Physiology and Biochemistry

Mechanism of Wilt Induction by Amylovorin in Cotoneaster Shoots and Its Relationto Wilting of Shoots Infected by Erwinia amylovora

Thomas M. Sjulin and Steven V. Beer

Former Liberty Hyde Bailey Research Assistant and Associate Professor, respectively, Department of PlantPathology, Cornell University, Ithaca, NY 14853.

Present address of senior author: Department of Plant Pathology, University of Illinois, Urbana, IL 61801.We thank R. N. Goodman, University of Missouri, Columbia, for gifts of amylovorin.Accepted for publication 20 June 1977.

ABSTRACT

SJULIN, T. M., and S. V. BEER. 1978. Mechanism of wilt induction by amylovorin in cotoneaster shoots and its relation to wilting ofshoots infected by Erwinia amylovora. Phytopathology 68:89-94.

The effects of amylovorin, a wilt-inducing polysaccaride higher than those of noninoculated shoots. Water potentialsisolated from fire blight ooze, on the water relations and cell of apparently healthy tissues distal from infected tissues inpermeability of Cotoneasterpannosa shoots were not related shoots inoculated 7 cm behind the shoot apex were notto changes observed in shoots infected by Erwinia amvlovora. significantly different from comparable tissues of non-Rates of solution uptake and transpiration by shoots in inoculated shoots. Electrolytes were lost at a much higheramylovorin solution were less than in water. Water potentials rate from wilted tissues of infected shoots than from healthyand water conductance of stem segments were reduced tissues of noninoculated shoots. We conclude thatsignificantly in amylovorin-wilted shoots relative to non- amylovorin induces wilt by restriction of water movement inwilted shoots. The effect of amylovorin solutions on water xylem, rather than by a directly toxic effect as previouslypotentials could be reproduced by dextran solutions or by suggested. However, the changes in water relations of shootssealing shoot bases with wax. Electrolyte loss from wilted infected by E. amy'lovora suggest that wilt of such shootstissues of shoots exposed to amylovorin was no different than results from a disruption of membrane integrity, rather thanloss from nonwilted tissues of shoots held in water. In by restriction of water movement.contrast, water potentials of infected shoots were significantly

Erwinia amylovora (Burrill) Winslow et al., causes E. amvlovora-infected shoots. Preliminary reports of thisrapid blighting of blossoms and vegetative shoots of work have been published (22, 23).susceptible plants. The first symptoms of the disease (fireblight) are water-soaking and wilting of affected tissues; MATERIALS AND METHODSnecrosis follows. A sticky exudatc or ooze containingbacteria often is associated with infected tissues (8). Production and isolation of amylovorin. - Amylovorin,

Previous workers have reported that bacteria-free which was used in all experiments unless stated otherwise,preparations from ooze or E. amvlovora-infected tissues was isolated from ooze produced on immature (2-to 3-cminduce wilting of detached shoots (9, 10, 11, 17). Goodman diameter) pear fruits (PFvrus communis 'Bartlett')et al. (10) isolated from ooze a high-molecular-weight inoculated with E. amy'lovora isolate 27-3 (20). Thepolysaccharide, amylovorin, which was characterized as a procedures of Goodman et al. (10) were used for isolationhost-specific toxin. When shoots of rosaceous plants and purification of amylovorin, with the followingwere placed in dilute amylovorin solution, they wilted in a modifications. Ooze was collected and diluted with 1.0 Mperiod of time that was negatively correlated with their sodium chloride, instead of water, to enhance thesusceptibility to E. amvlovora in the field. Shoots of six subsequent precipitation of polysaccharide with ethanol.nonrosaceous species tested did not wilt in amylovorin The polysaccharide solution was dialyzed against 20solutions. Based on observations of increased uptake of volumes of water (changed once) at 4 C for 48 hr prior toEvans' blue dye by cells of amylovorin-treated shoots, lyophilization.Huang and Goodman (12) suggested that wilting induced Comparison of amylovorin preparations. - Com-by amylovorin solutions resulted from increased parative analyses of the polysaccharide isolated frompermeability of membranes to solutes. infected pear fruits and of authentic amylovorin [isolated

Polysaccharides and glycopeptides induce wilting by from infected apple (Malus pumila 'Jonathan') fruits andeither disrupting membrane semipermeability or physi- kindly provided by R. N. Goodman] were made to deter-cally restricting water movement (6). The purposes of the mine if the two preparations were similar.present studies were to determine the mechanism by Polysaccharide samples in 1 mM Na_,EDTA (pH 7.0)which amylovorin induces wilting in detached shoots, and were applied to 2.5 x 40 cm columns of Sepharose 6Bto relate this mechanism to the water relations of (Pharmacia Fine Chemicals, Uppsala, Sweden). Samples

were eluted by gravity flow with I mM Na2EDTA (pH0032-949X/78/000013 803.00/0 7.0) at approximately 0.5 ml/min. Fractions were collectedCopyright © 1978 The American Phytopathological Society, 3340 at 2-min intervals and assayed for carbohydrate by thePilot Knob Road, St. Paul, MN 55121. All rights reserved. anthrone reaction (25). Column void volumes were

89

90 PHYTOPATHOLOGY [Vol. 68

determined with Blue Dextran 2000 (Pharmacia Fine extended through rubber seals to the outside of theChemcials). Elution peaks of the infected pear polysac- chamber. The water potentials of E. amilovora-infectedcharide were tested for biological activity. Fractions with shoots were determined 3 days after they had beenanthrone activity were pooled, dialyzed against 100 inoculated 0.5 cm from the apex.volumes of water (changed once) for 24 hr at 4 C, and Water conductance in stem segments. - Water con-lyophilized. Solutions of the recovered polysaccharide ductance by stem segments was measured by a method(100 pLg/ml) were compared with 100 tg/ ml solutions of similar to that used by Van Alfen and Turner (26).polysaccharide not subjected to gel filtration for the Immediately after a water potential determination, theability to wilt shoots as described below. 10-cm segment located 5 to 15 cm behind the apex was

The polysaccharides also were compared in com- removed under water and placed in the pressure chamberpositional studies to be published elsewhere (Beer et al., with the basal end immersed in water which had beenunpublished). Preparations of both polysaccharides were membrane-filtered (0.45-Am pore size). The apical endsubjected to methanolysis and the trimethylsilyl deriva- protruded from the chamber and was connected to atives of the resulting products were separated and esti- horizontal I-ml pipette (graduated in 10-pliter divisions)mated quantitatively by gas-liquid chromatography. An with a short length of tubing filled with water. Pressureantiserum prepared in rabbits against the polysaccharide within the chamber quickly was increased to 2.07 bars,from pear ooze was used to compare both polysac- and the time required to pass 10 pliters of water throughcharides in immunodiffusion assays. the segment was recorded after a constant rate was

Plant material. - Cotoneaster pannosa Franch. was achieved.used in all experiments because of its susceptibility to Cell permeability measurements. - The effect ofE. am'i'ovora (24) and the ease with which uniform amylovorin solutions on cell permeability was estimatedvegetative shoots could be produced in a greenhouse. by measuring electrolyte loss from leaf disks (4).Plants were grown from rooted cuttings in a peat: Cotoneaster leaf disks, 8mm in diameter (0.3 g fresh wt),vermiculite mix (1:1, v/v) at 24±3 C with a photoperiod of were bathed for 6 hr in 30 ml of 0. 1 mg/ ml amylovorin,at least 14 hr and sufficient water and fertilizer to 1.0mg/mlamylovorin, ordeionizedwaterat25-±0.5Conmaintain vegetative shoot extension. a rotary (180 rpm) platform shaker. After a quick rinse

Pairs of visually uniform shoots on individual plants with deionized water, disks were transferred to 30-mlwere selected for inoculation to obtain infected plant portions of deionized water and shaking was continued.material. One shoot of each pair was inoculated with a The electrical conductance of the bathing solution wassuspension of E. amy'lovora (isolate 27-3) from a 24-hr measured with a conductivity bridge and cell (Models 31nutrient agar slant culture. Bacteria were introduced into and 3403, respectively, Yellow Springs Instrument Co.,shoot apex or stem tissues with a No. 3 insect mounting Yellow Springs, OH 45387) at 30-min intervals..pin. The other shoot was wounded with a sterile pin in a Electrolytes that remained in the tissues after bathing insimilar fashion. water for 3.5 hr were liberated by freezing and thawing.

Assays of wilt induction by polysaccharide solutions. Shaking and conductance measurements then were re-- Solutions of amylovorin purified from infected apple sumed until a constant reading was obtained.and pear fruits were tested for their ability to induce wilt The permeability of stem tissues that had been wiltedof detached shoots. Solutions of T-500 Dextran and Blue by amylovorin or by infection with E. am vlovora wasDextran 2000 (Pharmacia Fine Chemicals) were also estimated similarly. Four 0.5-cm segments located 1.0 totested, since amylovorin eluted between these two poly- 3.0 cm behind the shoot apex were removed and placed insaccharides from columns of Sepharose 6B gels (21). 4 ml of deionized water on a wrist-action shaker atVisually uniform shoots (25-50 cm long) were removed 23 ± I C. Electrical conductance of the bathing solutionfrom plants, cut to desired length under water, and was measured at 30-min intervals for the next 4 hr.allowed to equilibrate in distilled water under assay Electrolytes remaining in the tissues were estimated asconditions (23± 1 C, 70% relative humidity and 17 klux) described above.for at least 1 hr before assays were begun. Shoots were RESULTSeach transferred into 2-ml amounts of test solution in a 5-ml vial; at least six shoots were tested for each solution. Comparison of amylovorin preparations. - The poly-Shoots were designated as wilted when the apex bent 90 saccharide isolated from infected pear fruits was eluteddegrees from normal. from gel filtration columns as a single peak which

Measurements of rates of water uptake and transpira- coincided with the peak of authentic amylovorin (Fig. 1).tion. - Transpiration rates were estimated by weighing This elution peak contained 99 ± 5% of the total anthrone-shoots plus their individual vials (± 10 mg) at 40-min positive material that had been applied to the column.intervals. Water uptake rates were estimated also at 40- Solutions of the polysaccharide from pear oozemin intervals by weighing vials after removal of shoots. (100 )ug/ml) recovered from the column induced wiltingEvaporative losses from vials without shoots were of 10-cm-long cotoneaster shoots in 95 min, whereas 100negligible throughout the experiment. yig/ml solutions not subjected to gel filtration induced

Water potential determinations. - Water potentials of wilting in 142 min (no significant difference, P=0.05).shoots were determined by the pressure chamber method Compositional studies (to be reported in detail else-(2, 19). Apical 5-cm portions were removed from shoots where) indicated that the methanolysis products ofand placed immediately into a pressure chamber (Model authentic amylovorin and the polysaccharide isolated1000, PMS Instrument Co., Corvallis, OR 97330) such from pear ooze did not differ. The chromatograms fromthat the lower 2-cm portion (with leaves removed) both preparations had seven major peaks. The retention

January 1978] SJULIN AND BEER: ERWINIA/COTONEASTER/AMY LOVORIN 91

times of each peak, relative to an internal standard, were bases with wax (Tissuemat, melting point 52.5 C, Fishersimilar for both preparations. The shape and corrected Scientific, Rochester, NY 14624).area of each of the seven peaks from authentic Wilting induced by amylovorin solutions was similar,amylovorin and from the polysaccharide from pear ooze but not identical, to wilting observed in shoots infectedwere similar. These results indicated that the two with E. amvlovora. Wilting of shoots inoculated withpreparations contain the same components and in the E. amylovora 0.5 cm from the apex was restricted to thesame relative proportion. Galactose, glucose, and succulent tissues of the stem. Loss of turgor and internalglucuronic acid were identified in both preparations by water-soaking of apical stem tissues, but not of leaf tissue,cochromatrography of the treated polysaccharides with were apparent in all shoots 3 days after inoculation. Whenauthentic sugars and uronic acids. Authentic amylovorin shoots were inoculated with E. amvilovora 7 cm from thereacted with antiserum produced against the polysac- apex, no wilting was observed in tissues distal to the pointcharide from pear ooze in a manner similar to that of the of inoculation after 5 days, despite the appearance ofhomologous combination, no spurs formed when the bacterial exudate at several locations on the stem and, inpolysaccharide from pear ooze was placed in wells some cases, partial collapse of the stem at the point ofadjacent to those that held authentic amylovorin in inoculation.microslide double-diffusion assays in agar gels. Effect of amylovorin on shoot water relations. - Rates

Appearance of amylovorin-wilted shoots and shoots of solution uptake and transpiration by shoots decreasedinfected with Erwinia amylovora. - Wilting induced by in amylovorin solutions relative to rates in water (Fig. 2).amylovorin solutions in cotoneaster shoots was visibly The mean rate of solution uptake by shoots placed in 1.0expressed as a loss of turgor by the succulent tissues of the mg/ml amylovorin solutions was significantly lowerstem. Although it was not visibly expressed, loss of leaf (P = 0.05) after 80 min of treatment than that of shootsturgor (as determined by touching the leaves) apparently that remained in water. Transpiration rates of amy-occurred in amylovorin-wilted shoots. Wilting induced lovorin-treated shoots were less than water-treated con-by amylovorin solutions was indistinguishable from trol shoots after 120 min; a significant differencewilting induced by dextran solutions or by sealing shoot (P = 0.01) was noted after 160 min. Shoots in amylovorin

solutions wilted in an average of 168 min during thisassay.

The wilt-inducing ability and effect on shoot water-0 E potentials of 100 ttg/ml amylovorin solutions were>0 compared to those of 100 Mig/ml solutions of dextrans

0.6 of similar size (Table I). There were no significantdifferences in the wilt-inducing ability of solutions ofamylovorin prepared from ooze from infected apple orpear fruits or of Dextran T-500. Water potentials of0.5 - o0 shoots wilted in all polysaccharide solutions tested were

E °cq significantly more negative (P = 0.01) than the watera 0o 0 potentials of nonwilted control shoots, but were not60.4 0

Lu 00 c 110 .110-Z . A Bca. 100 100

On 0 < 90- 90.co 0o

0.2 8 0 6 oo o80 80

00 0* 70. 700.1 ° 0 o

O 0 1uOO 60' 60

0 ao 50 500.0 0 *-, op IOQJ4 0H 2 0 0H 2 0

50 60 70 80 90 4061 o 40J040ELUTIO N VO LUM E (m l) 0 40 80 1.20 16"0'l • ! • - 0 40o 80 120 160

TIME IN MINUTES TIME IN MINUTESFig. 1. Elution profiles of authentic amylovorin (closed Fig. 2.-(A, B). Rates of A) water uptake and B) transpiration

circles) and the polysaccharide isolated from Erwinia amvlovora- by 10-cm Cotoneaster pannosa shoots in water or 1.0 mg/mlinfected P irus communis 'Bartlett' fruits (open circles). amylovorin solution. Rates are expressed as a percent of thePreparations (1.0 mg) were applied to a 2.5 x 40-cm column of mean rate of each shoot measured in water for the 3-hr periodSepharose 6B and eluted with I mM Na,EDTA (pH 7.0). before the start of the assay. Data points represent the mean rateColumn void volume was determined with Blue Dextran 2000. for the previous 40 min for four shoots in water and sixteenPolysaccharide content of fractions was determined by the shoots in amylovorin; vertical lines depict the standard error ofanthrone reaction. the means.

92 PHYTOPATHOLOGY [Vol. 68

TABLE I. Effect of amylovorin and dextran solutions, and different from water potentials of shoots wilted by sealing

wax on time to wilt and water potentials of Cotoneasterpannosa their bases with wax.shoots". The water conductance rate of stem segments from

Time to wilt Water potential amylovorin-wilted shoots (9.8 jliters sec-' bar-') was

Treatment (hr) (bars) significantly lower (P = 0.05) than the conductance rate of

Waterh . . . -8.6 A segments from water-treated control shoots (29.9 r/liters

W6Wax 0.'35 A -15.6 B sec-' bar-'). Furthermore, there was a highly significant

Amylovorinc 1.06 A B -15.6 B (P 0.01) and negative correlation between the resistance

Amylovorind 1.85 B -15.5 B to water flow through the stem segments (conductance -')

Dextran T-500 1.67 B -15.7 B and the water potential of the apical tissues of the same

Blue Dextran 2000 3.36 C -15.3 B shoots (Fig. 3).

aCotoneaster shoots (10-cm) were placed in water or 100 Effect of amylovorin on cell permeability. - After

pg/ml polysaccharide solutions or their bases were sealed with exposure of cotoneaster leaf disks to amylovorin solu-

wax. Shoots were held at 23 ± IC, 70% relative humidity and 17 tions for 6 hr, no alteration in their ability to retain solutes

klux. At the time of wilt (when the shoot apices bent 90 degrees could be detected. No significant differences in electolytes

from normal) water potentials were determined by the pressure remaining in tissues were observed among leaf disks

chamber method. Values in a column not followed by the same exposed to water (114 ± 2.8 /Amho), 0.1 mg/ml amy-letter were significantly different (P = 0.05) according to lovorin solutions (113 ± 5.7 /.tmho) or 1.0 mg/ml amy-Duncan's new multiple range test. lovorin solutions (114 + 3.0 pmho).

bOne of eight shoots in water wilted during the assay; the water The permeability of stem tissues wilted by amylovorinpotentials of the seven nonwilted shoots were determined after solutions was compared to that of stem tissues in water-3.0 to 3.5 hr.

clsolated from ooze produced on Erwinia amy*lovora-infected treated shoots (Fig. 4-A). No difference in electrolyte loss

Malus pumila 'Jonathan' fruits by R. N. Goodman. was detected between stem segments from the wilteddisolated from ooze produced on E. amylovora-infected P'rus region of amylovorin-treated shoots and segments from

eommunis 'Bartlett' fruits, comparable positions on water-treated shoots.Water relations of Erwinia amylovora-infected shoots.

- The mean water potential of infected shoots (-2.2 bars)

was significantly less negative (P = 0.01) than the mean

0.0 water potential of noninoculated shoots (-4. 1 bars) in asample of 10 pairs. Water potentials also were deter-mined in shoots 5 days after inoculation at a point 7 cm

y = -0.114x -1.80 behind the apex. The mean water potential of apparently

-2.5 r = -0.957** healthy tissues distal to infected tissues (-2.8 bars) wasnot significantly different (P = 0.05) from that of non-inoculated controls (-3.2 bars) in a sample of five pairs.

-5. Permeability of tissues infected with Erwinia amy-

< lovora. - The permeability of stem tissues from theC0 wilted region of infected shoots was determined 3 days

.-Z5

0B 0 HEALTHY

Z 25 - AMYLOVORIN 25 - INFECTED

0 0O

Ota.•20 20

I -12.15 - 15 -

0 10 -10 -4-15.0 1.-

ýe 5 5 ,-

-17.51 0 1 0 0

0 1 2 3 4 0 1 2 3 4

0 25 50 75 100 125 TIME IN HOURS TIME IN HOURS

RESISTANCE TO FLOW (sec bar /I"I) Fig. 4-(A, B). Electrolyte loss into 4 ml of water of four 5-mmCotoneasterpannosa stem segments from A) nonwilted shoots in

Fig. 3. Water potentials of the apical 5-cm portions of 20-cm water or shoots wilted in 0. 1 mg/ ml amylovorin; and B) healthyCotoneaster pannosa shoots as a function of the resistance to control shoots or shoots infected with Erwinia amylovora. Dataflow of stem segments located 5-15 cm behind shoot apices. Open points are the mean of six replicates [five replicates of healthy

circles represent data from nonwitled shoots in water and closed control in (B)] and the vertical lines depict standard errors. Totalcircles represent data from wilted shoots in amylovorin solutions electrolytes were determined by measuring the conductance of

(100 ygfml). the bathing solution after freezing and thawing the samples.

January 1978] SJULIN AND BEER: ERWINIA/COTONEASTER/AMYLOVORIN 93

after inoculation. Electrolyte loss from stem segments lations similar to those observed in shoots exposed tofrom infected shoots was much greater than that of amylovorin. However, the water relations of shootssegments from noninoculated shoots (Fig. 4-B). Infected exposed to amylovorin were not comparable to those oftissues had lost more than twice as many electrolytes as shoots infected with E. amylovora.had healthy tissues after 4 hr. The absence of wilting or decreased water potentials in

apparently healthy tissues distal to tissues infected byDISCUSSION E. amylovora indicates that significant restriction ofwater movement does not occur in the xylem of infectedThe decreases in water potential, rate of water uptake shoots. The increased electrolyte loss from infected stem

and transpiration, and water conductance of stem seg- tissues indicates that membrane integrity has beenments observed in shoots exposed to amylovorin indicate disrupted. Loss of membrane integrity of part or all of thethat amylovorin induces wilting by restricting water succulent tissues enclosed in the pressure chamber woulduptake, rather than by disrupting membrane semiperme- lower the balancing pressure required to force water in theability or by inducing uncontrolled loss of water from xylem back to the cut surface of the shoot base (3), thusleaves (26, 27). The similar appearance and water poten- explaining the apparent higher water potentialstials of shoots wilted by amylovorin, dextrans, or block- of infected shoots. Localized disruption of membraneage of shoot bases with wax further support the con- integrity in succulent stem tissues would result in theirclusion that amylovorin induces wilt by restricting water loss of turgor even though leaf tissue retained solutesuptake. normally and remained turgid.

The highly significant negative correlation observed The close association of bacteria to host cells showingbetween resistance to water movement in stem segments the first indications of symptoms (1, 13, 15, 16, 18)and the water potential of tissues distal to the segments suggests that factors involved in the early stages of(Fig. 3) suggests that resistance to water flow in stem pathogenesis are quite localized in their effect. Huang andxylem is an important factor in the wilting of cotoneaster Goodman (13) concluded that amylovorin was respons-shoots exposed to amylovorin. The mean resistance to ible for the initial ultrastructural changes observed inwater movement in stem segments from amylovorin- infected tissues, probably by affecting cellular mem-treated shoots was approximately three times greater branes. However, the absence of increased electrolytethan the mean resistance of segments from water-treated leakage from amylovorin-wilted tissues (Fig. 4-A)shoots. Increases in resistance of this magnitude have indicated that significant changes in membrane perme-been considered too small to significantly affect tissue ability had not occurred at the time wilting was apparent.water potentials in most plants studied previously (7). We observed no directly detrimental effects ofHowever, the resistance to water movement observed in amylovorin on host cells that could explain the changes10-cm segments of water-treated cotoneaster shoots observed during the early stages of pathogenesis.(33 sec bar pliter-') is much higher than the resistanceobserved in stem segments of similar length from other LITERATURE CITEDplants such as tomato (approximately 3.0 x 10-3 sec barMliter-') (5, 14). Thus, relatively small increases in xylem 1. BACHMANN, F. M. 1913. The migration of Bacillusresistance of cotoneaster stems might be sufficient to amylovorus in host tissues. Phytopathology 3:3-14.markedly affect shoot water potentials. Furthermore, a 2. BOYER, J. S. 1967. Leaf water potentials measured with arestriction of water movement through stem segments pressure chamber. Plant Physiol. 42:133-137.3.BOYER, J. 5. 1967. Matric potentials of leaves. Plantsuggests that similar restrictions may also occur in the PhYsio.S.4 1327.xylem of the petioles and leaves (26). Therefore, increases 4. DAMANN. K. E., JR., 4. M. GARDNER, and R.P.in resistance to water movement in the total pathway SCHEFFER. 1974. An assay for Helminthosporiumfrom shoot base to leaf cell are likely to be higher than victoriae toxin based on induced leakage of electrolytesthat indicated by measurements of only stem segment from oat tissue. Phytopathology 64:652-654.conductance. 5. DIMOND, A. E. 1966. Pressure and flow relations inConductance of water in stem segments from vascular bundles of the tomato plant. Plant Physiol.amylovorin-wilted shoots did not increase as water was 41:119-131.forced through stem segments. Thus, it would appear that 6. DIMOND, A. E. 1970. Biophysics and biochemistry of thefvascular wilt syndrome. Annu. Rev. Phytopathol.the increased resistance observed in those segments 8:301-322.results from an irreversible blockage of vascular elements 7. DUNIWAY, J. M. 1973. Pathogen-induced changes in hostrather than from transient viscosity effects of the water relations. Phytopathology 63:458-466.polysaccharide solution. Although the site of blockage is 8. EDEN-GREEN, S. J., and E. BILLING. 1974. Fireblight.unknown, direct occlusion of bordered pit membranes of Rev. Plant Pathol. 53:353-365.xylem vessels has been suggested as a likely site of 9. EDEN-GREEN, S. J., and M. KNEE. 1974. Bacterialrestriction of water movement by high-molecular-weight polysaccharide and sorbitol in fire blight exudate. J. Gen.compounds (6). Microbiol. 81:509-512.

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94 PHYTOPATHOLOGY [Vol. 68

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