International Journal of Systematic and Evolutionary Microbiology (2001), 51, 1011–1021 Printed in Great Britain Mesorhizobium chacoense sp. nov., a novel species that nodulates Prosopis alba in the Chaco Arido region (Argentina) 1 Departamento de Encarna Vela! zquez,1 Jose! Mariano Igual,2 Anne Willems,5 Microbiologı!a y Gene! tica, 4 6 1 Edificio Departamental, Marı!a P. Ferna! ndez, Estefanı!aMun4 oz, Pedro F. Mateos, Campus Miguel de Adriana Abril,3 Nicola! s Toro,6 Phillippe Normand,4 Unamuno, Universidad de 2 5 1 Salamanca, Salamanca, Emilio Cervantes, Monique Gillis and Eustoquio Martı!nez-Molina Spain 2 IRNA-CSIC, Apartado 257, Author for correspondence: Encarna Vela! zquez. Tel: j34 923 294532. Fax: j34 923 224876. Salamanca, Spain e-mail: evp!gugu.usal.es 3 Catedra de Microbiologı!a Agrı!cola, Facultad de Agronomı!a, Universidad Low-molecular-weight RNA analysis was performed for the identification and de Co! rdoba, Co! rdoba, classification of 20 Argentinian strains isolated from the root nodules of Argentina Prosopis alba. SDS-PAGE of total cellular proteins, determination of the DNA 4 Laboratoire d’Ecologie base composition, DNA–DNA reassociation experiments and physiological and Microbienne, Unite de biochemical tests were also carried out for these strains and the whole 16S Recherche Associee! au CNRS 697, Universite! Lyon rRNA gene was sequenced from one of the strains, strain LMG 19008T. Results 1, F-69622 Villeurbanne of the genotypic and phenotypic characterization showed that the strains cedex, France isolated in this study belong to a group that clustered in the genus 5 Laboratorium voor Mesorhizobium. The results of DNA–DNA hybridizations showed that this group Microbiologie, Vakgroep is a novel species of this genus. The name Mesorhizobium chacoense sp. nov. is Biochemie, Fysiologie en Microbiologie, K. L. proposed for this species. The type strain is LMG 19008T (l CECT 5336T). Ledeganckstraat 35, B-9000 Gent, Belgium 6 Departamento de Sistemas Keywords: taxonomy, Mesorhizobium, LMW RNA, 16S rRNA, Prosopis Simbio! ticos, Estacio! n Experimental del Zaidı!n, CSIC, Granada, Spain INTRODUCTION Acacia from Senegal resulted in the description of various species of Sinorhizobium (de Lajudie et al., The taxonomy of the family Rhizobiaceae has been the 1994) and Mesorhizobium (de Lajudie et al., 1998a; subject of important advances in recent years. Six Wang et al., 1999). The species Mesorhizobium pluri- genera are able to nodulate diverse species of legumes: farium contained strains isolated from nodules of Rhizobium, Bradyrhizobium, Azorhizobium, Sinor- Acacia, Leucaena, Prosopis and Chamaecrista in West hizobium, Mesorhizobium and Allorhizobium. In the Africa (Senegal), as well as East Africa (Sudan) and early years of Rhizobium taxonomy, species were, in South America (Brazil). Nevertheless, a large number general, described from legumes of agronomic im- of legumes exists whose bacterial symbionts have not portance, but a trend in recent years to investigate the been studied systematically. Thus, in general, isolation symbionts of exotic plants has resulted in important of their bacterial symbionts is a promising strategy to contributions to rhizobial taxonomy. Thus, Azor- increase our knowledge of the systematics and evol- hizobium was defined as a new genus based on strains ution of Rhizobium. Moreover, many of these rhizobia able to nodulate the stems of Sesbania rostrata that remain as yet unexamined have established (Dreyfus et al., 1988) and Allorhizobium was isolated symbiotic relationships with legumes that are of great from an aquatic legume, Neptunia natans, in Senegal importance in the maintenance and economy of large (de Lajudie et al., 1998b). Also, the analysis of and important ecosystems throughout the world. rhizobial symbionts of leguminous trees of the genus Therefore, knowledge and optimization in the man- agement of these ecosystems may benefit from the ................................................................................................................................................. study of these particular strains. Species of the genus Abbreviations: LMW RNA, low-molecular-weight RNA; PNP, para-nitro- Prosopis are important trees in many ecosystems phenyl substrates; SCE, staircase electrophoresis. throughout South America and some have been The GenBank accession number for the 16S rRNA sequence of Mesor- introduced into Africa, where they can be nodulated hizobium chacoense LMG 19008T is AJ278249. by indigenous rhizobia (de Lajudie et al., 1998a; 01473 # 2001 IUMS 1011 E. Vela! zquez and others Haukka et al., 1996; McInroy et al., 1999). Apart from plants growing in soil from two different locations. Nine the above references, little work has been described isolates were from Luque, located some 200 km east of concerning the taxonomy of American rhizobial sym- Cordoba Province in the Espinal region, and eleven isolates bionts of Prosopis. In this work, rhizobial strains have were from the Chancanı! reserve, in the north-west of been isolated from Prosopis chilensis plants growing in Cordoba Province in the Chaco Arido region (Argentina). Isolations were made according to Vincent (1970) using " diverse geographical locations in central Argentina, yeast mannitol agar [YMA, which contained (g l− ): including the Reserva Chancanı! in the south of El mannitol, 10; sodium glutamate, 0n5; K#HPO%,0n5; Chaco Arido, an important ecosystem, covering sev- MgSO%.7H#O, 0n2; NaCl, 0n05; CaCl#,0n04; FeCl$,0n004; eral thousands of hectares in Argentina. Based on a yeast extract, 1; agar, 20; pH 6n8] (Bergersen, 1961). The polyphasic taxonomy approach, including staircase cultures used in further studies were purified from single electrophoresis analysis (SCE) of low-molecular- colonies after 10 d incubation at 28 mC. weight (LMW) RNA, 16S rDNA sequencing, analysis Nodulation tests. Surface-sterilized seeds of P. chilensis, P. of total cell protein, determination of base composition alba, Prosopis flexuosa and Acacia sp. were heat treated and DNA–DNA hybridization and a series of bio- (50 mC for 5 min) and germinated axenically in Petri dishes. chemical tests, the novel species Mesorhizobium chaco- Seedlings were transferred to pots with sterile vermiculite ense is described. and watered with nitrogen-free nutrient solution (Rigaud & Puppo, 1975). Each plant was inoculated with 10 ml of a ) −" METHODS suspension of each strain containing 8i10 cells ml . The inoculated plants were placed for 30 d in a plant growth Bacterial strains. The reference strains and novel isolates chamber with mixed incandescent and fluorescent lighting # " used in this study are listed in Table 1. A total of 20 new (400 microeinsteins m− s− ; 400–700 nm), programmed for rhizobial isolates was obtained from young Prosopis alba a 16 h photoperiod day\night cycle, at a constant tem- Table 1. Strains used in this study ................................................................................................................................................................................................................................................................................................................. Abbreviations: ATCC, American Type Culture Collection, Manassas, VA, USA; LMG, Collection of Bacteria of the Laboratory voor Microbiologie, Gent, Belgium; CECT, Spanish Type Culture Collection, Valencia, Spain. Strain LMG no.* Host plant Geographical origin† Reference Mesorhizobium chacoense T T T",# Pr-5 (l CECT 5336 ) LMG 19008 Prosopis alba Chancanı! This study " Pr-11 LMG 19009 Prosopis alba Chancanı! This study " Pch-1 LMG 19010 Prosopis alba Chancanı! This study " Pch-2 LMG 19011 Prosopis alba Chancanı! This study " Pch-3 LMG 19012 Prosopis alba Chancanı! This study " Pch-4 LMG 19013 Prosopis alba Chancanı! This study " Pch-5 LMG 19014 Prosopis alba Chancanı! This study " Pch-6 LMG 19015 Prosopis alba Chancanı! This study " Pch-7 LMG 19016 Prosopis alba Chancanı! This study " Pch-8 LMG 19017 Prosopis alba Chancanı! This study " Pch-9 LMG 19018 Prosopis alba Chancanı! This study " Pl-1 LMG 19019 Prosopis alba Luque This study " Pl-3 LMG 19020 Prosopis alba Luque This study " Pl-5 LMG 19021 Prosopis alba Luque This study " # Pl-6 LMG 19022 , Prosopis alba Luque This study " Pl-7 LMG 19023 Prosopis alba Luque This study " Pl-8 LMG 19024 Prosopis alba Luque This study " Pl-10 LMG 19025 Prosopis alba Luque This study " Pl-11 LMG 19026 Prosopis alba Luque This study " Pl-12 LMG 19027 Prosopis alba Luque This study " # Mesorhizobium loti ATCC 33669T LMG 6125T , Lotus tenuis New Zealand Jarvis et al. (1982) # Mesorhizobium loti NZP 2014 LMG 6124 Lotus corniculatus New Zealand Jarvis et al. (1982) " # Mesorhizobium ciceri USDA 3383T LMG 14989T , Cicer arietinum Spain Nour et al. (1994) # Mesorhizobium mediterraneum UPM-CA142 LMG 14990 Cicer arietinum Spain Nour et al. (1995) Mesorhizobium mediterraneum USDA 3392T – Cicer arietinum Spain Nour et al. (1995) " # Mesorhizobium tianshanense USDA 3592T LMG 15767T , Glyzyrrhiza pallidiflora China Chen et al. (1995) " # Mesorhizobium huakuii USDA 4779T LMG 14107T , Astragalus sinicus China Chen et al. (1991) # Mesorhizobium plurifarium LMG 11892T LMG 11892T Acacia senegal Senegal de Lajudie et al. (1998a) " # Mesorhizobium amorphae ACCC 19665T LMG 18977T , Amorpha fruticosa China Wang et al. (1999) Sinorhizobium meliloti USDA 1002T LMG 6133T Medicago sativa USA de Lajudie et al. (1994) Rhizobium leguminosarum ATCC 10004T LMG 8817T Pisum sativum USA Skerman et al. (1980)