1975 42 +3 +2 Feb. 16, 1976 112 +3 +2 Mar. 15, 1976 140 +3 +1 * Three replicates per treatment were sampled at any given date. 77 var. at roseptica • — — • around tuber • • soil cell var. carotovora © Q around tuber o o soil cell ± E < u. Z < 40-30-20-X 10H 0 JUNE 3 0 T TIME (WEEKS) 8 l b AUG. 12 a, Fig. 12 Survival of Erwinia carotovora in artificially-infested fallow soil and around inoculated tubers in Pemberton during the summer of 1975. (a) Survival of the atroseptica and carotovora forms in soil cells and around decaying seed tubers buried in soil with time. (b) Air temperature during the study period. (c) Total precipitation during the experimental period. 78 Levels of. Erwinia carotovora around inoculated seed pieces peaked two weeks after inoculation (Fig. 12a). This increase was more marked around tubers inoculated with the carotovora form. No attempt was made to correlate this increase with the condition of the decaying seed piece. None of the atroseptica-inoculated tubers sprouted while only one of the ten carotovora-inoculated tubers failed to sprout. All but one of the control tubers produced healthy plants. Seed piece decay accounted for this single loss in the control plot. The atroseptica form populations declined rapidly until they could not be detected by soil dilution plating after 29 days. The carotovora varietal form was recovered from 1 of 5 replicates by dilution plating after 58 days and from 2 of 5 replicates after 70 days by baiting. The atroseptica varietal form, however, was not recovered beyond 29 days even with baiting. The carotovora isolates were serologically identical to the inoculum used. No Erwinia forms were recovered from the seed piece remnants or from the rhizospheres of weeds growing nearby at the conclusion of this experiment. Few traces remained of seed pieces inoculated with the atroseptica varietal form. However, 3 out of 10 control replicates in the carotovora plots were contaminated with var. carotovora but of a different serotype than that used in the experiment. The percentage recovery of the appropriate isolates from the carotovora and atroseptica plots totalled 84% and 88%(, respectively. Moderately high air temperatures were recorded during the summer (Fig. 12b). Temperatures exceeding 30° C were registered five weeks into the experiment. The average weekly minimum and maximum temperatures varied by as much as 15-20° C. Little precipitation was recorded in Pemberton during the study period (Fig. 12c) and what l i t t l e rain there was f e l l towards the end of the experimental period.-79 (5) Host Range Studies Considerable variability was apparent in the host reaction to the inoculation with representative isolates (Tables XIX and XX). Three groups of hosts were evident: (1) those that were susceptible to a l l isolates; (2) those that appeared to be resistant regardless of the isolate; and (3) those that were susceptible to some isolates and resistant to infection by others. The only hosts to respond by producing typical blackleg lesions on inoculation with atroseptica isolates were potatoes and faba beans (Fig. 13). These lesions became systemic and ultimately resulted in the death of the inoculated plants. Faba beans, also, produced identical symptoms with a l l carotovora isolates. Only two of the eight representative carotovora isolates, on the other hand, could incite such symptoms on potatoes. While cucumbers and pepper plants were susceptible to a l l the isolates tested, no black=pigment formation was evident. Only an extensive soft rot symptom was observed. Cereal and forage species were immune (Table XIX). The only weed to express symptoms was lady's thumb. Two of the isolates (# 316-2 arid WR19) produced large soft rot lesions at the site of inoculation but the plants continued to grow. A similar host response was noted on petunia plants. Some carotovora isolates (SR40, CH4, WA10-2 and MNP14-) had a very restricted host range. Others,Hike # 755 and WR19, were not only able to incite blackleg symptoms in inoculated potato plants but also had a much broader host range. None of the isolates produced blackleg symptoms on lady's thumb while a l l did on faba beans at 25° C. Only isolate SR40 failed to infect peas at this higher temperature. Table XIX Host.reactions at 18 C when inoculated with representative isolates of the atroseptica and carotovora varietal forms Hosts Reaction (1) Cucumber Blackleg symptoms or death (2) Faba bean 11 " " " (3) Pepper " 11 " " (4) Corn No symptoms (5) Oats " " (6) Wheat " 11 (7) Alfalfa No symptoms (8) Red clover " " (9) White clover " " (10) Lamb's quarters No symptoms (11) Black nightshade (12) Horsetail " " Table XX Host reactions at 18 C when inoculated with representative isolates of Erwinia carotovora varietal forms Host Symptom Production or Plant Death With 744 32-2 SR8 S13 S31 400-2 316-2 755 WR19 SR40 PR05 SM2 CH4 WA10-2 MNP14-C.quinoa - + - + + - + - - - + + - -Lady's thumb - - - - - - + - + - - - - -Onion - + + + + - - + + - + + - + -P i t + + - + + + + + + + + + + + Petunia + + + + - - + + + - + + - - -Potato + + + + + + + + + - - • - • - -Tobacco + + + + + - + + + - - + - - -Tomato - + - + - + + + - - - - - -82 Fig. 13 Erwinia carotovora infections in stem-prick inoculated faba beans. Isolates of both atroseptica and carotovora varietal forms were pathogenic to faba beans. 83 DISCUSSION • The levels of latent tuber contamination with Erwinia carotovora in B.C. seed potato stocks were low, even when tubers were sampled immediately after harvest. Only 2.1% and 11.3% of the tubers indexed in 1972 and-1975, respectively, were infected with Erwinia carotovora. These levels compare favorably with the 100% contamination reported in Scotland (108,109,114,121) and 27.6% reported in the U.S. (24) from stored tubers. The existence in B.C. of a virus-free stem cutting program may provide a partial explanation in that the use of stem cuttings has broken the tuber to tuber infection cycle. The trace levels of blackleg which have been reported in B.C. seed fields (B.M. Lawson, personal communication) since the program's inception probably result from recontamination. Perhaps because of the duration of the stem cutting program here, there is less inoculum generally available and the levels of recontamination are low. Whether the survey techniques used were sufficiently sensitive to detect a l l latent infections is a valid question in the case of the 1972 survey. Tubers were stored prior to indexing' and lenticels were not injured prior to incubation. Storage periods exceeding 6 months.have been shown to reduce the recovery efficiency of E_. carotovora by 50% (107,114). A 30% increase in the efficiency of isolation has been reported when lenticels were wounded prior to tuber incubation (24). If the value for the 1972 survey were doubled to account for these factors only a 4.2% recovery rate would be achieved. The 1975 survey is not subject to this criticism and the level of Erwinia-infection (11.3%) was s t i l l considerably lower than reported elsewhere (24,53), where similar techniques were used to recover 84 soft rot coliforms. The seed potato program in B.C. is s t i l l not totally blackleg or Erwinia-free but B.C. seed stocks are undoubtedly less infected with E_. carotovora than those available from other areas. The importance of recontamination was shown in the comparison of seed stocks which were grown out in different localities prior to-being planted under common conditions in Creston (Table VI). The recovery from Lot A, produced by an isolated grower growing primarily Erwinia-tested material, was only 13.3% compared to 35.0% for Lot B grown in a major Elite seed area. The introduction of tested stem cuttings into the Elite program is only the first step in the maintenance of clean seed potato stocks. ErWinia-tested seed material is destined to become recontaminated even i f special precautions are used. However, seed material produced in a "flush out" system is only as good as that produced by the worst grower in the program. In effect, the individual grower may be a limiting factor in the production of Erwinia-free seed potatoes. The field incidence of blackleg and the level of latent tuber contamination was found not to be correlated in B.C. seed potato stocks. This was perhaps most apparent in the seed lots indexed in 1975 (Table V) where levels of latent tuber infection ranged from 6-22% even though the field incidence seldom exceeded 0.1%. Nor does i t necessarily follow that tubers derived from potato crops with l i t t l e or no blackleg will be free of Erwinia contamination. Progeny tubers from fields with l i t t l e or no black-leg may produce crops with a high field incidence in the subsequent year (53,114). Roguing infected plants can only be considered a palliative measure as these plants do not constitute the major source of inoculum (108, 109,114,151). The lack of correlation between the field incidence of 85._; blackleg and the extent of Erwinia contamination of seed tubers casts some doubt on the value of the present certification levels. Blackleg tolerance levels perhaps have greater merit in a virus-free stem cutting program where a l l tubers are not 100% contaminated with Erwinia carotovora to start with. Nevertheless, a more logical approach would be to set tolerance levels to limit the extent of Erwinia-infection in seed tubers i f some practical method for tuber indexing could be developed. Both varietal forms of _E. carotovora were recovered from B.C. seed potato stocks in equal proportions, unlike the 4:1 (atroseptica: carotovora) recovery ratio observed in Scotland (114,115). The results of the B.C. surveys confirm the findings of DeBoer and Kelman (24) instead. Multiple isolate recovery studies indicated that tubers never harboured both varietal forms, though both pathogenic and nonpathogenic carotovora forms could be isolated from a single.tuber. A greater recovery rate of the carotovora form might have been expected as its recontamination potential is favourably influenced by its greater survival in soil and weed rhizospheres. There is also a distinct bias in favour of the carotovora varietal form in single colony isolations on the selective media used for these surveys. Bile salts in Stewart's medium (used in the 1972 survey) were shown to have a more adverse effect on the atroseptica form (120). Similarly, the atroseptica form was slower growing on CVP and thus would have been selected against in the 1975 survey. A more accurate assessment of the extent of tuber contamination with either form would have resulted i f several colonies had been characterized to varietal levels. This was not feasible due to the time-consuming nature of the biochemical tests. The argument as to the importance of the recovery ratio is largely 86, academic i n B.C. as 50% of the carotovora i s o l a t e s recovered were of the pathogenic type. The r e l a t i v e l y high recontamination of tubers from Erwinia-tested cuttings by var. carotovora a f t e r j u s t 1 year and the occasional recovery of var. carotovora from natural blackleg i n f e c t i o n s i n the f i e l d (R.J. Copeman, personal communication) emphasizes the d e s i r a b i l i t y of an Erwinia-free, not j u s t a blackleg fre e , seed program. Such a program w i l l l i k e l y be impossible considering the demonstrated a b i l i t y of var. carotovora to survive i n s o i l under B.C. conditions. S e r o l o g i c a l i d e n t i f i c a t i o n of atroseptica., i s o l a t e s recovered from B.C. seed potato surveys suggested a high degree of antigenic homo-geneity. Similar findings have been reported elsewhere (3,41,149). Vruggink and Maas Gesteranus (149) have used the micro-agglutination technique to detect the atro s e p t i c a v a r i e t a l form i n sap squeezed from diseased stems and rotted tubers. This detection technique may prove invaluable i n future surveys of B.C. seed potato stocks. I t i s more time-saving^and more r e l i a b l e than the standard p h y s i o l o g i c a l tests and can more r e a d i l y be used i n the f i e l d . Less antigenic homogeneity was observed among carotovora i s o l a t e s . Limited observations using only two an t i s e r a against var. carotovora i s o l a t e s would support the existence of several s e r o l o g i c a l s t r a i n s . Pathogenic i s o l a t e s appeared to be s e r o l o g i c a l l y d i s t i n c t because 73% of a l l i s o l a t e s i n t h i s group, reacted with the antiserum prepared against a pathogenic i s o l a t e . This high c o r r e l a t i o n observed may mean that the antiserum used was prepared against an i s o l a t e which was common i n a l l areas by v i r t u e of i t s being introduced on seed potatoes from a common source. However, both the weakly v i r u l e n t i s o l a t e s and the nonpathogenic i s o l a t e s f a i l e d to react 87 with antisera prepared against a pathogenic carotovora and a nonpathogenic carotovora isolate. The possible existence of an isolate specific relationship can, therefore, not be ruled out. Only additional serological work could determine this. Considerable variability in isolate virulence was demonstrated in the host range experiment. It was found that some carotovora isolates, like PRO-5 and SM-2, were pathogenic to,onions, cucumbers, petunias and peas among others without being pathogenic to potatoes. Others, including # 755 and WR 19, have a broader host range and can incite blackleg in potatoes. The latter group then mustl^ be considered more virulent and may play a more important role in the epidemiology of blackleg. Faba beansr,\ cucumbers and peppers were found to be susceptible to Erwinia infection but none of these crops 6-3* grown in rotation with seed potatoes in B.C. While these crops do not play a role in the survival of E_. carotovora in B.C., they may be important in the prairie provinces where faba beans are grown. Common rotation crops in B.C., like oats, red .and white clover and alfalfa, were found to be immune to infection. Though these crops are not likely to infest field soils with E_. carotovora, rhizosphere studies should be carried out to establish their importance as possible reservoirs for Erwinia survival. Alternate hosts are likely to have a more significant effect on the survival of Erwinia carotovora on intensively and continuously cropped land. The survival of Erwinia carotovora var. carotovora in fallow field soils appears a distinct possibility under B.C. conditions. Depending on the depth and duration of the snowpack in the winter, this varietal form i 1 88 was detected in fallow field soils for as long as 112-225 days using dilution plating and baiting techniques. The atroseptica form,: In contrast, was found to be shorter-lived in fallow soil, surviving for only 56 days. Similarly, var, carotovora was recovered from soils by baiting 58 days after the start of the oversummering experiment while the atroseptica form could not be detected after 29 days. Thus, i t seems unlikely that soil-borne atroseptica forms play a very important role in the epidemiology of the blackleg disease compared to infected tubers. The longevity of carotovora population levels in fallow field soils suggests that both pathogenic and nonpathogenic forms of this variant have the potential to be carried-over from one growing season to the next in soils. The survival of both varietal forms was enhanced by using the soil cells which permitted more natural conditions. Perombelon (108) was unable to detect either varietal form in artificially inoculated fallow field soil in plastic bags 12 weeks after the start of the experiment. Studies at U.B.C, however, indicated that the survival of. the carotovora form could be increased from 8 weeks to 16 weeks by using soil cells. No dramatic increase in longevity was indicated with the atroseptica form but less marked reductions in the population level were noted from one sampling date to the next in soil cells than in plastic bags. The existing selective media or baiting techniques used in Erwinia detection in soil have been shown to be insensitive to populations generally below 100 cells/g of soil (21,84). A more sensitive enrichment medium has recently been developed enabling detection of E_. carotovora at levels as low as 2 to 7 cells/g dry weight soil (90). However, i f levels are lower than can be detected by baiting, i t is unlikely that they will play as important V 89 a role in the spread of the disease as latent tuber infections. Reservoirs of inoculum probably play a more important role in the survival of the atroseptica form than the carotovora form. Studies at U.B.C. indicated that the rhizospheres of lamb's quarters, lady's thumb and horsetail plants could prolong the survival of the atroseptica form in fallow fields from 28 days to at least 42 days. Populations at that time were s t i l l considered moderately high as. the pathogen was recovered by dilution plating. The possibility that this varietal form may survive for at least 140 days under Lower Fraser Valley winter conditions was indicated by its recovery from 1 of 3 lamb's quarters replicates by baiting. The perennial growth habit of horsetail makes i t a logical choice for the over-wintering of the atroseptica form but i t could only be detected in 1 out of 3 horsetail replicates by dilution plating after 112 days. Early potatoes would likely not be planted soon enough in the spring for this reservoir of inoculum to constitute a problem in the Lower Fraser Valley. Based on the inoculum levels used in this experiment, i t seems certain that the atroseptica form population levels in soils would be too low for survival to be extended by weed rhizospheres. Definitive studies demonstrating that the carotovora form is better able to survive in rhizospheres of common weed species than in soils are s t i l l lacking. A comparative evaluation basing survival on a rhizo-sphere/soil (R/S) ratio (57) would be useful. Nevertheless, i t might s t i l l be argued that common weed species like horsetail,.lady's thumb and lamb's quarters maintain carotovora populations at higher inoculum potential levels. The carotovora form was recovered by serial dilution from 2 out of 3 lady's thumb and horsetail replicates, whereas i t was only detected in 1 c.-/ 90 out of 3 s o i l c e l l r e p l i c a t e s a f t e r 112 days at U.B.C. Bai t i n g was used at the end of the experiment (140 days) to recover t h i s pathogen from 1 out of 3 lady's thumb and h o r s e t a i l r e p l i c a t e s , suggesting that the inoculum p o t e n t i a l was s t i l l high enough to constitute a recontamination problem. The breakdown of seed pieces at the end of the growing season may play an important r o l e i n the contamination of weed rhizospheres. The carotovora v a r i e t a l form was recovered from the rhizospheres of several common weeds growing i n Pemberton seed potato f i e l d s i n the September 1974 survey (Table IX). I t was recovered from the rhizospheres of h o r s e t a i l , lady's thumb, lamb's quarters, nightshade and redroot pigweed using d i l u t i o n p l a t i n g . The lack of uniformity i n the timing of seed piece decay assures that the inoculum p o t e n t i a l i s maintained at high l e v e l s for longer periods. Thus, r e s e r v o i r s of the carotovora form i n weed rhizospheres may be important i n the carry-over of t h i s pathogen from one growing season to the next. I t i s noteworthy that neither v a r i e t a l form was recovered i n surveys c a r r i e d out i n seed potato f i e l d s e a r l i e r i n the growing, season. This suggests that seed piece decay had not yet occurred. The recovery of the carotovora form from lady's thumb rhizospheres i n the June 1974 survey does not nec e s s a r i l y imply that weeds play a r o l e i n the overwintering of Erwinia carotovora. The source of inoculum conceivably originated from a decomposing volunteer tuber; t h i s p a r t i c u l a r f i e l d was sown to oats and had no potatoes grown on i t f o r two years. Even i f Erwinia carotovora forms were able to overwinter i n s o i l s or i n the rhizospheres of weeds, i t seems u n l i k e l y that these s o i l 91 contaminants could survive through summers at Pemberton in the absence of susceptible tissue. The atroseptica form could not be detected in arti-ficially inoculated field soil 4 weeks after the start of the experiment (June 3) while the carotovora form could s t i l l be recovered by baiting at 58 days. When levels of these varietal forms were monitored around inoculated tubers, only the carotovora form was s t i l l detected by baiting after 70 days. The atroseptica form could not be detected after 4 weeks. Early blackleg (as incited by var. atroseptica) may thus play an insignificant role in the contamination of developing tubers later in the season or in late blackleg outbreaks. Pathogenic carotovora forms seem more likely to oversummer under Pemberton conditions. Although the role of tubers in the survival of Erwinia carotovora has been extensively documented (32,44,95,108,109,111), few studies have been undertaken to ascertain the importance of the level of burial on Erwinia survival. This study indicates that tubers left on the surface, unprotected by a long-lasting snowpack, were subject to extensive rotting, confirming work elsewhere (85,117). Significant differences were noted in the recovery percentages of carotovora-inoculated (83%) and atroseptica-inoculated (33%)' tubers left on the soil surface at U.B.C. after 6 weeks in the 1975-1976 winter. Burying tubers at 10 cm increased the recovery percentage in tubers inoculated with the atroseptica form to 50% but reduced the percentage in carotovora-inoculated tubers to 67% after 42 days. The marked discrepancy in the recovery percentages of these varietal forms in surface-borne tubers is likely related to the rapidity of decay in tubers inoculated with the atroseptica form. Increased recovery of both varietal forms was found when inoculated tubers were buried. This may explain why surface tubers indexed in the volunteer tuber surveys were found to be "clean". .4192 It is likely that any tubers containing Erwinia spp. had decayed over winter leaving only the non-infected to be picked-up in the spring surveys. A common practice in Scotland is plowing to 20 cm after harvest (117). This practice only serves to prolong the survival of Erwinia  carotovora on tubers and should be avoided. 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