Differentiation of rhizobia isolated from native legume trees in Uruguay

Applied Soil Ecology 16 (2001) 275–282

Differentiation of rhizobia isolated from native legume trees in Uruguay L. Frioni∗ , A. Rodr´ıguez, M. Meerhoff Microbiology, Facultad de Agronom´ıa, Av. Garzón 780, Montevideo, Uruguay Received 25 January 2000; received in revised form 27 July 2000; accepted 23 September 2000

Abstract Legume trees are symbiotically associated with rhizobia and mycorrhizal fungi, microorganisms that improve their growth. The objective of this work was to characterize 61 rhizobial isolates from eight species of native legume trees: Acacia caven, Inga urugüensis, Lonchocarpus nitidus, Prosopis nigra, Sesbania virgata, Peltophorum dubium, Enterolobium contortisiliquum and Erythrina crista-galli. The strains were isolated from nodules with high nitrogenase activity and their growth rate, antibiotic, salinity and acidity resistances were determined. Their relationships were analyzed building a matrix with the resistance characteristics. Most of the isolates were fast growers and acid-producing with high level of exopolysaccharides. In general, isolates were erythromycin resistant but sensitive to rifampicin. All the isolates grew well at pH 5.5 while 75% did so at pH 4.4. More than 60% of the isolates grew in 2% of NaCl but this declined to 21% of the isolates in 3% NaCl. This population showed high antibiotic, salinity and pH resistance, suggesting adaptability to major ecological environment stresses, and great saprohytic competence within soil environments. Isolates from the same host showed high homology between them. © 2001 Elsevier Science B.V. All rights reserved. Keywords: Antibiotic resistance; Acidity and salinity resistances; Rhizobia from Uruguay

1. Introduction Legumes are the third largest family of angiosperm plants, including 17 000–19 000 species distributed world-wide with nearly 3000 species identified as potential N2 fixers (Dommergues, 1995). The woody species have expanded out from their tropical origin and in the last 300 years there was an introduction of legume trees and shrubs to arid, semi-arid and temperate zones (Archer, 1994). These N2 -fixing trees (NFTs) are also the major source of N in tropical ecosystems: sequential cropping systems, agro∗ Corresponding author. Fax: +598-2-3093004. E-mail address: [email protected] (L. Frioni).

forestry and silvopastoril systems, providing timber, fuel, pulp, fodder and even human food. Excellent reviews and books have been published discussing the establishment and functioning of NFTs symbiosis (Brewbaker, 1990; Dommergues, 1995; Dommergues et al., 1999; Subba Rao and Rodr´ıguez-Barrueco, 1993; Faria et al., 1989). Many papers have been written in relation to rhizobia taxonomy and phylogeny (Jordan, 1984; Mart´ınez et al., 1990; Mart´ınez-Romero and CaballeroMellado, 1996); phenotypic characteristics (Sprent, 1997); evolution (Young, 1996), the effects of environmental factors such as salinity (Singleton et al., 1982; Cordovilla et al., 1999) and acidity (Keyser and Munns, 1979; Wood and Cooper,

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Table 1 Legume trees, zone of collection and number of rhizobial isolates Host

Characteristics

No. of isolates

Zone of collection

Erythrina crista-galli (Ec)

Trees or shrubs with glabrous deciduous leaves up to 20 m high Thorny trees or shrubs up to 5 m high growing in lowlands Trees 3–5 m high that can up to 25 m high Trees 5–8 m high with glabrous leaves Trees with rounded or flattened treetop with degradation resistant wood Shrubs 1–4 m high with a wide treetop Opulent trees up to 25 m high with a thick trunk Gigant trees that may reach up to 30 m high

12

Orange Island — Soriano

13

Native forest and nursery

4 7 10

Barrientos Island — Soriano Barrientos Island — Soriano Native forest —Flores

Acacia caven (Ac) Inga urugüensis (Iu) Lonchocarpus nitidus (Lo) Prosopis nigra (Pn) Sesbania virgata (Sv) Peltophorum dubium (Pd) Enterolobium contortisiliquum (En)

1985; Graham et al., 1994; Vance and Graham, 1995). Uruguay is a part of the Pampean region, with many ecotypes of N2 -fixing trees, which could be used for the improved production of several agrosystems. Native herbaceous and legume trees from Uruguay were studied in order to include them in agronomic and silvopastoril practices (Izaguirre and Beyhaut, 1998). Previous work (Frioni et al., 1998a,b) showed the nodulation and nitrogen fixing ability of rhizobial isolates in 17 species of legume trees. Milnitsky et al. (1997) characterized some of these isolates according to their physiological properties, protein and plasmid profiles. The characterization of indigenous populations of rhizobia for their resistances to stress environmental factors will be important before their selection for nursery inoculation. In temperate regions like our country, there are not many studies in this subject. The objectives of this work were to analyze the growth characteristics and the resistances to environmental stresses — antibiotic, salinity and acidity — of 61 rhizobial isolates from eight species of native legume trees from Uruguay and relate them by cluster analysis.

2. Materials and methods

7 3

Rocha Faculty of Agronomy forest and nursery

5

Faculty of Agronomy forest and nursery

and Beyhaut (1998) and Dommergues et al. (1999) (Table 1). 2.2. Rhizobial strains The survey was carried out in a previous work (Frioni et al., 1998b) in native forestry and nurseries, across the country, where nodules were collected and rhizobia strains were isolated and conserved in YEM (yeast extract-mannitol) liquid with 20% glycerol at −20◦ C until use (Frioni, 1999). 2.3. Growth characteristics of rhizobial isolates Growth was evaluated by measuring the size and time of appearance (days) of colonies in YEM (medium with agar). The mean generation time (MGT) was estimated from the growth curve in three isolates from each host by measuring the optical density (630 nm) in 25 ml of YEM inoculated with 1 ml culture with 1 × 109 ufc ml−1 and incubated at 28◦ C and 200 rev min−1 . The production of acid or alkali was determined in YEM medium with 25 ␮g bromothymol blue (BTB) ml−1 (Frioni, 1990). 2.4. Determination of intrinsic antibiotic resistance and salt tolerance level

2.1. Nodulate native legume trees The study was performed with eight tree native legume species that were described by Izaguirre

The intrinsic antibiotic resistance (IAR) was evaluated according to Eaglesham’s technique (Eaglesham, 1987) in plates of YEM with different concentra-

L. Frioni et al. / Applied Soil Ecology 16 (2001) 275–282

tions of antibiotics (10, 30, 50 and 100 ␮g ml−1 ): rifampicin (Rif), chloramphenicol (Chl), erythromycin (Ery), neomycin (Neo) and streptomycin sulfate (Str) (Sigma). Filter-sterilized aliquots of each antibiotic were added aseptically to sterile YEM medium at 50◦ C to give the final concentrations. Control plates contained no antibiotic. Each isolate was grown in YEM to late exponential phase and diluted to an inoculum size of 103 ufc ml−1 at the point of inoculation on the agar surface, transferred by a multipoint inoculator, in three replications. Plates were incubated at 28◦ C for 7 days and the highest concentration where colony’s diameter was similar to control assay was recorded as the resistance level. The salt tolerance level (STL) was determined with the same procedure and medium as antibiotic resistance, with 0, 0.1, 0.1, 0.5, 1, 2 and 3% of NaCl (Odee et al., 1997). 2.5. Acid resistance Each isolate was inoculated in Wood and Cooper (1985) solid medium acidified under sterile condition after autoclaving and sowed with rhizobial cultures from TY (tryptone-yeast extract medium) with less than 200 cfu in the inoculum (20 ␮l). The pH values tested were 7.0, 6.5, 5.5, 5.0, 4.8, 4.6 and 4.4, higher and lower than the soils were legumes were grown. The presence of more than 30 colonies after a week was considered as a tolerant strain (Graham et al., 1994).

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2.6. Analysis of results Results were analyzed using a generalized linear model (McCullagh and Nelder, 1989) with the GENMOD procedure. Data from IAR, STL and acid tolerance patterns of the isolates were used for cluster analysis, with the biostatistical analysis program NTSYS-pc (Rohlf, 1993).

3. Results 3.1. Growth characteristics of rhizobial isolates The majority of the isolates expressed milky-translucence with moderate to copious extracellular polysaccharide (EPS) colonies. The remaining few colonies were creamy or white opaque with little or moderate EPS production. The generation time varied from 1.5 to 5.8 h and produced acid in YEM with bromothymol blue (data not showed) and the time of appearance of colonies was between 1 and 3 days. The rhizobia isolate from native legume trees were mainly fast-growing strains, with an MGT average of 2.5 h, with the exception of some of the isolates from Acacia caven that showed an intermediate growth rate (5.1 h). 3.2. Tolerance to antibiotics and NaCl The results of the IAR assay are shown in Fig. 1. Isolates generally displayed resistance to erythromycin

Fig. 1. IAR of rhizobial isolates from native legume trees in Uruguay (two or more treatment with the same letter in both columns and rows are not significantly different at 5%).

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but were very susceptible to rifampicin. There were not significantly differences between the effect of other antibiotics. More than 60% of the isolates were resistant to 50 ␮g ml−1 of Ery, 36% to 50 ␮g ml−1 of Chl and 28% to a similar concentration of Str, but were more sensitive to Rif (only 8% of the isolates grew at 50 ␮g ml−1 ). Rhizobial isolated from different species of legume trees showed different behavior (Table 2). Rhizobial from Enterolobium contortisiliquum (En), Erythrina crista-galli (Ec) and Prosopis nigra (Pn) were more resistant to essayed antibiotics and the isolates from Acacia caven (Ac) and Lonchocarpus nitidus (Lo) were the most susceptibles. From the 61 rhizobia isolates 67% grew in YEM with 2% NaCl and only 21% did at salt concentration of 3% (data not shown). Table 2 shows the rhizobial STL of isolates from different hosts. Approximately a half of isolates from En, Iu, and Pn grew at 3%

Table 2 IAR and STL of rhizobial isolates from each native legume trees Antibiotic

Percentage of isolates resistance from each native legume treesa En

Sv

Pd

Streptomycin (␮g ml−1 ) 50 40 0 0 100 20 0 0 Chloramphenicol (␮g ml−1 ) 50 100 71 66 100 80 0 66

Pn

Iu

Ec

Ac

Lo

40 10

75 0

42 0

15 0

14 0

50 20

0 0

33 16

0 0

0 0

0 0

0 0

0 0

25 0

0 0

0 0

Erythromycin (␮g ml−1 ) 50 100 100 100 100 28

66 66

70 40

50 25

75 17

31 15

43 14

Neomycin (␮g ml−1 ) 50 20 43 100 0 43

0 0

40 20

0 0

50 0

15 0

0 0

100 100 0

100 100 40

100 100 50

92 75 17

92 31 0

86 71 28

Rifampicin 50 100

NaCl (%) 1 2 3 a

(␮g ml−1 ) 20 0

100 100 60

0 0

100 43 0

Native legume trees: Ac: Acacia caven; Ec: Erythrina crista-galli; En: Enterolobium contorstisiliquum; Iu: Inga urugüensis; Lo: Lonchocarpus nitidus; Pd: Peltophorum dubium; Pn: Prosopis nigra; Sv: Sesbania virgata.

of NaCl, those from Ac and Sv appeared to be more susceptible. 3.3. Acidity resistance All strains grew at pH 5.5; 87% grew at pH 4.6 and 76% did it at pH 4.4. The majority of those did not grow below pH 4.4. 3.4. Relationships between isolates according to their ecological characteristics Cluster analysis showed that there were four major groups separated by a similarity index of approximately of 60%. Cluster I included 12 isolates from Sesbania virgata (Sv), Erythrina crista-galli (Ec), and Prosopis nigra (Pn) legume host. Cluster II contained the bulk of isolates (35). This cluster can be split in two sub-clusters: one of them contained rhizobia isolated from Acacia caven (Ac) and Lonchocarpus nitidus (Lo). The other sub-cluster was very heterogeneous with five isolates from (Ec) and others from (Ac) and (Lo). The third cluster contained four of five isolates from Enterolobium contortisiliquum (En) and isolates from other hosts. The last cluster (IV) contained only two strains (Sv11, Ec8).

4. Discussion 4.1. Characterization of rhizobia according to growth morphology and growth rates Early reports of rhizobia associated with woody legumes described them as species that belong to the slow-growing, the cowpea miscellany (Jordan, 1984), but more recent reports (Zhang et al., 1991) have shown that this population includes a very diverse type of rhizobia including fast, intermediate and of slow-growing bacteria. These authors found that 29 isolates from Prosopis chilensis in Sudan and Kenya were fast-growing rhizobia, and very tolerant to salinity and high temperature. In this work, all the isolates were acid-producers but bacteria isolated from Acacia caven showed an intermediate growth rate (5.1 h). Barnet and Catt (1991) included two extra growth categories, intermediate and

L. Frioni et al. / Applied Soil Ecology 16 (2001) 275–282

279

Fig. 2. Phenogram showing clusters of rhizobial from native legume trees based on resistance characteristics.

very slow, to accommodate isolates from Australian Acacia spp. that did not fit into the traditional fastand slow-growing types. The scarce number of Bradyrhizobium strains isolated from woody legumes in Uruguay may suggest

that they are not as symbiotically competent as Rhizobium in nodulating with the majority of our woody species, as was found also by Odee et al. (1997) in Acacia and Abril and González (1994) in Prosopis from argentinian Chaco.

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4.2. Tolerance to antibiotics and salinity Fig. 1 shows that isolates were more sensitive to rifampicin than erythromycin and chloranphenicol. Döbereiner et al. (1981) related the increased resistance of rhizobial strains to the presence of antibiotics in the soil as a consequence of microbial activities such as Streptomyces, above all. This might have resulted in the isolation of rhizobial strains to a wide spectrum of antibiotics to be included in soil microbial analysis (Kremer and Peterson, 1982). Furthermore, the pattern of IAR is useful in the strain identification (Chanway and Holl, 1986). The isolates from Enterolobium contortisiliquum (En), Prosopis nigra (Pn) and Erythrina crista-galli (Ec) presented high tolerance to antibiotics and to NaCl concentration (Table 2). There could be very useful markers in following strains in the field. Highly antibiotic-resistant rhizobia have also been isolated from Malaysian soils (Roughley et al., 1992). Salt accumulation is a factor that may render soil unsuitable for agriculture. Increasing salt concentration from 50 to 200 mM NaCl significantly limits productivity by interfering with legume growth (Craig et al., 1991; Delgado et al., 1993). Unsuccessful symbiosis under salt-stress may be due to failure in the infection process because of the effect of salinity on the establishment of rhizobia (Singleton et al., 1982). Legumes and the process of nodule initiation are both more sensitive to osmotic stress. 4.3. Acidity resistance This rhizobial population seems to be resistant to pH as low as 4.4. Similar results were found with rhizobia that nodulate Lotus corniculatus in Uruguay (Baraibar et al., 1999). Rhizobia vary significantly in acid tolerance. Vance and Graham (1995) found that some isolates may grow even better in somewhat acid culture medium, but many S. meliloti do not grow below pH 5.6, and 62% of B. japonicum isolates evaluated by Keyser and Munns (1979) grew at pH 4.5 and some strains of Rhizobium tropici grow to pH 4.0 (Graham et al., 1994). The identification of glutamato as a compatible solute in acid-stressed cells and the demonstration that cell membrane differences could influence pH tolerance, have been found by these authors as responsible for the acid pH tolerance. But,

field studies are important to verify the in vitro results. Uruguay has important areas with acidic soils. Vargas and Graham (1989) found good correlation between growth in acidic medium and nodulation capacity of beans in acidic soils in Brazil. 4.4. Relationships between rhizobial isolates Based on the physiological and ecological characteristics analyzed we were able to identify four main cluster (Fig. 2). Some of these groups included isolates from the same host, as Sesbania virgata (Sv) in cluster I, Acacia caven (Ac) in cluster II and Enterolobium contortisiliquum (En) in cluster III. Zhang et al. (1991) analyzing 97 rhizobial strains isolated from trees (42 from Acacia senegal) from 115 phenotype characters, found 19 different groups. Many other methods are employing with the purpose to determine the filogenetic relationships between new rhizobial isolates (SDS-PAGE of total proteins, partial 16SrRNA gene sequence, FAME, enzymes homology (Moreira et al., 1998)). 5. Conclusions Most rhizobial isolates from legume trees from Uruguay belong to the fast and intermediate (isolates from Acacia caven) growing groups. The strains showed high antibiotic, salinity and pH resistance, typical of native isolates, suggesting adaptability to different ecological environments with many factors at stress levels. The ecological characteristics of the isolates allowed to group them. Acacia caven, E. cortortisiliquum and Sesbania virgata isolates showed high homology between them. Other methods are employing to group these strains and try to determine their taxonomic relationships, such as the genomic fingerprinting using rep-PCR and fatty acid methyl ester (FAME). The results obtained for comparison ecological characteristics could be confirmed by these approaches. Acknowledgements This work was supported by Conicyt-BID (Project No. 2129). The authors thank J. Franco for his assistance in statistical analysis.

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