Protoplasma (2010) 243:137–143 DOI 10.1007/s00709-009-0039-6

ORIGINAL ARTICLE

Damage in needle tissues after infection with Chrysomyxa rhododendri increases cuticular conductance of Picea abies in winter Stefan Mayr & Franziska Schwienbacher & Barbara Beikircher & Birgit Dämon

Received: 26 January 2009 / Accepted: 25 February 2009 / Published online: 17 March 2009 # Springer-Verlag 2009

Abstract Chrysomyxa rhododendri is a rust which infects Picea abies growing near the alpine timberline. Attacked needles are normally shed, but few remain on shoots. We hypothesised that these needles increase transpiration of Picea during winter. Partly damaged, completely damaged and healthy needles of an infected tree as well as healthy needles of a resistant tree were compared in a microscopy analysis, and needle conductance of shoots was measured gravimetrically. Despite needle shedding, more than 6% of needles remaining on infected tree shoots were damaged. Partly damaged needles showed local brownish areas in the periphery and completely damaged needles necrotic parenchyma and epidermal tissues. Cuticular conductance of affected shoots was up to 25.23±2.75 mmol m−2 s−1 at moderate water potential and thus twofold higher than in the resistant tree. Needle shedding reduces negative effects of Chrysomyxa infections during summer, but remaining damaged needles impair tree water relations in winter. Keywords Alpine timberline . Conifer . Frost drought . Needle tissues . Pathogen defence . Water relations

Introduction Chrysomyxa rhododendri (DC.) De Bary is a needle-rustinfecting Norway spruce (Picea abies (L.) Karst.), one of

This work is dedicated to Professor Cornelius Lütz on the occasion of his 65th birthday. S. Mayr (*) : F. Schwienbacher : B. Beikircher : B. Dämon Inst. f. Botanik, University Innsbruck, Sternwartestr. 15, A-6020 Innsbruck, Austria e-mail: [email protected]

the dominating conifer species in Alpine regions of Central Europe. Negative effects of this parasite on tree life and forest management were underestimated for a long time (Oechslin 1933; Butin 1989; Schmidt-Vogt 1989), but increasing infection rates during the last decades revealed that especially young trees may be seriously damaged upon heavy and repeated infection (Oberhuber et al. 1999; Plattner et al. 1999; Bauer et al. 2000). Chrysomyxa rhododendri is restricted to subalpine regions where trees are within the reach of the airborne basidiospores formed on the main (telio-) host Rhododendron spp. (De Bary 1879). These basidiospores penetrate into current-year developing needles of spruce while they are not able to attack older needles. Infected needles become yellow during early summer, and in August aeciospores are formed by the fungus. The discoloration of needles is related to chlorophyll breakdown as well as carotenoid formation by the fungus (Pfeifhofer 1989). When aecia develop, Norway spruce induces the formation of abscission zones at the base of infected needles which are shed in late summer. Shedding removes the fungi from infected shoots and reduces transpirational water losses over attacked needles. It was demonstrated that older needles can partly compensate for the loss in photosynthetic tissue by an up-regulation of photosynthetic activity (Mayr et al. 2001). Such a compensatory potential was also reported for other conifers (e.g. Gezelius et al. 1981; Weikert et al. 1989). In previous studies (e.g. Mayr et al. 2001), we often observed few needles completely or partly damaged remaining on shoots, which indicates that the described defence mechanism of Norway spruce may not always work perfectly. While this probably has little impact on carbon assimilation, it might be relevant for plant water relation as damaged needles and their impaired cuticular shield will cause increased transpirational water losses.

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Several authors reported a generally high cuticular transpiration of Norway spruce growing at high altitudes (Baig et al. 1974; Tranquillini 1974; Baig and Tranquillini 1976; Anfodillo et al. 2002; Mayr et al. 2003). According to the hypothesis of Michaelis (1934a, b), this is due to an incomplete maturation of the cuticula because of the short vegetation period (Günthardt and Wanner 1982; Tranquillini and Platter 1983). For tree water relations of evergreen conifers, winter is known to be the most critical period at higher altitudes. It was demonstrated that Norway spruce trees growing at the alpine timberline exhibit extremely low water potentials (Richards and Bliss 1986; Lindsay 1971) as well as excessive xylem embolism (Mayr et al. 2002, 2003, 2006) caused by frost drought (“Frosttrocknis”, e.g. Michaelis 1934a, b; Pisek and Larcher 1954; Larcher 1972; Tranquillini 1976). Winter drought occurs because the soil and basal tree parts are frozen for months while transpirational water losses over exposed crown parts accumulate. In addition, freeze–thaw stress impairs the water transport system of trees (e.g. Sparks et al. 2001; Pittermann and Sperry 2006). In Norway spruce, Chrysomyxa infections thus might contribute to water stress in the winter season. This should be most relevant for trees growing at the alpine timberline as (a) Chrysomyxa infection intensities are highest due to the abundance of Rhododendron stands and (b) winter drought stress is known to be most critical at highest tree stands (e.g. Mayr et al. 2002). In this study, we therefore mapped cellular damage caused by Chrysomyxa infections of needles not shed during the vegetation period. We compared partly and completely damaged needle tissues with healthy tissues of an infected tree as well as with healthy needles of a resistant tree. We analysed needle conductance (gN) of shoots hypothesising that needle damage caused by Chrysomyxa infection would lead to increased transpiration.

S. Mayr et al.

study had to be restricted to these well comparable specimens. Measurements were made on current-year shoots of twigs about 2 m above ground level. Microscopical studies were made on sun-exposed needles whereby healthy needles of the resistant tree were compared with healthy, partly damaged and completely damaged needles of the infected tree in winter 2008. Needle conductance was measured on shoots harvested in winter 2002 from sunexposed as well as shaded twigs of the infected and resistant tree, respectively.

Materials and methods Analysis of damage Terminal shoots of sun-exposed twigs of the resistant and infected tree were harvested in winter to count the number of already shed needles and the number of completely as well as partly damaged, but remaining needles. The insertion position of shed needles was still visible so that their number could be counted. Photos of representative shoots and needles were taken using an Olympus SZ61 Binocular (Olympus Austria, Vienna, Austria) and a Sony DSC-W17 digital camera (Sony, Vienna, Austria). Microscopical studies We analysed cross sections and longitudinal sections of needles cut with a microtome (Schlittenmikrotom OME, Reichert, Vienna, Austria) using an Olympus BX41 light microscope (Olympus Austria, Vienna, Austria) and a Sony DSC-W17 digital camera (Sony, Vienna, Austria). Within partly damaged needles, sections at the border between healthy and damaged regions of the needle were made. Photos were colour-corrected and the background was optimised with Adobe Photoshop 7.0 (Adobe Systems GmbH, München, Germany).

Investigation sites and plant material Needle conductance (gN) Samples were taken from trees growing on the upper edge of a closed Norway spruce stand at Praxmar at 1,650 m, about 350 m below the timberline in the Tyrolean Central Alps. Measurements were made on a pair of Norway spruce (Picea abies (L.) Karst.) specimens already used for the study of Mayr et al. (2001), in which the effects of Chrysomyxa infection on photosynthesis were analysed. One of these trees (termed “infected” throughout the text) was severely infected over years, while the other (termed “resistant”) hardly showed any infection even in years with intensive Chrysomyxa attack. These trees, about 15 m tall and 55 years old, grow at a distance of about 10 m from each other. Resistant trees are extremely rare so that this

Twigs were harvested, transported to the laboratory in a plastic bag and re-cut under water. To ensure complete stomatal closure, terminal shoots of twigs were placed in a 100 mM abscisic acid solution (2-cis, 4-trans-abscisic acid; Sigma-Aldrich GmbH, Vienna, A) at day light for 6 h and subsequently fully hydrated in a plastic bag over night. Test measurements of a previous study (Mayr et al. 2003) revealed that abscisic acid treatment of needles harvested from alpine Norway spruce substantially reduced transpiration (33 to 47% of control needles, unpublished). Thus, for a comparison of cuticular conductance, abscisic acid is necessary to induce complete stomata closure. It is

Chrysomyxa infection increases cuticular conductance of P. abies in winter

unknown, if stomata are already partly opened in situ or open during harvest and preparation, possibly due to changes in temperature. Weight of saturated shoots was determined (Sartorius BP61S, 0.0001 g precision, Sartorius AG, Göttingen, Germany). During the following dehydration (in a darkened room, shoots were exposed on a fine net and ventilated with a fan) fresh weight and corresponding water potential (see below) as well as air humidity, temperature and atmospheric pressure were measured. After reaching a water potential of about −5 MPa for each sample, dry weight to projected needle area (measured with a digital video camera, Leaf Area and Analysis System SL 721, Skye Instruments Ltd., Llandrindod Wells, UK) were determined for a representative amount of needles as well as dry weight of all needles of the sample to calculate needle area. Evapotranspiration (EV [mol m−2 s−1]) was calculated according to Eq. 1 EV ¼ðΔWÞ=ðΔt  LA  18:015Þ

ð1Þ

where ΔW [g] is the loss in weight (Sartorius BP61S, 0.0001 g precision, Sartorius AG, Göttingen, Germany) during the measurement interval Δt [s] and LA [m2] is the needle area. The molecular mass of water is required for the conversion to mol. Needle conductance gN [mol m−2 s−1 ] was calculated as given in Eq. 2 gN ¼ EV=ððSVP  VPÞ=PÞÞ

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tree. For measurements of needle conductance, five sunexposed and shaded shoots of the resistant tree, and four sun-exposed and shaded shoots of the infected tree were harvested, respectively. Cuticular conductance and corresponding water potential were measured 13 to 25 times per shoot. Mean values are given ±standard error throughout the text. Differences were tested at 5% probability level with Student’s t test after testing for normal distribution and variance of the data.

Results Extent of damage Shoots of the resistant tree showed no needle shedding and only few partly damaged needles (Fig. 1, Table 1). Shoots of the infected tree shed about a fourth of their current-year needles in summer (Table 1). While most of the remaining needles showed no damage (Fig. 1) more than 6% were partly or completely damaged (Table 1). Partly damaged needles showed sharp borders between intact (green) areas and damaged (brown) sections. Tissue and cell damage

ð2Þ

where SVP [Pa] is the saturated vapour pressure, VP the actual vapour pressure [Pa] and P [Pa] the atmospheric pressure. Please note that (although evapotranspiration of the whole shoot was measured) we use the term needle conductance gN in the text because shoots loose most water over their needles. We do not refer to cuticular conductance, even when abscisic acid caused complete stomata closure, as in damaged needles the cuticula layers might have been disrupted. Water potential Water potential of dehydrating shoots was measured with a pressure chamber (Model 1000 Pressure Chamber, PMS Instrument Company, Corvallis, OR, USA). After measurements, the pressure was released slowly and the sample was again exposed at a fine net for further dehydration. Number of samples and statistics Microscopical analysis was made on needles of ten shoots harvested from the resistant and the infected tree, respectively. Analysis of damage was calculated from ten shoots of the infected and six shoots of the resistant

Needles of the resistant tree had a cross sectional shape with recessed lower sides and clearly defined epidermis and cuticula structures (Fig. 2a, e, i). The palisade tissue below the epidermis often looked more rigid than in healthy needles of the infected tree, which developed needles with rhomb-like cross section (Fig. 2b, f, j). No difference in structure or thickness of the cuticula and epidermis was observed between these two needle types. Partly damaged needles showed intensively brown coloured areas in restricted regions often situated near the needle surface. Cell structures within these regions were not clearly visible while in intact regions, cells appeared greenish and tissues looked intact (Fig. 2c, g, k; note that presented sections were made at the border of damaged and healthy regions of needles). In the centre of damaged regions, cells and tissues looked similar to those of completely damaged needles where most of the cells were obviously dead with the cell wall structures remaining (Fig. 2d, h, l). Large cavities in parenchyma tissues indicate that these needle parts died some time ago. Frequently, cellular structures in the epidermis were destroyed (Fig. 2h, l). This was rarely found in partly damaged and never in healthy needles. Only in the corners of cross sections, less-damaged regions were observed. Other main structures of the needle, like the central cylinder and resin channels, were still visible (Fig. 2d).

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Fig. 1 Sun-exposed shoots and needles of a resistant and an infected Picea abies tree. Panels a and b show representative shoots harvested in December 2008. Panel c shows healthy needles of the resistant tree and panel d partly damaged needles of the infected tree

cally significant (see “Materials and methods”), except conductance values of shaded shoots at low water potential.

Needle conductance (gN) Needle conductance of all samples decreased asymptotically with increasing water potential (Fig. 3). At moderate water potentials (between 0 and −1 MPa), sun-exposed shoots of the infected tree showed an about twofold higher mean gN (25.23±2.75 mmol m−2 s−1) than samples of the resistant tree (12.94±0.72 mmol m−2 s−1). Mean gN of shaded shoots was overall higher and in the infected tree (34.45±6.92 mmol m−2 s−1) 1.8-fold that of the resistant tree (18.97±1.21 mmol m−2 s−1). At low water potential (between -3 and -6 MPa), the infected tree showed a mean gN of 5.37±0.620 and 8.01±1.17 mmol m−2 s−1 and the resistant tree 3.86±0.27 and 6.36±0.60 mmol m−2 s−1 in sun-exposed and shaded shoots, respectively. All differences between the infected and resistant tree were statisti-

Table 1 Extent of needle damage on sun-exposed shoots of a resistant and an infected Picea abies tree

Shed Unshed Healthy Partly damaged Dead

Resistant tree

Infected tree

0%

26.4±5.2%*

97.4±0.6% 3.3±1.0% 0%

93.1±1.5%* 2.6±0.6% 3.7±1.3%*

The proportion of shed needles related to the total number of needles as well as the proportion of healthy, partly damaged and completely damaged needles related to the number of needles which remained on shoots after shedding are given *P≤0.05 indicates significant differences. Mean±SE

Discussion The main defence reaction of Norway spruce upon Chrysomyxa infection is the formation of abscission zones at the base of attacked needles and, in consequence, the shedding of infected needles. In 2008, about every fourth needle was shed (Table 1). While shedding rates depend on the intensity of infection and vary site-specific and from year to year (Mayr et al. 2001), personal observations and presented data demonstrate that every year few partly or even completely damaged needles remain on shoots of infected trees (Fig. 1). It is unclear why these attacked needles could not be shed. It has to be mentioned that partly damaged needles were also found at the resistant tree (Table 1), but these injuries were normally restricted to smaller regions within needles and often looked different in shape and colour from Chrysomyxa infections. The infected Norway spruce tree obviously also started defence mechanisms within attacked but unshed needles as no fungal hyphae could be observed in microscopical sections (Fig. 2). In partly damaged needles, local brown areas with sharp borders to intact regions (Figs. 1d, and 2c) indicated that the pathogen was encapsulated by hypersensitive response reactions (e.g. Király et al. 2007; Kliebenstein and Rowe 2008). The more brownish colour of the needle parenchyma was probably related to beginning chlorophyll breakdown in these needle parts (Pfeifhofer 1989). For water losses over needles, within-needle conductivity and the cuticular shield may be of relevance. Both of these

Chrysomyxa infection increases cuticular conductance of P. abies in winter

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Fig. 2 Microscopical analysis on needles of sun-exposed shoots from a resistant and an infected Picea abies tree. In the first and second row, complete cross sections and tissues at the upper part of cross sections of healthy needles from the resistant tree (a, e) as well as of healthy, partly and completely damaged needles of the infected tree (b–d, f–h) are given, respectively. The third row shows according longitudinal section with cuticula, epidermis and parenchyma cells (i–l)

components were probably affected in damaged needles because parenchyma tissues as well as the epidermis were at least partly destroyed. In completely damaged needles, large cavities were visible between cells (Fig. 2l) which may have caused hydraulic changes like reported by Raimondo et al. (2005) for leaves of Aesculus hippo-

Fig. 3 Cuticular needle conductance. Panel a and c show the needle conductance (gN) of current-year shoots of sun-exposed (open symbols, a) and shaded (closed symbols, c) twigs from the resistant tree and panel b (sun-exposed) and d (shaded) from the infected tree at different water potentials during dehydration. Stomata closure was obtained with abscisic acid solution before measurements

castanum infested by Cameraria ohridella. In addition, former insertion areas of shed needles may contribute to water losses when bark tissue did not completely seal gaps in the transpiration shield. Needles, damaged by Chrysomyxa but not shed during the vegetation period, caused a remarkable increase in needle conductance (gN) of the infected tree (for the definition of gN see “Materials and methods”). Although there are annual differences between infection rates (note that gN was analysed in 2002), only few partly or completely damaged needles remain on twigs every year (personal observation). It is remarkable that the gN of shoots from the infected tree was up to twice that from the resistant tree: This difference was not related to differences in stomatal closure as use of abscisic acid in our experiments (see “Materials and methods”) ensured full stomata closure. We cannot completely exclude that the resistant tree had a generally lower gN but microscopical observations on healthy needles did not indicate differences in the transpiration shield, i.e. thickness of the epidermis or cuticlae (Fig. 2i, j). Thus, it is very probable that increased gN was an effect of Chrysomyxa infection. In sun-exposed needles, gN was lower as these needles have to be protected more efficiently from transpirational water losses due to overheating effects especially at the alpine timberline (Mayr et al. 2003; also see Baig and Tranquillini 1976; Anfodillo et al. 2002). Interestingly, shoots harvested at the upper border of the alpine timberline showed a higher gN (37.9±4.1 mmol m−2 s−1, Mayr et al. 2003) than samples of the presented study. This is due to the fact that trees analysed in the present study were growing about 350 m below the timberline. At lower altitude, full

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cuticula development is possible during summer, while the short vegetation period at the timberline causes an insufficient cuticula maturation (e.g. Michaelis 1934a, b, also see “Introduction”). Accordingly, Tranquillini (1976) reported a sevenfold higher cuticular transpiration of Picea abies needles growing at upper sites of the treeline compared to the valley. However, as Chrysomyxa infection rates are high at the timberline (see introduction) and nearly all trees are attacked, we assume that a part of the high gN measured on timberline trees was related to partly or completely damaged needles. Thus, on a needle area basis Chrysomyxa infections cause an increase in transpirational water losses at the timberline. On a tree level, effects are more difficult to estimate: during winter, ice blockages cause a complex and dynamic pattern of separated hydraulic sections within the crown (Mayr and Charra-Vaskou 2007) so that high gN of dead or partly damaged needles might cause critical drought stress in restricted regions, e.g. in distal shoot sections. On the other hand, needle area of infected trees is reduced due to needle shedding, which may counteract the effect of increased gN. In winter 2008, needle area of current-year shoots was reduced by about 26% (Table 1) so that a twofold increase in needle area based gN would result in an about 1.5-fold increase in shoot conductance. The effect on whole tree transpiration thus will strongly depend on intensities of infections and needle shedding which vary within trees. Many biotic and abiotic factors influence tree life at the alpine timberline whereby the limiting physiological components are still unknown. While defence mechanisms at a cellular, tissue and organ level help Norway spruce to reduce impairments of Chrysomyxa infections during the vegetation period, indirect negative effects on tree water relations during the consecutive winter season may contribute to overall high stress intensities. Acknowledgment This study was supported by APART (Austrian programme for advanced research and technology) and FWF, “Fonds zur Förderung der Wissenschaftlichen Forschung”.

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