Summary We examined manganese uptake and transloca-tion in 30-year-old silver fir trees (Abies alba Mill.) showing severe symptoms of needle chlorosis by analyzing both xylem and phloem sap of shoots and an extract of root sap originating from both xylem and phloem elements. Manganese concentra-tions in shoot xylem and phloem sap were significantly lower in chlorotic trees than in neighboring healthy trees. The Mn concentration of root sap was also lower in these Mn-deficient trees than in healthy trees, indicating reduced Mn uptake by Mn-deficient trees. Although Mn deficiency had no effect on the Mn concentration of whole roots, separation of root tissue into root cortex and stele (wood) suggested impaired translo-cation of Mn from the cortex to the stele in Mn-deficient trees. Triphenyltetrazolium chloride (TTC) tests indicated that there was no decrease in reducing capacity of the fine roots (< 1 mm in diameter) of Mn-deficient trees. Fine roots of Mn-deficient trees contained significantly more organic acids than fine roots of healthy trees, including increased concentrations of malic, quinic, trans-acontic and formic acid; however, concentrations of pyruvic and acetic acid were lower than in fine roots of healthy trees. The total amounts of organic acids in the rhi-zospheric soil were similar for healthy and Mn-deficient trees. Pyruvic acid concentration was significantly increased in the rhizospheric soil of Mn-deficient trees, and concentrations of simple aliphatic acids like formic and acetic acid also tended to be higher in the rhizospheric soil of Mn-deficient trees. Both pH and water content were higher in bulk soil and rhizospheric soil around Mn-deficient trees compared with soil around healthy trees. Although lower concentrations of exchangeable Mn were found in the soil around Mn-deficient trees, the active Mn concentration (sum of exchangeable and reducible Mn) did not differ between healthy and Mn-deficient trees. A consider-able proportion of manganese was in an oxidized form in the soil around Mn-deficient trees.
Keywords: needle chlorosis, organic acids, phloem exudation, xylem sap extraction.
Introduction
Needle chlorosis has attracted much attention because it is a common symptom of tree damage and forest decline. Needle yellowing in Picea abies L. Karst and Abies alba Mill. stands
has been associated with nutrient deficiencies and imbalances (Zöttl and Hüttl 1986, Ke and Skelly 1994). Magnesium defi-ciency has been identified as the main cause of needle yellow-ing in conifer trees growyellow-ing on acidic soil (Hüttl 1993), whereas potassium, iron and manganese deficiencies have been reported to induce needle chlorosis in trees growing mainly on limestone soils (Le Tacon and Miller 1970, Hant-schel et al. 1989). Several hypotheses have been proposed to explain these nutrient imbalances. For example, increasing acid and nitrogen deposition during the last decades may have contributed to nutrient imbalances in forest ecosystems (Goulding and Blake 1993). Furthermore, acid precipitation can have direct effects on nutrient status by promoting foliar leaching (Mengel et al. 1987). Acid precipitation can also alter soil conditions leading to either an increase or a reduction in plant manganese concentration, depending on the soil type and composition (Tveite et al. 1994).
In contrast to other conifer species, silver fir trees are known to have an enhanced sensitivity to biotic and abiotic factors (air pollutants, drought, forestry management) (Wentzel 1980, Schönhar 1985). Although many factors appear to be involved in the dieback of silver fir in Europe, the phenomenon is still not understood (review in Schütt 1994). Needle chlorosis of silver fir trees at a reforested site has been observed for the past decade (Flückiger et al. 1986), and in recent years, deficiency symptoms have become widespread within the stand. Foliar nutrient analysis revealed insufficient manganese concentra-tions in the chlorotic needles, but all other macro- and micro-nutrients were within the range of sufficiency. Needle chlorosis induced by manganese deficiency is frequently more pro-nounced in current-year needles than in older needles.
Because plant manganese is involved in the water-splitting reaction in photosynthesis and functions as a cofactor in many enzymatic reactions (Marschner 1986), manganese deficiency influences a variety of physiological and biochemical pro-cesses in plants (Burnell 1988). Few studies have focused on the physiological and biochemical changes in silver fir trees under conditions of manganese limitation; therefore, we have analyzed the causes of manganese deficiency by studying manganese uptake and translocation in trees, and plant-avail-able manganese in the soil. We also investigated the role of organic acids in uptake mechanisms under conditions of man-ganese deficiency.
Manganese deficiency of silver fir trees (
Abies alba
) at a reforested site
in the Jura mountains, Switzerland: aspects of cause and effect
ERIKA HILTBRUNNER and WALTER FLÜCKIGER
Institute for Applied Plant Biology, Sandgrubenstrasse 25, 4124 Schönenbuch, Switzerland
Received October 25, 1995
Materials and methods
General features of the site
The study was conducted in an approximately 30-year-old silver fir (Abies alba Mill.) plantation (located on former pasture land) in the Jura mountains, Switzerland (7°49′ N, 47°22′ W). Mean elevation of the northerly slope is 1000 m above sea level with an overall inclination of 20--27°. The site was influenced by landslides in the past (Geological Atlas of Switzerland 1977--1993). The soil, which is heterogeneous with a suspended water table, comprises a pseudogley-brown earth (gleyic cambisol) over dolomite.
Trees
In 1992, silver fir trees with severe needle chlorosis, presumed to be caused by Mn deficiency, were compared with adjacent trees that were not chlorotic. In 1993 and 1994, another group of healthy trees without neighboring chlorotic trees was in-cluded in the study. Trees were classified in four groups: Group A--healthy trees without neighboring Mn-deficient trees; Group B--healthy trees with neighboring Mn-deficient trees; Group C--Mn-deficient trees having shoots with green needles; and Group D--Mn-deficient trees having shoots with chlorotic needles. Groups C and D are subsamples taken from the same Mn-deficient trees. For studies of belowground biomass, Groups C and D were combined (Group C + D), because roots did not show any visible deficiency symptoms.
Manganese in tissue and sap
Manganese concentrations were determined in both xylem and phloem sap of current-year shoots and in root sap originating from both xylem and phloem elements. Manganese tissue concentrations in shoots, needles and roots were also meas-ured.
Extraction of xylem sap Extraction of xylem and phloem sap from shoots was conducted in June 1992 (Groups B--D) and August 1994 (Groups A--D). Shoots were harvested in the morning and placed in aluminum bags in the dark at 4 °C. Sap was extracted on the same day that shoots were harvested. Xylem sap was extracted by means of a Scholander pressure chamber. About 15 mm of the bark, phloem and cambium was removed from the cut end to prevent contamination of the sap by elements originating from these tissues. To minimize exclu-sion errors, the part of the shoot protruding from the pressure chamber ranged from 7 to 10 mm (Millar and Hansen 1975). The chamber pressure at which the cut surface began to wet was registered as the xylem water potential (Osonubi et al. 1988). Extruded sap was collected by a glass pipette, trans-ferred to Eppendorf tubes and stored at −20 °C.
Variation in shoot diameter among treatments was unavoid-able, because shoot diameters and lengths were significantly less in the Mn-deficient trees than in the apparently healthy trees (data not shown). Because of the small size of the chlo-rotic shoots (Group D), a pressure of between 1.0 and 2.0 MPa over the xylem water potential had to be applied to extract xylem sap.
Exudation of phloem sap Shoots were recut under deminer-alized water with a razor blade after needles had been removed from the cut end of the shoot. The freshly cut shoots were placed in 5 ml of 20 mM EDTA to prevent formation of callose in the sieve elements. Exudation was conducted in a dark, humid chamber at 10 °C and stopped after 7 h (Bolsinger 1987). The exuded sap was immediately frozen and stored at −20 °C. Chemical analysis of xylem and phloem saps Samples were thawed and diluted just before chemical analysis. Manganese concentration was determined by flameless atomic absorption spectrometry (Zeeman Varian SpectrAA 300/400, Varian Techtron, Australia). Ashing temperature was 500 °C and at-omization temperature was raised to 2500 °C; palladium was added as the matrix-modifier. Potassium concentration was measured by atomic absorption spectrometry (Varian Spec-trAA atomabsorber, Varian Techtron, Australia) with flame emission.
The manganese/potassium ratio Because the Mn-deficient trees had greatly reduced shoot and needle lengths, mor-phometric effects on the element concentrations in the ex-tracted saps could be assumed. The high pressure applied, in particular to the chlorotic shoots, could also influence element concentrations in the xylem sap. Moreover, varying amounts of exuded phloem sap may result in substantial variations in Mn concentration. To minimize these sources of variation, the Mn concentration in the sap was related to the dry weight of the shoot or root from which the sap was extracted. To account for the varying total volumes of saps exuded and collected, the Mn/K ratio (multiplied by 1000) was calculated.
Analyses of needles and shoots Needles and shoots were dried at 70 °C for two days, and then ground by a ball mill (Retsch, Germany) with millcups made of agate to prevent metal contamination. The samples were ashed at 500 °C, digested in HNO3 and HCl at 150 °C (dry digestion after Allen
1989), and Mn and K concentrations were determined as described for the xylem and phloem sap. Apple leaves from the US National Bureau of Standards were used as external stand-ards.
Sap extraction from roots Extraction of root sap, which originated from both tracheids and phloem elements, was carried out in August 1992. Roots were located by digging along main roots to ensure that sampled roots were from the tagged trees. Roots were packed in polyethylene bags and stored at 4 °C in the dark. Sap was extracted by means of a Scholander pressure chamber as described for shoot xylem sap. About 15 mm of root cortex was removed from the cut end before extraction. Only roots with a diameter greater than 1 mm (including cortex) could be inserted in the Scholander chamber. Measurements were made on roots in the following diameter classes: small roots (≥ 1 but < 2.5 mm); medium roots (≥ 2.5 but < 5 mm); and coarse roots (≥ 5 mm). Roots less than 1 mm in diameter are referred to as fine roots.
Root samples taken in 1993 were divided into cortex and stele by hand and the fractions were analyzed separately.
Reducing capacity of roots
Reducing activity of fine roots (< 1 mm in diameter) of healthy and Mn-deficient silver fir trees was assayed with triphenyl-tetrazolium chloride (TTC). Absorption of the red formazan (reduced TTC) was measured at 530 nm (UV/VIS spectro-photometer, Perkin Elmer, Germany). A maximum absorbance of 0.3 was obtained when dead root samples (1 mm in diame-ter) were assayed with TTC (Joslin and Henderson 1984). Therefore, to ensure that only living roots were tested, subsam-ples (six per tree) with an absorbance of less than 0.40 were excluded from the analysis.
Organic acids
To analyze the Mn mobilizing capacity of roots of Mn-defi-cient trees, we determined the concentrations of organic acids in roots both in bulk soil and in rhizospheric soil samples.
For analysis of organic acids, washed fine roots (< 1 mm) and medium roots (≥ 2.5 but < 5 mm) were frozen in liquid nitrogen, ground in prefrozen (−70 °C) steelcups of a ball mill and homogenized at high speed (Polytron, Kinematica, Switzerland) in 10 ml of 0.0045 N NaOH, then centrifuged twice for 10 min each time at 4000 rpm and filtered through a 0.2 µm sterile filter (FP 030/3, Schleicher and Schuell, Dassel, Germany). The filtrate was purified by passage through a C18-cartridge (Sep-Pak Cartridges, Waters, Division of
Mil-lipore, Milford, MA.). The organic acids were analyzed by high pressure liquid chromatography (Waters, Division of Mil-lipore, Milford, MA), separated on an Animex HPX-87H col-umn with a Microguard colcol-umn (Biorad®, Hercules, CA). Separation was performed isocratically at a flow rate of 0.6 ml min−1 with 0.009 N H
2SO4 as the mobile phase and a column
temperature of 30 °C. Organic acids were identified and quan-tified by comparing retention times and peak heights with those of external standards using a detection wavelength of 210 nm (Tuneable absorbance detector 486, Waters, Division of Millipore, Milford, MA).
Soil analysis
Plant-available Mn forms were measured in the soil. Soil parameters (pH, water content, organic matter content, cation exchange capacity (CEC)) that are known to affect manganese plant availability were also determined (Uren et al. 1988).
Soil samples (bulk soil) were taken in April and August 1994 at a depth of 0--20 cm. Several subsamples were taken in the crown periphery and mixed to give one sample per tree. Rhi-zospheric soil was sampled only in August. Soil adhering to roots (< 1 mm) after shaking, was defined as rhizospheric soil and separated by hand with a thin plastic rod (Pluemper, Boehringer, Mannheim, Germany).
For analysis of organic acids, rhizospheric and bulk soil samples were immediately frozen in liquid nitrogen. After extraction with 0.0045 N NaOH (shaking for 30 min at 150 rpm), the filtrate was processed as described for roots. As a reference parameter to the organic acid contents, the organic
matter content was measured in the soil samples by wet oxida-tion with potassium dichromate.
Soil water content was determined gravimetrically (dried at 105 °C) as well as volumetrically. Volumetric water measure-ment was conducted in situ by the TRIME method (IMKO, Ettlingen, Germany). Soil pH was measured in a suspension of 0.01 M CaCl2. For the analyses of Mn, samples were dried at
40 °C and passed through a plastic sieve (< 2 mm).
Exchangeable Mn was extracted with neutral 1 N NH4OAc,
and the easily reducible manganese was extracted with neutral 1 N NH4OAc containing 0.5 g l−1 hydroquinone. Manganese
oxides were extracted with hydroxylamine hydrochloroide as described by Chao (1972). Filtrates were analyzed directly by atomic absorption spectrometry. Additionally, Mn4+
concen-trations in soil samples were assayed spectrophotometrically by means of leucoberbelin blue.
Measurements of CEC were made as described by Trüby and Aldinger (1984). Individual cations were analyzed in the filtered extract (O.5 N NH4Cl) by atomic absorption
spec-trometry after addition of CsCl2-aluminum nitrate (Na, K) and
CsCl2-lanthane chloride (Ca, Mg) solutions as the ionization
buffers (2.5% v/v). The CEC (in meq kg−1) was calculated as
the sum of Mg, Ca, K, and Na contents and the exchangeable acid, which was measured by titration with 0.01 N NaOH.
Data analysis
Data were log transformed and subjected to analysis of vari-ance. Differences in Mn concentrations and in related variables (µg Mn g−1 shoot dry weight, Mn/K ratio) in the extracted saps
between single trees and tree groups were evaluated by nested design of analysis of variance (replicates per tree nested under tree group). Additionally, mean differences were compared by Tukey’s HSD test. All analyses were calculated with Systat®,
Version 5.03 (Systat, Inc., Evanston, IL).
Results
Xylem water potential in shoots and roots
In 1992 and 1994, shoots with visible Mn deficiency symp-toms had significantly lower xylem water potentials than green shoots of healthy trees (Table 1), and all values were signifi-cantly lower in 1994 than in 1992. The healthy Group A trees, which were only included in the 1994 measurements, had significantly less negative xylem water potentials than trees in the other groups. The differences in xylem water potential between trees in the four groups could not be explained by variations in soil water content (vol %) (regression coefficient for log-transformed data in 1994: R2 = 0.236, P = 0.164, see Table 7 for values of soil water content).
Higher water potentials were observed in roots than in shoots. Root water potential increased significantly with in-creasing root diameter, but there were no differences between the healthy and Mn-deficient trees.
Manganese concentration and related variables in xylem sap Because there were no differences in Mn concentrations in the xylem sap between shoots from the upper and lower parts of the crown, data from different crown layers were combined. Manganese concentrations in xylem sap varied significantly between individual trees (values of single trees are not shown, but note the high standard deviations from mean values) (Ta-ble 2). In 1992, healthy Group B trees had significantly higher Mn concentrations and Mn/K ratios in the xylem sap than Mn-deficient trees (Groups C and D). However, when Mn concentration was expressed on a shoot dry weight basis, the chlorotic shoots of Group D trees exhibited the highest Mn concentrations as a result of a substantial decrease in shoot dry weight in response to Mn deficiency.
All trees that were measured in both 1992 and 1994 had lower Mn concentrations in xylem sap in 1994 than in 1992, but differences in Mn concentration among trees in Groups B--D were similar in both years, with the chlorotic shoots of the Mn-deficient trees having the lowest xylem sap Mn con-centration. Healthy Group A trees, which were only included in the 1994 study, had much higher xylem sap Mn concentra-tions than the other group of healthy trees (Group B), as a result of differences in shoot dry weight between the two groups.
Although individual trees differed significantly in root sap Mn concentration (data for individual trees are not shown), higher Mn concentrations and Mn/K ratios were generally observed in root sap of healthy trees than in root sap from Mn-deficient trees (Table 2). The difference in root sap Mn concentration between healthy and Mn-deficient trees was less pronounced in small roots (≥ 1 but < 2.5 mm) than in larger roots. The Mn concentration of roots of Mn-deficient trees decreased with increasing root diameter.
In 1992, there was no correlation between xylem water potential and Mn concentration of xylem sap in either healthy or Mn-deficient trees, whereas in 1994, the Mn concentration of xylem sap of the Mn-deficient trees (Group C) was nega-tively correlated with xylem water potential (regression coef-ficient R = --0.366, P < 0.05). However, in chlorotic shoots of Group D trees, which had the lowest xylem water potential of
all tree groups, no relationship existed between these two parameters.
Although, root water potential did not differ between healthy and Mn-deficient trees, the Mn concentration of small root sap of Mn-deficient trees was weakly negatively corre-lated with root water potential (R = --0.450, P < 0.01).
Manganese concentration in phloem sap
Manganese concentrations in phloem exudates paralleled those in xylem sap, although the values were lower (Table 2). In both 1992 and 1994, the phloem of healthy trees contained significantly higher Mn concentrations than the phloem of Mn-deficient trees. Among the tree groups, Group A trees had the highest Mn concentration in phloem exudate.
The difference in Mn concentration of phloem exudate com-pared with that of xylem sap reflects the difference in method of obtaining sap from the two tissues and is minimized by comparing the Mn/K ratios rather than Mn concentrations. In the healthy Group A trees, which had the highest Mn/K ratios, there was no significant difference in Mn/K ratios between xylem and phloem saps.
Roots
In contrast to the Mn concentration of extruded root sap, the Mn concentration of whole roots was not affected by the presumed Mn status of the trees (Figure 1a). According to the multi-mean test, the Mn concentration of the fine roots of the Mn-deficient trees (Group C + D) was not significantly lower than that of the fine roots of the neighboring Group B healthy trees. When roots ≥ 1 mm were separated into cortex and stele we found that the root cortex generally had a higher Mn concentration than the stele (Figure 1b). Furthermore, the Mn concentrations in the cortex and stele of roots ≥ 1 mm of Mn-deficient trees were significantly lower than in those of healthy trees. In all trees, Mn concentration decreased with increasing root diameter; however, the effect was only signifi-cant in Mn-deficient trees.
Table 1. Xylem water potential (MPa) of healthy and Mn-deficient silver fir trees (mean ± SD). Means followed by the same letter within each row are not significantly different (log-transformed data). Differences in xylem water potential of shoots between healthy and Mn-deficient trees were significant at P < 0.05 and P < 0.001 in 1992 and 1994, respectively. For roots, Groups C and D were combined.
Year Plant part Healthy trees Mn-deficient trees P Value
Group A Group B Group C Group D
1992 (June--July) Shoots nd1 −0.57 a ± 0.12 −0.61 ab ± 0.17 −0.68 b ± 0.20 < 0.05
1994 (August) Shoots −0.62 a ± 0.19 −0.89 b ± 0.20 −0.86 b ± 0.17 −0.98 b ± 0.25 < 0.001
1992 (August) Roots
≥ 1 but < 2.5 mm nd −0.39 ± 0.23 −0.43 ± 0.19 ns2 ≥ 2.5 but < 5 mm nd −0.28 ± 0.27 −0.33 ± 0.25 ns
≥ 5mm nd −0.08 ± 0.12 −0.09 ± 0.13 ns
Needles and shoots
In both years, needles with visible Mn deficiency symptoms showed the lowest mean Mn concentrations (0.9--2.3 µg Mn gdm−1) (Table 3). In contrast, the healthy Group A trees had
more than tenfold higher foliar Mn concentration than the healthy Group B trees. In healthy trees, the Mn concentration in shoots was lower than in needles (Figures 2a and 2b), whereas in Mn-deficient trees the relative concentrations were reversed.
Foliar K concentration followed a trend opposite to that of Mn (Table 3), i.e., it was significantly higher in chlorotic needles in both years. However, the K concentration of shoots without needles did not vary between the tree groups. Because shoots are composed of the same tissues from which the xylem and phloem saps were extracted, Mn/K ratios of the saps were positively correlated with shoot Mn concentrations (Table 4), except for the Mn/K ratio of xylem sap in 1992.
Reducing capacity of fine roots
Only one root subsample had a formazan absorption value below 0.40, indicating that the amount of dead roots in the root samples was small. Roots of Mn-deficient trees showed a slightly higher capacity to reduce TTC than roots of healthy trees (mean absorption of TTC of 67.6% versus 57.3 and
60.9%; F-value = 0.880, P = 0.433), implying that the living fine roots of Mn-deficient trees were in a comparable physi-ological state to those of healthy trees. Root reducing ability was not correlated with either Mn concentration in the fine roots (R2 = 0.032, P = 0.494) or foliar Mn concentration (R2 = 0.047, P = 0.360).
Organic acids in roots and soil extracts
Organic acids identified in fine roots (≤ 1mm) included oxalic, tartaric, pyruvic, malic, quinic, trans-aconitic, succinic, for-mic, acetic, fumaric, shikifor-mic, isocitric and citric (isocitric and citric acid eluted together) acids. Three acids with retention times of 12.8, 14.5 and 15.1 min were not identifiable and were excluded from the data. Of the detected acids, quinic, isocitric, citric, malic, oxalic and shikimic acids predominated in fine roots (Table 5).
The total amount of acids detected in fine roots was signifi-cantly higher in Mn-deficient trees than in healthy trees. Quinic, malic, trans-aconitic and formic acids were increased in the fine roots of Mn-deficient trees, whereas pyruvic and acetic acids were reduced. The sum of organic acids in fine roots was positively correlated with pH of the rhizospheric soil (R2 = 0.798, P < 0.001), although individual organic acids were not necessarily related to rhizospheric soil pH. A similar pat-tern of organic acids was found in medium roots (Table 5). The Table 2. Manganese concentrations (µg ml−1) in xylem, phloem and root saps extracted from shoots and roots of healthy and Mn-deficient silver fir trees (mean ± SD). Manganese concentrations are related to dry weight of plant part (µg g−1 shoot) and to sap potassium concentration (× 1000). Means followed by the same letter within each row are not significantly different. Differences between tree groups are significant at P < 0.001, 0.01 and 0.05 (log-transformed data, nested design of ANOVA; Tukey). For root wood, Groups C and D were combined.
Year Origin of sap Unit Healthy trees Mn-deficient trees P Value
Group A Group B Group C Group D
1992 Shoot xylem µg ml−1 -- 0.52 a ± 0.48 0.23 b ± 0.21 0.29 b ± 0.15 0.001 µg g−1 shoot -- 7.98 a ± 6.32 4.27 b ± 5.90 11.18 a ± 8.41 0.001
Mn/K ratio 1 -- 2.75 a ± 2.92 1.47 bc ± 1.36 1.66 c ± 1.02 0.01 Shoot phloem µg ml−12 -- 0.19 a ± 0.08 0.05 b ± 0.04 0.03 c ± 0.01 0.001
µg g−1 shoot -- 1.96 a ± 1.34 0.53 b ± 0.31 1.04 c ± 0.50 0.001 Mn/K ratio -- 2.02 a ± 0.85 0.81 b ± 0.54 1.09 c ± 0.48 0.001
1994 Shoot xylem µg ml−1 63.18 a ± 71.34 0.14 b ± 0.09 0.02 c ± 0.02 0.01 c ± 0.01 0.001 µg g−1 shoot 481.00 a ± 569.17 0.85 b ± 0.68 0.08 c ± 0.10 0.12 c ± 0.06 0.001
Mn/K ratio 33.62 a ± 25.53 1.16 b ± 0.98 0.17 c ± 0.20 0.07 d ± 0.04 0.001 Shoot phloem µg ml−1 1.10 a ± 0.63 0.26 b ± 0.13 0.08 c ± 0.03 0.05 d ± 0.02 0.001 µg g−1 shoot 9.23 a ± 5.54 1.42 b ± 0.66 0.47 c ± 0.16 1.03 d ± 0.42 0.001 Mn/K ratio 40.91 a ± 17.27 7.26 b ± 4.71 2.05 c ± 1.08 2.14 c ± 0.77 0.001
1992 Root wood µg ml−1 -- 0.49 a ± 0.46 0.25 ab ± 0.29 0.05
≥ 1 but < 2.5 mm µg g−1 root -- 1.29 a ± 1.28 0.67 a ± 0.91 0.05
Mn/K ratio -- 1.67 a ± 1.37 0.72 ab ± 0.86 0.01
≥ 2.5 but < 5mm µg ml−1 -- 0.69 a ± 0.78 0.17 b ± 0.20 0.001 µg g−1 root -- 1.14 a ± 1.30 0.21 b ± 0.22 0.001
Mn/K ratio -- 3.45 a ± 4.88 0.60 b ± 0.50 0.001
≥ 5 mm µg ml−1 -- 0.30 a ± 0.24 0.11 b ± 0.08 0.001
µg g−1 root -- 0.23 a ± 0.20 0.09 b ± 0.09 0.001
Mn/K ratio -- 1.45 a ± 1.06 0.53 b ± 0.35 0.001
1 Mn/K ratio was multiplied by a factor of 1000. 2 Unit is µg ml−1 phloem sap in EDTA solution.
medium roots of Mn-deficient trees had higher concentrations of quinic acid and lower concentrations of pyruvic acid than medium roots of healthy trees; however, the total amount of identified organic acids in medium roots did not vary between Mn-deficient and healthy trees.
Generally, concentrations of organic acids were lower in rhizospheric soil than in roots (Table 6). The dominant acids in rhizospheric soil extracts were quinic acid and acetic acid, whereas trans-aconitic, oxalic and fumaric acids were not detectable. Four acids with retention times of 10.26, 13.70, 15.75 and 16.55 min could not be identified. Three of these unidentified acids were increased in the rhizospheric soil of Mn-deficient trees. The total amount of identified organic acids in rhizospheric soil was similar for healthy and
Mn-defi-cient trees. Total organic acid concentrations in rhizospheric soil were not correlated with rhizospheric soil pH or with organic C content. Higher concentrations of pyruvic, formic and acetic acids were found in the rhizospheric soil of Mn-de-ficient trees than in the soil of healthy trees, whereas quinic acid tended to be higher in the rhizospheric soil of healthy Group A trees. Only traces of quinic, isocitric/citric and formic acids were detected in bulk soil samples and there were no differences between healthy and Mn-deficient trees.
Chemical soil parameters
Differences in gravimetric soil water content (w %) between the tree groups were less pronounced in spring than later in the season. Volumetric water content (vol %) of the bulk soil, Table 3. Concentrations of Mn (µg gdm−1) and K (mg gdm−1) in needles and shoots of healthy and Mn-deficient silver fir trees (mean ± SD). Means
followed by the same letter within each row are not significantly different (log-transformed data). Differences between healthy and Mn-deficient trees are significant atP < 0.05 and < 0.001 (ANOVA; Tukey’s HSD).
Healthy trees Mn-deficient trees P Value
Year Plant part Element Group A Group B Group C Group D
1992 Needles Mn -- 30.29 a ± 12.49 2.35 b± 1.16 1.50 c ± 0.62 0.001 K -- 10.28 a ± 2.39 11.07 ac ± 2.16 12.60 c ± 2.54 0.05 Shoots1 Mn -- 18.47 a ± 7.67 3.47 ac ± 1.25 4.38 c ± 1.90 0.001
K -- 12.67 ± 1.62 12.51 ± 1.57 12.31 ± 1.85 ns2
1994 Needles Mn 526.43 a ± 237.80 46.83 b ± 23.40 3.76 c ± 1.46 1.82 c ± 0.45 0.001 K 8.27 a ± 1.86 8.72 ab ± 1.13 10.77 ab ± 2.20 12.23 b ± 2.39 0.001 Shoots1 Mn 478.93 a ± 277.39 23.36 b ± 13.77 2.81 c ± 1.78 3.02 c ± 1.20 0.001
K 12.20 ± 1.34 11.63 ± 1.06 12.20 ± 1.13 12.21 ± 2.00 ns
1 Shoots contain only woody parts (without needles). 2 ns = Not significant.
Figure 1. Manganese concentrations (µg gdm−1) in (a) whole roots and (b) root cortex and stele of roots of different root diameters. Finest roots (<
1 mm) were not separated. Filled stars indicate significant differences between healthy and Mn-deficient silver fir trees (wwwP < 0.001), open
stars indicate significant differences between root diameter classes (qqqP < 0.001) (log-transformed data). Vertical bars represent standard errors
which was measured in situ, was significantly greater for Mn-deficient trees than for healthy trees (Table 7). The pH of both bulk and rhizospheric soils differed substantially between healthy and Mn-deficient trees. The lowest pH values were measured in soil samples from around healthy trees and the highest pH values were in soil sampled from around Mn-defi-cient trees. The healthy Group B trees showed significantly lower soil pH values than the Mn-deficient trees (Group C + D). The mean pH of the rhizospheric soil was generally lower than that of the bulk soil. All soil samples had slightly higher pH values in April than in August.
There were significant differences in exchangeable Mn con-centration of both the bulk and rhizospheric soils between the healthy and Mn-deficient trees (Figure 3a). The concentration of exchangeable Mn was higher in rhizospheric soil than in bulk soil. Although the concentration of exchangeable Mn in rhizospheric soil of healthy Group A trees was higher than expected based on soil pH, the active Mn concentration, which includes exchangeable and readily reducible Mn, was similar for healthy and Mn-deficient trees.
There was a higher concentration of oxidized Mn in soils with a pH > 5.2 than in soils with a pH < 5.2. The Mn-oxide concentration of bulk soil was significantly higher for Mn-de-ficient trees than for healthy Group A trees (Figure 3b). There was a tendency toward higher Mn-oxide concentrations in rhizospheric soil of Mn-deficient trees compared with healthy
trees, but the difference was not statistically significant ( F-value = 3.02, P = 0.068).
In general, higher Mn concentrations were obtained by the method of Chao (1972) than by extraction with NH4OAc,
because the latter method dissolved less plant-available man-ganese concretions in the soil. The soil around healthy trees had significantly lower concentrations of exchangeable basic cations than soil around Mn-deficient trees (Table 7) as a result of the high concentrations of Mg and, to lesser extent, of Ca in soil around the Mn-deficient trees.
Discussion
Low xylem water potential in chlorotic shoots of Mn-deficient trees was not related to low soil water contents, indicating that the water relations of these trees might be affected by Mn deficiency. It is known that herbaceous and crop plants wilt under severe Mn limitation (Eltinge 1941); however, the devel-opment of these wilting symptoms is not well understood. Because Mn plays an important role in plant lignin and phenol synthesis, it has been proposed that structural components of the sclerenchyma are less developed under Mn-deficient con-ditions than under non-limiting concon-ditions (Campbell and Nable 1988). Polle and Chakrabarti (1994) found less needle cell wall material but similar lignin content in Mn-deficient Norway spruce trees compared to healthy trees.
Osonubi (1988) and Berger et al. (1994) both reported a decrease in the nutrient concentrations of pressure extracted xylem sap of Norway spruce trees with increasing applied pressure (>1 MPa over the xylem water potential). Berger et al. (1994) postulated that the decrease in nutrient concentration was caused by dilution with hyperfiltrated cell water. In the present study, the pressure applied to extract xylem sap, espe-cially in the case of chlorotic shoots, was within the range where effects on sap concentrations might have been expected. Simple regression analysis revealed a negative relationship between xylem water potential and xylem sap Mn concentra-tion of the green shoots of Mn-deficient trees. However, no such relation was found in chlorotic shoots in which the lowest Table 4. Correlation coefficients (Pearson’s R) for the relationship
between the Mn/K ratios in the shoot saps and the shoot manganese concentrations (log-transformed data); ns = not signifcant.
Year n Origin of sap R P Value
1992 62 Xylem 0.103 ns
58 Phloem 0.726 0.001
1994 109 Xylem 0.943 0.001
105 Phloem 0.926 0.001
Figure 2. Manganese concentra-tions (µg gdm−1) in needles and
shoots of healthy and Mn-deficient silver fir trees in (a) 1992 and (b) 1994. Differences between healthy and Mn-deficient trees in both plant parts were significant at www
P < 0.001 in both years. Open stars represent significant differences be-tween needles and shoots (qq =
P < 0.01 and qqq = P < 0.001)
(log-transformed data).Vertical bars represent standard deviations of means. A = healthy trees (n = 6) without neighboring Mn-deficient trees; B = healthy trees (n = 6) with neighboring Mn-deficient trees; C + D = Mn-deficient trees (n = 12).
xylem water potential was measured. Published reports on the influence of low water potential on xylem sap nutrient concen-trations are inconsistent. Osonubi (1988) reported a decline in N, Mg and K concentrations in the xylem with decreasing water potential, whereas Berger et al. (1994) found no signifi-cant relationship between nutrient concentration of xylem sap and xylem water potential. In our study, positive associations between the two parameters were not observed consistently either among tree groups or between years.
Because the efficiency of sap extraction varied with extrac-tion methods, we used the Mn/K ratio to compare the sap composition between plant parts and also between sampling years. Differences in Mn/K ratios of the saps closely paralleled differences in shoot Mn concentration between healthy and Mn-deficient silver fir trees. The difference in Mn/K ratios between the two years may have been the result of seasonal effects. In 1994, the investigation was conducted one month later in the growth period than in 1992. According to Dambrine et al. (1995), the xylem sap element concentrations in Norway spruce shoots were highest after bud break at the beginning of needle growth, and a second peak occurred when needle growth was completed. Therefore, it can be assumed that the
xylem extraction in 1994 took place after the elemental con-centration of xylem sap had peaked.
The lower Mn concentrations found in shoot xylem and phloem saps of trees in Groups C and D compared with healthy trees indicate that Mn deficiency can be explained by lower uptake of the micronutrient. Because the extruded root sap contained nutrients that passed the root endodermis, these elements originated from both the tracheids and sieve ele-ments. Therefore, the root sap is not entirely comparable with the shoot saps. However, the lower Mn concentration in root sap of Mn-deficient trees compared with healthy trees supports the results from the shoot sap analyses indicating that less Mn was taken up by Mn-deficient trees than by healthy trees.
Studies with split-root systems in crop plants have shown that Mn moves freely in the xylem, but poorly in the phloem, so no translocation between the split root parts is observed (Nable and Loneragan 1984, Loneragan 1988). A similar ex-planation may underlie the finding that the Mn status of the tree had no effect on the Mn concentration of whole roots. Thus, both healthy and Mn-deficient trees accumulated Mn in the roots, but the proportion of Mn that was translocated to the shoots was lower in Mn-deficient trees than in healthy trees. This explanation is supported by the finding that Mn translo-Table 5. Organic acids in roots of healthy and Mn-deficient silver fir trees (mean ± SE). Differences between healthy trees and Mn-deficient trees are significant at P < 0.05, P < 0.01 and P < 0.001 (log-transformed data); ns = not significant.
Acid Healthy trees Mn-deficient trees P Value
(µg gdm−1) Group A Group B Group C+D
(n = 5) (n = 6) (n = 8)
Fine roots Oxalic 60.3 ± 4.8 40.5 ± 5.9 55.6 ± 3.1 ns
(< 1mm ) Isocitric/citric1 170.7 ± 19.3 190.6 ± 21.5 226.4 ± 16.9 ns
Tartaric 6.1 ± 1.5 14.4 ± 3.1 14.1 ± 3.4 ns
Pyruvic 6.5 ± 1.4 2.2 ± 2.2 < 1 0.05
Malic 116.5 ± 10.1 153.0 ± 16.2 202.8 ± 25.5 0.01 Quinic 328.7 ± 83.9 574.2 ± 46.1 814.1 ± 45.9 0.001
trans-Aconitic 1.1 ± 0.2 2.1 ± 0.2 6.7 ± 1.2 0.001
Succinic 10.0 ± 8.2 11.8 ± 11.3 3.8 ± 2.1 ns
Shikimic 15.4 ± 6.5 20.2 ± 8.7 12.9 ± 1.6 ns
Formic < 1 13.5 ± 4.0 14.5 ± 1.5 0.01
Acetic 30.0 ± 15.3 < 1 < 1 0.05
Fumaric 4.3 ± 0.8 3.8 ± 0.3 3.7 ± 0.2 n.s
Total 752.7 ± 100.1 1028.6 ± 45.8 1357.0 ± 45.0 0.01
Medium roots Oxalic 67.0 ± 14.4 24.6 ± 15.2 31.5 ± 12.2 ns
(≥ 2. 5 but < 5mm) Isocitric/citric1 247.3 ± 23.7 205.6 ± 35.8 242.7 ± 15.0 ns
Tartaric 8.1 ± 2.1 12.1 ± 1.6 7.8 ± 1.6 ns
Pyruvic 7.3 ± 2.8 < 1 1.4 ± 1.4 0.05
Malic 248.4 ± 40.4 232.0 ± 26.8 239.0 ± 22.4 ns Quinic 323.4 ± 19.9 544.0 ± 90.7 582.0 ± 58.0 0.05
trans-Aconitic < 1 < 1 < 1 ns
Succinic 13.7 ± 6.8 5.5 ± 3.4 6.5 ± 2.7 ns
Shikimic 22.6 ± 6.1 17.0 ± 2.4 15.2 ± 2.2 ns
Formic 10.9 ± 4.2 22.3 ± 6.2 21.2 ± 3.6 ns
Acetic 88.5 ± 56.2 < 1 < 1 ns
Fumaric 8.0 ± 3.0 3.5 ± 0.2 3.3 ± 0.1 ns
Total 1047.6 ± 115.5 1069.0 ± 128.9 1153.0 ± 65.7 ns
Table 6. Organic acids in soil extracts of healthy and Mn-deficient silver fir trees (mean ± SE).
Acid Healthy trees Mn-deficient trees P Value
(µg gdm−1 soil) Group A Group B Group C + D
(n = 5) (n = 6) (n = 8)
Rhizospheric soil Oxalic nd2 nd nd
--Isocitric/citric1 12.5 ± 1.9 4.8 ± 2.0 9.0 ± 2.2 ns 3
Tartaric 6.6 ± 1.1 6.7 ± 0.9 7.6 ± 0.7 ns
Pyruvic 4.0 ± 0.1 6.3 ± 0.8 7.4 ± 0.5 0.01
Malic 2.0 ± 0.9 2.2 ± 1.4 8.7 ± 2.0 ns
Quinic 128.1 ± 64.6 39.8 ± 9.5 58.0 ± 15.1 ns
trans-Aconitic nd nd nd
--Succinic 2.2 ± 2.2 nd < 1 ns
Shikimic 1.6 ± 0.7 1.0 ± 0.2 < 1 ns
Formic 5.0 ± 2.6 8.2 ± 3.7 9.4 ± 1.6 ns
Acetic 36.5 ± 4.5 44.5 ± 15.1 99.6 ± 25.4 ns
Fumaric nd nd nd
-Total 199.3 ± 66.5 115.2 ± 25.8 203.0 ± 39.1 ns
Bulk Soil Oxalic nd nd nd
--Isocitric/citric1 nd 1.9 ± 1.9 nd
--Tartaric nd nd nd
--Pyruvic nd nd nd
--Malic nd nd nd
--Quinic 1.0 ± 1.0 4.0 ± 4.0 < 1 ns
trans-Aconitic nd nd nd
--Succinic nd nd nd
--Shikimic nd nd nd
--Formic 4.3 ± 3.0 4.1 ± 2.0 3.0 ± 1.5 n.s
Acetic nd nd nd
--Fumaric nd nd nd
--Total 5.3 ± 4.0 10.1 ± 5.1 3.9 ± 1.5 ns
1 Isocitric and citric acids eluted together. 2 nd = Not detected.
3 ns = Not significant.
Table 7. Chemical parameters of the soil. Soil samples were taken in April and August 1994 (mean ± SD). Means followed by the same letter within each row are not significantly different (log-transformed data before analysis). The values of P indicate significant differences between healthy and Mn-deficient trees (ANOVA; Tukey’s HSD). (w % = gravimetric soil water content; vol % = volumetric water content).
Sample Parameter Healthy trees Mn-deficient trees P values
Group C + D
Group A Group B
(n = 6) (n = 6) (n = 12)
Bulk soil pH 4.4 a ± 1.2 6.5 b ± 0.7 7.0 b ± 0.4 0.001
April Water content (w %) 35.1 ± 3.7 39.7 ± 7.1 40.8 ± 3.9 ns (P = 0.057)
Water content (vol %) -- -- --
--CEC (meq kg−1) 124.6 ± 29.8 340.8 b ± 67.5 406.8 b ± 46.2 0.001 Organic C (%) 3.8 a ± 0.3 5.0 ab ± 1.6 5.9 b ± 0.7 0.01
Bulk soil pH 3.7 a ± 0.1 6.2 b ± 0.8 7.1 c ± 0.1 0.001
August Water content (w %) 24.8 a ± 5.2 34.0 b ± 7.0 36.8 b ± 3.9 0.01 Water content (vol %) 21.9 a ± 5.3 26.1 a ± 3.0 36.1 b ± 6.2 0.001 CEC (meq kg−1) 118.2 a ± 23.9 335.6 b ± 41.9 364.4 b ± 29.6 0.001 Organic C (%) 3.2 a ± 0.5 4.5 b ± 1.1 5.9 b ± 0.7 0.01
Rhizospheric soil pH 3.9 a ± 0.4 5.8 b ± 0.5 7.0 c ± 0.1 0.001 August Water content (w %) 26.4 a ± 2.4 33.2 b ± 4.2 36.1 b ± 6.2 0.01
Water content (vol %) -- -- --
--CEC (meq kg−1) 131.2 a ± 41.3 335.8 b ± 50.7 437.4 b ± 40.7 0.001 Organic C (%) 4.65 a ± 1.0 6.0 b ± 0.7 6.5 b ± 0.3 0.01
cation from the cortex to the stele of the roots, and thus to the shoots, was reduced in Mn-deficient trees. However, because we did not differentiate between the various forms of Mn in roots, our explanation is based on the assumption that the amount of readily available Mn was reduced in the root cortex of the Mn-deficient trees.
Foliar Mn concentration was closely associated with the incidence of deficiency symptoms. Manganese concentrations of about 0.9--2.3 µg gdm−1 in the chlorotic needles are well
below the foliar Mn concentrations reported in other studies (Kreutzer 1970, Walker 1991). Bergmann (1993) concluded that a foliar Mn concentration of 50 µg gdm−1 was the lower
limit for adequate nutrition of silver fir trees. Our finding of higher Mn concentration in needles than in shoots of healthy trees is consistent with other studies showing that needles can accumulate high amounts of Mn (Trüby and Linder 1990, Saur et al. 1992). Berchtold et al. (1981) observed Mn concentra-tions of over 4000 µg gdm−1 in silver fir needles. In addition,
Mn concentration increases with increasing needle age (Vogt et al. 1987, Wyttenbach et al. 1995).
The potassium concentration was enhanced in chlorotic needles, but not in shoots bearing chlorotic needles. The K concentration in the Mn-deficient needles was slightly above the range of 5--10 mg gdm−1 for adequate K nutrition of conifers
(Bergmann 1993). Similar increases in K in response to Mn-deficient conditions have been reported for Norway spruce needles (Kreutzer 1970) and leaves of Betula pendula (Roth.) seedlings (Göransson 1994). These results indicate that the higher K concentration in chlorotic needles can be regarded as a direct effect of Mn deficiency. Because the shoots of Mn-de-ficient trees bearing chlorotic needles had higher Mn concen-trations than the needles, we conclude that Mn transport from the shoots to the needles was impaired in the Mn-deficient trees.
Based on the TTC test and the analysis of organic acids, we conclude that Mn-deficient trees generally had the same physi-ological potential to take up Mn from the soil solution as healthy trees. The total amount of organic acids was signifi-cantly increased in the fine roots of the Mn-deficient trees. Quinic acid, which was the predominant acid in fine roots, is the main component of organic acids in conifer needles (Agui-Figure 3. Concentrations of (a) active and (b) oxidized manga-nese (µg gdm−1) in bulk and
rhi-zospheric soils of healthy and Mn-deficient silver fir trees. Cross-hatched columns (a) rep-resent the exchangeable propor-tion of the active manganese content. Hatched columns (b) show the Mn4+ proportion of the Mn oxides. Significant dif-ferences between healthy and Mn-deficient trees are indi-cated by the letters in the col-umns. Open stars represent significant difference between bulk and rhizospheric soil (Au-gust) (qqq = P < 0.001).
nagalde and Hüser 1981, Dittrich et al. 1989). Kretz (1973) reported that quinic, shikimic and malic acids are the main acid components in the cambial sap of Abies alba Mill.Quinic and shikimic acids are important compounds in the shikimic acid pathway leading to the synthesis of lignin and phenolics (Dit-trich et al. 1989). Because Mn deficiency prevents plants from effectively synthesizing lignin and phenolics (Campbell and Nable 1988, Rengel et al. 1994), we conclude that the high concentrations of quinic acid found in fine roots of Mn-cient trees reflect an accumulation induced by the Mn defi-ciency. However, even when quinic acid was excluded from the data analysis, the difference in total amounts of organic acids in fine roots persisted between the healthy and Mn-deficient trees. Additionally, the total amount of organic acids in the fine roots was strongly correlated with rhizospheric pH. Clémant (1977) compared the organic acid concentrations of Pinus nigra (ssp. nigricans)and Picea excelsa (Link) growing on highly calcareous and non-calcareous soils and found that, in both species, needles from trees growing on the calcareous soil had increased concentrations of organic acids. Tyler and Ström (1995) found that species from limestone soils exuded substan-tially more organic acids than species native to acid soils.
The predominant organic acids (isocitric, malic, oxalic) in the fine roots of silver fir have been observed in roots of many tree species (Smith 1976, Grierson 1992). Malic, citric and oxalic acid are known to form stable complexes with metal cations. The exudation of organic acids by roots is suggested as a general mechanism to enhance the availability of metals and other nutrients, particularly under conditions of Fe and P deficiency. Compared with other micronutrients, Mn forms less stable complexes with chelators (Laurie and Manthey 1994), so the direct effects of organic acids on Mn mobilization may be less pronounced than on metals such as Fe, Cu and Zn. Jones and Darrah (1994) found that Mn was not mobilized by malic or citric acid in either acid or alkaline soil.
The pattern of organic acids in rhizospheric soil of the silver fir trees differed from the pattern of organic acids in fine roots. However, we did not measure the amount of organic acids that was released from fine roots to the rhizosphere. Furthermore, degradation effects in the rhizospheric soil caused by microor-ganisms were not taken into account. The rhizospheric soil of Mn-deficient trees was characterized by an increased concen-tration of pyruvic acid and possibly of acetic and formic acids also. Fox and Cromerford (1990) measured substantially higher formic acid concentrations in rhizospheric soil com-pared to concentrations observed in the present study. They attributed the high formic acid concentration to organic matter decomposition under anaerobic conditions. A comparative study with different forest soils in Japan revealed increasing concentrations of formic and acetic acids with increasing soil water content (Tani et al. 1993). Baziramakenga et al. (1995) reported acetic acid as the main degradation product of other aliphatic acids in the rhizospheric soil of quackgrass Elytrigia repens L. Similarly, the total amount of water-soluble organic acids in the rhizospheric soil was not correlated with the organic C content in our study. The increased concentration of pyruvic acid in the rhizospheric soil of Mn-deficient silver fir
trees is difficult to interpret. In the few studies on organic acids in rhizospheric soil, pyruvic acid has not been detected (review in Bolan et al. 1994). On the other hand, Jauregi and Reis-enauer (1982) showed that, under artificial conditions, pyruvic acid is formed as a degradation product of malic acid in the presence of hydrous δ-MnO2.
Because of varying soil pH conditions, there was a substan-tial difference in the exchangeable Mn concentration between healthy and Mn-deficient trees. Both soil pH and Mn defi-ciency influenced the amount of organic acids in the fine roots. Although the total amount of organic acids correlated posi-tively with the exchangeable Mn concentration in the soil, this correlation cannot be considered biologically relevant because each parameter was itself correlated with pH. Similarly, Pohlman and McColl (1988) found no correlation between Mn concentration and the organic acids released from litter leachates.
Based on the observed concentration range of active Mn and the high pH of the soil, we conclude that trees are suffering from Mn deficiency at the study site (Schachtschabel et al. 1984). Moreover, it is known that other basic cations notably Mg and Ca may interfere with the Mn uptake (Reisenauer 1994). Also, at high soil pHs, Mn-oxidizing organisms may play an important role in the supply of plant-available Mn (review in Ghiorse 1988).
Acknowledgments
We thank the cantonal forestry administrations of northwestern Swit-zerland for financial support. For technical assistance in the field we are grateful to U. Kühne and F. Mucklow. For helpful discussions on the manuscript we thank Dr. R. Quiring. C. Ziegler kindly improved the English of the manuscript.
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