host responses and metabolic profiles of wood components in

Host responses and metabolic profiles of wood components in Dutch elm hybridswith a contrasting tolerance to Dutch elm disease

Jaroslav Durkovic1,*, Frantisek Kacık2, Dusan Olcak3, Veronika Kucerova4 and Jana Krajnakova5,6

1Department of Phytology, Technical University, 96053 Zvolen, Slovakia, 2Department of Chemistry and Chemical Technologies,Technical University, 96053 Zvolen, Slovakia, 3Department of Physics, Technical University of Kosice, 04200 Kosice, Slovakia,

4Department of Forest Protection and Game Management, Technical University, 96053 Zvolen, Slovakia, 5Department ofAgricultural and Environmental Science, University of Udine, 33100 Udine, Italy and 6Department of Biology, University of Oulu,

90014 Oulu, Finland* For correspondence. E-mail [email protected]

Received: 12 February 2014 Returned for revision: 12 March 2014 Accepted: 24 March 2014 Published electronically: 22 May 2014

† Background and Aims Changes occurring in the macromolecular traits of cell wall components in elm wood fol-lowing attack by Ophiostoma novo-ulmi, the causative agent of Dutch elm disease (DED), are poorly understood. Thepurpose of this study was to compare host responses and the metabolic profiles of wood components for two Dutchelm (Ulmus) hybrids, ‘Groeneveld’ (a susceptible clone) and ‘Dodoens’ (a tolerant clone), that have contrasting sur-vival strategies upon infection with the current prevalent strain of DED.† Methods Ten-year-old plants of the hybrid elms were inoculated with O. novo-ulmi ssp. americana × novo-ulmi.Measurements were made of the content of main cell wall components and extractives, lignin monomer composition,macromolecular traits of cellulose and neutral saccharide composition.† Key Results Upon infection, medium molecular weight macromolecules of cellulose were degraded in both thesusceptible and tolerant elm hybrids, resulting in the occurrence of secondary cell wall ruptures and cracks in thevessels, but rarely in the fibres. The 13C nuclear magnetic resonance spectra revealed that loss of crystalline andnon-crystalline cellulose regions occurred in parallel. The rate of cellulose degradation was influenced by thesyringyl:guaiacyl ratio in lignin. Both hybrids commonly responded to the medium molecular weight cellulose deg-radation with the biosynthesis of high molecular weight macromolecules of cellulose, resulting in a significantincrease in values for the degree of polymerization and polydispersity. Other responses of the hybrids included anincrease in lignin content, a decrease in relative proportions of D-glucose, and an increase in proportions ofD-xylose. Differential responses between the hybrids were found in the syringyl:guaiacyl ratio in lignin.† Conclusions In susceptible ‘Groeneveld’ plants, syringyl-rich lignin provided a far greater degree of protectionfrom cellulose degradation than in ‘Dodoens’, but only guaiacyl-rich lignin in ‘Dodoens’ plants was involved in suc-cessful defence against the fungus. This finding was confirmed by the associations of vanillin and vanillic acid withthe DED-tolerant ‘Dodoens’ plants in a multivariate analysis of wood traits.

Key words: Cellulose degradation, crystallinity, Dutch elm disease, Ophiostoma novo-ulmi, syringyl to guaiacylratio, lignin, Ulmus.

INTRODUCTION

The ascomycetous fungus Ophiostoma novo-ulmi is the causa-tive agent of the current Dutch elm disease (DED) pandemic,which has ravaged both European and North American elmpopulations. This fungus is polytypic, spreading in the form oftwo subspecies, subsp. novo-ulmi and subsp. americana, previ-ously referred to as the Eurasian and North American races,respectively (Brasier and Kirk, 2001). Rapid emergence ofhybrids between these two subspecies has recently beenreported, and it is likely that complex hybrid swarms are nowexpanding across the European continent (Brasier and Kirk,2010). Several European elm breeding programmes wereinitiated in an attempt to combat the disease. Ulmus glabra ×U. minor hybrids are frequently included in these programmesas they often show lower susceptibility to the pathogen thanthat found in U. glabra trees. In addition, Asian elms, particularlyU. wallichiana and U. pumila, proved to be a useful additional

source of DED-resistance genes with the advent of the seconddisease pandemic in Europe during the 1970s (Heybroek,1983; Smalley and Guries, 1993; Santini et al., 2008, 2010).For better control of this disease, current elm breeding pro-grammes also integrate biotechnological approaches (Pijutet al., 1990; Fenning et al., 1996; Newhouse et al., 2007; Shuklaet al., 2012).

The pathogenic fungus spreads within the secondary xylemvessels of infected trees, causing the formation of vessel plugsdue to tyloses and gels (Ouellette et al., 2004), which ultimatelyresults in foliar wilting and subsequent tree death (Newbankset al., 1983). The wilt syndrome is apparently a result of interac-tions between fungal metabolites and the tree (Scheffer et al.,1987). The fungus produces hydrophobin cerato-ulmin (a para-sitic fitness factor; Temple et al., 1997), phytotoxic peptidor-hamnomannan (Strobel et al., 1978; Sticklen et al., 1991),tissue-invading structures that are thought to be involved in cavi-tation of the water column and alteration of parenchyma cells

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Annals of Botany 114: 47–59, 2014

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(Ouellette et al., 2004), and cell wall-degrading enzymes such asglucanases, glucosidases (Przybył et al., 2006), xylanases, lac-cases (Binz and Canevascini, 1996a, b), exo-glycanases and gly-cosidases (Svaldi and Elgersma, 1982). Several anatomicalparameters of wood have been found to be related to DED resist-ance, and their possible use in early screening has been dis-cussed. Unfortunately, the shortage of resistant trees tested hasfrequently made it difficult to obtain conclusive or reproducibleresults (Martın et al., 2008).

Plant interactions with pathogens are characterized by thedeployment of chemical signals and highly coordinated repro-gramming of metabolites (Allwood et al., 2006; Lloyd et al.,2011). Biochemical profiles of biomacromolecules and theirmetabolic fingerprinting provide rapid classification of plantsamples according to their origin and physiological state. Theability to obtain biochemical profiles and fingerprints for elmswould be useful in studies to characterize mechanisms of DEDor to identify resistant elms (Martın et al., 2005b). Changes inthe levels of cell wall components of elm wood after inoculationswith O. novo-ulmi isolates have been investigated for the mostpart by Fourier transform–infrared (FT-IR) spectroscopy to dis-criminate between resistant and susceptible elms, as well as toidentify metabolic profiles related to host resistance (Martınet al., 2005a, 2007, 2008). However, changes in the macromol-ecular traits of cell wall components of elm wood affected byDED are mostly unknown.

In this study we compared lignin monomer composition,macromolecular traits of cellulose and neutral saccharide com-position between two Dutch elm hybrids (‘Groeneveld’ and‘Dodoens’) that possess contrasting tolerance to DED. Weaddressed the following specific questions. (1) Is the ligninmacromolecular structure, quantified by the ratio of syringyl(S) to guaiacyl (G) subunits, involved in defence againstO. novo-ulmi and does the S/G ratio change upon infection? (2)How do the macromolecular traits of cellulose (polydispersity,degree of polymerization, crystallinity) change upon infection?(3) Is the content of neutral saccharides influenced by infection?

MATERIALS AND METHODS

Plant material and study site

The experiments were conducted on clonally micropropagated(Krajnakova and Longauer, 1996) plants of the Dutch elmhybrid cultivars ‘Groeneveld’ [(Ulmus × hollandica 49) ×U. minor ssp. minor 1] and ‘Dodoens’ (open-pollinatedU. glabra ‘Exoniensis’ × U. wallichiana P39) growing in anexperimental field plot at Banska Bela, Slovakia (48828′N,18857′E, 590 m a.s.l.). According to the meteorological stationat Arboretum Kysihybel in Banska Stiavnica (540 m a.s.l.),located 3.6 km south-west of the study site, the climate of thearea is characterized by a mean annual temperature of 7.7 8Cand a mean annual precipitation of 831 mm. The study sitesoil, which has a silt loam texture, is identified as an EutricCambisol formed from the slope deposits of volcanic rocks(andesite and pyroclastic materials).

Fungus identification and inoculation and wood sampling

Ten-year-old plants of similar size for each cultivar wereselected and inoculated with Ophiostoma novo-ulmi ssp.

americana × novo-ulmi isolate M3 according to the procedureof Solla et al. (2005). The spore suspension (1 × 107 sporesmL– 1) was inoculated into the current annual ring, 20 cmabove the base of the stem. Hybrid isolate M3 belongs tomating type B and was isolated from an infected elm tree inBrno, Czech Republic. This isolate proved to be ssp. americanain a fertility test and had a cerato-ulmi (cu) gene profile of ssp.americana, but also had a colony-type (col1) gene profile ofssp. novo-ulmi (Konrad et al., 2002; Dvorak et al., 2007). Theinfected ‘Groeneveld’ plants showed no tolerance to DED anddied at the end of the growing season after fungal inoculation(Dvorak et al., 2009). However, the infected ‘Dodoens’ plantsshowed high tolerance to O. novo-ulmi ssp. americana ×novo-ulmi (Durkovic et al., 2013). No signs of physiologicalweakening were found in these plants, and they grew for an add-itional 3 and a half years following fungal infection.

Discs 4 cm thick were sawn at a height of 1 m from the trunks ofboth hybrids. For ‘Groeneveld’ plants, the sampling was done halfa year after inoculation. For ‘Dodoens’ plants, the sampling wasdone 3 and a half years after inoculation. The discs were sym-metrical and visibly free of reaction wood and did not containknotwood. The infection zones, concentrated within the sixthannual ring at a height of 1 m, were separated from the discs ofinfected plants of both hybrids. The seventh and eighth annualrings (taken at a height of 1 m) were separated only from thediscs of infected ‘Dodoens’ plants (the two annual rings weremixed together because some portion of the seventh annual ringwas damaged and lost during separation of the sixth annualring). The sixth, seventh and eighth annual rings, respectively,separated from the discs of non-infected ‘Groeneveld’ and‘Dodoens’ plants, were used as controls. Figure 1A–I presentsscanning electron microscopy (SEM) images of plant materialresponding to the fungal inoculation that was used in this study.The experiments were conducted on wood samples taken fromfour infected and four non-infected plants per hybrid.

Scanning electron microscopy of wood samples

Wood sections (transverse, radial and tangential surfaces)were mounted on specimen stubs, sputter-coated with gold andobserved by high-vacuum SEM using a Vega TS 5130 instru-ment (Tescan, Brno, Czech Republic) operating at 15 kV.

Preparation and determination of extractives

Separated wood was mechanically disintegrated to sawdust,and a fraction size of 0.5–1.0 mm was extracted in a Soxhletapparatus with a mixture of ethanol and toluene accordingto the American Society for Testing and Materials (ASTM)International standard procedure D 1107-96. Measurementswere performed on four replicates of infected and four replicatesof non-infected plants per hybrid.

Determinations of lignin content and ligninmonomer composition

Lignincontentwasdeterminedaccording to theUSDepartmentof Energy, National Renewable Energy Laboratory analyticalprocedure (Sluiter et al., 2010). Measurements were performedon four replicates of infected and four replicates of non-infectedplants per hybrid.

Durkovic et al. — Host metabolic profiles in response to Dutch elm disease48


Nitrobenzene oxidation (NBO) was carried out using 2 M

NaOH in 10 mL stainless steel vessels at 180 8C for 2 h, andoxidation products were analysed by isocratic high-performanceliquid chromatography (HPLC) under the following conditions:LiChrospher 100 RP-18, 5 mm, 100 × 4 mm i.d. column(Merck, Darmstadt, Germany), mobile phase water:methanol:acetic acid (850:150:1), flow rate 1.0 mL min–1, column tempera-ture 35 8C, detection via diode array detector in the 210–360 nmregion (Durkovic et al., 2012). The S/G ratio in lignin was calcu-lated as (syringaldehyde + syringic acid) / (vanillin + vanillicacid).Measurementswereperformedonfour replicatesof infectedand four replicates of non-infected plants per hybrid.

Determinations of cellulose content and macromoleculartraits of cellulose

Cellulose content was determined by the method of Seifert(1956). Measurements were performed on four replicates ofinfected and four replicates of non-infected plants per hybrid.

Molecular weight distribution analysis of the cellulose sampleswas performed after their conversion into cellulose tricarbanilatesby a modified method described by other authors (Josefsson et al.,2001; Foston and Ragauskas, 2010). Briefly, the cellulose sampleswere dried over silica gel for several days. Anhydrous pyridine(8.0 mL), cellulose (50 mg) and phenyl isocyanate (1.0 mL)were sealed in a 50-mL dropping flask. The flasks were immersedin an oil bath at 70 8C for 72 h. At the end of the reaction, methanol(2.0 mL) was added to the mixture to eliminate excess phenyl iso-cyanate. The yellow solutions were then added dropwise into amethanol:water mixture (7:3, 150 mL), stirring rapidly with amagnetic stirrer. Cellulose tricarbanilates were dissolved in tetra-hydrofuran and filtered through a Puradisc 25 NYL filter(Whatman International, Maidstone, UK) with a pore size of0.45 mm. Size exclusion chromatography was performed at 358C with tetrahydrofuran at a flow rate of 1 mL min–1 on twoPLgel, 10 mm, 7.5 × 300 mm, MIXED-B columns (AgilentTechnologies, Santa Clara, CA, USA) preceded by a PLgel, 10mm, 7.5 × 50 mm, Guard-column (Agilent Technologies) as

A

D

G

E

H

F

I

B C

FI G. 1. Scanning electron microscopy images of wood samples separated from the sixth annual ring of infected ‘Groeneveld’ plants (A–C), the sixth annual ring ofinfected ‘Dodoens’ plants (D–F) and the seventh annual ring of infected ‘Dodoens’ plants (G–I). (A) Tyloses formed in latewood vessels in response to theOphiostoma novo-ulmi ssp. americana × novo-ulmi inoculation. Cross-section. (B) Tyloses occluding latewood vessel lumens. Tangential section. (C) Fungalhyphae growing inside latewood vessels. Cross-section. (D) Tyloses formed in earlywood vessels in response to fungal inoculation. Cross-section. (E) Formationof many narrowed latewood vessels in response to fungal inoculation. Cross-section. (F) Narrowed latewood vessels as a possible resistance factor limiting the move-ment of the fungus in the vascular system. Radial section. (G) Reduced formation of large earlywood vessels in the next growing season after fungal inoculation.Cross-section. (H) Continuation of narrowed latewood vessel formation in the next growing season after fungal inoculation. Cross-section. (I) Occasional occurrenceof fungal hyphae (arrows) growing inside earlywood vessel. Radial section. Scale bars: (A, D, E, G, H) ¼ 500 mm; (B) ¼ 200 mm; (C, I) ¼ 50 mm; (F) ¼ 100 mm.

Durkovic et al. — Host metabolic profiles in response to Dutch elm disease 49


described by Kacık et al. (2009). Data were acquired withChemStation software (Agilent Technologies) and calculationswere performed with the Clarity GPC module (DataApex,Prague, Czech Republic). Numerical outputs were obtained forMn (number-average molecular weight), Mw (weight-averagemolecular weight), Mz (z-average molecular weight), Mz+1 (z +1-average molecular weight) and Mp (peak molecular weight).The polydispersity index (PDI) of cellulose was calculated asthe ratio Mw/Mn. The degree of polymerization (DPw) was calcu-lated by dividing the molecular weightby the monomerequivalentweight of anhydroglucose (DPw ¼Mw/162). Measurementswere performed on four replicates of infected and four replicatesof non-infected plants per hybrid.

Solid-state 13C nuclear magnetic resonance and FT-IRmeasurements of cellulose crystallinity

The single-pulse 13C magic angle spinning (MAS) nuclearmagnetic resonance (NMR) measurements were performedwith a 400-MHz solid-state NMR spectrometer (Varian, PaloAlto, CA, USA). High-resolution 13C NMR spectra wererecorded at the resonance frequency of �100 MHz with theuse of 4-mm rotors and an MAS frequency of 10 kHz at a tem-perature of 30 8C inside the rotor. A free induction decay wasrecorded after a radio-frequency p/2 pulse of 2.0 ms duration,with high-power proton decoupling at 71 kHz and a recycledelay of 60 s. The 13C MAS NMR spectrum was obtained byFourier transformation of the free induction decay, which is anaverage of �3500 scans. The chemical shifts were referencedto tetramethylsilane using adamantane as an external standard.Deconvolutions were carried out with Mnova 8.1 software(Mestrelab Research, Santiago de Compostela, Spain). The crys-tallinity index (CI) of cellulose was determined by separatingthe C4 region of the spectrum into crystalline (89 p.p.m.) andamorphous (83 p.p.m.) peaks (Matulova et al., 2005; Vaneet al., 2006) and calculated according to a modified methoddescribed by Park et al. (2010). The height of the crystallinepeak was divided by the sum of the crystalline and amorphouspeak heights.

FT-IR spectra of wood powder samples were recorded on aNicolet iS10 FT-IR spectrometer equipped with a Smart iTRattenuated total reflectance sampling accessory (Thermo FisherScientific, Waltham, MA, USA). The spectra were acquiredby accumulating 64 interferograms at a resolution of 4 cm– 1 inan absorbance mode at wavenumbers from 4000 to 400 cm– 1.Spectral peaks were measured using OMNIC 8.0 software(Thermo Fisher Scientific). In order to normalize the infraredspectra obtained, we used the 1032 cm–1 band, assigned to theC–O stretching vibration in cellulose (Liang and Marchessault,1959; Colom et al., 2003). The lateral order index (LOI) of cellu-lose (A1422/A896) was calculated as described by O’Connor et al.(1958). Measurements were performed on four replicates ofinfected and four replicates of non-infected plants per hybrid.

Determinations of polysaccharide content and neutral sugarcomposition

Total content of polysaccharides (i.e. holocellulose) was deter-mined using the method of Wise et al. (1946). Measurements were

performed on four replicates of infected and four replicates ofnon-infected plants per hybrid.

Qualitative and quantitative analyses of saccharides werecarried out by HPLC according to the ASTM Internationalstandard procedure E 1758-01. The samples were hydrolysedin a two-stage process. In the first stage, 72 % (w/w) H2SO4 ata temperature of 30 8C was used for 1 h, and in the secondstage the formed oligomers were hydrolysed to monosaccharidesafter dilution to 4 % (w/w) H2SO4 at 121 8C for 1 h. The analyseswere performed with an Agilent 1200 HPLC chromatograph(Agilent Technologies) equipped with an Aminex HPX-87Pcolumn (Bio-Rad Laboratories, Hercules, CA, USA) at a tempera-ture of 80 8C and a mobile phase flow rate of 0.6 mL min–1.Measurements were performed on four replicates of infected andfour replicates of non-infected plants per hybrid.

Statistical analysis

Data showed a normal distribution and were therefore sub-jected to one-way analysis of variance. Duncan’s multiplerange tests were used for pairwise comparisons of means. ThePearson correlation coefficient was calculated for the relation-ship between cellulose content and DPw. The relationship wasconsidered significant if P , 0.05.

Multivariate association of the 25 examined wood traits wasanalysed by principal components analysis (PCA) to describepatterns of covariation among the contents of main cell wall com-ponents, lignin monomer composition, macromolecular traits ofcellulose and the neutral saccharide composition of wood.

RESULTS

Changes in contents of cell wall components

Data on the contents of main cell wall components for the Dutchelm hybrids are presented in Table 1. Within the sixth annual ringseparated from non-infected plants, significant differences werefound for the contents of lignin, cellulose, holocellulose andextractives. This result predominantly reflects genotypic differ-ences between the hybrids. On the other hand, the two hybridsshared some common trends in host responses to DED infection.Upon infection, in both hybrids, the lignin content increased, whilethe content of cellulose decreased. In the infected ‘Groeneveld’plants, a moderate decrease in the content of cellulose wasfound, whereas in the infected ‘Dodoens’ plants there was amarked decline in cellulose content. Strong dissimilaritiesbetween the hybrids were found for the content of extractives.Infected ‘Groeneveld’ plants responded with a significant decreasein the amount of extractives, whereas infected ‘Dodoens’ plantsmarkedly increased theircontentof thesesubstances.Whencompar-ing the sixth annual ring with the seventh and eighth annual ringswithin the infected ’Dodoens’ plants, the seventh and eighth ringscontained lower amounts of lignin, cellulose and extractives(Table 1). This implies that the content of hemicelluloses increasedat the expense of the above-mentioned components in successiveannual rings after fungal inoculation.

Changes in lignin monomer composition

Based on the analysis of NBO products, the hybrids showedstrong genotypic differences in lignin monomer composition



(Table 2). Within the sixth annual ring of the non-infected‘Groeneveld’ plants, the main constituents of lignin were Sunits; G units were much less abundant and the representationof p-hydroxyphenyl units was almost negligible. For non-infected ‘Dodoens’ plants, the prevailing constituents of ligninwere G units; S units were less abundant and the number ofp-hydroxyphenyl units was also very low. Upon infection, thehybrids showed completely different host responses in ligninmonomer composition. Within the sixth annual ring of infected‘Groeneveld’ plants, the S/G ratio decreased significantly but theabundance of S units was still twice as high as that of G units (S/G ¼ 2.06). On the contrary, the sixth annual ring of infected‘Dodoens’ plants was characterized by a significant increase inS units, which prevailed over G units within the lignin macromol-ecular structure (S/G ¼ 1.43). When comparing the sixth withthe seventh and eighth annual rings within the infected‘Dodoens’ plants, the seventh and eighth rings contained onlya slightly decreased number of S units (S/G ¼ 1.40).

Changes in macromolecular traits of cellulose

Changes in the macromolecular traits of cellulose samples aregiven in Table 3. The hybrids showed genotypic differences fordistributions of the molecular weights Mn, Mw, Mz and Mz+1.Upon infection, in both hybrids, the values for Mw, Mz andMz+1 significantly increased within the sixth annual ring.Figure 2 shows that both hybrids responded to infection with asignificant increase in the biosynthesis of high molecularweight macromolecules of cellulose, followed by a shift in the

peaks (Mp values) to the high molecular weight area. At thesame time, however, a substantial increase in low molecularweight macromolecules of cellulose occurred, which indicatesthat the medium molecular weight macromolecules weredegraded. Biodegradation affected mostly the macromoleculeswith DPw values of �202–206 (infected ‘Groeneveld’ plants)and those with DPw values of �235–238 (infected ‘Dodoens’plants).

In both hybrids, co-occurring biosynthetic and biodegradationprocesses resulted in a huge increase in the polydispersity indexof cellulose in the wood of infected plants (Table 3). Increasedvalues of DPw probably resulted from the biosynthesis of highmolecular weight macromolecules of cellulose, which remainedunaffected by the fungus. When comparing the sixth annual ringwith the seventh and eighth annual rings within the infected‘Dodoens’ plants, the seventh and eighth rings simultaneouslyshowed continuing biosynthesis of high molecular weightmacromolecules together with both the biodegradation ofmedium molecular weight macromolecules having DPw valuesof �186–189 and low molecular weight macromolecules(Fig. 2C). Biodegradation of medium and low molecularweight macromolecules of cellulose may explain the occurrenceof secondary cell wall ruptures (Fig. 3A) and cracks (Fig. 3B) inthe vessels but rarely in the fibres of infected plants.

For non-infected plants of both hybrids, the mean content ofcellulose in these samples was 41.01 % and the mean DPw

value was 406.17. Upon infection, the mean content of cellulosein the infected samples for both hybrids decreased to 30.54 %,whereas the mean DPw increased to 688.33. Figure 4A shows

TABLE 1. Contents of main cell wall components and extractives present in wood of Dutch elm hybrids (%)

Woodcomponent

‘Groeneveld’non-infected (sixth

annual ring)

‘Groeneveld’infected (sixthannual ring)

‘Dodoens’non-infected (sixth

annual ring)‘Dodoens’ infected(sixth annual ring)

‘Dodoens’ non-infected(seventh and eighth

annual rings)

‘Dodoens’ infected(seventh and eighth

annual rings)

Lignin 18.24+0.17e 22.85+0.28b 22.10+0.27c 24.05+0.22a 20.67+0.14d 22.92+0.23b

Cellulose 41.31+0.22b 37.03+0.30d 39.45+0.25c 28.68+0.12e 42.27+0.45a 25.91+0.40f

Holocellulose 80.40+0.23d 83.74+0.28a 82.31+0.24b 75.08+0.24e 81.58+0.29c 75.17+0.14e

Extractives 5.84+0.17b 3.51+0.14e 4.26+0.10d 7.68+0.15a 4.93+0.13c 4.93+0.09c

Data are means+ s.d.Mean values followed by the same letters within the same row across hybrids are not significantly different at P , 0.05.

TABLE 2. Nitrobenzene oxidation (NBO) products present in wood of Dutch elm hybrids (%)

NBO product


annual ring)



annual ring)

‘Dodoens’infected (sixthannual ring)

‘Dodoens’non-infected (seventh

and eighth annualrings)


annual rings)

p-Hydroxybenzoic acid 0.01+0.00cd 0.03+0.01a 0.02+0.00b 0.01+0.00d 0.01+0.00c 0.01+0.00d

p-Hydroxybenzaldehyde 0.04+0.00c 0.06+0.01a 0.06+0.01a 0.05+0.01b 0.05+0.00b 0.03+0.00c

Vanillic acid 0.19+0.01e 0.22+0.01d 0.31+0.01a 0.26+0.01c 0.29+0.01b 0.21+0.01d

Vanillin 2.43+0.02f 2.71+0.03d 3.96+0.03a 3.42+0.05c 3.82+0.01b 2.65+0.04e

Syringic acid 0.42+0.01a 0.41+0.02a 0.29+0.01c 0.38+0.01b 0.26+0.01d 0.29+0.01c

Syringaldehyde 5.61+0.04a 5.61+0.04a 3.75+0.08c 4.89+0.05b 3.42+0.02d 3.72+0.03c

Total yield in wood 8.70+0.08b 9.05+0.11a 8.39+0.12c 9.01+0.12a 7.85+0.06d 6.92+0.09e

S/G ratio 2.29+0.01a 2.06+0.02b 0.94+0.02e 1.43+0.01c 0.89+0.01f 1.40+0.01d




the close negative linear relationship between cellulose contentand DPw in non-infected plants. Upon infection, the slope ofthe descending line changed rapidly (Fig. 4B) but the Pearsoncorrelation value remained highly significant. This indicatescompensation for loss of cellulose through an increase in DPw,and thus a trade-off between these two traits to ensure thetensile strength of the wood.

In addition, 13C NMR spectra of the examined wood samplesdisplayed two signals at 83 and 89 p.p.m., corresponding to C4

carbon atoms of amorphous and crystalline cellulose, respective-ly. In infected plants of both hybrids, the decrease in signal inten-sities at both resonances revealed that losses of crystalline andnon-crystalline cellulose regions occurred in parallel. Within thesixth annual ring of the infected ‘Groeneveld’ plants, loss of theamorphous region was 3.13 % and that of the crystalline regionwas 5.26 % (Fig. 5A), and the CI of cellulose inferred from theNMR spectra decreased from 37.25 to 35.42 % (i.e. 1.83 % loss).Within the sixth annual ring of the infected ‘Dodoens’ plants,loss of the amorphous region was 5.26 % and that of the crystallineregion was 26.09 % (Fig. 5B), and the CI decreased from 37.70 to30.77 % (i.e. 6.93 % loss). When comparing the sixth annual ringwith the seventh and eighth annual rings within the infected‘Dodoens’ plants, the seventh and eighth rings contained a 2.78% lower proportion of the amorphous region and a 6.25 % lowerproportion of the crystalline region (Fig. 5C), and the CI decreasedfrom 30.77 to 29.41 % (i.e. 1.36 % loss).

With regard to the LOI of the cellulose crystallinity inferredfrom the FT-IR spectra, there were non-significant differencesbetween the infected and non-infected plants, except in thesixth annual ring of infected ‘Dodoens’ plants. In this case,LOI increased within the bulk of the cellulose macromolecules(Table 3).

Changes in saccharide contents

The absolute yields of saccharides and their relative contentsin the wood of the hybrids are presented in Table 4. We foundvery small genotypic differences between the hybrids for the con-tents of D-glucose, L-arabinose and D-galactose. Both hybridsshowed common trends in host responses to DED infection. Uponinfection, the relative proportions of D-glucose and D-mannosedecreased substantially within the sixth annual ring, whereas theproportions of D-xylose, L-arabinose and D-galactose significantlyincreased. The most substantial changes were related to the contentsof D-glucose and D-xylose. When comparing the sixth annual ringwith the seventh and eighth annual rings within the infected‘Dodoens’ plants, the seventh and eighth rings containedhigher absolute yields and relative proportions of D-glucoseand lower amounts of D-xylose, L-arabinose and D-mannose,while the content of D-galactose remained steady. The excessiveoccurrence of agglomerates of storage compounds and partiallygelatinized starch inside ray parenchyma cells and axial paren-chyma cells might contribute to the increased amounts ofD-glucose within the seventh and eighth annual rings of theinfected ‘Dodoens’ plants.

Associations among wood traits

A PCA was done to evaluate how wood traits were associated(Fig. 6). The first axis explained 47 % of the variation and showed

TA

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Macr

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ole

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ait

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seis

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‘Gro

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‘Dodoen

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Mn

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127+

98

d14

305+

86

c15

156+

163

a13

285+

146

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72

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327

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509+

636

b53

084+

76

f115

541+

541

a

Mz

185

817+

179

e343

385+

915

c272

344+

630

d348

548+

2328

b180

184+

464

f357

787+

1240

a

Mz+

1392

846+

779

f608

574+

4072

b513

384+

1378

d594

341+

5624

c423

274+

2149

e623

804+

4085

a

Mp

33

013+

218

e44

585+

386

c38

298+

132

d48

848+

721

b30

492+

105

f53

822+

807

a

PD

I4

. 50+

0. 0

3e

7. 5

8+

0. 0

3c

5. 6

3+

0. 0

5d

8. 3

2+

0. 0

6a

4. 3

4+

0. 0

2f

7. 8

2+

0. 0

7b

DP

w364+

1e

670+

2c

527+

2d

682+

4b

328+

1f

713+

3a

LO

I(A

1422/

A896)

1. 0

8+

0. 0

7b

1. 1

9+

0. 1

5b

1. 1

6+

0. 1

2b

1. 3

9+

0. 2

0a

1. 0

3+

0. 0

6b

1. 1

4+

0. 0

9b

Dat

aar

em

eans+

s.d.

Mea

nval

ues

foll

ow

edby

the

sam

ele

tter

sw

ithin

the

sam

ero

wac

ross

hybri

ds

are

not

signifi

cantl

ydif

fere

nt

atP

,0

. 05.



strong positive loadings for macromolecular traits of cellulose,such as polydispersity, degree of polymerization, Mw, Mz, Mz+1,Mp and the relative proportions of D-xylose and L-arabinose.The negative side of the axis indicated strong loadings for the CIof cellulose, the cellulose content and the relative proportion ofD-glucose. The second axis explained 21 % of the variation andshowed strong positive loadings for the S/G ratio in lignin, syrin-galdehyde, syringic acid and the relative proportion of D-mannose.The negative side of the axis indicated strong loadings for vanillicacid, vanillin, D-galactose and lignin content. The traits associated

with differential host responses to DED infection correlated withthe second PCA axis, whereas the traits associated withcommon host responses correlated with the first PCA axis. In add-ition, PCA showed that infected annual rings in both hybrids wereclearly separated from the non-infected control annual rings.

DISCUSSION

Metabolic fingerprinting of elm secondary xylem tissues toprovide bioindicators of chemical changes caused by DED

0·004A

B

C

‘Groeneveld’ non-infected(6th annual ring)

‘Dodoens’ non-infected(6th annual ring)

‘Dodoens’ non-infected(7th + 8th annual rings)

‘Dodoens’ infected(7th + 8th annual rings)

‘Dodoens’ infected(6th annual ring)


‘Groeneveld’ infected(6th annual ring)0·003

0·002dW

/ dL

ogM

wdW

/ dL

ogM

wdW

/ dL

ogM

w

0·001

0

0·004

0·003

0·002

0·001

0

0·004

0·003

0·002

0·001

03 4 5

LogMw

6 7

FI G. 2. Size exclusion chromatography of molecular weight distributions of cellulose tricarbanilates. (A) Sixth annual ring of ‘Groeneveld’ plants. (B) Sixth annualring of ‘Dodoens’ plants. (C) Seventh and eighth annual rings of ‘Dodoens’ plants.



infection is a recent tool in detecting progressive tree responses tothe pathogen. We found common host responses as well as differ-ential host responses to O. novo-ulmi ssp. americana ×novo-ulmi infection between the two Dutch elm hybrids exam-ined here. We relate these findings to previously publishedwork, highlighting the novel findings pertinent to biodegradationof medium molecular weight macromolecules of cellulose.

Host responses of lignin metabolism to O. novo-ulmi infection

Previous studies revealed increased levels of lignin in thebranches of U. minor (Martın et al., 2005a) and U. minor ×U. pumila hybrids (Martın et al., 2007) as an induced hostdefence response to inoculation with O. novo-ulmi. Our resultsregarding the changes in lignin content upon infection in

A B

FI G. 3. Scanning electron microscopy images of secondary cell wall ruptures and cracks. (A) Ophiostoma novo-ulmi ssp. americana × novo-ulmi hypha inside thevascular tracheid (arrowhead) and ruptures in secondary cell walls of wood fibres (arrows). Cross-section of sixth annual ring of an infected ‘Groeneveld’ plant. Scalebar ¼ 10 mm. (B) Fungal hyphae inside an earlywood vessel and radial cracks in the secondary cell wall (arrow). Radial section of sixth annual ring of an infected

‘Groeneveld’ plant. Scale bar ¼ 20 mm.

600

A

B

y = 3234·60 – 68·97xr = –0·96 P < 0·0001

y = 790·28 – 3·34xr = –0·86 P = 0·0003

500

‘Groeneveld’ non-infected (6th annual ring)

‘Dodoens’ non-infected (6th annual ring)

‘Dodoens’ non-infected (7th + 8th annual rings)

‘Groeneveld’ infected (6th annual ring)

‘Dodoens’ infected (6th annual ring)

‘Dodoens’ infected (7th + 8th annual rings)

Cellulose content (%)

DP

WD

PW

400

300

700

600

50025 30 35 40 45

FI G. 4. Relationship of cellulose content with the degree of polymerization (DPw) in (A) non-infected plants and (B) infected plants.



‘Groeneveld’ non-infected(6th annual ring)

A

B

C

‘Dodoens’ non-infected(6th annual ring)

120 110 100 90 80 70

Chemical shift (ppm)

60 50 40 30 20

‘Dodoens’ non-infected(7th + 8th annual rings)


‘Dodoens’ infected(7th + 8th annual rings)


‘Groeneveld’ infected(6th annual ring)

C4

crys

talli

ne c

ellu

lose

C4

amor

phou

s ce

llulo

se

C4

crys

talli

ne c

ellu

lose

C4

crys

talli

ne c

ellu

lose

C4

amor

phou

s ce

llulo

seC

4 am

orph

ous

cellu

lose

FI G. 5. Solid-state 13C MAS NMR spectra of wood sawdust samples. (A) Sixth annual ring of ‘Groeneveld’ plants. (B) Sixth annual ring of ‘Dodoens’ plants. (C)Seventh and eighth annual rings of ‘Dodoens’ plants.



Dutch elm hybrids ‘Dodoens’ and ‘Groeneveld’ support theabove observations. It is recognized that lignin provides woodytissues with protection against pathogens because of its resist-ance to biodegradation (Duchesne et al., 1992). Unfortunately,none of the above studies examined the influence of S/G ratioin lignin on cell wall degradation by O. novo-ulmi. The macro-molecular structure of lignin depends on the proportions of Sand G aromatic subunits, which determine the type andnumberof cross-links within the macromolecule as well as the re-activity of the lignin (S-rich lignin forms fewer cross-linkingstructures than G-rich lignin). NBO products originate fromthe corresponding phenylpropane units and their a(b)-O-4alkyl-arylethers due to the oxidation of lignin in an alkalineenvironment. Thus, the S/G ratio provides information regard-ing the relative amounts of uncondensed guaiacyl- and syringyl-propane units constituting the original lignin. In addition, theyield of these aldehydes reflects the degree of condensation ofthe lignin, because these uncondensed structures are cleavedby nitrobenzene oxidation, leaving condensed lignins as theresidue (Kacık et al., 2012). With regard to changes in the S/Gratio in lignin, our results showed differential host responses tofungal infection, but only the G-rich lignin of ‘Dodoens’ wasinvolved in successful defence against O. novo-ulmi ssp.americana × novo-ulmi. This finding was supported by the load-ings for vanillin and vanillic acid in the multivariate wood traitanalysis (Fig. 6). These two NBO products, typical of G lignin,were associated with the sixth annual ring of the non-infected,DED-tolerant ‘Dodoens’. On the other hand, D-mannose wasassociated with the sixth annual ring of the non-infected but sen-sitive ‘Groeneveld’. It is well established that lignin that is rich inG units has a greater degree of carbon–carbon bonding and amore condensed macromolecular structure than lignin rich in Sunits. Some monolignol precursors (for example ferulates andtheir dehydrodimers) can cross-link cell wall polysaccharides,forming an enhanced structural barrier to reduce biodegradabil-ity of the cell wall (Fry, 1986). Ferulate and monolignol metabo-lisms were found to be major sources of ethylene-mediatedresistance of Arabidopsis thaliana to the necrotrophic fungus

Botrytis cinerea (Lloyd et al., 2011). In addition, Quercus albabark lignin rich in G units showed a greater resistance to whiterot fungus (Lentinula edodes) decay compared with woodlignin rich in S units (Vane et al., 2006).

Changes in macromolecular traits of cellulose after attackby O. novo-ulmi

Owing to its high degree of polymerization and crystallinity,cellulose is considered to be primarily responsible for thetensile strength of wood. Therefore, reducing the length of thecellulose molecules (DPw) would cause a reduction in macro-strength properties (Sweet and Winandy, 1999; Vizarova et al.,2012). Upon infection, we observed the occurrence of secondarycell wall ruptures and cracks. From a mechanical point of view, adecrease in cellulose content was partially compensated for byanincrease in DPw of the cellulose chain to maintain the tensilestrength of the wood in the infected plants. Production of cellu-lolytic enzymes by O. ulmi and O. novo-ulmi isolates wasreported by Przybył et al. (2006). Scheffer and Elgersma(1982) documented that aggressive DED isolates caused severecell wall erosions in Ulmus americana secondary xylemvessels, whereas non-agressive isolates left cell walls intact. Inthis study, we used a highly aggressive isolate of the hybrid sub-species americana × novo-ulmi of O. novo-ulmi that is capableof severe cell wall degradation. We found that the primarytargets of fungal attack were medium molecular weight macro-molecules of cellulose. Kleman-Leyer et al. (1992) observed adifference in the kinetics of cotton cellulose depolymerizationby the brown rot fungus Postia placenta and the white rotfungus Phanerochaete chrysosporium. Cellulose attacked byP. placenta showed a broader molecular mass distribution withprogressively decreasing values of DPw over time. However, adifferent molecular mass distribution of cellulose was observedduring attack by P. chrysosporium. At first, the molecular mass dis-tribution curve broadened, but as attack progressed the curve nar-rowed. The DPw decreased during the initial period of fungal attackbut then increased gradually to near control values. Simultaneous

TABLE 4. Absolute yields of saccharides (% of oven-dry weight per unextracted wood) and their relative proportions (%) in wood ofDutch elm hybrids

Saccharide


annual ring)



annual ring)‘Dodoens’ infected(sixth annual ring)

‘Dodoens’ non-infected(seventh and eighth

annual rings)


annual rings)

Absolute yieldsD-Glucose 58.45+0.04b 53.41+0.06e 57.39+0.05c 48.73+0.26f 61.18+0.04a 54.57+0.08d

D-Xylose 13.92+0.05d 18.78+0.06a 14.64+0.05c 17.42+0.20b 12.74+0.04e 14.58+0.07c

L-Arabinose 1.25+0.02c 1.55+0.04a 1.22+0.01c 1.35+0.02b 1.04+0.01d 1.24+0.03c

D-Mannose 1.43+0.01a 0.41+0.02e 0.86+0.01b 0.69+0.02c 0.36+0.01f 0.52+0.02d

D-Galactose 0.22+0.01d 0.28+0.04c 0.22+0.02d 0.32+0.02b 0.38+0.02a 0.32+0.01b

Total yield 75.27+0.12b 74.42+0.18c 74.32+0.12c 68.50+0.44e 75.70+0.09a 71.23+0.13d

Relative proportionsD-Glucose 77.65+0.06b 71.76+0.09e 77.22+0.08c 71.14+0.18f 80.81+0.05a 76.61+0.04d

D-Xylose 18.49+0.04e 25.24+0.02b 19.70+0.04d 25.43+0.18a 16.84+0.04f 20.47+0.06c

L-Arabinose 1.66+0.02d 2.08+0.04a 1.64+0.01d 1.96+0.03b 1.37+0.02e 1.75+0.04c

D-Mannose 1.90+0.01a 0.55+0.02e 1.15+0.02b 1.01+0.02c 0.48+0.01f 0.73+0.02d

D-Galactose 0.30+0.01d 0.37+0.05c 0.29+0.03d 0.46+0.02ab 0.50+0.03a 0.44+0.02b




biodegradation of amorphous and crystalline forms of cellulose,examined using 13C NMR spectra, was also reported for Betulapapyrifera wood and Q. alba bark decayed by P. chrysosporium(Davis et al., 1994) and L. edodes (Vane et al., 2006). Seen fromthe viewpoint of cellulose depolymerization, the mechanism ofelm wood cell wall degradation by O. novo-ulmi ssp. americana ×novo-ulmi appears to resemble that caused by white rot fungi.

LOI represents the ordered regions of cellulose perpendicularto chain direction and is inferred from the ratio of infrared peakareas at 1422 and 896 cm– 1 (Siroky et al., 2010). The absorptionband at 1422 cm– 1 represents CH2 scissoring motion at C6

(Nelson and O’Connor, 1964a, b; Siroky et al., 2010). The 896cm– 1 band indicates the vibrational mode involving C1 and thefour atoms attached to it, which is characteristic of b-anomers

or b-linked glucose polymers (Nelson and O’Connor, 1964a,b; Siroky et al., 2010). A significant increase in LOI within thesixth annual ring of the infected ‘Dodoens’ plants may implythat either the crystalline regions parallel to the cellulose chaindirection were predominantly degraded or a recrystallizationeffect accompanied cellulose chain scission during fungalattack (Ibbett et al., 2008).

An interesting point in this study was how the S/G ratio inlignin affected cellulose degradability by O. novo-ulmi celluloly-tic enzymes in the infected plants. Recent studies revealed thatthe S/G ratio has a significant influence on the cross-linkingbetween lignin and other cell wall components, thus modifyingthe microscopic structure and topochemistry of the cell wall(Jung and Casler, 2006). Differences in S/G ratio affected the

0·4 G-n-6S/G

MAN

ARA

XYL

LOI

LIG

GAL

‘Groeneveld’ non-infected (6th annual ring)

‘Groeneveld’ infected (6th annual ring)‘Dodoens’ non-infected (6th annual ring)

‘Dodoens’ non-infected (7th + 8th annual rings)‘Dodoens’ infected (7th + 8th annual rings)

‘Dodoens’ infected (6th annual ring)

DPw

PDI

CEL

CI

GLC

HOL

HBAC

D-i-6

EXTHBAL

VAN VAC

D-n-6

D-n-7+8

D-i-7+8

MnMwMzMp

Mz+1

SYR SAC

G-i-6

0·3

0·2

0·1

PC

A a

xis

2 (2

1 %

)

0

–0·1

–5 –4 –3 –2 –1

–4

–3

–2

–1

1

1

2

2

3

3

4

4

50

0

–0·2

–0·3

–0·4

–0·5 –0·4 –0·3 –0·2 –0·1 0·1 0·2 0·3 0·4 0·50

PCA axis 1 (47 %)

FI G. 6. Positions of 25 wood traits on the first and second axes of the principal components analysis (PCA). Traits associated with differential host responses areindicated in grey. Bottom and left axes refer to wood traits; top and right axes refer to annual rings of Dutch elm hybrids. Trait abbreviations: ARA, L-arabinose;CEL, cellulose content; CI, crystallinity index of cellulose; DPw, degree of polymerization of cellulose; EXT, extractives content; GAL, D-galactose; GLC,D-glucose; HBAC, p-hydroxybenzoic acid; HBAL, p-hydroxybenzaldehyde; HOL, holocellulose content; LIG, lignin content; LOI, lateral order index; MAN,D-mannose; Mn, number-average molecular weight; Mp, peak molecular weight; Mw, weight-average molecular weight; Mz, z-average molecular weight; Mz+1,z + 1-average molecular weight; PDI, polydispersity index; S/G, syringyl:guaiacyl ratio in lignin; SAC, syringic acid; SYR, syringaldehyde; VAC, vanillic acid;

VAN, vanillin; XYL, D-xylose.



efficiency of kraft pulping (Lapierre et al., 1999; Li et al., 2008;Santos et al., 2013), as well as cell wall degradability duringchemical and hot water pretreatments, and the subsequent hy-drolysis of cellulose to glucose (Li et al., 2010; Studer et al.,2011; Papa et al., 2012). In the present study the lowest rate ofcellulose degradation was found in the infected ‘Groeneveld’plants. Syringyl-rich lignin in non-infected ‘Groeneveld’plants contained a 2.4-fold higher proportion of methoxylgroups than G-rich lignin in non-infected ‘Dodoens’ plants(or a 1.4-fold higher proportion of methoxyl groups for infected‘Groeneveld’ plants than for infected ‘Dodoens’ plants). Thus,the ‘Groeneveld’ cellulose microfibrils were provided withdenser steric protection by methoxyl groups and consequentlywith a decreased accessibility of the fungal cellulolyticenzymes, compared with ‘Dodoens’ microfibrils. This mightbe the reason why the medium molecular weight macromole-cules of ‘Groeneveld’ cellulose were degraded to a lesserextent than those of ‘Dodoens’. Similar results with respect tothe role of S-rich lignin in the resistance of hybrid poplar treesto degradation by wood decay fungi were recently reported bySkyba et al. (2013). In addition, it is well documented thatamorphous regions of cellulose microfibrils are more readily de-gradable to glucose than crystalline regions (Suchy et al., 2011;Papa et al., 2012). Cellulose degradation rates mediated byfungal cellulases are typically 3- to 30-fold faster for amorphouscellulose substrates than for high crystalline cellulose substrates(Zhang and Lynd, 2004). However, our results revealed that, inboth hybrids, crystalline regions of cellulose were degraded toa higher extent than amorphous regions. This suggests that, inboth hybrids, readily hydrolysable amorphous regions weremostly provided with steric protection by methoxyl groups ofboth S-rich and G-rich lignins. Adani et al. (2011) showed thatthe particular spatial disposition of cell wall polymers deter-mines the presence of microporous structures that modulate theaccessibility of enzymes to the cell wall. Steric protection (3-Dmolecular geometry) is the main mechanism proposed toexplain the biochemical recalcitrance of plant tissues (Papaet al., 2012).

To conclude, we have used metabolic profiles of wood compo-nents to find that, upon infection with O. novo-ulmi ssp.americana × novo-ulmi, medium molecular weight macromo-lecules of cellulose were degraded. The 13C NMR spectrarevealed that loss of crystalline and non-crystalline celluloseregions occurred in parallel. The rate of cellulose degradationwas influenced by the S/G ratio in lignin, but only the G-richlignin of ‘Dodoens’ plants was involved in successful defenceagainst DED. This finding was confirmed by the associationsof vanillin and vanillic acid with DED-tolerant ‘Dodoens’plants in the multivariate wood trait analysis.

ACKNOWLEDGEMENTS

We thank Drs M. Dvorak, I. Canova, M. Mamonova, R. Laganaand M. Moravcık for their technical assistancewith fungal inocu-lation, wood sampling and scanning electron microscopy, andMrs E. Ritch-Krc for language revision. We also wish to acknow-ledge the thoughtful comments and suggestions of the anonym-ous reviewers, which improved the quality of the paper. Thiswork was supported by funding from the Slovak scientificgrant agency VEGA (1/0132/12).

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