Tolerância ao sal e às altas temperaturas de estirpes de Sinorhizobium provenientes de zonas secas do Alentejo

Salt and temperature tolerance of Sinorhizobium strains isolated from dry environments in Alentejo

P. Fareleira¹, N. Matos¹, E. Ferreira² & J. F. Marques

 

RESUMO

O trabalho teve como principal objectivo a obtenção de estirpes de rizóbio adequadas à nodulação de luzernas anuais e adaptadas a sobreviver nas condições ambientais susceptíveis de ocorrer em solos degradados. Dado que as populações de rizóbio apresentam variabilidade considerável no que respeita à tolerância a factores ambientais, os estudos focaram-se na pesquisa e na selecção de estirpes resistentes a condições adversas. Efectuaram-se colheitas de solos em diversas zonas no sul do País, seleccionando-se locais afectados por secura, temperaturas elevadas e, pontualmente, salinidade. A partir destes solos, isolaram-se estirpes de rizóbio, usando como planta hospedeira a luzerna anual Medicago polymorpha.

Estudaram-se os efeitos de condições de stresse ambiental, como a salinidade e as altas temperaturas, no crescimento das estirpes isoladas. Dos 41 isolamentos analisados, 11 apresentaram crescimento em meio contendo 1,4 M de cloreto de sódio e suplementado com 10% de extracto de terra, e 22 cresceram quando incubadas a 45 ºC em meio sem aditivos. Três estirpes mostraram ter capacidade para crescer sob os efeitos conjuntos da salinidade e da alta temperatura.

A análise de extractos etanólicos de estirpes tolerantes à salinidade revelou, na maior parte dos casos, a acumulação, induzida pelo sal, dos solutos compatíveis de Sinorhizobium: o dipéptido N - acetilglutaminilglutamina amida, vários tipos de betaínas, trealose, glutamato e prolina.

A observação, por NMR de ³¹P in vivo, de uma estirpe tolerante ao sal, proveniente de um solo xistoso de baixo teor em fósforo, mostrou a presença de níveis elevados de reservas intracelulares de fosfato inorgânico (polifosfato), sugerindo um bom potencial para utilização em solos onde os riscos de salinização se conjuguem com deficiências em fósforo assimilável.

 

ABSTRACT

The main objective of this work was to obtain rhizobial strains able to nodulate annual medics and highly adapted to the environmental conditions present in degraded soils. Since populations of rhizobia vary in their tolerance to environmental factors, screening for resistant strains was pursued. Soil samples were collected in different regions in Southern Portugal, severely affected by drought, high temperatures, and, in some places, salinity; rhizobial strains were isolated using Medicago polymorpha as trap-host.

The effects of environmental stressful conditions, such as salinity or high temperatures, in the growth of the isolated strains were studied. Among the 41 isolates that were ana-lysed here, 11 showed good growth capacity in the presence of 1.4 M sodium chloride and 10% soil extract, and 22 could grow upon incubation at 45 ºC. Three strains were able to grow under the simultaneous effects of salinity and high temperature.

NMR analysis of ethanolic cell-free extracts of salt tolerant strains showed that, in most cases, cell growth in medium containing high salt concentrations resulted in the accumulation of the compatible solutes described for Sinorhizobium: the dipeptide N-acetylglutaminylglutamine amide, betaines, trehalose, glutamate, and proline.

Analysis by in vivo ³¹P-NMR of a salt tolerant strain originated from a schistous soil with low-phosphorus content, revealed the presence of high levels of intracellular inorganic phosphate reserves (polyphosphates). This suggests a high potential for the utilization of the strain in soils affected by both salinity and phosphorus deficiency.

 

 

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REFERÊNCIAS BIBLIOGRÁFICAS

Allen, O.N. & Allen, E.K. 1950. Biochemical and symbiotic properties of the rhizobia. Bacteriol. Rev., 14: 273-330.         [ Links ]

Bental, M., Pick, U., Avron, M. & Degani, H. 1991. Polyphosphate metabolism in the alga Dunaliella salina studied by ³¹P-NMR. Biochim. Biophys. Acta, 1092: 21-28.

Bieleski, R. 1973. Phosphate pool, phosphate transport and phosphate availability. Annu. Rev. Plant Physiol., 24: 225-252.

Cassman, K.G., Munns, D.N. & Beck. D.P. 1981. Phosphorus nutrition of R. japonicum: strain differences in phosphate storage and utilization. Soil Sci. Soc. Am. J., 45: 517-519.

Da Costa, M.S., Santos, H. & Galinsky, E. A. 1998. An overview of the role and diversity of compatible solutes in Bacteria and Archaea. Advances in Biochemical Engineering / Biotechnology, 61: 117-153.

Fareleira, P., Santos, B.S., António, C., Moradas-Ferreira, P., LeGall, J., Xavier, A.V. & Santos, H. 2003. Response of a strict anaerobe to oxygen: survival strategies in Desulfovibrio gigas. Microbiology, 149: 1513-1522.

Ferreira, E.M. & Marques, J.F. 1992. Selection of portuguese Rhizobium leguminosarum bv. trifolii strains for production of legume inoculants. Plant and Soil, 147: 151-158.

Graham, P.H. 1992. Stress tolerance in Rhizobium and Bradyrhizobium, and nodulation under adverse soil conditions. Can. J. Microbiol., 38: 475-484.

Lahav, R., Fareleira, P., Nejidat, A. & Abeliovich, A. 2002. The identification and characterization of osmotolerant yeast isolates from chemical wastewater evaporation ponds. Microbial Ecology, 43: 388-396.

Lamosa, P., Martins, L.O., Da Costa, M. S. & Santos, H. 1998. Effects of temperature, salinity and medium composition on compatible solute accumulation by Thermococcus spp. Appl. Environ. Microbiol., 64: 3591-3598.

Miller, K.J. & Wood, J.M. 1996. Osmoadaptation by rhizosphere bacteria. Annu. Rev. Microbiol., 50, 101-136.

O’Hara, G., Yates, R. & Howieson, J. 2002. Selection of strains of root nodule bacteria to improve inoculant performance and increase legume productivity in stressful environments. In: D. Herridge (ed) Inoculants and Nitrogen Fixation in Vietnam, pp. 75-80. ACIAR Proceedings 109e.

Reed, R.H., Richardson, D.L., Warr, S.R.C. & Stewart, W.D.P. 1984. Carbohydrate accumulation and osmotic stress in cyanobacteria. J. Gen. Microbiol., 130: 1-4.

Roberts, M.F. 1987. Polyphosphates. In: C. Tyler Burt (ed) Phosphorus NMR in Biology, Cap. 4, pp. 85-94. CRC Press, Inc., Boca Raton, Florida, USA.

Smith, R.S. 1992. Legume inoculant formulation and application. Can. J. Microbiol., 38: 485-492.

Soares, A.M. 1983. Carta Litológica de Portugal. Atlas do Ambiente, Comissão Nacional do Ambiente, Lisboa.

Vincent, J.M. 1970. A manual for the practical study of root-nodule bacteria. IBP Handbook nº15. Blackwell Scientific Publications, Oxford, United Kingdom.

Wacek, T.J. & Brill, W.J. 1976. Simple, rapid assay for screening nitrogen-fixing ability in soybean. Crop Science, 16: 519-523.

Zahran, H.H. 1999. Rhizobium-legume symbiosis and nitrogen fixation under severe conditions and in an arid climate. Microbiol. Mol. Biol. Rev., 63: 968-989.

Zuberer, D.A. 1994. Recovery and enumeration of viable bacteria. In R. W. Weaver et al. (eds) Methods of Soil Analysis, Part 2. Microbiological and Biochemical Properties – SSSA book Series, nº5, Soil Science Society of America, Madison, USA.

 

¹ Estação Agronómica Nacional, Departamento de Ciência do Solo, Quinta do Marquês, Av. da República, 2784-505 Oeiras, e-mail: paula.fareleira@oniduo.pt;

² Estação Florestal Nacional, Departamento de Ecologia, Recursos Naturais e Ambiente, Quinta do Marquês, Av. da República, 2784-505 Oeiras