Biological nitrogen fixation BY bicolor and A. macrostachya, potential forage species for arid and semi-arid environments

Darío Vileta, Luciana Bianco, Mónica Grosso and Rosana Malpassi

SUMMARY

In South American arid regions cattle feed lacks during win- tritive solution: “Without N”; “With N”; and “Inoculated seeds ter. Adesmia bicolor and A. macrostachya are promising forage with nutritive solution without N”. Nodules morpho-anatomy and species because of their growth habit and adaptability to dry win- spatio-temporal distribution; shoot-leaf, root, and nodule dry mat- ters and N poor soils. Information about their biological nitro- ter and N content were analyzed. The studied species showed a gen fixation (BNF) efficiencies is needed. The aim of this work mean BNF efficiency of ~60%, which is a relatively high efficien- is to determine their BNF efficiencies, nodule morpho-anatomy cy considering that alfalfa shows a 61% efficiency when grown in and spatio-temporal distribution, in symbiosis with rhizobia. In central Argentina. This attribute is desirable in wild species with a greenhouse, both species were seeded and irrigated with nu- forage potential in arid environments.

FIJACIÓN BIOLÓGICA DE NITRÓGENO DE Adesmia bicolor Y A. macrostachya, POTENCIALES ESPECIES FORRAJERAS PARA AMBIENTES ÁRIDOS Y SEMIÁRIDOS Darío Vileta, Luciana Bianco, Mónica Grosso y Rosana Malpassi RESUMEN

En regiones áridas de Sudamérica hay escasez de alimento y regadas con solución nutritiva: “Sin N”; “Con N”; y “Sin para el ganado durante el invierno. Adesmia bicolor y A. ma- N-semillas inoculadas”. Se analizaron la morfo-anatomía y dis- crostachya son prometedoras como forraje por sus hábitos de tribución espacio-temporal de nódulos, peso seco y contenido de crecimiento y adaptabilidad a inviernos secos y suelos pobres N de tallos-hojas, raíces y nódulos. Las especies mostraron una en N. Es esencial disponer de información de sus eficiencias eficiencia de BNF media de 60%, la cual es alta considerando de fijación biológica de N (BNF). El objetivo del presente tra- que alfalfa presenta un 61% cuando crece en Argentina central. bajo es determinar sus eficiencias de BNF, morfo-anatomía y Este atributo es deseable en especies silvestres con potencial fo- distribución espacio-temporal de los nódulos en simbiosis con rrajero en ambientes áridos. rizobios. En un invernadero, ambas especies fueron sembradas

Introduction expanding world population Dias et al., 2004; Veneciano winter species with high nutri- (Zahran, 1999; Howieson et et al., 2005). In these regions, tional quality that are already Arid and semi-arid climates al., 2008). many summer forage species adapted to local soil and cli- comprise about one third of Most of the arid and semi- are used, but there is a lack matic conditions (Tedesco et the total land area (Johnson arid regions of South America of cattle feed in winter. Dur- al., 2000; Scheffer-Basso et et al., 1981). These zones (southern Brazil, Uruguay, ing these months, food supple- al., 2001a; Speroni and Iza- were previously considered and central Argentina) have ment is not viable due to its guirre, 2003; Barreto Dias et as economically unimportant; extreme environmental condi- high cost. Consequently, as al., 2004). however, during the last three tions, such as dry and windy commercial forage species do Legume species are vital decades their economic and winters and low N content not grow, large areas remain in ecosystems characterized agricultural utilization has soils (Tedesco et al., 2000; unused for cattle production by N poor-soils (Howieson emerged as a critical element Scheffer-Basso et al., 2001a, (Döbereiner, 1997; Venecia- et al., 2008). In these eco- in maintaining and improv- 2002; Bianco, 2002; Speroni no et al., 2005). Therefore, systems, biological N2 fixa- ing the food supply for the and Izaguirre, 2003; Barreto the search is focused on wild tion (BNF) is the major form

KEYWORDS / Adesmia bicolor / Adesmia macrostachya / Arid Land / BNF Efficiency / Nitrogen Fixation / Nodule Distribution / Received: 03/03/2009. Modified: 05/20/2009. Accepted: 01/07/2010.

Darío G. Vileta. Ph.D. student Luciana Bianco. student in Bio- Argentina. Professor, UNRC, Argentina. Address: Ruta Nac. in Biological Sciences, Uni- logical Sciences, UNRC, Ar- Argentina. e-mail: mgrosso@ 36 Km 601, (5800) Río Cuar- versidad Nacional de Río Cu- gentina. e-mail: lbianco@ayv. ayv.unrc.edu.ar to, Córdoba, Argentina. e-mail: arto (UNRC), Argentina. e- unrc.edu.ar Rosana N. Malpassi. Ph.D. in [email protected] mail:[email protected] Mónica A. Grosso. Ph.D. in Biological Sciences, UNRC. Biological Sciences, UNRC. Argentina. Professor, UNRC,

120 0378-1844/10/02/120-06 $ 3.00/0 FEB 2010, VOL. 35 Nº 02 FIXAÇÃO BIOLÓGICA DE NITROGÊNIO DE Adesmia bicolor E A. macrostachya, POTENCIAIS ESPÉCIES FORRAGEIRAS PARA AMBIENTES ÁRIDOS E SEMIÁRIDOS Darío Vileta, Luciana Bianco, Mónica Grosso e Rosana Malpassi RESUMO

Em regiões áridas da América do Sul há escassez de alimento solução nutritiva: “Sem N”; “Com N”; e “Sem N-sementes ino- para o gado durante o inverno. Adesmia bicolor e A. macros- culadas”. Analisaram-se a morfo-anatomia e distribuição es- tachya são prometedoras como forragem por seus hábitos de paço-temporal de nódulos, peso seco e conteúdo de N de caule- crescimento e adaptabilidade a invernos secos e solos pobres folhas, raízes e nódulos. As espécies mostraram uma eficiência em N. É essencial dispor de informação de suas eficiências de de BNF média de 60%, a qual é alta considerando que alfafa fixação biológica de N (BNF). O objetivo do presente trabalho apresenta 61% quando cresce na Argentina central. Este atri- é determinar suas eficiências de BNF, morfo-anatomia e distri- buto é desejável em espécies silvestres com potencial forrageiro buição espaço-temporal dos nódulos em simbioses com rizóbios. em ambientes áridos. Em uma estufa, ambas espécies foram plantadas e regadas com

of N input and it represents carried out with different that A. bicolor has a high N fied Munns nutritive solution a renewable source for ag- Adesmia species and the re- fixation efficiency due to the with N. riculture (Borreani et al., sults are controversial. Bur- high dry matter accumula- Treatment 3. Inoculated 2003; Tabacco et al., 2003; kart (1952) and Date and tion obtained when it grows : Sterile vermiculite as Athar, 2005; Teixeira et al., Halliday (1980) included on sandy soils with low N substrate, seeds inoculated 2006; Armas et al., 2008). them among the ineffective content. Nothing is known with wild rhizobia (each spe- The significance of rhizobia species in response to Rhi- about A. macrostachya. cies had a specific inocu- of wild legumes is based on zobium. Scheffer-Basso et Therefore, it is necessary lant), irrigated with modi- their symbiotic N fixation al. (2000, 2001b) found that to know the quantity of N 2 fied Munns nutritive solution capacity and on other activi- A. araujoi has low BNF ef- that A. bicolor and A. mac- without N. ties in the soil that eventual- ficiency, though it develops rostachya are able to fix in The modified Munns nutri- ly improve fertility and excellent nodulation. Vidor symbiosis with native rhi- tive solution without N con- productivity (Zahran, 2001). and Neto (1992) observed zobia in order to evaluate tained 34g·l‑1 KH PO , 123g·l‑1 Even though most wild that A. tristis produces many their potential use as for- 2 4 MgSO ·7H O, 65g·l‑1 K SO , legumes that grow in cen- nodules also, but they did age species in the arid and 4 2 2 4 0.1g·l‑1 CaSO ·2H O, 1.4g·l‑1 tral Argentina have a spring- not study its BNF efficiency. semiarid regions of South 4 2 FeCl ·6H 0, 1.7g·l‑1 Na H EDTA, summer growth habit, some On the other hand, Scheffer- America. The objective of 3 2 2 2 0.75g·l‑1 KCl, 124mg·l‑1 of them develop in the fall Basso et al. (2001b) deter- this work was to determine H BO , 67mg·l‑1 MnSO ·H O, and fructify in early spring. mined that A. latifolia shows nodule morpho-anatomy, 3 3 4 2 46mg·l‑1 ZnSO ·7H O, 10mg·l‑1 Among the last ones, some an acceptable effectiveness nodule spatial and temporal 4 2 CuSO ·5H O and 2mg·l‑1 Adesmia species should be (37%) compared to plants distribution, dry matter ac- 4 2 H MoO ; whereas the nutri- highlighted because they supplemented with mineral cumulation, and N content 2 4 tive solution with N had the produce nutritive leaves, N after 65 post-germination of A. bicolor and A. mac- same chemical composition fruits and seeds for rumi- days. Golluscio et al. (2006) rostachya in symbiosis with plus 101g·l‑1 KNO and 133g·l‑1 nants (Maestri et al., 2002). observed that A. volckman- Rhizobium native strains. 3 Studies have been carried ni has ineffective nodules (NH4)2SO4 (Weaver and Freder- ick, 1982). out to domesticate many because of leghemoglobin Materials and Methods of them in different coun- absence, although they be- One rhizobia inoculant tries, such as New Zealand lieved that the study should Seeds of Adesmia bicolor specific for each species was (Dodd and Orr, 1995), Bra- continue. and Adesmia macrostachya obtained from nodules pro- zil (Scheffer-Basso et al., There are two species of were collected at the Uni- duced by the corresponding 2000; Tedesco et al., 2000; Adesmia that are particu- versidad Nacional de Río plants growing in situ at the Scheffer-Basso et al., 2001a, larly promising as forage Cuarto, Córdoba, Argentina same location. The nodules b, c; 2002; Barreto Dias et in Argentina, Brazil, and (33º05’S, 64º20’W). They were superficially steril- al., 2004; Dias et al., 2004), New Zealand, A. bicolor and were mechanically scari- ized and macerated. After Uruguay (Coll and Zarza, A. macrostachya, because fied and planted in Leonard that, bacteria were cultured 1992), and Argentina (Bi- of their growth habit and jars (Weaver and Frederick, in yeast extract agar with anco and Kraus, 2005; Vene- high adaptability to arid and 1982). Three treatments were congo red media until puri- ciano et al., 2005). semi-arid conditions (Coll applied: fication. Native rhizobia were counted in order to know One of the most important and Zarza, 1992; Dodd and Treatment 1. Plants with- the number of viable mi- characteristics that should Orr, 1995; Weberling et al., out N: Sterile vermiculite croorganisms present in the be known in a species that 2002; Barreto Dias et al., as substrate, irrigated with inoculant (Vincent, 1970). In is being domesticated or 2004; Bianco and Kraus, modified Munns nutritive introduced in an environ- 2005; Veneciano et al., the greenhouse, each strain solution without N. ment with arid or semi-arid 2005). However, informa- obtained from the isolations conditions is its efficiency tion related to their BNF Treatment 2. Plants with N: was confirmed in its ability to fix N2 in symbiosis with efficiency is scarce. Coll Sterile vermiculite as sub- to develop nodules in the rhizobia. Studies have been and Zarza (1992) inferred strate, irrigated with modi- corresponding host plants.

FEB 2010, VOL. 35 Nº 02 121 Five seeds were planted Adesmia macrostachya was in each Leonard jar. After in vegetative stage at 150 emergence, only one plant days post-germination, in was left. Each pot con- flowering stage at 270 days, stituted one experimental and in fructification stage at unit (EU), so that the to- 360 days post-germination. tal number of EU was 27. These plants were kept at Morpho-anatomical optimal environmental condi- characteristics of nodules tions (constant temperature of 25ºC and photoperiod of Typical mature nodules 12h) in order to determine of A. bicolor and A. mac- the potential BNF efficiency. rostachya were spherical, This exploratory study is to grew alone or in groups, be followed by studies under and had determinate growth. arid and semi-arid conditions The size of all nodules be- in the greenhouse and in the longing to one plant was field. uniform. In nodule cross At 150, 270, and 360 sections, two regions were days after germination, the observed, cortex and cen- aerial parts (leaves, shoots, tral zone (Figure 1). The flowers, and/or fruits) of first could be subdivided in three plants correspond- both species into cortical ing to each treatment were parenchyma, vascular bun- destructively sampled. dles, and a parenchymatous The samples were dried at sheath. The cortex was con- 55°C during seven days, stituted by ten cell layers. and weighed in an analyti- The outer four or five ones cal balance to determine had isodiametric cells with aerial dry matter. At the thin walls and no visible same sampling dates, the nucleus, and they sloughed root systems of the same Figure 1. Nodule cross sections. a: Adesmia bicolor showing cortex and central off during growth (Fig- three plants were also col- zone. The arrow indicates parenchymatic sheath. b: Adesmia macrostachya ures 1a and b). The inner showing the same zones. The arrow indicates parenchymatic sheath with starch. lected. In the samples cor- c: Vascular bundle in the inner nodule cortex of A. bicolor. d: Vascular bundle five or six layers showed responding to Treatment 3 of A. macrostachya. e: Nodule central zone of A. bicolor. The arrow indicates similar characteristics, and (inoculated plants), nodules cells infected with bacteroids. f: Nodule central zone of A. macrostachya. The contained abundant starch. were counted on main and arrow indicates uninfected cells with starch. C:= cortex, CZ:= central zone, Vascular bundles were em- lateral roots. After that, VB: vascular bundle. bedded in the inner cortex the nodules were separat- (Figures 1c and d). ed from the roots, dried and nodule dry matter after The experiment had a com- The nodule central zone at 55°C during seven days, Kjeldahl digestion follow- pletely randomized design was undivided and round in and weighed to obtain root ing the potentiometer meth- with three replications. Re- both species. In A. bicolor and nodule dry matter. The od (HANNA-pH 211 with sults were analyzed by analy- this zone was occupied by + effectiveness of BNF was 0.1 mV sensitivity and NH4 sis of variance (ANOVA) and parenchyma, with isodiamet- calculated with the follow- electrode; Bremner and Mul- Kruskal Wallis nonparametric ric cells, and nucleus usu- ing formula (Gibson, 1980): vaney, 1982). ANOVA, using INFOSTAT ally evident and centrally 2005/P.1 (Universidad Nacio- located. More than 90% of nal de Córdoba, Argentina; them were infected with Steel and Torrie, 1988; Ott, bacteroids (Figure 1e). On 1993). When the analysis of the other hand, the central where BNF Eff.: biologi- Nodules were also sam- variance showed significant zone of A. macrostachya cal N2 fixation effectiveness, pled for anatomical stud- differences among treatments, was also occupied by pa- symbiotic RDM: root dry ies. They were fixed the least significant differ- renchyma, but its cells were matter in inoculated plants in FAA (ethanol:acetic ences test (LSD test) was ap- infected in a lower propor- (Treatment 3), symbiotic acid:formaldehyde, 90:5:5), plied to make comparisons tion than in the other spe- ADM: aerial dry matter in dehydrated in an ethyl alco- among means (Steel and Tor- cies. Non-infected cells were inoculated plants (Treat- hol-xylol series, and embedded rie, 1988; Ott, 1993). isodiametric with thin walls, ment 3), mineral N RDM: in Histowax. Cross sections showed no visible nucle- root dry matter obtained in were cut at 10µm and stained Results us, and contained abundant plants with N (Treatment 2,) with safranin-fast green (Jo- starch granules (Figure 1f). and mineral N ADM: aerial hansen, 1940). Histological Plant growth stages in dry matter in plants with N slides were analyzed and pho- three sampling dates differed Spatio-temporal (Treatment 2). tographed with an Axiophot between species. Adesmia bi- distribution of nodules Nitrogen obtained from Zeiss microscope with Axiovi- color was in vegetative stage biological fixation was de- sion software and an Axiocam during the 360 days that the In both species, the total termined in aerial, root, HRC Zeiss camera. experiment lasted, whereas number of nodules increased

122 FEB 2010, VOL. 35 Nº 02 Table I ules of both species, they number of NODULES ON Adesmia bicolor and A. macrostachya (n= 3) in main, lat- were not statistically signifi- eral, and adventitious roots at three sampling dates cant (at 150 days: p= 0.14, 270 days: p= 0.74, and 360 Nodule number (mean ±SEM) Days after days post-germination: p= germination Adesmia bicolor A. macrostachya 0.64; Figure 2). There were Main root Lateral roots Adventitious Main root Lateral roots no significant differences in roots mean number of nodules on the main or lateral roots be- 150 9.67 ±6.11 a 3.33 ±2.89 a 0.00 46.33 ±25.70 a 51.33 ±54.27 a tween both species or each 270 167.00 ±109.42 a 101.33 ±52.60 a 24.00 ±41.57 a 105.00 ±58.81 a 295.00 ±429.97 a sampling date either (on 360 259.67 ±181.45 ab 354.00 ±170.01 b 45.00 ±56.93 a 244.33 ±145.11 a 333.00 ±124.58 a main root: at 150 days, p= 0.07; at 270 days, 0.44; and Different letters at the same sampling date in each species indicate significant differences among root types according at 360 days, 0.91; on lateral to Fisher LSD test (a>0.05). roots: at 150 days, p= 0.20; at 270 days, 0.50; and at as the plant grew (Figure 167 developed on the 360 days, 0.88). 2). In A. bicolor, the mean main root, 101 on lat- total number of nodules at eral roots, and 24 on ad- Dry matter 270 days post-germination ventitious ones, whereas increased 2248.69% with one year after the ex- Table II shows dr y regard to the previous sam- periment started more matter accumulation in pling date (150 days). Af- nodules were produced aerial, root, and nodule ter 360 days, the increase on lateral roots than on parts of both species. was 234.5% compared to the main root; the total At 150, 270, and 360 the previous one (270 days). number of nodules was days post-germination, From first to last sampling 659, 260 on the main there were no statisti- dates, the total number root, 354 on lateral roots cally significant dif- increased 5273.08%. A. and 45 on adventitious ferences among A. bi- macrostachya behaved in ones. color aerial dry weight Figure 2. Nodule total number at three different a similar way but the in- On the other hand, sampling dates (150, 270 and 360 days post- of the two treatments crease in nodule number nodules of A. mac- germination). Same letters in each sampling date that had plants alive was not so marked, because rostachya developed indicate non significant differences among treat- (plants with N -Treat- the plants developed nodules mainly on lateral roots ments according to Fisher LSD test (a>0,05). ment 2- and inoculated earlier than in A. bicolor from the beginning to plants - Treatment 3; (150 days post-germination). the end of the experi- with p= 0.08, 0.21 and From the first sampling date ment. At 150 days after 0.21, respectively). Fur- (vegetative growth stage) to germination, the total num- eral roots. After one year thermore, at the same sam- the second one (flowering ber of nodules was 98, 47 of experiment, 577 nodules pling dates, root dry matter growth stage), A. macro- developed on the main root were produced, 244 on the showed no significant dif- stachya nodule number in- and 51 on lateral roots. At main root and 333 on lateral ferences between treatments creased 307.19%. From the 270 days after germination, roots. either (p= 0.07, 0.16 and second to the third sampling there were 400 nodules, 105 Even though there were 0.20, respectively). Nod- date (fructification growth of them on the main root some differences between ule dry matter showed sig- stage) it increased 192.44%. and 295 distributed on lat- mean total number of nod- nificant differences among The total increase was 591.16%. Spatial distribu- Table II tion of nodules on Aerial, root, and nodule dry matter accumulation of Adesmia bicolor and the root system dif- A. macrostachya (n= 3) at three different sampling dates fered between spe- Days after Treatment Adesmia bicolor (g) A. macrostachya (g) germination cies. At the first Aerial Root Nodule Aerial Root Nodule two sampling dates, most A. bicolor Plants without N 0.005 ±0.002 a 0.005 ±0.001 b 0.000 0.010 ±0.002 a 0.008 ±0.002 a 0.000 nodules developed 150 Plants with N 0.050 ±0.040 b 0.011 ±0.004 ab 0.000 0.200 ±0.240 a 0.090 ±0.120 a 0.000 on the main root Inoculated plants 0.020 ±0.010 ab 0.008 ±0.001 a 0.002 0.130 ±0.090 a 0.030 ±0.020 a 0.020 (Table I). At 150 Plants without N* ------days after germina- Plants with N 8.350 ±0.792 a 0.540 ±0.390 a 0.000 5.640 ±1.690 b 1.120 ±0.280 b 0.000 tion, nodule total 270 Inoculated plants 1.550 0.98 a 0.130 0.120 a 0.200 0.340 0.080 a 0.080 0.020 a 0.080 number was 13, 10 ± ± ± ± on the main root Plants without N ------and 3 on lateral 360 Plants with N 7.690 ±3.120 a 1.750 ±1.640 a 0.000 9.050 ±5.360 a 2.800 ±1.300 a 0.000 roots. At 270 days Inoculated plants 4.970 ±0.78 a 0.290 ±0.010 a 0.340 1.870 ±0.740 a 0.360 ±0.210 a 0.330 post-ger m ination, Different letters in the same column and same sampling date indicate significant differences among treatments according to Fisher the total number of LSD test (a >0,05). nodules was 292, * This treatment does not have data at 270 and 360 days post-germination because plants without N die due to its absence.

FEB 2010, VOL. 35 Nº 02 123 sampling dates (p = Table III 0.002). Plants belong- Aerial, root, and nodule N content of Adesmia bicolor and A. macrostachya ing to plants without (n= 3) at three different sampling dates N -Treatment 1- died Days after Treatment Adesmia bicolor A. macrostachya because of N absence. germination (g. 100 g sample‑1) (g. 100 g sample‑1) Aerial and root dry matter of A. macro- Aerial Root Nodule Aerial Root Nodule stachya showed no 150 Plants without N ND ND ND ND ND ND significant differences Plants with N ND ND ND ND ND ND among treatments at 150 and 360 days post- Inoculated plants ND ND ND ND ND ND germination (aerial: p= 270 Plants without N* ------0.36 and 0.08, root: p= Plants with N 2.23 ±0.24 b 1.08 ±0.61 a 0.00 1.50 ±0.26 b 1.99 ±0.20 b 0.00 0.37 and 0.07, respec- Inoculated plants 1.23 ±0.49 a 0.79 ±0.46 a 2.40 ±1.43 0.68 ±0.37 a 1.11 ±0.33 a 2.30 ±0.16 tively), but they were 360 Plants without N ------significantly different Plants with N 2.57 ±0.21 a 1.78 ±0.14 a 0.00 1.86 ±0.26 a 1.29 ±0.05 a 0.00 at 270 days after ger- Inoculated plants 2.51 ±0.15 a 1.47 ±0.20 a 2.41 ±0.09 1.73 ±0.54 a 1.31 ±0.24 a 2.15 ±0.48 mination (aerial: p= Different letters in the same column and same sampling date indicate significant differences among treatments according to 0.006, root: p= 0.004). Fisher LSD test (a>0.05). There were significant * This treatment does not have data at 270 and 360 days post-germination because “Plants without nitrogen” die due to N differences in nodule absence. dry matter also among sampling dates in this spe- corresponding to both spe- 0.19 and 0.07, root: p=0.38 On the other hand, A. mac- cies (p=0.01). These dif- cies increased all along the and 0.39, nodule: p=0.80 and rostachya can be considered ferences arose at 360 days year of the experiment (Ta- 0.40, respectively). as an intermediate case as it post-germination, as the ble III). At 150 days post- had ~50% of its nodules on first two sampling dates germination, it could not be Discussion lateral roots from the start were not significantly dif- determined because the sam- of the experiment. ferent. ple size was too small and Even though there is a Even though leguminous A. bicolor effective- the technique was not sen- high degree of variability species that acquire N only ness of BNF was 40.32% sible enough. At 270 days, in the way nodule mor- through symbiosis usually de- at 150 days, 23% at 270, A. bicolor Inoculated plants phology and anatomy of crease their growth rates due and 59.19% at 360 days af- -Treatment 3- aerial dry mat- Adesmia are described in to morphological alterations ter germination (vegetative ter showed 52% of N and A. the literature (Rothschild, (Cassman et al., 1980), A. stage), whereas A. macro- macrostachya ones 45,3% 1967; Scheffer-Basso et al., bicolor and A. macrostachya stachya effectiveness was with regard to plants with N 2000), this study estab- still maintained a relatively 62% at 150 days (vegeta- -Treatment 2- aerial dry mat- lished that A. bicolor and high biomass accumulation tive stage), 7.4% at 270 days ter. These differences were A. macrostachya develop during the vegetative stage. (flowering stage), and 21.6% statistically significant (p= determinate nodules and, in Furthermore, these species at 360 days post-germination 0.03 for both species). How- cross section, they are only can be considered as highly (fructification stage). ever, at 360 days, there were divided in two regions: cor- effective to fix N in sym- Comparing only inoculat- no significant differences tex and central zone. The biosis with rhizobia. The ana- ed plants -Treatment 3- from between the treatments that novel finding was that A. lyzed species showed a mean both species, it could be have plants alive: A. bicolor macrostachya accumulates BNF efficiency of ~60% at observed that aerial, root, inoculated plants -Treatment abundant starch in inner vegetative stage, which is a and nodule dry matter at the 3- showed 97% of N con- cortical cells and central relatively high efficiency con- first two sampling dates did tent and A. macrostachya zone, whereas A. bicolor sidering that alfalfa shows not show significant differ- inoculated plants -Treatment only accumulates it in the 61% when it grows in central ences (150 days after germi- 3- showed 93% of N con- inner cortex. This charac- Argentina (Perticari, 2001). nation: aerial p= 0.25, root tent in the aerial dry mass in teristic was not found in According to Vance (2002), p= 0.10, nodule dry weight comparison to plants with N other Adesmia species ana- alfalfa is one of the most ef- p= 0.10; 270 days post-ger- -Treatment 2- (p= 0.69 and lyzed by Rothschild (1967). ficient forage legume. Among mination: aerial p= 0.10, 0.72, respectively). Root N Most nodules of A. bi- all Adesmia species studied root p= 0.65, and nodule content behaved in a similar color were found on the so far (Burkart, 1952; Coll dry matter p= 0.70). How- way, except at 270 days post- main root. Therefore, this and Zarza, 1992; Scheffer- ever, at 360 days aerial dry germination, when A. bicolor species resembles A. cap- Basso et al., 2000, 2001a), A. matter showed significant did not show significant dif- itellata, where almost all bicolor and A. macrostachya differences between the spe- ferences (p= 0.54). nodules developed on the would be the most efficient cies (p= 0.01), but root (p= Nitrogen content of in- main root, whereas other ones. This attribute is highly 0.50) or nodule dry matter oculated plants -Treatment species, like A. araujoi, de- desirable in wild species with did not (p= 0.90). 3- was similar in A. bicolor veloped nodules only on lat- forage potential in environ- and A. macrostachya as there eral roots (Scheffer Basso et ments under arid and semi- Nitrogen content were no significant differenc- al., 2000). At the end of the arid conditions. es in aerial, root or nodule N vegetative stage, nodules of On the other hand, during Nitrogen content in aerial, content at 270 and 360 days A. bicolor started to develop flowering and fructification, A. root, and nodule dry matter post-germination (aerial: p= on first degree lateral roots. macrostachya dry matter ac-

124 FEB 2010, VOL. 35 Nº 02 cumulation decreased because Borreani G, Tabacco E, Grignani EK, Sprent JI, Newton WE Steel RGD, Torrie JH (1988) flower and fruit development C (2003) Quantificazione (Eds.) Nitrogen-fixing Legumi- Bioestadística: Principios y dell’azotofissazione nelle legu- nous Symbioses. Springer. Dor- Procedimientos. 2nd ed. Mc- and ripening demanded an minose foraggere. Riv. Agron. drecht, Netherlands. pp. 363-393. Graw-Hill. México. 622 pp. important quantity of N. In 37: 21-31. Johansen DA (1940) Plant Micro- Tabacco E, Borreani G, Grignani order to reach growth rates Bremner JM, Mulvaney CS (1982) technique. McGraw-Hill. New C (2003) Azotofissazione close to potential ones, indi- Nitrogen-Total. En Page AL, York, USA. 523 pp. dell’erba medica e del trifoglio viduals should stay in vegeta- Miller RH, Keeney DR (Eds.) Johnson DA, Rumbaugh MD, Asay pratense stimata con il metodo tive stage or, otherwise, satis- Methods of Soil Analysis. Part KH (1981) Plant improvement della diluizione dell’isotopo 15N 2. Chemical and Microbiologi- nella Pianura Padana occiden- fy their N requirements from nd for semi-arid range lands: pos- cal Properties. 2 ed. Ameri- sibilities for drought resistance tale. Riv. Agron. 37: 93-97. the soil, although this latter can Society of Agronomy/Soil and nitrogen fixation. Plant Tedesco SB, Dall’Agnol M, Schifi- situation is hard to achieve in Science Society of America. Soil 58: 279-303. a N poor-environment. Madison, WI, USA. pp. 595- no-Wittmann MT, Valls JFM 624. Maestri DM, Fortunato RH, (2000) Mode of reproduction In conclusion, during the Guzmán CA, Torres MM, of Brazilian species of Ad- Burkart A (1952) Las Leguminosas vegetative stage, A. bicolor Lamarque AL (2002) Seed esmia (Leguminosae). Gen. Argentinas Silvestres y Culti- compositional studies of some Molec. Biol. 23: 475-478. and A. macrostachya fix- vadas. 2nd ed. ACM. Buenos species of Papilionoideae (Le- ing N in symbiosis with na- Aires, Argentina. 569 pp. Teixeira FCP, Reinert F, Rumjanek guminosae) native to Argen- tive rhizobia obtain enough Cassman KG, Whitney AS, Stock- NG, Boddey RM (2006) Quan- tina. J. Sci. Food Agric. 82: tification of the contribution of amounts of this nutrient in inger KR (1980) Root growth 248-251. and dry matter distribution of biological nitrogen fixation to order to grow and develop Ott RL (1993) An Introduction to 15 soybean as affected by phos- Cratylia mollis using the N in a similar way as do plants Statistical Methods and Data phorus stress, nodulation and natural abundance technique in Analysis. Duxbury. Pacific supplemented with mineral nitrogen source. Crop Sci. 20: the semi-arid Caatinga region Grove, CA, USA. 1051 pp. N. Therefore, these species 239-244. of Brazil. Soil Biol. Biochem. 38: 1989-1993. could be considered as highly Coll J, Zarza A (1992) Legumi- Perticari A (2001) Pasturas de beneficial in arid and semiarid nosas Nativas Promisorias: alfalfa: importancia de una Vance CP (2002) Root-bacteria adecuada inoculación. IMYZA- interactions: symbiotic N environments, because of their Trébol Polimorfo y Babositas. 2 CICV / A-INTA Castelar: 1-5. fixation. En Waisel Y, Eshel potential input of N and or- Instituto Nacional de Investiga- ciones Agropecuarias. Montevi- Rothschild DI (1967) Anatomía del A, Kafkafi U (Eds.) Plant ganic matter to the ecosystem deo, Uruguay. 19 pp. nódulo radical de las legumi- Roots. The Hidden Half. 3rd during winter. As results are nosas cultivadas. Rev. Inst. ed. Dekker. New York, USA. Date RA, Halliday J (1980). Rela- pp. 839-868. encouraging, this study should tionships between Rhizobium Munic. Bot. Jard. Bot. “Carlos continue with experiments in and tropical forage legumes. Thays” 3: 1-32. Veneciano JH, Frasinelli CA, Kraus the field and in a greenhouse En Summerfield RJ, Bunting Scheffer-Basso SM, Carneiro CM, TA, Bianco CA (2005) Do- under dry conditions. AH (Eds.) Advances in Le- Voss M (2000) Nodulaçâo e mesticación de Especies For- gume Science. University of fijaçâo biológica de nitrogênio rajeras. Universidad Nacional Reading, Kew, UK. pp. 597- em Adesmia araujoi Burk. Rev. de Río Cuarto. Córdoba, Ar- Acknowledgements 601. Bras. Zootec. 6: 16-18. gentina. 60 pp. Dias PM, Schifino-Wittmann MT, Scheffer-Basso SM, Ávila Jacques Vidor MA, Neto JS (1992) Lages The authors thank María Dall’Agnol M (2004) Adesmia AV, Dall’Agnol M, Riboldi J, preserva espécies vegetais for- Cristina Más for laboratory DC: Estado Atual do conheci- Jesuz Castro SM (2001a) Dis- rageiras. Agropec. Catar. 2: assistance and Susan Vilor mento e perspectivas de uso de ponibilidade e valor nutritivo 13-15. uma forrageira nativa de alta de forragem de Legumino- for English revision. qualidade. Rev. Cient. Rural sas nativas (Adesmia DC.) e Vincent JM (1970) A Manual for 9: 60-71. exóticas (Lotus L.). Rev. Bras. the Practical Study of the References Zootec. 30: 975-982. Root-nodule Bacteria. IBP Döbereiner J (1997) Biological ni- Handbook Nº 15. Blackwell. trogen fixation in the tropics: Scheffer-Basso SM, Vendrus- Oxford. UK. 200 pp. Armas C, Pugnaire FI, Sala OE Social and economic contribu- culo MC, Baréa R, Benincá (2008) Patch structure dynam- tions. Soil Biol. Biochem. 29: C, Lubenow R, Cecchetti C Weaver RW, Frederick LR (1982) ics and mechanisms of cycli- 771-774. (2001b) Comportamiento de Rhizobium. En Page AL, Miller RH, Keeney DR (Eds.) cal succession in a Patagonian Dodd MB, Orr SJ (1995) Seasonal Leguminosas (Adesmia, Lotus, Methods of Soil Analysis. Part steppe (Argentina). J. Arid growth, phosphate response, Trifolium) em mistura com 2. Chemical and Microbiologi- Env. 72: 1552-1561. and drought tolerance of 11 pe- festuca. Rev. Bras. Zootec. 31: cal Properties. 2nd ed. Amer- Athar M (2005) Nodulation of rennial legume species grown 2197-2203. ican Society of Agronomy/ native legumes in Pakistani in a hill-country soil. New Scheffer-Basso SM, Voss M, Ávila Soil Science Society of Amer- rangelands. Agric. Consp. Sci- Zeal. J. Agric. Res. 38: 7-20. Jacques AV (2001c) Nodula- ica. Madison, WI, EEUU. pp. ent. 70: 49-54. Gibson AH (1980) Methods for le- çâo e fixaçâo biológica de ni- 1043-1070. Barreto Dias PM, Dall’Agnol M, gumes in glasshouses and con- trogênio de Adesmia latifolia Schifino-Wittmann MT (2004) trolled environment cabinets. e Lotus corniculatus em vasos Weberling F, Kraus T, Bianco C, Genetic diversity in the Bra- En Bergensen FL (Ed.) Meth- de Leonard. Rev. Bras. Zootec. Malpassi R (2002) Variación zilian species of Adesmia DC ods for Evaluating Biological 30: 1-12. y estrategias adaptativas de los sistemas de ramificación (Leguminosae) as assessed by Nitrogen Fixation. CSIRO/ Scheffer-Basso SM, Ávila Jacques RAPD. Plant Gen. Resour. 2: Willey. Canberra, Australia. de Fabáceas herbáceas. Feddes AV, Dall Agnol M (2002) Repertorium 113: 342-353. 43-50. pp. 139-184. Alocâção da biomassa e cor- Bianco CA (2002) Growth Forms, Golluscio R, Faigón A, Tanke M relaçôes morfofisiológica em Zahran HH (1999) Rhizobium- , Distribution, and (2006) Spatial distribution of leguminosas forrageiras com legume symbioses and nitrogen Uses of Adesmia Species (Le- roots and nodules, and δ15N hábitos de crescimento con- fixation under severe condi- guminosae) in Central Argen- evidence of nitrogen fixation in trastantes. Rev. Sci. Agríc. 59: tions and in an arid climate. tina. Cramer. Stuttgart, Ger- Adesmia volkmanni: a Patago- 629-634. Microbiol. Molec. Biol. Rev. many. 156 pp. nian leguminous shrub. J. Arid Speroni G, Izaguirre P (2003) Car- 70: 968-989. Bianco CA, Kraus TA (2005) De- Env. 67: 328-335. acterísticas biológicas de la Zahran HH (2001) Rhizobia from sarrollo y estructura de la se- Howieson JG, Yates RJ, Foster KJ, leguminosa nativa promisoria wild legumes: diversity, taxon- milla y el fruto de Adesmia Real D, Besier RB (2008)Pros- forrajera Trifolium polymor- omy, ecology, nitrogen fixation bicolor (Poir.) DC. (). pects for the future use of le- phum Poir. (Fabaceae, Faboi- and biotechnology. J. Biotech- Φyton: 71-77. gumes. En Dilworth MJ, James deae). Agrociencia 7: 68-76. nol. 91: 143-153.

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