Introduction.
The Salado River Basin is an extensive area in Buenos Aires
province (Argentina). Soils in this area are characterized by
a high salinity, being poorly drained and periodically exposed
to waterlogged conditions. The region is mainly utilized for beef
and dairy cattle production, with principal feed for these purposes
represented by native grassland and naturalized Lotus glaber
(Montes 1987). Lotus is a legume associated, in a symbiotic
way, with bacteria belonging to the genus Rhizobium loti,
thus producing root nodules where the nitrogen fixation process
occurs. R. loti strains commonly found in soils of the
Salado River Basin are capable of forming nodules quite specifically
with L. glaber (Estrella et al. 1997).
However, the characterization of native R. loti strains
was not performed and data concerning the potential symbiotic
capacity of these strains are not currently available.
Rhizobium loti-legume host association shows some characteristics which are different from those exhibited by other nitrogen fixation systems. Rhizobia nodulating Lotus species include both fast-and slow-growing strains (Irisarri et al. 1996; Jordan et al. 1984), and those strains of R. loti so far examined exhibit symbiotic promiscuity as they establish N2-fixing association not only with Lotus spp., but also with a variety of other legumes (Sullivant et al. 1996).
The purpose of this work was to obtain information about native
R. loti population found in Chascomús, a representative
area of the Salado River Basin region, to determine the symbiotic
potential of different isolate strains and to characterize them
by establishing a comparison with other isolates from the region
and with collection strains. The long range objective of our work
is to develop technological tools to obtain good forage species
and the corresponding Rhizobium strain to optimize productivity
and economic efficiency in the zone.
Material and Methods
Organisms and maintenance of cultures
Strains of Rhizobium were originally isolated from
nodules of L. glaber plants naturally growing in: i) the
outskirts of Chascomús city, strains 1-INTECh, 2-INTECh,
3-INTECh, 4-INTECh; ii) two different sites of the Salado River
Basin area, strains Pirán (from Pirán) and Ayac
(from Ayacucho); and iii) reference strains of Rhizobium Culture
Collection (kindly gifted by Dr. Esperanza Martinez-Romero, Centro
de Investigación sobre Fijación de Nitrógeno,
UNAM, Cuernavaca, Mexico). All bacteria were cultured in YEM medium
(Vincent 1970), at 28 ºC with rotator shaking.
Generation times and acid production
Doubling times were calculated from the exponential growth
phase of cultures according to Martínez de Drets et
al. (1974), by reading optical density (A= 620 nm) every 1
h. Native isolates and reference strains were examined for acid
or alkaline production after growth for 2 days (fast-growing isolates)
or 6 days (slow-growing isolates) as indicated earlier (Monza
et al. 1992).
Carbon source utilization
For determination of carbon source utilization, cultured cells
were collected by 4 min centrifugation at 8000 rmp (4ºC),
washed twice with PBS buffer (20 mM sodium phosphate, pH 7.0,
150 mM NaCl and 3 mM KCl) and finally resuspended in the mineral
medium (MM) described by Vincent (1970), containing appropriate
carbon source at a final concentration of (5 mg/ml). Utilization
of monosaccharides (D-glucose, D-galactose), polyols (glycerol
and mannitol), disaccharides (raphinose and sucrose), carboxylic
acids (succinate, citrate) and aromatic compounds (ferulic acid
and cumaric acid) was determined as described by Arias et al.
(1979). When aromatic compounds were used as carbon sources, stock
solutions were prepared in ethanol and added to the mineral medium
at a final concentration of 20 mM.
Results and discussion.
The generation time of 6 native isolates from nodulated L.
glaber are presented in Table 1. Rhizobium loti NZP2227
and Bradyrhizobium loti NZP 2309 were used as
reference strains for fast and slow-growing rhizobia, respectively.
R. loti strain used as commercial inoculant for Lotus
spp. was also assayed for comparative studies. As shown, the reference
strain NZP2227 and two of the isolates (1-INTECh and 4-INTECh)
exhibited fast growth rates, ranging from 2.4 to 5.4 h. The isolates
2-INTECh and 3-INTECh showed intermediate growth rates of 8.3
and 7.2, respectively. The remaining isolates from the region
(Ayac and Pirán) and the commercial R. loti showed
growth rates ranging from 10.5 to 11.9 h, and thus included with
the slow-growing reference strain NZP2309 (16.3 h) (Table 1).
Table 1. Generation time and carbon utilization for the different
rhizobia isolates from Chascomús and the Salado River Basin
as well as for reference strains and commercial R. loti
utilized for a comparison. Abbreviations are: Glc, glucose; Gal,
galactose; Gly, glycerol; Man, mannitol; Suc, sucrose; Rap, raphinose;
Citr, citrate; Succ, succinate; Fer, ferulic acid; Cum, cumaric
acid.
Growth (+); no growth (-); nd, not determined.
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On the other hand, only the strain NZP2227 and the isolates 1-INTECh and 4-INTECh produced an acid reaction in YEM medium (data not shown). This is consistent with previous results indicating that fast-growing R. loti strains are able to acidify the growth medium (Monza et al. 1992). All other isolates, as well as the slow-growing reference strain NZP2309 did not produce any significant change in pH the medium (data not shown).
As also shown in Table 1, no differences in the utilization of monosaccharides were observed among isolates. All of them grew well with either D-glucose or D-galactose, as the only carbon source. Similarly, all rhizobial cells tested were able to grow on mineral medium with either glycerol or mannitol (Table 1). It has been shown that fast- and slow-growing species of the Rhizobiaceae can use a variety of disaccharides, being the utilization of this carbon source a possible criterion to distinguish between Rhizobium and Bradyrhizobium strains (Jordan et al. 1984; Martinez de Drets et al. 1974). In our hands, all the isolates utilized both, sucrose and raphinose; except for the strain Ayac, which behaved as the reference Bradyrhizobium NZP2309 according to this criterion (Table 1).
Among carboxylic acids, succinate allowed growing of all the isolates, while citrate supported growing of slow-growing but not of the fast-growing strains (Table 1). It is interesting to note that the slow-growers strains Pirán and Ayac and the intermediate-grower 2-INTECh utilized cumaric acid, thus exhibiting a similar behaviour than the strain Bradyrhizobium loti NZP2309 (Table 1). This is consistent with other reports indicating that the ability to use aromatic compounds is widespread among slow-growing species of the Rhizobiaceae (Stower et al. 1995; Irisarri et al. 1996).
Our results, based on plant specificity, doubling time, carbon
source utilization and acid production indicate that native isolates
from the Chascomús area are predominantly intermediate
and fast-growers, in difference with strains from other zones
of the Salado River Basin region and the commercial R. loti
utilized as inoculant, which are slow-growers. Although more information
about the native isolates is required, preliminary results indicate
that from them and from new isolates it is possible to obtain
adequate strains to improve N2-fixation in Lotus
cultivars in the region.
Acknowledgments.
This work was supported, in part, by grants from the Comisión
de Investigaciones Científicas (CIC, Buenos Aires). MMF
and AAI are research career members from Consejo Nacional de Investigaciones
Científicas y Técnicas (CONICET, Argentina).
References.
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