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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 MICROPROPAGATEDPOTATO PLANTLETS AND GREENHOUSE PLANTS ANDCHARACTERIZATION OF A FACTOR INHIBITING ROOT ELONGATIONbyXUESONG LANB.Sc., The Heilongjiang August First Land ReclamationUniversity ( People’s Republic of China), 1984A THESIS SUBMITTED IN PARTIAL FULFILMENT OF THE REQUIREMENTSFOR THE DEGREE OF MASTER OF SCIENCEinTHE FACULTY OF GRADUATE STUDIES(Department of Plant Science)We accept this thesis as conforming—the required standardTHE UNIVERSITY OF BRITISH COLUMBIAJune 1992© Xuesong Lan, 1992In presenting this thesis in partial fulfilment of the requirements for an advanceddegree at the University of British Columbia, I agree that the Library shall make itfreely available for reference and study. I further agree that permission for extensivecopying of this thesis for scholaily purposes may be granted by the head of mydepartment or by his or her representatives. It is understood that copying orpublication of this thesis for financial gain shall not be allowed without my writtenpermission.Department of Pt C-I € fl L.The University of British ColumbiaVancouver, CanadaDate____________DE-6 (2/88)iiABS TRACTSymptoms induced by soft rot erwinias were observed inmicropropagated potato plantlets of cultivar Russet Burbank,Teton, and Urgenta grown from nodal cuttings inoculated withErwinia carotovora subsp. atroseptica strain 31 (Eca 31) inpreliminary experiments. The development of short roots,stunting, and yellowing in inoculated plantlets was evident after25—30 days of growth in a tissue culture growth chamber.Two strains from each of E. c. subsp. atroseptica andcarotovora, and E. chrysanthemi were tested individually fortheir effect on the growth of micropropagated potato plantlets bya medium—inoculation method. All strains tested significantlyinhibited root growth of micropropagated plantlets of cv.Kennebec. Most strains, except for E. c. subsp. atrosepticastrain 196 and E. c. subsp. 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 lesionswhich appeared as black necrotic dots also occurred, although thefrequencies were low within each experiment.Root growth inhibition, stem length reduction and severityof yellowing increased as the inoculum concentration increased.Root elongation was dramatically inhibited by Eca 31 at inoculumconcentrations between 0.2—20 colony forming units (cfu)/mL iniiitissue culture growth medium. All four cultivars Kennebec,Russet Burbank, Red Pontiac, and Red Lasoda, responded similarlyto the low bacterial concentration with respect to stem length,but differently to the high inoculum concentration. The roots ofcv. Red Lasoda were more susceptible to Eca 31 at 0.2 cfu/mL, butwere more resistant at 20 cfu/mL than those of the three othercultivars. The concentration of bacterial antigens at stem endsof the four cultivars increased directly with an increase ininoculum concentration as indicated by the indirect enzyme—linkedimmunosorbent assay (ELISA) . Electron microscopy revealed that alarge population of Eca 31 cells covered the epidermal surface ofthe root, and that a large number of bacteria was present in theintercellular space of root epidermal cells and in the vascularbundles of roots.The inhibition of root elongation and reduction in root dryweight occurred in potato plants grown from seed pieces in soilinoculated with E. c. subsp. atroseptica after 6 days of growthat 18°C in a greenhouse. The roots were inhibited by up to 50%at an inoculum concentration of 1.1 x iO cfu/g soil. Stuntingoccurred, but no other foliage symptoms were observed.The root inhibitory factor was shown to be present inbacterial supernatant of 5-day old nutrient broth cultures of Eca31. The inhibition of root elongation was directly correlatedwith supernatant concentration in a “Petri dish bioassay”. Theivfactor responsible for root inhibition was relatively resistantto heat, of high molecular weight (> 12000—14000),and precipitable by ammonium sulphate at 70% saturation.VTABLE OF CONTENTSPAGEAbstract iiTable of Contents vList of Tables viList of Figures viiAcknowledgement xiCHAPTER1. General Introduction 12. Symptoms Caused by Three Soft Rot Erwinias andOther Bacterial Species in Micropropagated PotatoPlantlets 123. Response of Micropropagated Potato Plantlets toErwinia carotovora subsp. atroseptica 434. Effect of Erwinia carotovora subsp. atrosepticaon Potato Plants Grown in Soil 695. Characterization of a Factor Responsible forInhibition of Root Elongation 836. General Discussion 97Literature Cited 103viLIST OF TABLESTABLE PAGE1. Bacterial cultures used in the study of symptomexpression of micropropagated potato plantlets ofcv. Kennebec 152. Effect of Erwinia carotovora subsp. atrosepticastrain 198 on micropropagated potato plantletsof cv. Kennebec 203. Effect of Erwinia carotovora subsp. atrosepticastrain 196 on micropropagated potato plantletsof cv. Kennebec 214. Symptoms of micropropagated potato plantlets ofcv. Kennebec caused by Erwinia carotovora subsp.carotovora strain 21 235. Symptoms of micropropagated potato plantlets ofcv. Kennebec caused by Erwinia carotovora subsp.carotovora strain 71 246. Effect of Erwinia chrysanthemi strain 573 onmicropropagated potato plantlets of cv. Kennebec 257. Effect of Erwinia chrysanthemi strain 574 onmicropropagated potato plantlets of cv. Kennebec 268. Comparison of symptoms expressed by micropropagatedpotato plantlets of cv. Kennebec caused byCorynebacterium sepedonicum and Erwinia carotovoraatroseptica strain 31 279. Effect of Pseudomonas marginalis and Erwiniaamylovora on micropropagated potato plantlets ofcv. Kennebec 2910. Comparison of response of micropropagated potatoplantlets of cv. Kennebec, Russet Burbank, RedPontiac, and Red Lasoda to Erwinia carotovora subsp.atroseptica strain 31 55viiLIST OF FIGURESFIGURE PAGE1. Root length and stem length of micropropagatedpotato plantlets of cv. Kennebec grown for21 days on Murashige and Skoog’s mediuminoculated with Erwinia carotovora subsp.atroseptica strain 31 (right) in comparison tothose of control (left) 302. Root symptoms on plantlets of cv. Kennebecafter 26 days of growth. A, Browning andblackening of root tips caused by Erwiniachrysanthemi strain 573. B, Aerial root formationinduced by Erwinia carotovora subsp.carotovorastrain 71 313. Stem symptoms on plantlets of cv. RussetBurbank growing for 30 days on Murashige andSkoog’s medium inoculated with Erwinia chrysanthemistrain 576. A,Blackening, yellowing, and blacknecrotic dots on stems. B, Rotting of stem tips 334. Leaves of micropropagated potato plantlets of cv.Kennebec after 26 days of growth. A, Leaves of thecontrol plantlets. B, Leaf necrotic spots, rot, andyellowing developed in treatments inoculated withErwinia carotovora subsp. atroseptica strain 22 345. Black necrotic dots on leaf of micropropagatedpotato plantlets of cv. Russet Burbank grown for30 days on Murashige and Skoog’s medium inoculatedwith Erwinia chrysanthemi strain 576 356. Curling of leaves of micropropagated potatoplantlets of cv. Kennebec after 26 days of growthon Murashige and Skoog’s medium inoculated withErwinia carotovora subsp. atroseptica strain 31 367. Leaf tip necrosis of plantlets of cv. Kennebecafter 26 days of growth on Murashige and Skoog’smedium inoculated with Erwinia chrysanthemi strain576 378. Mean of maximum root length of plantlets ofcv. Russet Burbank (RB) and Red Pontiac (RP) grownfor 18 days on Murashige and Skoog’s mediuminoculated with Erwinia carotovora subsp.atroseptica strain 31 49viiiFIGURE PAGE9. Mean number of roots per plantlet grown for 18days on Murashige and Skoog’s medium inoculatedwith different concentrations of Erwiniacarotovora subsp. atroseptica strain 31 5010. Mean length of stems of micropropagated potatoplantlets of cv. Russet Burbank (RB) and RedPontiac (RP) grown for 18 days on Murashige andSkoog’s medium inoculated with differentconcentrations of Erwinia carotovora subsp.atroseptica strain 31 5111. Mean number of leaves of plantlets grown for 18days on Murashige and Skoog’s medium inoculatedwith different levels of Erwinia carotovora subsp.atroseptica strain 31 5212. Severity of yellowing of plantlets of cv. RussetBurbank (RB) and Red Pontiac (RP) grown for 18days on Murashige and Skoog’s medium inoculatedwith different concentrations of Erwinia carotovorasubsp. atroseptica strain 31 5313. Response in stem length of plantlets of cv.Russet Burbank to different concentrations ofErwinia carotovora subsp. atroseptica strain 31 5614. Severity of yellowing of plantlets of cv. RussetBurbank in response to different inoculumconcentrations of Erwinia carotovora subsp.atroseptica strain 31 5715. Effect of different inoculum concentrations ofErwinia carotovora subsp. atroseptica strain 31on root elongation of plantlets of cv. RussetBurbank 5816. Mean absorbance values (A405nm) for enzyme—linkedimmunosorbant assay conducted on stem end portionsof plantlets of cv. Kennebec (D), Russet Burbank(+), Red Pontiac (0), and Red Lasoda () inResponse to different inoculum concentrations ofErwinia carotovora subsp. atroseptica strain 31 6017. Electron micrograph of root section of cv. RedPontiac grown for 26 days on inoculated Murashigeand Skoog’s medium. A huge population ofbacterial cells of Erwinia carotovora subsp.atroseptica strain 31 covered on rootsurface (X 8750) 61ixFIGURE PAGE18. Electron micrograph of longitudinal section of aroot nearthe edge showing the presence ofbacterial cells of Erwinia carotovora subsp.atroseptica strain 31 in intercellular spacesbetween the epidermal cells. Plantlets ofcv. Red Pontiac grown for 26 days on Murashigeand Skoog’s medium (X 3750) 6219. Electron micrograph of cross section of a root ofcv. Red Pontiac grown for 26 days on Murashige andskoog’s medium inoculated with Erwinia carotovorasubsp. atroseptica strain 31. Bacterial cells invascular tissue of the root (X 10000) 6320. Electron micrograph of cross section of upperportion of a root of cv. Red Pontiac grown for26 days on Murashige and Skoog’s medium inoculatedwith Erwinia carotovora subsp. atroseptica strain31. Bacterial cells present inside a root cell(X 7500) 6421. Mean of maximum root length per plant grown insoil inoculated with Erwinia carotovora subsp.atroseptica strain 31 at concentrations of 1.1,110, and 11000 x i0 cfu/g soil compared tocontrol 7422. Healthy (left) and diseased (right) plants of cvRusset Burbank after 6 days of growth at 18°C ina greenhouse. The seed piece was grown in soilinoculated with Erwinia carotovora subsp.atroseptica strain 31 at 1.1 x iO cfu/g soil 7523. Mean dry weight of root systems per plant grownfrom a seed piece in soil inoculated with Erwiniacarotovora subsp. atroseptica strain 31 atconcentrations of 1.1, 110, and 11000 x iO cfu/gsoil compared to control 7624. Mean length of stem per plant grown from a seedpiece in soil inoculated with Erwinia carotovorasubsp. atroseptica strain 31 at concentrations of1.1, 110, and 11000 x 103 cfu/g soil compared tocontrol 7725. Mean number of leaves per plant grown from a seedpiece in pot soil inoculated with Erwiniacarotovora subsp. atroseptica strain 31 atconcentrations of 1.1, 110, and 11000 x iO cfu/gsoil compared to control 79xFIGURE PAGE26. Effect of supernatant concentrations on rootelongation of cuttings of cv. Kennebec 8927. Effect of heated bacterial supernatant on rootelongation of nodal cuttings of cv. Kennebec 9128. Effect of dialysed bacterial supernatant on rootelongation of nodal cuttings of cv. Kennebec 9229. Effect of bacterial supernatant of Erwiniacarotovora subsp. atroseptica strain 31 on rootelongation of nodal cuttings of cv. Kennebec 9330. Effect of four fractions of bacterial supernatanton root elongation of nodal cuttings of cv.Kennebec 94xiACKNOWLEDGEMENTSThe author wish to express his sincere appreciation to Dr. SolkeH. De Boer for his guidance, valuable suggestions, andencouragement given during the research and writing of thisthesis.I also thank Dr. R. J. Copeman, Department of Plant Science, UBC;Dr. I. E. P. Taylor, Department of Botany, UBC; and Dr. P. M.Townsley, Department of Food Science, UBC for serving on mycommittee.Sincere thanks are extended to Mr. D. Kirkham, Mr. K. Turner andMrs. E. Sela for supplying disease-free potato tissue culturematerials and allowing me to use their Laboratory facilities.I extend my thanks to Dr. F. Leggett for preparing the electronmicrographs; and Mr. W. MacDiarmid for taking photographs ofinoculated micropropagated potato plantlets.I also thank Dr. J. W. Hall for his suggestions in analyzingdata; Dr. B. D. Frazer and Dr. R. I. Hamilton for lending metheir growth chambers; and other members of the staff at theAgriculture Canada Vancouver Research Station, for theirxiiassistance.Support for this study was from New Brunswick Department ofAgriculture sponsored by the Canadian International DevelopmentAgency.1CHAPTER 1 - GENERAL INTRODUCTIONPRACTICE AND PROBLEMS IN POTATO TISSUE CULTUREMeristem tip (MT) culture is widely used to obtain virus—free stocks of vegetatively propagated species such as carnation,chrysanthemum, orchid, potato, etc. (Hollings 1965; Kassanis1967; Hollings and Stone 1968). The technique was first appliedto potato (Solanum tuberosum L.) 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 thenmultiplied in vitro to produce plantlets. The stock materialsare tested against major viruses by ELISA. ELISA utilizes thespecific binding characteristics between an antibody and anantigen to detect the presence of the antigen of a pathogen intest samples. Disease—free plantlets are multiplied in largenumbers in vitro and subsequently transplanted to soil in pots inthe greenhouse. The plants can be regenerated both by stemcuttings and tubers in the greenhouse. After quarantine testing,the disease—free potatoes are distributed to Elite seed farms toproduce seed potatoes. A seed lot is grown in the field beforeit is released to commercial growers.2In vitro meristem tip cultures do not always producedisease—free plants (Wang 1985) . Contamination ofmicropropagated plantlets by bacteria and bacteria—like organismsis a big problem in many research and commercial laboratories.Indexing programs have to be carried out frequently at differentstages of in vitro mass cloning to exclude bacteria, bacteria-like organisms, fungi or even mycoplasma (Wang 1985; Debergh1987)Three types of contamination of plant tissue cultures arerecognized. The first, acute contamination at the establishmentstage is due to incomplete surface sterilisation of the explant.The second, contamination that occurs post-establishment ispossibly due to endogenous microflora or to poor technique at thesubculture stage. The third, chronic contamination arisesapparently simultaneously in a batch of cultures after anextended period of supposedly axenic growth (Long et al. 1988).De Forssard (1977) stated that the contamination that occurs postestablishment can cause a slowing of the growth rate of theplantlets.The interaction of phytopathogenic bacteria with tissuecultures provides a useful system to study mechanisms of plantpathogenesis. In tissue culture systems, the types and numbersof organisms in an interaction can be easily and accuratelycontrolled. Since the growth medium is chemically defined, the3composition is easily adjusted to suit the objective of theexperiment. Treatments are relatively easy to handle in an invitro system by adding and removing certain compounds at aparticular time during the experiment. Uniform treatments can beachieved with great ease. Growth conditions in vitro can beaccurately controlled to avoid the unpredictable and severeconditions found in the field. Outside factors influencinggrowth and interactions of plant and pathogens, such as,activities of humans and animals, variation in soil properties,and soil microbial numbers and composition, can be eliminated.Biochemical and molecular biological techniques have beenfrequently used in phytopathology. It is much easier to studythe pathogenic interactions at the molecular level in planttissue cultures than in field—grown plants. Moreover, someexperiments which utilized tissue cultures are difficult orimpossible with field plants. For example, the synthesis of“amylovorin” by the host in response to Erwinia amylovorainfection was studied using apple cell suspension culture (Hsuand Goodman 1978). This study could not have been done usingapple trees because of the difficulties in handling them in thefield. Similarly, tissue culture and tissue slices are used tostudy the effect of pectolytic enzymes produced by Erwiniacarotovora on plant tissue. The effect of bacterial metaboliteson plants can be examined easily in tissue culture rather thanunder field conditions. In these cases the experimental4manipulations and measurements could not have been carried out inthe field.BACTERIAL DISEASES OF POTATOBLACKLEG AND SOFT ROT DISEASES. The blackleg and soft rotdiseases of potato are caused by three soft rot erwinias [Erwiniacarotovora subsp. atroseptica (Van Hall) Dye, E. carotovorasubsp. carotovora (Jones) Bergey et al., and E. chrysanthemiBurkholder et al.]. The pectolytic erwinias have a worldwidedistribution, but contrasting host ranges and host specificitythat are reflected in their serological and optimum temperaturefor growth characteristics. E. c. subsp. atroseptica (Eca) isrestricted mostly to potatoes in temperate regions, where thecrops are grown most extensively and the strains isolated usuallybelong to one serogroup (Serogroup I) (De Boer et al. 1979;Mather et al. 1986) . However two other soft rot erwinias, E. c.subsp. carotovora (Ecc) and E. chrysanthemi (Echr), can infectpotato plants in the field and cause symptoms similar to thoseassociated with E. c. subsp. atroseptica infection, includingbasal stem rot (Cother 1980) . E. c. subsp. carotovora has aworld—wide distribution in both temperate and tropical zones andis pathogenic to a wide range of plants. E. chrysanthemi is alsowidely distributed in tropical, subtropical and warm temperatezones where it is a pathogen of many crops as well as those grownin greenhouses in temperate regions. E. chrysanthemi can also5cause stem rot under field conditions in some cool temperateregions (Tominaga et al. 1979). Potato crops in some countriesare frequently infected by more than one soft rot erwinia strainsince most of them are not host specific (De Boer et al. 1975; DeLindo 1978)Nonemergence can result from early decay of the seed tubersor the death of sprouts at or soon after emergence due toinfection. Initial symptoms can be quite diverse on growingplants. Wilting and yellowing of leaves are usually associatedwith an early attack by pectolytic erwinias. The basal stem rot,known as blackleg, results from decay of the seed tubers.However, seed piece decay does not always result in blackleg.The basal stem rot symptoms caused by three soft rot erwiniasunder field conditions optimal for each pathogen are essentiallyindistinguishable (Stanghellini and Meneley 1975).Although soft rot erwinias are facultative anaerobes, theygrow better in vitro under aerobic than anaerobic conditions(Meneley and Stanghellini 1976; Wells 1974). Oxygen, carbondioxide, and water are essential factors influencing diseaseexpression. Factors that assist the growth and spread of theprimary inoculum that, in turn, contribute to the build—up ofhigh bacterial densities in stems are likely to favour expressionof the disease (Perombelon and Icelman 1980)6BACTERIAL RING ROT DISEASE. Bacterial ring rot caused byCorynebacterium sepedonicum (Spieck. & Kotth. [Skapt. & Burkhl;syn: Clavibacter michiganensis subsp. sepedonicus [Spieck. &Kotth.] Davis et al.) is one of the most important diseases ofpotato because the bacteria may spread rapidly among tubers inplanting operations and all tubers from infected plants may rot(De Boer and Slack 1984). This disease occurs in both field andstorage conditions (Sletten 1985; Easton 1979; Lelliott 1988)The symptoms of diseased plants include wilting, chlorosis, andstunting (Nelson and Torfason 1974). Diseased tubers initiallyshow a characteristic “ring rot” symptom. Transverse sectionshave a creamy yellow and cheesy texture in the vascular ring andlater the affected tuber tissues become brown and necrotic (DeBoer and Slack 1984). Symptomless infection of potato plantsalso occurs in the field and is attributed to low inoculumlevels, late season—infection, or environmental conditions thatsuppress or mask symptom expression (De Boer and Slack 1984).Low light intensity and short photoperiod increase the severityof ring rot symptoms of potato plants grown in greenhouses(Nelson 1980)CAUSAL ORGANISMSSOFT ROT ERWINIAS. Soft rot erwinias are the majorbacterial pathogens of potato. The main characteristicdistinguishing soft rot erwinias from other Erwinia species is7the ability to produce large quantities of pectolytic enzymes(mainly pectic lyase) that enable the bacteria to macerateparenchymatous tissue of a wide range of plant species(Perombelon and Kelman 1980)Although the pectolytic Erwinia species and subspecies cansometimes cause the same symptoms in potato, the bacteria can bedifferentiated from one another by specific biochemical tests(Slade and Tiffin 1984) . For instance, E. chrysanthemi can bedifferentiated easily from E. c. subsp. atroseptica andcarotovora because it exhibits phosphatase activity during growth(Graham 1971) . Furthermore, E. c. subsp. atroseptica producesacid from a—methylglucoside and reducing substances from sucrose,but E. c. subsp. carotovora does not. Strains of E. c. subsp.atroseptica do not grow at 37°C; in contrast E. c. subsp.carotovora grows at this temperature. Most E. c. subsp.carotovora strains do not grow at 39°C, whereas strains of E.chrysanthemi can grow relatively well at 39°C. (Perombelon andKelman 1980)There is a controversy over the longevity of soft roterwinias in soil. The number of E. carotovora declined rapidlyin naturally and artificially inoculated nonsterile field soils(Perombelon and Kelman 1980). Soil temperature was not alimiting factor in determining survival of soft rot erwinias insome soils. (Perombelon and Kelman 1980) . E. c. subsp.8atroseptica was found to survive longer in water—saturated thanin air—dried soil, but the reverse was true for E. chrysanthemi.E. carotovora survived best at most temperatures in wet soilcollected in winter or in moist soils collected in summer(Perombelon and Kelman 1980) . Addition of nutrients to the soilextended the longevity of E. carotovora significantly (Perombelonand Kelman 1980) . Soft rot erwinias can overwinter incontaminated plant residue remaining in the soil. In someregions, a high proportion of tubers left in the field afterharvest is likely to be contaminated by soft rot erwinias(Perombelon 1975) . The bacteria may also overwinter inassociation with volunteer plants and certain weeds (Perombelonand Kelman 1980)Soft rot erwinias are located superficially on soilparticles (Perombelon and Kelman 1980) . They are moved readilyin soil water from decayed seed tubers to soil and to the progenytubers. Soil water can carry a large number of bacteria forseveral meters in soil (Graham and Harper 1967) . Large numbersof soft rot erwinias are leached after a heavy rain from therotting seed tubers to adjacent zones (Perombelon 1976). On theother hand, movement of bacteria in soil is impeded bydiscontinuity of the water film around soil particles.(Perombelon and Kelman 1980)CORYNEBACTERIUM SEPEDONICUM. C. sepedonicum is classified9as a Gram—positive, nonmotile, obligate aerobic rod—shapedorganism. The slow growth rate and a requirement for severalgrowth factors are the characteristics of this bacterial species,which make it a difficult organism to isolate and study. Underfield conditions, C. sepedonicum is reported to cause diseaseonly in potatoes, but can also infect other plants by artificialinoculation (De Boer and Slack 1984) . The major source ofinoculum for bacterial ring rot of potato is infected tubers.Nelson (1979) reported that survival of C. sepedonicum in non—sterile soil was poor. Survival was better at a low moistureregime and frozen conditions. C. sepedonicum can overwinter incontaminated seed tubers left in the field (Bonde 1942)EFFECTS OF HARNFUL RHIZOSPHERE MICROORGANISMS ON POTATO PLANTSPlant growth-inhibiting microorganisms can be divided intothree groups: 1) plant pathogens parasitizing plant tissue,thereby causing distinct macroscopic disease symptoms, 2) minorpathogens or subclinical pathogens that parasitize root cellswithout causing distinct macroscopic disease symptoms, and 3)microorganisms that do not parasitize plant tissue but as aresult of their metabolic activities may become harmful to rootdevelopment and plant growth (harmful rhizosphere microorcTanisms)(Schippers et al. 1985)Long—term crop rotation experiments in the Netherlands10indicated that frequent potato cropping caused significant yieldreduction which was more severe with increasing potato croppingfrequencies (Schippers et al. 1985). Analysis of the cause ofpotato yield reduction led to a conclusion that an unidentifiedmicrobial factor in short potato—rotation soils was involved.The analysis of nutrient contents showed that nitrogen,phosphorus, and potassium did not differ significantly betweendifferent soils (short and long potato—rotation soils) to accountfor the yield reduction observed (Bakker and Schippers 1987).Bakker et al. (1987) proposed that potato yield reduction inshort potato rotations was due to impaired root function causedby harmful rhizosphere microorganisms, rather than by directdamage caused by invasive pathogens.In pot experiments, plant growth and tuber production weresuppressed in comparison to those in soil from the same fieldwith little or no history of potato cropping (Geels and Schippers1983; Scholte et al. 1985) . Increasing potato croppingfrequencies were favourable for enhancing the activities ofmicroorganisms harmful to root development. In addition,microbial metabolites harmful to root cell activitiesprogressively accumulated in the soil (Schippers et al. 1985).The alleviation of potato yield reduction and root growthinhibition in short potato—rotation soil by selected Pseudomonasspp. (Schippers et al. 1985; Bakker et al. 1987) suggested thatthe numbers of harmful microorganisms were significantly reduced11by the fluorescent pseudomonads.OBJECTIVES OF THE RESEARCH PROJECTThe objectives of this study were: (1) to evaluate theeffect of the bacterial pathogens on micropropagated potatoplantlets, (2) to determine the responses of micropropagatedplantlets to different inoculum concentrations of E. carotovoraand the response of different cultivars, (3) to illustrate theeffects of E. carotovora on the development of potato plants insoil, and (4) to characterize a factor inhibiting root growth.12CHAPTER 2 - SYMPTOMS CAUSED BY THREE SOFT ROT ERWINIAS AND OTHERBACTERIAL SPECIES IN MICROPROPAGATED POTATOPLANTLETSINTRODUCTIONThe contamination of commercial tissue cultures by differentbacterial pathogens has been reported repeatedly. Long et al.(1988) demonstrated that micropropagated potato plantlets thathad undergone a number of subcultures contained a variety ofbacterial contaminants. A fluorescent pseudomonad and acoryneform bacterium were most numerous. Leggatt et al. (1988)isolated 31 microorganisms from ten different micropropagatedplant cultivars and reported that the most common isolates wereyeasts, Corynebacterium spp., and Pseudomonas spp.E. carotovora has been isolated from commercial plant tissuecultures of Nephrolepsis exaltata “Bostoniensis” (L.) Schott(Boston fern), Saxifraga sarmentosa L. (Strawberry begonia), andPteris spp. (Pteris fern) (Knauss and Miller 1978). Five out often bacterial isolates were identified as E. carotovora, fourbelonged to the genus Bacillus, and one was a pseudomonad. Thesecontaminants caused reduced vigour and chiorosis in plant tissuecultures. Debergh and Vanderschaeghe (1988) reported that thepresence of brown spots on the petioles and leaf blades of13Gerbera indicated bacterial contamination. Bacterialcontamination often caused dark brown bracts at the base ofshoots of tissue cultured Calathea makoyana.The description of many subtle symptoms of bacterialcontamination in potato tissue cultures has also been published.A contaminant pseudomonad, isolated by Long et al. (1988), aloneand in combination with a coryneform bacterium was reinoculatedinto disease—free potato plantlets of cv. British Queen. Thecontaminated plantlets were smaller than control plantlets anddeveloped a large number of necrotic tips.The objectives of this study were: (1) to illustrate theeffect of different strains of soft rot erwinias, and otherphytopathogenic bacterial species on micropropagated potatoplantlets, (2) to document the symptoms of the plantletsinoculated with these bacterial species.MATERIALS AND METHODSMICROPROPAGATED PLANTLETS. Micropropagated potato plantletsof cv. Kennebec obtained originally from the Virus—free PotatoLaboratory at the Agriculture Canada Vancouver Research Stationwere free of contamination by potato virus X, potato virus Y,potato virus S, potato leaf roll virus, and potato spindle tuberviroid. Bacterial contamination was examined by plating sap14expressed from the stem end of the plantlets onto nutrient agarplates. Any colonies that developed on the nutrient agar platesduring fourteen days of incubation were considered to becontaminants. The plantlets, used as source materials forfurther multiplication, were completely free from viral andbacterial contamination. The plantlets were multiplied bygrowing nodal cuttings in 250 mL jars containing 30 mL Murashigeand Skoog’s (MS) medium (Murashige and Skoog 1962) each at 21°C(dark) and 23°C (light) with 16 h photoperiod in a tissue culturegrowth chamber. Only vigorously growing plantlets were used assources of cuttings for inoculation.PREPARATION OF NODAL CUTTINGS. Nodal cuttings,approximately 1 cm in length, were excised from vigorouslygrowing plantlets with sterile scissors. All leaflets wereremoved from plantlets. Both the top and the lowest nodes ofeach plantlet as well as nodes with aerial roots were discarded.The slender and weak plantlets, which were observed to havedifferent growth rates in subsequent culture in comparison tothose of normal cuttings, were excluded.BACTERIAL CULTURES. Three strains of E. c. subsp.atroseptica, two strains each of E. c. subsp. carotovora, and E.chrysanthemi, and one strain each of C. sepedonicum, Pseudomonasmarginalis and E. amylovora, were used in this study (Table 1).15Table 1. Bacterial cultures used in the study of symptomexpression of micropropagated potato plantlets of cv. KennebecSpecies Sero—(Subsp.) Strain group Source Origin HostErwinia 31 I H.P. Maas Nether— Potatocarotovora Geesteranus landssubsp. 161atroseptica198 XXII R.J. Copeman Canada PotatoE17196 XX S.H. De Boer Canada PotatoErwinia 21 II H.P. Maas Nether— Potatocarotovora Geesteranus landssubsp. 139Fcarot ovora71 III H.P. Maas Nether— PotatoGeesteranus lands226Erwinia 573 R.S. Dickey U.S.A. Guayulechrysan themi574 R. Cother Austra— PotatohaErwinia SR5 P. Sholberg Canada Pearamyl ovoraCoryrie— R8 S.H. De Boer Canada Potatobacteriumsepedoni cumPseudomonas 90—521 L. MacDonald Canada Potatomarginalis16Serogroup differentiation of the soft rot erwinias was based ontheir serological relationships determined by Ouchterlony doublediffusion (De Boer et al. 1979)Cultures of each of the soft rot erwinia strains were storedin a solution containing 60% nutrient broth and 40% glycerol at—80°C. A nutrient agar slant was streaked with a culture fromstorage and subsequently incubated at 23°C. A single colony foreach strain showing typical pitting on crystal violet, pectate(CVP) medium was selected.C. sepedonicum strain R8 (Cs—R8) was obtained from theVancouver Research Station collection and maintained on yeast,glucose medium (YGM) (De Boer 1989) slants at 23°C. A singlecolony of Cs—R8 was selected from a YGM plate, and subsequentlystreaked onto YGM slants.One fluorescent strain of P. marginalis 90—521 and onestrain of E. amylovora were obtained from the culture collectionat the British Columbia Ministry of Agriculture and Fisheries(BCMAF). Cultures of both species were grown on nutrient agarslants initially. Kings Medium B (King et al. 1954) and a highsucrose medium (Crosse and Goodman 1973) were use for theselection of a single colony for P. marginalis and E. amylovora,respectively. Both cultures were grown on nutrient agar slantsat 23°C.17EXPERIMENTAL TREATMENTS. Each of the pectolytic Erwiniastrains, and one strain of P. marginalis and E. amylovora wereindividually grown on nutrient agar slants at 23°C for 48 hours.Four—day—old freshly growing cultures of Cs—R8 were used forinoculum preparation. Freshly grown cells were washed andsuspended in sterile Ringers solution. A stock suspension ofeach strain was prepared by dilution of the washed cells toobtain an absorbance value of 0.4 at 660 nm with aspectrophotometer (Spec 20). A series of dilutions were madefrom each stock and used as inoculum. Actual inoculumconcentrations were determined by a standard plate count method.Two inoculum concentrations, ten—fold apart, were prepared forall strains of soft rot erwinias, and one concentration for P.marginalis and E. amylovora. Inoculum of Eca 31 prepared in thesame way in all experiments was used as a positive control.Sterile Ringers solution was used as a negative control. Aseptictechniques were used and the experiments were repeated twice.A modified MS medium with 0.75 % agar was prepared in flasksat 79 mL/flask and maintained at 40°C in a water bath afterautoclaving. One mL of each inoculum preparation (including bothpositive and negative controls) was added to the 79 mL molten MSmedium and was mixed thoroughly in each jar. The jars were leftopen in the laminar flowhood until the medium was completelysolidified.18For the ring rot pathogen, the nodal cuttings were dipped ineach inoculum suspension of Cs—R8 for 10 seconds and then blottedon dry sterile filter paper to eliminate the extra inoculumaround the cuttings. Five inoculated cuttings of the sametreatment were transferred into a jar. Five treatments, two forCs R8, two for Eca 31, and one for Ringers control, were employedand each was replicated three times. However, treatments with P.marginalis and E. amylovora were inoculated in the same way asthose of the soft rot erwinia strains.Freshly cut nodal cuttings were collected in a sterilePetri—dish and randomized by shaking. The cuttings werecarefully laid flat on top of the agar medium and then pushed 5mm into the agar. Six cuttings were used per jar and each jarwas replicated three times.All inoculated plantlets were incubated at 18°C (dark) and23°C (light) with a photoperiod of 16 hours for 25—30 days.Illumination was at 194.5 lIE m2s’ provided by eight F48T12/CW/HO Phillips fluorescent tubes.SAI4PLING The plantlets and the medium were taken out of thejars together and separated carefully. Length and number of allroots and stem of each plantlet were measured with a ruler. Thenumber of roots of a plantlet was counted for those roots over0.5 mm in length. Discoloration of plantlets was measured on a19scale of 1—5 (Table 2) in comparison to the normal leaf colour ofcontrol plantlets which was set at 0. Symptoms of the inoculatedplantlets were photographed at sampling time.RESULTSE. c. subsp. atroseptica. Eca 31 was selected as arepresentative strain of soft rot erwinias to demonstrate itseffect on the growth of micropropagated potato plantlets of Tetonand Russet Burbank in preliminary experiments. Root growthinhibition and stunting were observed when the plantlets weregrown on MS medium for 25—30 days in a growth chamber.Elongation of roots was significantly inhibited by Eca 198and 196 only at 41 and 40 cfu/mL, respectively (Table 2, 3) . Nosignificant difference in the number of roots was found for anytreatments with either Eca 198 or 196 (Table 2, 3) . Stem lengthwas significantly shorter in the treatment with Eca 198 atinoculum concentration of 41 cfu/mL compared to buffer control(Table 2) . Stem length did not differ significantly betweentreatments with Eca 196 and buffer (Table 3), but Eca 31 causedstunting (Table 2, 3). E. c. subsp. atroseptica strain 198caused reduction in the number of leaves and yellowing of theplantlets (Table 2)E. c. subsp. carotovora. Root elongation was inhibited by20Table 2. Effect of Erwinia carotovora subsp. atrosepticastrain 198 on micropropagated potato plantlets of cv. KennebecInoc.1 Stem No. Root No.Treat— concn length of length ofment (cfu/mL) (cm) leaves Color2 (cm) rootsBuffer 0 7.33a3 8.50a 0.lla 9.53a 7.67aEca 31 29 5.06b 6.28b l.56b 2.94c 9.28aEca 198 4 7.62a 7.61c 2.33b 7.26ab 8.OOa41 5..74b 6.92c 2.34b 5.85b 8.93aStnd.error 0.30 0.15 0.21 0.51 0.54‘One mL inoculum suspension was mixed with 79 mL Murashigeand Skoog’s medium. Numbers indicated final concentrations.2Six numerical classes of discoloration of plantlets wereused: 0, normal; 1, 1—2 top leaves yellowed; 2, 3—4 topleaves yellowed; 3, over 5 leaves yellowed; 4, topblackened and/or all leaves yellowed; 5, stem rotted.3Numbers followed by the same letter within each column donot differ significantly (p=0.05) according to Tukey’smultiple range test. The value is the mean of 18 observations.21Table 3. Effect of Erwinia carotovora subsp. atrosepticastrain 196 on micropropagated potato plantlets of cv. KennebecInoc.1 Stem No. Root No.Treat— concn length of length ofment (cfu/mL) (cm) leaves color2 (cm) rootsBuffer 0 6.27a3 8.44a 0.lla 9.49a 9.17aEca 31 29 4.28b 6.89b l.17a 3.28c 11.17aEca 196 4 5.9Oab 7.72ab 2.67b 6.68ab 8.89a40 5.l7ab 6.94ab 3.44b 4.38c 9.83aStnd.error 0.44 0.33 0.28 0.64 0.67‘One mL inoculum suspension was mixed with 79 mL Murashige andSkoog’s medium. Numbers indicated final concentration.2Six numerical classes of discoloration of plantlets were used:0, normal; 1, 1—2 top leaves yellowed; 2, 3—4 top leavesyellowed; 3, over 5 leaves yellowed; 4, top blackened and/or allleaves yellowed; 5, stem rotted.3Numbers followed by the same letter within each column do notdiffer significantly (p=0.05) according to Tukey’s multiplerange test. The value is the mean of 18 observations.22Ecc 71 at both inoculum concentrations, but only at the highinoculum level by Ecc 21 (Table 4, 5). Stem length and thenumber of leaves of plantlets growing in the medium inoculatedwith Ecc 21 did not differ significantly from those of buffercontrol at both 3 and 27 cfu/mL (Table 4). Ecc 71 causedstunting and reduction in the number of leaves only at 68 cfu/mL(Table 5). Both E. c. subsp. carotovora strains causeddiscoloration of plantlets at low inoculum levels (Table 4, 5).E. chrysanthemi. Significant inhibition of root elongation,stem growth, and the number of leaves occurred at both inoculumconcentrations for E. chrysanthemi strain 573 and 574 (Table 6,7). Eca 31, used as a positive control over a range of inoculumconcentrations between 29-53 cfu/mL, consistently causedinhibition of root elongation and reduction in stem growth(Tables 2—7)C. sepedonicum, P. marcina1is, and E. amylovora Nosignificant effect of Cs—R8 was found on any plantletcharacteristics of cv. Kennebec after incubation for 25 days,although the effect of Eca 31 on all except the number of rootswas significant (Table 8). A large number of bacterial cells ofCs—R8 was recovered on YGM plates from stems of plantlets 2 cmabove the agar surface.23Table 4. Symptoms of micropropagated potato plantlets of cv.Kennebec caused by Erwinia carotovora subsp. carotovora strain 21Inoc.’ Stem No. Root No.Treat— concn length of length ofment (cfu/mL) (cm) leaves color2 (cm) rootsBuffer 0 7.57a3 9.61a O.28a 9.Ola 7.56aEca 31 32 5.06b 7.28b 1.78b 2.02c 8.33aEcc 21 3 6.88ab 8.56ab 1.22b 7.95a 6.67a27 6.98ab 9.17a 1.50b 6.OOb 7.41aStnd.error 0.49 0.31 0.13 0.38 0.39‘One mL inoculum suspension was mixed with 79 mL Murashige andSkoog’s medium. Numbers indicated final concentrations.2Six numerical classes of discoloration of plantlets were used:0, normal; 1, 1—2 top leaves yellowed; 2, 3—4 top leavesyellowed; 3, over 5 leaves yellowed; 4, top blackened and/or allleaves yellowed; 5, stem rotted.3Numbers followed by the same letter within each column do notdiffer significantly (p=0.05) according to Tukey’s multiplerange test. The value is the mean of 18 observations.24Table 5. Symptoms of micropropagated potato plantlets of cv.Kennebec caused by Erwinia carotovora subsp. carotovora strain 71Inoc.’ Stem No. Root No.Treat— concn length of length ofment (cfu/mL) (cm) leaves Color2 (cm) rootsBuffer 0 7.54a3 9.06a 0.17a 8.62a 8.17aEca3l 32 4.56b 6.94ab 2.llc 2.33bc 7.llaEcc7l 7 7.16a 8.39ab l.06b 4.32b 9.28a68 2.96b 6.39b 2.33c 0.80c 6.56aStnd.error 0.45 0.59 0.19 0.69 0.64‘One mL inoculum suspension was mixed with 79 mL Murashige andSkoog’s medium. Numbers indicated final concentrations.2Six numerical classes of discoloration of plantlets were used:0, normal; 1, 1—2 top leaves yellowed; 2, 3—4 top leavesyellowed; 3, over 5 leaves yellowed; 4, top blackened and/or allleaves yellowed; 5, stem rotted.3Numbers followed by the same letter within each column do notdiffer significantly (p=O.O5) according to Tukey’s multiplerange test. The value is the mean of 18 observations.25Table 6. Effect of Erwinia chrysanthemi strain 573 onmicropropagated potato plantlets of cv. KennebecInoc.’ Stem No. Root No.Treat— concn length of length ofment (cfu/mL) (cm) leaves Color2 (cm) rootsBuffer 0 7.55a3 8.06a 0.lla 7.80a 7.33aEca 31 53 4.29b 6.28b 2.17b l.95b 5.56aEchr 4 2.37c 3.89c 1.44ab 0.63b 3.llb57341 1.56c 2.52c l.66ab O.19b 1.19bStnd.error 0.34 0. 31 0.36 0.39 0.54‘One mL inoculum suspension was mixed with 79 mL Murashige andSkoog’s medium. Numbers indicated final concentrations.2Six numerical classes of discoloration of plantlets were used:0, normal; 1, 1—2 top leaves yellowed; 2, 3—4 top leavesyellowed; 3, over 5 leaves yellowed; 4, top blackened and/or allleaves yellowed; 5, stem rotted.3Numbers followed by the same letter within each column do notdiffer significantly (p=0.05) according to Tukey’s multiplerange test. The value is the mean of 18 observations.26Table 7. Effect of Erwinia chrysanthemi strain 574 onmicropropagated potato plantlets of cv. KennebecInoc.’ Stem No. Root No.Treat— concn length of length ofment (cfu/mL) (cm) leaves Color2 (cm) rootsBuffer 0 7.76a3 7.89a 0.OOa 8.62a 7.33aEca 31 40 3.06b 3.94b 2.61b 1.36b 4.22aEchr 574 4 2.74b 4.OOb 0.83a 2.24b 6.50a37 2.65b 4.56b l.06a 1.18b 5.56aStnd.error 0.28 0.45 0.25 0.40 0.83‘One mL inoculum suspension was mixed with 79 mL Murashigeand Skoog’s medium. Numbers indicated final concentrations.2Six numerical classes of discoloration of plantlets wereused: 0, normal; 1, 1—2 top leaves yellowed; 2, 3—4 topleaves yellowed; 3, over 5 leaves yellowed; 4, top blackenedand/or all leaves yellowed; 5, stem rotted.3Numbers followed by the same letter within each column do notdiffer significantly (p=0.05) according to Tukey’s multiplerange test. The value is the mean of 18 observations.27Table 8. Comparison of symptoms expressed by micropropagatedpotato plantlets of cv. Kennebec caused by Corynebacteriumsepedonicum strain R8 and Erwinia carotovora subsp. atrosepticastrain 31’Inoc. Stem No. Root No.Treat— concn length of length ofment (cfu/mL)2 (cm) leaves color3 (cm) rootsBuffer 0 7.51a4 7.13a 0.20a 8.33a 7.2OabEca3l 296 4.52b 4.73b 1.27a 2.24b 5.33b29600 1.38c 2.07c 3.27b 0.66c 0.67cCs—R8 640 8.21a 7.33a 0.13a 8.95a 7.73a64000 6.92a 7.40a 0.80a 8.77a 7.33aStnd.error 0.46 0.25 0.39 0.33 0.41‘Nodal cuttings of all treatments were inoculated by dipping inthe treatment solutions for 10 seconds, followed by beingtransferred to a sterile filter paper to remove surface liquidinoculum, then transferred to jars.2Two inoculum concentrations, 100—fold apart were used for eachorganism.3Six numerical classes of discoloration of plantlets were used:0, normal; 1, 1—2 top leaves yellowed; 2, 3—4 top leavesyellowed; 3, over 5 leaves yellowed; 4, top blackened and/or allleaves yellowed; 5, stem rotted.4Numbers followed by the same letter within each column do notdiffer significantly (p=0.05) according to Tukey’s multiplerange test. The value is the mean of 18 observations.28One strain from each of P. marginalis and E. amylovora wastested and both significantly inhibited plantlet root growthof cv. Kennebec (Table 9). Stem lengths in treatments with bothbacterial species were not significantly different from that ofthe buffer control. P. marginalis and E. amylovora did not causeyellowing of the plantlets, however Eca 31 did (Table 9)Root growth of the cuttings was observed 6 days afterincubation. The soft rot erwinia strains tested varied in growthrate in MS medium. Colonies of some strains developed in MSmedium within 3 days of incubation. The inhibition of rootelongation in the treatments inoculated with the high inoculumconcentration was observed within 7—8 days for all strainstested. The degree of root inhibition depended on the rate ofbacterial growth in the medium. In addition, bacterial coloniesgrew faster in association with the roots of the plantlets.The major and minor symptoms of the plantlets inoculatedwith the soft rot erwinia strains became apparent during thecourse of incubation. The symptoms were associated with roots,stems and leaves. Root symptoms consisted of small roots (Fig.1), blackening and browning of root tips (Fig. 2. A), and aerialroot formation (Fig. 2. B) . Inhibition of root elongation wasassociated with all strains of soft rot erwinias and occurred asearly as the noticeable root growth. The browning of root tipswas observed in all strains tested and occurred during later29Table 9. Effect of Pseudomonas marginalis and Erwiniaamylovora on micropropagated potato plantlets of cv.Kennebe c’Inoc. Stem No. Root No.Treat— concn4 length of length ofment (cfu/mL) (cm) leaves color2 (cm) rootsBuffer 0 4.80a3 8.56a 0.OOa 12.97a 6.28abEca 31 46 3.40b 6.64a 2.78b 5.91b 5.llaPm5 31 3.55ab 6.46a 0.83ab 6.12b 7.68bEa6 69 3.63ab 7.56a 0.OOa 6.16b 6.OlaStnd.error 0.28 0.76 0.49 1.02 0.31‘Treatments were incubated at 18—22°C for 30 days.2Six numerical classes (0 — 5) of discoloration of plantletswere used: 0, normal; 1, 1—2 top leaves yellowed; 2, 3—4 topleaves yellowed; 3, over 5 leaves yellowed; 4, top blackenedand/or all leaves yellowed; 5, stem rotted.3Numbers followed by the same letter within each column do notdiffer significantly (p=0.05) according to Tukey’s multiplerange test.4One mL inoculum suspension was mixed with 79 mL Murashige andSkoog’s medium. Numbers indicated final concentrations.5’6One strain of P. marginalis and one potato strain of E.amylovora was grown at 23°C for 48 h before being used ininoculum preparation.30Fig. 1. Root length and stem length of micropropagated potatoplantlets of cv. Kennebec grown for 21 days on Murashige andSkoog’s medium inoculated with Erwinia carotovora subsp.atroseptica strain 31 (right) in comparision to those of control(left)31Fig. 2. Root symptoms on plantlets of cv. Kennebec after 26 daysof growth. A, Browning and blackening of root tips caused by E.chrysanthemi strain 573. B, Aerial root formation induced byErwinia carotovora subsp. carotovora strain 71.32stage of growthi although the frequency was low within eachexperiment. Aerial root formation was also a common effectcaused by soft rot erwinias regardless of inoculum concentration.The stem symptoms consisted of stunting (Fig. 1), blackeningat the stem base, black necrotic dots, stem yellowing (Fig. 3.A), and decay (Fig. 3. B). Blackening at the stem base waspossibly caused by bacterial growth on the medium at the base ofthe stem. In contrast, stem rot usually started at the tip ofthe stem which first turned black and progressed downward. Mostleaves of a plant]-et turned yellow when stem rot occurred,usually at the later stage of growth.The leaf symptoms consisted of necrotic lesions, leaf rotand yellowing (Fig. 4. B), black necrotic dots (Fig. 5), leafcurling (Fig. 6), and leaf tip necrosis (Fig. 7) . Leaf yellowingwas common and its severity was proportional to the inoculumconcentration.Some minor symptoms became apparent on plantlets grown for30 days on the medium inoculated with P. marginalis and E.amylovora. Small black localized lesions on the underside ofleaves were occasionally observed in the plantlets on mediuminoculated with E. amylovora. P. margirialis caused browning ofsome of stems and the underside of leaves of the plantlets.33Fig. 3. Stem symptoms on plantlets of cv. Russet Burbank grownfor 30 days on Murashige and Skoog’s medium inoculated withErwiriia chrysanthemi strain 576. A, Blackening, yellowing, andblack necrotic dots on stems. B, Rotting of stem tips.jBcv)‘cia)0•Ha>0>C)-ia)4)‘ciI’4-ioobU)C)U)4J0)(I)0).0H-IJHC54)HS.-1c9-iCi)0Ce0>1H0Cs)‘ci4-)0)0o>CeS-i4)ceCeCi)-C)4)i-i-I-)ooCt324%S.-iHl0)•4),4iS.-iCe-ii0Ce0U),c:S.-i4)obC)-H-H9-44)oo0‘ciS-iS-i(1)C)U)C)-I-)-H>0)CeECecH‘ciz4-i4-iC)o‘.-oeo(NCl)U)i-i-H0)::‘0)-U)Ce-I-)-I-)0)4-4Ce(I)U)EC).1-)4)•ci)(1)Ce‘,QH0)Ci)-iiS-i•c::cb’e•r40)H-H•-(N(N-riCeS-i4-)C,)(13C-)I35Fig. 5. Black necrotic dots on the leaf of micropropagatedpotato plantlets of cv. Russet Burbank grown for 30 days onMurashige and Skoog’s medium inoculated with Erwinia chrysanthemistrain 576.36,f/ I, \ I,0‘I‘IFig. 6. Curling of leaves of micropropagated potato plantlets ofcv. Kennebec grown for 26 days on Murashige and Skoog’s mediuminoculated with Erwinia carotovora subsp. atroseptica strain 31.37/7Fig. 7. Leaf tip necrosis of plantlets of cv. Kennebec grown for26 days on Murashige and Skoog’s medium inoculated with Erwiniachrysanthemi strain 576.:::NA38DISCUSSIONAlthough many symptoms developed in plantlets of cv.Kennebec infected by soft rot erwinias, root growth inhibitionwas the only one consistently exhibited by all strains tested.It is possible that all strains of soft rot erwinias possess thecapacity for root growth inhibition. Moreover, when theelongation of roots was inhibited earlier in the growth, rootdevelopment was more severely affected. The fact that inhibitionof root growth was consistent in all experiments also indicatedthat the tissue culture system was a reliable model for studyingthe root inhibitory effect by soft rot erwinias.Although both E. chrysanthemi strains inhibited root growth,strain 573, but not strain 574, significantly inhibited rootinitiation of the plantlets (Table 6, 7). The reason for thisdiscrepancy was not known. Perhaps high amounts of a rootinitiation inhibitor were produced by Echr 573 at inoculumconcentration as low as 4 cfu/mL in comparison to other soft roterwinia strains.My observation that root inhibition by some soft rot erwiniastrains occurred soon after the start of root elongation may berelated to the finding by Taylor and Secor (1985) that the growthand survival of potato callus tissue were dramatically inhibitedby Ecc 71. Most calli were killed within 5 days of growth on the39inoculated medium. The factor that inhibited root elongation ofmicropropagated potato plantlets may be the same as that whichkilled the calli.Perombelon and Kelman (1980) stated that a commoncharacteristic of soft rots and associated disorders was the lackof specificity of the host—pathogen interaction. The effect ofroot growth inhibition on micropropagated potato plantlets by allstrains of soft rot erwinias tested is consistent with this lackof specificity.The response of stem growth to soft rot erwinias varied whendifferent strains were used. Only two out of seven strains, Eca196 and Ecc 21, did not cause stunting even at high inoculumlevels, although both inhibited root growth significantly. Thereason for this difference might be due to the difference ingrowth rates of the strains in MS medium.At high inoculum concentration, or with bacterial strainswith high growth rates in MS medium, the plantlets rotted anddied at an early stage of growth. Decay of stem tips was alsofrequently observed in different experiments. Although manybacteria do produce tissue—macerating enzymes, only Erwinia spp.,P. marginalis, and a few others have been associated with decayof living plant tissue (Lund 1979; Rudd-Jones and Dowson 1950)These tissue macerating enzymes from soft rot erwinias probably40played a role in decay development. Some symptoms might be dueto nutrient limitation in the medium or the blocking or breakdownof the nutrient transport systems of the plantlets. In eithercase, the essential nutrients failed to reach the growing pointsand the plantlets eventually died.The number of leaves and discoloration of plantletsdeveloping on the media inoculated with Eca 31 were not alwaysconsistent in different experiments, suggesting that the responseof both characters to soft rot erwinias might not be stable invitro.In spite of the frequent reoccurrence of some minor symptomsin different experiments, the frequency of a particular symptomwas usually low within each experiment. It would be necessary toemploy other strategies, such as modifying growth conditions ormedium composition to increase the frequency of the minorsymptoms, to study them in greater detail.The browning of tissues, together with a loss ofelectrolytes and an increased rate of respiration of tobaccosuspension cultures were described by Matthysse (1983) as anincompatible response of these cultured cells to P. pisi. Sheconcluded that tissue browning and loss of electrolytes were dueto damage to the plasmalemma. Stephens and Wood (1975) reportedthat endo—polygalacturonate trans—eliminase recovered from41infection of tissue slices by E. carotovora caused the release ofpolyphenol oxidase and thus tissue browning. The root tipbrowning (blackening) was frequently observed in the plantletsgrowing in the medium inoculated with soft rot erwinias. It ispossible that root tip browning of the plantlets in response tosoft rot erwinias was an incompatible response to the presence ofthe pathogen and might be due to polyphenol oxidase activity.Although inoculum concentrations were different, P.marginalis and E. amylovora caused inhibition of root elongationto a similar extent as Eca 31. Large E. amylovora colonies werefound scattered around the small roots of a cutting. Long rootsgrew within 1 cm to the agar surface and some roots developed inthe 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 sloughedcells of root caps which were capable of diffusing through theagar medium. The scattered appearance of bacterial coloniesaround roots was unique for E. amylovora among the differentbacterial species tested. The root inhibition might be caused bya toxic secondary metabolite secreted by E. amylovora coloniesaround the roots. Possibly a gradient of toxic metabolite thatdecreased with distance from the centre of those colonies wasbuilt up and the roots were less inhibited at distance furtheraway from those colonies.42Different factors that were either directly or indirectlyproduced by the bacteria possibly contributed to root inhibitionof micropropagated potato plantlets. The inoculated media onwhich 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, lackof 02, or changes in medium pH. However other factors, forexample, toxic metabolites, may also be involved.No apparent growth of Cs-R8 on/in MS medium could bedetected after incubation at 19°C (day) and 23°C (night) for 15days. For this reason, direct inoculation of nodal cuttings witha bacterial suspension was employed. Cs—R8 colonies wereoccasionally observed growing around the roots of Kennebec nodalcuttings during incubation. It is proposed that the lack ofsymptoms in plantlets inoculated with Cs—R8 was probably due tothe slow growing characteristic of this bacterial species.The cause of the inhibition of root elongation, stunting,and yellowing of micropropagated potato plantlets by soft roterwinias may not be the same. A metabolite from the pectolyticerwinias may be involved in root inhibition. The minor symptomsmay be due to the physiological disorders of the plantlets inresponse to the change in their growth medium.43CHAPTER 3 — RESPONSE OF MICROPROPAGATED POTATO PLANTLETS TOERWINIA CARO TOVORA SUB SP. A TROSEP TICAINTRODUCTIONRoot growth inhibition and stem length reduction of in vitrogrown potato plantlets occurred in MS medium inoculated with thesoft rot erwinia strains. The extent of changes in tissueculture medium is determined by the size of the bacterialpopulation. Inoculum concentration controls the rate at whichthe bacteria alter the culture medium. Consumption rate ofnutrients, the oxygen and pH levels change faster and to agreater degree at higher bacterial concentration than at a lowerone. Moreover, more toxic bacterial metabolites accumulate athigher bacterial levels. It is quite possible that plantlets mayrespond differently to varying concentrations of bacteria in themedium.Introduction of bacteria into a new environment results inalteration in bacterial metabolism. Some new metabolites may besecreted; others might be repressed. Moreover, the associationof soft rot erwinias with nodal cuttings or only with MS mediumrepresents two different environments which would result indifferent kinds and amounts of enzymes released from bacterialcells. The fact that cells of Eca 31 grew faster once they were44associated with the plants indicates that some factors from nodalcuttings promoted the growth of the bacteria. The bacterialpopulation density associated with the plantlets was a functionof the method of inoculation. This would result in differentresponses of the plantlets depending on the inoculation procedurethat was used.Genetic control of host plants over the number and type ofmicroorganisms was demonstrated by Atkinson et al. (1975). Theyshowed that resistant varieties of wheat supported lowerpopulations of bacteria than did susceptible ones andsubstitution of a single resistance chromosome into an otherwisesusceptible variety changed the rhizosphere microflora.Moreover, the disease development in tubers caused by E. c.subsp. atroseptica was also reported to be affected by cultivarsusceptibility which varied greatly (Bourne et al. 1981)Similarly, it is proposed here that, after identical experimentaltreatment, micropropagated plantlets of different cultivars mightharbour different numbers of bacteria of the same species due togenetic differences among cultivars used.The blackleg disease of potato occurring in temperateregions is caused mainly by E. c. subsp. atroseptica which isoften present on seed tubers. Most E. c. subsp. atrosepticastrains, irrespective of the country of origin, form aserologically homogeneous group (Allan et al. 1977; De Boer et45al. 1979). Eca 31 is a typical strain of serogroup I whichrepresent 96% of the strains of this subspecies (De Boer andMcNaughton 1987). Therefore, Eca 31 was used in this study.The objectives of this study were: (1) to measure thesensitivity of micropropagated plantlets to varying inoculumconcentrations; (2) to compare the responses of differentcultivars to the same pathogen; (3) to illustrate the effect ofdifferent inoculation methods on the responses of micropropagatedplantlets.MATERIALS AND METHODSMICROPROPAGATED PLANTLETS. Disease—free micropropagatedpotato plantlets of cv. Kennebec, Russet Burbank, Red Pontiac andRed Lasoda were obtained from the Virus—free Potato Laboratory atAgriculture Canada, Vancouver Research Station. The examinationof contamination, growth, and multiplication of the plantlets invitro were the same as that reported in Chapter 2.ECA 31 CULTURE. Cultures of Eca 31, stored at —80°C, werestreaked on casamino acid, peptone, glucose (CPG) plates. Thecultures from a typical Eca 31 colony on CPG was streaked oncrystal violet, pectate (CVP) medium and a single colony with theunique pitting characteristic on the medium was streaked onnutrient agar slants for inoculum preparation. Cells from 2-day46old slant cultures were suspended in sterile Ringers solution andthe stock solution was made by adjusting the cell suspension toan absorbance value of 0.4 at 660 nm using a spectrophotometer(Spec 20) . Three inoculum concentrations, estimated at 10, iO,and iO cfu/mL determined by a standard plate count method, wereused.INOCULATION METHOD. A modified MS medium which contained0.7% agar was adopted to reduce damage to root systems ofplantlets during sampling. Forty-nine mL molten MS medium at40°C was added to each of thirty-two 500 mL—jars and mixedthoroughly with 1 mL of each of the three inoculum preparationsand Ringers solution. The cuttings were prepared in the same wayas described before and eight cuttings/jar were planted after themedium was completely solidified. Each treatment was replicatedtwice. The treatments were randomized and incubated for 18 daysat 19°C(dark) and 23°C (light) with a photoperiod of 16 hours,under an illumination of 194.5 LE m2s’ provided by eigth F48T12/CW/HO Phillips fluorescent tubes. Tukey’s multiple range testwas used to analyze the data.ELISA METHOD. A 1.5 cm long stem was cut from the lower endof the plantlet and transferred into a sterile plastic bagcontaining 1 mL sterile distilled water. After homogenization,the tissue sap was assayed by indirect ELISA according to theprocedure by De Boer et al. (1988), except that polyclonal E5447IgG and monoclonal 4F6 antisera were used for detection of E.carotovora in this study.AN ALTERNATIVE INOCULATION PROCEDURE. The bacterial stocksolution (0D660 = 0.1) was prepared as above. Three levels ofbacterial concentrations, 6 x 102, 6 x iO, and 6 x 106 cfu/mL,were used as inoculum, and sterile Ringers solution was acontrol. The nodal cuttings were inoculated by immersing themdirectly in the bacterial suspensions for 10 seconds and thendipping them in Ringers solution briefly to rinse away extrasuspension adhering to the cuttings. The cuttings were blottedon dry sterile filter paper and planted into the jars. Eachtreatment was replicated six times. The treatments wereincubated for 31 days at 19°C(dark) and 23°C (light) with 16 hphotoperiod, under an illumination of 196.7 .LE m2s1 provided byeigt F48 T12/CW/HO Phillips fluorescent tubes and four 25 W lightbulbs.ELECTRON MICROSCOPIC EXAMINATION. Samples of roots ofmicropropagated potato plantlets of cv. Red Pontiac were fixed in4% glutaraldehyde (v/v) in 0.1 M cacodylate buffer pH 7.2 for 2 hand washed in 0.1 M cacodylate buffer. They were postfixed in1% Os04 for 1 h in buffer, washed in water, dehydrated in aseries of alcohols, and embedded in propylene oxide infiltratedEPON 812 mixture.48The roots were sectioned and stained for 20 mm in 4%uranylacetate, and lead citrate ( diluted 1:1 0.1 N NaOH) for 5mm. The preparations were viewed in an Hitachi 600 electronmicroscope.RESULTSSENSITIVITY TEST. The roots and stems responded differentlyto the varying inoculum concentrations. Root elongation of cv.Russet Burbank and Red Pontiac in vitro was dramaticallyinhibited by Eca 31 at an inoculum concentration of 20 cfu/mL(Fig. 8) . The inhibition of root initiation occurred as theinoculum concentration increased from 0.2 to 2000 cfu/mL, butsignificantly only at the highest inoculum level for bothcultivars (Fig. 9) . Root elongation was more sensitive thaninitiation to inhibition (Fig. 8, 9)Stunting of plantlets occurred at inoculum concentrationhigher than 20 cfu/mL (Fig. 10). The plantlets of both cultivarsdeveloped significantly fewer leaves at an inoculum concentrationover 20 cfu/mL compared to control (Fig. 11) . Cultivar RedPontiac developed more leaves per unit stem length than cv.Russet Burbank. The plantlets of both cultivars turned yellow at20 cfu/mL and the yellowing become more severe for cv. Russetthan for cv. Red Pontiac when inoculum concentration increasedfrom 20 to 2000 cfu/mL (Fig. 12)497I Aa RB >: RP-J AaIzI-(9zw ‘-JF0I__ I___________xu..20z BWi0— 0 0.2 20 2000INOCULUM 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 andSkoog’s medium inoculated with E. carotovora subsp. atrosepticastrain 31. Means (n=1 6) (bars) for each cultivar having the same letterdo not differ significantly (p=0.05) according to Tukey’s multiple range test508I—Lii-JIz0I-00ccU-04ccLiiDzzw00.2 20 2000INOCULUM CONCENTRATION (cfu/ml)Fig. 9. Mean number of roots per plantlet grown for 18 days on Murashige andSkoog’s medium inoculated with different concentrations of Erwinia carotovorasubsp. atroseptica strain 31. Means (n=1 6) (bars) for each cultivar having thesame letter do not differ significantly (p=0.05) according to Tukey’s multiplerange test. RB: Russet Burbank, RP: Red Pontiac.05150F—w4-JIza-0I—0zUI-JUII—C/)ziUI00 0.2 20 2000INOCULUM 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’smedium inoculated with different concentrations of Erwinia carotovorasubsp. atroseptica strain 31. Means (n=1 6) (bars) for each cultivarhaving the same letter do not differ significantly (p=0.05) accordingto Tukey’s multiple range test.5210ILii-Jz0Cl)LU><6LU-JU0UiDzz0Fig. 11. Mean number of leaves per plantlet grown for 18 days onMurashige and Skoog’s medium inoculated with different levels ofErwinia 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 BurbankRP: Red Pontiac.0 0.2 20 2000INOCULUM CONCENTRATION (cfulml)5365(!3Z40-J—j3w>-2100 0.2 20 2000INOCULUM 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 andSkoog’s medium inoculated with different concentrations of Erwiniacarotovora subsp. atroseptica strain 31. Six numerical classes ofyellowing: 0, normal; 1, 1-2 top leaves yellowed; 2,3-4 top leavesyellowed; 3, over 5 leaves yellowed; 4, top blackened and/orall leaves yellowed; 5, stem rotted. Means (n=1 6) (bars) for eachcultivar followed by the same letter do not differ significantly(p=0.05) according to Tukey’s multiple range test.54VARIETY RESPONSE. The roots of cv. Red Lasoda were shorterat the low, but longer at the high inoculum concentration thanthose of the other three cultivars (Table 10). Root elongationresponses in cv. Kennebec, Red Pontiac, and Red Lasoda weresimilar to one another at both inoculum concentrations. The rootinitiation response of cv. Red Lasoda was significantly differentfrom those of cv. Red Pontiac and Russet Burbank at 20 cfu/mL(Table 10)The response of stem growth of cv. Kennebec, Russet Burbank,Red Pontiac, and Red Lasoda to E. c. subsp. atroseptica wassimilar at 0.2 cfu/mL (Table 10) . The stems of cv. RussetBurbank were significantly longer than those of Red Pontiac andRed Lasoda at 20 cfu/mL and were only slightly shorter at thehigh in comparison to the low inoculurn concentration (Table 10).ALTERNATIVE INOCULATION METHOD. Inhibition of rootelongation, stunting, and yellowing occurred in plantlets grownfrom cuttings directly inoculated with Eca 31. The stem lengthof cv. Russet Burbank was increasingly smaller as the inoculumconcentration increased from 0 to 6 x 106 cfu/mL (Fig. 13) . Theseverity of yellowing increased as the inoculum concentrationincreased (Fig. 14). The roots were significantly inhibited byEca 31 in all inoculated treatments (Fig. 15)55Table 10. Comparison of response of micropropagated potatoplantlets of cv. Kennebec, Russet Burbank, Red Pontiac, and RedLasoda to Erwinia carotovora subsp. atroseptica strain 31Percentage of controlCharacter Potato cultivar 0.2 20of the (cfu/mL) (cfu/mL)plantletsStem Kennebec 98.38 a 73.29 ablength Russet Burbank 110.25 a 103.71 bRed Pontiac 105.88 a 50.50 aRed Lasoda 91.25 a 67.00 aRoot Kennebec 78.63 ab 15.29 alength Russet Burbank 85.75 a 7.86 aRed Pontiac 87.13 a 8.50 aRed Lasoda 65.63 b 42.71 bNumber Kennebec 107.38 a 100.71 abof Russet Burbank 100.00 a 72.86 aroots Red Pontiac 90.50 a 64.13 aRed Lasoda 115.50 a 139.71 b‘Numbers followed by the same letter for each character withineach column do not differ significantly (p=0.05) according toTukey’s multiple range test.5612a 10I—UI-JIz<8-JI0Z6LII-JUII-Cl)4zUI200 6 600 60000INOCULUM CONCENTRATION (x 102 cfu/mI)Fig. 13. Response in stem length of plantlets of cv. Russet Burbankto different concentrations of Erwinia carotovora subsp. atrosepticastrain 31. The nodal cuttings were inoculated directly with thebacteria. Means (n=6) (bars) having the same letter do not differsignificantly (p=0.05) according to Tukey’s multiple range test.5732.52z0l.5Iii>-10.500 6 600 60000INOCULUM CONCENTRATION (x i? cfu/ml)Fig. 14. Severity of yellowing of plantlets of cv. Russet Burbank in responseto 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 leavesyellowed; 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.bbba6 600 60000INOCULUM CONCENTRATION (x lO2cfu/ml)Fig. 15. Effect of different inoculum concentrations of Erwinia carotovorasubsp. atroseptica strain 31 on root elongation of plantlets of cv. RussetBurbank. The nodal cuttings were inoculated directly with bacteria and weregrown on sterile Murashige and Skoog’s medium. Means (n=6) (bars) havingthe same letter do not differ significantly (p=0.05) according to Tukey’smultiple range test.58aC.)IuJ-JI—z00zw-JI00><LI0zw14121086420bC0C59ELISA DATA. The concentrations of the bacterial antigen atthe stem end of the plantlets of four cultivars rose as theinoculum 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 examinationrevealed that bacterial cells of Eca 31 were present in threemajor locations in root systems of the plantlets of cv. RedPontiac. A large population of bacterial cells covered theepidermal surface of the root (Fig. 17). A large number ofbacterial cells was present in the xylem of the root (Fig. 18),and in the intercellular spaces of the parenchyma (Fig. 19). Inaddition, a few bacterial cells were discovered inside a rootcell (Fig. 20). The electron micrographs showed that a slimelayer was always associated with aggregated bacterial cells.DISCUSSIONAlthough an effect of bacterial contamination on tissuecultures of ornamental plants by E. carotovora was previouslydemonstrated (Knauss and Miller 1978), this study was the firstattempt to elucidate dose response of micropropagated potatoplantlets to Erwinia spp. Since the culture medium of theplantlets was chemically defined, and the growth conditions in a601-0.9 -0.80.7cin 0,60w 0.5 -0z40.4U)m40.3 -0.2 -0.1 -0— I I I I I I I I0 0.4 0.8 1.2 1.6 2 2.4 2.8 3.2INOCULUI CONCENTRATION Clog cfu/ml)Fig. 16. Mean absorbance values (A405 nm) for enzyme—linkedimmunosorbent assay conducted on stem end portions of plantletsof cv. Kennebec (D), Russet Burbank (+), Red Pontiac (0), and RedLasoda (v) in response to different inoculum concentrations ofErwinia carotovora subsp. atroseptica strain 31.61Fig. 17. Electron micrograph of root section of cv. Red Pontiacgrown for 26 days on inoculated Murashige and Skoog’s medium. Alarge 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 rootnear the edge showing the presence of bacterial cells of Erwiniacarotovora subsp. atroseptica strain 31 in intercellular spacesbetween the epidermal cells. Plantlets of cv. Red Pontiac grownfor 26 days on Murashige and Skoog’s medium (x 3705)..: )‘63Fig. 19. Electron micrograph of cross section of a root of cv.Red Pontiac grown for 26 days on Murashige and Skoog’s mediuminoculated with Erwinia carotovora subsp. atroseptica strain 31.Bacterial cells in vascular tissue of the root (x 10000)•H•—-r - -*5-S(/4d-Fig. 20. Electron micrograph of cross section of uper portion ofa root of cv. Red Pontiac grown for 26 days on Murashige andSkoog’s medium inoculated with Erwinia carotovora subsp.atroseptica strain 31. Bacterial cells present inside a root cell(x 7500)64---—---—-.‘ e/1/V.. ‘-••. •..65growth chamber were set at a constant level, the dose response ofplantlets to the pathogen in vitro may be different from that inthe field. Nevertheless, the response of the plantlets to softrot erwinias in vitro should provide a valuable indication of theresponse of potato to the same pathogen in the field. Thesensitivity tests utilizing the method of medium inoculationshowed that two cultivars behaved similarly except that thenumber of roots of cv. Russet Burbank increased, but decreasedfor cv. Red Pontiac between 0—0.2 cfu/mL. The reason for thisdifference is not known.Eca 31 colonies in the inoculated MS medium grew so fastthat the effect of bacterial metabolites was exerted on theinitial growth of the cuttings. It was predicted that the amountof bacterial metabolites secreted into the medium would beproportional to the initial inoculum concentration. The severityof inhibition of root elongation was related to the inoculumlevel.The inoculum concentration necessary to inhibit elongationof roots was lower than that to inhibit root initiation.Therefore, inhibition of root elongation and initiation by E. C.subsp. atroseptica should be considered separately.The fact that root elongation of cv. Red Lasoda wasinhibited more at the low, than at the high inoculum66concentration compared to other cultivars indicated that theroots of plantlets of this cultivar were comparatively tolerantto Eca 31 at the high inoculum concentration. However, roots andstems behaved differently for cv. Red Lasoda, indicating thatdifferent mechanisms in the plantlets might control the responsesof root and stem. My results are in agreement with Lyon’s (1989)suggestion that several mechanisms may operate within potato toeither directly inhibit growth of Erwinia spp. or indirectlyinhibit the enzymes involved in pathogenesis. Differentmechanisms may be important in different parts of the plant (Lyon1989)The plantlets of cv. Red Lasoda developed more roots thanthose of the three other cultivars at both inoculumconcentrations and more roots developed at high than at lowinoculum concentration for cv. Red Lasoda. It is evident thatroot initiation of cv. Red Lasoda was promoted in the presence ofa high number of Eca 31 cells.The conjugated monoclonal antibody used in the ELISA wasproduced against lipopolysaccharide of the outer membrane of E.c. subsp. atroseptica. The higher the ELISA values, the higherthe LPS concentrations in the stem end of the plantlets andprobably the higher the Eca 31 cell population. The fact thatthe ELISA values for cv. Red Lasoda were consistently lower thanthose for three other cultivars provided additional evidence that67this cultivar supported the growth of fewer bacteria than othercultivars.The effect of soft rot erwinias on potato plants grown invitro was diverse and the symptoms in treatments with highinoculum concentrations were easily observed during the growth ofthe cuttings. However the plantlets often appeared normal intreatments with lower inoculum levels. My observation was inagreement with that reported by Weber and Schenk (1988) who foundthat there were no symptoms or growth retardation on theplantlets with low bacterial density.The population of soft rot erwinias decline rapidly innaturally and artificially infested soils (Perombelon and Kelman1980) . However, in common with many other plant pathogenicbacteria, soft rot erwinias can overwinter in contaminated plantresidue left in the soil after harvest. These contaminatedmaterials together with diseased seed tubers serve as the mostimportant inoculum sources for the next growing season. Thepurpose of different inoculation methods used in this study wasto create different inoculum sources for the plantlets to studythe individual responses to the pathogen. Root elongation,stunting, and the severity of yellowing of cv. Russet Burbankwere affected progressively by the increased inoculum dose inboth inoculation methods. Moreover, root elongation was sharplyinhibited when certain inoculum concentrations were reached.68Both inoculation methods gave similar results.Rhizosphere microorganisms are extensively associated withroots of plants and the root exudate provides various nutrientsto support the growth of the bacteria. Bowen and Rovira (1976)reported that almost the entire surface of roots growing in soilis covered with microorganisms. Newman and Bowen (1974) foundthat rhizosphere bacteria tended to aggregate on the surface ofplant roots. Immunofluorescence and immunogold stainingtechniques revealed dense populations of E. chrysanthemi alongthe longitudinal cell walls of potato roots, at the points wherethe lateral roots were formed, and sometimes close to the roottips (Underberg and Van Vuurde 1989). In my study, a largenumber of bacterial cells was present on the surface of the root,in the intercellular spaces of the parenchyma, and in the xylemof the root. In addition, most surface cells of the root of themicropropagated plantlets had either died or lost their normalcell shape probably due to the toxic metabolites of theaggregated bacterial colonies. The higher the amount ofbacterial metabolites released, the more severe the damage to theroot system of the plants.69CHAPTER 4 - EFFECT OF ERWINIA CAROTOVORA SUBSP. ATROSEPTICA ONPOTATO PLANTS GROWN IN SOILINTRODUCT IONE. 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 thepredominant blackleg pathogen in temperate regions, whereas athigher soil temperatures E. c. subsp. carotovora and also E.chrysantheini have been shown to be the cause of blackleg(Staghellini and Menely 1975; Molina and Harrison 1977, 1980;Perombelon 1985) . Blackleg infected plants, a few weeks afteremergence, are smaller than healthy ones (Persson 1988)Root growth of potato stem cuttings is inhibited in shortpotato—rotation soils in comparison to that in long potato—rotation soil (Bakker et al. 1987) . Root systems of potato stemcuttings grown for 2 weeks in soil with 1:1 potato frequencyattained less than half the weight of those grown in soil with1:6 potato frequency. The fact that the absence of symptoms,such as discoloration or lesions on roots of potato plants grownfrom short potato-rotation soil, indicates that an unknownmicroorganism affects root functioning without being parasitic(Bakker et al. 1987)70Many differences between the soil and tissue culture systemsexist in terms of nutrients, growing conditions and environment,and the growth of the pathogen used in the experiments. Responseof micropropagated plantlets to the pathogen in tissue culturesystems may or may not represent those of the plants in thefield. Fett and Zacharius (1982, 1983) reported that some tissueculture lines lose their ability to synthesize phytoalexins withincreasing time in culture. The comparison for the response ofpotato plants to the same pathogen between culture in vitro andin a soil environment is necessary to make sure that the tissueculture is a suitable model for studying certain aspects of thedisease in soil.The purpose of this study was to elucidate the effect of E.c. subsp. atroseptica on the symptom expression of potato plantsgrowing in soil.MATERIALS AND METHODSPREPARATION OF SEED TUBERS. Disease—free seed potato tubersof cv. Russet Burbank were obtained from seed farms in thePernberton Valley of British Columbia. The tubers were free frompotato virus X (PVX), potato virus Y (PVY), potato leaf rollvirus (PLRV), potato spindle tuber viroid (PLRV), and C.sepedonicum. Tubers were tested for contamination by soft roterwinias by combining portions of tuber tissues at stolen ends71and 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 42.5-113.4grams. The tubers were stored in the cold room at 4°C beforeuse. The tubers were held in an open box in a greenhouse at 18°Cfor 3 weeks to break dormancy. Individual seed pieces with oneeye each were obtained by cutting the seed tubers with a melonballer (diameter = 2.9 cm) . The seed pieces were stored underthe same conditions overnight.The seed pieces were planted, 2.5 cm deep and 10 cm apart,in regular soil mix in a plastic tray. The trays were wateredthoroughly. The seed pieces were grown at 18°C for 2 days in agreenhouse.INOCULUM PREPARATION. A 2—litre flask containing 1.25 Lnutrient broth was inoculated with 2—day—old Eca 31 cultures andwere incubated at 23°C with constant shaking. Bacterial cellswere harvested at the exponential growth phase by centrifugationat 10700 g for 20 mm. The supernatant was discarded and thepellets were suspended in 1.8 L sterile distilled water. Thebacterial suspension was mixed thoroughly by stirring continuallyfor half an hour. Then the number of cells was determined by astandard plate count method. Two series of 100—fold dilutionswere prepared subsequently by transferring 18 mL bacterial72suspension to 1782 mL sterile distilled water to obtainadditional inoculum preparations.EXPERIMENTAL TREATMENTS. Sixty-four 4-inch pots were eachfilled with 450 g steamed soil mix. Dry weight of the soil mixwas determined after heating to 8000 for 24 h. Four treatmentpreparations, including one water control and three inoculumlevels consisting of 3.3 x iOn, 3.3 x iO, and 3.3 x iO cfu/mL,were employed. There were 4 treatments, 16 replicates in eachtreatment, and one seed piece in each replicate (pot).INOCULATION, GROWTH, AND SANPLING. Before inoculation, eachseed piece was taken carefully out of the tray and planted in apot, 2.5 cm below the soil line. A few seed pieces with nogrowth were discarded. The pots for each treatment were randomlyselected and the inoculum at 110 mL/pot was added evenly to thepots. Four treatments with 0, 1.1, 110, and 11000 x iO cfu/gsoil were used. The pots were completely randomized on thebench. The maximum water capacity of the soil in each pot waspremeasured by recording the volume of water retained by 450 gsoil per pot. The plants were grown at 18°C for 6 days afterinoculation. All treatments were watered once every two days.After 6 days of growth, the length of stems was measured andthe number of leaves was recorded. The entire plants wereremoved carefully from the pots. The root systems were washedwith tap water and the length of the longest root per plant was73measured. The roots of each plant were kept separately andheated at 80°C for 24 h in an oven to determine dry weight. Theanalysis of variance was carried out to measure the treatmenteffects. The greenhouse experiment was repeated twice.RESULTSInhibition of root elongation occurred in potato plantsgrown from seed pieces in soils inoculated with Eca 31. Rootgrowth was sensitive to the increase in inoculum concentration.Roots of the plants at inoculum concentrations higher than 1.1 xiO cfu/g soil, were significantly shorter than those of controltreatments (Fig. 21). Root growth was inhibited by 50% at aninoculum concentration of 1.1 x lO7cfu/g soil. Decay of seedpieces were observed in treatments with inoculum concentrationshigher than 1.1 x 10 cfu/g soil and small roots were oftenassociated with rotted seed pieces (Fig. 22).Dry weights of roots in treatments inoculated with Eca 31was lower than that of the control only when inoculumconcentration was higher than 1.1 x iO cfu/g soil (Fig. 23).The dry weight of root systems at the two highest inoculumconcentrations did not differ significantly from each other.0I.z-J0FzLU-JF00cc><LL0zLii742.221.8 -1.61.4 -1.21—0.8 i i0 1 2 3 4 5 6 7INOCULUM CONCENTRATION (log cfu/g soil)Fig. 21. Mean of maximum root length per plant grown in soil inoculated withErwinia carotovora subsp. atroseptica strain 31 at concentrations of 1.1, 110,and 11000 x 10fu/g soil compared to control. The plants were grownat l8b for 6 days in pots in a greenhouse. Bars represent standard error ofmeans of 16 replicates.G)HU)-H00Cl)CiwG)Z0tyi-HU)4)-IcDIIIr1H4)C)HHo4)HHG)4-)-H4)(I.44)Cii•(iiHH5c)C_)ci)4-)0U)-H(110-H(11(I)I-)U)H4-)-H-HU)•,11I0oCl)(iiC)U)--1(ii>1-H4.)(iici)-I-)U)4-I‘D00ci)-IS-iH-il:y).1-)—Cl)dl4)U)>14-4(Ii.cr1:4)U)Hci)Qcr1C):iCl)ciici)U)Q-H(11•d0C41Cl)>C14-)CI)0ci)Cl)4)•U)0bU)(I)-i•1-4ciiElC)76110c,)100-J0o 80Fz0W 70>-60zw500 1 2 3 4 5 6 7INOCULUM CONCENTRATION (log cfu/g soil)Fig. 23. Mean dry weight of root systems per plant grown from seed piecein soil inoculated with Erwinia carotovora subsp. atroseptica strain 31 atconcentrations of 1.1, 110, and 11000 x 1O3cfu/g soil compared to control.The plants were grown at 18°C for 6 days in pots in a greenhouse. Dry weightwas determined after drying at 8cfC for 24 h. Bars represent standard errorof means of 16 replicates.7712 -C.) -Hz-LUHCl)LL 9-0H(9Z 8-LU-JzLU 7-6 i0 1 2 3 4 5 6 7INOCULUM CONCENTRATION (log cfu/g soil)Fig. 24. Mean length of stem per plant grown from a seed piece in soilinoculated with Erwinia carotovora subsp. atroseptica strain 31 atconcentrations of 1.1, 110, and 11000 x 1O3cfu/g soil compared to control.The plants were grown at 18°C for 6 days in a greenhouse. Bars representstandard error of means of 16 replicates.78Stem lengths were significantly shorter in treatments withEca 31 at both 1.1 x iO, and 1.1 x iO cfu/g soil than that ofthe control after 6 days of growth (Fig. 24) . The stems lengthswere 95, 63, and 62% of the controls at 1.1 x iO, 1.1 x iO, and1.1 x iO cfu/g soil, respectively. The number of leaves perplant became smaller as the inoculum concentrations increased(Fig. 25) . No other visible foliage symptoms were observed.DISCUSSIONThe inhibition of root elongation of cv. Russet Burbankgrowing in soil inoculated with Eca 31 suggested that the samephenomenon of root growth inhibition observed in vitro also canoccur in soil and the factor inhibitory to root growth wasproduced by the bacteria in soil environments. Inhibition ofroot elongation and occurrence of short stems in the inoculatedsoils suggest that tissue culture systems can be used reliably asa model system for studying symptoms of diseased plants infectedby soft rot erwinias. However the response of plants to E. c.subsp. atroseptica in soil was less uniform than that ofmicropropagated plantlets. The greatest variations of theresponse in both stem and root in soil occurred in treatmentswith an inoculum concentration of 1.1 x iO cfu/g soil.The inhibition of root elongation was related to thezcC-J0C’)U]>cCU]-JU0U]DzzcCU]796.56-5.5 -5-4.5INOCULUM CONCENTRATION (log cfu/g soil)Fig. 25. Mean number of leaves per plant grown from a seed piece in pot soilinoculated with Erwinia carotovora subsp. atroseptica strain 31 atconcentrations of 1.1, 110, and 11000 x 1O3cfu/g soil compared to control.The plants were grown at 18°C for 6 days in a green house. Bars representstandard error of means of 16 replicates.I I I I I I I I0 1 2 3 4 5 6 780reduction in dry weight of the root systems since both weresignificantly different from the control at inoculumconcentrations higher than 1.1 x iO cfu/g soil. However, theroot elongation was also inhibited at a higher inoculumconcentration but the dry weight was not. The reason for thisdifference is not known. The fact that a sharp reduction in dryweight, stem length, and number of leaves occurred when inoculumconcentration increased from 1.1 x iO to 1.1 x iO cfu/g soilsuggested that the inoculum concentration of 1.1 x iO cfu/g soilplayed a crucial role in causing a significant effect on thedevelopment of potato plants by E. c. subsp. atroseptica by thismethod of inoculation.The frequent watering to maintain high levels of waterpotential in pot soils met the requirement for the bettersurvival of E. c. subsp. atroseptica. Seed piece decayfrequently occurred in treatments with inoculum concentrationshigher than 1.1 x iO cfu/g soil. However, non—emergence did notoccur in any of the treatments. This might be due to the factthat the seed pieces were sprouted before inoculation and theysurvived to produce a plant before being decayed by Eca 31.Profound root growth inhibition frequently occurred inplants with decaying seed pieces. Large numbers of bacteria werereleased from the decaying seed pieces into the rhizosphere,resulting in inhibition of root elongation. The inhibited81root development could in turn limit the nutrient uptake by rootsresulting in stunting and wilting of plants. This speculation isstrengthened by the findings of Lapwood et al. (1985) whoreported that seed tubers inoculated with E. c. subsp.atroseptica produced plants with less vigour and reduced yield.Stunting of potato plants caused by E. c. subsp. atrosepticawas quite dramatic after 6 days of growth. I observed that alarge number of the small plants contributed to the significantreduction in average stem length and the length of stems becamemore uniform as the inoculum concentration increased. Thesesmall plants usually had rotted seed pieces attached. My resultsconfirm the findings by Rhodes and Logan (1986) and Lapwood andGans (1984) who reported that seed tubers inoculated with E.carotovora produced small plants in the field.Three soft rot erwinias cause major bacterial diseases inpotato and most seed stocks are contaminated by more than onestrain of them (Perombelon 1972; Nielson 1978). Blackleg is awidespread disease and is found in all potato—producing areas ofthe world (Peltzer and Sivasithamparam 1985) . Bain et al. (1990)inoculated the seed tubers with 106 E. c. subsp. atrosepticacells per tuber at planting time and found that yield lossescould be as high as 60% when the inoculated tubers were grownunder conditions favourable for blackleg. Yield of symptomlesspotato plants grown from seed inoculated with E. c. subsp.82atroseptica decreased as much as 25% in comparison to that ofuninoculated plants (Perombelon and Hyman 1987, 1988). Bakkerand Schippers (1987) proposed that potato yield reduction inshort potato—rotation soils may originate partially from impairednutrient uptake brought about by depressed root cell energymetabolism caused by harmful microorganisms. My experimentalresults revealed that both root elongation and dry weight weresignificantly inhibited by E. c. subsp. atroseptica in soil.Potato yield reduction in both diseased and symptomless plantsmight be the result of inhibited root growth by the erwinias.This study showed that root length was shorter and root dryweight decreased as inoculum concentrations increased. Bain etal. (1990) reported that blackleg incidence and tuber yield werecorrelated with the population of E. c. subsp. atroseptica onseed tubers. The correlation between potato yield reduction andinhibition of root elongation and dry weight in response to theincrease in inoculum dose in different experiments suggested thatthe yield reduction in potato caused by soft rot erwinias mightbe related to the inhibited root growth.83CHAPTER 5 - CHABACTERIZATION OF A FACTOR RESPONSIBLE FORINHIBITION OF ROOT ELONGATIONINTRODUCTIONThe inhibition of energy metabolism of root cells may be onemechanism of the microbial inhibition of root growth in potato(Bakker and Schippers 1987). The inhibition may be due to theaction of metabolites from rhizosphere microorganisms. Most ofthe metabolites are N—containing heterocyclic compounds, amongthese are phenazine and indole derivatives (Leisinger andMargraff 1979). Cytochrome oxidase respiration of roots isinhibited at least 40% by cyanide at a concentration of 4 jIM(Schippers et al. 1985) . Cyanide is known to be a secondarymetabolite of many microbes, including pseudomonads. Other knownsecondary metabolites antagonistic to root growth are someunusual amino acids and peptides (Schippers et al. 1987).Although the metabolites from soft rot erwinias that affectroot growth have not been identified, those from some otherbacterial species were shown to inhibit root growth of theplants. Fredrickson and Elliott (1985) demonstrated that a toxinproduced by root-colonizing pseudomonads significantly inhibitswinter wheat seedling root growth. Indole acetic acid (IAA) fromAzospirillum brasilense was reported to inhibit root elongation84of Panicum miliaceum (Harari et al. 1988).Taylor and Secor (1985) studied the effect of culturefiltrates produced from 5, 11, and 18-day—old shake cultures ofE. c. subsp. carotovora on the survival and growth of protoplast—derived potato calli and found that both growth and survival wereinhibited by all filtrates. Stephens and Wood (1975) showed thatcrude dialysed extracts from rots of cucumber fruit infected withE. carotovora caused cell separation and death of protoplasts innormal and plasmolysed tissues. They further tested differentfactors purified from rot extracts and found that pectate trans—eliminase, proteinase and phosphatidase in crude extracts killedprotoplasts of normal tissue, but not those in unpiasmolysedtissue.A number of bacterial metabolites with different physical,chemical, and biochemical properties exhibit pathogenic effectson tissue cultured cells as well as causing root inhibition inplants. Therefore, a series of experiments was designed in thisstudy to characterize the factor responsible for inhibition ofroot elongation of potato plantlets by E. c. subsp. atroseptica.MATERIALS AND METHODSPETRI DISH BIOASSAY. A piece of sterile filter paper wastransferred to a sterile Petri—dish and 4 mL of the treatment85solution were added. Five cuttings were laid on the filter paperpad inside the Petri—dish which was sealed completely withparafilm. All these procedures were carried out in a flowhoodand aseptic techniques were used throughout the experiments.The treatments were incubated at 23°C for 11—12 days in agrowth chamber with 16 h photoperiod, under the light intensityof 196.7 lIE m2s1 provided by 8 F48 T12/CW/HO Phillipsfluorescent tubes and four 25 W light bulbs. The incandescentlights were on half an hour before the fluorescent lights andhalf an hour after they were off. After the incubation period,the length of the maximum root and number of roots per cuttingwere measured. The stem lengths were also recorded as referencefor each treatment. Plant symptoms were recorded duringsampling. Multiple comparisons of the treatment means were madeby using Tukey’s multiple range test.SUPERNATANT PRODUCTION AND TESTING. A single colony of Eca31 showing the typical pitting characteristics on CVP wasselected to inoculate two nutrient agar slants. Two—day oldnutrient agar slant cultures were used to inoculate 100 mLsterile nutrient broth to give an absorbance value of 0.03 at 660nm in a spectrophotometer (Spec 20) . After growth at 23°C for 5days in shake culture, bacterial cells were pelleted bycentrifugation at 8000 g for 20 mm. The supernatant wascollected and sterilized by filtering through 0.2 j.m pore size86filter.The sterile supernatant was diluted three—fold in sterilenutrient broth and a dilution series containing 33.3 %, 11.1 %,3.7 %, and 1.2 % supernatant was obtained. The supernatantpreparations, sterile nutrient broth, and water were eachcombined with an equal volume of double—strength liquid MS mediumand mixed thoroughly. The following concentrations of thesupernatant in the final mixtures were obtained as individualtreatments, 50 % (bacterial supernatant), 16.7 %, 5.6 %, 1.9 %,0.6 %, and 0 % (nutrient broth). Each treatment was replicatedthree times. Five nodal cuttings were used in each replicate.HEAT TREATMENT. The same method was adopted for theproduction of bacterial supernatant in all subsequentcharacterization procedures. The filter—sterilized supernatantin test tubes was heated to 100°C for 15 mm. and sterilizedagain through a 0.2 p.m pore size filter after cooling to roomtemperature. In addition, the unheated bacterial supernatant andnutrient broth were also filter—sterilized twice.The heated and unheated supernatant, nutrient broth, andsterile distilled water each was mixed thoroughly with an equalvolume of double-strength MS medium to obtain four treatments.Three replicates in each treatment and six nodal cuttings in eachreplicate (Petri dish) were used in the “Petri dish bioassay”.87DIALYSIS. Standard cellulose dialysis tubing (35 mm indiameter; mol wt cutoff: 12,000—14,000) was used for dialysis.Dialysis tubing pieces (approximately 30 cm long) were boiled indistilled water and cooled to room temperature. Filter—sterilized supernatant (25 mL) was transferred to a dialysis bagand dialysed against 4 L sterilized nutrient broth at 4°C for 24h. The dialysis medium was changed once. The dialysed solutionwas filter sterilized again.Four treatments, consisting of dialysed and undialysedsupernatant, nutrient broth, and distilled water, were tested inthe “Petri dish bioassay”. Five replicates in each treatment and5 nodal cuttings in each Petri dish were used.FRACTIONATION WITH ANMONIUM SULPHATE. Ammonium sulphate wasadded to a flask containing 100 mL supernatant to give 70 %saturation. This solution was maintained in a cooler at 4°C for2 hours with constant stirring. The precipitate was pelleted bycentrifugation at 8000 g for 20 minutes. Both pellets andsupernatant were retained. The precipitate in the original flaskand the pellets in the centrifuge tubes were rinsed four timesand finally dissolved in 50 mL phosphate buffer (pH 7.3) . A 100mL sample of nutrient broth was given exactly the same treatment.The above two fractions were the first fractions of bacterialsupernatant and nutrient broth. The second and the thirdfractions were obtained one after another in the same manner by88adding ammonium sulphate to the remaining liquid to give 85% and95% saturation. The final remaining liquid was the fourthfraction.The residual ammonium sulphate in each fraction waseliminated by dialysis at 4°C against phosphate buffer for 36hours for fraction 1 and 2, and for 60 hours for fraction 3, 4(nutrient broth and bacterial supernatant). The phosphate bufferin each container was changed every 12 hours. All dialysedfractions were filter sterilized.The dialysed fractions were tested for their effect on rootelongation of nodal cuttings by the “Petri dish bioassay”.Eleven treatments, 4 replicates in each treatment, and 6 nodalcuttings in each replicate were used.RESULTSSENSITIVITY TEST. The culture supernatant of Eca 31 grownin nutrient broth for 5 days inhibited root elongation of stemcuttings of micropropagated potato plantlets of cv. Kennebec.Inhibition of root elongation was proportional to the logsupernatant concentration (Fig. 26). Regression analysis showedthat the treatment effect on root length of nodal cuttings fitted89550zDC)I—40Lii35Cl) 30Cl)025U020LU150.5 1 1.5LOG SUPERNATANT CONCN. (%)Fig. 26. Effect of supernatant concentrations on root elongation of cuttingsof cv. Kennebec. Supernatant was produced from 5-day old nutrient brothcultures of Erwinia carotovora subsp. atrosetica strain 31 at 2’C withconstant shaking.0 290a straight line having a coefficient of determination of 0.95which indicated a good fit (Fig. 26) . Root elongation in thetreatment with the highest supernatant concentration wasinhibited 59% compared to the control.HEAT TREATMENT. The mean root length in treatment withheated bacterial supernatant was not significantly different fromthose of both nutrient broth and unheated supernatant, but wassignificantly lower than that of water control (Fig. 27) . Theeffect of nutrient broth and water on root elongation was notsignificantly different.DIALYSIS. The dialysed bacterial supernatant inhibited rootelongation to an extent similar to that of the undialysedsupernatant (Fig. 28), indicating that the root inhibitory factorwas retained in the dialysis tubing.ANMONIUM SULPHATE FRACTIONATION. Phosphate buffer did notsignificantly affect root elongation (Fig. 29) . The rootinhibitory factor was precipitated by ammonium sulphate at 70%saturation only (Fig. 30). Four fractions of nutrient broth didnot significantly inhibit root elongation (Fig. 30).C.)0zDQ0><0zI—0zuJ-JzuJ916543210Fig. 27. Effect of heated bacterial supernatant on root growth of nodalcuttings 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’smultiple range test. NB, BS, and BS (H) represent nutrient broth, bacterialWATER NB BS(H) BSTREATMENTsupernatant and heated supernatant, respectively.C.)0z1::C)00xU0zI—0zw-Jzw92543210Fig. 28. Effect of dialyzed bacterial supernatant on root elongation of nodalcuttings 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 nutrientbroth, bacterial supernatant, and dialyzed bacterial supernatant, respectively.WATER NB BS(D) BSTREATMENT936C.)CD 5zD00cr><3U0I-CD 2zw-Jzw0CONTROL TREATMENTSFig. 29. Effect of bacterial supernatant of Erwinia carotovora subsp.atroseptica strain 31 on root elongation of nodal cuttings of cv. Kennebec. Thesupernatant was produced from 5-day old nutrient broth cultures at 2C withconstant shaking. Means (n=24) (bars) having the same letter do not differsignificantly (p=0.05) according to Tukey’s multiple range test.SUPERNATANT BROTH BUFFER94E0040W -4—IlZI zC)wI!LI02 0zI- I-o 0z zWi Liiz zNB BUFFERTREATMENTFig. 30. Effect of four fractions of bacterial supernatant on root elongationof 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, andFraction 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.NBTREATMENTNBTREATMENTBS BS NB BUFFERTREATMENT95DISCUSSIONRoot elongation of cuttings was linearly inhibited inresponse to increasing bacterial supernatant concentration. Thistrend was similar to that observed in pot experiments. Howeverthe plantlets grown in inoculated medium had a different patternof root length reduction in response to varying inoculum levels.One explanation for the discrepancy is the fact that, in Petridish experiments, the amount of root inhibitory substances may bereduced gradually, due to biological degradation. However in theinoculated medium bacteria were present to continually exerttheir effect and produce metabolites. Moreover, other indirectinfluences of bacterial growth in vitro, for example, competitionfor nutrients, decreasing 02 concentration in the medium, and thechange in pH, might also affect root growth. The observed rootgrowth inhibition by the bacterial supernatant was caused mainlyby the toxic metabolite(s) rather than other indirect factors.This difference might also contribute to the different resultsobtained by the medium inoculation method and the “Petri dishbioassay’t.Although plant growth substances of bacterial origin areknown to inhibit root growth of many plant species (Loper andSchroth 1986; Harari et al. 1988), the results of this studyindicated that the factor inhibiting root elongation ofmicropropagated potato plantlets was not a phytohormone. It was96retained in the dialysis bag, indicating that a relatively largemolecular weight molecule (>12000—14000) was involved. Inaddition, the fact that the effect of heated bacterialsupernatant on root elongation was not significantly differentfrom those of nutrient broth and unheated bacterial supernatantled to three possible explanations about the nature of theinhibitory factor. The first, two molecules were involved andone was destroyed by heat treatment; the second, one molecule waspartially destroyed; the third, the heat destruction of theinhibitory factor was reversible in the bioassay.Although the first supernatant fraction significantlyinhibited root growth, the second fraction may also have someinhibitory effect on root elongation (Fig. 30) . It is possiblethat the saturation point of ammonium sulphate for the firstfraction was not high enough to precipitate all of the rootinhibitory factor in the supernatant.Since the root inhibitory factor was partially destroyed byheat, retained in the dialysis tubing, and precipitated byammonium sulphate at 70% saturation which included most proteinmolecules, the factor may have a protein or a peptide moiety.Perhaps, the active molecule is a glycoprotein or a lipoprotein.97CHAPTER 6 — GENERAL DISCUSSIONThis study has established that various symptoms can beinduced by soft rot erwinias in micropropagated potato plantletsas well as in potato plants grown in the artificially inoculatedsoil. Similarity in the symptoms expressed by the potato plantsgrown in both systems existed. Moreover, a compound from E.carotovora which inhibited root elongation of the stem cuttingsof the micropropagated potato plantlets was characterized byusing the “Petri dish bioassay” system.Inhibition of root elongation and stunting of plants in bothin vitro and soil environments (established only for Eca 31) wasthe most common effect demonstrated by the soft rot erwiniastrains, although the effects on stem growth by two of sevenstrains tested were not significant. The consistency of theeffect of root growth inhibition exhibited by soft rot erwiniasin both systems indicate that the production and secretion of aroot growth inhibitory factor were not significantly affected bythe different growth environments of the pathogen. Althoughfield experiments to determine if root growth inhibition occursto field potato crops by soft rot erwinias are still necessary,the inhibitory effect could happen if the different conditionsbetween the field and green house did not significantly influencethe production and function of the inhibitory factor in its98interaction with root growth. Soft rot erwinias are well knownto associate with potato crops wherever they are grown and theroot systems are extensively covered with the bacteria (Peltzerand Sivasithamparam 1985; Kloepper 1983). A combination of allthese factors will result in root growth inhibition in the fieldplants. Although stunting occurred in both systems, the factorscausing the recorded effect may not be the same. This conclusionwas based on my observation that, in the tissue culture system,the cuttings and roots were often covered with bacterial oozewhich definitely affected the nutrient uptake by the cuttings,perhaps contributing to a certain extent to the stunting ofplantlets. In my pot experiments, stunting and dramaticinhibition of root elongation appeared to be associated with seedpiece decay. If it is assumed that the nutrient deficiency ofplants was the cause of small plants, then, stunting may be theresult of inhibited root elongation. However, we can not ignoreanother possibility that a toxin from soft rot erwinias could beinvolved in the induction of the retarded stem growth at the sametime.The other minor symptoms induced in micropropagated potatoplantlets by the pectolytic erwinia strains, including aerialroot formation, root tip browning and blackening, rotting andlesions on foliage, were not evident in potato plants growing insoil inoculated with E. c. subsp. atroseptica after 6 days ofgrowth. This discrepancy in symptom expression in potato in99different systems could be the result of the differentinteractions and responses of the plants and the bacteria to eachother.The results of my study provide a possible solution to theunknown cause of the well established yield reduction of potatocrops growing in short potato—rotation soil in comparison tolong—potato rotation soil. In the Netherlands, the analysis ofthe cause of yield reduction in short potato rotation reached theconclusions that the increased potato cropping frequency promotedmicrobial activities harmful to potato root growth and a unknownmicrobial factor was involved in yield reduction (Schippers etal. 1985) . A bioassay carried out in the greenhouse by usingpotato stem cuttings revealed that root growth was inhibitedwithin two weeks in short potato—rotation soil in comparison tolong potato—rotation soil (Bakker et al. 1987) . Bakker andSchippers (1987) discovered that potato root growth was verysensitive to cyanide produced by soil fluorescent pseudomonadsand that 5 pM HCN inhibited cytochrome oxidase respiration by atleast 40 % in intact potato roots in vitro. My results obtainedfrom both in vitro and soil experiments indicate that roots ofpotato plants can be dramatically inhibited by soft rot erwiniastrains. In considering the fact that the pectolytic Erwiniaspp. are widely distributed in the potato production areas of theworld and most seed stocks are contaminated by more than onestrain of these (Perombelon 1972; Nielson 1978), it is very100likely that soft rot erwinias are responsible for the yieldreduction by directly inhibiting root growth.Plant growth promotion by tuber bacterization withfluorescent pseudomonads has been attributed to the suppressionof growth inhibiting rhizosphere microorganisms (Schippers et al.1985; Geels and Schippers 1983) . Some of these pseudomonads areantagonistic to erwinias (Rhodes and Logan 1986). Kloepper(1983) reported that E. carotovora is a common root zoneinhabitant of potato and seed tuber bacterization withfluorescent pseudomonad PGPR strains, which were previouslydemonstrated to increase significantly potato yield in fieldtests, causes significant reduction of root zone population of E.carotovora. Rhodes and Logan (1986) demonstrated that the effectof E. carotovora on the development of small plants and theincidence of blackleg were partially reversed by treatments withfluorescent pseudomonads and suggested that the enhanced plantgrowth may be due to a reduction in numbers and activity of E.carotovora. Furthermore, based on my results, I suggest that thepromotion of plant growth is accounted for by the reversal of theroot growth inhibition resulting from the growth suppression ofsoft rot erwinias on plant roots by antagonistic pseudomonads,resulting in an increase in tuber production. Furtherexperiments are still necessary to be certain that the growthpromoting pseudomonads can alleviate the inhibition of rootelongation by soft rot erwinias in soil environments.101Nevertheless, my experiments provide a possible new solution tothe mechanism of plant growth promotion by fluorescentpseudomonads.My experiments have demonstrated that root growth of potatoplants growing in both in vitro and in soil was inhibited by softrot erwinias. Moreover, it is a bacterial metabolite of Eca 31that inhibits root growth of potato plants. I recognize anotherpossible role of soft rot erwinias acting as a deleteriousrhizosphere microorganism inhibiting root growth by a toxicmetabolite without parasitizing root systems. At the same time,pectolytic erwinias may also function as a pathogen inside theplants causing foliage symptoms. This assumption is supported bymy electron microscopic finding that a large bacterial populationwas present on the root surface of the micropropagated potatoplantlets. Moreover, by using immunofluorescent and irnmunogoldstaining techniques, Underberg and Van Vuurde (1989) detectedlarge populations of E. chrysanthemi on root surface of fieldgrown potato plants. The toxic metabolite from the pectolyticErwinia spp. may serve as a “pathogenicity factor” to inducedisease in potato. Furthermore, it would be very interesting toknow whether the metabolites from other plant pathogenicbacterial species inhibit root growth. Nevertheless root growthinhibition would be recognized as a new symptom of the diseasedpotato plants by soft rot erwinias, once it was confirmed byfield experiments.102The symptoms in micropropagated plantlets caused by soft roterwinias have been documented extensively. The symptomdescription will be very helpful for identifying and screeningout the contaminated potato tissue cultures in research andcommercial laboratories where bacteria and bacteria—likecontaminants are a big problem (Debergh and Vanderschaeghe 1988).This study focused on the response of micropropagated potatoplantlets to a particular bacterial species in vitro in order tobetter understand the in vitro capacity of a pathogen in causinga disease. The comparison of symptoms induced by soft roterwinias in vitro and in soil environments indicates the extentto which tissue culture systems could be used to study thediseases of the plants in soil. Further search into the propertyof a factor causing disease symptoms at the molecular level couldonly be easily carried out by using plant tissue culture systemsand would contribute to our knowledge and understanding of therole of the microbial factor in plant pathogenesis.103LITERATURE CITEDAllan, E., Kelman, A. 1977. 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