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Effect of soft rot erwinias on micropropagated potato plantlets and greenhouse plants and characterization… Lan, Xuesong 1992

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EFFECT OF SOFT ROT ERWINIAS ON MICROPROPAGATED POTATO PLANTLETS AND GREENHOUSE PLANTS AND CHARACTERIZATION OF A FACTOR INHIBITING ROOT ELONGATION by XUESONG LAN  B.Sc.,  The Heilongjiang August First Land Reclamation  University  ( People’s Republic of China), 1984  A THESIS SUBMITTED IN PARTIAL FULFILMENT OF THE REQUIREMENTS FOR THE DEGREE OF MASTER OF SCIENCE in THE FACULTY OF GRADUATE STUDIES (Department of Plant Science)  We accept this thesis as conforming —the required standard  THE UNIVERSITY OF BRITISH COLUMBIA June 1992 © Xuesong Lan,  1992  In presenting this thesis in partial fulfilment of the requirements for an advanced degree at the University of British Columbia, I agree that the Library shall make it freely available for reference and study. I further agree that permission for extensive copying of this thesis for scholaily purposes may be granted by the head of my department  or  by  his  or  representatives.  her  It  is  understood  that  copying  or  publication of this thesis for financial gain shall not be allowed without my written permission.  Department of  Pt  C-I € fl L.  The University of British Columbia Vancouver, Canada  Date  DE-6 (2/88)  ii ABS TRACT  Symptoms induced by soft rot erwinias were observed in micropropagated potato plantlets of cultivar Russet Burbank, Teton,  and Urgenta grown from nodal cuttings inoculated with  Erwinia carotovora subsp.  atroseptica strain 31  preliminary experiments.  The development of short roots,  stunting,  (Eca 31)  in  and yellowing in inoculated plantlets was evident after  25—30 days of growth in a tissue culture growth chamber.  Two strains from each of E. carotovora,  and E.  c.  subsp.  atroseptica and  chrysanthemi were tested individually for  their effect on the growth of micropropagated potato plantlets by a medium—inoculation method.  All strains tested significantly  inhibited root growth of micropropagated plantlets of cv. Kennebec.  Most strains,  strain 196 and E.  c.  except for E.  subsp.  c.  subsp.  atroseptica  carotovora strain 21 caused stunting.  Aerial root formation was caused by all soft rot erwinia strains. Root tip browning,  stem tip blackening,  leaf curling and lesions  which appeared as black necrotic dots also occurred,  although the  frequencies were low within each experiment.  Root growth inhibition,  stem length reduction and severity  of yellowing increased as the inoculum concentration increased. Root elongation was dramatically inhibited by Eca 31 at inoculum concentrations between 0.2—20 colony forming units  (cfu)/mL in  iii tissue culture growth medium. Russet Burbank, Red Pontiac,  All four cultivars Kennebec, and Red Lasoda,  responded similarly  to the low bacterial concentration with respect to stem length, but differently to the high inoculum concentration.  The roots of  cv. Red Lasoda were more susceptible to Eca 31 at 0.2 cfu/mL, but were more resistant at 20 cfu/mL than those of the three other cultivars.  The concentration of bacterial antigens at stem ends  of the four cultivars increased directly with an increase in inoculum concentration as indicated by the indirect enzyme—linked immunosorbent assay  (ELISA)  .  Electron microscopy revealed that a  large population of Eca 31 cells covered the epidermal surface of the root,  and that a large number of bacteria was present in the  intercellular space of root epidermal cells and in the vascular bundles of roots.  The inhibition of root elongation and reduction in root dry weight occurred in potato plants grown from seed pieces in soil inoculated with E.  c.  subsp.  at 18°C in a greenhouse.  atroseptica after 6 days of growth  The roots were inhibited by up to 50%  at an inoculum concentration of 1.1 x iO cfu/g soil.  Stunting  occurred, but no other foliage symptoms were observed.  The root inhibitory factor was shown to be present in bacterial supernatant of 5-day old nutrient broth cultures of Eca 31.  The inhibition of root elongation was directly correlated  with supernatant concentration in a “Petri dish bioassay”.  The  iv factor responsible for root inhibition was relatively resistant to heat,  of high molecular weight  (> 12000—14000),  and precipitable by ammonium sulphate at 70% saturation.  V  TABLE OF CONTENTS  PAGE Abstract Table of Contents List of Tables List of Figures Acknowledgement  ii v vi vii xi  CHAPTER 1.  General Introduction  2.  Symptoms Caused by Three Soft Rot Erwinias and Other Bacterial Species in Micropropagated Potato Plantlets  12  Response of Micropropagated Potato Plantlets to Erwinia carotovora subsp. atroseptica  43  Effect of Erwinia carotovora subsp. on Potato Plants Grown in Soil  69  3.  4.  5.  6.  1  atroseptica  Characterization of a Factor Responsible for Inhibition of Root Elongation  83  General Discussion  97  Literature Cited  103  vi LIST OF TABLES  TABLE  1.  2.  3.  4.  5.  6. 7.  8.  9.  10.  PAGE  Bacterial cultures used in the study of symptom expression of micropropagated potato plantlets of cv. Kennebec  15  Effect of Erwinia carotovora subsp. atroseptica strain 198 on micropropagated potato plantlets of cv. Kennebec  20  Effect of Erwinia carotovora subsp. atroseptica strain 196 on micropropagated potato plantlets of cv. Kennebec  21  Symptoms of micropropagated potato plantlets of cv. Kennebec caused by Erwinia carotovora subsp. carotovora strain 21  23  Symptoms of micropropagated potato plantlets of cv. Kennebec caused by Erwinia carotovora subsp. carotovora strain 71  24  Effect of Erwinia chrysanthemi strain 573 on micropropagated potato plantlets of cv. Kennebec  25  Effect of Erwinia chrysanthemi strain 574 on micropropagated potato plantlets of cv. Kennebec  26  Comparison of symptoms expressed by micropropagated potato plantlets of cv. Kennebec caused by Corynebacterium sepedonicum and Erwinia carotovora atroseptica strain 31  27  Effect of Pseudomonas marginalis and Erwinia amylovora on micropropagated potato plantlets of cv. Kennebec  29  Comparison of response of micropropagated potato plantlets of cv. Kennebec, Russet Burbank, Red Pontiac, and Red Lasoda to Erwinia carotovora subsp. atroseptica strain 31  55  vii LIST OF FIGURES  PAGE  FIGURE  1.  2.  3.  4.  5.  6.  7.  8.  Root length and stem length of micropropagated potato plantlets of cv. Kennebec grown for 21 days on Murashige and Skoog’s medium inoculated with Erwinia carotovora subsp. atroseptica strain 31 (right) in comparison to those of control (left)  30  Root symptoms on plantlets of cv. Kennebec after 26 days of growth. A, Browning and blackening of root tips caused by Erwinia chrysanthemi strain 573. B, Aerial root formation induced by Erwinia carotovora subsp.carotovora strain 71  31  Stem symptoms on plantlets of cv. Russet Burbank growing for 30 days on Murashige and Skoog’s medium inoculated with Erwinia chrysanthemi strain 576. A,Blackening, yellowing, and black necrotic dots on stems. B, Rotting of stem tips  33  Leaves of micropropagated potato plantlets of cv. Kennebec after 26 days of growth. A, Leaves of the control plantlets. B, Leaf necrotic spots, rot, and yellowing developed in treatments inoculated with Erwinia carotovora subsp. atroseptica strain 22  34  Black necrotic dots on leaf of micropropagated potato plantlets of cv. Russet Burbank grown for 30 days on Murashige and Skoog’s medium inoculated with Erwinia chrysanthemi strain 576  35  Curling of leaves of micropropagated potato plantlets of cv. Kennebec after 26 days of growth on Murashige and Skoog’s medium inoculated with Erwinia carotovora subsp. atroseptica strain 31  36  Leaf tip necrosis of plantlets of cv. Kennebec after 26 days of growth on Murashige and Skoog’s medium inoculated with Erwinia chrysanthemi strain 576  37  Mean of maximum root length of plantlets of cv. Russet Burbank (RB) and Red Pontiac (RP) for 18 days on Murashige and Skoog’s medium inoculated with Erwinia carotovora subsp. atroseptica strain 31  49  grown  viii PAGE  FIGURE 9.  10.  11.  12.  13.  14.  15.  16.  17.  Mean number of roots per plantlet grown for 18 days on Murashige and Skoog’s medium inoculated with different concentrations of Erwinia carotovora subsp. atroseptica strain 31  50  Mean length of stems of micropropagated potato plantlets of cv. Russet Burbank (RB) and Red Pontiac (RP) grown for 18 days on Murashige and Skoog’s medium inoculated with different concentrations of Erwinia carotovora subsp. atroseptica strain 31  51  Mean number of leaves of plantlets grown for 18 days on Murashige and Skoog’s medium inoculated with different levels of Erwinia carotovora subsp. atroseptica strain 31  52  Severity of yellowing of plantlets of cv. Russet Burbank (RB) and Red Pontiac (RP) grown for 18 days on Murashige and Skoog’s medium inoculated with different concentrations of Erwinia carotovora subsp. atroseptica strain 31  53  Response in stem length of plantlets of cv. Russet Burbank to different concentrations of Erwinia carotovora subsp. atroseptica strain 31  56  Severity of yellowing of plantlets of cv. Russet Burbank in response to different inoculum concentrations of Erwinia carotovora subsp. atroseptica strain 31  57  Effect of different inoculum concentrations of Erwinia carotovora subsp. atroseptica strain 31 on root elongation of plantlets of cv. Russet Burbank  58  Mean absorbance values (A nm) for enzyme—linked 405 immunosorbant assay conducted on stem end portions of plantlets of cv. Kennebec (D), Russet Burbank (+), Red Pontiac (0), and Red Lasoda () in Response to different inoculum concentrations of Erwinia carotovora subsp. atroseptica strain 31  60  Electron micrograph of root section of cv. Red Pontiac grown for 26 days on inoculated Murashige and Skoog’s medium. A huge population of bacterial cells of Erwinia carotovora subsp. atroseptica strain 31 covered on root surface (X 8750)  61  ix PAGE  FIGURE 18.  Electron micrograph of longitudinal section of aroot near the edge showing the presence of bacterial cells of Erwinia carotovora subsp. atroseptica strain 31 in intercellular spaces between the epidermal cells. Plantlets of cv. Red Pontiac grown for 26 days on Murashige 62 and Skoog’s medium (X 3750)  19.  Electron micrograph of cross section of a root of cv. Red Pontiac grown for 26 days on Murashige and skoog’s medium inoculated with Erwinia carotovora Bacterial cells in subsp. atroseptica strain 31. vascular tissue of the root (X 10000)  63  Electron micrograph of cross section of upper portion of a root of cv. Red Pontiac grown for 26 days on Murashige and Skoog’s medium inoculated with Erwinia carotovora subsp. atroseptica strain 31. Bacterial cells present inside a root cell (X 7500)  64  Mean of maximum root length per plant grown in soil inoculated with Erwinia carotovora subsp. atroseptica strain 31 at concentrations of 1.1, 110, and 11000 x i0 cfu/g soil compared to control  74  Healthy (left) and diseased (right) plants of cv Russet Burbank after 6 days of growth at 18°C in The seed piece was grown in soil a greenhouse. inoculated with Erwinia carotovora subsp. atroseptica strain 31 at 1.1 x iO cfu/g soil  75  Mean dry weight of root systems per plant grown from a seed piece in soil inoculated with Erwinia carotovora subsp. atroseptica strain 31 at concentrations of 1.1, 110, and 11000 x iO cfu/g soil compared to control  76  Mean length of stem per plant grown from a seed piece in soil inoculated with Erwinia carotovora subsp. atroseptica strain 31 at concentrations of 1.1, 110, and 11000 x 103 cfu/g soil compared to control  77  Mean number of leaves per plant grown from a seed piece in pot soil inoculated with Erwinia carotovora subsp. atroseptica strain 31 at concentrations of 1.1, 110, and 11000 x iO cfu/g soil compared to control  79  20.  21.  22.  23.  24.  25.  x PAGE  FIGURE 26. 27. 28. 29.  30.  Effect of supernatant concentrations on root elongation of cuttings of cv. Kennebec Effect of heated bacterial supernatant on root elongation of nodal cuttings of cv. Kennebec  89 91  Effect of dialysed bacterial supernatant on root elongation of nodal cuttings of cv. Kennebec  92  Effect of bacterial supernatant of Erwinia carotovora subsp. atroseptica strain 31 on root elongation of nodal cuttings of cv. Kennebec  93  Effect of four fractions of bacterial supernatant on root elongation of nodal cuttings of cv. Kennebec  94  xi  ACKNOWLEDGEMENTS  The author wish to express his sincere appreciation to Dr. H.  De Boer for his guidance,  valuable suggestions,  Solke  and  encouragement given during the research and writing of this thesis.  I also thank Dr. Dr.  I.  E. P.  Townsley,  R.  Taylor,  J.  Copeman,  Department of Plant Science,  Department of Botany,  Department of Food Science,  UBC;  UBC;  and Dr. P. M.  UBC for serving on my  committee.  Sincere thanks are extended to Mr. D. Mrs.  E.  Kirkham,  Mr. K.  Turner and  Sela for supplying disease-free potato tissue culture  materials and allowing me to use their Laboratory facilities.  I extend my thanks to Dr. F. micrographs;  Leggett for preparing the electron  and Mr. W. MacDiarmid for taking photographs of  inoculated micropropagated potato plantlets.  I also thank Dr. data; Dr. B.  J. W.  Hall for his suggestions in analyzing  D. Frazer and Dr. R.  their growth chambers;  I. Hamilton for lending me  and other members of the staff at the  Agriculture Canada Vancouver Research Station,  for their  xii assistance.  Support for this study was from New Brunswick Department of Agriculture sponsored by the Canadian International Development Agency.  1 CHAPTER 1  -  GENERAL INTRODUCTION  PRACTICE AND PROBLEMS IN POTATO TISSUE CULTURE  Meristem tip  (MT)  culture is widely used to obtain virus—  free stocks of vegetatively propagated species such as carnation, chrysanthemum, 1967;  orchid,  potato,  etc.  Hollings and Stone 1968).  to potato  (Solanum tuberosum L.)  (Hollings 1965; Kassanis  The technique was first applied by Norris  (1954).  Since then,  MT culture has been used in production of virus—free potatoes.  Meristematic tips are excised from axillary buds of heat— treated plants or sprouts of disease—free tubers and then multiplied in vitro to produce plantlets. are tested against major viruses by ELISA.  The stock materials ELISA utilizes the  specific binding characteristics between an antibody and an antigen to detect the presence of the antigen of a pathogen in test samples.  Disease—free plantlets are multiplied in large  numbers in vitro and subsequently transplanted to soil in pots in the greenhouse.  The plants can be regenerated both by stem  cuttings and tubers in the greenhouse.  After quarantine testing,  the disease—free potatoes are distributed to Elite seed farms to produce seed potatoes.  A seed lot is grown in the field before  it is released to commercial growers.  2 In vitro meristem tip cultures do not always produce  disease—free plants  (Wang 1985)  .  Contamination of  micropropagated plantlets by bacteria and bacteria—like organisms is a big problem in many research and commercial laboratories. Indexing programs have to be carried out frequently at different stages of in vitro mass cloning to exclude bacteria, bacterialike organisms,  fungi or even mycoplasma  (Wang 1985; Debergh  1987)  Three types of contamination of plant tissue cultures are The first,  recognized.  acute contamination at the establishment  stage is due to incomplete surface sterilisation of the explant. The second,  contamination that occurs post-establishment is  possibly due to endogenous microflora or to poor technique at the subculture stage.  The third,  chronic contamination arises  apparently simultaneously in a batch of cultures after an extended period of supposedly axenic growth De Forssard  (1977)  (Long et al.  1988).  stated that the contamination that occurs post  establishment can cause a slowing of the growth rate of the plantlets.  The interaction of phytopathogenic bacteria with tissue cultures provides a useful system to study mechanisms of plant pathogenesis.  In tissue culture systems,  the types and numbers  of organisms in an interaction can be easily and accurately controlled.  Since the growth medium is chemically defined,  the  3 composition is easily adjusted to suit the objective of the experiment.  Treatments are relatively easy to handle in an in  vitro system by adding and removing certain compounds at a particular time during the experiment.  Uniform treatments can be  Growth conditions in vitro can be  achieved with great ease.  accurately controlled to avoid the unpredictable and severe conditions found in the field.  Outside factors influencing  growth and interactions of plant and pathogens, activities of humans and animals,  such as,  variation in soil properties,  and soil microbial numbers and composition,  can be eliminated.  Biochemical and molecular biological techniques have been frequently used in phytopathology.  It is much easier to study  the pathogenic interactions at the molecular level in plant tissue cultures than in field—grown plants.  Moreover,  some  experiments which utilized tissue cultures are difficult or impossible with field plants.  For example,  the synthesis of  “amylovorin” by the host in response to Erwinia amylovora infection was studied using apple cell suspension culture and Goodman 1978).  (Hsu  This study could not have been done using  apple trees because of the difficulties in handling them in the field.  Similarly, tissue culture and tissue slices are used to  study the effect of pectolytic enzymes produced by Erwinia carotovora on plant tissue.  The effect of bacterial metabolites  on plants can be examined easily in tissue culture rather than under field conditions.  In these cases the experimental  4 manipulations and measurements could not have been carried out in the field.  BACTERIAL DISEASES OF POTATO  BLACKLEG AND SOFT ROT DISEASES.  The blackleg and soft rot  diseases of potato are caused by three soft rot erwinias carotovora subsp.  subsp.  (Van Hall)  atroseptica  (Jones)  carotovora  Burkholder et al.].  Bergey et al.,  Dye, E. and E.  [Erwinia  carotovora chrysanthemi  The pectolytic erwinias have a worldwide  distribution, but contrasting host ranges and host specificity that are reflected in their serological and optimum temperature for growth characteristics.  E.  c.  subsp.  atroseptica  restricted mostly to potatoes in temperate regions,  (Eca)  is  where the  crops are grown most extensively and the strains isolated usually belong to one serogroup Mather et al. subsp.  1986)  carotovora  .  (Serogroup I)  (De Boer et al.  1979;  However two other soft rot erwinias, E.  (Ecc)  and E.  chrysanthemi  (Echr),  c.  can infect  potato plants in the field and cause symptoms similar to those associated with E. basal stem rot  c.  subsp.  (Cother 1980)  atroseptica infection, .  E.  c.  subsp.  including  carotovora has a  world—wide distribution in both temperate and tropical zones and is pathogenic to a wide range of plants. widely distributed in tropical,  E.  chrysanthemi is also  subtropical and warm temperate  zones where it is a pathogen of many crops as well as those grown in greenhouses in temperate regions.  E.  chrysanthemi can also  5 cause stem rot under field conditions in some cool temperate regions  (Tominaga et al.  1979).  Potato crops in some countries  are frequently infected by more than one soft rot erwinia strain since most of them are not host specific  (De Boer et al.  1975; De  Lindo 1978)  Nonemergence can result from early decay of the seed tubers or the death of sprouts at or soon after emergence due to infection. plants.  Initial symptoms can be quite diverse on growing  Wilting and yellowing of leaves are usually associated  with an early attack by pectolytic erwinias. known as blackleg, However,  The basal stem rot,  results from decay of the seed tubers.  seed piece decay does not always result in blackleg.  The basal stem rot symptoms caused by three soft rot erwinias under field conditions optimal for each pathogen are essentially indistinguishable  (Stanghellini and Meneley 1975).  Although soft rot erwinias are facultative anaerobes,  they  grow better in vitro under aerobic than anaerobic conditions (Meneley and Stanghellini 1976; Wells 1974). dioxide,  Oxygen,  carbon  and water are essential factors influencing disease  expression.  Factors that assist the growth and spread of the  primary inoculum that,  in turn,  contribute to the build—up of  high bacterial densities in stems are likely to favour expression of the disease  (Perombelon and Icelman 1980)  6 Bacterial ring rot caused by  BACTERIAL RING ROT DISEASE.  & Kotth.  Corynebacterium sepedonicum (Spieck. syn:  Clavibacter michiganensis subsp.  Kotth.]  Davis et al.)  [Skapt.  sepedonicus  & Burkhl;  [Spieck.  &  is one of the most important diseases of  potato because the bacteria may spread rapidly among tubers in planting operations and all tubers from infected plants may rot (De Boer and Slack 1984). storage conditions  This disease occurs in both field and  (Sletten 1985;  Easton 1979;  Lelliott 1988)  The symptoms of diseased plants include wilting, stunting  (Nelson and Torfason 1974).  chlorosis,  and  Diseased tubers initially  show a characteristic “ring rot” symptom.  Transverse sections  have a creamy yellow and cheesy texture in the vascular ring and later the affected tuber tissues become brown and necrotic Boer and Slack 1984).  (De  Symptomless infection of potato plants  also occurs in the field and is attributed to low inoculum levels,  late season—infection,  or environmental conditions that  suppress or mask symptom expression  (De Boer and Slack 1984).  Low light intensity and short photoperiod increase the severity of ring rot symptoms of potato plants grown in greenhouses (Nelson 1980)  CAUSAL ORGANISMS  SOFT ROT ERWINIAS.  Soft rot erwinias are the major  bacterial pathogens of potato.  The main characteristic  distinguishing soft rot erwinias from other Erwinia species is  7 the ability to produce large quantities of pectolytic enzymes (mainly pectic lyase)  that enable the bacteria to macerate  parenchymatous tissue of a wide range of plant species (Perombelon and Kelman 1980)  Although the pectolytic Erwinia species and subspecies can sometimes cause the same symptoms in potato,  the bacteria can be  differentiated from one another by specific biochemical tests (Slade and Tiffin 1984)  .  For instance,  differentiated easily from E.  c.  E.  chrysanthemi can be  atroseptica and  subsp.  carotovora because it exhibits phosphatase activity during growth  (Graham 1971)  .  Furthermore,  E.  c.  subsp.  atroseptica produces  acid from a—methylglucoside and reducing substances from sucrose, but E.  c.  subsp.  carotovora does not.  atroseptica do not grow at 37°C;  Strains of E.  in contrast E.  carotovora grows at this temperature.  Most E.  carotovora strains do not grow at 39°C,  c. c.  c.  subsp.  subsp. subsp.  whereas strains of E.  chrysanthemi can grow relatively well at 39°C.  (Perombelon and  Kelman 1980)  There is a controversy over the longevity of soft rot erwinias in soil.  The number of E.  carotovora declined rapidly  in naturally and artificially inoculated nonsterile field soils (Perombelon and Kelman 1980).  Soil temperature was not a  limiting factor in determining survival of soft rot erwinias in some soils.  (Perombelon and Kelman 1980)  .  E.  c.  subsp.  8 atroseptica was found to survive longer in water—saturated than  in air—dried soil, but the reverse was true for E. E.  chrysanthemi.  carotovora survived best at most temperatures in wet soil  collected in winter or in moist soils collected in summer (Perombelon and Kelman 1980) extended the longevity of E. and Kelman 1980)  .  Addition of nutrients to the soil carotovora significantly  Soft rot erwinias can overwinter in  .  contaminated plant residue remaining in the soil. regions,  (Perombelon  In some  a high proportion of tubers left in the field after  harvest is likely to be contaminated by soft rot erwinias (Perombelon 1975)  .  The bacteria may also overwinter in  association with volunteer plants and certain weeds  (Perombelon  and Kelman 1980)  Soft rot erwinias are located superficially on soil particles  (Perombelon and Kelman 1980)  .  They are moved readily  in soil water from decayed seed tubers to soil and to the progeny tubers.  Soil water can carry a large number of bacteria for  several meters in soil  (Graham and Harper 1967)  .  Large numbers  of soft rot erwinias are leached after a heavy rain from the rotting seed tubers to adjacent zones  (Perombelon 1976).  On the  other hand, movement of bacteria in soil is impeded by discontinuity of the water film around soil particles. (Perombelon and Kelman 1980)  CORYNEBACTERIUM SEPEDONICUM.  C.  sepedonicum is classified  9 as a Gram—positive, nonmotile, organism.  obligate aerobic rod—shaped  The slow growth rate and a requirement for several  growth factors are the characteristics of this bacterial species, which make it a difficult organism to isolate and study. field conditions,  C.  Under  sepedonicum is reported to cause disease  only in potatoes, but can also infect other plants by artificial inoculation  (De Boer and Slack 1984)  .  The major source of  inoculum for bacterial ring rot of potato is infected tubers. Nelson  (1979)  reported that survival of C.  sterile soil was poor.  sepedonicum in non—  Survival was better at a low moisture  regime and frozen conditions.  C.  sepedonicum can overwinter in  contaminated seed tubers left in the field  (Bonde 1942)  EFFECTS OF HARNFUL RHIZOSPHERE MICROORGANISMS ON POTATO PLANTS  Plant growth-inhibiting microorganisms can be divided into three groups:  1)  plant pathogens parasitizing plant tissue,  thereby causing distinct macroscopic disease symptoms,  2)  minor  pathogens or subclinical pathogens that parasitize root cells without causing distinct macroscopic disease symptoms,  and 3)  microorganisms that do not parasitize plant tissue but as a result of their metabolic activities may become harmful to root development and plant growth (Schippers et al.  (harmful rhizosphere microorcTanisms)  1985)  Long—term crop rotation experiments in the Netherlands  10 indicated that frequent potato cropping caused significant yield reduction which was more severe with increasing potato cropping frequencies  (Schippers et al.  1985).  Analysis of the cause of  potato yield reduction led to a conclusion that an unidentified microbial factor in short potato—rotation soils was involved. The analysis of nutrient contents showed that nitrogen, phosphorus,  and potassium did not differ significantly between  different soils  (short and long potato—rotation soils)  for the yield reduction observed Bakker et al.  (1987)  to account  (Bakker and Schippers 1987).  proposed that potato yield reduction in  short potato rotations was due to impaired root function caused by harmful rhizosphere microorganisms,  rather than by direct  damage caused by invasive pathogens.  In pot experiments, plant growth and tuber production were suppressed in comparison to those in soil from the same field with little or no history of potato cropping 1983;  Scholte et al.  1985)  .  (Geels and Schippers  Increasing potato cropping  frequencies were favourable for enhancing the activities of microorganisms harmful to root development.  In addition,  microbial metabolites harmful to root cell activities progressively accumulated in the soil  (Schippers et al.  1985).  The alleviation of potato yield reduction and root growth inhibition in short potato—rotation soil by selected Pseudomonas spp.  (Schippers et al.  1985; Bakker et al.  1987)  suggested that  the numbers of harmful microorganisms were significantly reduced  11 by the fluorescent pseudomonads.  OBJECTIVES OF THE RESEARCH PROJECT  The objectives of this study were:  (1)  to evaluate the  effect of the bacterial pathogens on micropropagated potato plantlets,  (2)  to determine the responses of micropropagated  plantlets to different inoculum concentrations of E. and the response of different cultivars, effects of E. soil,  and  (4)  (3)  carotovora  to illustrate the  carotovora on the development of potato plants in to characterize a factor inhibiting root growth.  12 CHAPTER 2  SYMPTOMS CAUSED BY THREE SOFT ROT ERWINIAS AND OTHER  -  BACTERIAL SPECIES IN MICROPROPAGATED POTATO PLANTLETS  INTRODUCTION  The contamination of commercial tissue cultures by different bacterial pathogens has been reported repeatedly. (1988)  Long et al.  demonstrated that micropropagated potato plantlets that  had undergone a number of subcultures contained a variety of bacterial contaminants.  A fluorescent pseudomonad and a  coryneform bacterium were most numerous.  Leggatt et al.  (1988)  isolated 31 microorganisms from ten different micropropagated plant cultivars and reported that the most common isolates were yeasts,  E.  Corynebacterium spp.,  and Pseudomonas spp.  carotovora has been isolated from commercial plant tissue  cultures of Nephrolepsis exaltata “Bostoniensis” (Boston fern), Pteris spp.  Saxifraga sarmentosa L.  (Pteris fern)  (L.)  (Strawberry begonia),  (Knauss and Miller 1978).  ten bacterial isolates were identified as E. belonged to the genus Bacillus,  Schott and  Five out of  carotovora,  four  and one was a pseudomonad.  These  contaminants caused reduced vigour and chiorosis in plant tissue cultures.  Debergh and Vanderschaeghe  (1988)  reported that the  presence of brown spots on the petioles and leaf blades of  13  Gerbera indicated bacterial contamination.  Bacterial  contamination often caused dark brown bracts at the base of shoots of tissue cultured Calathea makoyana.  The description of many subtle symptoms of bacterial contamination in potato tissue cultures has also been published. A contaminant pseudomonad,  isolated by Long et al.  (1988),  alone  and in combination with a coryneform bacterium was reinoculated into disease—free potato plantlets of cv. British Queen.  The  contaminated plantlets were smaller than control plantlets and developed a large number of necrotic tips.  The objectives of this study were:  (1)  to illustrate the  effect of different strains of soft rot erwinias,  and other  phytopathogenic bacterial species on micropropagated potato plantlets,  (2)  to document the symptoms of the plantlets  inoculated with these bacterial species.  MATERIALS AND METHODS  MICROPROPAGATED PLANTLETS.  Micropropagated potato plantlets  of cv. Kennebec obtained originally from the Virus—free Potato Laboratory at the Agriculture Canada Vancouver Research Station were free of contamination by potato virus X, potato virus Y, potato virus S, viroid.  potato leaf roll virus,  and potato spindle tuber  Bacterial contamination was examined by plating sap  14 expressed from the stem end of the plantlets onto nutrient agar plates.  Any colonies that developed on the nutrient agar plates  during fourteen days of incubation were considered to be contaminants.  The plantlets,  further multiplication,  used as source materials for  were completely free from viral and The plantlets were multiplied by  bacterial contamination.  growing nodal cuttings in 250 mL jars containing 30 mL Murashige and Skoog’s (dark)  (MS)  and 23°C  medium (light)  growth chamber.  (Murashige and Skoog 1962)  each at 21°C  with 16 h photoperiod in a tissue culture  Only vigorously growing plantlets were used as  sources of cuttings for inoculation.  PREPARATION OF NODAL CUTTINGS. approximately 1 cm in length,  Nodal cuttings,  were excised from vigorously  growing plantlets with sterile scissors. removed from plantlets.  All leaflets were  Both the top and the lowest nodes of  each plantlet as well as nodes with aerial roots were discarded. The slender and weak plantlets,  which were observed to have  different growth rates in subsequent culture in comparison to those of normal cuttings,  BACTERIAL CULTURES. atroseptica, chrysanthemi,  were excluded.  Three strains of E.  two strains each of E.  c.  and one strain each of C.  marginalis and E.  amylovora,  subsp.  c.  subsp.  carotovora,  sepedonicum,  were used in this study  and E.  Pseudomonas  (Table 1).  15  Table 1. Bacterial cultures used in the study of symptom expression of micropropagated potato plantlets of cv. Kennebec Species (Subsp.) Erwinia carotovora subsp. atroseptica  Erwinia carotovora subsp. carot ovora  Erwinia chrysan themi  Erwinia amyl ovora Coryrie— bacterium sepedoni cum Pseudomonas marginalis  Strain  Sero— group  31  I  Origin  Host  Nether— lands  Potato  Copeman  Canada  Potato  Source H.P. Maas Geesteranus 161  198  XXII  196  XX  S.H. De Boer  Canada  Potato  21  II  H.P. Maas Geesteranus 139F  Nether— lands  Potato  71  III  H.P. Maas Geesteranus 226  Nether— lands  Potato  573  R.S.  U.S.A.  Guayule  574  R.  Cother  Austra— ha  Potato  SR5  P.  Sholberg  Canada  Pear  De Boer  Canada  Potato  L. MacDonald  Canada  Potato  R8  90—521  R.J. E17  S.H.  Dickey  16 Serogroup differentiation of the soft rot erwinias was based on their serological relationships determined by Ouchterlony double diffusion  (De Boer et al.  1979)  Cultures of each of the soft rot erwinia strains were stored in a solution containing 60% nutrient broth and 40% glycerol at A nutrient agar slant was streaked with a culture from  —80°C.  storage and subsequently incubated at 23°C.  A single colony for  each strain showing typical pitting on crystal violet, pectate (CVP)  medium was selected.  C.  sepedonicum strain R8  (Cs—R8)  was obtained from the  Vancouver Research Station collection and maintained on yeast, glucose medium  (YGM)  (De Boer 1989)  slants at 23°C.  colony of Cs—R8 was selected from a YGM plate,  A single  and subsequently  streaked onto YGM slants.  One fluorescent strain of P. marginalis 90—521 and one strain of E.  amylovora were obtained from the culture collection  at the British Columbia Ministry of Agriculture and Fisheries (BCMAF).  Cultures of both species were grown on nutrient agar  slants initially. sucrose medium  Kings Medium B  (King et al.  (Crosse and Goodman 1973)  1954)  were use for the  selection of a single colony for P. marginalis and E. respectively. at 23°C.  and a high  amylovora,  Both cultures were grown on nutrient agar slants  17 Each of the pectolytic Erwinia  EXPERIMENTAL TREATMENTS. strains,  and one strain of P. marginalis and E.  amylovora were  individually grown on nutrient agar slants at 23°C for 48 hours. Four—day—old freshly growing cultures of Cs—R8 were used for inoculum preparation.  Freshly grown cells were washed and  suspended in sterile Ringers solution.  A stock suspension of  each strain was prepared by dilution of the washed cells to obtain an absorbance value of 0.4 at 660 nm with a spectrophotometer  (Spec 20).  A series of dilutions were made  from each stock and used as inoculum.  Actual inoculum  concentrations were determined by a standard plate count method. Two inoculum concentrations, ten—fold apart, all strains of soft rot erwinias, marginalis and E.  amylovora.  were prepared for  and one concentration for P.  Inoculum of Eca 31 prepared in the  same way in all experiments was used as a positive control. Sterile Ringers solution was used as a negative control.  Aseptic  techniques were used and the experiments were repeated twice.  A modified MS medium with 0.75 % agar was prepared in flasks at 79 mL/flask and maintained at 40°C in a water bath after autoclaving.  One mL of each inoculum preparation  positive and negative controls)  (including both  was added to the 79 mL molten MS  medium and was mixed thoroughly in each jar.  The jars were left  open in the laminar flowhood until the medium was completely solidified.  18 For the ring rot pathogen,  the nodal cuttings were dipped in  each inoculum suspension of Cs—R8 for 10 seconds and then blotted on dry sterile filter paper to eliminate the extra inoculum around the cuttings.  Five inoculated cuttings of the same  treatment were transferred into a jar. Cs R8,  two for Eca 31,  and one for Ringers control,  and each was replicated three times. marginalis and E.  Five treatments, two for were employed  However, treatments with P.  amylovora were inoculated in the same way as  those of the soft rot erwinia strains.  Freshly cut nodal cuttings were collected in a sterile Petri—dish and randomized by shaking.  The cuttings were  carefully laid flat on top of the agar medium and then pushed 5 mm into the agar.  Six cuttings were used per jar and each jar  was replicated three times.  All inoculated plantlets were incubated at 18°C 23°C  (light)  (dark)  and  with a photoperiod of 16 hours for 25—30 days.  s’ provided by eight F48 2 Illumination was at 194.5 lIE m T12/CW/HO Phillips fluorescent tubes.  SAI4PLING  The plantlets and the medium were taken out of the  jars together and separated carefully.  Length and number of all  roots and stem of each plantlet were measured with a ruler.  The  number of roots of a plantlet was counted for those roots over 0.5 mm in length.  Discoloration of plantlets was measured on a  19 scale of 1—5  (Table 2)  in comparison to the normal leaf colour of  control plantlets which was set at 0.  Symptoms of the inoculated  plantlets were photographed at sampling time.  RESULTS  E.  c.  subsp.  atroseptica.  Eca 31 was selected as a  representative strain of soft rot erwinias to demonstrate its effect on the growth of micropropagated potato plantlets of Teton Root growth  and Russet Burbank in preliminary experiments.  inhibition and stunting were observed when the plantlets were grown on MS medium for 25—30 days in a growth chamber.  Elongation of roots was significantly inhibited by Eca 198 and 196 only at 41 and 40 cfu/mL,  (Table 2,  respectively  3)  .  No  significant difference in the number of roots was found for any treatments with either Eca 198 or 196  (Table 2,  3)  .  Stem length  was significantly shorter in the treatment with Eca 198 at inoculum concentration of 41 cfu/mL compared to buffer control (Table 2)  .  Stem length did not differ significantly between  treatments with Eca 196 and buffer (Table 2,  stunting  3).  E.  c.  (Table 3),  subsp.  but Eca 31 caused  atroseptica strain 198  caused reduction in the number of leaves and yellowing of the plantlets  E.  c.  (Table 2)  subsp.  carotovora.  Root elongation was inhibited by  20  Table 2. Effect of Erwinia carotovora subsp. atroseptica strain 198 on micropropagated potato plantlets of cv. Kennebec Treat— ment  1 Inoc. concn (cfu/mL)  Stem length (cm)  No. of leaves  2 Color  Root length (cm)  No. of roots  Buffer  0  3 7.33a  8.50a  0.lla  9.53a  7.67a  Eca 31  29  5.06b  6.28b  l.56b  2.94c  9.28a  Eca 198  4  7.62a  7.61c  2.33b  7.26ab  8.OOa  41  5..74b  6.92c  2.34b  5.85b  8.93a  0.30  0.15  0.21  0.51  0.54  Stnd. error  ‘One mL inoculum suspension was mixed with 79 mL Murashige and Skoog’s medium. Numbers indicated final concentrations. Six numerical classes of discoloration of plantlets were 2 used: 0, normal; 1, 1—2 top leaves yellowed; 2, 3—4 top leaves yellowed; 3, over 5 leaves yellowed; 4, top blackened and/or all leaves yellowed; 5, stem rotted. Numbers followed by the same letter within each column do 3 not differ significantly (p=0.05) according to Tukey’s multiple range test. The value is the mean of 18 observations.  21  Effect of Erwinia carotovora subsp. atroseptica Table 3. strain 196 on micropropagated potato plantlets of cv. Kennebec Treat— ment  Inoc. 1 concn (cfu/mL)  Stem length (cm)  No. of leaves  2 color  Root length (cm)  No. of roots  Buffer  0  3 6.27a  8.44a  0.lla  9.49a  9.17a  Eca 31  29  4.28b  6.89b  l.17a  3.28c  11.17a  Eca 196  4  5.9Oab  7.72ab  2.67b  6.68ab  8.89a  40  5.l7ab  6.94ab  3.44b  4.38c  9.83a  0.44  0.33  0.28  0.64  0.67  Stnd. error  ‘One mL inoculum suspension was mixed with 79 mL Murashige and Numbers indicated final concentration. Skoog’s medium. Six numerical classes of discoloration of plantlets were used: 2 0, normal; 1, 1—2 top leaves yellowed; 2, 3—4 top leaves yellowed; 3, over 5 leaves yellowed; 4, top blackened and/or all leaves yellowed; 5, stem rotted. Numbers followed by the same letter within each column do not 3 differ significantly (p=0.05) according to Tukey’s multiple range test. The value is the mean of 18 observations.  22 Ecc 71 at both inoculum concentrations,  inoculum level by Ecc 21  (Table 4,  5).  but only at the high Stem length and the  number of leaves of plantlets growing in the medium inoculated with Ecc 21 did not differ significantly from those of buffer control at both 3 and 27 cfu/mL  (Table 4).  Ecc 71 caused  stunting and reduction in the number of leaves only at 68 cfu/mL (Table 5).  Both E.  subsp.  c.  carotovora strains caused  discoloration of plantlets at low inoculum levels  E.  and the number of leaves occurred at both inoculum  concentrations for E. 7).  Eca 31,  5).  Significant inhibition of root elongation,  chrysanthemi.  stem growth,  (Table 4,  chrysanthemi strain 573 and 574  (Table 6,  used as a positive control over a range of inoculum  concentrations between 29-53 cfu/mL,  consistently caused  inhibition of root elongation and reduction in stem growth (Tables 2—7)  C.  sepedonicum,  P. marcina1is,  and E.  amylovora  No  significant effect of Cs—R8 was found on any plantlet characteristics of cv. Kennebec after incubation for 25 days, although the effect of Eca 31 on all except the number of roots was significant  (Table 8).  A large number of bacterial cells of  Cs—R8 was recovered on YGM plates from stems of plantlets 2 cm above the agar surface.  23  Symptoms of micropropagated potato plantlets of cv. Table 4. Kennebec caused by Erwinia carotovora subsp. carotovora strain 21 Treat— ment  Inoc.’ concn (cfu/mL)  Stem length (cm)  No. of leaves  2 color  Root length (cm)  No. of roots  Buffer  0  3 7.57a  9.61a  O.28a  9.Ola  7.56a  Eca 31  32  5.06b  7.28b  1.78b  2.02c  8.33a  Ecc 21  3  6.88ab  8.56ab  1.22b  7.95a  6.67a  27  6.98ab  9.17a  1.50b  6.OOb  7.41a  0.49  0.31  0.13  0.38  0.39  Stnd. error  ‘One mL inoculum suspension was mixed with 79 mL Murashige and Numbers indicated final concentrations. Skoog’s medium. Six numerical classes of discoloration of plantlets were used: 2 0, normal; 1, 1—2 top leaves yellowed; 2, 3—4 top leaves yellowed; 3, over 5 leaves yellowed; 4, top blackened and/or all leaves yellowed; 5, stem rotted. Numbers followed by the same letter within each column do not 3 differ significantly (p=0.05) according to Tukey’s multiple range test. The value is the mean of 18 observations.  24  Symptoms of micropropagated potato plantlets of cv. Table 5. Kennebec caused by Erwinia carotovora subsp. carotovora strain 71 Treat— ment  Inoc.’ concn (cfu/mL)  Buffer  0  2 Color  Root length (cm)  No. of roots  9.06a  0.17a  8.62a  8.17a  Stem length (cm)  No. of leaves  3 7.54a  Eca3l  32  4.56b  6.94ab  2.llc  2.33bc  7.lla  Ecc7l  7  7.16a  8.39ab  l.06b  4.32b  9.28a  68  2.96b  6.39b  2.33c  0.80c  6.56a  0.45  0.59  0.19  0.69  0.64  Stnd. error  ‘One mL inoculum suspension was mixed with 79 mL Murashige and Numbers indicated final concentrations. Skoog’s medium. Six numerical classes of discoloration of plantlets were used: 2 0, normal; 1, 1—2 top leaves yellowed; 2, 3—4 top leaves yellowed; 3, over 5 leaves yellowed; 4, top blackened and/or all leaves yellowed; 5, stem rotted. Numbers followed by the same letter within each column do not 3 differ significantly (p=O.O5) according to Tukey’s multiple range test. The value is the mean of 18 observations.  25  Effect of Erwinia chrysanthemi strain 573 on Table 6. micropropagated potato plantlets of cv. Kennebec Treat— ment  Inoc.’ concn (cfu/mL)  Stem length (cm)  No. of leaves  2 Color  Root length (cm)  No. of roots  Buffer  0  3 7.55a  8.06a  0.lla  7.80a  7.33a  Eca 31  53  4.29b  6.28b  2.17b  l.95b  5.56a  Echr 573  4  2.37c  3.89c  1.44ab  0.63b  3.llb  41  1.56c  2.52c  l.66ab  O.19b  1.19b  0.34  0.  0.36  0.39  0.54  Stnd. error  31  ‘One mL inoculum suspension was mixed with 79 mL Murashige and Numbers indicated final concentrations. Skoog’s medium. Six numerical classes of discoloration of plantlets were used: 2 0, normal; 1, 1—2 top leaves yellowed; 2, 3—4 top leaves yellowed; 3, over 5 leaves yellowed; 4, top blackened and/or all leaves yellowed; 5, stem rotted. Numbers followed by the same letter within each column do not 3 differ significantly (p=0.05) according to Tukey’s multiple range test. The value is the mean of 18 observations.  26  Effect of Erwinia chrysanthemi strain 574 on Table 7. micropropagated potato plantlets of cv. Kennebec Treat— ment  Inoc.’ concn (cfu/mL)  Stem length (cm)  No. of leaves  2 Color  Root length (cm)  No. of roots  Buffer  0  3 7.76a  7.89a  0.OOa  8.62a  7.33a  Eca 31  40  3.06b  3.94b  2.61b  1.36b  4.22a  Echr 574  4  2.74b  4.OOb  0.83a  2.24b  6.50a  37  2.65b  4.56b  l.06a  1.18b  5.56a  0.28  0.45  0.25  0.40  0.83  Stnd. error  ‘One mL inoculum suspension was mixed with 79 mL Murashige and Skoog’s medium. Numbers indicated final concentrations. Six numerical classes of discoloration of plantlets were 2 used: 0, normal; 1, 1—2 top leaves yellowed; 2, 3—4 top leaves yellowed; 3, over 5 leaves yellowed; 4, top blackened and/or all leaves yellowed; 5, stem rotted. Numbers followed by the same letter within each column do not 3 differ significantly (p=0.0 ) according to Tukey’s multiple 5 range test. The value is the mean of 18 observations.  27  Comparison of symptoms expressed by micropropagated Table 8. potato plantlets of cv. Kennebec caused by Corynebacterium sepedonicum strain R8 and Erwinia carotovora subsp. atroseptica strain 31’ Treat— ment  Inoc. concn 2 (cfu/mL)  Stem length (cm)  No. of leaves  3 color  Root length (cm)  No. of roots  Buffer  0  4 7.51a  7.13a  0.20a  8.33a  7.2Oab  Eca3l  296  4.52b  4.73b  1.27a  2.24b  5.33b  29600  1.38c  2.07c  3.27b  0.66c  0.67c  640  8.21a  7.33a  0.13a  8.95a  7.73a  64000  6.92a  7.40a  0.80a  8.77a  7.33a  0.46  0.25  Cs—R8  Stnd. error  0.39  0.33  0.41  ‘Nodal cuttings of all treatments were inoculated by dipping in the treatment solutions for 10 seconds, followed by being transferred to a sterile filter paper to remove surface liquid inoculum, then transferred to jars. Two inoculum concentrations, 100—fold apart were used for each 2 organism. Six numerical classes of discoloration of plantlets were used: 3 0, normal; 1, 1—2 top leaves yellowed; 2, 3—4 top leaves yellowed; 3, over 5 leaves yellowed; 4, top blackened and/or all leaves yellowed; 5, stem rotted. Numbers followed by the same letter within each column do not 4 differ significantly (p=0.05) according to Tukey’s multiple range test. The value is the mean of 18 observations.  28 One strain from each of P. marginalis and E.  amylovora was  tested and both significantly inhibited plantlet root growth of cv. Kennebec  Stem lengths in treatments with both  (Table 9).  bacterial species were not significantly different from that of P. marginalis and E.  the buffer control.  yellowing of the plantlets,  amylovora did not cause  however Eca 31 did  (Table 9)  Root growth of the cuttings was observed 6 days after incubation.  The soft rot erwinia strains tested varied in growth  rate in MS medium.  Colonies of some strains developed in MS  medium within 3 days of incubation.  The inhibition of root  elongation in the treatments inoculated with the high inoculum concentration was observed within 7—8 days for all strains tested.  The degree of root inhibition depended on the rate of  bacterial growth in the medium.  In addition,  bacterial colonies  grew faster in association with the roots of the plantlets.  The major and minor symptoms of the plantlets inoculated with the soft rot erwinia strains became apparent during the course of incubation. stems and leaves.  The symptoms were associated with roots,  Root symptoms consisted of small roots  1), blackening and browning of root tips root formation  (Fig. 2. B)  .  (Fig.  2. A),  (Fig.  and aerial  Inhibition of root elongation was  associated with all strains of soft rot erwinias and occurred as early as the noticeable root growth.  The browning of root tips  was observed in all strains tested and occurred during later  29  Effect of Pseudomonas marginalis and Erwinia Table 9. amylovora on micropropagated potato plantlets of cv. Kennebe c’ Treat— ment  Inoc. 4 concn (cfu/mL)  2 color  Root length (cm)  No. of roots  8.56a  0.OOa  12.97a  6.28ab  Stem length (cm)  No. of leaves  3 4.80a  Buffer  0  Eca 31  46  3.40b  6.64a  2.78b  5.91b  5.lla  5 Pm  31  3.55ab  6.46a  0.83ab  6.12b  7.68b  6 Ea  69  3.63ab  7.56a  0.OOa  6.16b  6.Ola  0.28  0.76  0.49  1.02  0.31  Stnd. error  ‘Treatments were incubated at 18—22°C for 30 days. 5) of discoloration of plantlets 2 S ix numerical classes (0 were used: 0, normal; 1, 1—2 top leaves yellowed; 2, 3—4 top leaves yellowed; 3, over 5 leaves yellowed; 4, top blackened and/or all leaves yellowed; 5, stem rotted. Numbers followed by the same letter within each column do not 3 differ significantly (p=0.05) according to Tukey’s multiple range test. One mL inoculum suspension was mixed with 79 mL Murashige and 4 Skoog’s medium. Numbers indicated final concentrations. One strain of P. marginalis and one potato strain of E. 6 ’ 5 amylovora was grown at 23°C for 48 h before being used in inoculum preparation. —  30  Fig.  1.  Root length and stem length of micropropagated potato  plantlets of cv. Kennebec grown for 21 days on Murashige and Skoog’s medium inoculated with Erwinia carotovora subsp. atroseptica strain 31  (left)  (right)  in comparision to those of control  31  Fig.  2.  Root symptoms on plantlets of cv. Kennebec after 26 days  of growth. A,  Browning and blackening of root tips caused by E.  chrysanthemi strain 573. B, Aerial root formation induced by Erwinia carotovora subsp.  carotovora strain 71.  32 stage of growthi  although the frequency was low within each  Aerial root formation was also a common effect  experiment.  . caused by soft rot erwinias regardless of inoculum concentration  The stem symptoms consisted of stunting at the stem base, black necrotic dots, A),  and decay  (Fig.  3. B).  (Fig.  1), blackening  stem yellowing  (Fig.  3.  Blackening at the stem base was  possibly caused by bacterial growth on the medium at the base of the stem.  In contrast,  stem rot usually started at the tip of  the stem which first turned black and progressed downward.  Most  leaves of a plant]-et turned yellow when stem rot occurred, usually at the later stage of growth.  The leaf symptoms consisted of necrotic lesions, and yellowing curling  (Fig.  (Fig. 6),  4. B), black necrotic dots  and leaf tip necrosis  (Fig.  leaf rot  (Fig. 5), 7)  .  leaf  Leaf yellowing  was common and its severity was proportional to the inoculum concentration.  Some minor symptoms became apparent on plantlets grown for 30 days on the medium inoculated with P. marginalis and E. amylovora.  Small black localized lesions on the underside of  leaves were occasionally observed in the plantlets on medium inoculated with E.  amylovora.  P. margirialis caused browning of  some of stems and the underside of leaves of the plantlets.  33  j B  Fig. 3.  Stem symptoms on plantlets of cv. Russet Burbank grown  for 30 days on Murashige and Skoog’s medium inoculated with Erwiriia chrysanthemi strain 576. A,  black necrotic dots on stems. B,  Blackening,  yellowing,  Rotting of stem tips.  and  cv)  •  -  I’  •  > C) o  4-i U)  J 4 0)  c  H 4)  Ce H o  4)  Ce  4)  o  24  0)  0  H  -i 4)  o  C)  0)  .  a)  ‘ci  a  0  > a) ‘ci  b  H H Ci)  0  9-i  >1  -IJ  0  ‘ci  U)  (I)  C5  1 S.-  0  0 4-)  S-i  0  C)  H  Ct3  S-i  0  (1) -I-)  ‘ci  4) -H  ,c:  S.-i  S.-i  C)  U)  0  4i  o  -I-)  Ce  Cs)  0) >  ce  Ci)  %  i-i  l  •  -ii  ,  0  Ce  4)  Ce  b  o  C) 0)  Ce H  -H 4)  U) >  c  C)  -H  z  Ce  o  Cl) i-i  ‘.-o  ‘ci  e  4-i  9-4  S.-i o  S-i  o  C) -H  E 4-i  (N  o  -  U)  0)  0) ::‘  (I)  U) -I-)  -I-)  E  4)  S-i  0)  Ce  H  c  H  -H  -ii  U) .1-) (1)  Ce  0)4-4 Ce  ci)  C) •  c::  ,Q  0)  Ci)  •  ‘  b’e  •r4  (N (N  Ce  -ri  S-i 4-) C,)  (13  C-)  I  35  Fig.  5.  Black necrotic dots on the leaf of micropropagated  potato plantlets of cv. Russet Burbank grown for 30 days on Murashige and Skoog’s medium inoculated with Erwinia chrysanthemi strain 576.  36  I  ‘I  ,f/  I,  \  I ,0  ‘  Fig.  6.  Curling of leaves of micropropagated potato plantlets of  cv. Kennebec grown for 26 days on Murashige and Skoog’s medium inoculated with Erwinia carotovora subsp.  atroseptica strain 31.  37  /7  N  :::  Fig.  7.  A  Leaf tip necrosis of plantlets of cv. Kennebec grown for  26 days on Murashige and Skoog’s medium inoculated with Erwinia chrysanthemi strain 576.  38 DISCUSSION  Although many symptoms developed in plantlets of cv. Kennebec infected by soft rot erwinias,  root growth inhibition  was the only one consistently exhibited by all strains tested. It is possible that all strains of soft rot erwinias possess the capacity for root growth inhibition.  Moreover,  when the  elongation of roots was inhibited earlier in the growth, development was more severely affected.  root  The fact that inhibition  of root growth was consistent in all experiments also indicated that the tissue culture system was a reliable model for studying the root inhibitory effect by soft rot erwinias.  Although both E. strain 573,  chrysanthemi strains inhibited root growth,  but not strain 574,  significantly inhibited root 7).  The reason for this  initiation of the plantlets  (Table 6,  discrepancy was not known.  Perhaps high amounts of a root  initiation inhibitor were produced by Echr 573 at inoculum concentration as low as 4 cfu/mL in comparison to other soft rot erwinia strains.  My observation that root inhibition by some soft rot erwinia strains occurred soon after the start of root elongation may be related to the finding by Taylor and Secor  (1985)  that the growth  and survival of potato callus tissue were dramatically inhibited by Ecc 71.  Most calli were killed within 5 days of growth on the  39 inoculated medium.  The factor that inhibited root elongation of  micropropagated potato plantlets may be the same as that which killed the calli.  Perombelon and Kelman  (1980)  stated that a common  characteristic of soft rots and associated disorders was the lack of specificity of the host—pathogen interaction.  The effect of  root growth inhibition on micropropagated potato plantlets by all strains of soft rot erwinias tested is consistent with this lack of specificity.  The response of stem growth to soft rot erwinias varied when different strains were used. 196 and Ecc 21, levels,  Only two out of seven strains, Eca  did not cause stunting even at high inoculum  although both inhibited root growth significantly.  The  reason for this difference might be due to the difference in growth rates of the strains in MS medium.  At high inoculum concentration, with high growth rates in MS medium, died at an early stage of growth.  or with bacterial strains the plantlets rotted and  Decay of stem tips was also  frequently observed in different experiments.  Although many  bacteria do produce tissue—macerating enzymes,  only Erwinia spp.,  P.  marginalis,  and a few others have been associated with decay  of living plant tissue  (Lund 1979; Rudd-Jones and Dowson 1950)  These tissue macerating enzymes from soft rot erwinias probably  40 played a role in decay development.  Some symptoms might be due  to nutrient limitation in the medium or the blocking or breakdown of the nutrient transport systems of the plantlets.  In either  the essential nutrients failed to reach the growing points  case,  and the plantlets eventually died.  The number of leaves and discoloration of plantlets developing on the media inoculated with Eca 31 were not always consistent in different experiments,  suggesting that the response  of both characters to soft rot erwinias might not be stable in vitro.  In spite of the frequent reoccurrence of some minor symptoms in different experiments,  the frequency of a particular symptom  was usually low within each experiment. employ other strategies,  It would be necessary to  such as modifying growth conditions or  medium composition to increase the frequency of the minor symptoms, to study them in greater detail.  The browning of tissues,  together with a loss of  electrolytes and an increased rate of respiration of tobacco suspension cultures were described by Matthysse  (1983)  as an  incompatible response of these cultured cells to P. pisi.  She  concluded that tissue browning and loss of electrolytes were due to damage to the plasmalemma.  Stephens and Wood  (1975)  reported  that endo—polygalacturonate trans—eliminase recovered from  41 infection of tissue slices by E.  carotovora caused the release of  polyphenol oxidase and thus tissue browning. browning  (blackening)  The root tip  was frequently observed in the plantlets  growing in the medium inoculated with soft rot erwinias.  It is  possible that root tip browning of the plantlets in response to soft rot erwinias was an incompatible response to the presence of the pathogen and might be due to polyphenol oxidase activity.  Although inoculum concentrations were different, P. marginalis and E.  amylovora caused inhibition of root elongation  to a similar extent as Eca 31.  Large E.  amylovora colonies were  found scattered around the small roots of a cutting.  Long roots  grew within 1 cm to the agar surface and some roots developed in the medium but the growth was drastically reduced by E. amylovora.  This phenomenon may be due to stimulation of E.  amylovora cell growth by the nutrients released from sloughed cells of root caps which were capable of diffusing through the agar medium.  The scattered appearance of bacterial colonies  around roots was unique for E. bacterial species tested.  amylovora among the different  The root inhibition might be caused by  a toxic secondary metabolite secreted by E. around the roots.  amylovora colonies  Possibly a gradient of toxic metabolite that  decreased with distance from the centre of those colonies was built up and the roots were less inhibited at distance further away from those colonies.  42 Different factors that were either directly or indirectly produced by the bacteria possibly contributed to root inhibition of micropropagated potato plantlets.  The inoculated media on  which the plantlets grew were cloudy as a result of growth of P. rnarginalis.  It is likely that root growth inhibition by P.  marginalis was caused indirectly by nutrient deficiencies, of 02,  However other factors,  or changes in medium pH.  example,  lack  for  toxic metabolites, may also be involved.  No apparent growth of Cs-R8 on/in MS medium could be detected after incubation at 19°C days.  For this reason,  (day)  and 23°C  (night)  for 15  direct inoculation of nodal cuttings with  a bacterial suspension was employed.  Cs—R8 colonies were  occasionally observed growing around the roots of Kennebec nodal cuttings during incubation.  It is proposed that the lack of  symptoms in plantlets inoculated with Cs—R8 was probably due to the slow growing characteristic of this bacterial species.  The cause of the inhibition of root elongation,  stunting,  and yellowing of micropropagated potato plantlets by soft rot erwinias may not be the same.  A metabolite from the pectolytic  erwinias may be involved in root inhibition.  The minor symptoms  may be due to the physiological disorders of the plantlets in response to the change in their growth medium.  43 CHAPTER 3  RESPONSE OF MICROPROPAGATED POTATO PLANTLETS TO  —  ERWINIA CARO TOVORA SUB SP.  A TROSEP TICA  INTRODUCTION  Root growth inhibition and stem length reduction of in vitro grown potato plantlets occurred in MS medium inoculated with the soft rot erwinia strains.  The extent of changes in tissue  culture medium is determined by the size of the bacterial population.  Inoculum concentration controls the rate at which  the bacteria alter the culture medium. nutrients,  Consumption rate of  the oxygen and pH levels change faster and to a  greater degree at higher bacterial concentration than at a lower one.  Moreover,  more toxic bacterial metabolites accumulate at  higher bacterial levels.  It is quite possible that plantlets may  respond differently to varying concentrations of bacteria in the medium.  Introduction of bacteria into a new environment results in alteration in bacterial metabolism. secreted;  others might be repressed.  Some new metabolites may be Moreover,  the association  of soft rot erwinias with nodal cuttings or only with MS medium represents two different environments which would result in different kinds and amounts of enzymes released from bacterial cells.  The fact that cells of Eca 31 grew faster once they were  44 associated with the plants indicates that some factors from nodal cuttings promoted the growth of the bacteria.  The bacterial  population density associated with the plantlets was a function of the method of inoculation.  This would result in different  responses of the plantlets depending on the inoculation procedure that was used.  Genetic control of host plants over the number and type of microorganisms was demonstrated by Atkinson et al.  (1975).  They  showed that resistant varieties of wheat supported lower populations of bacteria than did susceptible ones and substitution of a single resistance chromosome into an otherwise susceptible variety changed the rhizosphere microflora. the disease development in tubers caused by E.  Moreover, subsp.  c.  atroseptica was also reported to be affected by cultivar  susceptibility which varied greatly Similarly,  it is proposed here that,  (Bourne et al.  1981)  after identical experimental  treatment, micropropagated plantlets of different cultivars might harbour different numbers of bacteria of the same species due to genetic differences among cultivars used.  The blackleg disease of potato occurring in temperate regions is caused mainly by E.  c. subsp. atroseptica which is  often present on seed tubers.  Most E.  strains,  c. subsp. atroseptica  irrespective of the country of origin,  serologically homogeneous group  (Allan et al.  form a  1977; De Boer et  45 al.  1979).  Eca 31 is a typical strain of serogroup I which  represent 96% of the strains of this subspecies McNaughton 1987).  Therefore,  (De Boer and  Eca 31 was used in this study.  The objectives of this study were:  (1)  to measure the  sensitivity of micropropagated plantlets to varying inoculum concentrations;  (2)  to compare the responses of different  cultivars to the same pathogen;  (3)  to illustrate the effect of  different inoculation methods on the responses of micropropagated plantlets.  MATERIALS AND METHODS  MICROPROPAGATED PLANTLETS. potato plantlets of cv. Kennebec,  Disease—free micropropagated Red Pontiac and  Russet Burbank,  Red Lasoda were obtained from the Virus—free Potato Laboratory at Agriculture Canada, Vancouver Research Station. of contamination,  growth,  The examination  and multiplication of the plantlets in  vitro were the same as that reported in Chapter 2.  ECA 31 CULTURE.  Cultures of Eca 31,  streaked on casamino acid,  peptone,  stored at —80°C,  glucose  (CPG)  were  plates.  The  cultures from a typical Eca 31 colony on CPG was streaked on crystal violet,  pectate  (CVP)  medium and a single colony with the  unique pitting characteristic on the medium was streaked on nutrient agar slants for inoculum preparation.  Cells from 2-day  46 old slant cultures were suspended in sterile Ringers solution and the stock solution was made by adjusting the cell suspension to an absorbance value of 0.4 at 660 nm using a spectrophotometer (Spec 20)  .  Three inoculum concentrations,  estimated at 10,  and iO cfu/mL determined by a standard plate count method,  iO, were  used.  INOCULATION METHOD.  A modified MS medium which contained  0.7% agar was adopted to reduce damage to root systems of plantlets during sampling.  Forty-nine mL molten MS medium at  40°C was added to each of thirty-two 500 mL—jars and mixed thoroughly with 1 mL of each of the three inoculum preparations and Ringers solution.  The cuttings were prepared in the same way  as described before and eight cuttings/jar were planted after the medium was completely solidified. twice.  Each treatment was replicated  The treatments were randomized and incubated for 18 days  at 19°C(dark)  and 23°C  (light)  with a photoperiod of 16 hours,  s’ provided by eigth F48 2 under an illumination of 194.5 LE m T12/CW/HO Phillips fluorescent tubes.  Tukey’s multiple range test  was used to analyze the data.  ELISA METHOD.  A 1.5 cm long stem was cut from the lower end  of the plantlet and transferred into a sterile plastic bag containing 1 mL sterile distilled water.  After homogenization,  the tissue sap was assayed by indirect ELISA according to the procedure by De Boer et al.  (1988),  except that polyclonal E54  47 IgG and monoclonal 4F6 antisera were used for detection of E.  carotovora in this study.  The bacterial stock  AN ALTERNATIVE INOCULATION PROCEDURE. solution  660 (0D  =  0.1)  was prepared as above.  bacterial concentrations, were used as inoculum, control.  6 x 102,  6 x iO,  Three levels of  and 6 x 106 cfu/mL,  and sterile Ringers solution was a  The nodal cuttings were inoculated by immersing them  directly in the bacterial suspensions for 10 seconds and then dipping them in Ringers solution briefly to rinse away extra The cuttings were blotted  suspension adhering to the cuttings.  on dry sterile filter paper and planted into the jars. treatment was replicated six times. incubated for 31 days at 19°C(dark) photoperiod,  Each  The treatments were and 23°C  (light)  with 16 h  1 provided by s 2 under an illumination of 196.7 .LE m  eigt F48 T12/CW/HO Phillips fluorescent tubes and four 25 W light bulbs.  ELECTRON MICROSCOPIC EXAMINATION.  Samples of roots of  micropropagated potato plantlets of cv. Red Pontiac were fixed in 4% glutaraldehyde and  (v/v)  in 0.1 M cacodylate buffer pH 7.2 for 2 h  washed in 0.1 M cacodylate buffer.  1% Os0 4 for 1 h in buffer, series of alcohols, EPON 812 mixture.  They were postfixed in  washed in water,  dehydrated in a  and embedded in propylene oxide infiltrated  48 The roots were sectioned and stained for 20 mm uranylacetate, mm.  and lead citrate  in 4%  ( diluted 1:1 0.1 N NaOH)  for 5  The preparations were viewed in an Hitachi 600 electron  microscope.  RESULTS  The roots and stems responded differently  SENSITIVITY TEST.  to the varying inoculum concentrations.  Root elongation of cv.  Russet Burbank and Red Pontiac in vitro was dramatically inhibited by Eca 31 at an inoculum concentration of 20 cfu/mL (Fig.  8)  .  The inhibition of root initiation occurred as the  inoculum concentration increased from 0.2 to 2000 cfu/mL, but significantly only at the highest inoculum level for both cultivars  (Fig.  9)  .  Root elongation was more sensitive than  initiation to inhibition  (Fig.  8,  9)  Stunting of plantlets occurred at inoculum concentration higher than 20 cfu/mL  (Fig.  10).  The plantlets of both cultivars  developed significantly fewer leaves at an inoculum concentration over 20 cfu/mL compared to control  (Fig.  11)  .  Cultivar Red  Pontiac developed more leaves per unit stem length than cv. Russet Burbank.  The plantlets of both cultivars turned yellow at  20 cfu/mL and the yellowing become more severe for cv. Russet than for cv. Red Pontiac when inoculum concentration increased from 20 to 2000 cfu/mL  (Fig.  12)  49  7  I -J  I  z  I-  (9 z w -J  F  0  ‘  Aa  RB >: RP  Aa  x 2 u.. 0  z  Wi  0 —  0  I_ I 0.2  B  20  2000  INOCULUM CONCENTRATION (cfu/mI)  Fig.8. Mean of maximum root length of plantlets of cv. Russet Burbank (RB) and Red Pontiac (RP) grown for 18 days on Murashige and Skoog’s medium inoculated with E. carotovora subsp. atroseptica strain 31. Means (n=1 6) (bars) for each cultivar having the same letter do not differ significantly (p=0.05) according to Tukey’s multiple range test  50 8 I—  Lii -J  I  z 0  I-  0 0 cc U-  04 cc Lii  D  z z  w  0 0  0.2  20  2000  INOCULUM CONCENTRATION (cfu/ml)  Fig. 9. Mean number of roots per plantlet grown for 18 days on Murashige and Skoog’s medium inoculated with different concentrations of Erwinia carotovora subsp. atroseptica strain 31. Means (n=1 6) (bars) for each cultivar having the same letter do not differ significantly (p=0.05) according to Tukey’s multiple range test. RB: Russet Burbank, RP: Red Pontiac.  51  5 0  F—  w4 -J  I  z  a0 I—  0  z  UI -J  UI I— C/)  zi UI  0 0  0.2  20  2000  INOCULUM CONCENTRATION (cfu/mI) Fig. 10. Mean stem length of plantlets of cv. Russet Burbank (RB) and Red Pontiac (RP) grown for 18 days on Murashige and Skoog’s medium inoculated with different concentrations of Erwinia carotovora subsp. atroseptica strain 31. Means (n=1 6) (bars) for each cultivar having the same letter do not differ significantly (p=0.05) according to Tukey’s multiple range test.  52  10 I Lii  -J  z 0 Cl) LU  > <6 LU -J U  0 Ui  D  z z  0 0  0.2  20  2000  INOCULUM CONCENTRATION (cfulml)  Fig. 11. Mean number of leaves per plantlet grown for 18 days on Murashige and Skoog’s medium inoculated with different levels of Erwinia carotovora subsp. atroseptica strain 31. Means (n=1 6) (bars) for each cultivar having the same letter do not differ significantly (p=0.05) according to Tukey’s multiple range test. RB: Russet Burbank RP: Red Pontiac.  53  6  5  (!3 Z4  0 -J —j3  w  >2  1  0 0  0.2  20  2000  INOCULUM CONCENTRATION (cfu/mI) Fig. 12. Severity of yellowing of plantlets of cv. Russet Burbank (RB) and Red Pontiac (RP) grown for 18 days on Murashige and Skoog’s medium inoculated with different concentrations of Erwinia carotovora subsp. atroseptica strain 31. Six numerical classes of yellowing: 0, normal; 1, 1-2 top leaves yellowed; 2,3-4 top leaves yellowed; 3, over 5 leaves yellowed; 4, top blackened and/or all leaves yellowed; 5, stem rotted. Means (n=1 6) (bars) for each cultivar followed by the same letter do not differ significantly (p=0.05) according to Tukey’s multiple range test.  54  VARIETY RESPONSE. at the low,  The roots of cv. Red Lasoda were shorter  but longer at the high inoculum concentration than  those of the other three cultivars responses in cv. Kennebec,  Root elongation  (Table 10).  Red Pontiac,  and Red Lasoda were  similar to one another at both inoculum concentrations.  The root  initiation response of cv. Red Lasoda was significantly different from those of cv. Red Pontiac and Russet Burbank at 20 cfu/mL (Table 10)  The response of stem growth of cv. Kennebec, Red Pontiac,  and Red Lasoda to E.  similar at 0.2 cfu/mL  (Table 10)  c.  .  subsp.  Russet Burbank,  atroseptica was  The stems of cv.  Russet  Burbank were significantly longer than those of Red Pontiac and Red Lasoda at 20 cfu/mL and were only slightly shorter at the high in comparison to the low inoculurn concentration  ALTERNATIVE INOCULATION METHOD. elongation,  stunting,  (Table 10).  Inhibition of root  and yellowing occurred in plantlets grown  from cuttings directly inoculated with Eca 31.  The stem length  of cv. Russet Burbank was increasingly smaller as the inoculum concentration increased from 0 to 6 x 106 cfu/mL  (Fig.  13)  .  The  severity of yellowing increased as the inoculum concentration increased Eca 31  (Fig.  14).  The roots were significantly inhibited by  in all inoculated treatments  (Fig.  15)  55  Table 10. Comparison of response of micropropagated potato plantlets of cv. Kennebec, Russet Burbank, Red Pontiac, and Red Lasoda to Erwinia carotovora subsp. atroseptica strain 31 Percentage of control Character of the plantlets  Potato cultivar  Stem length  Kennebec Russet Burbank Red Pontiac Red Lasoda  Root length  Kennebec Russet Burbank Red Pontiac Red Lasoda  Number of roots  Kennebec Russet Burbank Red Pontiac Red Lasoda  0.2 (cfu/mL) 98.38 110.25 105.88 91.25  a a a a  78.63 ab 85.75 a 87.13 a 65.63 b 107.38 100.00 90.50 115.50  a a a a  20 (cfu/mL) 73.29 ab 103.71 b 50.50 a 67.00 a 15.29 7.86 8.50 42.71  a a a b  100.71 ab 72.86 a 64.13 a 139.71 b  ‘Numbers followed by the same letter for each character within each column do not differ significantly (p=0.05) according to Tukey’s multiple range test.  56  12  a  10  I— UI -J I  z  <8 -J I  0 Z6 LII  -J  UI I4 Cl)  z UI  2  0 0  6  600  60000  INOCULUM CONCENTRATION (x 102 cfu/mI) Fig. 13. Response in stem length of plantlets of cv. Russet Burbank to different concentrations of Erwinia carotovora subsp. atroseptica strain 31. The nodal cuttings were inoculated directly with the bacteria. Means (n=6) (bars) having the same letter do not differ significantly (p=0.05) according to Tukey’s multiple range test.  57  3  b 2.5  2  b  z  b  0 l.5 Iii  >-  1  0.5  a  0 0  6  600  60000  INOCULUM CONCENTRATION (x i? cfu/ml) Fig. 14. Severity of yellowing of plantlets of cv. Russet Burbank in response to different inoculum concentrations of Erwinia carotovora subsp. atroseptica strain 31. Six numerical classes of yellowing: 0, normal; 1, 1-2 top leaves yellowed; 2, 3-4 top leaves yellowed; 3, over 5 leaves yellowed; 4, top blackened and/or all leaves yellowed; 5, stem rotted. Means (n=6) (bars) having the same letter do not differ significantly (p=0.05) according to Tukey’s multiple range test.  58  14  a  C.)  I 12 uJ -J I—  z  10 0  0 z w  8  I 0 0  6  -J  b  >< 4 LI  0  z  C  2  C  w 0 0  6  600  60000  INOCULUM CONCENTRATION (x lO cfu/ml) 2 Fig. 15. Effect of different inoculum concentrations of Erwinia carotovora subsp. atroseptica strain 31 on root elongation of plantlets of cv. Russet Burbank. The nodal cuttings were inoculated directly with bacteria and were grown on sterile Murashige and Skoog’s medium. Means (n=6) (bars) having the same letter do not differ significantly (p=0.05) according to Tukey’s multiple range test.  59 ELISA DATA.  The concentrations of the bacterial antigen at  the stem end of the plantlets of four cultivars rose as the inoculum concentration increased  (Fig.  16)  .  The values for cv.  Red Lasoda were consistently lower than those for other cultivars (Fig.  16)  ELECTRON MICROSCOPY.  The electron microscopic examination  revealed that bacterial cells of Eca 31 were present in three major locations in root systems of the plantlets of cv. Red Pontiac.  A large population of bacterial cells covered the  epidermal surface of the root  (Fig.  17).  A large number of  bacterial cells was present in the xylem of the root and in the intercellular spaces of the parenchyma addition, cell  (Fig.  (Fig.  (Fig.  18),  19).  In  a few bacterial cells were discovered inside a root 20).  The electron micrographs showed that a slime  layer was always associated with aggregated bacterial cells.  DISCUSSION  Although an effect of bacterial contamination on tissue cultures of ornamental plants by E. demonstrated  carotovora was previously  (Knauss and Miller 1978),  this study was the first  attempt to elucidate dose response of micropropagated potato plantlets to Erwinia spp.  Since the culture medium of the  plantlets was chemically defined,  and the growth conditions in a  60  1-  0.9  -  0.8  0.7 c in 0  0,6  w  0.5  0  -  z  4  0.4 U)  m 4  0.3  0.2  0.1  -  -  -  0—  I  0  0.4  I  I  0.8  I  1.2  I  I  1.6  I  I  2  2.4  2.8  3.2  INOCULUI CONCENTRATION Clog cfu/ml)  Fig.  16.  Mean absorbance values  405 nm) (A  for enzyme—linked  immunosorbent assay conducted on stem end portions of plantlets of cv. Kennebec Lasoda  (v)  (D), Russet Burbank (+), Red Pontiac (0), and Red  in response to different inoculum concentrations of  Erwinia carotovora subsp.  atroseptica strain 31.  61  Fig.  17. Electron micrograph of root section of cv. Red Pontiac  grown for 26 days on inoculated Murashige and Skoog’s medium. A large population of bacterial cells of Erwinia carotovora subsp. atroseptica strian 31 on root surface  (x 8750)  62  ,  ..:  ‘  )  Fig. 18. Electron micrograph of longitudinal section of a root near the edge showing the presence of bacterial cells of Erwinia carotovora subsp.  atroseptica strain 31  in intercellular spaces  between the epidermal cells. Plantlets of cv. Red Pontiac grown for 26 days on Murashige and Skoog’s medium  (x 3705)  63  •H•  —  -r  -  -  *5  -S  Fig. 19. Electron micrograph of cross section of a root of cv. Red Pontiac grown for 26 days on Murashige and Skoog’s medium inoculated with Erwinia carotovora subsp.  atroseptica strain 31.  Bacterial cells in vascular tissue of the root  (x 10000)  64  ---—---—-  .‘ e  /  1/  (/4  d  -  -••. V..  •..  ‘  Fig. 20. Electron micrograph of cross section of uper portion of a root of cv. Red Pontiac grown for 26 days on Murashige and Skoog’s medium inoculated with Erwinia carotovora subsp. atroseptica strain 31.  (x 7500)  Bacterial cells present inside a root cell  65 growth chamber were set at a constant level,  the dose response of  plantlets to the pathogen in vitro may be different from that in the field.  Nevertheless, the response of the plantlets to soft  rot erwinias in vitro should provide a valuable indication of the response of potato to the same pathogen in the field.  The  sensitivity tests utilizing the method of medium inoculation showed that two cultivars behaved similarly except that the number of roots of cv. Russet Burbank increased, but decreased for cv. Red Pontiac between 0—0.2 cfu/mL.  The reason for this  difference is not known.  Eca 31 colonies in the inoculated MS medium grew so fast  that the effect of bacterial metabolites was exerted on the initial growth of the cuttings.  It was predicted that the amount  of bacterial metabolites secreted into the medium would be proportional to the initial inoculum concentration.  The severity  of inhibition of root elongation was related to the inoculum level.  The inoculum concentration necessary to inhibit elongation of roots was lower than that to inhibit root initiation. Therefore, subsp.  inhibition of root elongation and initiation by E.  atroseptica should be considered separately.  The fact that root elongation of cv. Red Lasoda was inhibited more at the low,  than at the high inoculum  C.  66 concentration compared to other cultivars indicated that the roots of plantlets of this cultivar were comparatively tolerant However,  to Eca 31 at the high inoculum concentration. stems behaved differently for cv. Red Lasoda,  roots and  indicating that  different mechanisms in the plantlets might control the responses of root and stem.  My results are in agreement with Lyon’s  (1989)  suggestion that several mechanisms may operate within potato to either directly inhibit growth of Erwinia spp.  or indirectly  inhibit the enzymes involved in pathogenesis.  Different  mechanisms may be important in different parts of the plant  (Lyon  1989)  The plantlets of cv. Red Lasoda developed more roots than those of the three other cultivars at both inoculum concentrations and more roots developed at high than at low inoculum concentration for cv. Red Lasoda.  It is evident that  root initiation of cv. Red Lasoda was promoted in the presence of a high number of Eca 31 cells.  The conjugated monoclonal antibody used in the ELISA was produced against lipopolysaccharide of the outer membrane of E. c.  subsp.  atroseptica.  The higher the ELISA values,  the higher  the LPS concentrations in the stem end of the plantlets and probably the higher the Eca 31 cell population.  The fact that  the ELISA values for cv. Red Lasoda were consistently lower than those for three other cultivars provided additional evidence that  67 this cultivar supported the growth of fewer bacteria than other cultivars.  The effect of soft rot erwinias on potato plants grown in vitro was diverse and the symptoms in treatments with high inoculum concentrations were easily observed during the growth of the cuttings.  However the plantlets often appeared normal in  treatments with lower inoculum levels.  My observation was in  agreement with that reported by Weber and Schenk  (1988)  who found  that there were no symptoms or growth retardation on the plantlets with low bacterial density.  The population of soft rot erwinias decline rapidly in naturally and artificially infested soils 1980)  .  However,  bacteria,  (Perombelon and Kelman  in common with many other plant pathogenic  soft rot erwinias can overwinter in contaminated plant  residue left in the soil after harvest.  These contaminated  materials together with diseased seed tubers serve as the most important inoculum sources for the next growing season.  The  purpose of different inoculation methods used in this study was to create different inoculum sources for the plantlets to study the individual responses to the pathogen. stunting,  Root elongation,  and the severity of yellowing of cv. Russet Burbank  were affected progressively by the increased inoculum dose in both inoculation methods.  Moreover,  root elongation was sharply  inhibited when certain inoculum concentrations were reached.  68 Both inoculation methods gave similar results.  Rhizosphere microorganisms are extensively associated with roots of plants and the root exudate provides various nutrients to support the growth of the bacteria.  Bowen and Rovira  (1976)  reported that almost the entire surface of roots growing in soil is covered with microorganisms.  Newman and Bowen  (1974)  found  that rhizosphere bacteria tended to aggregate on the surface of plant roots.  Immunofluorescence and immunogold staining  techniques revealed dense populations of E. the longitudinal cell walls of potato roots, the lateral roots were formed, tips  chrysanthemi along  at the points where  and sometimes close to the root  (Underberg and Van Vuurde 1989).  In my study,  a large  number of bacterial cells was present on the surface of the root, in the intercellular spaces of the parenchyma, of the root.  and in the xylem  In addition, most surface cells of the root of the  micropropagated plantlets had either died or lost their normal cell shape probably due to the toxic metabolites of the aggregated bacterial colonies.  The higher the amount of  bacterial metabolites released,  the more severe the damage to the  root system of the plants.  69 CHAPTER 4  EFFECT OF ERWINIA CAROTOVORA SUBSP. ATROSEPTICA ON  -  POTATO PLANTS GROWN IN SOIL  INTRODUCT ION  E.  c.  subsp.  atroseptica causes tuber decay,  nonemergence,  and blackleg of potato and is present on most seed tubers (Perombelon and Kelman 1980; Bain et al.  1990)  .  It is the  predominant blackleg pathogen in temperate regions, higher soil temperatures E.  c.  subsp.  whereas at  carotovora and also E.  chrysantheini have been shown to be the cause of blackleg (Staghellini and Menely 1975; Molina and Harrison 1977, Perombelon 1985) emergence,  .  Blackleg infected plants,  are smaller than healthy ones  1980;  a few weeks after  (Persson 1988)  Root growth of potato stem cuttings is inhibited in short potato—rotation soils in comparison to that in long potato— rotation soil  (Bakker et al.  1987)  .  Root systems of potato stem  cuttings grown for 2 weeks in soil with 1:1 potato frequency attained less than half the weight of those grown in soil with 1:6 potato frequency.  The fact that the absence of symptoms,  such as discoloration or lesions on roots of potato plants grown from short potato-rotation soil,  indicates that an unknown  microorganism affects root functioning without being parasitic (Bakker et al.  1987)  70 Many differences between the soil and tissue culture systems exist in terms of nutrients,  growing conditions and environment,  and the growth of the pathogen used in the experiments.  Response  of micropropagated plantlets to the pathogen in tissue culture systems may or may not represent those of the plants in the field.  Fett and Zacharius  (1982,  1983)  reported that some tissue  culture lines lose their ability to synthesize phytoalexins with increasing time in culture.  The comparison for the response of  potato plants to the same pathogen between culture in vitro and in a soil environment is necessary to make sure that the tissue culture is a suitable model for studying certain aspects of the disease in soil.  The purpose of this study was to elucidate the effect of E.  c. subsp. atroseptica on the symptom expression of potato plants growing in soil.  MATERIALS AND METHODS  PREPARATION OF SEED TUBERS.  Disease—free seed potato tubers  of cv. Russet Burbank were obtained from seed farms in the Pernberton Valley of British Columbia. potato virus X virus  (PLRV),  sepedonicum.  (PVX),  potato virus Y  The tubers were free from (PVY),  potato spindle tuber viroid  potato leaf roll  (PLRV),  and C.  Tubers were tested for contamination by soft rot  erwinias by combining portions of tuber tissues at stolen ends  71 and homogenizing them in sterile Ringers solution with a hammer. The homogenate was streaked onto CVP plates.  The weight of seed tubers used was in the range 42.5-113.4 grams.  The tubers were stored in the cold room at 4°C before The tubers were held in an open box in a greenhouse at 18°C  use.  for 3 weeks to break dormancy.  Individual seed pieces with one  eye each were obtained by cutting the seed tubers with a melon baller  (diameter  =  2.9 cm)  .  The seed pieces were stored under  the same conditions overnight.  The seed pieces were planted,  2.5 cm deep and 10 cm apart,  in regular soil mix in a plastic tray. thoroughly.  The trays were watered  The seed pieces were grown at 18°C for 2 days in a  greenhouse.  INOCULUM PREPARATION.  A 2—litre flask containing 1.25 L  nutrient broth was inoculated with 2—day—old Eca 31 cultures and were incubated at 23°C with constant shaking.  Bacterial cells  were harvested at the exponential growth phase by centrifugation at 10700 g for 20 mm.  The supernatant was discarded and the  pellets were suspended in 1.8 L sterile distilled water.  The  bacterial suspension was mixed thoroughly by stirring continually for half an hour.  Then the number of cells was determined by a  standard plate count method.  Two series of 100—fold dilutions  were prepared subsequently by transferring 18 mL bacterial  72 suspension to 1782 mL sterile distilled water to obtain additional inoculum preparations.  EXPERIMENTAL TREATMENTS.  Sixty-four 4-inch pots were each  filled with 450 g steamed soil mix.  Dry weight of the soil mix  was determined after heating to 8000 for 24 h. preparations,  including one water control and three inoculum  levels consisting of 3.3 x iOn, were employed. treatment,  Four treatment  3.3 x iO,  There were 4 treatments,  and 3.3 x iO cfu/mL, 16 replicates in each  and one seed piece in each replicate  INOCULATION, GROWTH, AND SANPLING.  (pot).  Before inoculation,  each  seed piece was taken carefully out of the tray and planted in a pot,  2.5 cm below the soil line.  growth were discarded.  A few seed pieces with no  The pots for each treatment were randomly  selected and the inoculum at 110 mL/pot was added evenly to the Four treatments with 0,  pots.  soil were used. bench.  1.1,  110,  and 11000 x iO cfu/g  The pots were completely randomized on the  The maximum water capacity of the soil in each pot was  premeasured by recording the volume of water retained by 450 g soil per pot. inoculation.  The plants were grown at 18°C for 6 days after All treatments were watered once every two days.  After 6 days of growth, the length of stems was measured and the number of leaves was recorded. removed carefully from the pots.  The entire plants were The root systems were washed  with tap water and the length of the longest root per plant was  73  measured.  The roots of each plant were kept separately and  heated at 80°C for 24 h in an oven to determine dry weight.  The  analysis of variance was carried out to measure the treatment effects.  The greenhouse experiment was repeated twice.  RESULTS  Inhibition of root elongation occurred in potato plants grown from seed pieces in soils inoculated with Eca 31.  Root  growth was sensitive to the increase in inoculum concentration. Roots of the plants at inoculum concentrations higher than 1.1 x iO cfu/g soil, treatments  (Fig.  were significantly shorter than those of control 21).  Root growth was inhibited by 50% at an  cfu/g soil. 7 inoculum concentration of 1.1 x lO  Decay of seed  pieces were observed in treatments with inoculum concentrations higher than 1.1 x 10 cfu/g soil and small roots were often associated with rotted seed pieces  (Fig. 22).  Dry weights of roots in treatments inoculated with Eca 31 was lower than that of the control only when inoculum concentration was higher than 1.1 x iO cfu/g soil  (Fig.  23).  The dry weight of root systems at the two highest inoculum concentrations did not differ significantly from each other.  74  2.2 0  I.  2  z  -J 0  1.8  -  F  z LU -J F  1.6  cc  1.4  0 0  -  >< LL  1.2  0  z Lii  1—  0.8  i  i  0  1  2  3  4  5  6  7  INOCULUM CONCENTRATION (log cfu/g soil) Fig. 21. Mean of maximum root length per plant grown in soil inoculated with Erwinia carotovora subsp. atroseptica strain 31 at concentrations of 1.1, 110, and 11000 x 10fu/g soil compared to control. The plants were grown at l8b for 6 days in pots in a greenhouse. Bars represent standard error of means of 16 replicates.  Ci 0 U) 4) H  Cl)  H -H 0  0 > 0 4) 0 -i cii C)  (11  :i U)  Q  U)  ci) U) 0 S-i .1-) dl  (ii C) --1 4.)  -H (11 I-) 4-) U)  c)  H  4-) (I  H  H  r1  cD  Z  G) U)  -H  4) H  -I III  0  w  G)  tyi  C)  o  4) (ii  G) 4) Cii H 5 C_) 0  4) -H .4 •  4-)  -H  H  ci)  0  U) (11  H -H 0  d  El  (I)  Cl) CI) Cl)  ci) C) ci) -H  :  U) (Ii  0 -I l:y)  -H  Cl)  11I  4-) ci) U) U)  Q  cii  cr1  -i Cl) 4) 4-4  ‘D  (ii  >1  U)  o  (I) U) -H •,  (ii -I-) 4-I ci) H —  >1 .  4) H cr1 Cl)  • C41 C1 •  b •1-4  76  110  c,) 100 -J 0  o  80  F  z 0 W 70  >-  z  60  w 50 0  1  2  3  4  5  6  7  INOCULUM CONCENTRATION (log cfu/g soil) Fig. 23. Mean dry weight of root systems per plant grown from seed piece in soil inoculated with Erwinia carotovora subsp. atroseptica strain 31 at cfu/g soil compared to control. 3 concentrations of 1.1, 110, and 11000 x 1 O The plants were grown at 18°C for 6 days in pots in a greenhouse. Dry weight was determined after drying at 8cfC for 24 h. Bars represent standard error of means of 16 replicates.  77  12  C.)  -  -  H  z  -  LU H Cl) LL  0  9-  H  (9 Z LU -J  8-  LU  7-  z  6  i  0  1  2  3  4  5  6  7  INOCULUM CONCENTRATION (log cfu/g soil) Fig. 24. Mean length of stem per plant grown from a seed piece in soil inoculated with Erwinia carotovora subsp. atroseptica strain 31 at concentrations of 1.1, 110, and 11000 x 1 O cfu/g soil compared to control. 3 The plants were grown at 18°C for 6 days in a greenhouse. Bars represent standard error of means of 16 replicates.  78 Stem lengths were significantly shorter in treatments with Eca 31 at both 1.1 x iO,  and 1.1 x iO cfu/g soil than that of  the control after 6 days of growth were 95,  63,  (Fig.  24)  .  The stems lengths  and 62% of the controls at 1.1 x iO,  1.1 x iO cfu/g soil,  respectively.  1.1 x iO,  and  The number of leaves per  plant became smaller as the inoculum concentrations increased (Fig. 25)  .  No other visible foliage symptoms were observed.  DISCUSSION  The inhibition of root elongation of cv. Russet Burbank growing in soil inoculated with Eca 31 suggested that the same phenomenon of root growth inhibition observed in vitro also can occur in soil and the factor inhibitory to root growth was produced by the bacteria in soil environments.  Inhibition of  root elongation and occurrence of short stems in the inoculated soils suggest that tissue culture systems can be used reliably as a model system for studying symptoms of diseased plants infected by soft rot erwinias. subsp.  However the response of plants to E.  c.  atroseptica in soil was less uniform than that of  micropropagated plantlets.  The greatest variations of the  response in both stem and root in soil occurred in treatments with an inoculum concentration of 1.1 x iO cfu/g soil.  The inhibition of root elongation was related to the  79  6.5  z  cC  -J  0 C’) U]  6-  > cC U]  -J  U  0  5.5  -  U] D  z z  cC  5-  U]  4.5  I  I  I  I  I  I  I  I  0  1  2  3  4  5  6  7  INOCULUM CONCENTRATION (log cfu/g soil) Fig. 25. Mean number of leaves per plant grown from a seed piece in pot soil inoculated with Erwinia carotovora subsp. atroseptica strain 31 at concentrations of 1.1, 110, and 11000 x 1 O cfu/g soil compared to control. 3 The plants were grown at 18°C for 6 days in a green house. Bars represent standard error of means of 16 replicates.  80 reduction in dry weight of the root systems since both were significantly different from the control at inoculum concentrations higher than 1.1 x iO cfu/g soil.  However,  the  root elongation was also inhibited at a higher inoculum concentration but the dry weight was not. difference is not known. weight,  stem length,  The reason for this  The fact that a sharp reduction in dry  and number of leaves occurred when inoculum  concentration increased from 1.1 x iO to 1.1 x iO cfu/g soil suggested that the inoculum concentration of 1.1 x iO cfu/g soil played a crucial role in causing a significant effect on the development of potato plants by E.  c.  subsp.  atroseptica by this  method of inoculation.  The frequent watering to maintain high levels of water potential in pot soils met the requirement for the better survival of E.  c.  subsp.  atroseptica.  Seed piece decay  frequently occurred in treatments with inoculum concentrations higher than 1.1 x iO cfu/g soil. occur in any of the treatments.  However,  non—emergence did not  This might be due to the fact  that the seed pieces were sprouted before inoculation and they survived to produce a plant before being decayed by Eca 31.  Profound root growth inhibition frequently occurred in plants with decaying seed pieces.  Large numbers of bacteria were  released from the decaying seed pieces into the rhizosphere, resulting in inhibition of root elongation.  The inhibited  81 root development could in turn limit the nutrient uptake by roots This speculation is  resulting in stunting and wilting of plants. strengthened by the findings of Lapwood et al.  (1985)  who  c. subsp.  reported that seed tubers inoculated with E.  atroseptica produced plants with less vigour and reduced yield.  c. subsp. atroseptica  Stunting of potato plants caused by E.  I observed that a  was quite dramatic after 6 days of growth.  large number of the small plants contributed to the significant reduction in average stem length and the length of stems became more uniform as the inoculum concentration increased.  These  small plants usually had rotted seed pieces attached.  My results  confirm the findings by Rhodes and Logan Gans  (1984)  and Lapwood and  (1986)  who reported that seed tubers inoculated with E.  carotovora produced small plants in the field.  Three soft rot erwinias cause major bacterial diseases in potato and most seed stocks are contaminated by more than one strain of them  Blackleg is a  (Perombelon 1972; Nielson 1978).  widespread disease and is found in all potato—producing areas of the world  (Peltzer and Sivasithamparam 1985)  inoculated the seed tubers with 106 E.  .  Bain et al.  (1990)  c. subsp. atroseptica  cells per tuber at planting time and found that yield losses could be as high as 60% when the inoculated tubers were grown under conditions favourable for blackleg.  Yield of symptomless  potato plants grown from seed inoculated with E.  c. subsp.  82  atroseptica decreased as much as 25% in comparison to that of uninoculated plants and Schippers  (1987)  (Perombelon and Hyman 1987,  1988).  Bakker  proposed that potato yield reduction in  short potato—rotation soils may originate partially from impaired nutrient uptake brought about by depressed root cell energy metabolism caused by harmful microorganisms.  My experimental  results revealed that both root elongation and dry weight were significantly inhibited by E.  c. subsp. atroseptica in soil.  Potato yield reduction in both diseased and symptomless plants might be the result of inhibited root growth by the erwinias.  This study showed that root length was shorter and root dry weight decreased as inoculum concentrations increased. al.  (1990)  Bain et  reported that blackleg incidence and tuber yield were  correlated with the population of E. seed tubers.  c. subsp. atroseptica on  The correlation between potato yield reduction and  inhibition of root elongation and dry weight in response to the increase in inoculum dose in different experiments suggested that the yield reduction in potato caused by soft rot erwinias might be related to the inhibited root growth.  83 CHAPTER 5  -  CHABACTERIZATION OF A FACTOR RESPONSIBLE FOR INHIBITION OF ROOT ELONGATION  INTRODUCTION  The inhibition of energy metabolism of root cells may be one mechanism of the microbial inhibition of root growth in potato (Bakker and Schippers 1987).  The inhibition may be due to the Most of  action of metabolites from rhizosphere microorganisms. the metabolites are N—containing heterocyclic compounds, these are phenazine and indole derivatives Margraff 1979).  among  (Leisinger and  Cytochrome oxidase respiration of roots is  inhibited at least 40% by cyanide at a concentration of 4 jIM (Schippers et al.  1985)  .  Cyanide is known to be a secondary  metabolite of many microbes,  including pseudomonads.  Other known  secondary metabolites antagonistic to root growth are some unusual amino acids and peptides  (Schippers et al.  1987).  Although the metabolites from soft rot erwinias that affect root growth have not been identified,  those from some other  bacterial species were shown to inhibit root growth of the plants.  Fredrickson and Elliott  (1985)  demonstrated that a toxin  produced by root-colonizing pseudomonads significantly inhibits winter wheat seedling root growth.  Indole acetic acid  (IAA)  from  Azospirillum brasilense was reported to inhibit root elongation  84 (Harari et al.  of Panicum miliaceum  Taylor and Secor  (1985)  filtrates produced from 5, E.  c.  subsp.  1988).  studied the effect of culture  11,  and 18-day—old shake cultures of  carotovora on the survival and growth of protoplast—  derived potato calli and found that both growth and survival were inhibited by all filtrates.  Stephens and Wood  (1975)  showed that  crude dialysed extracts from rots of cucumber fruit infected with E.  carotovora caused cell separation and death of protoplasts in  normal and plasmolysed tissues.  They further tested different  factors purified from rot extracts and found that pectate trans— eliminase, proteinase and phosphatidase in crude extracts killed protoplasts of normal tissue, but not those in unpiasmolysed tissue.  A number of bacterial metabolites with different physical, chemical,  and biochemical properties exhibit pathogenic effects  on tissue cultured cells as well as causing root inhibition in plants.  Therefore,  a series of experiments was designed in this  study to characterize the factor responsible for inhibition of root elongation of potato plantlets by E.  c.  subsp.  atroseptica.  MATERIALS AND METHODS  PETRI DISH BIOASSAY.  A piece of sterile filter paper was  transferred to a sterile Petri—dish and 4 mL of the treatment  85 solution were added.  Five cuttings were laid on the filter paper  pad inside the Petri—dish which was sealed completely with parafilm.  All these procedures were carried out in a  flowhood  and aseptic techniques were used throughout the experiments.  The treatments were incubated at 23°C for 11—12 days in a growth chamber with 16 h photoperiod,  under the light intensity  1 provided by 8 F48 T12/CW/HO Phillips s 2 of 196.7 lIE m fluorescent tubes and four 25 W light bulbs.  The incandescent  lights were on half an hour before the fluorescent lights and half an hour after they were off.  After the incubation period,  the length of the maximum root and number of roots per cutting were measured.  The stem lengths were also recorded as reference  for each treatment. sampling.  Plant symptoms were recorded during  Multiple comparisons of the treatment means were made  by using Tukey’s multiple range test.  SUPERNATANT PRODUCTION AND TESTING.  A single colony of Eca  31 showing the typical pitting characteristics on CVP was selected to inoculate two nutrient agar slants.  Two—day old  nutrient agar slant cultures were used to inoculate 100 mL sterile nutrient broth to give an absorbance value of 0.03 at 660 nm in a spectrophotometer  (Spec 20)  .  After growth at 23°C for 5  days in shake culture, bacterial cells were pelleted by centrifugation at 8000 g for 20 mm.  The supernatant was  collected and sterilized by filtering through 0.2 j.m pore size  86 filter.  The sterile supernatant was diluted three—fold in sterile nutrient broth and a dilution series containing 33.3 %, 3.7 %,  and 1.2 % supernatant was obtained.  preparations,  sterile nutrient broth,  11.1 %,  The supernatant  and water were each  combined with an equal volume of double—strength liquid MS medium and mixed thoroughly.  The following concentrations of the  supernatant in the final mixtures were obtained as individual 50 %  treatments, 0.6 %,  and 0 %  three times.  (bacterial supernatant),  (nutrient broth).  16.7 %,  5.6 %,  1.9 %,  Each treatment was replicated  Five nodal cuttings were used in each replicate.  HEAT TREATMENT.  The same method was adopted for the  production of bacterial supernatant in all subsequent characterization procedures.  The filter—sterilized supernatant  in test tubes was heated to 100°C for 15 mm.  and sterilized  again through a 0.2 p.m pore size filter after cooling to room temperature.  In addition,  the unheated bacterial supernatant and  nutrient broth were also filter—sterilized twice.  The heated and unheated supernatant,  nutrient broth,  and  sterile distilled water each was mixed thoroughly with an equal volume of double-strength MS medium to obtain four treatments. Three replicates in each treatment and six nodal cuttings in each replicate  (Petri dish)  were used in the “Petri dish bioassay”.  87 DIALYSIS.  Standard cellulose dialysis tubing  diameter; mol wt cutoff: Dialysis tubing pieces  12,000—14,000)  was used for dialysis. were boiled in  (approximately 30 cm long)  Filter—  distilled water and cooled to room temperature. sterilized supernatant  (25 mL)  (35 mm in  was transferred to a dialysis bag  and dialysed against 4 L sterilized nutrient broth at 4°C for 24 h.  The dialysis medium was changed once.  The dialysed solution  was filter sterilized again.  Four treatments, supernatant,  consisting of dialysed and undialysed  nutrient broth,  the “Petri dish bioassay”.  and distilled water,  were tested in  Five replicates in each treatment and  5 nodal cuttings in each Petri dish were used.  FRACTIONATION WITH ANMONIUM SULPHATE.  Ammonium sulphate was  added to a flask containing 100 mL supernatant to give 70 % saturation.  This solution was maintained in a cooler at 4°C for  2 hours with constant stirring.  The precipitate was pelleted by  centrifugation at 8000 g for 20 minutes. supernatant were retained.  Both pellets and  The precipitate in the original flask  and the pellets in the centrifuge tubes were rinsed four times and finally dissolved in 50 mL phosphate buffer  (pH 7.3)  .  A 100  mL sample of nutrient broth was given exactly the same treatment. The above two fractions were the first fractions of bacterial supernatant and nutrient broth.  The second and the third  fractions were obtained one after another in the same manner by  88 adding ammonium sulphate to the remaining liquid to give 85% and 95% saturation.  The final remaining liquid was the fourth  fraction.  The residual ammonium sulphate in each fraction was eliminated by dialysis at 4°C against phosphate buffer for 36 hours for fraction 1 and 2,  and for 60 hours for fraction 3,  (nutrient broth and bacterial supernatant). in each container was changed every 12 hours.  4  The phosphate buffer All dialysed  fractions were filter sterilized.  The dialysed fractions were tested for their effect on root elongation of nodal cuttings by the “Petri dish bioassay”. Eleven treatments,  4 replicates in each treatment,  and 6 nodal  cuttings in each replicate were used.  RESULTS  SENSITIVITY TEST.  The culture supernatant of Eca 31 grown  in nutrient broth for 5 days inhibited root elongation of stem cuttings of micropropagated potato plantlets of cv. Kennebec. Inhibition of root elongation was proportional to the log supernatant concentration  (Fig.  26).  Regression analysis showed  that the treatment effect on root length of nodal cuttings fitted  89  55 0  z D C) I—  40 Lii  35 Cl) Cl)  30  0  25 U  0 20 LU  15 0.5  0  1  1.5  2  LOG SUPERNATANT CONCN. (%) Fig. 26. Effect of supernatant concentrations on root elongation of cuttings of cv. Kennebec. Supernatant was produced from 5-day old nutrient broth cultures of Erwinia carotovora subsp. atrosetica strain 31 at 2’C with constant shaking.  90 a straight line having a coefficient of determination of 0.95 which indicated a good fit  (Fig. 26)  Root elongation in the  .  treatment with the highest supernatant concentration was inhibited 59% compared to the control.  HEAT TREATMENT.  The mean root length in treatment with  heated bacterial supernatant was not significantly different from those of both nutrient broth and unheated supernatant, but was (Fig.  significantly lower than that of water control  27)  .  The  effect of nutrient broth and water on root elongation was not significantly different.  DIALYSIS.  The dialysed bacterial supernatant inhibited root  elongation to an extent similar to that of the undialysed supernatant  (Fig.  28),  indicating that the root inhibitory factor  was retained in the dialysis tubing.  ANMONIUM SULPHATE FRACTIONATION. significantly affect root elongation  Phosphate buffer did not (Fig.  29)  .  The root  inhibitory factor was precipitated by ammonium sulphate at 70% saturation only  (Fig.  30).  Four fractions of nutrient broth did  not significantly inhibit root elongation  (Fig.  30).  91  6  C.)  5  0  z  D  Q  4  0 ><  0 z I— 0 z uJ  3  2  -J  z  1  uJ 0 WATER  NB  BS(H)  BS  TREATMENT Fig. 27. Effect of heated bacterial supernatant on root growth of nodal cuttings of cv. Kennebec. Bacterial supernatant of Erwinia carotovora subsp. atroseptica strain 31 was heated to 100°C for 15 mm. Means (n=1 8) (bars) having the same letter do not differ significantly (p=0.05) according to Tukey’s multiple range test. NB, BS, and BS (H) represent nutrient broth, bacterial supernatant and heated supernatant, respectively.  92  5 C.)  0  z  1::  4  C) 0 0  3  x U  0  2  z  I— 0  z w -J  z  1  w 0 WATER  NB  BS  BS(D)  TREATMENT Fig. 28. Effect of dialyzed bacterial supernatant on root elongation of nodal cuttings of cv. Kennebec. The supernatant of Erwinia carotovora subsp. atroseDtica strain 31 was dialyzed against nutrient broth at  .  C for 24 h.  Means (n=25) (bars) having the same letter do not differ significantly (p=0.05) according to Tukey’s multiple range test. NB, BS, and BS (D) represent nutrient broth, bacterial supernatant, and dialyzed bacterial supernatant, respectively.  93  6 C.)  CD  z  5  D 0 0  cr ><  3 U  0  ICD  z w -J z w  2  0 SUPERNATANT  BROTH  BUFFER  CONTROL TREATMENTS Fig. 29. Effect of bacterial supernatant of Erwinia carotovora subsp.  atroseptica strain 31 on root elongation of nodal cuttings of cv. Kennebec. The supernatant was produced from 5-day old nutrient broth cultures at 2 C with  constant shaking. Means (n=24) (bars) having the same letter do not differ significantly (p=0.05) according to Tukey’s multiple range test.  94 E 0  04  0  W  -4  ZI  z  —Il  C)  w  NB  NB  TREATMENT  TREATMENT  I! LI  0  02  z I-  I-  o z  z  Wi  Lii  z  z  0  BS  NB  BUFFER  TREATMENT  BS  NB  BUFFER  TREATMENT  Fig. 30. Effect of four fractions of bacterial supernatant on root elongation of nodal cuttings of cv. Kennebec. The supernatant (BS) and nutrient broth (NB) were fractionated with ammonium sulphate at 70, 85, and 95% saturation, resulting in three fractions designated as Fraction 1, Fraction 2, and Fraction 3, respectively. Fraction 4 is the remaining fraction. Means (n=24) (bars) for each fraction having the same letter do not differ significantly (p=0.05) according to Tukey’s multiple range test.  95 DISCUSSION  Root elongation of cuttings was linearly inhibited in response to increasing bacterial supernatant concentration.  This  However  trend was similar to that observed in pot experiments.  the plantlets grown in inoculated medium had a different pattern of root length reduction in response to varying inoculum levels. One explanation for the discrepancy is the fact that, dish experiments, reduced gradually,  in Petri  the amount of root inhibitory substances may be However in the  due to biological degradation.  inoculated medium bacteria were present to continually exert their effect and produce metabolites.  Moreover,  influences of bacterial growth in vitro, for nutrients,  decreasing  02  other indirect  for example,  competition  concentration in the medium,  change in pH, might also affect root growth.  and the  The observed root  growth inhibition by the bacterial supernatant was caused mainly by the toxic metabolite(s)  rather than other indirect factors.  This difference might also contribute to the different results obtained by the medium inoculation method and the “Petri dish . t bioassay’  Although plant growth substances of bacterial origin are known to inhibit root growth of many plant species Schroth 1986;  Harari et al.  (Loper and  1988), the results of this study  indicated that the factor inhibiting root elongation of micropropagated potato plantlets was not a phytohormone.  It was  96 retained in the dialysis bag, molecular weight molecule  indicating that a relatively large  (>12000—14000)  was involved.  In  the fact that the effect of heated bacterial  addition,  supernatant on root elongation was not significantly different from those of nutrient broth and unheated bacterial supernatant led to three possible explanations about the nature of the inhibitory factor.  The first,  two molecules were involved and  one was destroyed by heat treatment; the second, partially destroyed; the third,  one molecule was  the heat destruction of the  inhibitory factor was reversible in the bioassay.  Although the first supernatant fraction significantly inhibited root growth,  the second fraction may also have some  inhibitory effect on root elongation  (Fig.  30)  .  It is possible  that the saturation point of ammonium sulphate for the first fraction was not high enough to precipitate all of the  root  inhibitory factor in the supernatant.  Since the root inhibitory factor was partially destroyed by heat,  retained in the dialysis tubing,  and precipitated by  ammonium sulphate at 70% saturation which included most protein molecules, Perhaps,  the factor may have a protein or a peptide moiety.  the active molecule is a glycoprotein or a lipoprotein.  97 CHAPTER 6  —  GENERAL DISCUSSION  This study has established that various symptoms can be induced by soft rot erwinias in micropropagated potato plantlets as well as in potato plants grown in the artificially inoculated soil.  Similarity in the symptoms expressed by the potato plants  grown in both systems existed.  Moreover,  a compound from E.  carotovora which inhibited root elongation of the stem cuttings  of the micropropagated potato plantlets was characterized by using the “Petri dish bioassay” system.  Inhibition of root elongation and stunting of plants in both in vitro and soil environments  (established only for Eca 31)  was  the most common effect demonstrated by the soft rot erwinia strains,  although the effects on stem growth by two of seven  strains tested were not significant.  The consistency of the  effect of root growth inhibition exhibited by soft rot erwinias in both systems indicate that the production and secretion of a root growth inhibitory factor were not significantly affected by the different growth environments of the pathogen.  Although  field experiments to determine if root growth inhibition occurs to field potato crops by soft rot erwinias are still necessary, the inhibitory effect could happen if the different conditions between the field and green house did not significantly influence the production and function of the inhibitory factor in its  98 interaction with root growth.  Soft rot erwinias are well known  to associate with potato crops wherever they are grown and the root systems are extensively covered with the bacteria and Sivasithamparam 1985; Kloepper 1983).  (Peltzer  A combination of all  these factors will result in root growth inhibition in the field plants.  Although stunting occurred in both systems,  This conclusion  causing the recorded effect may not be the same. was based on my observation that,  the factors  in the tissue culture system,  the cuttings and roots were often covered with  bacterial ooze  which definitely affected the nutrient uptake by the cuttings, perhaps contributing to a certain extent to the stunting of plantlets.  In my pot experiments,  stunting and dramatic  inhibition of root elongation appeared to be associated with seed piece decay.  If it is assumed that the nutrient deficiency of  plants was the cause of small plants, then, result of inhibited root elongation.  stunting may be the  However,  we can not ignore  another possibility that a toxin from soft rot erwinias could be involved in the induction of the retarded stem growth at the same time.  The other minor symptoms induced in micropropagated potato plantlets by the pectolytic erwinia strains, root formation,  root tip browning and blackening,  lesions on foliage,  rotting and  were not evident in potato plants growing in  soil inoculated with E. growth.  including aerial  c. subsp. atroseptica after 6 days of  This discrepancy in symptom expression in potato in  99 different systems could be the result of the different interactions and responses of the plants and the bacteria to each other.  The results of my study provide a possible solution to the unknown cause of the well established yield reduction of potato crops growing in short potato—rotation soil in comparison to long—potato rotation soil.  In the Netherlands, the analysis of  the cause of yield reduction in short potato rotation reached the conclusions that the increased potato cropping frequency promoted microbial activities harmful to potato root growth and a unknown microbial factor was involved in yield reduction al.  1985)  .  (Schippers et  A bioassay carried out in the greenhouse by using  potato stem cuttings revealed that root growth was inhibited within two weeks in short potato—rotation soil in comparison to long potato—rotation soil Schippers  (1987)  (Bakker et al.  1987)  .  Bakker and  discovered that potato root growth was very  sensitive to cyanide produced by soil fluorescent pseudomonads and that 5 pM HCN inhibited cytochrome oxidase respiration by at least 40 % in intact potato roots in vitro.  My results obtained  from both in vitro and soil experiments indicate that roots of potato plants can be dramatically inhibited by soft rot erwinia strains.  In considering the fact that the pectolytic Erwinia  spp. are widely distributed in the potato production areas of the world and most seed stocks are contaminated by more than one strain of these  (Perombelon 1972; Nielson 1978),  it is very  100 likely that soft rot erwinias are responsible for the yield reduction by directly inhibiting root growth.  Plant growth promotion by tuber bacterization with fluorescent pseudomonads has been attributed to the suppression of growth inhibiting rhizosphere microorganisms 1985;  Geels and Schippers 1983)  reported that E.  Some of these pseudomonads are  (Rhodes and Logan 1986).  antagonistic to erwinias (1983)  .  (Schippers et al.  Kloepper  carotovora is a common root zone  inhabitant of potato and seed tuber bacterization with fluorescent pseudomonad PGPR strains,  which were previously  demonstrated to increase significantly potato yield in field tests,  causes significant reduction of root zone population of E.  carotovora. of E.  Rhodes and Logan  (1986)  demonstrated that the effect  carotovora on the development of small plants and the  incidence of blackleg were partially reversed by treatments with fluorescent pseudomonads and suggested that the enhanced plant growth may be due to a reduction in numbers and activity of E.  carotovora.  Furthermore,  based on my results,  I suggest that the  promotion of plant growth is accounted for by the reversal of the root growth inhibition resulting from the growth suppression of soft rot erwinias on plant roots by antagonistic pseudomonads, resulting in an increase in tuber production.  Further  experiments are still necessary to be certain that the growth promoting pseudomonads can alleviate the inhibition of root elongation by soft rot erwinias in soil environments.  101 Nevertheless, my experiments provide a possible new solution to the mechanism of plant growth promotion by fluorescent pseudomonads.  My experiments have demonstrated that root growth of potato plants growing in both in vitro and in soil was inhibited by soft rot erwinias.  Moreover,  it is a bacterial metabolite of Eca 31  that inhibits root growth of potato plants.  I recognize another  possible role of soft rot erwinias acting as a deleterious rhizosphere microorganism inhibiting root growth by a toxic metabolite without parasitizing root systems.  At the same time,  pectolytic erwinias may also function as a pathogen inside the plants causing foliage symptoms.  This assumption is supported by  my electron microscopic finding that a large bacterial population was present on the root surface of the micropropagated potato plantlets.  Moreover,  staining techniques,  by using immunofluorescent and irnmunogold Underberg and Van Vuurde  large populations of E. grown potato plants.  (1989)  detected  chrysanthemi on root surface of field  The toxic metabolite from the pectolytic  Erwinia spp. may serve as a “pathogenicity factor” to induce  disease in potato.  Furthermore,  it would be very interesting to  know whether the metabolites from other plant pathogenic bacterial species inhibit root growth.  Nevertheless root growth  inhibition would be recognized as a new symptom of the diseased potato plants by soft rot erwinias, field experiments.  once it was confirmed by  102 The symptoms in micropropagated plantlets caused by soft rot erwinias have been documented extensively.  The symptom  description will be very helpful for identifying and screening out the contaminated potato tissue cultures in research and commercial laboratories where bacteria and bacteria—like contaminants are a big problem  (Debergh and Vanderschaeghe 1988).  This study focused on the response of micropropagated potato plantlets to a particular bacterial species in vitro in order to better understand the in vitro capacity of a pathogen in causing a disease.  The comparison of symptoms induced by soft rot  erwinias in vitro and in soil environments indicates the extent to which tissue culture systems could be used to study the diseases of the plants in soil.  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