Effects of nodular extracts of Alnus glutinosa (L.) Gaertn. on nitrogen fixation (Acetylene reduction assay) and denitrification in different soils

Orsis 10. 1995 17-23 Effects of nodular extracts of Alnus glutinosa (L.) Gaertn. on nitrogen fixation (Acetylene reduction assay) and denitrification in different soils F.J. Gutiérrez Mañero, J.M. Pozuelo González, J.A. Lucas and A. Probanza Universidad San Pablo CEU. Unidad de Biologia Vegetal 28660 Boadilla del Monte (Madrid). Spain Abstract European alder (Alnus glutinosa [L.] Gaertn) nodules were maintained under conditions that allow their exudation (water stress). The effect of this nodular extracts colected on free nitrogen fixation (acetylene reduction assay [ARA]) and denitrification, was evaluated in three soils of significatively different physico-chemical characteristics. The comparisons with controls having different concentrations of glucose as carbon source suggest that, in the assayed conditions, the nodular european alder extracts activate ARA (up to 9.97% respect water control) and produces a partial inhibition of denitrification. The effects do not seem to be due neither to organic carbon nor to nitrate, but rather to specific activator(s) and inhibitor(s) of the processes considered. Key words: Alnus glutinosa (L.) Gaertn., ARA, Denitrification, Exudates, nitrogen fixation, nodular extracts, water stress. Resumen. Efectos de 10s extractos nodulares de Alnus glutinosa (L.) Gaertn. Sobre la jijación de nitrógeno (ensayo de la reducción de acetileno) y desnitrificación en distintos suelos Nódulos radicales de aliso europeo [Alnus glutinosa (L.) Gaertn.] fueron mantenidos en condiciones que optimizan el proceso de exudación (estrés hídrico). Se evaluó el efecto de dichos extractos sobre la fijación libre de nitrógeno (ARA) y desnitrificación en tres suelos con diferencias significativas en sus características físico-químicas. Las comparaciones con diferentes controles, muestran que 10s extractos activan (en un 9.97%) el ARA y promueven una inhibición parcial de la desnitrificación. Los efectos no parecen causados por el aporte de carbono orgánico o de nitrato por parte de 10s exudados, sino por algdn activador o inhibidor especifico de 10s procesos considerados. Palabras clave: Alnus glutinosa (L.) Gaertn., ARA, desnitrificación, exudados, fijación de nitrógeno, extractos nodulares, estrés hidrico. Introduction The rhizosphere is a region of intense microbial activity, where plant root exudates stimulate the development of a distinct rhizosphere comrnunity (Curl & Truelove, 18 Orsis 10, 1995 F.J. Gutiérrez Maiiero, J.M. Pozuelo Gonzáiez, J.A. Lucas and A. Probanza 1986; Whipps & Lynch, 1986). The rhizosphere microbial community transforms and selectively uses exudates, causing changes in residual carbon and nitrogen in the root environment. The role of the rhizosphere microorganisms in the processing and retention of nutrients in the plant-soil systems has been considered in general tems and in relation to broad attributes of ecosystem functioning (O'Neill & Reichele, 1980). In the root exudates there are not only compounds like aminoacids, carbohydrates and lipids (Barber & Gunn, 1974; Vancura, 1967), but also a number of different substances that affect rhizosphere microorganisms: allelopatic compounds (Rice, 1984), phytoalexines (Ebel, 1986) or agglutinines (Mansfield & Brown, 1985). Many authors have studied the effect of some diazotrophic plants on the nitrogen cycle microorganisms (Bollen & Lu, 1968; Tarrant & Trappe, 1971) in field conditions. Turner & Frantz (1985), identifyed four factors that may affect microorganisms: wash of leaves and stems, addition of organic matter by the litterfall, canopy effect, and excretion of different sustances by the roots. The mediterranean alder riparian copse has two important characteristics during the dry season: letting down of water table (that implies exudation due to water stress) and more intensive plant growth. The aim of the present work is to study of the effect of nodular extracts (obtained under water stress conditions) of european alder (Alnus glutinosa) both on free-living nitrogen fixing and on denitrifying microorganisms, the main responsible agents of nitrogen inputs and outputs to the soil. Materials and methods Nodules were collected from a 20 years old Alnus glutinosa stand near Madrid. Surface of nodules was sterilized with NaClO (10 mgll) and washed with sterile water. To obtain the exudates, collected nodules were stressed by keeping them in sterile conditions for 7 days. This conditions promove the maximal exudation due to water and photosintates stress (Klein et al., 1989). The nodules were then divided in three fractions of 100,50 and 20 g of dry weight, each of wich were added to 1000 cm3 of sterile water. After 12 h, the three aqueous fractions were filtred and stored at -20 OC; each fraction was the nodular extracts at 10%,5% and 2%. Physico-chemical characteristics are represented in table 2. To evaluate the effect of the nodular extracts on nitrogen fixiation (ARA) and denitrification, three soils of different geomorphological zones of Madrid were selected (table 1). These soils were collected near from the two dominant plants species of each zone, at 10-20 cm depth, taking 6 subsamples in each zone. The subsamples sf each zone were mixed paired to obtain 3 replicates. All soils were dried at 25 OC, sieved through a 2 rnm diameter sift, and stored at 4 OC. Soils shows different physico-chemical characteristics (table 2). Total nitrogen was determined following Kjeldhal method (Bremner, 1965) using 10 g soils and 1 cm3 of nodule extract. The organic carbon was determined following the Walkley & Black (1934) titulation method also using 10 g of soil and 1 cm3 of nodule extracts. Nitrate was measured following Milham et al. (1970) method, in 5 g of soil and in 2 cm3 of extract. This method uses a CRISON Alder root-nodule extracts and N cycle Orsis 10, 1995 19 digit 501 mvIpH meter with nitrate specific electrode (ORION 930700) and a ORION 900200 reference electrode. Ammoniurn was measured with a specific electrode ORION 95 1000 and the same mvIpH meter was used as in previous case; 5 g of soil and 2 cm3 of extracts were also used. The pH was directly measured in the extracts using an pH meter BECKMAN Fi-12 with an electrode RADIOMETER GK 2401 C; the pH of soil was measured in destilled water (1:3). All measurements of the physico-chemical parameters were made in three replicates. Nitrogen fixation was measured by the acetylene reduction assay (ARA), following Llinares et al. (1991) method: the different concentrations of nodular extracts were added at 100% Water Holding Capacity (WHC) of 100 g of each soil, in a flask of 375 cm3 of volume. 10% of the flasks atmosphere was replaced by acetylene. Since the most important requirement of free-living nitrogen fixing bacteria is the source of organic carbon, four controls were made of each soil where extracts were replaced by the following: (1) sterile water at 100% WHC Table 1. Characteristics of the zones where soils were collected. Parent Soil Texture (%) Dominant material (USDA) Sand Clay Silt vegetation Soil A Arkose Inceptisol 65 13 21 Quercusrotundifolia Retama sphaerocarpa Soil B Methaplutonic Ustic 23 52 23 Pinus sylvestris Cytisus purgans Soil C Gypsum-Clays Entisol 52 18 28 Quercus coccifera Stipa tenacissama Table 2. Results of characterization of soils and nodular extracts (means + standard error), and differences of the corresponding LSDs are representated by differents letters when there were statistical differences. Units: Organic C (%), Total N (mglg), N-NH,' (pglg), N-NO3- (~g/g). Soil A Soil B Soil C PH 7.30 + .Ola 4.30 + .OOb 8.01 + .02C Organic C 1.84 + .04a 6.36 + .12b 0.32 + .05a Total N 12.5 + .22a 9.54 + .22b 1.19 + .04C N-NH: 2.68 + .31a 4.71 + .31b 0.78 + .Ogc N-NO; 1.53 + .lga 2.45 + .25b 1.17 & .04a Extract 10% Extract 5% Extract 2% PH 4.66 + .OIa 5.20 + .OOb 5.51 & .0OC Organic C 2.66 & .Ola 1.92 + .OOb 1.20 + .OIC Total N 1.30 k .07a 0.81 + .07b 0.46 + .06C N-NH: 0.44 + .OOa 0.16 + .OOb 0.10 + .OOb N-NO; 3.57 + .OOa 1.73 + .Ogb 0.58 + .OOC 20 Orsis 10. 1995 F.J. Gutikrrez Maiiero, J.M. Pozuelo Gonzáiez, J.A. Lucas and A. Probanza (CW); (2) glucose in the same concentration of organic carbon found in the most concentrated extracts (10%) --that is 2.66%- plus sterile water at 100% WHC (CG); (3) twice the concentration of glucose in (2) -5.32%- ( CDG ); and (4) a control having both 2.66% of glucose and extracts at 100% WHC (CGE). The flasks were incubated 24 h at 25 OC . The ethylene produced was determinated by a KONIK 3000 HRGC gas chromatograph equipped with a flame ionization detector KNK 019-421, and PORAPAK R column, together with a KONIK 825-3 18 integrator. A control without acetylene was made to prove that spontaneous emission of ethylene did not occur. Three replicates of each treatment and control were made. The potential of denitrification was estimated by the method of acetylene inhibition (Yoshinari & Knowles, 1976), in the conditions proposed by Llinares et al. (1991), that are at 100% WHC of soils. Different concentrations of nodular extracts were added to 375 cm3 flasks, containing 50 g of soil. 10% of the headspace was replaced by acetylene. Controls identical to those for ARA were employed. After 96 h of incubation at 25", the nitrous oxide produced was measured by a gas chromatograph KONIK 3000 HRGC with a termal conductivity detector KNK 019-501, and a CROMOSORB 101 column together with a KNK 825-3 18 integrator. Three replicates of each treatment and control were made. The data were subjet to analysis of variance (ANOVA), two-way with three replicates in the experiments or one-way three replicates in the characterizations (Sokal & Rohlf, 1979). Differences between treatrnents were recognized by multiple comparisons employing the Least Significant Diference (LSD) procedure. Results and discussion The characterization of extracts and soils (Table 2) showed strong differences in the physico-chernical parameters. The extracts have an important amount of organic carbon and nitrate (2.66% and 3.57 yglg respectively), in the most concentrated extracts (10%). The results of nitrogen fixation are shown in Table 3. The ANOVA revealed significant differences between treatments and controls (6.42 d.f., F = 310.6, p < 0.01) and also between soils (2.42 d.f., F = 8.941, p < 0.01) but not for their interaction (12.42 d.f., F = 1.229). The fact that ARA increases both with the concentration of extracts and with that of glucose, suggests that this increment may have been caused by the source of organic carbon as pointed out by many (Child, 1981). However the existence of significant differences between control CDG and that CGE (60.33% of increase in the second case), indicates that the increase in ARA depends not only of the total amount of organic carbon because it is identical for both controls but it is only in form of glucose in CDG and in glucose plus exudate in the second one. These results point out that the nodular extracts of european alder have some specific activator(s) of freeliving nitrogen fixing bacteria. This is not in accordance with previous studies which show that the increase of the ARA in the rhizosphere is simply caused by the presence of organic matter (Beck & Gilmour, 1983; Bermúdez de Castro & Gutiérrez Mañero, 1987). Nevertheless, other authors pointed out that the ARA in soils increased with sustances like molybdenum, cofactor of nitrogenase (Grant & Binkley, 1987). Alder root-nodule extracts and N cycle Orsis 10, 1995 21 It stands to reason that the presence of such element (molybdenum) in the nodular extracts of Alnus which come from the nitrogenase of the endophyte Frankia, could be the cause of the increase observed in the ARA. Since it was found in the three soils assayed, the activator effect on the ARA must exist independently from the physico-chemical characteristics of the soil. Agreing with this, it must be pointed out that the interaction between soil and treatments is significative. The results of denitrification appears in Table 3. The ANOVA showed sigruficant differences between soils (2.42 d.f., F = 170.1, p < 0.01) as well as arnong treatments and controls (6.42 d.f., F = 8.876, p < 0.01), soil x treatrnent interaction was non significant (12.42 d.f., F = 1.954).The denitrification decreases with the increasing concentration of extracts, and increases with the concentration of glucose in the controls, except for CDG. The production of nitrous oxide in the treatments with extracts is higher than in the controls. Presumably the presence of the sustrate of reaction (nitrate) allows a higher activity than in the controls with glucose. The increase of denitrification with a source of carbon and nitrate has already been reported (Standford et al., 1975; Star & Parlange, 1975; Smith & Tiedje, 1979). On the other hand, when comparing the control CDG and CGE, the denitrification is significantly higher in the first than in the second one (43.87% plus). We think that this difference could be caused by the existence of a specific inhibitor(s) of the denitrifying microorganisms. Inhibitory effects on denitrification in soil contacting the roots have not been described, but it could answer to strategies similar to those pointed out in other processes (e.g. inhibition of nitrification, Moore & Waid, 1971; Baldwin et al., 1983). Besides this, it is possible that Table 3. ARA and denitrification in the different soils and treatments (means + standard error). Three soils mean for each treatment appears in each column (x). The LSD (treatrnents and controls) values appears as well. 10%, 5% and 2% nodular exudates at 10%, 5% and 2% respectively (assayed at 100% WHC); CW sterile water at 100% WHC; CG glucose at 2.66% plus sterile water at 100% WHC; CDG twice the concentration of glucose in CG -5.32%-; CGE control having both 2.66% of glucose and extracts at 100% WHC (CGE) . Treatments and controls 10% 5% 2% CW CG CDG CGE ARA (nmol ethylene g-I h-l) Soil A 0.352 k.045 0.056 k.002 0.030 2.005 0.077 k.000 0.701 k.088 1.383 k.180 2.332 k.166 Soil B 0.505 k.155 0.049 k.004 0.033 k.005 0.017 2.002 0.808 k.004 1.501 k.001 2.399 k.019 Soil C 0.192 k.034 0.030 k.003 0.016 k.001 0.016 k.000 0.381 k.009 1.326 k.035 2.059 +.O16 x = 0.349 0.045 0.027 0.035 0.631 1.412 2.264 0.200 0.145 Denitrification (nmol N20 g1 h-l) Soil A 4.133 k.359 4.858 k.262 5.889 k.446 3.523 2.160 3.877 k.047 6.160 +.O51 4.070 k.023 Soil B 2.976 k.000 4.372 k.030 4.636 k.038 1.910 k.067 2.230 2.029 4.213 k.023 3.080 k.012 Soil C 0.223 k.030 0.300 2.006 1.277 k.088 0.413 k.021 0.490 k.070 0.303 k.007 0.270 k.009 x = 2.444 3.176 3.884 1.949 2.199 3.558 2.473 1.031 0.748 22 Orsis 10, 1995 F.J. Gutiérrez Maiiero, J.M. Pozuelo Gonzáiez, J.A. Lucas and A. Probanza exists a lirnitation in growth caused by other microbial groups. 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