... determined by optical density at 600nm. RHIOO RH44 A6. AlO CM2. 1003 ANU843. 7·5. 5. 7·5. 7-5 >10. 7·5. 7-5. 10. 5. 10. 5. 1. 10. 10. SO. >50. >50 >50 >50.
Journal of Applied Microbiology 1998.84.81-89
Metabolic properties, stress tolerance and macromolecular profiles of rhizobia nodulating Hedysarum coronarium P. Struffi, V. Corich, A. Giacomini, A. 8enguedouar, A. Squartlnl, S. Casella and M.P. Nutl Dipartimento di Biotecnologie Agrarie, Universita di Padova, Agripolis, Legnaro (Padova), Italy 6032112196: received 9 December 1996. revised 25 April 1997 and accepted 29 April 1997
P. STRUFFI. V. CORICH. A. GIACOMINI, A. BENGUEDOUAR. A. SaUARTINI, S. CASELLA AND M.P. NUTI. 1998. The drought-tolerant legume lIedysarum
coronarium is a Mediterranean species valued as a forage crop for its high performance in stressful conditions. The plant shows peculiar capabilities of nodulating above pH 9 and thriving in highly calcareous soils. With the aim of providing an adequate characterization of its bacterial symbiotic partner, a study was undertaken, approaching from several viewpoints the physiology and structural features of bacteria isolated from nodules of JJ. coronarium. Tests involved trophic capabilities on different carbon and nitrogen sources, vitamin requirements, and resistance to factors including antibiotics, heavy metals, salinity, pH, and temperature. Enzyme activities, including those of cellulase, pectinase, urease, fJ-galactosidase, nitrate and nitrite reductase, were evaluated. The DNA G + C percentage content was determined. Species-specific bacteriophages were isolated and a strain-typing grid established. In order to characterize further and fingerprint the different Rhizobium 'hedysari' isolates, electrophoretic pattern of proteins, plasmid DNA, and digested genomic DNA (in pulsed-field gel separation) were compared. Adansonian taxonomy yielded similarity clusters of the different isolates.
INTRODUCTION
The perennial legume, lIedysarum coronarium L. (sulla, zulla, Spanish sainfoin, Spanish esparcet, French honeysuckle, halmenthopf), is widely used as a forage crop in the Mediterranean basin because of its drought resistance, alkaline soil tolerance (Lupi et aI. 1988), and good agronomical traits both in terms of yield, up to 60 tons of green forage ha- I yr- t, and protein content, up to 24% of leaf dry weight (Dalla tore 1963; Sarno and Stringi 1981). The plant has been successfully introduced in marginal areas including highly calcareous and semi-arid soils (Nuti and Casella 1989). Nitrogen-fixing bacteria nodulating this plant have been isolated and referred to as Rhizobium 'hedysari' (Cabrera and Ruiz Argtieso 1979). Strains have been characterized as fast growing and highly host-specific for II. coronarium; no crossnodulation was observed with Trifolium repms, T. subterraneum, Pisum sativum, Vida Jaba, Glycine max, Vigna unguiculata and Cjcer arietinum (Cabrera and Ruiz Argoeso Correspondence to: Dr Andrea Squart;",', Dipartimenlo di B;olecnolog;e Agrarie. Un;versila di Padova. Agripolis. Strada ROlllea /6, 35020 Legnaro (Pad()va). Italy (e-Illail: squart@agripo!is.unipd.it). C 1998 The Society for Applied Microbiology
1979}. Casella et al. (1984) reported that strains specific for II. coronar;um could only sporadically nodulate Onohrychis t'idaeJalia. Symbiotic incomp;ltibility between two species of IIedysarum, namely II. cor01l0r;UII/ and II.flexllosum, has been found in Morocco; strains nodulating one species can only form ineffective nodules on the other (Glatzle tt al. 1986). Unlike several different legume species, II. coronarimn cannot be nodulated by the bro,ld host range Rhizobium strain NGR234 (\V.]. Broughton, personal communication). Symbiotic genes are carried on large plasmids in Rh. 'hrdysar;' (Espuny et al. 1987). The bacterial infection process, occurring through infection threads in deformed root hairs and generating indeterminate nodules with cross-shaped baeteroids, has been previously reported (Squartini tt al. 1993). In addition, the surface glycoeonjugates of Rh. 'hedysarj' (Orgambide et al. 1996) were studied, different isolates of Rh. 'lIrdysar;' were characterized by DNA fingerprinting (Se\enska-PobeU el al. 19%). and a genetic analysis was carried out by molecular cloning and sequencing of nod genes (Pasti el al. 1993; Meneghetti tf al. 1996). The remarkable features of the host plant which enable it to thrive and be nodulated in saline soils, and at a pH as high as 9·3 (Lupi tt
82 P. STRUFFI ET AL.
al. 1988) at which other rhizobial symbioses cannot occur, have prompted efforts towards an understanding of the two partners' peculiarities. With respect to the plant, its capability of modifying the pH of its root environment, starting from values at which the growth of other legumes is prevented (Henriques-Saba et al. 1996), was recently reported. With respect to the bacteria, the present work is providing an analysis, from physiological and metabolic standpoints, of a number of strains of R. 'hedysari', isolated from nodules of N. coronarium from different locations throughout the Mediterranean basin. This paper complements parallel studies characterizing these bacteria by l6S rRNA sequencing (Se1enska et al., manuscript in preparation), whose results indicate that strains of R. 'hedysari' cluster independently within the alpha proteobacteria in close proximity to Rh. leguminosarum and Rh. elli.
Utilization of carbon sources
Bacteria were streaked on DIll plates containing 0'1% of different sugars or salts as sole carbon source (Tabl~ 2). Growth was compared to that obtained on the manmtolbased minimal medium.
METHODS
Vitamin requirements
Vitamin-free Bill liquid medium was supplemented with IOOng ml- I of thiamin or calcium pantothenate, both separately and in combination. Growth was compared to that attained in regular Dm. Utilization of nitrogen sources
Defined medium 8 without sodium glutamate was supplemented with different amino acids (Table 2). Bacteria were streaked on plates and growth was compared to that on regular defined medium 8.
Bacterial strains and growth conditions
3-ketolactose assay
Strains are listed in Table 1. Bacteria were routinely grown at 28 °C in BIn minimal medium (Dazzo 1984), or in defined medium 8 (Lindstrt>m and Lehtomaki 1988), modified according to the specific test as described below. TY medium (Deringer 1974) was used as an alternative where indicated. YS broth (Vincent 1970) was used for bacteriophage sensitivity tests. Media were solidified where required by the addition of 18 g 1-1 agar. Plates were routinely incubated for at least
The test was performed as described by Dernaerts and DeLey (1963).
5 d.
Hydrolysis of urea, salt tolerance, preCipitation of calcium glycerophosphate, reduction of nitrate, reaction in litmus milk
These tests were performed as decsribed by Lindstrom and Lehtomaki (1988).
Table 1 Dacterial strains
Strain
Geographical origin
Rhizobium 'hedsari' CC133S IS 123
J. Brockwell, Canberra Southern Spain Cadiz, Southern Spain F.J. Ollcro, Seville
llCNT-l (ATCC 43676) RIIF IUIl9
RlIlOO lUH4
A6 AlO Rh. leguminosarum bv. viciae 1003 Rh. leguminosarum bv. trifolii 843 SinIJrhizobium melilol; CM2 Agrobatlerium lume/aciens LDA202 Escherichia coli DMS 2840
Source
S. Casella, Pisa S. Casella, Pisa S. Casella, Pisa A. Toffanin, Madrid A. Toffanin, Madrid A. Denguedouar, Constantine
Volterra, Italy
Pisa, Italy Sicily, Italy Dalearic Islands Southern Spain Eastern Algeria Eastern Algeria
A. Dcnguedouar, Constantine Rothamstead Collection D.J. Rolfe, Canberra F. O'Gara, Cork P.].J. Hooykaas, Leiden K. Lindstrom, Helsinki ~
1998 The Society for Applied Microbiology, Journal of Applied Microbiology 84,81-89
CHARACTERIZATION OF RH. 'HEOYSARI' 83
Table 2 Physio-metabolic properties and stress tolerance of Rhizobium strains
Strain
CC1335 IS123
Utilization of carbon sources D( + )arabinose Sodium citrate D( - )fructose + D( + )galactose + Sodium glutamate Lactose + Maltose + Mannitol + Raffinose + Rhamnose + D( + )saccharose D( + )glucose Sorbitol D( + )xylose Utilization of nitrogen sources Valine + Sodium glutamate + Tyrosine + Leucine + Proline + Threonine Isoleucine + Phenylalanine + Tryptophan + Lysine + Glycine ± Serine + Histidine + Arginine + Methionine + Alanine ± Asparagine + Cysteine + Litmus milk test + NaCI tolerance 1% Hydrolysis of urea + Calcotluor binding Maximum growth T 37
HCNTI RHF
RHl9
RlllOO
RIH4
A6
AIO
CM2
1003
ANU843
+
+
+
+
+
+
+
+ +
+ +
+ +
+ + + + + + + + + +
+ + + + + + + + + +
+ +
+
+ + + + + + + + +
+ + + + +
+ + + + +
+ + + + +
+ + + +
+ + + +
+
+ + + + + +
+
+ + + + + + + + + + + +
+
+
+
+
+
+
+
+ + + + + +
+ + +
+ +
+
+ +
+ + + +
+
+
+
+
+ +
+ + +
+ + +
+ + + + + +
±
± +
+ +
± +
+ +
+ +
+ + +
+ + +
+ +
+ + +
+
+
+
+
+ + + +
±
+ + + +
+ + +
±
+
+ + + +
+ + +
+
±
+ + + +
+ + + +
+ + + +
+ + + +
+ + + +
+
+
+
+ +
+ + + + + + +
±
± ±
+
±
± +
±
+
0·5%
+ +
+ +
+ + +
+
+ + + + + +
+ + +
+
±
+
±
+ + + + +
± ±
+ + +
±
± +
±
±
±
+
+
± ± ±
+ + +
± ±
+
+ + +
+
+ +
+
+ +
+
±
+
+ +
+
+ + + + +
+
+
± ±
+ + + +
±
+
±
+ + + +
+
+
+
±
+
0·5%
0·5%
0·5%
+
1%
1%
+(acic.l) + 3% 0'5%
+
+ +
+
+
+
+
+
37
32
32
34
39
32
5 5 5
