RESEARCH ARTICLE
Genetic diversity of Mimosa pudica rhizobial symbionts in soils of French Guiana: investigating the origin and diversity of Burkholderia phymatum and other beta-rhizobia Ravi P.N. Mishra1,5, Pierre Tisseyre1, Re´my Melkonian1, Cle´mence Chaintreuil1, Lucie Miche´1, Agnieszka Klonowska1, Sophie Gonzalez2, Gilles Bena1,4, Gise`le Laguerre3 & Lionel Moulin1 1
IRD, UMR LSTM, Montpellier, France; 2IRD, Herbier de Guyane, Cayenne, French Guiana; 3INRA, UMR LSTM, Montpellier, France; and Laboratoire de Microbiologie et de Biologie Mole´culaire, University Mohammed V Agdal, Agdal, Morocco
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Correspondence: Lionel Moulin, Laboratoire des Symbioses Tropicales et Me´diterrane´ennes, TA-A82/J Campus de Baillarguet 34398 Montpellier Cedex 5, France. Tel.: +33 4 67 59 37 63; fax: +33 4 67 59 38 02; e-mail:
[email protected]
MICROBIOLOGY ECOLOGY
Present address: Ravi P.N. Mishra, Novartis Vaccines Research Centre, Via Fiorentina 1, Siena, 53100, Italy. Received 25 July 2011; revised 14 October 2011; accepted 19 October 2011. Final version published online 21 November 2011. DOI: 10.1111/j.1574-6941.2011.01235.x Editor: Philippe Lemanceau Keywords rhizobia; symbiosis; Mimosa; Burkholderia; Cupriavidus; biodiversity.
Abstract The genetic diversity of 221 Mimosa pudica bacterial symbionts trapped from eight soils from diverse environments in French Guiana was assessed by 16S rRNA PCR-RFLP, REP-PCR fingerprints, as well as by phylogenies of their 16S rRNA and recA housekeeping genes, and by their nifH, nodA and nodC symbiotic genes. Interestingly, we found a large diversity of beta-rhizobia, with Burkholderia phymatum and Burkholderia tuberum being the most frequent and diverse symbiotic species. Other species were also found, such as Burkholderia mimosarum, an unnamed Burkholderia species and, for the first time in South America, Cupriavidus taiwanensis. The sampling site had a strong influence on the diversity of the symbionts sampled, and the specific distributions of symbiotic populations between the soils were related to soil composition in some cases. Some alpha-rhizobial strains taxonomically close to Rhizobium endophyticum were also trapped in one soil, and these carried two copies of the nodA gene, a feature not previously reported. Phylogenies of nodA, nodC and nifH genes showed a monophyly of symbiotic genes for beta-rhizobia isolated from Mimosa spp., indicative of a long history of interaction between beta-rhizobia and Mimosa species. Based on their symbiotic gene phylogenies and legume hosts, B. tuberum was shown to contain two large biovars: one specific to the mimosoid genus Mimosa and one to South African papilionoid legumes.
Introduction Rhizobia are a functional class of soil bacteria able to develop a nitrogen-fixing symbiosis with legumes, and are also termed legume nodulating bacteria (or LNB). The legume nodulation ability is spread among the Alphaand Beta- subclasses of Proteobacteria, and the names alpha- and beta-rhizobia have been proposed for convenience (Gyaneshwar et al., 2011). Beta-rhizobia were originally described in 2001 in two parallel studies: one describing two Burkholderia strains (STM678 and STM815) isolated from Aspalathus carnosa (Papilionoideae) in South Africa and Machaerium lunatum (Papilionoideae) in French Guiana, respectively (Moulin et al., 2001), which were subsequently named Burkholderia tuberum and Burkholderia phymatum FEMS Microbiol Ecol 79 (2012) 487–503
(Vandamme et al., 2002); and one describing Ralstonia taiwanensis from two Mimosa (Mimosoideae) species in Taiwan (Chen et al., 2001), which was later renamed Cupriavidus taiwanensis (Vandamme & Coenye, 2004). Since then, several diversity studies have demonstrated the widespread occurrence of beta rhizobia as symbionts of Mimosa species in Costa Rica and Texas (Barrett & Parker, 2006; Andam et al., 2007), Panama (Barrett & Parker, 2005; Parker, 2008), Brazil and Venezuela (Chen et al., 2005b; Bontemps et al., 2010), Taiwan (Chen et al., 2005a), India (Verma et al., 2004), Papua New Guinea (Elliott et al., 2009), Australia (Parker et al., 2007), and China (Liu et al., 2011). To date, several nodulating beta-rhizobia species have been described, most of them belonging to the Burkholderia genus. The B. tuberum type strain (STM678T) is able ª 2011 Federation of European Microbiological Societies Published by Blackwell Publishing Ltd. All rights reserved
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to nodulate a range of Cyclopia species (Elliott et al., 2007b). Strains affiliated to B. tuberum were also isolated from Mimosa pigra in Panama (Barrett & Parker, 2005), Mimosa spp. in Costa Rica (Barrett & Parker, 2006) and from several diverse Mimosa species in Central Brazil (Bontemps et al., 2010). Burkholderia mimosarum has been isolated from M. pigra nodules in Taiwan, Venezuela, Brazil, China and Australia (Chen et al., 2006; Parker et al., 2007; Liu et al., 2011), while Burkholderia nodosa was isolated from nodules on Mimosa scabrella and Mimosa bimucronata in Brazil (Chen et al., 2007), and Burkholderia sabiae from Mimosa caesalpiniifolia nodules, also in Brazil (Chen et al., 2008). So far, only one nodulating species has been described in the Cupriavidus genus, namely C. taiwanensis (Chen et al., 2001), though other nodulating strains have been isolated but not yet described to species level. Cupriavidus strains were isolated from nodules of Mimosa pudica, Mimosa diplotricha and M. pigra in Taiwan (Chen et al., 2003, 2005a), Costa Rica (Barrett & Parker, 2006), Texas (Andam et al., 2007) and India (Verma et al., 2004). The species B. phymatum was originally described based on a single strain, STM815T, which was isolated from M. lunatum nodules collected in French Guiana (Moulin et al., 2001; Vandamme et al., 2002). This species has since been reported as being isolated from M. pudica nodules in Papua New Guinea (two strains, Elliott et al., 2007a) and China (four strains, Liu et al., 2011), but has not yet been found as a symbiont of Mimosa in Australia, or in Central and South America (Chen et al., 2003, 2005b; Barrett & Parker, 2005, 2006; Bontemps et al., 2010). It is relevant to note that the Bontemps et al. (2010) diversity study was performed on 47 native (mainly endemic) Mimosa species in Central Brazil, and not a single strain among the 148 isolates belonged to B. phymatum. On the other hand, this is not so surprising, as the preferred symbiont of B. phymatum, M. pudica, was only infrequently encountered in Central Brazil by Bontemps et al. (2010), as its niche was occupied by the other 200+ Mimosa spp. that are native/endemic to these centres of Mimosa diversity (Simon & Proenc¸a, 2000; Simon et al., 2011). In addition, Central Brazilian endemics appear not to nodulate effectively with B. phymatum (dos Reis Junior et al., 2010). Nevertheless, although the Cerrado/Caatinga biomes appear not to contain it, until further sampling of M. pudica is performed in these regions, the occurrence of B. phymatum in Central Brazil cannot be excluded. The type strain of B. phymatum, STM815, was originally isolated from a M. lunatum nodule in a plant nursery in Paracou (French Guiana). The demonstration of nodulation ability of M. lunatum by STM815 has proven inconclusive because of the difficulties in germinating its seeds, and growing this tree under ª 2011 Federation of European Microbiological Societies Published by Blackwell Publishing Ltd. All rights reserved
R.P.N. Mishra et al.
greenhouse conditions (L. Moulin, unpublished data), although nodulation tests on a different species of Machaerium (Machaerium brasilense) were also unsuccessful (Elliott et al., 2007b). On the other hand, Elliott et al. (2007b, 2009) and dos Reis Junior et al. (2010) showed that STM815 has a very large host range on Mimosa species, with higher symbiotic capacities (competition for nodulation, nitrogen fixation within nodules) than C. taiwanensis LMG19424T. The question thus remains open about the occurrence and diversity of B. phymatum in South America. Previous diversity studies have thus shown an affinity between beta-rhizobia and Mimosa species. Some Mimosa species also appear to have a broad host range for betarhizobial strains. For example, in Bontemps et al. (2010), among 118 strains recovered from 47 Mimosa species and belonging to at least seven Burkholderia species, 98 were able to nodulate M. pudica. Mimosa pudica thus appears to be highly promiscuous, capable of nodulating with a very diverse range of beta-rhizobial species. Based on this fact, the aim of this study was to uncover the diversity of beta-rhizobia using a soil-trapping strategy with M. pudica as a trap host in French Guiana, a country located in South America, which contains the principal centre of diversification of the Mimosa genus, that is the Cerrado region of Brazil (Simon & Proenc¸a, 2000; Simon et al., 2011). The specific questions we adressed were the following: (i) What is the intra and interspecific diversity of beta-rhizobia trapped by M. pudica in diverse soils of French Guiana? (ii) Is B. phymatum predominant in French Guiana? (iii) Is there a link between soil parameters and beta-rhizobial diversity? (iv) What are the origins of the symbiotic genes in the beta-rhizobial populations? To isolate M. pudica compatible symbionts, we used a trapping method on dilutions of soils recovered from eight sites mainly around Cayenne with M. pudica as a trap host. Strains were isolated from the nodules, tested for their nodulation ability, and further characterized at the genetical and genomic level. To determine the specificity of the symbiosis between beta-rhizobia and M. pudica, we conducted the same trapping strategy on four soils using Siratro (Macroptilium atropurpureum), a broad-host-range legume that is able to trap both alpha and beta-rhizobia (Lima et al., 2009). The diversity of symbionts trapped by Siratro was then compared with that of the symbionts trapped by M. pudica. Rhizobial genetic diversity was assessed using 16S rRNA PCR-RFLP, REP-PCR genomic fingerprints, as well as by phylogenies based upon the 16S rRNA and recA housekeeping genes. Phylogenies of the symbiosis-related genes nodA, nodC and nifH were also performed in order to determine the origin of symbiosis in the beta-rhizobial strains that we isolated in the coastal lowlands of French Guiana. FEMS Microbiol Ecol 79 (2012) 487–503
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Investigating the origin and diversity of beta-rhizobia
Materials and methods Soil sampling and characteristics
Two hundred grams of soil was sampled under M. pudica plants on eight sites in French Guiana (see Table 1), mainly on Cayenne island (on the coastal side east of Cayenne, near the towns of Remire and Montjoly) and on the roadside between Cayenne and Kourou cities. GPS coordinates and Flora composition of sampled soils are given in Table 1 and Supporting Information Fig. S1. Altitude of sampled sites ranged from 7 to 21 m. Soil parameters were analysed by ‘Laboratoire d’analyse des sols d’Arras’, INRA, France: pH (water and KCL method), granulometry (five fractions), humidity (at 105 °C), C/N, CEC (Metson method), total nitrogen and organic carbon, P2O5 (Joret–He´bert method), total CaCO3, Na and H+ (BaCl2 extraction), Cu, Fe, Zn and Mn contents (by ICP-AES). Plant trapping of rhizobia
Mimosa pudica (B&T world seeds; ecotype M. pudica var. hispida) was used as a legume host for trapping and for nodulation tests of bacterial strains. Siratro (M. atropurpureum; UCAD, Dakar, Senegal) was also used as a trap host in parallel experiments. Mimosa pudica seeds were scarified and surface sterilized with 96% H2SO4 and 3% Calcium hypochlorite (15 min each treatment, followed by five or six washes with sterile dH2O); while Siratro seeds were immersed for 3 min in 96% H2SO4 followed
by thorough washing. The seeds were then soaked overnight in sterile dH2O, transferred to water agar plates (0.8% Agar) and incubated overnight at 37 °C for germination. Seedlings were transferred to Gibson tubes or to deep-well microplates, and incubated in a tropical plant growth chamber (30 °C, 80% humidity, 16 h day/8 h night). Five grams of soil was suspended in 50 mL of sterile water and vortexed thoroughly. Serial dilutions up to 10 2 were performed, and 0.7 mL of diluted soil suspension was inoculated in to either Gibson tubes (80 mL) or to deep-well microplates (8 mL per well) filled with Jensen’s nutrient medium (Vincent, 1970). Eight replicates were used for each dilution level. Nodules were harvested at 28 days post-inoculation. Nodules of varying sizes were selected for isolation (60 nodules for soils S1–S4, and 20 nodules for soils S5–S8). Nodules were thoroughly washed in running tap water, sterilized by immersing in 3% calcium hypochlorite for 5 min and washed five or six times with sterile dH2O. Surface-sterilized nodules were individually crushed in 20 lL dH2O. Nodule contents were streaked on yeast mannitol agar plates (Vincent, 1970) and incubated at 28 °C for 48 h. All colony types were picked and purified twice by repeated streaking on YMA plates. All isolates were retested for nodulation with M. pudica (or M. atropurpureum) using Gibson tubes under the growth conditions described earlier, except that 1 mL of exponential bacterial culture was inoculated into the tubes instead of soil suspension. All strains isolated from M. pudica and M. atropurpureum nodules are listed in Table S1. For long-term
Table 1. Characteristics of soils used for rhizobial trapping
Soil
Site characteristics and flora at proximity
S1 S2
East Cayenne, Coastal gardens, Mimosa pudica and Machaerium lunatum East Cayenne, Montjoly town, M. pudica
S3
East Cayenne, Coastal path, M. pudica
S4 S5
North Cayenne, Elyse´e savannah, M. pudica Cayenne South, Roadside, M. pudica
S6
Cayenne North, Roadside, M. pudica
S7
West of Remire town, M. pudica, Aeschynomene sp. Rochambeau savannah, M. pudica
S8
GPS coordinates 4°56′46″N 52°18′03″W 4°55′19″N 52°16′38″W 4°56′25″N 52°17′41″W 4°55′32″N; 52°24′50″W 4°52′51″N; 52°18′46″W 5°02′50″N; 52°36′20″W 4°53′03″N; 52°17′34″W 4°49′26″N; 52°21′20″W
Altitude (m)
Soil parameters Gr
pH
Corg
Ntot
8
Loamy sand
7.04
26.2
1.57
45.3
