Chloroplast DNA phylogeography of Alnus glutinosa (L.) Gaertn.

Traditionally, information on the postglacial history of plant species has been gained from the analysis of fossil pollen data. More recently, surveys of present patterns of genetic variation have given valuable insights into species phylogeography. The genus Alnus , based on fossil data, is known to have had at least four glacial refugia. A survey of chloroplast DNA (cpDNA) diversity in populations of black alder ( A. glutinosa ) was undertaken in order to gain more insight into its postglacial history. This revealed a high degree of structuring of 13 cpDNA haplotypes on a European scale which indicated that most of northern and central Europe was colonized from a refuge in the Carpathian Mountains. Based on the distribution of two common cpDNA haplotypes, colonization routes from this refuge can be determined. The locations of other previously identified refugia are confirmed and two formerly unconfirmed refugial areas for alder (southern Spain and Turkey) are proposed.     

Cryopreservation of in vitro-grown shoot tips of Alnus glutinosa (L.) Gaertn.

In vitro-grown shoot tips of Alnus glutinosa (L.) Gaertn. were successfully cryopreserved by vitrification. Shoot tips (0.5–1 mm) excised from 6-week-old shoots were precultured in hormone-free Woody Plant Medium (WPM) supplemented with 0.2 M sucrose, for 2 days at 4 °C in the dark, and then treated with a mixture of 2 M glycerol plus 0.4 M sucrose, for 20 min at 25 °C. Osmoprotected shoot tips were first dehydrated with 50 % vitrification solution (PVS2), for 30 min at 0 °C, and then placed in 100 % PVS2, for 30 min at 0 °C. The solution was replaced with fresh 100 % PVS2, and the shoot tips were plunged directly into liquid nitrogen. The shoot tips were rewarmed in a water bath at 40 °C for 2 min, and then washed twice, for 10 min at 25 °C, with 1.2 M sucrose solution, before being transferred onto WPM supplemented with 0.5 mg l^?1 N ⁶-benzyladenine, 0.5 mg l^?1 indole-3-acetic acid, 0.2 mg l^?1 zeatin, 20 g l^?1 glucose and 6 g l^?1 Difco Bacto agar. The shoot tips were kept in darkness for 1 week and under dim lighting for another week, before being exposed to standard culture conditions (16 h photoperiod). This protocol was successfully applied to three alder genotypes, with recovery rates higher than 50 %.     

Characterization of a thermo-osmotic gas transport mechanism in Alnus glutinosa (L.) Gaertn.

Summary A gas transport system based upon the physico-chemical effect of thermo-osmosis of gases in described for the black alder, Alnus glutinosa (L.) Gaertn. Air is transported through the alder's stem to the roots, thus improving O₂ supply to respiring tissues of the root system. The gas transport system is investigated by means of a tracer gas technique (11% ethane in air, v/v). Gas transport depends on any source of radiant heat generating a temperature difference between the tree's stems and the atmosphere. The amount of gas transported in leafless trees is four times higher than the amount of gas reaching the roots by gas diffusion. Two-thirds of the gas is transported in the wood, only one-third in the bark. Intercellular spaces inside the porous lenticels of the bark are responsible for this kind of gas transport. Their diameters are estimated by the effusion rates of different tracer gases to be in the range of 1 m.     

Morphological and anatomical characterisation of black alder Alnus glutinosa (L.) Gaertn. ectomycorrhizas

 Ectomycorrhizal types of black alder [Alnus glutinosa (L.) Gaertn.] collected over a 3-year period within an alder forest were characterised by morphological and anatomical features. Of the total of 16 types, 14 are described for the first time in this paper. Eight identified types belong to the genera Russula, Lactarius, Naucoria, and Cortinarius, while eight further types remained unidentified. In some cases, similarities of mantle features indicate relationships to identified mycorrhizas. Mycorrhizas of Naucoria escharoides and N. subconspersa were not distinguished. Two unidentified mycorrhizal types exhibited hyphal mantle structures very similar to these Naucoria species. Within the genus Cortinarius, mycorrhizas of C. cf. helvelloides were easily distinguished from all other Cortinarius-like mycorrhizas described on Alnus, which in general showed little anatomical variation. Two further unidentified mycorrhizas, "Alnirhiza lilacina" and "A. violacea", probably also belong to Cortinarius. The ectomycorrhiza of Russula pumila was the only identified type within the genus Russula, but the unidentified type "Alnirhiza cremicolor" also likely belongs to this genus. Three Lactarius species were present in the experimental plot. Two species (L. obscuratus and L. omphaliformis) had indistinguishable mycorrhizal types, but were easily differentiated from the mycorrhizas of L. lilacinus, which caused intracellular penetration of Hartig net hyphae into epidermal and cortical cells. All other mycorrhizal types of black alder exhibited a paraepidermal Hartig net without penetration of root cells. Two unidentified mycorrhizal types "Alnirhiza atroverrucosa" and "A. cystidiobrunnea", already described from North American Alnus rubra as unnamed morphotypes, showed no similarity to identified mycorrhizas. All 16 mycorrhizal types appeared to be specific or at least typical for alders, since they have not yet been reported from other tree species. Accepted: 29 August 1997     

Effects of nickel on Frankia and its symbiosis with Alnus glutinosa (L.) Gaertn

The tolerance of nickel by Frankia in culture and in symbiosis with Alnus was determined. Yield of three Frankia strains was not affected significantly by 2.25 mM nickel when cultured in propionate medium containing hydolysed casein as nitrogen source. Yield of two strains in medium without combined nitrogen, and thus reliant on fixed nitrogen, was stimulated markedly by the same nickel concentration. Utilisation of nickel for synthesis of uptake hydrogenases is presumed to be the cause of enhanced nitrogenase activity.Although growth was reduced, treatment of 2-month-old seedlings with 0.025 mM nickel for 4 weeks did not affect nodulation significantly while nitrogenase activity was doubled. Nodulation and nitrogenase activity of seedlings receiving 0.075 mM nickel were inhibited markedly, while 0.5 mM nickel was lethal to all seedlings after 4 weeks of treatment. A few small, ineffective nodules were initiated early on some of the latter seedlings, suggesting that effects of nickel on host plant processes rather than Frankia are the primary cause of inhibition of nodulation. This interpretation is supported by the retention of substantial nitrogenase activity in 10-month-old plants 1 day after the treatment with 0.59 mM nickel, when the nickel content of roots and nodules was already maximal. No nitrogenase activity was detected after 3 days, by which time the leaves were almost completely necrotic. Over a 4 day period, most nickel was retained in the roots and nodules. Supplying histidine simultaneously at concentrations equal to, or in excess of, nickel prevented wilting and leaf necrosis, but did not increase translocation of nickel to the shoot.     

Seasonal fluctuations of the mineral concentration of alder (Alnus glutinosa (L.) Gaertn.) from the field

Summary The seasonal fluctuation of N, P, K, Ca, Mg, Fe, Mn, Mo, and Co, in leaves, roots and nodules of 40–50 year oldAlnus glutinosa trees growing at four different locations along the banks of the Tormes river, in the province of Salamanca, was studied. Also, the evolution of the soil organic matter under the trees sampled was evaluated. The data obtained for the various nutrient elements in the three plant parts are statistically treated at the significance levels of 99–95 per cent, and some remarks as to the nutritional status of the European alder in respect to the nutrients and its contribution to soil nutrient-cycling are provided. A positive correlation was found between N–P, N–K, N–Mg, and N–Mo, in leaves, and between N–P, N–K, N–Fe, N–Mn, and N–Mo in root nodules. In roots only, no significance at any level was obtained between N and any of the elements analyzed.     

Growth and nitrogen accretion of dinitrogen-fixing Alnus glutinosa (L.) Gaertn. under elevated carbon dioxide

Short-term studies of tree growth at elevated CO₂ suggest that forest productivity may increase as atmospheric CO₂ concentrations rise, although low soil N availability may limit the magnitude of this response. There have been few studies of growth and N₂ fixation by symbiotic N₂-fixing woody species under elevated CO₂ and the N inputs these plants could provide to forest ecosystems in the future. We investigated the effect of twice ambient CO₂ on growth, tissue N accretion, and N₂ fixation of nodulated Alnus glutinosa (L.) Gaertn. grown under low soil N conditions for 160 d. Root, nodule, stem, and leaf dry weight (DW) and N accretion increased significantly in response to elevated CO₂. Whole-plant biomass and N accretion increased 54% and 40%, respectively. Delta-¹⁵N analysis of leaf tissue indicated that plants from both treatments derived similar proportions of their total N from symbiotic fixation suggesting that elevated CO₂ grown plants fixed approximately 40% more N than did ambient CO₂ grown plants. Leaves from both CO₂ treatments showed similar relative declines in leaf N content prior to autumnal leaf abscission, but total N in leaf litter increased 24% in elevated compared to ambient CO₂ grown plants. These results suggest that with rising atmospheric CO₂ N₂-fixing woody species will accumulate greater amounts of biomass N through N₂ fixation and may enhance soil N levels by increased litter N inputs.     

Observations on the Formation and Function of the Root Nodules of Alnus glutinosa (L.) Gaertn

Nodulated alder plants grow vigorously in water culture withoutcombined nitrogen. Evidence is advanced to show that the fixationof atmospheric nitrogen thus implied occurs actually withinthe nodulated plant and probably in the nodule. Nodule formation occurred most freely over the pH range 5•4to 7•0, while subsequent to nodulation the best growthof plants was in the pH range 4•2 to 5•4. The capacityof the host plant to tolerate relatively low pH levels considerablyexceeds that of the nodule organism. The oxygen requirementof the nodules appears to be relatively high. The fixation ofnitrogen per unit dry weight of nodule tissue exceeds that oflegumes grown under comparable conditions.     

Phylogenetic characterization of ineffective Frankia in Alnus glutinosa (L.) Gaertn. nodules from wetland soil inoculants

Ineffective Frankia endophytes were retrieved from various wet soils by using Alnus glutinosa clones as trapping plants. No pure cultures could be isolated from these ineffective nodules. Therefore, the phylogenetic position of these endophytes was determined by sequence analysis of cloned PCR products of bacterial 16S rDNA, derived from nodules. The results showed that all nodule endophytes belong to a hitherto undescribed cluster of the Frankia phylogenetic tree. The position of these uncultured ineffective Frankia nodule endophytes is different from that of the ineffective Frankia isolates derived from A. glutinosa nodules, even when originating from the same geographical location. This suggests a bias in current isolation techniques.     

The occurrence of amoeboid plastids in the actinorhizal root nodules of Alnus glutinosa (L.) Gaertn.

Abstract. The invasion of the actinomycete Frankia into the root cells of Alnus glutinosa with subsequent nodule formation effects a number of ultrastructural changes in the host cell cytoplasm. Among other changes the amyloplasts rapidly lose their starch and acquire an amoeboid or pleomorphic form. Such plastids occur predominantly in the mature vesicle-containing, nitrogen-fixing cells of the nodule. They lack starch, have an electron dense stroma and a complex lamellar system. This last would appear to be associated with a distinct membranous reticulum which can be extensive. The flexible form of these plastids is mirrored in their ability to enclose portions of host cytoplasm together with organelles and even other plastids. Their close association with cristate mitochondria suggests an active metabolic role in the nodule symbiosis.     

Drought-Induced Xylem Dysfunction in Petioles,Branches, and Roots of Populus balsamifera L. and Alnus glutinosa (L.) Gaertn

Variation in vulnerability to xylem cavitation was measured within individual organs of Populus balsamifera L. and Alnus glutinosa (L.) Gaertn. Cavitation was quantified by three different techniques: (a) measuring acoustic emissions, (b) measuring loss of hydraulic conductance while air-dehydrating a branch, and (c) measuring loss of hydraulic conductance as a function of positive air pressure injected into the xylem. All of these techniques gave similar results. In Populus, petioles were more resistant than branches, and branches were more resistant than roots. This corresponded to the pattern of vessel width: maximum vessel diameter in 1- to 2-year-old roots was 140 [mu]m, compared to 65 and 45 [mu]m in rapidly growing 1-year-old shoots and petioles, respectively. Cavitation in Populus petioles started at a threshold water potential of -1.1 MPa. The lowest leaf water potential observed was -0.9 MPa. In Alnus, there was no relationship between vessel diameter and the cavitation response of a plant organ. Although conduits were narrower in petioles than in branches, petioles were more vulnerable to cavitation. Cavitation in petioles was detected when water potential fell below -1.2 MPa. This value equaled midday leaf water potential in late June. As in Populus, roots were the most vulnerable organ. The significance of different cavitation thresholds in individual plant organs is discussed.     

The effect of sea shore displacement on population age structure of coastal Alnus glutinosa (L.) Gaertn.

The age structure of Alnus glutinosa (L.) Gaertn. populations was studied in two areas on the coast of the Baltic Sea. One area, situated in Upplatid, eastern central Sweden, at 60°07'N, has a relative land uplift of 5.3 mm yr⁻¹; the other area, situated in Blekinge, southeastern Sweden, at 56°10'N, is nearly stable. The spatial distribution of 200 Alnus individuals of various age classes at nine sites was analyzed in order to reconstruct establishment history. In the land uplift area, the youngest individuals were found in the lowest parts of the sites, and age and elevation were significantly correlated. In the area without land uplift, no such pattern was found. Regression analysis of the time/space pattern of establishment in the land uplift area showed that the zone where individuals establish follows the moving shore line with roughly constant speed. Alnus glutinosa uses two different regenerative strategies: (1) continuous colonization of emerging land from seeds (with subsequent mortality of the older trees) in the land uplift area, and (2) regeneration from old stem bases, forming multistemmed trees, in the area without land uplift.     

Hormones in Plants Bearing Nitrogen-fixing Root Nodules: Cytokinin Transport from the Root Nodules of Alnus glutinosa (L.) Gaertn.

The movement and metabolism of [8-¹⁴C]zeatin applied to theroot nodules of Alnus glutinosa (L.) Gaertn, was investigated.Twenty-four hours after the start of uptake, zeatin and a numberof its metabolites were detected in all parts of the plant.The major radioactive compounds present in a cationic fractionof different plant parts at this time co-chromatographed onSephadex LH20 with zeatin (in nodules, stems, and leaves) andwith zeatin riboside (in roots, stems, and buds). In the roots,in addition to the peak co-chromatographing with zeatin riboside,there was also a prominent unidentified polar peak. The presence of zeatin and zeatin riboside in the stems andleaves was indicated also by chromatographic behaviour in othersystems, effects of permanganate oxidation, and cocrystallisationwith the authentic unlabelled compounds. Biological activitywas exhibited by both peaks in the soybean callus bioassay.Other metabolites in the shoot, possibly active as cytokinins,had the characteristics of dihydrozeatin, zeatin or dihydrozeatin-5'-nucleotide(s),and zeatin or dihydrozeatin glucosides. The gradual disappearancewith time of zeatin and its riboside from the shoot was accompaniedby an increase in the proportion of more polar metabolites. These results are discussed in relation to the possible exportof endogenous cytokinins by the nodules.     

Hormones in Plants Bearing Nitrogen-fixing Root Nodules: Partial Characterisation of Cytokinins from Root Nodules of Alnus glutinosa (L.) Gaertn

The nature of the substances responsible for the major cytokininactivity in extracts of Alnus glutinosa (L.) Gaertn. root noduleswas investigated by means of chromatographic, chemical, andenzymic methods. Five cytokinins were demonstrated and a furthertwo compounds were probably present in trace amounts. The propertiesof the cytokinins were consistent with their being identicalor closely similar to trans-zeatin, trans-zeatin riboside, zeatin-O-ß-D-glucoside,and a ß-D -glucoside of zeatin riboside together withcertain of the corresponding dihydrozeatin compounds. The greatestpart of the cytokinin activity was represented by the glucosides.