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Annals of Botany logoLink to Annals of Botany
. 2012 Dec 21;111(2):215–227. doi: 10.1093/aob/mcs274

Leaf trait dissimilarities between Dutch elm hybrids with a contrasting tolerance to Dutch elm disease

Jaroslav Ďurkovič 1,*, Ingrid Čaňová 1, Rastislav Lagaňa 2, Veronika Kučerová 3, Michal Moravčík 1, Tibor Priwitzer 4, Josef Urban 5, Miloň Dvořák 5, Jana Krajňáková 5
PMCID: PMC3555532  PMID: 23264236

Abstract

Background and Aims

Previous studies have shown that Ophiostoma novo-ulmi, the causative agent of Dutch elm disease (DED), is able to colonize remote areas in infected plants of Ulmus such as the leaf midrib and secondary veins. The objective of this study was to compare the performances in leaf traits between two Dutch elm hybrids ‘Groeneveld’ and ‘Dodoens’ which possess a contrasting tolerance to DED. Trait linkages were also tested with leaf mass per area (LMA) and with the reduced Young's modulus of elasticity (MOE) as a result of structural, developmental or functional linkages.

Methods

Measurements and comparisons were made of leaf growth traits, primary xylem density components, gas exchange variables and chlorophyll a fluorescence yields between mature plants of ‘Groeneveld’ and ‘Dodoens’ grown under field conditions. A recently developed atomic force microscopy technique, PeakForce quantitative nanomechanical mapping, was used to reveal nanomechanical properties of the cell walls of tracheary elements such as MOE, adhesion and dissipation.

Key Results

‘Dodoens’ had significantly higher values for LMA, leaf tissue thickness variables, tracheary element lumen area (A), relative hydraulic conductivity (RC), gas exchange variables and chlorophyll a fluorescence yields. ‘Groeneveld’ had stiffer cell walls of tracheary elements, and higher values for water-use efficiency and leaf water potential. Leaves with a large carbon and nutrient investment in LMA tended to have a greater leaf thickness and a higher net photosynthetic rate, but LMA was independent of RC. Significant linkages were also found between the MOE and some vascular traits such as RC, A and the number of tracheary elements per unit area.

Conclusions

Strong dissimilarities in leaf trait performances were observed between the examined Dutch elm hybrids. Both hybrids were clearly separated from each other in the multivariate leaf trait space. Leaf growth, vascular and gas exchange traits in the infected plants of ‘Dodoens’ were unaffected by the DED fungus. ‘Dodoens’ proved to be a valuable elm germplasm for further breeding strategies.

Keywords: Adhesion, atomic force microscopy, gas exchange, leaf mass per area, modulus of elasticity, Ophiostoma novo-ulmi, tracheary element

INTRODUCTION

Ophiostoma novo-ulmi, the causative agent of current Dutch elm disease (DED) pandemics, is highly pathogenic to both native European and North American elm trees. This ascomycetous fungus is polytypic, spreading in the form of two subspecies, subsp. novo-ulmi and subsp. americana, previously referred to as the Eurasian and North American races, respectively (Brasier and Kirk, 2001). A rapid emergence of hybrids between these two subspecies has recently been reported, and it is likely that complex hybrid swarms are now expanding across the European continent (Brasier and Kirk, 2010). The initial elm breeding programmes, launched in The Netherlands, were focused on the identification of resistant elms to replace the popular but susceptible cultivar Ulmus × hollandica ‘Belgica’, and emphasized the native European elms, especially U. glabra and U. minor. Asian elms, particularly U. wallichiana, proved to be a useful additional source of DED resistance genes with the advent of the second disease pandemics in Europe during the 1970s (Heybroek, 1983; Smalley and Guries, 1993). The Dutch elm cultivar releases of the 1960s and 1970s, such as ‘Groeneveld’, ‘Commelin’, ‘Dodoens’, ‘Lobel’, ‘Plantyn’ and others, were widely planted in western Europe and show a varying degree of resistance to O. novo-ulmi isolates. For better control of this disease, current elm breeding programmes also integrate biotechnological approaches (Pijut et al., 1990; Fenning et al., 1996; Corredoira et al., 2002).

Fungal metabolites associated with vascular wilt disease affect the plasma membrane function, and cause an interference with the stomatal regulation of transpiration and a reduction of flow through the stem due either to vascular plugging or to an increase in the viscosity of the xylem sap (Van Alfen and Turner, 1975; Van Alfen, 1989). The fungus produces hydrophobin cerato-ulmin (a parasitic fitness factor) (Temple et al., 1997), phytotoxic peptidorhamnomannan (Strobel et al., 1978; Sticklen et al., 1991), cell wall-degrading enzymes such as xylanases, laccases (Binz and Canevascini, 1996a, b), exo-glycanases and glycosidases (Svaldi and Elgersma, 1982), and tissue-invading structures, which are thought to be involved in cavitation of the water column and alteration of parenchyma cells (Ouellette et al., 2004). Several species of elm bark beetles, primarily in the genus Scolytus and Hylurgopinus, have been recognized as the major transmission vectors of DED (Webber, 2004).

Leaf traits are often closely associated with plant growth, survival and light requirement, and thus may be good predictors of plant performance across a diverse range of plant communities (Poorter and Bongers, 2006; Sterck et al., 2006). Although leaves vary considerably in area, thickness, shape, nutrient concentrations and their capacity for gas exchange, the intercorrelation of many traits places some bounds on this diversity, particularly among those related to carbon economy, including leaf mass per area (LMA), leaf life span, nitrogen concentration per mass and net photosynthetic rate per mass. The LMA is a key trait in plant growth and a significant descriptor of plant strategies that can contribute to the success of a given plant species in the field (Westoby et al., 2002; Poorter et al., 2009). In its own right, LMA is a hub trait interlinked with a disproportionate number of other traits (Sack et al., 2003; Sack and Holbrook, 2006). Variation in leaf traits of Ulmus species has been the focus of several studies with respect to their resistance to elm leaf beetle defoliation (Young and Hall, 1986; Bosu and Wagner, 2007, 2008). However, not so widely covered is the research aimed at leaf trait variation in elms with a varying degree of resistance to DED.

The mechanical properties of the cellular microenvironment, notably its rigidity and stiffness, play a critical regulatory role for a variety of fundamental cell behaviours and responses (Janmey et al., 2009). In the case of tracheary elements of leaf primary xylem – highly specialized cells for transporting water and nutrients to the leaf lamina – knowledge of the in situ stiffness of the cell wall quantified by the modulus of elasticity (MOE) should be of great importance because it can be used either to assess stress values resulting in cell wall deformations or to evaluate the risk of tracheary element implosion when an embolism spreads and cavitation occurs under stressful conditions. Although the internal organization of the cell wall architecture of tracheary elements has been the focus of structural studies (Nakashima et al., 1997; Lacayo et al., 2010), information about the nanomechanical properties of the cell walls of tracheary elements is largely lacking.

The objective of this study was to compare the performances in leaf traits, including an in situ assessment of nanomechanical properties of cell walls of tracheary elements, between two Dutch elm hybrids (‘Groeneveld’ and ‘Dodoens’) which possess a contrasting tolerance to DED. ‘Dodoens’ plants tolerant to DED respond to O. novo-ulmi inoculation with an altered pattern of secondary xylem annual ring organization. These plants tolerant to DED show the formation of many narrowed vessels with small lumen areas in the successive annual rings (Fig. 1A–C). Thus, we hypothesized that performances in leaf traits of infected plants of ‘Dodoens’ related to leaf growth (leaf area, LMA), nanomechanical properties of primary xylem cell walls (MOE), and gas exchange and chlorophyll a fluorescence (net photosynthetic rate, transpiration, stomatal conductance, variable-to-initial fluorescence ratio) will be significantly decreased due to the altered morphology of vessels and the occasional occurrence of fungal hyphae in water-conducting cells, when compared with the non-infected plants. Additionally, we tested trait relationships with LMA and MOE. We expected correlations with these traits as a result of structural, developmental or functional linkages, including leaf dimensions, net photosynthetic rate and other vascular traits (Sack and Holbrook, 2006; Dunbar-Co et al., 2009; Bartlett et al., 2012; Ďurkovič et al., 2012).

Fig. 1.

Fig. 1.

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Scanning electron microscopy images of leaf and wood samples of infected plants of ‘Dodoens’ (A–F), non-infected plants of ‘Dodoens’ (G–I) and non-infected plants of ‘Groeneveld’ (J–L) used in this study. Ophiostoma novo-ulmi ssp. americana × novo-ulmi hyphae (white arrows) inside the secondary xylem vessel, radial section (A). The formation of narrowed secondary xylem vessels as a response to the fungus incoculation, radial section (B) and cross-section (C). Mesophyll tissue, cross-section (D, G, J). Midrib and primary xylem area, cross-section (E, H, K). Fungal hyphae inside primary xylem tracheary elements, cross-section (F). Unaffected, natural secondary xylem vessel grouping, cross-section (I, L). Scale bars: (A) = 50 µm, (B, D, G, J) = 100 µm, (C, E, H, I, K, L) = 500 µm, (F) = 10 µm.

MATERIALS AND METHODS

Plant material and study site

The experiments were conducted on clonally micropropagated (Krajňáková and Longauer, 1996), mature flowering plants of the Dutch elm hybrid cultivars ‘Groeneveld’ [(Ulmus × hollandica 49) × U. minor ssp. minor 1] and ‘Dodoens’ (open pollinated U. glabra ‘Exoniensis’ × U. wallichiana P39) growing in the experimental field plot at Banská Belá, Slovakia (48°28′N, 18°57′E, 590 m a.s.l.). According to the meteorological station at Arboretum Kysihýbel in Banská Štiavnica (540 m a.s.l.), located 3·6 km south-west of the study site, the climate of the area is characterized by a mean annual temperature of 7·7 °C and a mean annual precipitation of 831 mm. The study site soil which has a silt loam texture is identified as an Eutric Cambisol formed from the slope deposits of volcanic rocks (andesite and pyroclastic materials).

Fungus identification and inoculation, and plant source selection

Five plants of each cultivar, at least 10 years of age, were inoculated with O. novo-ulmi ssp. americana × novo-ulmi isolate M3 according to the procedure of Solla et al. (2005). The spore suspension (1 × 107 spores mL−1) was inoculated into the current annual ring, 20 cm above the base of the stem. Hybrid isolate M3 belongs to mating type B, and was isolated from an infected elm tree in Brno, Czech Republic. This isolate proved to be ssp. americana in the fertility test and had a cerato-ulmi (cu) gene profile of ssp. americana but a ssp. novo-ulmi colony type (col1) gene profile (Konrad et al., 2002; Dvořák et al., 2007).

The infected plants of ‘Groeneveld’ do not show any tolerance toward O. novo-ulmi ssp. americana × novo-ulmi, and they die at the end of the growing season after a fungus inoculation (Dvořák et al., 2009). Thus, leaf traits coming from this plant source were not investigated in our study. However, the infected plants of ‘Dodoens’ do show a high tolerance to DED. Although scanning electron microscopy (SEM) confirmed the occasional occurrence of fungal hyphae inside both the secondary xylem vessels of successive annual rings after a fungus inoculation (Fig. 1A) and the primary xylem tracheary elements of current-year leaves (Fig. 1F), no visible evidence of DED was observed on the infected plants of ‘Dodoens’ in the following growing seasons. Thereby, due to their high tolerance to the pathogen, the infected plants of ‘Dodoens’ were included in the experiments described herein. SEM images of the plant material used in this study are presented in Fig. 1D–L. The experiments were conducted on fully expanded leaves which were measured directly in the field and sampled for the further laboratory procedures and analyses in June 2011.

Scanning electron microscopy of leaf and wood samples

Leaf cross-sections from the centre of the leaf blade were immersed in 5 % glutaraldehyde in a 0·1 m cacodylate buffer at pH 7·0, dehydrated in ethanol and acetone, and dried in liquid CO2 using a Leica EM CPD030 critical point drier (Leica Microsystems, Wetzlar, Germany). Leaf and wood sections were mounted on specimen stubs, sputter-coated with gold, and observed by high-vacuum SEM using a VEGA TS 5130 instrument (Tescan, Czech Republic) operating at 15 kV.

Leaf growth

Leaf growth characteristics were assessed on the third fully expanded leaf from the apex that was sampled from five-leaved current-year shoots. Leaf sizes (length, width, area) were measured with an LI-3000A leaf area meter (LI-COR, Lincoln, NE, USA). Leaf slenderness was calculated as the ratio between leaf length and leaf width. Leaves were dried at 65 °C for 72 h, then weighed. The variable LMA was calculated as the ratio between leaf dry mass and leaf area. Measurements were performed on eight plants of the non-infected ‘Groeneveld’ (five sun leaves per plant), four plants of the non-infected ‘Dodoens’ (five sun leaves per plant) and four plants of the infected ‘Dodoens’ (five sun leaves per plant). Two repetitions of the measurements were carried out.

Leaf histology

Leaf lamina samples were cut with a razor blade, fixed in 5 % glutaraldehyde in a 0·1 m cacodylate buffer at pH 7·0, dehydrated in ethanol and propylene oxide, and embedded in Spurr embedding medium. Sections approx. 1·5 µm thick were cut using the automated rotary microtome Leica RM2255 (Leica Biosystems, Nussloch, Germany) and glass knives, and stained with toluidine blue and basic fuchsin as described by Lux (1981). Sections were observed using an Olympus BX50F light microscope (Olympus Europa, Hamburg, Germany). The thickness of the leaf, mesophyll and palisade parenchyma was measured using NIS-Elements AR 3·0 image analysis software (Laboratory Imaging, Prague, Czech Republic). Measurements were performed on eight plants of the non-infected ‘Groeneveld’ (two sun leaves per plant, two sections per leaf, one measurement per section), four plants of the non-infected ‘Dodoens’ (two sun leaves per plant, two sections per leaf, one measurement per section) and four plants of the infected ‘Dodoens’ (two sun leaves per plant, two sections per leaf, one measurement per section). Two repetitions of the measurements were carried out.

Primary xylem conduit density and relative conductivity

Conduit density characteristics of the midrib primary xylem [tracheary element lumen area (A) and tracheary element densities (N) per 0·1 mm2 of the primary xylem area] were determined using NIS-Elements AR 3·0 image analysis software. Measurements of A and calculations of N were made on the population of tracheary elements, which ranged from 118 (the non-infected ‘Dodoens’) to 373 (the non-infected ‘Groeneveld’) tracheary elements per 0·1 mm2 of the primary xylem area within the examined midrib sections. In addition, the tracheary element lumen fraction (F) and the tracheary element size:number ratio (S) were calculated as described in Zanne et al. (2010). Total theoretical relative conductivity (RC) per 0·1 mm2 of the primary xylem area was calculated as the sum of individual RCs divided by the area of a cross-section of primary xylem (Ďurkovič et al., 2012), whereas the individual RC was calculated according to Zimmermann (1983) as the fourth power of the equivalent circle diameter of the tracheary element lumen. Lignin autofluorescence in cell walls of tracheary elements was detected by excitation at 360 nm using a barrier filter with a transmission cut-off at 470 nm, and photographed using a Leica DM4000 B microscope equipped with a Leica DFC490 digital colour CCD camera (Leica Microsystems). Primary xylem conduit density and RC measurements were performed on eight plants of the non-infected ‘Groeneveld’ (two sun leaves per plant, one section per leaf midrib), four plants of the non-infected ‘Dodoens’ (two sun leaves per plant, one section per leaf midrib) and four plants of the infected ‘Dodoens’ (two sun leaves per plant, one section per leaf midrib). Two repetitions of the measurements were carried out.

Atomic force microscopy and nanomechanical properties of cell walls of tracheary elements

Deparaffinized midrib cross-sections, approx. 15 µm thick, were mounted on glass slides coated with (3-aminopropyl)triethoxy-silane and allowed to air-dry in sterile Petri dishes. PeakForce QNM (quantitative nanomechanical) measurements were done using a MultiMode 8 atomic force microscope (AFM) with a Nanoscope V controller (Bruker Nano Surfaces, Santa Barbara, CA, USA). Cell walls of tracheary elements were indented by silicon cantilevers MPP-12120, model TAP150A (Bruker AFM Probes, Camarillo, CA, USA) with a spring constant between 4·7 and 5·3 N m−1, deflection sensitivity between 33·1 and 42·8 nm V−1, and resonance frequency between 141 and 144 kHz, at 25 °C and ambient air pressure. Prior to the measurements, the tip radius and geometry were controlled using a commercial grid for 3-D visualization. PeakForce QNM measurements of the reduced Young's MOE, adhesion and dissipation were performed at low approach tip velocities of 0·3–0·5 µm s−1. To achieve accurate and reliable calculations of MOE, a sufficient sample deformation of 2 nm was ensured by adjusting the PeakForce setpoint to 25 nN. Measurements were performed on eight plants of the non-infected ‘Groeneveld’ (one sun leaf per plant, four cell walls of tracheary elements per leaf midrib), four plants of the non-infected ‘Dodoens’ (one sun leaf per plant, four cell walls of tracheary elements per leaf midrib) and four plants of the infected ‘Dodoens’ (one sun leaf per plant, four cell walls of tracheary elements per leaf midrib). Two repetitions of the measurements were carried out.

Atomic force microscopy data processing

The initial data of MOE, coming from the PeakForce QNM mapping (represented by 256 × 256 matrices), were analysed by NanoScope Analysis software, version 1·40r2 (Bruker AXS, Santa Barbara, CA, USA), which uses the DMT model (Derjaguin et al., 1975). The raw data were subsequently imported into the MATLAB software, version 7 (MathWorks, Natick, MA, USA). A slippery effect on the steep surface topography strongly influenced the measurement of MOE. Thus, the height gradient was calculated for each image pixel. Values corresponding to ‘steep’ points, where the surface slope exceeded the value of 30 °, were not used (Ďurkovič et al., 2012).

Gas exchange

An open portable photosynthesis system with infra-red gas analyser LI-6400 XTR (LI-COR) was used for in situ measurements of gas exchange characteristics. Net photosynthetic rate (PN), transpiration rate (E), stomatal conductance (gs) and intercellular CO2 concentration (ci) were measured on leaves fully exposed to the sun under a saturating photosynthetic photon flux density of 1200 ± 5 µmol m−2 s−1 and an ambient CO2 concentration of 370 ± 5 µmol mol−1 using the 6400-08 standard leaf chamber with the 6400-02B LED red/blue light source (LI-COR). Instantaneous water-use efficiency (WUE), giving information on the photosynthetic carbon gain per unit transpirational water loss, was calculated as the ratio of PN to E (Campbell et al., 2005). During the described measurements, microclimatic conditions inside the assimilation chamber were kept constant (leaf temperature TL 21 ± 1 °C, relative air humidity 70 ± 5 %). The vapour pressure deficit ranged from 0·9 to 1·3 kPa. Measurements were performed on eight plants of the non-infected ‘Groeneveld’ (five sun leaves per plant, three measurements per leaf), four plants of the non-infected ‘Dodoens’ (five sun leaves per plant, three measurements per leaf) and four plants of the infected ‘Dodoens’ (five sun leaves per plant, three measurements per leaf). Two repetitions of the measurements were carried out.

Chlorophyll fluorescence and chlorophyll content

Chlorophyll a fluorescence yields were measured on both adaxial and abaxial surfaces of sun-exposed leaves using a portable fluorometer Plant Efficiency Analyser (Hansatech Instruments Ltd, Kings Lynn, UK). Leaves were kept for 30 min under leaf clamps for dark adaptation. After the initial measurement of dark-adapted minimum fluorescence (F0), leaves were exposed to a saturating irradiance of 2100 µmol m−2 s−1 for 1 s to measure the maximal fluorescence of dark-adapted foliage (Fm). Maximal photochemical efficiency of photosystem II [Fv/Fm = (Fm – F0)/Fm], variable-to-initial fluorescence ratio (Fv/F0) and potential electron acceptor capacity – ‘area’ (i.e. area above the induction curve between F0 and Fm) were determined. Measurements were performed on eight plants of the non-infected ‘Groeneveld’ (ten sun leaves per plant), four plants of the non-infected ‘Dodoens’ (ten sun leaves per plant) and four plants of the infected ‘Dodoens’ (ten sun leaves per plant). Two repetitions of the measurements were carried out.

Relative chlorophyll content was estimated with a portable chlorophyll meter CL-01 (Hansatech Instruments Ltd), and the results were expressed as the chlorophyll index (Cassol et al., 2008). Measurements were performed on eight plants of the non-infected ‘Groeneveld’ (seven sun leaves per plant, three measurements per leaf), four plants of the non-infected ‘Dodoens’ (seven sun leaves per plant, three measurements per leaf) and four plants of the infected ‘Dodoens’ (seven sun leaves per plant, three measurements per leaf). Two repetitions of the measurements were carried out.

Water potential

Leaf water potential (ΨL) was measured using the Scholander pressure chamber technique. After cutting, the leaves were immediately placed into a sealed plastic bag and then quickly measured (no more than 10 min elapsed between the time of leaf collection and measurement). Leaf samples were stuck through the head of a pressure chamber, model 1000 (PMS Instrument Co., Albany, OR, USA), which was slowly pressurized at a rate <0·02 MPa s−1 until a droplet of liquid occurred on the leaf cut surface. Chamber pressure at the moment of liquid emergence was recorded as a negative value of leaf water potential. Measurements were done from 0600 to 1800 h in 2 h resolution on eight plants of the non-infected ‘Groeneveld’ (one sun leaf per plant at each measuring interval), four plants of the non-infected ‘Dodoens’ (one sun leaf per plant at each measuring interval) and four plants of the infected ‘Dodoens’ (one sun leaf per plant at each measuring interval). Two repetitions of the measurements were carried out.

Statistical analysis

Data showed a normal distribution and thus were subjected to a one-way analysis of variance (ANOVA). Duncan's multiple range tests were used for pairwise comparisons of means. The effects of leaf surface on the yields of chlorophyll a fluorescence were tested by two-way ANOVA. The Pearson correlation coefficients were calculated for trait–trait linkages. The relationships were considered significant if P < 0·05.

Multivariate association of the 27 examined leaf traits was analysed with a principal component analysis (PCA) to describe patterns of covariation among leaf growth, vascular and ecophysiological traits.

RESULTS

Leaf growth

Data on the leaf growth traits are presented in Table 1. For leaf length, width, area and dry mass, there were no statistically significant differences found between non-infected plants of ‘Groeneveld’ and ‘Dodoens’. ‘Dodoens’ performed better than ‘Groeneveld’ for LMA, leaf thickness, mesophyll thickness and palisade parenchyma thickness. Infected plants of ‘Dodoens’ had a higher value of leaf slenderness than non-infected plants of ‘Dodoens’. For the remaining eight leaf growth variables, however, the differences between infected and non-infected plants of ‘Dodoens’ were negligible.

Table 1.

Leaf growth characteristics in the examined Dutch elm hybrids

Trait ‘Groeneveld’ non-infected ‘Dodoens’ non-infected ‘Dodoens’ infected
LL (cm) 8·36 ± 0·19a 7·79 ± 0·20a 7·96 ± 0·17a
LW (cm) 4·97 ± 0·12a 4·83 ± 0·12ab 4·57 ± 0·11b
LS (cm cm−1) 1·69 ± 0·01b 1·62 ± 0·02c 1·76 ± 0·03a
LA (cm2) 27·6 ± 1·3a 24·4 ± 1·2ab 22·9 ± 1·0b
LDM (g) 0·12 ± 0·01a 0·14 ± 0·01a 0·13 ± 0·01a
LMA (g m−2) 44·9 ± 1·0b 55·8 ± 1·7a 57·3 ± 1·3a
LT (μm) 136 ± 3b 166 ± 6a 165 ± 5a
MT (μm) 102 ± 2b 127 ± 5a 129 ± 4a
PPT (μm) 54·5 ± 1·6b 64·9 ± 3·0a 68·4 ± 3·3a
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LL, leaf length; LW, leaf width; LS, leaf slenderness; LA, leaf area; LDM, leaf dry mass; LMA, leaf mass per area; LT, leaf thickness; MT, mesophyll thickness; PPT, palisade parenchyma thickness.

Data represent means ± s.e. Mean values followed by the same letters within the same row across hybrids are not significantly different at P < 0·05.

Primary xylem conduit density and nanomechanical properties of cell walls of tracheary elements

‘Dodoens’ had significantly higher values than ‘Groeneveld’ for primary xylem conduit density components such as A and S, as well as for RC per unit area (Table 2). With regard to the components N and F, there were no significant differences found between the two Dutch elm hybrids. Interestingly, the differences between infected and non-infected plants of ‘Dodoens’ were negligible for all the examined primary xylem conduit density components.

Table 2.

Primary xylem conduit density characteristics and nanomechanical properties of cell walls of tracheary elements in the examined Dutch elm hybrids

Trait ‘Groeneveld’ non-infected ‘Dodoens’ non-infected ‘Dodoens’ infected
A (10−5 mm2) 13·8 ± 0·7b 17·3 ± 0·9a 17·3 ± 0·9a
N 202 ± 13a 168 ± 15a 165 ± 8a
F (10−3 mm2) 26·1 ± 0·4a 27·8 ± 1·1a 27·8 ± 1·0a
S (10−7 mm4) 7·8 ± 0·7b 11·4 ± 1·3a 11·0 ± 0·9a
RC (10−6 mm4) 7·27 ± 0·44b 9·62 ± 0·50a 9·78 ± 0·77a
MOE (MPa) 2809 ± 185a 1960 ± 199b 2097 ± 235b
ADH (nN) 9·6 ± 0·6a 10·1 ± 1·3a 10·6 ± 0·9a
DIS (eV) 485 ± 41a 515 ± 69a 521 ± 45a
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A, tracheary element lumen area; N, number of tracheary elements per 0·1 mm2 of the primary xylem area; F, tracheary element lumen fraction; S, tracheary element size:number ratio; RC, total theoretical relative conductivity per 0·1 mm2 of the primary xylem area; MOE, modulus of elasticity; ADH, adhesion; DIS, energy dissipation.

Data represent means ± s.e. Mean values followed by the same letters within the same row across hybrids are not significantly different at P < 0·05.

‘Groeneveld’ had significantly higher values than ‘Dodoens’ for MOE of cell walls of midrib tracheary elements. However, for this trait, there was no significant difference found between infected and non-infected plants of ‘Dodoens’. In addition, cell walls shared the same similarities for other nanomechanical properties, i.e. differences between non-infected plants of ‘Groeneveld’ and ‘Dodoens’, as well as between infected and non-infected plants of ‘Dodoens’, were negligible for both adhesion and energy dissipation (Table 2). Lignin autofluorescence imaging of the primary xylem structure in the midrib, and AFM peak force error and height images of the cell wall surfaces of tracheary elements are presented in Fig. 2.

Fig. 2.

Fig. 2.

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Lignin autofluorescence images of the primary xylem in the midrib (left images), AFM peak force error images (middle images) and AFM flatten height images (right images) of the cell wall surfaces of tracheary elements. Scale bars for non-infected plants of ‘Groeneveld’ and ‘Dodoens’: 100 µm for fluorescence microscopy images and 1·7 µm for AFM images. Scale bars for infected plants of ‘Dodoens’: 100 µm for the fluorescence microscopy image and 1·6 µm for AFM images.

Ecophysiological traits

‘Dodoens’ had significantly higher values than ‘Groeneveld’ for PN, E, gs and ci (Table 3). ‘Groeneveld’ performed better than ‘Dodoens’ for WUE and ΨL. There were no significant differences found between non-infected and infected plants of ‘Dodoens’ for the above five gas exchange variables and leaf water potential. The highest relative chlorophyll content was determined in non-infected plants of ‘Dodoens’. The infected plants of ‘Dodoens’ still had a significantly higher value of chlorophyll index than non-infected plants of ‘Groeneveld’.

Table 3.

Ecophysiological characteristics in the examined Dutch elm hybrids

Trait ‘Groeneveld’ non-infected ‘Dodoens’ non-infected ‘Dodoens’ infected
PN (μmol CO2 m−2 s−1) 14·6 ± 0·2b 21·5 ± 0·5a 22·0 ± 0·2a
E (mmol H2O m−2 s−1) 2·33 ± 0·03b 3·76 ± 0·08a 3·72 ± 0·03a
gs (mmol H2O m−2 s−1) 200 ± 4b 380 ± 14a 392 ± 6a
ci (μmol CO2 mol−1) 238 ± 2b 253 ± 1a 255 ± 2a
WUE (μmol CO2 mmol H2O−1) 6·34 ± 0·09a 5·70 ± 0·05b 5·94 ± 0·08b
ΨL (MPa) 1·76 ± 0·05a 1·55 ± 0·03b 1·54 ± 0·05b
CHLI 10·4 ± 0·2c 14·1 ± 0·2a 11·9 ± 0·3b
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PN, net photosynthetic rate; E, transpiration; gs, stomatal conductance; ci, intercellular CO2 concentration; WUE, instantaneous water-use efficiency; ΨL, leaf water potential; CHLI, chlorophyll index.

Data represent means ± s.e. Mean values followed by the same letters within the same row across hybrids are not significantly different at P < 0·05.

The results of chlorophyll a fluorescence yields for both leaf surfaces are given in Fig. 3A–F. Two-way ANOVA showed that elm cultivar and leaf surface significantly contributed to the differences that were found for the three examined variables (Table 4). Abaxial surfaces had significantly higher values of Fv/Fm and Fv/F0 ratios than adaxial surfaces. With regard to the variable ‘area’, higher values were recorded on adaxial surfaces. Taken together for both leaf surfaces, ‘Dodoens’ performed better than ‘Groeneveld’ for Fv/Fm and Fv/F0 ratios as well as for the area above the induction curve between F0 and Fm. Interestingly, the infected plants of ‘Dodoens’ had significantly higher values of the three chlorophyll fluorescence variables than non-infected plants of ‘Dodoens’.

Fig. 3.

Fig. 3.

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Chlorophyll a fluorescence yields in the examined Dutch elm hybrids determined on adaxial (A, C, E) and abaxial (B, D, F) leaf surfaces. (A, B) Results of the maximal photochemical efficiency of photosystem II (Fv/Fm). (C, D) Results of the variable-to-initial fluorescence ratio (Fv/F0). (E, F) Results of the potential electron acceptor capacity (‘area’). Histograms represent means ± s.e. Mean values followed by the same letters across hybrid cultivars are not significantly different at P < 0·05.

Table 4.

Two-way ANOVA of chlorophyll a fluorescence variables in the examined Dutch elm hybrids

Source of variation Degrees of freedom
 Sum of squares
 Mean square
 F-test
Fv/Fm Fv/F0 ‘Area’ Fv/Fm Fv/F0 ‘Area’ Fv/Fm Fv/F0 ‘Area’ Fv/Fm Fv/F0 ‘Area’
Elm 2 2 2 0·026 17·753 0·210 0·0132 8·877 0·105 71·84*** 74·09*** 44·53***
Leaf surface 1 1 1 0·001 0·584 0·655 0·0010 0·584 0·655 5·91* 4·88* 277·48***
Elm × surface 2 2 2 0·001 0·718 0·046 0·0005 0·359 0·023 2·79 NS 3·00 NS 9·73***
Error 314 314 314 0·058 37·623 0·741 0·0001 0·120 0·002
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***P < 0·001; *P < 0·05; NS, non-significant.

Correlated and independent traits

As expected, several traits were correlated with LMA and MOE, respectively. Leaves with a large carbon and nutrient investment in LMA tended to have a shorter leaf length (r = –0·58, P = 0·018), and a greater leaf thickness (r = 0·70, P = 0·002, Fig. 4A), mesophyll thickness (r = 0·73, P = 0·001) and palisade parenchyma thickness (r = 0·67, P = 0·005, Fig. 4B), as well as higher rates of PN (r = 0·83, P < 0·001, Fig. 4C), E (r = 0·82, P < 0·001), gs (r = 0·87, P < 0·001) and ‘area’ (r = 0·65, P = 0·007). On the other hand, LMA was independent of RC per unit area (r = 0·18, P = 0·51, Fig. 4D).

Fig. 4.

Fig. 4.

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Trait linkages with (A–D) leaf mass per area (LMA) and with (E–H) modulus of elasticity (MOE), identified in the examined Dutch elm hybrids. Relationships of LMA to leaf thickness (A), palisade parenchyma thickness (B), net photosynthetic rate (C) and relative hydraulic conductivity per 0·1 mm2 of the primary xylem area (D). Relationships of MOE to relative hydraulic conductivity per 0·1 mm2 of the primary xylem area (E), tracheary element lumen area (F), number of tracheary elements per 0·1 mm2 of the primary xylem area (G) and leaf area (H). Non-infected plants of ‘Groeneveld’, non-infected plants of ‘Dodoens’ and infected plants of ‘Dodoens’ are as indicated in the key in (E).

Significant linkages were also found between cell wall stiffness of tracheary elements and other vascular traits. RC per unit area (r = –0·74, P = 0·001, Fig. 4E), tracheary element lumen area (r = –0·76, P < 0·001, Fig. 4F) and tracheary element size to number ratio (r = –0·75, P < 0·001) were greater in midribs with lower MOE values of cell walls of tracheary elements. A positive correlation has been found between MOE and the number of tracheary elements per unit area (r = 0·68, P = 0·004, Fig. 4G). We found no significant support for a negative correlation of MOE with leaf area (r = –0·08, P = 0·78, Fig. 4H).

Associations among leaf traits

A PCA was done to evaluate how leaf traits were associated (Fig. 5). The first axis explained 37 % of the variation and showed strong positive loadings for E, gs, PN, A, S and the leaf tissue thickness traits. The negative side of the axis indicated strong loadings for N, MOE, WUE and ΨL. The second axis explained 18 % of the variation and showed strong positive loadings for leaf growth variables such as leaf area, length, width and dry mass. The negative side of the axis indicated strong loadings for WUE, LMA, MOE and N. In addition, PCA showed that both examined hybrids formed compact homogeneous clusters, clearly separated from each other, except for the single outlier specimen from each hybrid cultivar. These two specimens were placed outside their own clusters, extending to the clusters of their counterparts. The positions of infected plants of ‘Dodoens’ were placed almost within the range of non-infected plants of ‘Dodoens’.

Fig. 5.

Fig. 5.

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Positions of 27 leaf traits on the first and second axes of the principal component analysis (PCA). Trait abbreviations are found in Tables 1–3 and Fig. 3. Non-infected plants of ‘Groeneveld’, non-infected plants of ‘Dodoens’ and infected plants of ‘Dodoens’ are as indicated in the key. The bottom and left-hand axes refer to the examined leaf traits, whereas the top and right-hand axes refer to the examined trees.

DISCUSSION

We found definite differences in leaf traits between the two Dutch elm hybrids with a contrasting tolerance to DED. Furthermore, we found an unexpected number of similarities between the infected and non-infected plants of ‘Dodoens’. We relate these findings to previously published works, highlighting the novel findings pertinent to nanomechanical properties of the cell walls of midrib tracheary elements.

Leaf trait dissimilarities between the examined Dutch elm hybrids

For the whole complex of leaf growth, vascular and ecophysiological traits, trait dissimilarities reached a frequency of 70·4 %. Dissimilarities were particularly dominant among ecophysiological traits, where 100 % of the examined traits showed significant differences. Dissimilarities were also found, but to a lesser extent, among leaf growth (55·6 %) and vascular traits (50 %). We found that ‘Dodoens’ leaves had significantly higher gas exchange rates than ‘Groeneveld’ leaves. Thus, the enhanced photosynthetic capacity accumulated more carbon biomass per unit leaf area (LMA), which would contribute to the faster relative growth rate of ‘Dodoens’ (Shipley, 2002; Kruger and Volin, 2006; Pasquet-Kok et al., 2010). In addition, the greater thickness of leaf, mesophyll and palisade parenchyma may also contribute to the faster growth of ‘Dodoens’ due to their positive correlation with LMA that was found in this study (Niinemets, 1999; Vendramini et al., 2002).

Vascular traits

Tracheary element size and number are the primary indicators of leaf vascular strategy. Tracheary element lumen area strongly affects hydraulic conductivity, whereas conduit density influences bulk xylem composition (Preston et al., 2006). The influence of tracheary elements on xylem density can be decomposed into two additional significant components of vascular strategy, tracheary element lumen fraction and tracheary element size to number ratio (Zanne et al., 2010). These primary xylem conduit density components were used in the previous comparative study of leaf vascular strategy among parental and hybrid species of Sorbus, where they exhibited parental-like or transgressive phenotypic expression in the examined hybrids (Ďurkovič et al., 2012). In this study, ‘Dodoens’ performed better than ‘Groeneveld’ for the components A and S. Moreover, the significantly lower value for relative hydraulic conductivity per unit area found in ‘Groeneveld’ limited its performance for the examined gas exchange variables such as PN, E, gs and ci (Tombesi et al., 2010).

Nanomechanical properties of cell walls of tracheary elements

There are a few studies in which AFM mapping has been used to examine the stiffness of vascular cells, e.g. stiffness of smooth muscle cells in monkey (Qiu et al., 2010) and coronary atherosclerotic plaque components in mouse (Tracqui et al., 2011). In a study carried out by Lacayo et al. (2010), AFM images were used to characterize the topography of the cell wall surface of single tracheary elements of Zinnia elegans after the transdifferentiation of cultured leaf mesophyll cells into tracheary elements. Unfortunately, no nanomechanical data for cell walls were provided. In this study, we were able to provide unique quantitative nanomechanical data through the application of PeakForce QNM measurements. The data of the reduced Young's MOE for Dutch elm hybrids lay fully within the range that was reported for cell walls of midrib tracheary elements of five Sorbus species (Ďurkovič et al., 2012). The cell walls of tracheary elements of ‘Groeneveld’ were stiffer as their MOE values were significantly higher than those of ‘Dodoens’. Possible indirect reasons for the above observations could be that the MOE was inversely correlated with tracheary element lumen area. ‘Groeneveld’ had significantly smaller free cavity space for water flow than ‘Dodoens’, hence ‘Groeneveld’ should have a thicker cell wall mass for water-conductive cells. The higher stiffness is usually caused by the thicker cell wall mass.

In addition, de Farias Viégas Aquije et al. (2010) examined the adhesion forces in leaves of Ananas comosus cultivars infected by the fungus Fusarium subglutinans using the AFM contact mode. The fusariosis-susceptible cultivar had a significantly higher frequency of high adhesion force measurements for soft mesophyll tissue cell walls than the resistant cultivar. However, this was not the case for the stiff, lignified cell walls of the Ulmus hybrid cultivars in this study. We found no significant support for the more adhesive surface of cell walls of tracheary elements in infected plants of ‘Dodoens’. This result may reflect a different nature and response of soft and stiff cells to fungus infection, as well as different effects of the fungi examined. Adhesion is a measure of a force interaction between the tip and the sample cell wall surface. Changes in adhesion may be a good indicator of alterations in cell wall biopolymer distribution and orientation as well as chemical alterations in cell walls. Dissipation deals with a deformation resistance of a material. Energy dissipated between the tip and the sample during each tap on the cell wall surface is related to the toughness of a material, and thus may be used to evaluate a degree of cell wall degradation by fungal hyphae. Biodegradation rapidly decreases toughness of secondary xylem measured by dynamic tests (Clausen, 2010). To the best of our knowledge, this study is the first to report quantitative data for adhesion and dissipation of the walls of conductive cells of leaves.

Leaf trait similarities between the infected and non-infected plants of ‘Dodoens’

For the whole complex of leaf growth, vascular and ecophysiological traits, trait similarities reached a frequency of 81·5 %. Strong similarities were found among vascular traits where 100 % of the examined traits showed non-significant differences. The proportion of trait similarities was also very high among leaf growth traits (88·9 %), with the only exception being the infected plants of ‘Dodoens’ with their more slender leaves. Although some differences were found among ecophysiological traits, the similarities were proportionally greater (60 %). Previous studies have shown that DED fungus is able to colonize remote areas in the plant such as the leaf midrib and secondary veins (Pomerleau and Mehran, 1966; Nasmith et al., 2008). Thus, we hypothesized that leaf trait performances of infected plants of ‘Dodoens’ related to gas exchange rates, chlorophyll fluorescence yields, leaf growth and nanomechanical properties of primary xylem cell walls would be significantly decreased. Unexpectedly, the infected plants had significantly higher values for chlorophyll a fluorescence variables, but these higher chlorophyll fluorescence yields did not have a direct impact on the more effective biological functioning of photosystem II in comparison with the non-infected plants. The reaction centres of photosystem II were intact functionally in both types of ‘Dodoens’; moreover, their Fv/Fm ratios were far higher than the threshold value of 0·725 that indicates the onset of reversible changes in reactions centres of photosystem II (Čaňová et al., 2012). In addition, the infected plants had a significantly lower chlorophyll index, but, again, no impact on the reduced rates of PN was observed in comparison with the non-infected plants. In U. minor plants inoculated with O. novo-ulmi ssp. americana, Oliveira et al. (2012) observed a significant decrease in chlorophyll content that was accompanied by a decrease in the Fv/Fm ratio. This discrepancy may be explained by the plant material used in the experiment and the experimental conditions. The authors used 2-month-old in vitro plants sensitive to DED, and the experiment was completed 42 d after inoculations with a suspension of blastospores. However, no work with the tolerant or resistant elm clones accompanied the above experiment. Also, no information about the destiny and the performances of infected plants in the next growing season was provided. In our experiment, however, mature plants of ‘Dodoens’ survived inoculations without severe damage, their leaves were fully functional in the following growing seasons, and no decreases in the Fv/Fm ratio and PN rate were observed in comparison with the non-infected plants.

Taken together, except for two traits (leaf slenderness and relative chlorophyll content), we found no evidence of a decrease in leaf trait performances among infected plants of ‘Dodoens’, despite the persistence of O. novo-ulmi hyphae in the lumens of midrib tracheary elements. This result implies that leaf growth, vascular and gas exchange traits in mature plants of ‘Dodoens’ were unaffected by the DED fungus.

Correlated and independent traits

The LMA, an important plant carbon economy trait, shows frequent linkages with other leaf functional ecology traits that together shape the performance of plants. In this study, we noted that LMA was interlinked with leaf lamina thickness and other examined leaf growth traits (Niinemets, 1999; Vendramini et al., 2002). Plants with higher values of LMA (here it was ‘Dodoens’) usually have a better leaf persistence and defence against herbivores and physical hazards (Poorter et al., 2009). We also observed that LMA strongly influenced gas exchange variables, mostly the net photosynthetic rate (Shipley, 2002; Wright et al., 2004). Thus, photosynthetic capacity scales linearly to the biomass investment in the leaf, making leaf anatomy the main driver of photosynthesis (Poorter et al., 2009). In addition, water flux-related traits including RC are also a potentially fundamental determinant of species performance differences (Sack and Holbrook, 2006). In this study, the RC of the examined Dutch elm hybrids was orthogonal to LMA, which supports the independence of these two hub traits (Brodribb et al., 2005; Sack et al., 2005).

Furthermore, we noted that MOE of the cell walls of tracheary elements was inversely correlated with tracheary element lumen area. Woodrum et al. (2003) and Jacobsen et al. (2005) observed a similar trend between the MOE of bulk secondary xylem and fibre lumen diameter. The lesser stiffness was caused by a thinner mass of supportive cell walls. In our case, the higher tracheary element lumen area caused higher stress in the cell walls due to the leaf lamina mass, and consequently caused a decrease in the MOE of cell walls, an observation also reported by Kern et al. (2005). On the other hand, MOE was positively related to the number of tracheary elements per unit area. Thus, the stiffness of the primary xylem, weakened by the higher tracheary element lumen area and the lower MOE of cell walls, was supported by the higher number of tracheary elements to achieve a trade-off. Lastly, in contrast to the previously reported negative correlation of MOE with leaf area in diverse Sorbus species (Ďurkovič et al., 2012), no relationship was found in this study due to insufficient variation in leaf area between the examined Dutch elm hybrids.

Conclusions

Strong dissimilarities in leaf trait performances (70·4 % in total) were observed between the examined Dutch elm hybrids. Both hybrids were clearly separated from each other in the multivariate leaf trait space. ‘Dodoens’ had significantly higher values for LMA, leaf tissue thickness variables, tracheary element lumen area, RC, gas exchange variables, relative chlorophyll content and chlorophyll a fluorescence yields. On the other hand, ‘Groeneveld’ had stiffer cell walls of tracheary elements, and also higher values for WUE and ΨL. Unexpectedly, we found a very high proportion of leaf trait similarities between the infected and non-infected plants of ‘Dodoens’ (81·5 % in total). Leaf growth, vascular and gas exchange traits in the infected plants of ‘Dodoens’ were unaffected by the DED fungus. Leaves with a large carbon and nutrient investment in LMA tended to have a greater leaf thickness and higher rates of PN, but LMA was independent of RC. Significant linkages were also found between the MOE of cell walls of tracheary elements and some vascular traits such as RC, tracheary element lumen area and the number of tracheary elements per unit area.

ACKNOWLEDGEMENTS

The authors thank Professor D. Gömöry for statistical advice, Dr A. Cicák and Mr M. Mamoň for technical assistance, and Mrs E. Ritch-Krč for language revision. This work was financed by the Slovak scientific grant agency VEGA (1/0132/12).

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