TENTATIVE IDENTIFICATION AND ITS RELATION TO STUNTING INDUCED BYPSEUDOMONAS SOLANACEARUM'
Received for publication January 12, 1970
J. R. STEADMAN2 AND Luis SEQUEIRADepartment of Plant Pathology, University of Wisconsin, Madison, Wisconsin 53706
ABSTRACT
In tobacco plants inoculated with the wilt-inducing bac-terium, Pseudomonas solanacearum, there was a correla-tion between decreased internode elongation, maximummultiplication of the bacterium, and an increase in thegrowth inhibitor content of stems 4 to 12 days after inocu-lation, as determined by a wheat coleoptile assay. Initialwilting of the upper leaves was also correlated with an in-crease in inhibitor content of these tissues.Application of either the partially purified inhibitor from
tobacco or pure (+)-abscisic acid to roots, terminal buds,or petioles of tobacco plants caused a reduction of internodelength which lasted from 8 to 10 days following a singletreatment. Repeated treatment was necessary to obtaingrowth retardation over a longer period of time.The tobacco inhibitor was tentatively identified as ab-
scisic acid, based on a comparison with authentic abscisicacid on paper, thin layer, column, and gas-liquid chroma-tography. On the basis of optical rotatory dispersion,circular dichroism, and ultraviolet spectra, the tobaccoinhibitor was indistinguishable from abscisic acid. In-creases in the inhibitor content of infected tissues areattributed primarily to abscisic acid although other sub-stances, not separable from abscisic acid by the proceduresused, could also play a role. The inhibitor was not foundin P. solanacearum culture medium.
Stunting is one of the earliest symptoms of many plant diseasescaused by vascular wilt fungi and bacteria and by systemic viruses.Reduced growth of infected plants has generally been attributedto mechanical, nutritional, or toxic factors. Little effort has beendirected toward determining the relationship of endogenous hostgrowth regulators to this phenomenon. Numerous instances ofreversal by gibberellic acid of the stunting which accompanisemany virus infections of plants have been reported (7, 11, 16, 18,25), which suggests that these pathogens interfere with synthesisof gibberellins. An increase in the growth inhibitor content ofplants exhibiting stunting associated with infection by fungi,bacteria, or viruses has been reported (2, 10, 20, 23, 24, 26). In all
1 Supported in part by a United States Public Health Service Fellow-ship to the senior author and by Research Grant GB-8288 from theNational Science Foundation. Published with the approval of theDirector, Wisconsin Agricultural Experiment Station. Project 1474.
2 Present address: Department of Plant Pathology, University ofNebraska, Lincoln, Nebraska 68503.
these instances, the inhibitor had properties similar to that of theinhibitor-d described by Bennet-Clark and Kefford (4). None ofthe studies with plant parasites, however, has presented evidencefor a correlation between stages of stunting and inhibitor contentor on the nature of the inhibitor or inhibitors involved. Mil-borrow (17) has demonstrated that (+)-ABA3 is the majorcomponent of the inhibitor-: zone in chromatograms of extractsfrom many plant species on the basis of chromatographic be-havior and optical rotatory dispersion spectra.We have reported (24) that increases in the content of an
inhibitor, with properties similar to those of inhibitor-fl, appearedto be responsible for the stunting of tobacco induced by Pseudo-monas solanacearwn E.F.Sm. during early stages of pathogenesis.As a continuation of these investigations, the objectives of thisstudy were to determine: (a) if the inhibitor-fl increases are cor-related with the degree of stunting in tobacco infected by P.solanacearum, (b) if inhibitor-,B from tobacco or a known plantgrowth inhibitor, ABA, can cause stunting of tobacco, and (c)the nature of the inhibitor-fl from tobacco.
MATERIALS AND METHODS
Growth of Plants. Seeds of Nicotiana tabacum L. var. BottomSpecial were sown on vermiculite, and seedlings were grown for30 days. Seedlings were selected for uniform height and trans-planted in coarse silica sand in 6-inch pots. Distilled water orHoagland's nutrient solution was applied on alternate days fromthe time of seeding. All plants were grown in growth chambers at28 C, 50%7 relative humidity, and 2000 ft-c provided by SylvaniaGrolux and General Electric cool white fluorescent lamps andMazda incandescent bulbs on a 12-hr photoperiod.
Inoculation Procedures. An isolate of P. solanacearum (K-60)pathogenic to tomato, tobacco, and other solanaceous hosts wasgrown for 48 hr on TZC at 30 C (13, 14). A distilled water sus-pension (109 cells per ml) from this growth was used to inoculatetobacco plants 30 days after transplanting (approximately 24 cmtall) by injecting 0.1 ml of the suspension with a hypodermicneedle inserted at the fifth node at two points 90 degrees apart.Plants were harvested at 2-day intervals during a 12-day incuba-tion period, and each was divided into: (a) apical section (upper3 cm of stem including leaf primorida and all the leaves above thefifth node), (b) lower leaves (leaves below the fifth node), and(c) stem. The tissues were frozen and stored at -20 C untilextracted.
Bacterial Cell Count Procedure. At various intervals during a3Abbreviations: ABA: abscisic acid; BEA: benzene-ethyl acetate-
12-day inoculation period, fresh sections of stems (10-20 cmlong, including the points of inoculation) from three separateplants were weighed and ground in a Waring Blendor for 1 min.The suspension was allowed to settle for1 hr, and the supernatantfluid was diluted with sterile distilled water in a standard logarith-mic series. Two 0.1-ml samples from each dilution up to 10-l'were each pipetted onto the surface of a TZC agar plate. Theliquid was spread over the surface of the medium with a bentglass rod, and individual colonies were counted after 48-hr incuba-tion at 30 C.
Extraction. The procedure for extraction of the growth inhibitorfrom tobacco was adapted from Sequeira and Kelman (23). Forsmall scale extraction, frozen plant tissue (100 g) was ground to afine powder with mortar and pestle at Dry Ice temperature. Thepowdered tissue was slowly added to boiling ethyl acetate (1:1,w/v) and boiled for 15 min. The suspension was strained throughcheesecloth, and the solids were ground in distilled water with aWaring Blendor. After removing the debris by filtration, the waterextract was adjusted to pH 3.7 with 8 N sulfuric acid and extractedwith ethyl acetate. The ethyl acetate fractions were combined, andacidic substances were extracted with 5% (w/v) NaHCO3. TheNaHCO3 extract was adjusted to pH 3.0 and extracted withdiethyl ether. The ether extract was evaporated under vacuum toapproximately 1 ml and stored at -10 C.For large scale extractions, 22 kg of leaves from field-grown
tobacco plants were ground in 1-kg batches in ethyl acetate atDry Ice temperature (1 :1, w/v) with a Waring Blendor, and themixture was allowed to stand at 4 C for 24 hr. The suspension wasthen strained through cheesecloth and extracted as above.
Paper Chromatography. For chromatographic analysis, eachether extract prepared as above was dissolved in an appropriatevolume of methanol-ether (1 :1, v/v) so that 1 ,ul of extract rep-resented 1 g fresh weight of tissue. Samples (1-100 Al) of theextract were spotted on Whatman No. 1 filter paper and separatedwith isopropyl alcohol-concentrated ammonium hydroxide-water (10:1:1) solvent by the descending technique. After thesolvent front had moved 40 cm from the origin, the paper wasdried, and the inhibitor or inhibitors were located by bioassay.The inhibitory zone from each chromatogram was cut into smallpieces, and substances were eluted from the paper by stirring withhot absolute ethanol. The elution procedure was repeated fourtimes, and the ethanol was removed under vacuum. The residuewas dissolved in chloroform, filtered to remove insoluble material,and evaporated to dryness under a nitrogen stream. At this stage,the original 22 kg of tissue yielded 240 mg of crude inhibitorymaterial.Column Chromatography. The crude inhibitory material was
further purified by eluting through a silicic acid column, asmodified from Bulen et al. (6). A 30-g sample of dried silicic acidwas suspended in 120 ml of chloroform and poured into a glasscolumn (2.1 cm inside diameter); after settling, this resulted in abed 17 cm long. After the bed had been washed with redistilledchloroform and benzene, a sample (50 mg) of the crude inhibitorwas applied to the top of the column in a small volume of diethylether. The material was eluted with successive 18-ml volumes ofbenzene mixed with increasing amounts of ether (0-100% in 10%increments). The eluted fractions were evaporated under nitrogen,and each residue was assayed for inhibitory activity. Of the 240mg of crude material, only 8 mg of active inhibitory substanceswere recovered in the 7:3, 8:2, and 9:1 ether-benzene fractions.
Thin Layer Chromatography. Two milligrams of the activefractions obtained by column chromatography were dissolved inmethanol-ether (1:1) and streaked on four 20- X 20-cm platescoated with a 250-nm layer of Silica Gel GF254 (Merck). Pure,synthetic (=)-ABA (obtained through the courtesy of Dr.B. V. Milborrow) was chromatographed on each side of thestreak. The chromatograms were developed in n-butanol:n-
propanol-concentrated ammonium hydroxide-water (2:6:1:2)solvent and inhibitory zones were located by bioassay of a smallportion of the chromatogram.The remaining inhibitory zone was scraped off the plate,
eluted with ethanol, and dried under a nitrogen stream. The inhibi-tor was further purified by TLC with benzene-ethyl acetate-aceticacid (50:5:2) solvent followed by successive (five times) develop-ment with chloroform-benzene-acetic acid (100:100:1) solvent.The inhibitory zone was eluted and dried as before. Three millili-ters of water, adjusted to pH 3.0 with 8 N sulfuric acid, wereadded, and organic acids were extracted with ether.
Gas-Liquid Chromatography. Two to 3 Mg of crystalline (i)-ABA, or of the highly purified inhibitory material from tobacco,were placed in a 2-ml test tube with 0.1 ml bis(trimethylsilyl)-trifluoroacetamide and incubated for 20 min. Gas-liquid chro-matography was carried out with a Packard model 7834 chro-matograph equipped with an electron capture detector and a 4-ftX 1/8-inch Pyrex column packed with 5% Dow Corning 11silicone grease on 60/80 mesh Chromosorb W. A carrier gas(N2) flow of 120 ml/min was used. The silylated extract (0.2 Mul)was injected while the column was at 190 C, the injector at 260C, and the detector at 215 C; development was isothermal at190 C.
Optical Rotatory Dispersion and Circular Dichroism. Theoptical rotatory dispersion and circular dichroism of the purifiedinhibitory substance dissolved in 0.005 N sulfuric acid in ethanolwere determined with a Cary model 60 spectrophotopolarimeterequipped with a model 6002 CD attachment.
Bioassay Procedures. After separation of crude or purifiedextracts from tobacco by paper or TLC, the chromatograms weredivided horizontally into 20 (paper) or 10 (TLC) sections fromthe origin to the solvent front. Sections of similar size were cut orscraped above the line of origin to serve as controls. Each chro-matogram section was placed directly in an assay vial containing2 ml of 2% sucrose solution. In the case of separations by columnchromatography, each fraction was evaporated to dryness,redissolved, and dispensed into assay vials in diethyl ether. Theether was evaporated before adding 2% sucrose. Although thesucrose solution was not buffered, the pH remained close to 5.5throughout each assay.A wheat coleoptile straight growth test, adapted from Walker
et al., (27), was used for the bioassay. Seeds of wheat (Triticumaestivum L. var. Atlas 66) were washed in running tap water for2 min and soaked in distilled water for 2 to 3 hr. The seeds werethen planted in moist, muck soil and incubated in the dark at24 C and 90% relative humidity for 70 hr. Coleoptiles (25-30mm long) were placed in a Van der Weij microtome (19) and 2mm at the apices were cut off; the 4-mm portions immediatelybelow were used for the bioassay. Cutting was done under a greenfluorescent safe-light. Coleoptile sections were floated in distilledwater, and 2 to 3 sections, selected at random, were placed in eachassay vial. At this time, the average initial length of the coleop-tiles was recorded. The vials were stoppered and placed horizont-ally on a wheel rotating at 1 rpm.
After 20 hr of incubation in the dark at 24 C the coleoptileswere removed from the vials and measured under a dissectingmicroscope fitted with an ocular micrometer. Percentage inhibi-tion of growth was calculated by reference to growth of the con-trols. From a statistical analysis (least significant difference) ofseveral experiments, it was determined that a reduction in coleop-tile extension of 20% below the control value was significant at the1 % level of confidence. In this assay, a parabola described therelationship between coleoptile response and increasing concen-trations of the tobacco inhibitor (extractable from 2-100 g freshwt) (24), or pure (±)-ABA (from 10-300 ng/ml).
Culture Techniques. P. solanacearum was grown in a tobaccomedium prepared by steaming 400 g (fresh wt) of stems and leaves
FIG. 1. A: Reduction in stem length from the fifth node to the apex in P. solanacearum-infected tobacco plants at various time intervals afterinoculation. Each point is an average of eight determinations; the vertical bars represent the range in values obtained. B: Populations of P.solanacearum in tobacco stem sections (10-20 cm long) at various time intervals after inoculation. Each point is an average of four determina-tions; the vertical bars represent the range in values obtained.
of 6-week-old plants for 30 min. The tissue was ground in 2000 mlof double distilled water for 3 min in a Waring Blendor. Theresulting suspension was filtered through four layers of cheese-cloth and centrifuged for 20 min at 25,000g. One gram each ofcasamino acids (Difco) and yeast extract was added to the super-natant fluid which was then filtered through a Millipore HA (0.45ju pore) filter and dispensed in 400-ml aliquots in Fernbach flasks.The medium was seeded with a loopful of bacteria from a TZCplate and incubated for 48 hr on a rotary shaker at 32 C in dark-ness. Bacterial cells were removed from the culture medium bycentrifuging at 4,100g for 30 min. Inhibitors were extracted fromthe supernatant fluid by the small scale procedure outlined above.
RESULTS
Stunting and Pathogen Multiplication. Two days after tobaccoplants had been inoculated with P. solanacearwn, the onlyobservable symptom was a slight, but measurable, reduction instem length above the fifth node (Fig. IA). Height differencesbetween healthy and diseased plants became more pronouncedfrom 2 to 8 days after inoculation; by 10 to 12 days, infectedupper stem internodes were only 37% as long as healthy inter-nodes.The number of bacteria present in tobacco stems increased
logarithmically during the early stages of infection. From 2 to 8days after inoculation, there was a rapid increase in bacterialnumbers resulting in more than a 1000-fold increase over theinitial population (Fig. IB). This increase in numbers of bacteriaappeared correlated with increased stunting of the plants.
Inhibition by Extracts from P. solanacearum-infected TobaccoPlants. The data in Table I provide an example of the type ofcalculations and statistical analysis that were required to compareinhibition caused by extracts from healthy and diseased tissues.For simplification of these data, an arbitrary unit basis was usedas in Figure 2. A 25% reduction in coleoptile growth was con-sidered 1 inhibition unit; since assays from four samples from eachextract were pooled, maximum inhibition was 16 units. Inhibitionof coleoptile growth was detected only at RF values between 0.60
and 0.75 in chromatograms of extracts developed with IAWsolvent.
Healthy or infected tobacco plant parts were harvested at 0, 2,4, 6, 8, and 12 days after inoculation, and, after extraction, theinhibitor content was determined by bioassay. When lower leaves
Table I. Growth of Wheat Coleoptile Sections in Eluates from FiveRF Positions of Chromatograms (JAW Solvent) from Healthyand P. solanacearum-infected Upper Leaves and Terminal Budsof Tobacco Plants 4, 6, and 8 Days after Inoculation
4 Days after 6 Days after 8 Days afterInoculation2 Inoculation2 Inoculation2
FIG. 2. Effect of P. solanacearum infection on inhibition of wheat
coleoptiles by extracts from various parts of tobacco plants at various
time intervals after inoculation. One unit = 25% inhibition in eluates
of chromatograms at four RF values from 0.60 to 0.75, after separationwith IAW solvent. A: Stems; B: lower leaves; C: upper leaves plusterminal bud. Data partially from Table I.
only were analyzed, no statistically significant differences in
inhibitor content were found between healthy and inoculated
plants at any interval after inoculation (Fig. 2B). However, there
was a significant (1% level) increase in inhibitor content in
terminal buds and upper leaves of diseased plants by 6 days after
inoculation. A significant difference in inhibition of coleoptilegrowth over the control remained through 12 days after inocula-
tion (Table I and Fig 2C). Similarly, the inhibitor content of stem
tissue, where the bacterium multiplied most extensively, increased
significantly after infection (Fig. 2A). This increase was significantby 4 days after infection and was of a greater magnitude by 8 and
12 days. No significant changes in inhibitor content were detectedin healthy tobacco tissues during the 12-day incubation period.
When the data were calculated on a whole plant (above groundplant parts) basis, an increasing trend in inhibitor content, evenduring the early stage of infection, was evident.
Production of Inhibitor-,B by P. solanacearum in Culture. After48-hr growth of the bacterium in a tobacco medium, the inhibitorcontent of the culture filtrate was compared with that of sterilemedium by the standard extraction and bioassay procedures.Although the bacterium grew profusely in this medium, no sig-nificant changes in the concentration of inhibitor-,l were ob-served, and no additional inhibitors were found. The resultsindicate that P. solanacearum does not release inhibitors similarto inhibitor-fl, at levels which are detectable by the assay used,in the culture medium provided.
Effect of Inhibitors on the Growth of Tobacco. The effect ofexogenous application of inhibitors on the growth of tobaccoplants was determined by three separate methods, as follows.
1. Uniform 27-day-old seedlings were held upright over rec-tangular trays with the roots immersed in one-fifth strengthHoagland's solution, After 3 days the nutrient solution wasreplaced with either a solution of the crude inhibitor from tobacco(equivalent to 5 g of extracted tissue per ml), abscisic acid (1,ug/ml), or distilled water. Trays were covered with aluminumfoil to protect from light; water lost from the tray was replaceddaily with nutrient solution or distilled water. A second inhibitortreatment at the same concentrations was applied 8 days later.The height of each seedling from the base of the stem to the apexwas measured at 2-day intervals for 16 days. Stem lengths weresignificantly shorter than controls (t test, 5% level) between the2nd and the 10th day in seedlings treated with the tobacco inhibi-tor and with ABA between the 4th and 10th day after initial treat-ment (Fig. 3A). Relative decrease in stem length reached a maxi-mum by 6 days in both cases, but growth was resumed at a ratefaster than normal between the 6th and the 12th day. The secondapplication of inhibitor, however, resulted in a significant decreasein stem elongation only at 4 to 6 days after treatment.
2. A piece of Tygon tubing covered with aluminum foil wasattached to the petiole of the fifth leaf from the base of a 25-day-old tobacco plant and held vertically. Three milliliters of a solu-tion of ABA (9 ,4g/ml), the tobacco inhibitor (equivalent to50 g of extracted tissue per ml), or distilled water was introducedinto the tubing. Uptake of the solution occurred within 2 hr inmost plants. A significant decrease (t test, 5% level) in inter-node elongation was evident by 4 days, and persisted for at least10 days, after treatment with either inhibitor (Fig. 3B). There-after, growth of treated plants resumed at a rate faster thannormal.
3. External application of water solutions of tobacco inhibi-tor (equivalent to 300 g of extracted tissue per plant) or ABA(25 ug/plant) to the terminal buds of 20-day-old tobacco plantsresulted in growth inhibition similar to that obtained with thepetiole treatment (Fig. 3B). When amounts of either inhibitorwere increased 2-fold, the reduction in stem length after 6 dayswas nearly twice that reported for petiole treatment. The resultsof these three separate methods for exogenous application ofrelatively low concentrations of both ABA and the tobaccoinhibitor, subject to considerable dilution effects, suggest thatalterations in the normal content of inhibitors can result inmarked stunting of tobacco plants.
Identification of the Inhibitor from Tobacco. The inhibitor intobacco extracts had an RF of 0.60 to 0.75 after paper chroma-tography (IAW solvent); that of ABA was 0.68 to 0.73. This is inagreement with previously reported RF values for ABA (17).Both ABA and the tobacco inhibitor were eluted from a silicicacid column with 7:3, 8:2, and 9:1 ether-benzene. Furtherpurification of the tobacco inhibitor from these column eluatesby TLC (BPAW solvent) indicated an RF of 0.50 to 0.70, whichwas the range reported for pure ABA (17). A second TLC
Plant Physiol. Vol. 45, 1970 ABSCISIC ACID IN TOBACCO PLANTS
2 4 6 8 10 12 14 16
DAYS AFTER TREATMENT
-
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Z
UI °
-i
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0 2 4 6 8 10 12
DAYS AFTER TREATMENT
FIG. 3. A: Growth of tobacco seedlings treated with ABA (1 pg/ml solution) and partially purified inhibitor extracts (equivalent of 5 g oftissue extracted per ml of solution). Seedlings were 27 days old when transferred to one-fifth strength Hoagland's solution and 30 and 38 daysold when inhibitors were added. Each point is the average of 12 measurements; data are cumulative from day zero. B: Reduction in stem lengthof tobacco plants from the fifth node to the apex at various times after treatment with ABA (27 Og per plant) or partially purified tobacco in-hibitor extracts (equivalent of 150 g of tissue extracted per plant). Tobacco plants were 24 days old from date of transplanting, when the in-hibitors were applied through the fifth petiole. Each point is an average of four determinations.
RETENTION TIME (M N)
FIG. 4. Gas chromatograms obtained after injection of 0.2 PI of thesilylated derivatives of: a: pure (+)-ABA (3 ,ug); b: tobacco inhibitor(2 IAg) eluted from TLC plates Rp 0.11 to 0.16 developed with BEAsolvent; c: tobacco inhibitor (2 ,ug) eluted from TLC plates RF 0.17to 0.22 developed with BEA solvent. Five per cent Dow 11 on Chromo-sorb W; 190 C; 120 ml N2/min; electron capture detector.
separation of the tobacco inhibitor (BEA solvent) resulted intwo inhibitory zones which absorbed short wave ultravioletlight; that at RF 0.11 to 0.16 was nearly 20 times as active bio-logically as that at RF 0.17 to 0.22. The same solvent system hasbeen reported (21) to separate the cis,trans-isomer of ABA(RF 0.15) from the less inhibitory trans,trans-isomer (RF 0.21).Gas chromatograms of the silylated derivative of the tobacco
inhibitor eluted from thin layer plates at RF 0.11 to 0.16 (BEAsolvent), as well as of pure ABA, showed a sharp peak (presum-ably due to the cis,trans-isomer of ABA) at a retention time of4.8 min (Fig. 4, a and b, respectively). The silylated derivative
of the inhibitory substance eluted at RF 0.17 to 0.22 (presumablythe trans, trans-isomer) showed only small peaks at 4.8 and 6.8min (Fig. 4c). Davis et al. (9) have shown that gas-liquid chroma-tography of the silylated ABA can be used to separate these twoisomers, although by means of different liquid phases. Thesilylating agent used (BSTFA) showed a small peak at a reten-tion time of 6.7 min, which interfered with resolution of thetrans, trans-isomer.The ultraviolet absorption spectrum of the inhibitory material
eluted from the final thin layer separation (CBA solvent) anddissolved in 0.005 N ethanolic sulfuric acid exhibited a broadpeak at 263 nm, with a shoulder at approximately 245 nm.Pure ABA, dissolved in the same solvent, had a peak at 260 nmwith a shoulder at 245 nm, in agreement with previous reports(8, 17).The optical rotatory dispersion curve of the inhibitory material
from tobacco eluted from thin layer plates at either RF 0.11 to0.16 or 0.17 to 0.22 (BEA solvent) and dissolved in acidifiedethanol showed a strong positive cotton effect. The peaks oc-curred at 289 nm (positive rotation) and 245 nm (negative rota-tion) with zero rotation at 267 and 219 nm. The profile corre-sponds quite well with that of (+)-ABA reported by Milborrow(17). Since very few substances possess an optical activity ap-proaching that of ABA, these results constitute the most posi-tive evidence regarding the identity of the tobacco inhibitor.The circular dichroism curve of the tobacco inhibitor dis-
solved in acidified ethanol indicated a positive maximum at 262nm and two negative maxima at 230 and 318 nm. The maximumat 318 nm exhibited approximately 8% of the rotational strengthof that at 230 nm. The relative rotational strength and wavelength of the maxima are in agreement with those reported byMilborrow (17) for ABA.
DISCUSSIONThe stunted growth characteristic of tobacco plants during
early stages of pathogenesis by P. solanacearum appears to be
correlated with significant increases in the content of a growthinhibitor. There was an apparent correlation between decreasedinternode elongation and an increased inhibition obtained withextracts from stems 4 to 12 days after inoculation. In the lowerleaves, on the other hand, no symptoms were present in the earlystages of the disease, and, correspondingly, there was no detect-able change in inhibitor content. These observations suggestthat an increase in inhibitor content could cause the decreasedinternode elongation observed during early stages of infection.The appearance of the first symptoms of wilting of tobacco
plants was also correlated with an increase in inhibitor contentin the upper leaves plus terminal bud. Pegg and Selman (20)also observed a marked increase in the inhibitor-3 content oftomato plants which had wilted as a result of infection by Ver-ticillium albo-atrum. Wright (28) noted an increase in the in-hibitor content of detached wheat leaves 40 min after the onsetof wilting caused by passage of a warm air stream over the leaves.The inhibitor increase was correlated with the degree of wilting.It is possible, therefore, that part of the inhibitor increase indiseased tobacco plants may have been due to wilting. How-ever, the inhibitor increased in stem tissues 2 days before theonset of wilting and the increases in leaves appeared correlatedwith movement of the pathogen into those tissues. It is conceiv-able that responses to both the pathogen and to wilting per secontribute to the inhibitor increase in tobacco plants.The lack of a significant increase in inhibitor content of a
tobacco medium after growth of the bacterium is in agreementwith Sequeira and Kelman (23) and suggests that synthesis ofinhibitor by the pathogen does not occur in vitro under the con-ditions used. Similarly, production of inhibitory substances byV. albo-atrwn and Penicillium italicum in Czapek's medium couldnot be demonstrated (20, 22). These few results suggest thatpathogens may not contribute directly to the observed increasesin inhibitor content of host tissues, although it should be em-phasized that direct evidence from experiments in vivo is notavailable.
Inhibition of stem growth by exogenous application of ABAhas been reported for various plants (1). Our results indicatethat a single application of low levels of either the partiallypurified tobacco inhibitor or ABA can cause a significant re-duction of internode elongation in tobacco plants. Similarly, areduction in seedling growth, which reached a maximum in 6days, was obtained after a single treatment with either inhibitor.Plants appeared to grow at a faster than normal rate after theinitial effects of the inhibitor. When inhibitors were again appliedto the same plants, considerable lag in the response of the plantsfollowed. More mature tobacco plants resumed normal growth12 days after initial treatment with either inhibitor. This is con-sistent with reports that shoot growth can be retarded for pro-longed periods only by repeated exposure to ABA (1).
Progress has been made recently in understanding the physio-logical changes associated with advanced and intermediatestages of pathogenesis by P. solanacearwn in tobacco. Thechanges at early stages remain largely unknown, however.Stunting, one of the earliest symptoms observed, has beenattributed to an interaction of several factors, such as: (a) inter-ference with water movement; (b) decreased supply of nutrients;(c) toxic effects of host or pathogen metabolites; and (d) in-hibitory levels of IAA (5). However, stunting occurs as early as4 days after infection, before any wilting is evident, and neithertoxins nor high IAA levels seem to play an important role atthis stage. As Husain and Kelman (12) have shown, wilting oftobacco occurs 6 to 7 days after infection and is due to accumu-lation of a polysaccharide produced by P. solanacearum whichinterferes with water movement in the vessels. Beckman et al.(3) have shown that before wilting is observed water movementmay be significantly affected. Whether this interference results
in stunting by itself, or whether it creates a sufficient nutrientdeficit to account for stunting, however, has not been demon-strated. A more reasonable explanation for stunting is that it isthe result of an increase in content of natural growth inhibitors.The close agreement of the physical and biological properties
of the inhibitor from tobacco and ABA, especially the ORDspectra, strongly suggests that the two inhibitors are identical.Calculation of the concentration of ABA in tobacco tissuesbased on the first extremum of the ORD curve of purifiedpreparations showed that all of the inhibitory activity could beattributed to ABA. Since no inhibitory activity, other than thatin the inhibitor-,B zone, was evident during chromatographicpurification from healthy or diseased tissues, the major com-ponent of the inhibitor from tobacco must be ABA. It is possible,however, that compounds not separable from ABA by themethods used in this study may contribute to the inhibition inboth healthy and diseased tissues.The concept that growth in plants is under the control of a
balance between inhibiting and promoting hormones is an at-tractive one (24), but conclusive evidence to support it is lackingat present. A recent report by Madison and Rappaport (15)indicates that ABA applications reduce the gibberellin contentof tissues, which suggests a possible mode of action for increasedlevels ofABA during pathogenesis. It is evident that the complexinterrelationships between ABA, ethylene, gibberellins, cyto-kinin, and IAA following infection would have to be elucidatedbefore we can attempt to understand the effects on internodeelongation.
Acknowledgments-The authors wish to express appreciation to Dr. V. Perrin forassistance in obtaining ORD and CD spectra, Mr. Kenneth Schultz for assistance inobtaining gas chromatograms, Mr. Thomas Barbour for assistance in tissue extractionand Mr. Steven Vicen for assistance in the preparation of illustrations.
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