8
In situ analysis of introduced Frankia populations in root nodules of Alnus glutinosa grown under different water availability 1 Anja Nickel, Dittmar Hahn, Kornelia Zepp, and Josef Zeyer Abstract: The competitive ability for nodulation of Alnus glutinosa (L.) Gaertn. plants by Frankia strains inoculated into soil with indigenous Frankia populations was studied at two matric potentials representing “dry” (–0.016 MPa) and “wet” (–0.001 MPa) conditions. In pots kept at a matric potential of –0.001 MPa, nitrate concentrations decreased within 3 weeks more than 10-fold to an average of approx. 200 µ mol·(g soil dry wt.) –1 . After 4 months, nitrate concentrations in these pots were 16 ± 16 and 277 ± 328 µ mol·(g soil dry wt.) –1 (mean ± SD) for non-inoculated and inoculated soils, respectively. At a matric potential of –0.016 MPa, nitrate concentrations for non-inoculated and inoculated soils were 687 ± 491 and 1796 ± 1746 µ mol·(g soil dry wt.) –1 , respectively. Inoculated plants always grew better than their non-inoculated counterparts. The largest plants were found on inoculated soil at a matric potential of –0.001 MPa, whereas the smallest plants were found on non-inoculated soil at the same matric potential. At a matric potential of –0.016 MPa, plants grown on non-inoculated soil were not as tall as those grown on inoculated soil and were slightly chlorotic, indicating that the high level of nitrate in the soil was not providing optimal plant growth conditions. The number of nodule lobes formed on plants was not significantly different among treatments, though size and weight of lobes differed. Nodules from plants grown on inoculated soils always harbored vesicle-producing Frankia populations, while nodules from plants grown on non-inoculated soils harbored only Frankia with distorted vesicles or no Frankia at all. All strains in nodules from plants grown on non-inoculated soil were of Alnus host infection group IIIa. Nodules from plants grown on soil inoculated with strains ArI3 (group IIIa), Ag45/Mut15 (group IV), and AgB1.9 (group I) were also infected by Frankia strain Ag45/Mut15. These results indicate that by inoculation, Frankia populations could be established under conditions that did not favour vesicle formation in root nodules formed by the indigenous Frankia population. Inoculation even in soils with high nitrogen content might therefore be an appropriate strategy to enhance plant growth. Key words: competition, fluorescent oligonucleotide probes, inoculation, in situ hybridization, matric potential, nitrate, rRNA. 1238 Résumé : Les auteurs ont étudié la capacité compétitive de plants d’Alnus glutinosa (L.) Gaertn. inoculés avec des souches de Frankia et plantés dans du sol contenant des populations indigènes de Frankia, sous deux conditions d’humidité du sol, soient « sèche » (–0,016 MPa) et « humide » (–0,001 MPa). Dans les pots maintenus sous des conditions relativement humides avec –0,001 MPa, les teneurs en nitrates diminuent de 10 fois en moins de trois semaines, pour atteindre une moyenne de 200 µ mol·(g de sol sec) –1 . Après 4 mois, les teneurs en nitrates dans ces pots sont de 16 ± 16 et 277 ± 328 µ mol·(g de sol sec) –1 pour les sols non inoculés et inoculés, respectivement. Sous les conditions relativement sèches avec –0,016 MPa, les teneurs en nitrates des sols pour les plants non inoculés et inoculés étaient de 687 ± 491 et 1796 ± 1746 µ mol·(g de sol sec) –1 , respectivement. Les plants inoculés poussent toujours mieux que les témoins non inoculés. On retrouve les plants les plus gros en sol inoculé avec les conditions relativement humide de –0,001 MPa, alors que les plantes les plus petites se retrouvent en sol non inoculé sous les mêmes conditions d’humidité. Sous les conditions sèches à –0,016 MPa, les plantes cultivées en sol non inoculé ne sont pas aussi hautes que celles cultivées en sol inoculé et sont faiblement chlorotique, ce qui indique que la teneur élevée du sol en nitrates ne fournit pas des conditions optimales de croissance pour les plants. Le nombre de lobes nodulaires formés sur les plants ne diffère pas selon les traitements, bien que la grosseur et le poids des lobes diffèrent. Les nodules provenant de plants cultivés en sols inoculés comportent toujours des populations de Frankia produisant des vésicules, alors que chez les nodules de plants cultivés en sol non inoculé, on détecte seulement des vésicules difformes ou pas de Frankia du tout. Toutes les souches obtenues des plantes cultivées en sol non inoculé Can. J. Bot. 77: 1231–1238 (1999) © 1999 NRC Canada 1231 Received June 25, 1998. A. Nickel, D. Hahn, 2 K. Zepp, and J. Zeyer. Swiss Federal Institute of Technology (ETH), Institute of Terrestrial Ecology, Soil Biology, Grabenstrasse 3, CH-8952 Schlieren, Switzerland. 1 This paper was presented at the 11th International Conference on Frankia and Actinorhizal Plants, June 7–11, 1998, University of Illinois at Urbana–Champaign. 2 Author to whom all correspondence should be addressed. Present address: Department of Chemical Engineering, Chemistry and Environmental Science, New Jersey Institute of Technology, University Heights, Newark, NJ 07102-1811, U.S.A. e-mail: [email protected]

In situ analysis of introduced Frankia populations in root nodules of Alnus glutinosa grown under different water availability

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Page 1: In situ analysis of introduced Frankia populations in root nodules of Alnus glutinosa grown under different water availability

In situ analysis of introduced Frankiapopulations in root nodules of Alnus glutinosagrown under different water availability1

Anja Nickel, Dittmar Hahn, Kornelia Zepp, and Josef Zeyer

Abstract: The competitive ability for nodulation of Alnus glutinosa (L.) Gaertn. plants by Frankia strains inoculatedinto soil with indigenous Frankia populations was studied at two matric potentials representing “dry” (–0.016 MPa)and “wet” (–0.001 MPa) conditions. In pots kept at a matric potential of –0.001 MPa, nitrate concentrations decreasedwithin 3 weeks more than 10-fold to an average of approx. 200 µmol·(g soil dry wt.)–1. After 4 months, nitrateconcentrations in these pots were 16 ± 16 and 277 ± 328 µmol·(g soil dry wt.)–1 (mean ± SD) for non-inoculated andinoculated soils, respectively. At a matric potential of –0.016 MPa, nitrate concentrations for non-inoculated andinoculated soils were 687 ± 491 and 1796 ± 1746 µmol·(g soil dry wt.)–1, respectively. Inoculated plants always grewbetter than their non-inoculated counterparts. The largest plants were found on inoculated soil at a matric potential of–0.001 MPa, whereas the smallest plants were found on non-inoculated soil at the same matric potential. At a matricpotential of –0.016 MPa, plants grown on non-inoculated soil were not as tall as those grown on inoculated soil andwere slightly chlorotic, indicating that the high level of nitrate in the soil was not providing optimal plant growthconditions. The number of nodule lobes formed on plants was not significantly different among treatments, though sizeand weight of lobes differed. Nodules from plants grown on inoculated soils always harbored vesicle-producing Frankiapopulations, while nodules from plants grown on non-inoculated soils harbored only Frankia with distorted vesicles orno Frankia at all. All strains in nodules from plants grown on non-inoculated soil were of Alnus host infection groupIIIa. Nodules from plants grown on soil inoculated with strains ArI3 (group IIIa), Ag45/Mut15 (group IV), and AgB1.9(group I) were also infected by Frankia strain Ag45/Mut15. These results indicate that by inoculation, Frankiapopulations could be established under conditions that did not favour vesicle formation in root nodules formed by theindigenous Frankia population. Inoculation even in soils with high nitrogen content might therefore be an appropriatestrategy to enhance plant growth.

Key words: competition, fluorescent oligonucleotide probes, inoculation, in situ hybridization, matric potential, nitrate,rRNA.

1238Résumé : Les auteurs ont étudié la capacité compétitive de plants d’Alnus glutinosa (L.) Gaertn. inoculés avec dessouches de Frankia et plantés dans du sol contenant des populations indigènes de Frankia, sous deux conditionsd’humidité du sol, soient « sèche » (–0,016 MPa) et « humide » (–0,001 MPa). Dans les pots maintenus sous desconditions relativement humides avec –0,001 MPa, les teneurs en nitrates diminuent de 10 fois en moins de troissemaines, pour atteindre une moyenne de 200 µmol·(g de sol sec)–1. Après 4 mois, les teneurs en nitrates dans ces potssont de 16 ± 16 et 277 ± 328 µmol·(g de sol sec)–1 pour les sols non inoculés et inoculés, respectivement. Sous lesconditions relativement sèches avec –0,016 MPa, les teneurs en nitrates des sols pour les plants non inoculés etinoculés étaient de 687 ± 491 et 1796 ± 1746 µmol·(g de sol sec)–1, respectivement. Les plants inoculés poussenttoujours mieux que les témoins non inoculés. On retrouve les plants les plus gros en sol inoculé avec les conditionsrelativement humide de –0,001 MPa, alors que les plantes les plus petites se retrouvent en sol non inoculé sous lesmêmes conditions d’humidité. Sous les conditions sèches à –0,016 MPa, les plantes cultivées en sol non inoculé nesont pas aussi hautes que celles cultivées en sol inoculé et sont faiblement chlorotique, ce qui indique que la teneurélevée du sol en nitrates ne fournit pas des conditions optimales de croissance pour les plants. Le nombre de lobesnodulaires formés sur les plants ne diffère pas selon les traitements, bien que la grosseur et le poids des lobesdiffèrent. Les nodules provenant de plants cultivés en sols inoculés comportent toujours des populations de Frankiaproduisant des vésicules, alors que chez les nodules de plants cultivés en sol non inoculé, on détecte seulement desvésicules difformes ou pas de Frankia du tout. Toutes les souches obtenues des plantes cultivées en sol non inoculé

Can. J. Bot. 77: 1231–1238 (1999) © 1999 NRC Canada

1231

Received June 25, 1998.

A. Nickel, D. Hahn,2 K. Zepp, and J. Zeyer. Swiss Federal Institute of Technology (ETH), Institute of Terrestrial Ecology, SoilBiology, Grabenstrasse 3, CH-8952 Schlieren, Switzerland.

1This paper was presented at the 11th International Conference on Frankia and Actinorhizal Plants, June 7–11, 1998, University ofIllinois at Urbana–Champaign.

2Author to whom all correspondence should be addressed. Present address: Department of Chemical Engineering, Chemistry andEnvironmental Science, New Jersey Institute of Technology, University Heights, Newark, NJ 07102-1811, U.S.A.e-mail: [email protected]

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correspondent au groupe hôte d’infection Alnus IIIa. Les nodules provenant de plantes cultivées en sol inoculé avec lessouches Ar13 (groupe IIIa), Ag45/Mut15 (groupe IV) et AgB1.9 (groupe I) comportaient également la souche deFrankia Ag45/Mut15. Ces résultats indiquent que suite à l’inoculation, des populations de Frankia ont pu s’établirdans des nodules racinaires sous des conditions qui ne favorisent pas la formation des vésicules dans les nodulesvenant de la population indigène de Frankia. L’inoculation même dans des sols contenant beaucoup d’azote, pourraitêtre par conséquent une stratégie valable pour augmenter la croissance des plants.

Mots clés : compétition, sondes à oligonucléotides fluorescentes, inoculation, hybridation in situ, potentiel hydrique dusol, nitrate, rARN.

[Traduit par la Rédaction] Nickel et al.

Introduction

Actinorhizal plants are characterized by their ability toform root nodules in symbiosis with the nitrogen-fixingactinomycete Frankia, which enables them to grow on siteswith restricted nitrogen availability (Bond 1983). Econom-ically, actinorhizal plants are interesting for reforestation andreclamation of depauperate, nitrogen-limited soils, especiallyin developing countries in subtropical regions in which, inaddition to their use for reforestation and reclamation ofpoor soils, Casuarina species are an important source offirewood (Dawson 1986; Wheeler et al. 1986). In temperateregions Alnus species have the highest potential for use inforestry (Gordon 1983; Gordon and Dawson 1979). They areused as “nurse” trees in mixed plantations with valuable treespecies (i.e., by interplanting them with suitable tree cropssuch as walnut), for production of fuelwood, and as a sourceof timber in monocultures (Fessenden 1979; Gordon 1983;Teissier du Cros et al. 1984; Zavitkovski et al. 1979).

The efficiency of root nodule formation on actinorhizalplants is largely determined by environmental factors such asthe soil pH (Crannell et al. 1994; Griffiths and McCormick1984; Zitzer and Dawson 1992), the soil matric potential(Dawson et al. 1989; Schwintzer 1985), and the availabilityof elements such as nitrogen (Kohls and Baker 1989;Thomas and Berry 1989) or phosphorus (Sanginga et al.1989; Yang 1995), but it is also determined by the source ofgenotypes of both partners of this symbiosis (Hall et al.1979; Prat 1989). An improvement in the symbiosis for eco-nomic purposes therefore requires the selection of optimalgrowth sites, but also an optimal combination of plants of in-terest (e.g., forest ecotypes of Alnus glutinosa (L.) Gaertn.)and superior genotypes of Frankia as inoculum (Hall et al.1979; Hilger et al. 1991; Wheeler et al. 1991). Criteria suchas nitrogen-fixing capacity and compatibility of Frankiastrains and the ability of introduced strains to form nodulespromptly, to persist in soil, and to compete with indigenousFrankia populations must be considered for efficient inocu-lation programs with Frankia strains on alders.

The aim of our study was to investigate the competitiveability of introduced Frankia strains with indigenous Frankiapopulations in soil for nodulation on A. glutinosa plants.Non-inoculated soil or soil inoculated with Frankia strainsArI3 (group IIIa), Ag45/Mut15 (group IV), and AgB1.9(group I) was planted with A. glutinosa seedlings and kept attwo matric potentials representing “dry” (–0.016 MPa) and“wet” (–0.001 MPa) conditions found in natural stands of A.glutinosa. Matric potentials were maintained for 4 months

and their impact was determined afterwards with respect tosoil, plant, and nodulation parameters. In root nodules ob-tained on plants grown on inoculated and non-inoculatedsoils, Frankia populations were analyzed by in situ hybrid-ization (Zepp et al. 1997a, 1997b).

Materials and methods

Experimental set-upSurface samples (down to a depth of 20 cm) were collected from

a sandy loam at a natural stand of A. glutinosa (Ettiswil, Switzer-land) (Zepp et al. 1997a). This soil was characterized by high ni-trate concentrations (6 to 7 mM), a low content of organic material(0.02%), a high matric potential (–0.016 MPa), and the presence ofFrankia subgroups I, IIIa, and IV of the Alnus host infection group.At the natural site, however, nodules were only formed by sub-group IIIa (Zepp et al. 1997a, 1997b). Freshly sampled soil wascleared of larger particles (e.g., roots and stones), sieved (meshsize 5 mm), and stored at 12°C. After 4 weeks of storage, a part ofthe soil was inoculated with a mixture of pure cultures of Frankiastrains AgB1.9, ArI3, and Ag45/Mut15. Frankia strains weregrown for 4 weeks in P + N medium (Meesters et al. 1985) con-taining sodium propionate and ammonium chloride as C and Nsources, respectively. Cultures were harvested by centrifugation,washed twice in phosphate buffered saline (PBS, composed of0.13 M NaCl, 7 mM Na2HPO4, and 3 mM NaH2PO4, pH 7.2 inwater) (Hahn et al. 1992), and homogenized in PBS by repeatedpassages through a needle (0.6 mm in diameter) with a sterile sy-ringe (Hahn et al. 1990). Cell numbers were calculated from freshweight determination. Five millilitres of the homogenized cultureswere sprayed onto thin layers of 800-g subsamples of soil (freshweight) followed by careful mixing to achieve an even distributionof Frankia strains each at an estimated density of 107 cells·(g soilwet wt.)–1. Non-inoculated soil samples were only mixed.

Each 800-g sample of inoculated (n = 40) and non-inoculated(n = 20) soil was put into 800-cm3 pots. Pots were planted with ap-proximately 4-week-old seedlings of A. glutinosa that had beengerminated and grown in Perlite supplemented with a modifiedHeller salt solution (Heller 1953) containing 0.075 µM nitrate asthe nitrogen source at pH 5.4 (Hahn et al. 1988). Plants were main-tained in a growth chamber with a photoperiod of 16 h light : 8 hdark and a thermoperiod of 24:18°C (light:dark). Half of the sam-ples (i.e., 20 pots with inoculated soil and 10 pots with non-inoculated soil) were adjusted to, and maintained at, a matric po-tential of –0.016 MPa and the other half, at –0.001 MPa. Thematric potential was separately maintained in every pot via suctioncups and controlled at the end of the experiment by determinationof water contents and comparison to a moisture release characteris-tic determined for the original soil at the appropriate bulk density.Plants in pots were grown under natural light and temperature con-ditions in a greenhouse for 4 months (March 14 to July 14).

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Analysis of soil parametersConcentrations of NO3

–, NO2–, SO4

2–, PO43–, and Cl– were ana-

lyzed in pore water of soil samples obtained at the beginning of thestudy, after 1, 2, and 3 weeks of growth, and at the end of the ex-periment after 4 months. Pore water was obtained by centrifugationof approximately 12 g of soil put into 5-mL plastic syringes thatwere plugged with silane-treated glass wool (Supelco Inc.,Bellefonte, U.S.A.). The filled syringes were placed in 15-mL Fal-con tubes and centrifuged at 4°C and 2500 × g for 10 min. Fifteen-microlitre samples of pore water were analyzed by ion chroma-tography (Dionex DX-100 ion chromatograph equipped with anIonPac AS4A-SC column; Dionex, Sunnyvale, U.S.A.) using aneluent of 1.8 mM Na2CO3 and 1.7 mM NaHCO3 (Hess et al.1996). Data from ion chromatography were analyzed with Chrom-Card for Windows (Fison Instruments, Rodano, Italy) (Hess et al.1996). Concentrations in pore water (µM) were correlated to watercontents and expressed in µmol·(g soil dry wt.)–1.

Analysis of plant parametersPlant height was monitored monthly. At the end of the 4-month

experiment, shoots, roots, and nodules of the plants were harvestedseparately. Shoots and roots were dried at 105°C for 24 h to deter-mine dry weights and to calculate shoot/root ratios. C/N ratios inleaves were determined after the analysis of C and N contentsin dried and ground leaves using a CHNS-932 analyzer (Leco,Kirchheim, Germany). Numbers of nodule lobes were counted andfresh weights of nodules were determined.

Analysis of Frankia populations in root nodulesFor the analysis of Frankia populations in root nodules, all nod-

ules were harvested, split into lobes, and fixed in 4% parafor-maldehyde in PBS at 4°C for 16 h (Hahn et al. 1993). Lobes weresubsequently washed in PBS and about two-thirds of the lobeswere ground in a mortar. Lobe homogenates and remaining nodulelobes were stored in 96% ethanol at –20°C (Hahn et al. 1993). Allremaining nodule lobes were covered with and soaked in embed-ding medium (No. 350100; Microm, Walldorf, Switzerland) for2 days and subsequently sectioned longitudinally through the mainaxis in a HM 500 OM cryostat (Microm). Sections between 10 and14 µm as well as 3-µL samples of lobe homogenates were air driedon gelatin-coated slides (0.1% gelatin, 0.01% KCr(SO4)2) for atleast 2 h (Zarda et al. 1997). After dehydration in 50, 80, and96% ethanol for 3 min each, the preparations were pretreated withSDS/DTT (10 mg·mL–1 SDS, 50 mM dithiothreitol (DTT, Fluka)in water, freshly prepared) at 65°C for 30 min followed by anincubation with lysozyme (Fluka, Buchs, Switzerland 1 mg corre-sponding to 37 320 U dissolved in 1 mL of 100 mM Tris-HCl,pH 7.5, 5 mM EDTA) at 37°C for 10 min (Zepp et al. 1997a).Afterwards, the samples were rinsed with distilled water and dehy-drated as described above.

Oligonucleotide probes targeting 16S rRNA of the Domain Bac-teria (Eub338; (Amann et al. 1990)) or specific sequences on the23S rRNA insertion of Frankia strains AgB1.9 (probe B1.9; 5′ACCACC TCA ACC CCC GAA), ArI3 (probe 23ArI3) (Zepp et al.1997a), and Ag45/Mut15 (probe 23Mut(II)) (Zepp et al. 1997a)representing Alnus host infection groups I, IIIa, and IV, respec-tively (Hönerlage et al. 1994), were synthesized with a primaryamino group at the 5′ end (C6-TFA, MWG, Ebersberg, Germany).The fluorescent dye Cy3 (Amersham, Zurich, Switzerland) wascovalently bound to the amino group of the oligonucleotide probe.The dye–oligonucleotide conjugate (1:1) was purified from un-reacted components and stored at –20°C in distilled water at a con-centration of 25 ng·µL–1 (Amann et al. 1990). This solution wasamended with the DNA-specific dye 4′6-diamidino-2-phenylindole(DAPI) (Sigma, Buchs, Switzerland) to give a final concentrationof 10 µg·mL–1.

Hybridizations were performed in 9 µL of hybridization buffer(900 mM NaCl, 20 mM Tris-HCl, 0.01% SDS, pH 7.2) in the pres-ence of 30% formamide (Manz et al. 1992) and 1 µL of oligo-nucleotide probe (25 ng) at 42°C for 2 h (Zepp et al. 1997a,1997b). After hybridization, the slides were washed in hybridiza-tion buffer without formamide at 48°C for 20 min, rinsed with dis-tilled water, and air dried. Preparations were mounted withCitifluor solution (Citifluor, Canterbury, U.K.) and examined witha Zeiss Axiophot microscope (Zeiss, Oberkochen, Germany) fittedfor epifluorescence detection with a high-pressure mercury bulb(50 W). DAPI-stained bacteria were analyzed with filter set 02(Zeiss; G365, FT395, LP420), whereas binding of Cy3-labeledprobes was detected with filter set HQ-Cy3 (AHF Analy-sentechnik, Tübingen, Germany; G535/50, FT565, BP610/75).

Statistical analysesAll data were expressed as mean ± standard deviation and as-

sessed by multiple pairwise comparisons with Tukey’s honestlysignificant difference test (SYSTAT). The significance level was setat α = 0.05.

Results

Soil parametersDifferences in water contents developed between non-

inoculated and inoculated pots kept at the same matric po-tential. The water contents of non-inoculated soils withmatric potentials of –0.001 and –0.016 MPa were, respec-tively, 14% and 43% higher than those of inoculated soilsof the same matric potentials (Table 1). At the end of the4-month experiment, pots kept at a matric potential of−0001. MPa had an average water content of 29 ± 3%, whilethose kept at –0.016 MPa had an average water contentof 16 ± 4%, which corresponded to matric potentials of−0005. MPa and less than –0.02 MPa, respectively. At thebeginning of the experiment the pore water had a pH of 7.1.During and at the end of the experiment, no major changesin pH were observed (Table 1). Nitrate concentrations inthe pore water changed significantly during the experiment.In pots where soil matric potential was maintained at−0001. MPa, nitrate concentration in pore water decreased13-fold within 3 weeks to an average of 200 µmol·(g soil drywt.)–1. After 4 months, the nitrate concentrations in thesepots, when non-inoculated or inoculated with Frankia, were1% and 11% of the original value, respectively (Table 1).This compares with significantly higher nitrate concentra-tions of 26% and 69% of the original value that remained innon-inoculated and inoculated pots, respectively, when soilmatric potential was –0.016 MPa. Concentrations of nitriteincreased from below the detection limit (1 µM) at the be-ginning of the experiment to values of up to 35 µmol·(g soildry wt.)–1. Sulfate concentrations (191 µmol·(g soil dry wt.)–1)remained in the same range in pots with a matric potential of–0.016 MPa, but were reduced about 3-fold in pots kept at amatric potential of –0.001 MPa (Table 1). In all pots chlo-ride concentrations remained in the same range of 311 ±414 µmol·(g soil dry wt.)–1. Phosphate concentrations werealways below the detection limit at 20 µM.

Plant parametersMonthly plant height measurements showed a faster

growth of inoculated plants compared with non-inoculated

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Nickel et al. 1233

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plants (Fig. 1). After 4 months of growth, plants on the non-inoculated soil kept at a matric potential of –0.001 MPawere the smallest plants, at 18 ± 10 cm (Table 2). Those oninoculated soil at the same matric potential were 3-foldlarger, and those on non-inoculated and inoculated soil at amatric potential of –0.016 MPa were about 1.5-fold and 2-fold larger, respectively. The statistically significant differ-ences of the latter, however, were apparent only towards theend of the experiment (Fig. 1). Comparable patterns to plantheight measurements were found for plant dry weights (Ta-ble 2). The highest values for shoot and root dry weightswere obtained with plants growing on inoculated soil at amatric potential of –0.001 MPa; they were about 4-foldhigher than those for plants on non-inoculated soil at thesame matric potential (1.5 ± 0.9 g dry wt.). Plants grown ata matric potential of –0.016 MPa showed values of 2.5 ± 0.8and 3.8 ± 1.8 g dry wt., again with higher values for the in-oculated soil.

Root/shoot ratios between plants grown at matric poten-tials of –0.001 and –0.016 MPa were not significantly differ-ent. No differences were apparent between plants grown oninoculated and non-inoculated soils (Table 2). The carboncontent in leaves was similar in all treatments (47 ± 1%),while the content of nitrogen differed. Highest nitrogen con-tents were obtained in leaves of plants grown on inoculatedsoil (2.9 ± 0.4 and 3.0 ± 0.3% at matric potentials of –0.001and –0.016 MPa, respectively). Leaves from plants grown onnon-inoculated soils were chlorotic and consequently hadmuch lower nitrogen contents (2.2 ± 0.4 and 1.9 ± 0.2% atmatric potentials of –0.001 and –0.016 MPa, respectively).Consequently, C/N ratios were lower for leaves of plantsgrown on inoculated soils than for leaves of plants grown onnon-inoculated soils.

Populations of Frankia in root nodulesWith an average of about 20 lobes per plant, the number

of nodule lobes formed on plants was not significantly dif-ferent among all treatments, though size and weight of lobesdiffered (Table 3). Lobes formed on plants grown on inocu-lated soil at a matric potential of –0.001 MPa had a totalweight of 358 ± 211 mg fresh wt. and an average weight perlobe of 22 ± 11 mg fresh wt. These values were reduced 18-fold and 11-fold, respectively, on plants grown on non-ino-culated soil of the same matric potential. At a matric poten-tial of –0.016 MPa, lobes on plants grown on inoculated soilhad a total weight of 166 ± 116 mg fresh wt. and an averagelobe weight of 9 ± 4 mg fresh wt. Both values were reducedabout 4-fold for lobes on plants grown on non-inoculatedsoil (Table 3).

Nodule lobes from plants grown on inoculated soils usu-ally contained Frankia populations, though occasionallynodule lobes without Frankia were found. All lobes fromplants grown on inoculated soils contained nitrogen-fixingFrankia, except for one in which the non-nitrogen-fixingFrankia strain AgB1.9 was detected (Table 3). In these treat-ments Frankia strains Ag45/Mut15 and ArI3 were identifiedin high percentages with probes 23Mut(II) and 23ArI3, re-spectively. Nodule lobes on plants grown at a matric poten-tial of –0.001 MPa contained comparable percentages ofAg45/Mut15 (45 ± 27%) and ArI3 (49 ± 28%), and thosefrom plants grown at a matric potential of –0.016 MPa hadslightly fewer nodules infected by Ag45/Mut15 (38 ± 25)than by ArI3 (62 ± 24).

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1234 Can. J. Bot. Vol. 77, 1999

Matric potential (–0.001 MPa) Matric potential (–0.016 MPa)

Soil parameters Initial conditionsa Non-inoculatedb Inoculatedc Non-inoculatedb Inoculatedc

Water content (%) 23±1 32±1 28±2 20±1 14±3pH (in water) 7.1±0.1 7.4±0.2 7.3±0.2 7.2±0.2 7.3±0.2NO3

– (µmol·(g soil dry wt.)–1) 2609±1906 16±16 277±329 687±491 1796±1746NO2

– (µmol·(g soil dry wt.)–1) <1 1±3 11±27 8±23 35±64SO4

2– (µmol·(g soil dry wt.)–1) 191±101 58±97 83±54 214±220 138±94Cl– (µmol·(g soil dry wt.)–1) 311±414 136±88 244±208 407±406 313±226

Note: Concentrations of phosphate were always below the detection limit of 20 µM. Only values for water content and nitrateconcentrations are significantly different (α = 0.05) between non-inoculated and inoculated soils at the same matric potential.

aSoil Ettiswil at the beginning of the experiment (n = 5).bSoil Ettiswil after 4 months (n = 10).cSoil Ettiswil after 4 months, inoculated with Frankia strains ArI3, Ag45/Mut15, and AgB1.9 each at a density of approximately 107

cells·(g soil fresh wt.)–1 (n = 20).

Table 1. Soil parameters at the beginning and at the end of the growth experiment (non-inoculated and inoculated soils).

Fig. 1. Height of A. glutinosa seedlings grown on inoculatedor non-inoculated soils at two matric potentials (–0.001 and–0.016 MPa), respectively.

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On non-inoculated soils, about 90% of the lobes on plantsgrown at a matric potential of –0.016 MPa and 40% of thoseon plants grown at a matric potential of –0.001 MPa did notcontain filaments, vesicles, or spores typical for Frankia.Vesicles in the remaining nodules were usually muchsmaller than those in nodules from plants on the inoculatedsoils (Fig. 2). The majority of plants grown at a matric po-tential of –0.016 MPa (8 of 10) did not have any Frankia-containing nodules. In contrast, 8 of 10 plants grown at amatric potential of –0.001 MPa had nodules with Frankia,though vesicles were usually distorted. Frankia populationsin vesicle-containing lobes of plants grown on non-inoculatedsoils at either matric potential were all detectable with probe23ArI3 (55 ± 37% and 13 ± 28%). None of the otherFrankia-specific probes (23B1.9 and 23Mut(II)) showed hy-bridization signals.

Discussion

The major objective of the experiment required the main-tenance of different matric potentials for two sets of plantsgrowing on soils non-inoculated or inoculated with Frankiastrains. Although the aim to match the matric potentialsfound at two natural stands of A. glutinosa was not achieved,different growth conditions that simulated “dry” and “wet”

environmental conditions were obtained. The establishmentof dry and wet environmental conditions was demonstratedby changes in concentration of soil chemicals, particularlynitrate, in soils with different matric potentials (Table 1). Ata matric potential of –0.001 MPa, nitrate concentrations de-creased by one order of magnitude within the first 3 weeksof incubation, resulting in nitrogen-limited growth condi-tions for plants. The loss of nitrate could be due to highdenitrification activity resulting from the high water contentand the concomitant reduction of O2 availability in thesesoils. The establishment of at least partially anaerobic condi-tions is indicated by the decrease in sulfate concentrations.At a matric potential of –0.016 MPa, nitrate decreased innon-inoculated soils to approximately half the original con-centration, whereas in inoculated soil, nitrate concentrationsremained comparable to those at the beginning of the experi-ment. At this matric potential, nitrogen availability initiallywas not limiting plant growth. However, towards the end ofthe experiment (after 4 months) leaves on plants grown onnon-inoculated soil became slightly chlorotic, suggesting areduced nitrogen supply (Dawson et al. 1989; Hahn et al.1990), although nitrate concentrations in soil were still quitehigh. Plant growth of A. glutinosa and nodulation by Frankiapopulations in non-inoculated and inoculated soils kept attwo different matric potentials might therefore be influenced

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Nickel et al. 1235

Matric potential (–0.001 MPa) Matric potential (–0.016 MPa)

Nodulation parameters Non-inoculateda Inoculatedb Non-inoculateda Inoculatedb

Number of lobes 19±20 18±12 30±28 20±11Total lobe weight (mg fresh wt.) 20±35 358±211 38±43 166±116Average lobe weight (mg fresh wt.) 2±4 22±11 2±1 9±4Group IIIa (%) 55±37 49±28 13±28 62±24Group IV (%) 0 45±27 0 38±25Group I (%) 0 <1c 0 0Lobes without Frankia (%) 43±36 2±5d 87±28 1±2e

Note: All values except for the number of lobes are significantly different (α = 0.05) between non-inoculated and inoculated soilsat the same matric potential.

aSoil Ettiswil (n = 10).bSoil Ettiswil, inoculated with Frankia strains ArI3, Ag45/Mut15, and AgB1.9 each at a density of approximately 107 cells·(g soil

fresh wt.)–1 (n = 20).cDetection in only one nodule.dSix nodules without Frankia on five plants.eTwo nodules without Frankia on one plant.

Table 3. Nodulation parameters.

Matric potential (–0.001 MPa) Matric potential (–0.016 MPa)

Plant parameters Non-inoculateda Inoculatedb Non-inoculateda Inoculatedb

Plant height (cm) 18±10 49±11 28±4 35±8Plant weight (g dry wt.) 1.5±0.9 5.9±3.2 2.5±0.8 3.7±1.8Shoot (g dry wt.) 0.8±0.5 3.2±1.8 1.5±0.3 2.1±1.0Root (g dry wt.) 0.7±0.5 2.7±1.5 1.0±0.5 1.6±0.9Root/shoot ratio 0.9±0.4 0.9±0.4 0.7±0.3 0.7±0.3C/N ratio of leaves 21±4 16±2 25±3 16±2

Note: Only values for plant height, plant weight, shoot weight, and C/N ratio are significantly different (α = 0.05)between non-inoculated and inoculated soils at the same matric potential.

aSoil Ettiswil (n = 10).bSoil Ettiswil, inoculated with Frankia strains ArI3, Ag45/Mut15, and AgB1.9 each at a density of approximately

107 cells·(g soil fresh wt.)–1 (n = 20).

Table 2. Plant parameters after 4 months of growth.

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not only by the matric potentials but also by different redoxconditions, the availability of O2, and the availability of ni-trate as a source of nitrogen.

In our study, inoculated plants always grew better thantheir non-inoculated counterparts (Table 2 and Fig. 1), indi-cating a significant effect of inoculation on plant growthindependent of the environmental conditions. Although thenumber of nodule lobes was not significantly different onplants grown on non-inoculated or inoculated soils, largedifferences in size and weight of lobes as well as in theinhabiting Frankia populations were obtained (Table 3).Lobes from plants grown on non-inoculated soils were al-ways much smaller than those from plants grown on inocu-lated soils. The Frankia populations in lobes from plants onnon-inoculated soil hybridized only to probe 23ArI3, whichtargets the Alnus host infection group IIIa, confirming earlierstudies on field-collected nodules (Zepp et al. 1997a) andnodules from a previous competition experiment (Zepp et al.1997b). In those studies nodules usually contained vesicle-producing Frankia. In contrast, only nodules that had dis-torted vesicles or no Frankia at all were found in our study,indicating that even under conditions of limited nitrogenavailability for plants, the indigenous Frankia population onlyformed non-functional nodules at both matric potentials. Un-der the same environmental conditions, nodules from plantsgrown on inoculated soils always contained vesicle-producingFrankia populations. Because of the lack of nitrogen-deficiency symptoms on the host plants grown at both matricpotentials, the vesicles seemed to be functional. When soilwas inoculated with the nitrogen-fixing Frankia strains ArI3and Ag45/Mut15, both strains outcompeted the indigenous

Frankia populations and showed comparable competitiveabilities to each other at both matric potentials. They alsoindicate that by inoculation, Frankia populations could beestablished under conditions that did not obviously favourefficient nitrogenase activity in nodules formed by the indig-enous Frankia population. Inoculation even in soils withhigh nitrogen contents might therefore be an appropriatestrategy to enhance plant growth when soils contain onlypoorly effective Frankia.

The result of the inoculation, however, might be influ-enced by the experimental set-up. Comparative analysis ofnodulation units with genomic units of soils determined bythe polymerase chain reaction – most probable number(PCR–MPN) technique using nested PCR (Myrold et al.1994; Myrold and Huss-Danell 1994) indicated that only asmall portion of the total population of Frankia was able tonodulate. It was suggested that the nodulation capacity of asoil was controlled largely by the physiological status of theFrankia populations rather than by the total population size(Myrold and Huss-Danell 1994). This suggestion was sup-ported by studies in which only one population of Frankiawas detected in nodules of the host plant at the respectivesite by in situ hybridization, though different Frankia popu-lations were detected in soil by PCR (Maunuksela et al.1999; Zepp et al. 1997b). The inoculated strains that arephysiologically active pure cultures therefore might havea competitive advantage over the indigenous populations,which may be largely inactive. Both strains ArI3 andAg45/Mut15 form nodules promptly, whereas the non-nitrogen-fixing strain AgB1.9, and probably the indigenousFrankia population, require much more time for nodule for-

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1236 Can. J. Bot. Vol. 77, 1999

Fig. 2. Section of nodules of A. glutinosa grown either on inoculated soil showing normal vesicles (a) or on non-inoculated soilshowing distorted vesicles (b). Frankia cells were detected by in situ hybridization with fluorescent (Cy3-labeled) oligonucleotideprobe 23ArI3. Scale bar = 25 µm.

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mation (Hahn et al. 1988). It was shown that a systemicinhibition of nodulation operated in plants of A. incana 3 to6 days after an initial infection with Frankia (Wall andHuss-Danell 1997). A similar effect was observed on plantsof Hippophaë rhamnoides, providing evidence that sea buck-thorn had an active, systemic mechanism for feedback con-trol of nodulation that suppressed further nodule formationand prevented excessive nodulation (Dobritsa and Novik 1992).

Acknowledgements

This work was supported by grants from the Swiss Na-tional Science Foundation (Priority Program Biotechnology),and the Swiss Federal Office of Environment, Forests andLandscape (BUWAL).

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