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

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  • 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 mol(g soil dry wt.)1. After 4 months, nitrateconcentrations in these pots were 16 16 and 277 328 mol(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 mol(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 of0.001 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.

    1238Rsum : Les auteurs ont tudi la capacit comptitive de plants dAlnus glutinosa (L.) Gaertn. inoculs avec dessouches de Frankia et plants dans du sol contenant des populations indignes de Frankia, sous deux conditionsdhumidit du sol, soient sche (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 mol(g de sol sec)1. Aprs 4 mois, les teneurs en nitrates dans ces potssont de 16 16 et 277 328 mol(g de sol sec)1 pour les sols non inoculs et inoculs, respectivement. Sous lesconditions relativement sches avec 0,016 MPa, les teneurs en nitrates des sols pour les plants non inoculs etinoculs taient de 687 491 et 1796 1746 mol(g de sol sec)1, respectivement. Les plants inoculs poussenttoujours mieux que les tmoins non inoculs. On retrouve les plants les plus gros en sol inocul avec les conditionsrelativement humide de 0,001 MPa, alors que les plantes les plus petites se retrouvent en sol non inocul sous lesmmes conditions dhumidit. Sous les conditions sches 0,016 MPa, les plantes cultives en sol non inocul nesont pas aussi hautes que celles cultives en sol inocul et sont faiblement chlorotique, ce qui indique que la teneurleve du sol en nitrates ne fournit pas des conditions optimales de croissance pour les plants. Le nombre de lobesnodulaires forms sur les plants ne diffre pas selon les traitements, bien que la grosseur et le poids des lobesdiffrent. Les nodules provenant de plants cultivs en sols inoculs comportent toujours des populations de Frankiaproduisant des vsicules, alors que chez les nodules de plants cultivs en sol non inocul, on dtecte seulement desvsicules difformes ou pas de Frankia du tout. Toutes les souches obtenues des plantes cultives en sol non inocul

    Can. J. Bot. 77: 12311238 (1999) 1999 NRC Canada


    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 711, 1998, University ofIllinois at UrbanaChampaign.

    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:

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  • correspondent au groupe hte dinfection Alnus IIIa. Les nodules provenant de plantes cultives en sol inocul avec lessouches Ar13 (groupe IIIa), Ag45/Mut15 (groupe IV) et AgB1.9 (groupe I) comportaient galement la souche deFrankia Ag45/Mut15. Ces rsultats indiquent que suite linoculation, des populations de Frankia ont pu stablirdans des nodules racinaires sous des conditions qui ne favorisent pas la formation des vsicules dans les nodulesvenant de la population indigne de Frankia. Linoculation mme dans des sols contenant beaucoup dazote, pourraittre par consquent une stratgie valable pour augmenter la croissance des plants.

    Mots cls : comptition, sondes oligonuclotides fluorescentes, inoculation, hybridation in situ, potentiel hydrique dusol, nitrate, rARN.

    [Traduit par la Rdaction] Nickel et al.


    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) andwet (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 12C. 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 M 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 24:18C (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).

    1999 NRC Canada

    1232 Can. J. Bot. Vol. 77, 1999

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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 4C 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 (M) were correlated to watercontents and expressed in mol(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 105C 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 4C 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 20C (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 m as well as 3-L 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 mgmL1 SDS, 50 mM dithiothreitol (DTT, Fluka)in water, freshly prepared) at 65C 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 37C 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; 5ACCACC 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, III...


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