New Phytol. (1993), 124, 505-513

Colonization of wheat {Triticum vulgare L.)by N2-fixing cyanobacteria: III. The roleof a hormogonia-promoting factor

BY M. G A N T A R \ N. W. K E R B Y AND P. ROWELL*

Department of Biological Sciences, University of Dundee DDl 4HN, UK'^Institute of Biology, University of Novi Sad, 21000 Novi Sad, Yugoslavia

{Received 16 November 1992; accepted 8 March 1993)

SUMMARY

Initial colonization of the roots of wheat plants by Nostoc 2S9B in liquid culture was by motile hormogonia,irrespective of whether co-culture was started using a cyanobacterial inoculum consisting of hormogonia or ofaseriate packages. In the latter case the aseriate packages differentiated into hormogonia and this was most rapidand extensive when co-culture was carried out in NO3 -free BG-11 medium. Hormogonia-promoting activityappears to be due to a plant product that can be extracted from roots and is released into the growth medium inthe presence or absence of the cyanobacterium. Induction of hormogonia was inhibited by 17 mM NaNOj, theconcentration in BG-11 medium. This effect was apparently not due to the presence of a combined N source(NO3") per se but appears to be an ionic effect on the functioning of the hormogonia-promoting factor, since othersalts (NaCl and KNO3) had the same effect as NaNO3. The presence in aseriate packages of an autoinhibitor ofhormogonia differentiation is inferred from the effects on hormogonia differentiation of washing aseriate packages.The hormogonia-promoting factor from wheat roots appears to act antagonistically with respect to theautoinhibitor of hormogonia differentiation. Following initial colonization of plants roots by hormogonia, thecyanobacterial filaments progressed through a complete developmental cycle of hormogonia, long filaments andtightly bound aseriate packages, with subsequent differentiation of hormogonia from these aseriate packages givingrise to further colonization of the growing roots. The attachment of Nostoc 2S9B to the root surface may involvean initial attachment of hormogonia via fimbriae, and a subsequent tight binding of the cyanobacterium by meansof the mucilaginous sheath.

Key words: Colonization, cyanobacterium, differentiation-promoter, hormogonia, Nostoc, wheat.

attachment of cyanobacteria to the root surface, asINTRODUCTION ^^jj ^^ penetration into the roots, was characteristic

Cyanobacteria are phototrophic prokaryotes that of some Nostoc isolates and that hormogonia wereoccur in both free-living and symbiotic forms. They involved in the initial colonization of roots,form a few symbiotic associations with plants. In most of the symbiotic associations betweenincluding some species of bryophytes, pteridophytes, cyanobacteria and plants it appears that every plantgymnosperms and angiosperms (Stewart, Rowell & has to be infected with the cyanobacterium de novo.Rai 1980) ^0o//«, where the cyanobacterial symbiont is present

There is much interest in creating artificial throughout the life cycle, may be an exceptionassociations between higher plants and N.j-fixing (Braun-Howland & Nierzwicki-Bauer, 1990). Thus,microorganisms (Al-Mallah, Davey & Cocking, the infection process will probably depend on1989; Cockmget al., 1990; Quispel, 1990), including motility in the cyanobacterium, which may, at leastcyanobacteria (Gusev & Korzhenevskaya, 1990). We partly, explain why cyanobacteria associated sym-have described the colonization of wheat roots by biotically with plants are almost invariably Nostoccertain N.^-fixing cyanobacteria when co-cultivated species (Peters, 1990; Vagnoli et al., 1992). Thein a liquid medium (Gantar, Kerby & Rowell, possession of hormogonia, which is the main charac-1991 a; Gantar et al., 1991 b). It was shown that firm teristic that separates the genus Nostoc from Ana-

baena, may be important in this respect, since

* To whom correspondence should be addressed. hormogonia are usually motile. Hormogonia are

34-2

506 M. Gantar, N. W. Kerby and P. Rowell

filaments that are distinguishable from the parentaltrichome by at least some of the following character-istics: cell size, cell shape, gas vacuolation and theabsence of heterocysts even when grown without asource of combined nitrogen (Herdman & Rippka,1988).

In this paper we report on the process ofcolonization of wheat roots in relation to thedevelopmental cycle of Nostoc 2S9B, with emphasison the role of hormogonia and effects of a hormo-gonia-promoting factor produced by the plant.

MATERIALS AND METHODS

Organisms and growth conditions

Nostoc strain 2S9B was isolated from solonetz soil inthe province of Vojvodina, Yugoslavia (Gantar,Obreht & Fojkar, 1991 c). It was routinely grown inbatch culture in BGll medium with or without17 7mMNaNO3 (Rippka et al., 1979), at roomtemperature (approximately 24 °C), in continuouswhite light at a photon fluence rate of 20 /imol m"'̂ s~̂and harvested after 20 d. The wheat (Triticumvulgare L.) was a gift from the Institute of Field andVegetable Crops, Faculty of Agriculture, Universityof Novi Sad, Yugoslavia. Three days after ger-mination, wheat seedlings were suspended with theirroots in 600ml glass vessels containing BGllmedium with or without NOg". Cultivation wasthen continued in a growth cabinet with con-stant illumination at a photon fluence rate of40//mol m"'̂ s"̂ and at a temperature of 24 °C.

Colonization of roots

Wheat seedlings were grown in NOg'-containingBGll medium and, when the roots were approxi-mately 10 cm long, the plants were transferred tofresh medium which was inoculated at the same timewith Nostoc 2S9B. The inoculum of Nostoc 2S9Bwas from a culture (grown in NOg"-containing BG-11 medium) shown microscopically to consist pre-dominantly of aseriate packages (referred to through-out as a preparation of aseriate packages) and wasadded to give a concentration of 3-5 //g chl a ml"^To obtain a measurement of associated cyano-bacterial biomass, plant roots were excised andextracted with methanol for chlorophyll a deter-mination (MacKinney, 1941). After chlorophyllextraction, the roots were air dried and weighed.Fach result is the mean of measurements made onsix plants.

Hormogonia preparations and pronase treatment

In some experiments colonization was carried outusing hormogonia preparations. These wereobtained by repeatedly washing and resuspending

aseriate packages in N-free BG-11 medium, thenincubating under white light for 24 h. The numberof hormogonia was determined by counting thefilaments using a Neubauer haemocytometer. Inorder to determine the role of fimbriae in attachmentof hormogonia to plant roots, hormogonia prepara-tions were incubated with 0-5 mg ml"^ pronase(Sigma Chemical Co. Ltd., Poole, Dorset) for 1 h,washed and resuspended in N-free medium. Theroots of the wheat plants were suspended in thesepreparations, incubated for 1 h to allow initialattachment of hormogonia, and then washed byrepeated immersion in water. The wheat plants werethen grown for a further 6 d in NOg'-containingBG-11 medium to allow the attached hormogonia todevelop into colonies, before the extent of colon-ization of the roots was determined.

Removal of cyanobacterial biomass from the roots

To assess how tightly cyanobacteria were attached towheat roots at different stages of colonization, thecolonized roots were sonicated in an ultrasonic bath(Cole Parmer, Chicago, Illinois) for 1 min, thenwashed in water. Cyanobacterial biomass remainingattached to the roots was then determined byestimation of chlorophyll a content.

Scanning electron microscopy

Roots were excised, cut into 1 cm lengths andprepared for microscopy as previously described(Gantar et al., 1991a). In the final step of de-hydration, ethanol was replaced by acetone forcritical point drying.

Transmission electron microscopy

In order to observe the presence of fimbriae on thesurface of cyanobacterial cells, hormogonia wereobtained by incubating Nostoc 2S9B in the presenceof a root extract for 24 h, and samples were preparedfor microscopy as described by Vaara & Vaara(1988).

Preparation of root extracts

Root extracts were prepared from the roots of wheatplants grown for 14 d in BG-11 medium with orwithout NOg . Roots were homogenized, using apestle and mortar, in a small volume of the samemedium at a concentration of 0-1 g root wet weightml̂ ^ medium. The homogenate was centrifuged at15 000^ for 20 min and filter-sterilized. The proteinconcentration of the extract was estimated by themethod of Bradford (1976). Samples were dialyzed(12000-14000 M, cut-off) for 24 h against 200volumes of water, taken to dryness using a rotary

Colonization of wheat by N^-fixing cyanobacteria 507

1 3 5 7 10Time after start of co-culture (d)

Figure 1. Progress of colonization of wheat roots byNostoc 2S9B as chlorophyll a associated with roots (#)and the extent of removal of associated cyanobacteria bysonication ( • ) . The plants were placed with their rootssubmerged in a suspension of Nostoc 2S9B consistingpredominantly of aseriate packages in fresh, NO3 -containing BG-11 medium. In each figure, the data shownare the mean + SE for a representative experiment (in somecases error bars are covered by the symbols and are notshown).

evaporator and resuspended in fresh medium to avolume equal to that of the original extract used.

RESULTS

Adhesion of Nostoc 2S9B to wheat roots

Colonization of wheat roots by Nostoc 2S9B in liquidculture, as indicated by the rapid rise in amount ofchlorophyll a per unit of root dry weight (Fig. 1),occurred during the first 3 d of co-culture, afterwhich there was only a slight increase in associatedcyanobacterial biomass, as indicated by the chloro-phyll a content per unit of root dry weight. It should,however, be noted that cyanobacterial growth oc-curred in this latter period with a correspondingincrease in root dry weight (data not shown). Inorder to monitor the adhesion of the cyanobacteriumto the root surface, the roots were subjected to a briefperiod of sonication. Most (66%) of the associatedcyanobacterial biomass could be removed in thisway, one day after the start of co-culture. However,this figure had decreased to only 8 % by the fifth dayof co-culture, indicating a tighter binding to the rootsurface. The readiness with which cyanobacterialbiomass could be removed from the roots increasedthereafter, 25 % of the chlorophyll a being removedby sonication on the tenth day.

Sequence of root colonization

Scanning electron microscopy was used to inves-tigate the involvement of the different stages of theNostoc developmental cycle in the process of colon-ization of the root surface (Fig. 2). Within one day,most of the root surface was covered with motilehormogonia. The hormogonia of Nostoc 2S9B (Fig.

2 a) are straight, non-heterocystous filaments con-sisting of cylindrical cells with conspicuous gasvacuoles that were evident on light microscopy andcollapsed on centrifugation resulting in a loss ofbuoyancy of the filaments. Gliding motility of thehormogonia was observed on light microscopy. Theinitial attachment of hormogonia of Nostoc 2S9B tothe root surface may involve fimbriae, which wereobserved on the surface of hormogonia (Fig. 3).After 2 d, the hormogonia had developed into longfilaments (Fig. 2 b) composed of more or lessspherical cells that were immotile and did not containgas vacuoles. At the next stage, the filaments becameembedded in mucilaginous envelopes (Fig. 2 c) andwere beginning to distort (Fig. 2d). After 7 d, thecyanobacterium was almost exclusively in the formof tightly bound aseriate packages, with firm, well-defined mucilaginous sheaths (Fig. 2e, see also Fig.1). When viewed using the light microscope, hetero-cysts were observed at this stage, although thefilamentous nature of the cyanobacterium was noteasily discerned, due to the tight packing of the cells.After 10 d, the envelopes surrounding the aseriatepackages were disintegrating and hormogonia wereliberated (Fig. 2f), giving rise to further colonizationof the growing roots.

When the co-culture was initiated using Nostoc2S9B predominantly in the form of hormogoniathere was, on average, 20% more cyanobacterialbiomass associated with the roots throughout theperiod of colonization, when compared to inocu-lation with a suspension of aseriate packages. Thefinding that there was only a small difference inthe extent of colonization between these two treat-ments may be explained by our observation thatthere was a rapid liberation of hormogonia fromthe aseriate packages during co-culture. Pre-treat-ment of hormogonia preparations with pronase toremove fimbriae, as evidenced by electron micro-scopy (Fig. 3 b), resulted in a 40% decrease,from 0-123 ±0-012 fig chl a mg"^ dry weight in con-trols without pronase treatment to 0-080 ± 0-006/igchl a mg^^ dry weight, in the extent of colonizationof roots.

Promotion of hormogonia differentiation by a plantproduct

In order to assess the effects of roots or of rootexudate on the liberation of hormogonia fromaseriate packages, the numbers of hormogonia liber-ated were determined after suspending a culture ofNostoc 2S9B predominantly in the form of aseriatepackages in fresh medium, in medium pre-con-ditioned by the previous growth of wheat plants andin fresh medium together with the roots of wheatplants. A rapid and extensive liberation of hormo-gonia occurred under conditions of co-cultivationwith wheat roots in N-free medium (Figs 4 & 5).

508 M. Gantar, N. W. Kerby and P. Rowell

Figure 2. For legend see opposite.

Colonization of wheat by N^-fixing cyanobacteria 509

Figure 5 shows that a maximum concentration of1-38x10' hormogonia ml"' medium was reachedafter 24 h. This was 70-fold higher than the initialconcentration of hormogonia in the preparation ofaseriate packages used and a 10-fold increase com-pared with the maximum concentration reachedwhen the preparation of aseriate packages wasresuspended in fresh N-free medium. Under condi-tions of co-cultivation in NOg'-containing medium,the liberation of hormogonia followed a similar timecourse but was less extensive (50%) than in N-freemedium in the presence of wheat roots. The pre-conditioned, N-free medium promoted the liberationof hormogonia at a lower rate, but to almost the sameextent, as the roots of wheat plants in co-culture(Fig. 5). In contrast, the pre-conditioned, NOg -containing medium was ineffective.

Like the roots of intact wheat plants and pre-conditioned N-free medium, cell-free extracts ofwheat roots also promoted the liberation of hormo-gonia, and the effect was concentration-dependentover the range tested (Fig. 6). Since stimulation ofhormogonia liberation by wheat roots was muchmore pronounced in the N-free medium than in thepresence of NO3", the hormogonia-promoting ac-tivities of root extracts from plants grown in N-freeor NOg'-containing medium, were compared. Onthe basis of the protein concentrations of rootextracts, extracts of the roots of plants grown on N-free medium had a ' specific hormogonia promotingactivity' that was 2-2-fold higher than those from theroots of NOg'-grown plants (data not shown). Afterextensive dialysis of root extracts about 50 % of theactivity was lost, but this lost activity was notrecovered in the dialysate (Table 1).

Although root extracts from wheat plants grown ineither N-free or NOg^-containing medium hadhormogonia-promoting activity, the subsequent ad-dition of NaNOg inhibited this activity (Table 2).The effect of NaNOg therefore appears to beprimarily on the response of Nostoc 2S9B to theplant product, rather than on its synthesis orliberation by the plant. In addition, the effect ofNaNOg is not specifically related to its use as acombined N source, as equal concentrations of theother salts tested had the same inhibitory effect asNaNOg (Table 2).

Autoinhibition of hormogonia differentiation fromaseriate packages

It has been suggested (Herdman & Rippka, 1988)that the liberation of hormogonia is prevented by an

inhibitory compound produced by the cyano-bacterium. Repeated washing of Nostoc 2S9B aseri-ate packages, by centrifugation and resuspension infresh N-free medium, progressively enhanced theliberation of hormogonia up to a maximum con-centration of 1-3x10' hormogonia ml"' (Table 3),which could be explained by the removal of such aninhibitor.

DISCUSSION

The Nostoc strain 2S9B used in this work showed acomplete developmental cycle consisting of hormo-gonia, long heterocystous filaments and aseriatepackages (see Lazaroff, 1973), although in matureliquid culture aseriate packages predominated. Whenthese were co-cultivated with wheat plants in liquidculture, the aseriate packages were to some extenttrapped between the root hairs. However, within24 h, the trichomes in these packages had beentransformed almost completely into hormogoniawhich were attracted towards and migrated over theroots. The hormogonia of Nostoc 2S9B could bedistinguished from the long filaments into whichthey subsequently developed by the absence ofheterocysts, the presence of gas vacuoles and motilityand, at this stage, could easily be removed from theroot surface by mild sonication (Fig. 1). Once thehormogonia had lost their motility and the filamentswere undergoing transition to the aseriate stage,there was a build up of a mucilaginous envelopeaccompanied by a much tighter adhesion to the rootsurface. The mucilaginous sheath appeared to be ofcyanobacterial origin and to be responsible for thefirm attachment of aseriate packages to the rootsurface, since hormogonia do not produce abundantmucilage and were easily removed. The initialattachment of the hormogonia may involve thefimbriae on their cell surfaces. Dick & Stewart(1980) suggested possible roles of fimbriae in motilityand specificity in the Nostoc-Peltigera symbiosis andfimbriae were recently reported to occur on someNostoc isolates from cycads (Lindblad & Bergman,1990). Treatment with the proteolytic enzymepronase, which resulted in the removal of fimbriae,reduced the extent to which hormogonia of Nostoc2S9B colonized the roots of wheat plants. Thecolonization of wheat roots by Nostoc 2S9B thusappears to proceed in two phases: in the first phasehormogonia are weakly attached by means of fim-briae and, in the second phase, the heterocystousfilaments become firmly attached by the mucilagin-ous sheath. Two modes of attachment of Azo-

Figure 2. Scanning electron micrographs of the surface of wheat roots colonized by Nostoc 2S9B, showing therelationship between the developmental cycle of the cyanobacterium and the progress of colonization, at 1,2,3, 5, 7 and 10 d (a-f, respectively) after the start of co-culture in NO., -containing BG-11 medium, a, motilehortnogonia; b, long filaments; c, long filaments developing a mucilaginous sheath ; d, early development of theaseriate stage; e, aseriate packages; f, release of hormogonia (arrow) from aseriate packages. Bar, 10//m.

510 M. Gantar, N. W. Kerby and P. Rowell

(a)

Figure 3. Transmission electron micrographs showing (a) fimbriae on the cell surface of hormogonia of Nostoc2S9B and (b) surface of hormogonia after pronase treatment. Bar, 0 2 //m.

spirillum brasilense to wheat roots have also beenreported, with the first step being dependent onbacterial surface protein and the second step de-pendent on bacterial extracellular polysaccharide(Michiels, Croes & Vanderleyden, 1991).

Motile hormogonia may function in colonizationin many cyanobacteria-plant associations (Stewart etal. 1980; Bonnett & Silvester, 1981; Campbell &Meeks, 1989) and our data indicate their importancein the colonization of the roots of wheat plants inliquid culture. In assessing the ability of isolates ofNostoc and Anabaena to infect Gunnera, Bonnett &Silvester (1981) showed that highest gland infectionper plant was obtained with the Nostoc isolate that

was the most prolific producer of hormogonia.Campbell & Meeks (1989) found that Nostoc isolatescapable of reconstituting the Anthoceros punctatussymbiosis all formed motile hormogonia and alsothat extensive conversion of Nostoc vegetative fila-ments into hormogonia occurred in the presence ofeither Anthoceros punctatus itself or A. punctatus-conditioned N-free medium. On the other hand,medium conditioned by A. punctatus in the presenceof combined nitrogen had no hormogonia-inducingactivity.

Our observations on hormogonia induction duringco-cultivation of wheat roots and Nostoc 2S9B aresimilar, in some respects, to those on A. punctatus

Colonization of wheat by N,^-Jixing cyanobacteria 511

Figure 4. Aseriate packages of Nostoc 2S9B (a) and hormogonia (b) formed after 24 h incubation of aseriatepackages with wheat roots in fresh N-free medium. Bar, 50

(Campbell & Meeks, 1989). A hormogonia-pro-moting factor was synthesized and liberated by theroots of wheat plants during growth in the presenceor absence of NaNOg. The higher ' specific activity'of this factor in extracts from plants grown in N-freeBG-11 medium may indicate increased synthesis inresponse to N-limitation or may simply reflect alower protein content of the roots. The NaNOgpresent in NOg'-containing BG-11 medium in-hibited the activity of the hormogonia-promotingfactor in both cell-free extracts of wheat roots andmedium preconditioned by the growth of the plants.However, it is not clear why NaNOg only partiallyinhibited the induction of hormogonia in co-culture.

It is possible that the concentration of NaNOg in themedium was lowered as a consequence of uptake bythe roots and the cyanobacterium. Alternatively, thefactor may have been more effective in co-culturebecause direct transfer from the roots to thecyanobacterium was possible.

Differentiation of hormogonia is photoregulatedand dependent on light quality (Lazaroff, 1973;Damerval et al,, 1991). In addition, an activesubstance (' motility factor') that can promote hormo-gonia differentiation under conditions when they arenot normally formed, such as darkness, has beendetected in supernatants from motile cultures(Lazaroff & Vishniac, 1961; P^binson & Miller,

512 M. Gantar, N. W. Kerby and P. Rowell

160

140-

12 24 36 48 60

Time after start of co-cultivation (h)

Figure 5. Liberation of hormogonia from aseriate pack-ages of Nostoc 2S9B resuspended in N-free medium (a) orNO.J -containing medium (b). The aseriate packages wereresuspended in fresh medium (#) , in medium which hadbeen pre-conditioned by the growth of wheat plants for10 d (A), or in fresh medium together with the roots ofwheat plants ( • ) .

0 10 20 30 40 50

Protein concentration of root extract (//g mf^)

Figure 6. P̂ ffect of the concentration of wheat root extracton hormogonia liberation. Fresh N-free medium, con-taining root extract to the protein concentrations shown,was inoculated with a preparation of aseriate packages andincubated for 24 h.

Table 1. Effect of dialysis on the hormogonia-promoting activity of root extract of wheat plantsgrown in N-free medium

Extract or dialvsate used' Hormogonia x 10"* ml"

NoneExtract before dialysisDialyzed extractConcentrated dialysate^

0-32 + 0-10*3-60±0-161-60 + 0120-07-F 0-05

' Aseriate packages were resuspended in fresh N-freemedium with root extract or dialysate added as indicatedand the concentration of hormogonia was determined after24 h incubation under white light.

^ Dialysate concentrated to a volume equal to that of theextract before dialysis.

* Mean + S.E.

Table 2. Effects of NaNO^ and other salts on thehormogonia-promoting activity of wheat root extracts.Aseriate packages zvere resuspended in fresh N-freemedium before adding the salt to a concentration of17-7 mM. The concentration of hormogonia wasdetermined after 24 h incubation under white light

Additions Hormogonia x 10 * ml

NoneRoot extractRoot extract+ NRoot extract 4-NaClRoot extract 4-KNO,,

0-10 + 0-02*2-50 + 0-230-25 ±0-030-20 + 0-010-08 4-0-01

* Mean + SE.

Table 3. Effect, on hormogonia liberation, of washingNostoc 2S9B aseriate packages by repeatedcentrifugation and resuspension in fresh N-freemedium. Following each wash, the concentration ofhormogonia was determined after 24 h incubation underwhite light

Washes Hormogonia x 10" ml'^

01234

N.D.0-1 ±0-02*0-8±0-17

13-O + O-2513-0-1-0-25

ND, not detectable.* Mean-I-SE.

1970). Our data do not provide any evidence forproduction of such a motility factor by Nostoc 2S9Bitself, but its occurrence cannot be ruled out.However, mature cultures of this strain containedvery low numbers of hormogonia, especially whengrown in medium containing NaNOg. Repeatedwashing of aseriate packages with N-free mediumresulted in increased hormogonia liberation (Table3), up to a maximum hormogonia concentration

Colonization of wheat by N^-fixing cyanobacteria 513

similar to that observed in co-culture with roots ofwheat plants (Fig. 5). It is possible therefore thataseriate packages produce an autoinhibitor of hormo-gonia differentiation (see Herdman & Rippka, 1988)that can be released into the medium.

Further study is needed to elucidate the identity,synthesis, release and mode of action of the hormo-gonia-promoting factor that is produced by the rootsof wheat plants. The findings may contribute to abetter understanding of the control of hormogoniaformation, which may be an important property, ifnot a prerequisite, in the formation of associationswith plants. The results obtained to date indicatethat the wheat root factor is not a small molecule, asit is to a large extent retained on dialysis. The partialloss of activity, which is not accompanied byappearance of activity in the dialysate, may suggest alabile or complex structure or that the factor isadsorbed onto the dialysis membrane. The wheatroot factor appears either to have a direct stimulatoryeffect on hormogonia differentiation from aseriatepackages or to block the synthesis or functioning ofan autoinhibitor produced by Nostoc 2S9B. Theapparently non-specific inhibitory effect of NaNOgand other salts indicates an involvement of ionicinteractions in the functioning of the hormogonia-promoting factor or at some stage in the developmentof hormogonia. This latter possibility is also con-sistent with similar inhibitory effects of salts onhormogonia induction in cultures of Nostoc mus-corum (Armstrong, Hayes & Walsby, 1983).

A C K N O W L E D G E M E N T S

This work was supported by the Royal Society, the Societyfor General Microbiology and the University of DundeeResearch Initiatives Fund. M.G. is grateful to the RoyalSociety for the award of a Visiting Research Fellowshipand the Institute of Biology, Novi Sad for the award of atravel grant.

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