Arid Land Research and Management, 19:307–326, 2005 Copyright # Taylor & Francis Inc. ISSN: 1532-4982 print/1532-4990 online DOI: 10.1080/15324980500299649

The Rhizobia Nodulating for Revegetation of Arid Lands: Isolation of Native Strains and Specificity of the – Rhizobia Interaction by Cross Inoculation Tests

Fernando Gonzaalez-Andre ´s Escuela Universitaria de Ingenierı´aTe´cnica Agrı´cola INEA, Valladolid, Spain

Jesu´s Alegre Instituto Madrile, Madrid, Spain

Jose´-Luis Ceresuela Escuela Te´cnica Superior de Ingenieros Agroonomos, Universidad Polite´cnica de Madrid, Madrid, Spain

Medicago strasseri, M. citrina, arborescens, and Dorycnium pentaphyllum are legume shrubs potentially useful for the revegetation of semiarid Mediterranean ecosystems and for animal grazing. As nodulation with specific rhizobia and=or mycorrhizal fungi, may become essential for the settlement and growth of legumes, the objective was to ascertain the specificity=promiscuity of the plant–rhizobia inter- action, by detecting the occurrence of infective rhizobia in the soils to be revegetated and by cross inoculation tests. Native rhizobia for the target species, were isolated from nondisturbed native areas. The four species were inoculated with four semiarid soils located in central Spain, that we intend to revegetate. Specific rhizobia for M. strasseri and M. citrina were detected in three of the four soils. The soil that did not show infective rhizobia had received sewage-sludge from a waste-water treatment plant, and levels of heavy metals were high. None of the 4 soils had specific rhizobia for C. arborescens and D. pentaphyllum. The cross inoculation tests revealed that M. strasseri and M. citrina belonged to the same symbiotic grouping as alfalfa. On the other hand C. arborescens and D. pentaphyllum only nodulated with their own specific native rhizobia, and the rhizobia from C. arborescens and D. pentaphyl- lum were not infective with any of the tested species. For M. strasseri the effective- ness of the native rhizobia was significantly higher than the effectiveness of the infective rhizobia from the soils to be revegetated. Consequently for M. strasseri, C. arborescens and D. pentaphyllum, inoculation with native strains (I.MsS1, I.CA and I.DP respectively) would be advisable.

Received 17 September 2004; accepted 19 January 2005. This work has been financially supported by projects 06M=016=96, 07M=0076=98, and 07M=0023=2000 from Comunidad de Madrid, Spain. We want to thank INEA students Ana-Esther Fernaandez, Ana Cuadrado, and David Meneses for their help. Address correspondence to Fernando Gonzaalez-Andre ´s, Escuela Superior y Te´cnica de Ingenierı´a Agraria. Universidad de Leoon. Avda. de Portugal, 41. 24071 Leoon, Spain. E-mail: [email protected]

307 308 F. Gonzaalez-Andre ´s et al.

Keywords shrubby legumes, N-fixation, rhizobia

Shrubby have a double interest in the degraded semiarid Mediterranean ecosystems that usually present severe water stress, scarcity of plant available nutri- ents, and low microbiological activity (Caravaca et al., 2002). First, it has been demonstrated that drought tolerant shrubby species are of great interest for the recovery of degraded, or even desertified, Mediterranean ecosystems, because they are able to reestablish functional shurblands (Francis & Thornes, 1990; Alegre et al. 2004.). On the other hand, in the extensive livestock production systems of the Mediterranean basin, woody plants are considered important contributors to grazing animal nutrition (Dumont et al., 1995; Espejo Dı´az, 1996; Papachristou et al., 1999), because they are able to grow when herbaceous plants are dormant during the summer and=or winter period, and provide green foliage rich in nutrients to animals (Papachristou & Papanastasis, 1994). Legumes are able to fix atmospheric nitrogen in association with rhizobia. This process accounts for the ability of legumes to colonize N–deficient soils, and to enhance the fertility of the soils where they grow, because of the increment of the edaphic N content (Haystead et al., 1989; Dart, 1998). Woody legumes have become an essential part in the revegetation projects because of their ecological importance (Ndiaye & Ganry, 1997; Rodrı´guez-Echeverrı´a&Pe´rez-Fernaandez, 2003), due to the biotic N fixation in the one hand, and to the occurrence of mycorrhizas in the other, which improves nutrient acquisition and help plants to become established and cope with stress (Herrera et al., 1993; Requena et al., 2001). In some cases, the relationships between woody legumes and rhizobia has been proved highly specific (Lafay & Burdon, 1998; Zahran, 2001). However in other cases, a high degree of promiscuity can be achieved by both plant and microbes (Young & Haukka, 1996; Zahran, 2001; Pe´rez-Fernaandez & Lamont, 2003). For dif- ferent herbaceous and shrubby legume taxa, nodulation with specific rhizobia and=or mycorrhizal fungi, may become essential for the settlement and growth of the plants, and therefore, if the infective rhizobia are not present in the soil, inocu- lation may be necessary (Herrera et al., 1993; Jha et al., 1995; Lal & Khanna, 1996; Gonzaalez-Andre ´s & Ortiz, 1999). For the revegetation of a degraded area, two main strategies can be used. The first one is to reintroduce the preexisting species, which is called reclamation, and the second one is to introduce species from different ecosystems, which is called rehabilitation. In the last case, new stable and sustainable ecosystems are established, although having different land uses (Herrera et al., 1993). Four legume shrubs, potentially useful for reclamation or rehabilitation of soils in seasonally dry desertification-threatened areas, have been selected for the present study: Medicago strasseri Greuter, Mattha¨s & Risse; Medicago citrina (Font Quer) Greuter; Colutea arborescens L.; and Dorycnium pentaphyllum Scop. They are well adapted to the semiarid Mediterranean climatic conditions of central Spain, characterized by dry and hot summers, and cold winters. In addition, they produce forage which is useful for animal feed during the shortage from conventional pastures. Several studies, some of them are listed in the following lines, support the mentioned properties of the studied species: M. strasseri (Alegre et al., 1994; Gonzaalez-Andres & Ceresuela, 1998), M. citrina (Font Quer) Greuter (Gonzaalez-Andres & Ceresuela, 1998), C. arborescens L. (Papanastasis et al., 1998; Papachristou et al., 1999, Papachristou, Rhizobia Nodulating Mediterranean Shrubby Legumes 309

2000), Dorycnium sp. (Davies & Lane, 2003; Wills et al., 1999). In addition, when Dorycnium pentaphyllum Scop. is included in lamb diets, egg hatching and larval development for the parasite Trhichostongylus coumbriformis, decreases due to the action of the condensed tannins (Niezen et al., 2002). The starting hypothesis for this work is that prior to revegetation of land, it is necessary to assess the specificity or promiscuity of the plant–rhizobia interaction and the occurrence of symbiotically effective rhizobia in soils in order to determine if it is necessary to inoculate with selected rhizobia strains. The starting point is the isolation of infective rhizobia strains obtained from seedlings growing in non- disturbed native areas. The general objective is to ascertain the specificity or promiscuity of the plant–rhizobia interaction for the species Medicago strasseri, M. citrina, Colutea arborescens, and Dorycnium pentaphyllum. The tasks to fulfill this objective were: (a) To test several soils, in order to detect the presence of natural populations of rhizobia capable of effectively nodulating the studied species and (b) To assess the range of rhizobia that effectively nodulates the target legume spe- cies, as well as the host range of the rhizobia isolated from those species to define cross-inoculation groups for the studied species.

Material and Methods A list of the rhizobial accessions included in this research, their parent hosts and ori- gin or donor institutions is presented in Table 1. Stock cultures were maintained on yeast extract mannitol agar (YMA) medium.

Isolation of Nodulating Rhizobia Strains I.MsS1, I.McSC, I.McSM, I.CA, and I.DP were isolated from root nodules of the parent hosts indicated in Table 1, from seedlings growing in a native habitat in the location cited in the column ‘‘origin.’’ An area of 4 km2 was sampled, and one plant was randomly selected from each km2. One random nodule was selected from every plant and the isolates were authenticated by inoculation of seedlings of the host species. Strains I.M3 and I.M4 were each isolated from root nodules of M. citrina formed after inoculation with a 10-fold dilution from two soils intended to be revegetated (Tables 1 and 2), following the procedure described in the next subsection. They were authenticated as well. In both cases, the nodules were surface sterilized with 0.1% HgCI2, thoroughly washed, individually crushed following the crushed nodule method (Beck et al., 1993), plated onto YMA medium containing 25 mg litre1 Congo Red, and incubated at 28C for 10 days. Bacteria were isolated from a single typical colony on the assumption that this would represent the domi- nant strain in any nodule (Johnston & Beringer, 1975). Isolates were characterized by color, colony appearance, extracellular polysaccharide production (EPS), and size of the first discrete colonies appearing away from the confluent growth on YMA plates (Odee et al., 1997). Colony size was measured as diameter to the nearest millimeter, using a 10X magnifying glass with graticule, after 3, 5, 7, and 10 days’ incubation. These data were used to categorize the isolates according to growth rates, following the description of Jordan (1984) modified by Odee et al. (1997). The growth cate- gories were as follows: very fast colonies were 5 mm in diameter after 3 days’ incubation; fast colonies were 2 mm in diameter after 5 days’ incubation; inter- mediate colonies were 1–2 mm in diameter after 7 days’ incubation; slow colonies Table 1. Rhizobia strains used in this study Strain Parent host Origin Donor institution

ISM16 Sinorhizobium meliloti Medicago sativa Estacioon El Zaidı´n, Granada, Spain ISL18 Rhizobium leguminosarum Vicia sativa INIA, Seville, Spain bv. vicieae ISLU21 Bradyrhizobium sp. Lupinus luteus INIA, Seville, Spain ISP.74 Rhizobium etli Phaseolus vulgaris INIA, Seville, Spain IST79 Rhizobium leguminosarum Trifolium repens Rothamsted Collection bv. trifolii of Rhizobium. USDA110 Bradyrhizobium japonicum Glycine max USDA, Beltsville, USA 310 BV.GL1 Bradyrhizobium sp.a Genista linifolia La Almoraima (Caadiz, Spain) BV.GM1 Bradyrhizobium sp.a Genista monspessulana La Almoraima (Caadiz, Spain) I.MsS1 Not determinedb Medicago strasseri Petre`s Gorge (Crete Island, Greece) I.McSC Not determinedb Medicago citrina Ferrera (Columbretes Islands, Castelloon, Spain) I.McSM Not determinedb Medicago citrina Ses Bledes (Cabrera Island, Baleares, Spain) I.M3 Not determinedb Medicago citrina ETSIA-UPM (Madrid, Spain)c I.M4 Not determinedb Medicago citrina Torozos (Valladolid, Spain) I.CA Not determinedb Colutea arborescens Tendilla (Guadalajara, Spain) I.DP Not determinedb Dorycnium pentaphyllum Los Santos de la Humosa (Madrid, Spain)

a Tentative taxonomy on the basis of phenotypic characteristics of the colonies in YMA medium and cross inoculation data (Gonzaalez-Andre ´s & Ortiz 1999). b The taxonomy of these organism is not the purpose of this work. c ETSIA-UPM: Escuela Te´cnica Superior de Ingenieros Agroonomos. Universidad Polite´cnica de Madrid. Rhizobia Nodulating Mediterranean Shrubby Legumes 311 were 1 mm after 7 days’ incubation, and very slow growers were colonies appearing after 10 days.

Inoculation of Plants with Soils M. strasseri, M. citrina, C. arborescens, and D. pentaphyllum were inoculated with suspensions from several soils (Table 2), following the most probable number (MPN) plant infection technique (Beck et al., 1993) to ascertain the number of infec- tive rhizobia in each soil. Every species was inoculated with its native soil, and with four other soils from central Spain, intended to be revegetated. The soil from San Fernando de Henares (Madrid, Spain) (Table 2) received 800 t=ha of sewage-sludge from a waste-water treatment plant, and it had the following contents of heavy metals: Zn 28.8 mg kg1; Pb 8.9 mg kg1; Cd 1.85 mg kg1; Ni 0.83 mg kg1; and Cu 32.3 mg kg1. In all the cases, contents were significantly higher than in the con- trol. Seedlings were obtained from seeds individually scarified by immersion in H2SO4 (95%) for 30 min for M. strasseri and M. citrina, in HCl (37%) for 15 min for D. pentaphyllum and by immersion in boiling water for 2 min for C. arborescens. After scarification, seeds were sterilized for 2 min in HgCl2, and washed with sterile distilled water. They were sown in sterile Petri dishes containing 1% sterile water agar, and incubated at 21C in 16 h of light and at 16C in 8 h of darkness. When the radicles reached 10 mm in length, seedlings were transferred to sterile test tubes, 15 180 mm, filled with a vermiculite substrate containing about 2 cm of gravel at the bottom for drainage. The test tubes were closed with sterile cotton plugs and watered with sterile N free Leonard solution (Beck et al., 1993). Plants were inocu- lated with 1 ml of appropriate soil dilution after transplanting. Six soil dilutions (101–106) were prepared, and four plants were inoculated with each dilution. For the first dilution, the soil was sieved, and 10 g samples suspended in 90 ml sterile Leonard solution. The protocol was adapted from Beck et al. (1993). After inocu- lation, plants were grown at 23C in 16 h of light and at 16C in 8 h of darkness. They were watered with sterile Leonard solution as needed and, after 60 days, examined for the presence of nodules with pink-red interior which may indicate active fixation. From the number of plants forming nodules with pink-red interior at each dilution level, the MPN of rhizobia in the original sample was calculated using a modified version of the Fischer and Yates (1963) tables (Beck et al., 1993).

Determination of the Range of Rhizobia that Effectively Nodulate Medicago strasseri, M. citrina, Colutea arborescens, and Dorycnium pentaphyllum The rhizobial strains in Table 1 were used to inoculate seedlings of the studied spe- cies, to ascertain infectivity, and effectiveness for those strains that produce infective nodules. Seedlings were obtained following the protocol described above. Four plants for every species were inoculated with the strains indicated in Table 4, using 1 ml of yeast extract mannitol broth (YMB) culture obtained after cultivation of the strain, at 28C. Four plants were used as a non inoculated control, and another four plants as an N-supplied non inoculated control. Plants were grown as described above, except for the N-supplied control, which was watered with sterile Leonard 1 solution, supplemented with 70 mg liter of N from CaNO3. After the 60–day growth period, presence or absence of nodules with pink-red interior, stem height, and above-ground dry mass (DM) were determined for each plant. The 312 F. Gonzaalez-Andre ´s et al.

Table 2. Location, and ecological, geographical, edafological and climatological Colutea arborescens and Dorycnium pentaphyllum

Reason for inclusion in the study Origin Observations Latitude

Garganta Petre`s M. strasseri is native from 35190 N (Crete Island; Greece) this location. Illa Grossa M. citrina is native from 39520 N (Columbretes Islands; Columbretes Islands. It Castelloon; Spain) disappeared for 20 years from this location and the plant was reintroduced from Ferrera (Columbretes Islands). Native soils Ferrera (Columbretes M. citrina is native from 39520 N Islands; Castelloon; this location. Electric Spain) conductivity is high: 5dSm1 in relation soil=water 1=2, because of influence from the sea. Ses Bledes (Cabrera M. citrina is native from 3990 N Island; Baleares; this location. Spain) Tendilla (Guadalajara; C. arborescens is native from 40330 N Spain) this location. Los Santos de la D. pentaphyllum is native 40300 N Humosa (Madrid; from this location. Spain) Torozos (Valladolid; Some herbaceous wild 41450 N Spain) Medicago and Melilotus species grew among the spontaneous flora. ETSIA-UPMa Alfalfa was an habitual crop 40260 N (Madrid, Spain) in the crops rotation. Soils from San Fernando de Received 800 t=ha of 40260 N central Spain Henares (Madrid; sewage-sludge from a intended to Spain) waste-water treatment be revegetated plant and it had with the significantly high studied species contents of heavy metals (see text for details). Aranjuez (Madrid; Some herbaceous wild 4020 N Spain) Medicago species and Retama sphaerocarpa grew among the spontaneous flora. Electric conductivity is high: 6 dS m1 in relation soil=water 1=2.

aExperimental fields at ETSIA-UPM: Escuela Te´cnica Superior de Ingenieros Agroonomos. bU.S.D.A. (1975). Rhizobia Nodulating Mediterranean Shrubby Legumes 313 characterisitics of the soils used for inoculation of Medicago strasseri, Medicago citrina,

Av. Av. Elevation winter summer Av. winter Av. summer above sea PH soil= USDA rainfall rainfall temperature temperature Longitude level (m) water 1=2 classificationb (mm) (mm) (C) (C)

25100 E 30 7.2 368 58 15.0 22.8

0400 E 25 7.1 248 198 10.2 17.3

0400 E 35 7.3 248 198 10.2 17.3

2560 E 2 7.4 378 172 10.2 16.8

2570 W 789 7,8 214 163 8.8 19.5

3150 W 906 8.1 214 163 8.8 19.5

4530 W 840 8.5 246 205 6.4 15.8

3440 W 595 8 Entisol; Typic 259 177 9.3 19.9 Xerorthent 3320 W 585 6.7 214 163 8.8 19.5

3360 W 494 7.8 227 161 9.1 20.0

Universidad Polite´cnica de Madrid. Table 3. Most Probable Number (MPN) of infective rhizobia per gram of soil for M. strasseri, M. citrina, C. arborescens, and D. pentaphyllum in native soils of these species, and other semiarid soils located in central Spain, intended to be revegetated with the mentioned species Medicago Medicago Colutea Dorycnium Origin Native species strasseri citrina arborescens pentaphyllum

Petre`s Gorge (Crete Island; Greece) M. strasseri 6.9 105 ND ND ND Illa Grossa (Columbretes Islands; M. citrina ND 1.8 105 ND ND Castelloon; Spain) Ferrera (Columbretes Islands; M. citrina ND 7 105 ND ND Castelloon; Spain) 314 Ses Bledes (Cabrera Island; M. citrina ND 1 105 ND ND Baleares; Spain) Tendilla (Guadalajara; Spain) C. arborescens ND ND 1.8 105 ND Los Santos de la Humosa D. pentaphyllum ND ND ND 3.4 105 (Madrid; Spain) Torozos (Valladolid; Spain) None of the studied 1.8 105 6.9 104 00 ETSIA-UPMa (Madrid, Spain) None of the studied 6.9 105 5.9 104 00 San Fernando de Henares None of the studied 0 0 0 0 (Madrid; Spain) Aranjuez (Madrid; Spain) None of the studied 1.7 103 1.0 104 00

ND: Not determined; 0: not found. aExperimental fields at: ETSIA-UPM: Escuela Te´cnica Superior de Ingenieros Agroonomos. Universidad Polite´cnica de Madrid. Rhizobia Nodulating Mediterranean Shrubby Legumes 315 above-ground DM of the nodulated plants in comparison with the plants receiving sufficient combined nitrogen, was used as a first approximation of the symbiotic effectiveness of the infective strains (Beck et al., 1993), and was calculated based on average data of the four plants per treatment, using the following formula (Beck et al., 1993):

Strain effectiveness DM of the plant inoculated with the strain DM of the noninoculated control ¼ 100 DM of the N-supplied control DM of the noninoculated control

One-way analysis of variance (ANOVA) was used to detect differences in growth parameters among plants inoculated with different rhizobial strains and the controls. The Duncan multiple range test was used to compare the means (P<0.05).

Determination of the Host Range of Isolated Rhizobia The infectivity of rhizobia isolated from M. strasseri, M. citrina, C. arborescens and D. pentaphyllum was estimated in the following host species: Glycine max (L.) Merr, Lupinus albus L., Medicago sativa L., Phaseolus vulgaris L., Trifolium repens L., and Vicia sativa L. (commercial origin), and Genista monspessulana L. (from the germplasm bank, Departamento de Biologı´a Vegetal, Universidad Polite´cnica de Madrid, Madrid, Spain). Seedlings were obtained following the same protocol used for the other species, but Genista monspessulana seeds were scarified for 40 min in H2SO4 (95%), and the other five species did not require scarification. Small-seeded species were grown in sterile 15 180 mm test tubes, and large-seeded ones in 250- ml flasks. When transplanted, four plants per species were inoculated with 1 ml of broth culture of its specific rhizobial strain (Table 1), and another four plants with each of the rhizobial accessions, I.MsS1, I.McSC, I.McSM, I.CA, I.DP (Table 4). Four plants per species were used as non inoculated controls. The parameters eval- uated and the statistical analysis performed were the same as in the experiment described above.

Results

Characterisation of Isolated Rhizobia The strains isolated from Medicago strasseri and M. citrina in their native areas (I.MsS1, I.McSC, and I.McSM in Table 1) presented the following characteristics: Colonies moderately absorbed the dye (Congo Red), and they were translucent, circular, shiny, and slightly raised. They produced moderate extracellular polysac- charide (EPS), with a slightly gummy consistency. The colonies reached a diameter >2 mm after 5 days of incubation at 28C. Therefore, they were classified as fast growing colonies, according to the description of Jordan (1984), modified by Odee et al. (1997). The strain isolated from Dorycnium pentaphyllum (I.DP in Table 1) dif- fered from the previous description in the growth rate, and it was classified as a slow grower because colonies were 1 mm in diameter after 7 days growing. On the con- trary, the strain isolated from Colutea arborescens (I.CA in Table 1) was fast grower but showed a slightly higher dye absorption than the isolates from Medicago spp. 316 F. Gonzaalez-Andre ´s et al.

Table 4. Data obtained from M. strasseri, M.citrina, C. arborescens,andD. pentaphyllum in aseptic conditions. (Stem height and above-ground dry mass are mean data of four ANOVA. analysis (Duncan’s test P < 0.05). The effectiveness was estimated from

Medicago strasseri Medicago citrina

Rhizobia Above- Above- strains Stem ground Effective- Stem ground Effective- used for height dry mass ness height dry mass ness inoculation Nodules (cm) (mg) (%) Nodules (cm) (mg) (%)

ISM16 yes 6.1 b 26.2 b 62.0 yes 6.0 b 83.3 b 75.8 ISL18 no 4.3 a 18.5 a NA no 3.3 a 51.2 a NA ISLU21 no 4.3 a 18.6 a NA no 3.7 a 51.6 a NA IST79 no 4.1 a 17.8 a NA no 3.4 a 50.7 a NA USDA110 no 4.3 a 18.4 a NA no 3.3 a 50.9 a NA BV.GL1 þ no 4.2 a 18.0 a NA no 3.2 a 52.8 a NA BV.GM1 I.MsS1 yes 7.3 c 31.4 c 100.5 yes 6.2 bc 85.3 bc 80.7 I.McSC yes 7.1 c 30.3 bc 84.4 yes 7.4 d 96.3 d 92.7 I.McSM yes 6.8 bc 29.2 bc 92.4 yes 6.8 bcd 90.3 bcd 107.3 I.M3 yes 6.2 b 26.6 b 65.2 yes 6.2 bc 84.5 bc 78.9 I.M4 yes 6.1 b 26.3 b 62.9 yes 6.4 bc 87.2 bcd 85.3 I.CA no 4.3 a 19.1 a NA no 3.1 a 49.9 a NA I.DP no 4.2 a 18.5 a NA no 3.8 a 55.2 a NA Noninoculated no 4.1 a 17.7 a 0 no 3.5 a 51.9 a 0 control N-supplied no 7.3 c 31.3 c 100 no 7.0 cd 93.3 cd 100 noninoculated control

ND: Not determined; NA: Not applicable. aStrain effectiveness DM of the plant inoculated with the accession DM of the noninoculated control ¼ 100: DM of the N-supplied control DM of the noninoculated control Rhizobia Nodulating Mediterranean Shrubby Legumes 317 plantlets after the inoculation with the different rhizobia strains, and 60 days’ growing plants. Values followed by the same letter were not significantly different in the the dry mass of the aerial parta.)

Colutea Dorycnium arborescens pentaphyllum

Above- Above- Stem ground Effective- Stem ground Effective- height dry mass ness height dry mass ness Nodules (cm) (mg) (%) Nodules (cm) (mg) (%)

no 3.0 a 17.1 a NA no 6.7 a 12.6 a NA no 2.7 a 15.4 a NA no 6.1 a 11.1 a NA no 3.1 a 17.9 a NA no 6.8 a 12.4 a NA no 2.3 a 13.1 a NA no 6.2 a 11.3 a NA no 3.0 a 17.8 a NA no 6.5 a 12.8 a NA no 2.9 a 16.8 a NA no 7.0 a 13.0 a NA

no 3.0 a 16.7 a NA no 6.3 a 11.0 a NA no 2.8 a 16.2 a NA no 6.5 a 12.4 a NA no 2.6 a 15.2 a NA no 6.0 a 11.3 a NA ND ND ND ND ND ND ND ND ND ND ND ND ND ND ND ND yes 5.5 b 32.4 b 105.0 no 7.0 a 13.1 a NA no 2.7 a 16.6 a yes 10.6 b 21.7 b 131.2 no 5.5 b 17.9 a 0 no 6.9 a 12.2 b 0

no 3.2 a 31.7 b 100 no 10.5 b 19.4 b 100 318 F. Gonzaalez-Andre ´s et al.

Inoculation of Plants with Soils The four species under investigation (M. strasseri, M. citrina, C. arborescens, and D. pentaphyllum), formed nodules with pink-red interior after the inoculation with 10-fold dilutions from their native soils (Table 3). The most probable number of infective rhizobia ranged from 1 105 rhiobia per g of soil (M. citrina inoculated with soil from Ses Bledes, Cabrera Island, Baleares, Spain) to 7 105 (M. citrina inoculated with soil from Ferrera, Columbretes Islands, Castellon, Spain). When plant species were inoculated with decimal dilutions from other soils located in central Spain intended to be revegetated with them (Torozos, Valladolid; ETSIA-UPM, Madrid; Aranjuez, Madrid; San Fernando de Henares, Madrid), only M. strasseri and M. citrina formed nodules with pink-red interior after the inoculation with the first three soils (Table 3). The most probable number of rhizobia per g of soil ranged from 1.7 103 (Medicago strasseri inoculated with soil from Aranjuez) to 6.9 105 (M. strasseri inoculated with soil from ETSIA-UPM). The remaining soil that had received sewage-sludge, had no infective rhizobia for Medicago species. None of the soils had infective rhizobia for C. arborescens or D. pentaphyllum.

Determination of the Range of Rhizobia that Effectively Nodulate Medicago strasseri, M. citrina, Colutea arborescens and Dorycnium pentaphyllum Out of the 13 strains used for inoculation of M. strasseri and M. citrina, the follow- ing produced infective nodules (Table 4): ISM16, an isolation from alfalfa (Medicago sativa L.); I.MsS1, native rhizobia from M. strasseri; I.McSC and I.McSM, native rhizobia from M. citrina; and I.M3 and I.M4, isolated from root nodules formed in M. citrina when it was inoculated with soils from central Spain (Torozos and ETSIA-UPM, respectively). For M. strasseri and M. citrina, there were no signifi- cant differences in stem height or above-ground dry matter production among the noninoculated control and the plants inoculated with strains that did not produce nodules (Table 4). Conversely, plants inoculated with the rhizobia that produced nodules, and the N-supplied noninoculated control were significantly taller and pro- duced more above-ground dry mass than the treatments that did not produce nodu- lation and the noninoculated control. Finally, there were no significant differences of stem height and above-ground dry mass among the N-supplied noninoculated con- trol and the treatments inoculated with their own native rhizobia. Some differences were found among the strains that formed nodules (Table 4): M. strasseri had signifi- cantly greater stem height and produced more above-ground dry mass in symbiosis with the native rhizobia from Crete (I.MsS1), than in symbiosis with alfalfa rhizobia (ISM16) and the rhizobia isolated from central Spain soils (I.M3, I.M4). Moreover, stem height and above-ground dry mass, did not significantly differ among plants inoculated with I.MsS1, and with M. citrina native rhizobia (I.McSC and I.McSM). In M. citrina there were no significant differences, for stem height and above- ground dry mass, among plants inoculated with the two native rhizobia: From Columbretes Islands (I.McSC) and from Cabrera Island (I.McSM). Nevertheless, the dry mass did not significantly differ from that obtained after inoculation with the strain from Torozos (I.M4) in central Spain. Symbiosis with native rhizobia from Columbretes (I.McSC) produced taller plants and more above-ground dry mass than symbiosis with the commercial strain ISM16, with M. strasseri’s native rhizobia (I.MsS1), and with the isolate from ETSIA-UPM (I.M3). However, the two Rhizobia Nodulating Mediterranean Shrubby Legumes 319 parameters did not differ among plants inoculated with the native rhizobia from Cabrera Island (I.McSM), and with the strains ISM16, I.MsS1 and I.M3. Out of the 13 strains used for inoculation of C. arborescens and D. pentaphyllum, only their own native rhizobia was infective (Table 4). There were no significant differences in stem height or above-ground dry mass production among the nonino- culated control and the plants inoculated with strains that did not form nodules (Table 4). On the other hand, plants inoculated with native rhizobia and the N-supplied noninoculated control did not differ for the mentioned parameters, and produced significantly higher values than the rest of the treatments. In all the species, the highest percent effectiveness was obtained for the inocu- lation with native rhizobia. It was near 100% (92.8% for I.McSM in M. citrina and 100% for I.MsS1 in M. strasseri) or even over 100% (107% for I.McSC in M. citrina, 105% for I.CA in C. arborescens, and 131% for I.DP in D. pentaphyllum).

Determination of the Host Range of Isolated Rhizobia Six of the seven species analyzed did not produce nodules when they were inoculated with the native rhizobia of M. strasseri (I.MsS1), M. citrina (I.McS5, I.McSM), C. arborescens (I.CA), and D. pentaphyllum (I.DP) (Table 5). The six species were Genista monspessulana, Glycine max, Lupinus albus, Phaseolus vulgaris, Trifolium repens,andVicia sativa. As a consequence, no significant differences were found between the noninoculated control and the plants inoculated with the above men- tioned rhizobia isolates, neither for stem height nor for above-ground dry mass. Conversely, when all those species were inoculated with their specific rhizobia strains, they produced nodules with pink-red interiors, and significantly higher values of stem height and above-ground dry mass, which is considered as an authen- tification of the strain. On the contrary, Medicago sativa L. produced nodules when it was inoculated with the isolates from M. strasseri and M. citrina (I.MsS1, I.McS5 and I.McSM) and with their own strain ISM16 (Table 5), but not when it was inoculated with the native rhizobia from C. arborescens and D. pentaphyllum. No significant differ- ences regarding stem height, or above-ground dry mass, were found in M. sativa for the N-supplied noninoculated control, the plants inoculated with ISM16, and the plants inoculated with the isolates I.MsS1, I.McSC and I.McSM. On the other hand there were no differences for either parameter, among the noninoculated control and the plants inoculated with noninfective rhizobia. Plants that produced nodules showed significantly higher mean values than plants that did not produce nodules.

Discussion Five rhizobia strains were isolated in four shrubby legume species, from seedlings growing in five different native soils located in Mediterranean semiarid lands: I.MsS1 from M. strasseri, I.McSC and I.McSM from M. citrina, I.CA from C. arborescens and I.DP from D. pentaphyllum. We consider that the isolated strains are adequate for the inoculation of the host species on the basis of the following rea- sons: (a) the number of infective rhizobia per gram of native soil was high, more than 1.8 105; (b) there was an important presence of nodulated seedlings in the native soil, more than 5 per m2; (c) the general condition of the seedlings and adult plants living in the native soils was healthy, and (d) the effectiveness of the strains in vitro Table 5. Data obtained from plantlets belonging to the species indicated in the first column, after the inoculation with the mentioned rhizobia strains, and 60 days’ growing in aseptic conditions (mean values standard deviation). Strains used for the inoculation Stem heightb Dry mass aerial partb Plant species (No. according to Table 1) Nodulesa (cm) (mg)

Genista monspessulana L. BV.GL1 þ BV.GM1 y 9.0 b 32.8 b I.MsS1 n 4.76 a 16.1 a I.McSC n 4.7 a 17.1 a I.McSM n 4.9 a 17.1 a I.CA n 4.9 a 15.8 a I.DP n 4.8 a 17.3 a N-supplied noninoculated control n 9.0 b 33.8 a Noninoculated control n 4.5 a 15.8 a Glycine max (L.) Merr USDA 110 y 70.0 b 750.4 b 320 I.MsS1 n 56.3 a 555.0 a I.McSC n 56.0 a 576.1 a I.McSM n 56.7 a 571.8 a I.CA n 56.0 a 574.5 a I.DP n 55.4 a 551.6 a N-supplied noninoculated control n 67.3 b 754.6 b Noninoculated control n 56.1 a 554.0 a Lupinus albus L. ISLU 21 y 22.8 c 212.4 b I.MsS1 n 17.0 b 155.2 a I.McSC n 15.8 ab 159.2 a I.McSM n 15.0 a 154.7 a I.CA n 15.6 ab 153.9 a I.DP n 17.0 b 160.3 a N-supplied noninoculated control n 23.3 c 216.4 b Noninoculated control n 16.1 ab 159.6 a Medicago sativa L. ISM 16 y 11.7 b 28.3 b I.MsS1 y 12.1 b 28.6 b I.McSC y 11.6 b 28.3 b I.McSM y 11.9 b 29.4 b I.CA n 4.2 a 3.9 a I.DP n 4.2 a 5.3 a N-supplied noninoculated control n 11.7 b 30.3 b Noninoculated control n 4.1 a 4.5 a Phaseolus vulgaris L. ISP 74 y 34.8 b 693.0 b I.MsS1 n 24.2 a 531.7 a I.McSC n 25.2 a 520.7 a I.McSM n 24.3 a 530.8 a I.CA n 24.0 a 530.6 a I.DP n 24.0 a 526.4 a 321 N-supplied noninoculated control n 35.2 b 707.7 b Noninoculated control n 24.7 a 520.8 a Trifolium repens L. IST 79 y 6.0 a 3.0 c I.MsS1 n 2.9 a 2.0 ab I.McSC n 3.2 a 2.0 ab I.McSM n 3.2 a 2.1 b I.CA n 3.0 a 1.8 a I.DP n 3.7 a 2.0 ab N-supplied noninoculated control n 5.5 b 2.8 c Noninoculated control n 3.0 a 1.9 ab

(Continued) Table 5. Continued

Strains used for the inoculation Stem heightb Dry mass aerial partb Plant species (No. according to Table 1) Nodulesa (cm) (mg)

Vicia sativa L. ISL 18 y 58.1 b 122.1 b I.MsS1 n 49.8 a 93.9 a I.McSC n 48.6 a 92.3 a 322 I.McSM n 49.5 a 94.6 a I.CA n 48.6 a 92.9 a I.DP n 48.6 a 99.4 a N-supplied noninoculated control n 55.1 b 120.1 b Noninoculated control n 48.9 a 92.1 a

a Presence (þ) or absence () of nodules. b Stem height and dry mass of the aerial part are mean data of four plants. Values followed by the same letter were not significantly different in the ANOVA analysis (Duncan’s test P < 0.05). Rhizobia Nodulating Mediterranean Shrubby Legumes 323 was close to 100% or even over 100% compared with a noninoculated control sup- plemented with sufficient mineral N. Inoculation would be necessary if the specific rhizobia had disappeared in the soil, as recommended by Herrera et al. (1993). The survival of these strains in the soils to be revegetated should be studied in the future to ascertain competitiveness and adaptation. In the soils from semiarid central Spain, on lands intended to be revegetated (Torozos, Valladolid; ETSIA-UPM, Madrid; Aranjuez, Madrid; San Fernando de Henares, Madrid), we detected infective rhizobia for M. strasseri and M. citrina, except for the soil, that had received sewage-sludge from the waste-water treatment plant, in which no rhizobia for any of the studied species was found. The levels of heavy metals were high, mainly Zn and Cu, and this could be the reason for the lack of infective rhizobia. The cross inoculation analysis revealed that M. strasseri, M. citrina, and alfalfa (M. sativa) belong to the same symbiotic grouping. The study of two woody species of the section Dendrotelis is reported for the first time, and supports Young’s (1996) statement about the cross-inoculation group for alfalfa, consisting of species of Medicago, Melilotus and Trigonella. In the tested soils, from central Spain, Medicago spp. grew as spontaneous flora in Torozos and Aranjuez. Moreover alfalfa was an habitual crop in the usual crop rotation of the soil from ETSIA. Consequently the presence of infective rhizobia in those soils might be due to the presence of Medicago spp. In M. strasseri the strain from the native soil (I.MsS1), was significantly more effective in N fixation (estimated from above-ground dry mass) than the strains that occurred in the soils intended to be revegetated, and the alfalfa specific strain ISM16. M. strasseri is an edemism from Crete (Greuter et al., 1982; Robledo et al., 1993) and this could explain the specialization of the plant-rhizobia interaction. However, I.MsS1 was as much effective because the native isolates from M. citrina (I.McSC and I.McSM). M. strasseri and M. citrina belong to the same botanical section Dendrotelis (Gonzaalez-Andre ´s et al., 1999) found on shrubby Medicago species, and this could explain the lack of observed differences. Consequently, when M. strasseri is utilized for the revegetation of soils that are not located in the native area, inoculation with strain I.MsS1 should be recommended. However, M. citrina, presents a wider range of occurrence within the Mediterranean basin (Robledo et al., 1993), and this could explain why this species is more promiscuous in the plant- rhizobe interaction, as there were not clear differences of effectiveness among the strains that produced nodules in M. citrina. Using Duncan as post-hoc statistical treatment, it was not possible to distinguish among the above-ground dry mass produced after nodulation with the rhizobia from native soils, and the rhizobia preexisting in some of the soils to be revegetated. For C. arborescens and D. pentaphyllum, out of the 13 strains tested, only the isolates from their own native areas were infective, and conversely the rhizobia iso- lated from C. arborescens and D. pentaphyllum did not infect any of the plant species tested. For C. arborescens, our results are different from those cited by (Allen & Allen, 1981) that described this species and C. cilicica versatile in symbiotic perform- ance with Trifolium and Vicia species, both studied in our work. From our results C. arborescens and D. pentaphyllum require quite specific rhizobial strains, and this may be the reason for the lack of infective rhizobial strains in non-native soils, and consequently for the revegetation of soils, it would be necessary to inoculate seedlings with the strains I.CA and I.DP, respectively. 324 F. Gonzaalez-Andre ´s et al.

This work has ascertained the specificity=promiscuity of the legume-bacteria symbiosis for woody legumes. This information is important because the degree of specificity or promiscuity shows wide variations in woody legumes depending on the taxa: Gonzaalez-Andre ´s and Ortiz (1998), Zahran (2001), Lafay and Burdon (1998) described highly specific relations, whereas Olivares et al. (1988), Batzli et al. (1992), Zahran (2001), and Pe´rez-Fernaandez and Lamont (2003) indicated that relationships between woody legumes and rhizobia are rather promiscuous. As a conclusion of this work, C. arborescens and D. pentaphyllum are highly spe- cific in the plant-rhizobia interaction because only the rhizobia isolated from their native areas was infective. Inoculation with strains I.CA and I.DP, respectively, would be advisable prior to revegetation of degraded lands, if these species are not autochthonous. Conversely M. strasseri and M. citrina belong to the same sym- biotic grouping as alfalfa, and therefore infective rhizobia seem to be present in a wide range of soils. However, for M. strasseri the effectiveness of the native rhizobia was significantly higher than the effectiveness of the infective rhizobia from the soils to be revegetated, and therefore inoculation with strain IMsS1 would be advisable as well.

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