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Native bradyrhizobia from Los Tuxtlas in Mexico are symbionts of Phaseolus lunatus (Lima bean)
Systematic and Applied Microbiology 36 (2013) 33–38
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Native bradyrhizobia from Los Tuxtlas in Mexico are symbionts of Phaseolus lunatus (Lima bean) a ˜ Aline López-López a , Simoneta Negrete-Yankelevich b , Marco A. Rogel a , Ernesto Ormeno-Orrillo , a a,∗ Julio Martínez , Esperanza Martínez-Romero a b
Centro de Ciencias Genómicas, UNAM, Cuernavaca, Morelos CP 62210, Mexico Red de Ecología Funcional, INECOL, Xalapa, Veracruz, Mexico
a r t i c l e
i n f o
Article history: Received 6 September 2012 Received in revised form 12 October 2012 Accepted 15 October 2012 Keywords: Symbiosis Rain forest Nitrogen fixation
a b s t r a c t Los Tuxtlas is the northernmost rain forest in North America and is rich in Bradyrhizobium with an unprecedented number of novel lineages. ITS sequence analysis of legumes in polycultures from Los Tuxtlas led to the identification of Phaseolus lunatus and Vigna unguiculata in addition to Phaseolus vulgaris as legumes associated with maize in crops. Bacterial diversity of isolates from nitrogen-fixing nodules of P. lunatus and V. unguiculata was revealed using ERIC-PCR and PCR-RFLP of rpoB genes, and sequencing of recA, nodZ and nifH genes. P. lunatus and V. unguiculata nodule bacteria corresponded to bradyrhizobia closely related to certain native bradyrhizobia from the Los Tuxtlas forest and novel groups were found. This is the first report of nodule bacteria from P. lunatus in its Mesoamerican site of origin and domestication. © 2012 Elsevier GmbH. All rights reserved.
Introduction Nitrogen (N)-fixing bacteria from different genera (collectively designated rhizobia) form nodules on legumes and allow plants to grow in N deficient soils, such as rain forest soil that becomes N deficient after a few years of tilling and cropping. Legumes and rhizobia coexist at their sites of origin and domestication, for example, there is a large diversity of bradyrhizobia and rhizobia nodulating soybean in Asia. In contrast, in Africa and Mexico there is a need to inoculate introduced soybeans with Bradyrhizobium, since soybeans are not native and there are no soybean nodulating bacteria. There has been an agricultural selection of soybeans that could form nodules with native African bradyrhizobia but this has had limited success [18]. In Mexico, there is a large diversity of Phaseolus vulgaris and Phaseolus coccineus nodulating bacteria [21,31] because Mexico is the center of origin and domestication of many Phaseolus species [3,6,10], including Phaseolus lunatus (Lima bean). P. lunatus is a widely consumed grain legume for human nutrition in the USA and other countries. There are many studies on P. vulgaris nodule bacteria [2,15], but very few on P. lunatus symbionts. P. vulgaris forms nodules with Rhizobium (reviewed in [15,21]) while P. lunatus forms them with
∗ Corresponding author at: Centro de Ciencias Genómicas, UNAM, Av. Universidad SN, Chamilpa, Cuernavaca, Morelos CP 62210, Mexico. Tel.: +52 777 329 1692. E-mail addresses: [email protected], [email protected] (E. Martínez-Romero). 0723-2020/$ – see front matter © 2012 Elsevier GmbH. All rights reserved. http://dx.doi.org/10.1016/j.syapm.2012.10.006
bradyrhizobia [27,35]. P. lunatus isolates had not been studied in its Mesoamerican site of origin and domestication [19,20,23], although P. lunatus nodule bacteria were described from Peru, which is another domestication center where new bradyrhizobial groups were found [27]. Legumes were domesticated in association with cereals in different geographical regions worldwide [1]. Simultaneous growth of crops in traditional agriculture had the practical value of providing complementary and diverse nutrition to sustain families and communities. Maize and P. vulgaris (bean) have been grown in association for several thousand years. This agricultural practice is still maintained in Mexico, Peru and North Spain where beans and maize were introduced several hundred years ago. In Mesoamerica, polycultures are called milpa, a slash and burn system, that commonly includes maize, squash, P. vulgaris (common bean), and sometimes perennials. It is estimated that agriculture started in Los Tuxtlas at least 4000 years ago [9]. Evidence suggests that P. vulgaris and maize were probably introduced there from their centers of origin in Central-West Mexico [11]. Rhizobia from Los Tuxtlas have been studied in relation to land use, with forest, secondary forest, pasture and maize crop soils sampled using Macroptilium atropurpureum, P. vulgaris and Vigna unguiculata as trap plants [25]. A large diversity of lineages seemingly corresponding to new Bradyrhizobium species were described in the sampled areas [25]. Polycultures were not studied then. Therefore, the aim of this work was to describe the nodule bacteria both from native P. lunatus and introduced V. unguiculata legumes in polycultures from previously unstudied Los Tuxtlas regions.
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Materials and methods
Nodule bacteria isolation, PCR and sequencing
Composite samples
Root nodules were surface disinfected with 1.5% sodium hypochlorite, and nodule extracts were grown on YM [37] and PY media [36]. Purified colonies were cultured on PY or YM at 28 ◦ C. DNA extraction was with the DNA Isolation Kit for Cells and Tissues (Roche). 16S rRNA, recA and rpoB gene sequences were obtained after PCR, as previously described [17,29]. PCR-RFLP patterns were obtained by restriction of rpoB with the enzymes HindIII, MspI, HhaI and RsaI. Sequencing (Sanger) was performed by Macrogen. Alignments were performed with Clustal W [13] and manually revised. Phylogenetic trees were obtained with neighborjoining [30] and maximum likelihood [8] analyses using MEGA5 [34].
No specific permits were required for the described studies and no endangered or protected species were involved. Three soil samples from each of the 12 farmers from Ocotal Chico and Mazumeapan municipalities (described in [24]) were mixed and used as substrate in small pots. Six different bean types commonly used in Los Tuxtlas, which were identified as distinct by farmers, were surface disinfected and grown. The surface of pots was covered with paper and cotton to prevent contamination. Irrigation was with sterile water. Plants were harvested 26 days after emergence.
95
Phaseolus maechalii (AF_115198) Phaseolus maechalii (AF_115197) Phaseolus sinuatus (AF_115194) Phaseolus salicifolius (AF_115182) 64 Phaseolus sonoensis (AF_115183) Phaseolus itensis (AF_115185) 51 99 Phaseolus itensis (AF_115186) 80 Phaseolus itensis (AF_115184) 73 Phaseolus macvaughii (AF_115200) Phaseolus leptostachyus (AF_115204) 82 Phaseolus micanthus (AF_115205) 99 81 Phaseolus leptostachyus (AF_115202) 73 Phaseolus leptostachyus (AF_115203) 54 Phaseolus augus (AF_115180) 88 Phaseolus pachyrhizoides (AF_115178) 98 Phaseolus augus (AF_115179) Phaseolus bolivianus (AF_115181) Phaseolus lignosus (AF_115177) 99 Phaseolus mollis (AF_115170) 55 Phaseolus lunatus (AF_115171) 95 Phaseolus lunatus (AF_115172) 65 Phaseolus lunatus (AF_115173) 89 Coopik JQ974393 96 Phaseolus tueckheimi (AF_115231) Phaseolus tueckheimii (AF_115248) 100 Phaseolus oligospermus (AF_115233) 99 Phaseolus oligospermus (AF_115234) Phaseolus oligospermus (AF_115232) 99 Phaseolus chiapasanus (AF_115223) 99 Phaseolus chiapasanus (AF_115222) 99 Phaseolus esquincensis (AF_115225) 92 Phaseolus xanthochus (AF_115224) 59 Phaseolus hintonii (AF_115226) Phaseolus zimapanensis (AF_115230) 98 Phaseolus zimapanensis (AF_115228) 93 Phaseolus zimapanensis (AF_115229) 100 Phaseolus acufolius (AF_115143) 100 Phaseolus acufolius (AF_115144) Phaseolus acufolius (AF_069126) 98 Phaseolus vulgaris (AF_115161) 66 Phaseolus vulgaris (AF_115162) 56 Phaseolus vulgaris (AF_115163) 91 Phaseolus vulgaris (AF_115169) 100 Bejuco Colorado JQ974392 Bejuco Negro JQ974390 59 Bejuco Pinto JQ974391 63 Phaseolus albescens (AF_115150) 99 100 Phaseolus albescens (AF_115148) Phaseolus albescens (AF_115152) 56 Phaseolus coccineus (AF_115156) 95 Phaseolus coccineus (AF_115157) 80 Phaseolus coccineus (AF_069130) Phaseolus costaricensis (AF_115147) 80 Phaseolus polyanthus (AF_115151) 100 Phaseolus dumosus (AF_069127) 69 Phaseolus polyanthus (AF_115149) 95 60 Vigna aconifolia (AF_069118) 100 Vigna adenantha (AY_583526) Vigna adenantha (AF_069119) Vigna caacalla (AF_069124) Vigna populnea (AF_115136) Vigna linearis (AF_069123) Vigna peduncularis (AF_069122) Vigna speciosa (AF_069121) 78 Vigna radiata voucher PI 425754 (MONT) (DQ_445738) 98 Vigna radiata voucher BJW0403 (JF_430409) Vigna radiata RMG 62 (HQ_148147) Vigna mungo var. mungo J6 (GQ275366) Vigna angularis var. nipponensis voucher BJW0312 (JN_418209) Vigna angularis voucher BJW0401 (JF_430407) 100 100 Vigna subterranea J2 (GQ_275362) 100 Vigna subterranea J3 (GQ_275363) Vigna minima J12 (GQ_275371) Vigna vexillata J8 (GQ_275368) Chipo Beige JQ974394 Vigna unguiculata subsp. Sesquipedalis (EU_727148) 100 54 Vigna unguiculata voucher Vu40 (GQ_411778) 74 Vigna unguiculata culvar HH-WD8 (FJ_176926) Vigna unguiculata IT87D-885.2 (AY_748431) Chipo Negro JQ974395 Vigna vexillata voucher BJW0407 (JF_430413) 79 97
100
ITS NJ Tamura-Nei (757 nt)
61
Phaseolus lunatus
100
Phaseolus vulgaris
100
76 98 100 53
100 73
Vigna unguiculata
0.05
Fig. 1. Phylogenetic relationships of Phaseolus species based on ITS sequences. Sequences obtained in this work are shown bold. Bootstrap values >70% are indicated at the nodes. Bar, 5 nt substitutions per 100 nt.
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Plant DNA extraction and ITS analysis To identify the Phaseolus species, a molecular approach was followed because only native seeds were available. Young leaves from the local bean plants germinated in the laboratory were macerated with liquid nitrogen and DNA was extracted with the same Roche DNA isolation kit. The internal transcribed spacer (ITS) was amplified with ITS primers PhaseoF and PhaseoR, and PCR conditions were as previously described [6]. PCR products were Sanger sequenced. Phylogenetic analyses were performed as described above.
Accession numbers The GenBank accession numbers for the recA gene sequences determined in this study are JQ966948–JQ966967 and JX943615– JX943617. The nodZ and nifH gene sequences have been assigned accession numbers JX645470–JX645478 and JX943618–JX943622, respectively.
Results Beans from Los Tuxtlas were classified into three different species, P. vulgaris, P. lunatus and V. unguiculata, according to ITS sequences (Fig. 1). Morphologically, P. lunatus seeds from Los Tuxtlas resembled the most common type of P. lunatus seeds grown in Southern Mexico [J. Martínez-Castillo, personal communication]. By plant trap experiments with local P. lunatus and V. unguiculata seeds, nodule bacteria were sampled from milpa soils from a region of Los Tuxtlas where a large diversity of crops have been preserved [4]. P. lunatus and V. unguiculata isolates were slow growing bacteria. A total of 157 bradyrhizobia isolates were obtained, 98 from P. lunatus and 59 from Vigna (Supplementary Table S1). Isolates from Vigna were grouped into eight ERIC profiles, with two of them representing almost 80% of the profiles. From P. lunatus isolates, 52 ERIC profiles were identified and most of them were represented only by a single strain, and only three profiles had five or more isolates. Only one ERIC profile was found in common among isolates from both legumes. PCR-RFLP patterns of rpoB genes, previously used to characterize bradyrhizobial isolates from P. lunatus in Peru [27], showed four distinct patterns in Vigna isolates, and two of them included 95% of the isolates. The dominant ERIC profiles were included in these rpoB-based genotypes. On the other hand, P. lunatus isolates corresponded to eight patterns by rpoB PCR-RFLP analysis, one of which had 91% of the isolates and most ERIC profiles. The remaining rpoB patterns had one or two isolates. It is known that ERIC profiles may distinguish different strains within a single species that may be represented by single rpoB gene sequences. The bradyrhizobial populations of V. unguiculata and P. lunatus were different and only one rpoB PCR-RFLP pattern was shared. recA gene phylogenies have been used to describe bradyrhizobial diversity [38], and the current study showed that, on this basis, most P. lunatus symbionts were closely related to previously described isolates from Los Tuxtlas (Fig. 2). Bradyrhizobial isolates from P. lunatus corresponded to reported genospecies TUXTLAS-17, TUXTLAS-4, TUXTLAS-20 and TUXTLAS-2, to three novel genospecies (TUXTLAS-33, -34 and -35) and to Bradyrhizobium japonicum (Fig. 2). P. lunatus isolate Pop235 was closely related to Inga isolate Inga3-1a, while Pop363 was related to Inga32d. Inga species are native trees in Los Tuxtlas forest and their nodule bacteria were isolated and characterized previously [25]. Mexican isolates from P. lunatus were different to those from Peru (Fig. 2). Most isolates from Vigna grouped with Bradyrhizobium
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elkanii, whereas others grouped with genospecies TUXTLAS-17 and TUXTLAS-21. Representatives of all rpoB restriction patterns from Vigna and P. lunatus isolates were tested in the laboratory for nodulation of both legumes (Supplementary Table S3). All P. lunatus isolates could nodulate and fix nitrogen in Vigna and P. lunatus. Vigna isolates were capable of nodulating both legumes but only one (Pop352) could fix nitrogen in P. lunatus, which indicated that P. lunatus is a more restricted host while Vigna is considered promiscuous [14,28]. B. japonicum USDA 110 and B. elkanii USDA 76 nodulated and fixed nitrogen in Vigna but failed to nodulate in P. lunatus. B. elkanii USDA 76 formed only root bumps in P. lunatus. Additionally some bradyrhizobial strains previously described, representing native TUXTLAS genospecies [25] were tested in nodulation assays in the laboratory. All tested strains were found to nodulate P. lunatus with some of them fixing nitrogen (Supplementary Table S2). Based on nodZ sequences, most V. unguiculata isolates corresponded to nodZ gene group III (Fig. 3), according to a classification proposed earlier [33]. Group III is considered a pantropical ancient group of bradyrhizobial nodulation genes and most Vigna bradyrhizobia from Africa possess these genes [33]. Pop352, the only Vigna isolate able to fix nitrogen with P. lunatus, clustered in nodZ clade V together with Pop367 from P. lunatus and other bradyrhizobial isolates previously isolated from Los Tuxtlas (Fig. 3). Group V was proposed earlier based on a single sequence from a Brazilian Lupinus bradyrhizobia [33]. In this study, it was found that bradyrhizobia from Los Tuxtlas, as well as Brazilian strains isolated from different plants, had nodZ genes corresponding to group V, which seems to have an American origin. nifH gene phylogeny also separated isolates from groups III and V (Supplementary Fig. S1) and showed the same close relationship between Brazilian group V isolates and bradyrhizobia from Los Tuxtlas.
Discussion As polycultures in Mexico commonly include P. vulgaris it was surprising to find P. lunatus and V. unguiculata in milpas from Los Tuxtlas. In comparison to P. vulgaris and V. unguiculata, P. lunatusnodulated roots with Los Tuxtlas rhizobia exhibited the highest levels of acetylene reduction activity (not shown) and highest symbiont diversity. Of all Phaseolus species, P. lunatus seems to have the broadest geographical distribution. It is widely cultivated in the tropics and subtropics, possibly in relation to its capacity to form nodules with bradyrhizobia that are abundant in tropical soils [22,25]. Wild P. lunatus are found in Mexico [19,20,23] and have been described in Los Tuxtlas, but wild P. vulgaris have not been found in this area [Jaime Martínez-Castillo, personal communication]. Mexico, together with Peru, is a P. lunatus domestication site [10,23,27] but P. lunatus cultivars grown in Peru and Mexico are different and their bradyrhizobial symbionts seem to be specific for each domestication site ([27] and this work). Supposedly, there is a domestication origin of P. lunatus in Southern Mexico but it has not been located yet. Most P. lunatus isolates were found to correspond to genospecies TUXTLAS-17 that was reported as abundant in crop fields [25,26]. Genospecies TUXTLAS-20 and TUXTLAS-22 were less frequently isolated from P. lunatus, which corresponds to the observation in previous studies that they were less abundant in crop fields [25,26]. P. lunatus forms nodules with native bradyrhizobia in tropical Los Tuxtlas soils, whereas P. vulgaris symbionts are exotic and have been introduced to Los Tuxtlas [25,26]. Therefore, it is concluded that P. lunatus is a more adequate crop legume for Los Tuxtlas than P. vulgaris.
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Bradyrhizobium genosp. TUXTLAS-4 85v (FJ970368) Bradyrhizobium genosp. TUXTLAS-4 Inga3-1a (FJ970372) Pop235 [Phaseolus lunatus] (JQ966951) Bradyrhizobium genosp. TUXTLAS-5 1246v (FJ970345) Bradyrhizobium genosp. TUXTLAS-1 1681v (FJ970354) 99 Bradyrhizobium genosp. TUXTLAS-1 1041v (FJ970331) Bradyrhizobium genosp. TUXTLAS-8 35v (FJ970361) Bradyrhizobium genosp. TUXTLAS-8 42v (FJ970363) 99 Bradyrhizobium genosp. TUXTLAS-15 1028v (FJ970329) Bradyrhizobium genosp. TUXTLAS-10 1288v (FJ970346) 99 Bradyrhizobium genosp. TUXTLAS-10 1736m (FJ970357) Bradyrhizobium genosp. TUXTLAS-11 8v (FJ970369) 92 84 Bradyrhizobium genosp. TUXTLAS-11 44m (FJ970364) B. iriomotense EK05 (AB300996) Bradyrhizobium genosp. TUXTLAS-12 1648v (FJ970353) 72 Bradyrhizobium genosp. TUXTLAS-12 1784m (FJ970358) 98 Pop370 [Phaseolus lunatus] (JQ966967) Bradyrhizobium genosp. TUXTLAS-7 1018v (FJ970327) 99 Bradyrhizobium genosp. TUXTLAS-7 1595v (FJ970351) Bradyrhizobium genosp. TUXTLAS-6 1609v (FJ970352) 99 Bradyrhizobium genosp. TUXTLAS-6 1020v (FJ970328) B. yuanmingense LMTR 28 [Phaseolus lunatus] (AY591573) B. liaoningense LMG18230 (AY591564) Bradyrhizobium genosp. TUXTLAS-9 1031v (FJ970330) Bradyrhizobium genosp. TUXTLAS-14 1064v (FJ970333) B. canariense BTA-1 (AY591553) B. betae LMG21987 (AB353734) B. japonicum DSMZ30131 (AY591555) Pop363 [Phaseolus lunatus] (JQ966957) 99 95 B. japonicum Inga3-2d (FJ970374) 99 Pop231 [Phaseolus lunatus] (JQ966949) Pop271 [Phaseolus lunatus] (JQ966953) Pop306 [Vigna unguiculata] (JQ966955) 99 B. elkanii USDA76 (AY591568) 99 Pop255 [Phaseolus lunatus] (JQ966951) Pop253 [Phaseolus lunatus] (JQ966952) 93 Bradyrhizobium genosp. TUXTLAS-20 1017v (FJ970326) 99 Bradyrhizobium genosp. TUXTLAS-20 66v (FJ970367) Pop338 [Vigna unguiculata] (JQ966960) 95 82 Bradyrhizobium genosp. TUXTLAS-21 1325v (FJ970348) Bradyrhizobium genosp. TUXTLAS-18 1090v (FJ970335) 99 Pop234 [Phaseolus lunatus] (JQ966964) Bradyrhizobium genosp. TUXTLAS-22 1234v (FJ970344) 76 Pop321 [Vigna unguiculata] (JQ966956) 98 Pop304 [Vigna unguiculata] (JQ966959) Bradyrhizobium sp. Tpma1 -5 (EU099330 ) 71 Bradyrhizobium genosp. TUXTLAS-17 1110v (FJ970339) Bradyrhizobium genosp. TUXTLAS-17 109m (FJ970336) 91 78 Pop352 [Vigna unguiculata] (JQ966962) Bradyrhizobium sp. Dr3b-11 (EU099332) Bradyrhizobium sp. Cj3-3 (EU099333) Pop366 [Phaseolus lunatus] (JQ966958) Pop229 [Phaseolus lunatus] (JQ966948) Pop238 [Phaseolus lunatus] (JQ966950) Pop288 [Phaseolus lunatus] (JQ966954) Pop263 [Phaseolus lunatus] (JQ966966) Bradyrhizobium genosp. TUXTLAS-17 1730m (FJ970356) Pop218 [Phaseolus lunatus] (JQ966963) Pop342 [Vigna unguiculata] (JQ966961) Bradyrhizobium sp. LMTR 13 [ Phaseolus lunatus] (JX943615) Bradyrhizobium sp. LMTR 3 [ Phaseolus lunatus] (JX943616) Bradyrhizobium sp. LMTR 21 [ Phaseolus lunatus] (JX943617) Rhodopseudomomas palustris CGA009 (BX571963) Nitrobacter hamburgensis X14 (CP000319) 89 99
73
0.01
76
88
71
97
TUXTLAS-4
TUXTLAS-33
B. japonicum TUXTLAS-35 B. elkanii TUXTLAS-34
TUXTLAS-21 TUXTLAS-22
TUXTLAS-17
Fig. 2. recA gene phylogenetic relationships of Bradyrhizobium from Phaseolus lunatus and Vigna unguiculata isolated in this work (Pop strains). Geno(species) affiliations of Pop strains are indicated on the right-hand side. Novel genospecies are shown in bold. Bootstrap values >70% are indicated at the nodes. Bar, 1 nt substitution per 100 nt.
Vigna includes more than 200 species distributed throughout the tropics, some of them were domesticated and are widely used in tropical agriculture. The geographical origin of cowpea (V. unguiculata) is Africa and the bacteria associated with this legume have been reported as bradyrhizobia and some fast growing rhizobia. Cowpea bradyrhizobia isolated in Africa [33,39], China [40,41] and Brazil [42] were identified as B. elkanii, B. japonicum, Bradyrhizobium liaoningense, Bradyrhizobium yuanmingense and other unnamed Bradyrhizobium genospecies. In this work, the symbionts of Vigna were identified as B. elkanii and two previously reported genospecies (TUXTLAS-17 and TUXTLAS-21) [25,26]. Symbionts of V. unguiculata had already been studied using Vigna as
a trap plant from soils of another region in Los Tuxtlas [25]. B. elkanii and the three novel genospecies found here were not previously reported in that study [25,26]. This indicates that there is geographical heterogeneity of soil bacteria, which was not unexpected in view of the different landscape and environmental conditions therein [5,12]. However, it is remarkable that rhizobia similar to those found in Los Tuxtlas also exist in Central America and a microbiological corridor comprising such regions has been identified [16]. Approximately 40% of the forest cover has been lost in Los Tuxtlas [32] and soils are nutrient depleted [7]. Knowledge of the legume species grown in the Los Tuxtlas milpas is essential for
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99 ORS170 [Faidherbia albida, Senegal] (AJ431114) 98 SEMIA6160 [Albizia lebeck, Brazil] (HQ259524)
SEMIA6152 [Calopogonium sp., Brazil] (HQ259523) USDA3259 [Phaseolus lunatus, USA] (AM168393) R5 [Vigna unguiculata, Botswana] (EU364746) Pop321[Vigna sp.] (JX645470) 99 Pop304 [Vigna sp.] (JX645472)
99
81 USDA3139 [Astragalus canadensis, USA] (AJ431120)
SEMIA695 [Neonotonia wightii, Australia] (HQ259512) SEMIA6053 [Clitoria ternatea, Malaysia] (HQ259501) 99
SEMIA5026 [Glycine max, Thailand] (HQ259497) USDA76 [Glycine max, USA] (AM168395)
III.1
99 SEMIA5019 [Glycine max, Brazil] (HQ259496)
USDA135 [Glycine max, USA] (AJ431119) ORS130 [Faidherbia albida, Senegal] (AJ431111) SEMIA6028 [Desmodium uncinatum, Zimbabwe] (HQ259518)
98 82
SEMIA6146 [Centrosema sp., Brazil] (HQ259522)
93
SEMIA6148 [Neonotonia wightii, Brazil] (HQ259505) 98 Pop342 [Vigna sp., Mexico] (JX645473) SEMIA6069 [Leucaena leucocephala, Brazil] (HQ259502)
III.2
99
99
III.1 1234v [Vigna unguiculata, Mexico] (JX645477) SEMIA6101 [Dalbergia nigra, Brazil] (HQ259504)
99
CH2493 [Lupinus paraguariensis, Brazil] (AM168382) 96 SEMIA6099 [Dimorphandra exaltata, Brazil] (HQ259521)
SEMIA6440 [Arachis pintoi, Brazil] (HQ259509)
99
V
109m [Macroptilium atropurpureum, Mexico] (JX645478) 99
Pop352 [Vigna sp.] (JX645474) Pop367 [Phaseolus lunatus] (JX645471) 107m [Macroptilium atropurpureum, Mexico] (JX645476)
99 1017v [Vigna unguiculata, Mexico] (JX645475)
IV 99 88
II
VII A. caulinodans ORS571 (AP009384)
0.1 Fig. 3. Maximum likelihood phylogeny of nodZ gene sequences. Sequences of strains isolated from Los Tuxtlas are shown in bold. Those obtained in this study have the Pop prefix. nodZ clades are indicated with Roman numerals. Bootstrap values >70% are indicated at the nodes. Bar, 1 nt substitution per 10 nt.
determining which rhizobia could be used as inoculants for fulfilling our agro-ecological project aimed at maintaining soil fertility through symbiosis with native microorganisms. Acknowledgements To the memory of Jose Luis Blanco who provided seeds and contacts with farmers and was a wonderful friend. This work was part of the BioPop Project, FOMIX-CONACYT-Veracruz (Fondos Mixtos de Fomento a la Investigación Científica y Tecnológica – Consejo Nacional de Ciencia y Tecnología) 94427. We thank farmers in Ocotal Chico and Mazumeapan: Marcelino Gutiérrez Matias, Angel Gutiérrez Zacarías, Leopoldo González Mateo, Wenseslao Duarte González, Juan Santiago, Pedro González
Santiago, Roberto García Arias, Josafath Gutiérrez Matías, León Hernández Gutiérrez, Angel González Santiago, Isaías González Rodríguez, Diego González Mateo. To Luis E. Servín for providing primers, to Isis de la Rosa for technical assistance and to Michael Dunn for reading the paper. Funding was provided by UNAMPAPIIT (Universidad Nacional Autónoma de México – Programa de Apoyo a Proyectos de Investigación e Innovación Tecnológica) IN205412. Appendix A. Supplementary data Supplementary data associated with this article can be found, in the online version, at http://dx.doi.org/10.1016/j.syapm. 2012.10.006.
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References [1] Abbo, S., Saranga, Y., Peleg, Z., Kerem, Z., Lev-Yadun, S., Gopher, A. (2009) Reconsidering domestication of legumes versus cereals in the ancient near east. Q. Rev. Biol. 84, 29–50. [2] Aserse, A.A., Rasanen, L.A., Assefa, F., Hailemariam, A., Lindstrom, K. (2012) Phylogeny and genetic diversity of native rhizobia nodulating common bean (Phaseolus vulgaris L.) in Ethiopia. Syst. Appl. Microbiol. 35, 120–131. [3] Bitocchi, E., Nanni, L., Bellucci, E., Rossi, M., Giardini, A., Zeuli, P.S., Logozzo, G., Stougaard, J., McClean, P., Attene, G., Papa, R. (2012) Mesoamerican origin of the common bean (Phaseolus vulgaris L.) is revealed by sequence data. Proc. Natl. Acad. Sci. U.S.A. 109, E788–E796. [4] Blanco, J.L. (2006) Erosión de la agrodiversidad en la milpa zoque popoluca de Soteapan: Xutuchincon y Aktevet. Ph.D. Thesis, Universidad Iberoamericana, Mexico City. [5] Campos, C. (2004) El suelo. In: Guevara, S., Laborde, J., Sanchez-Ríos, G. (Eds.), Los Tuxtlas, El paisaje de la Sierra, Instituto de Ecología, A.C. & European Union, Xalapa, pp. 181–192. [6] Delgado-Salinas, A., Turley, T., Richman, A., Lavin, M. (1999) Phylogenetic analysis of the cultivated and wild species of Phaseolus (Fabaceae). Syst. Bot. 24, 438–460. [7] Eilittä, M., Arteaga, F., Diaz, M., Guerrero, C., Herrera, B., Narvaez, G., Paré, L., Robles, G., Triomphe, B. (2004) Cultivating maize with Mucuna in the Los Tuxtlas region of South-Eastern Veracruz, Mexico. In: Eilittä, M., Mureithi, J., Derpsch, R. (Eds.), Green Manure/Cover Crop Systems of Smallholder Farmers, Springer, Dordrecht, Netherlands, pp. 99–127. [8] Felsenstein, J. (1981) Evolutionary trees from DNA sequences: a maximum likelihood approach. J. Mol. Evol. 17, 368–376. [9] Goman, M., Byrne, R. (1998) A 5000-year record of agriculture and tropical forest clearance in the Tuxtlas, Veracruz, Mexico. Holocene 8, 83–89. [10] Kaplan, L. (1965) Archeology and domestication in American Phaseolus (Beans). Econ. Bot. 19, 358–368. [11] Kwak, M., Kami, J.A., Gepts, P. (2009) The putative mesoamerican domestication center of Phaseolus vulgaris is located in the Lerma-Santiago basin of Mexico. Crop Sci. 49, 554–563. [12] Laborde, J. (2004) Los Habitantes. In: Guevara, S., Laborde, J., Sanchez-Ríos, G. (Eds.), Los Tuxtlas, El paisaje de la Sierra, Instituto de Ecología, A.C. & European Union, Xalapa, pp. 61–84. [13] Larkin, M.A., Blackshields, G., Brown, N.P., Chenna, R., McGettigan, P.A., McWilliam, H., Valentin, F., Wallace, I.M., Wilm, A., Lopez, R., Thompson, J.D., Gibson, T.J., Higgins, D.G. (2007) Clustal W and Clustal X version 2.0. Bioinformatics 23, 2947–2948. [14] Lewin, A., Rosenberg, C., Meyer, H., Wong, C.H., Nelson, L., Manen, J.F., Stanley, J., Dowling, D.N., Denarie, J., Broughton, W.J. (1987) Multiple host-specificity loci of the broad host-range Rhizobium sp. NGR234 selected using the widely compatible legume Vigna unguiculata. Plant Mol. Biol. 8, 447–459. ˜ [15] López-Guerrero, M.G., Ormeno-Orrillo, E., Velázquez, E., Rogel, M.A., Acosta, J.L., Gónzalez, V., Martínez, J., Martínez-Romero, E. (2012) Rhizobium etli taxonomy revised with novel genomic data and analyses. Syst. Appl. Microbiol. 35, 353–358. [16] Lopez-Lopez, A., Rogel-Hernandez, M.A., Barois, I., Ortiz Ceballos, A.I., Martinez, ˜ J., Ormeno-Orrillo, E., Martínez-Romero, E. (2012) Rhizobium grahamii sp. nov., from nodules of Dalea leporina, Leucaena leucocephala and Clitoria ternatea, and Rhizobium mesoamericanum sp. nov., from nodules of Phaseolus vulgaris, siratro, cowpea and Mimosa pudica. Int. J. Syst. Evol. Microbiol. 62, 2264–2271. ˜ [17] Lloret, L., Ormeno-Orrillo, E., Rincón, R., Martínez-Romero, J., Rogel-Hernández, M.A., Martínez-Romero, E. (2007) Ensifer mexicanus sp. nov. a new species nodulating Acacia angustissima (Mill.) Kuntze in Mexico. Syst. Appl. Microbiol. 30, 280–290. [18] Maingi, J.M., Gitonga, N.M., Shisanya, C.A., Hornetz, B., Muluvi, G.M. (2006) Population levels of indigenous bradyrhizobia nodulating promiscuous soybean in two Kenyan soils of the semi-arid and semi-humid agroecological zones. J. Agric. Rural Dev. Trop. Subtrop. 107, 149–159. [19] Martinez-Castillo, J., Zizumbo-Villarreal, D., Gepts, P., Delgado-Valerio, P., Colunga-GarciaMarin, P. (2006) Structure and genetic diversity of wild populations of Lima bean (Phaseolus lunatus L.) from the Yucatan Peninsula, Mexico. Crop Sci. 46, 1071–1080. [20] Martínez-Castillo, J., Zizumbo-Villarreal, D., Perales-Rivera, H., ColungaGarcíamarin, P. (2004) Intraspecific diversity and morpho-phenological variation in Phaseolus lunatus L. from the Yucatan Peninsula, Mexico. Econ. Bot. 58, 354–380. [21] Martínez-Romero, E. (2003) Diversity of Rhizobium-Phaseolus vulgaris symbiosis: overview and perspectives. Plant Soil 252, 11–23. [22] Moreira, F.M., Haukka, K., Young, J.P. (1998) Biodiversity of rhizobia isolated from a wide range of forest legumes in Brazil. Mol. Ecol. 7, 889–895.
[23] Motta-Aldana, J.R., Serrano-Serrano, M.L., Hernandez-Torres, J., CastilloVillamizar, G., Debouck, D.G., Chacon, M.I. (2010) Multiple origins of Lima bean landraces in the Americas: evidence from chloroplast and nuclear DNA polymorphisms. Crop Sci. 50, 1773–1787. [24] Negrete-Yankelevich, S., Maldonado-Mendoza, I., Lázaro-Castellanos, J., Sangabriel-Conde, W., Martínez-Álvarez, J. Arbuscular mycorrhizal root colonization and soil P availability are positively related to agrodiversity in Mexican maize polycultures. Biol. Fertil. Soils, http://dx.doi.org/10.1007/s00374-0120710-5 ˜ [25] Ormeno-Orrillo, E., Rogel-Hernandez, M.A., Lloret, L., Lopez-Lopez, A., Martinez, J., Barois, I., Martínez-Romero, E. (2012) Change in land use alters the diversity and composition of Bradyrhizobium communities and led to the introduction of Rhizobium etli into the tropical rain forest of Los Tuxtlas (Mexico). Microb. Ecol. 63, 822–834. ˜ E., Rogel, M.A., Lloret, L., López-López, A., Martínez, J., Vin[26] Ormeno-Orrillo, uesa, P., Martinez Romero, E. (2009) Rhizobial diversity in different land use systems in the rain forest of Los Tuxtlas, Mexico. In: Barois, I., Huising, E.J., Ojosth, P., Trejo, D., De los Santos, M. (Eds.), Below-Ground Biodiversity in Sierra de Santa Marta, Los Tuxtlas, Veracruz, Mexico, Instituto de Ecología, Xalapa, pp. 65–84. ˜ ˜ [27] Ormeno-Orrillo, E., Vinuesa, P., Zúniga-Dávila, D., Martínez-Romero, E. (2006) Molecular diversity of native bradyrhizobia isolated from Lima bean (Phaseolus lunatus L.) in Peru. Syst. Appl. Microbiol. 29, 253–262. [28] Ren da, W., Chen, W.F., Sui, X.H., Wang, E.T., Chen, W.X. (2011) Rhizobium vignae sp. nov., a symbiotic bacterium isolated from multiple legume species. Int. J. Syst. Evol. Microbiol. 61, 580–586. [29] Rincón-Rosales, R., Lloret, L., Ponce, E., Martínez-Romero, E. (2009) Rhizobia with different symbiotic efficiencies nodulate Acaciella angustissima in Mexico, including Sinorhizobium chiapanecum sp. nov. which has common symbiotic genes with Sinorhizobium mexicanum. FEMS Microbiol. Ecol. 67, 103–117. [30] Saitou, N., Nei, M. (1987) The neighbor-joining method: a new method for reconstructing phylogenetic trees. Mol. Biol. Evol. 4, 406–425. [31] Silva, C., Vinuesa, P., Eguiarte, L.E., Martínez-Romero, E., Souza, V. (2003) Rhizobium etli and Rhizobium gallicum nodulate common bean (Phaseolus vulgaris) in a traditionally managed milpa plot in Mexico: population genetics and biogeographic implications. Appl. Environ. Microbiol. 69, 884–893. [32] Soto, M. (2004) El clima. In: Guevara, S., Laborde, J., Sanchez-Ríos, G. (Eds.), Los Tuxtlas., El paisaje de la Sierra, Instituto de Ecología, A.C. & European Union, Xalapa, pp. 195–199. [33] Steenkamp, E.T., Stepkowski, T., Przymusiak, A., Botha, W.J., Law, I.J. (2008) Cowpea and peanut in southern Africa are nodulated by diverse Bradyrhizobium strains harboring nodulation genes that belong to the large pantropical clade common in Africa. Mol. Phylogenet. Evol. 48, 1131–1144. [34] Tamura, K., Peterson, D., Peterson, N., Stecher, G., Nei, M., Kumar, S. (2011) MEGA5: molecular evolutionary genetics analysis using maximum likelihood, evolutionary distance, and maximum parsimony methods. Mol. Biol. Evol. 28, 2731–2739. [35] Thies, J.E., Bohlool, B.B., Singleton, P.W. (1991) Subgroups of the cowpea miscellany: symbiotic specificity within Bradyrhizobium spp. for Vigna unguiculata, Phaseolus lunatus, Arachis hypogaea, and Macroptilium atropurpureum. Appl. Environ. Microbiol. 57, 1540–1545. [36] Toledo, I., Lloret, L., Martínez-Romero, E. (2003) Sinorhizobium americanum sp. nov., a new Sinorhizobium species nodulating native Acacia spp. in Mexico. Syst. Appl. Microbiol. 26, 54–64. [37] Vincent, J.M. 1970 A Manual for the Practical Study of Root Nodule Bacteria, Blackwell Scientific Publications, Oxford. [38] Vinuesa, P., Silva, C., Werner, D., Martínez-Romero, E. (2005) Population genetics and phylogenetic inference in bacterial molecular systematics: the roles of migration and recombination in Bradyrhizobium species cohesion and delineation. Mol. Phylogenet. Evol. 34, 29–54. [39] Wolde-Meskel, E., Terefework, Z., Frostegard, A., Lindstrom, K. (2005) Genetic diversity and phylogeny of rhizobia isolated from agroforestry legume species in southern Ethiopia. Int. J. Syst. Evol. Microbiol. 55, 1439–1452. [40] Zhang, W.T., Yang, J.K., Yuan, T.Y., Zhou, J.C. (2007) Genetic diversity and phylogeny of indigenous rhizobia from cowpea [Vigna unguiculata (L.) Walp.]. Biol. Fertil. Soils 44, 201–210. [41] Zhang, Y.F., Wang, E.T., Tian, C.F., Wang, F.Q., Han, L.L., Chen, W.F., Chen, W.X. (2008) Bradyrhizobium elkanii, Bradyrhizobium yuanmingense and Bradyrhizobium japonicum are the main rhizobia associated with Vigna unguiculata and Vigna radiata in the subtropical region of China. FEMS Microbiol. Lett. 285, 146–154. [42] Zilli, J.E., Valisheski, R.R., Freire, F.R., Neves, M.C.P., Rumjanek, N.G. (2004) Assessment of cowpea rhizobium diversity in Cerrado areas of Northeastern Brazil. Braz. J. Microbiol. 35, 281–287.
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Systematic and Applied Microbiology journal homepage: www.elsevier.de/syapm
Native bradyrhizobia from Los Tuxtlas in Mexico are symbionts of Phaseolus lunatus (Lima bean) a ˜ Aline López-López a , Simoneta Negrete-Yankelevich b , Marco A. Rogel a , Ernesto Ormeno-Orrillo , a a,∗ Julio Martínez , Esperanza Martínez-Romero a b
Centro de Ciencias Genómicas, UNAM, Cuernavaca, Morelos CP 62210, Mexico Red de Ecología Funcional, INECOL, Xalapa, Veracruz, Mexico
a r t i c l e
i n f o
Article history: Received 6 September 2012 Received in revised form 12 October 2012 Accepted 15 October 2012 Keywords: Symbiosis Rain forest Nitrogen fixation
a b s t r a c t Los Tuxtlas is the northernmost rain forest in North America and is rich in Bradyrhizobium with an unprecedented number of novel lineages. ITS sequence analysis of legumes in polycultures from Los Tuxtlas led to the identification of Phaseolus lunatus and Vigna unguiculata in addition to Phaseolus vulgaris as legumes associated with maize in crops. Bacterial diversity of isolates from nitrogen-fixing nodules of P. lunatus and V. unguiculata was revealed using ERIC-PCR and PCR-RFLP of rpoB genes, and sequencing of recA, nodZ and nifH genes. P. lunatus and V. unguiculata nodule bacteria corresponded to bradyrhizobia closely related to certain native bradyrhizobia from the Los Tuxtlas forest and novel groups were found. This is the first report of nodule bacteria from P. lunatus in its Mesoamerican site of origin and domestication. © 2012 Elsevier GmbH. All rights reserved.
Introduction Nitrogen (N)-fixing bacteria from different genera (collectively designated rhizobia) form nodules on legumes and allow plants to grow in N deficient soils, such as rain forest soil that becomes N deficient after a few years of tilling and cropping. Legumes and rhizobia coexist at their sites of origin and domestication, for example, there is a large diversity of bradyrhizobia and rhizobia nodulating soybean in Asia. In contrast, in Africa and Mexico there is a need to inoculate introduced soybeans with Bradyrhizobium, since soybeans are not native and there are no soybean nodulating bacteria. There has been an agricultural selection of soybeans that could form nodules with native African bradyrhizobia but this has had limited success [18]. In Mexico, there is a large diversity of Phaseolus vulgaris and Phaseolus coccineus nodulating bacteria [21,31] because Mexico is the center of origin and domestication of many Phaseolus species [3,6,10], including Phaseolus lunatus (Lima bean). P. lunatus is a widely consumed grain legume for human nutrition in the USA and other countries. There are many studies on P. vulgaris nodule bacteria [2,15], but very few on P. lunatus symbionts. P. vulgaris forms nodules with Rhizobium (reviewed in [15,21]) while P. lunatus forms them with
∗ Corresponding author at: Centro de Ciencias Genómicas, UNAM, Av. Universidad SN, Chamilpa, Cuernavaca, Morelos CP 62210, Mexico. Tel.: +52 777 329 1692. E-mail addresses: [email protected], [email protected] (E. Martínez-Romero). 0723-2020/$ – see front matter © 2012 Elsevier GmbH. All rights reserved. http://dx.doi.org/10.1016/j.syapm.2012.10.006
bradyrhizobia [27,35]. P. lunatus isolates had not been studied in its Mesoamerican site of origin and domestication [19,20,23], although P. lunatus nodule bacteria were described from Peru, which is another domestication center where new bradyrhizobial groups were found [27]. Legumes were domesticated in association with cereals in different geographical regions worldwide [1]. Simultaneous growth of crops in traditional agriculture had the practical value of providing complementary and diverse nutrition to sustain families and communities. Maize and P. vulgaris (bean) have been grown in association for several thousand years. This agricultural practice is still maintained in Mexico, Peru and North Spain where beans and maize were introduced several hundred years ago. In Mesoamerica, polycultures are called milpa, a slash and burn system, that commonly includes maize, squash, P. vulgaris (common bean), and sometimes perennials. It is estimated that agriculture started in Los Tuxtlas at least 4000 years ago [9]. Evidence suggests that P. vulgaris and maize were probably introduced there from their centers of origin in Central-West Mexico [11]. Rhizobia from Los Tuxtlas have been studied in relation to land use, with forest, secondary forest, pasture and maize crop soils sampled using Macroptilium atropurpureum, P. vulgaris and Vigna unguiculata as trap plants [25]. A large diversity of lineages seemingly corresponding to new Bradyrhizobium species were described in the sampled areas [25]. Polycultures were not studied then. Therefore, the aim of this work was to describe the nodule bacteria both from native P. lunatus and introduced V. unguiculata legumes in polycultures from previously unstudied Los Tuxtlas regions.
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Materials and methods
Nodule bacteria isolation, PCR and sequencing
Composite samples
Root nodules were surface disinfected with 1.5% sodium hypochlorite, and nodule extracts were grown on YM [37] and PY media [36]. Purified colonies were cultured on PY or YM at 28 ◦ C. DNA extraction was with the DNA Isolation Kit for Cells and Tissues (Roche). 16S rRNA, recA and rpoB gene sequences were obtained after PCR, as previously described [17,29]. PCR-RFLP patterns were obtained by restriction of rpoB with the enzymes HindIII, MspI, HhaI and RsaI. Sequencing (Sanger) was performed by Macrogen. Alignments were performed with Clustal W [13] and manually revised. Phylogenetic trees were obtained with neighborjoining [30] and maximum likelihood [8] analyses using MEGA5 [34].
No specific permits were required for the described studies and no endangered or protected species were involved. Three soil samples from each of the 12 farmers from Ocotal Chico and Mazumeapan municipalities (described in [24]) were mixed and used as substrate in small pots. Six different bean types commonly used in Los Tuxtlas, which were identified as distinct by farmers, were surface disinfected and grown. The surface of pots was covered with paper and cotton to prevent contamination. Irrigation was with sterile water. Plants were harvested 26 days after emergence.
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Phaseolus maechalii (AF_115198) Phaseolus maechalii (AF_115197) Phaseolus sinuatus (AF_115194) Phaseolus salicifolius (AF_115182) 64 Phaseolus sonoensis (AF_115183) Phaseolus itensis (AF_115185) 51 99 Phaseolus itensis (AF_115186) 80 Phaseolus itensis (AF_115184) 73 Phaseolus macvaughii (AF_115200) Phaseolus leptostachyus (AF_115204) 82 Phaseolus micanthus (AF_115205) 99 81 Phaseolus leptostachyus (AF_115202) 73 Phaseolus leptostachyus (AF_115203) 54 Phaseolus augus (AF_115180) 88 Phaseolus pachyrhizoides (AF_115178) 98 Phaseolus augus (AF_115179) Phaseolus bolivianus (AF_115181) Phaseolus lignosus (AF_115177) 99 Phaseolus mollis (AF_115170) 55 Phaseolus lunatus (AF_115171) 95 Phaseolus lunatus (AF_115172) 65 Phaseolus lunatus (AF_115173) 89 Coopik JQ974393 96 Phaseolus tueckheimi (AF_115231) Phaseolus tueckheimii (AF_115248) 100 Phaseolus oligospermus (AF_115233) 99 Phaseolus oligospermus (AF_115234) Phaseolus oligospermus (AF_115232) 99 Phaseolus chiapasanus (AF_115223) 99 Phaseolus chiapasanus (AF_115222) 99 Phaseolus esquincensis (AF_115225) 92 Phaseolus xanthochus (AF_115224) 59 Phaseolus hintonii (AF_115226) Phaseolus zimapanensis (AF_115230) 98 Phaseolus zimapanensis (AF_115228) 93 Phaseolus zimapanensis (AF_115229) 100 Phaseolus acufolius (AF_115143) 100 Phaseolus acufolius (AF_115144) Phaseolus acufolius (AF_069126) 98 Phaseolus vulgaris (AF_115161) 66 Phaseolus vulgaris (AF_115162) 56 Phaseolus vulgaris (AF_115163) 91 Phaseolus vulgaris (AF_115169) 100 Bejuco Colorado JQ974392 Bejuco Negro JQ974390 59 Bejuco Pinto JQ974391 63 Phaseolus albescens (AF_115150) 99 100 Phaseolus albescens (AF_115148) Phaseolus albescens (AF_115152) 56 Phaseolus coccineus (AF_115156) 95 Phaseolus coccineus (AF_115157) 80 Phaseolus coccineus (AF_069130) Phaseolus costaricensis (AF_115147) 80 Phaseolus polyanthus (AF_115151) 100 Phaseolus dumosus (AF_069127) 69 Phaseolus polyanthus (AF_115149) 95 60 Vigna aconifolia (AF_069118) 100 Vigna adenantha (AY_583526) Vigna adenantha (AF_069119) Vigna caacalla (AF_069124) Vigna populnea (AF_115136) Vigna linearis (AF_069123) Vigna peduncularis (AF_069122) Vigna speciosa (AF_069121) 78 Vigna radiata voucher PI 425754 (MONT) (DQ_445738) 98 Vigna radiata voucher BJW0403 (JF_430409) Vigna radiata RMG 62 (HQ_148147) Vigna mungo var. mungo J6 (GQ275366) Vigna angularis var. nipponensis voucher BJW0312 (JN_418209) Vigna angularis voucher BJW0401 (JF_430407) 100 100 Vigna subterranea J2 (GQ_275362) 100 Vigna subterranea J3 (GQ_275363) Vigna minima J12 (GQ_275371) Vigna vexillata J8 (GQ_275368) Chipo Beige JQ974394 Vigna unguiculata subsp. Sesquipedalis (EU_727148) 100 54 Vigna unguiculata voucher Vu40 (GQ_411778) 74 Vigna unguiculata culvar HH-WD8 (FJ_176926) Vigna unguiculata IT87D-885.2 (AY_748431) Chipo Negro JQ974395 Vigna vexillata voucher BJW0407 (JF_430413) 79 97
100
ITS NJ Tamura-Nei (757 nt)
61
Phaseolus lunatus
100
Phaseolus vulgaris
100
76 98 100 53
100 73
Vigna unguiculata
0.05
Fig. 1. Phylogenetic relationships of Phaseolus species based on ITS sequences. Sequences obtained in this work are shown bold. Bootstrap values >70% are indicated at the nodes. Bar, 5 nt substitutions per 100 nt.
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Plant DNA extraction and ITS analysis To identify the Phaseolus species, a molecular approach was followed because only native seeds were available. Young leaves from the local bean plants germinated in the laboratory were macerated with liquid nitrogen and DNA was extracted with the same Roche DNA isolation kit. The internal transcribed spacer (ITS) was amplified with ITS primers PhaseoF and PhaseoR, and PCR conditions were as previously described [6]. PCR products were Sanger sequenced. Phylogenetic analyses were performed as described above.
Accession numbers The GenBank accession numbers for the recA gene sequences determined in this study are JQ966948–JQ966967 and JX943615– JX943617. The nodZ and nifH gene sequences have been assigned accession numbers JX645470–JX645478 and JX943618–JX943622, respectively.
Results Beans from Los Tuxtlas were classified into three different species, P. vulgaris, P. lunatus and V. unguiculata, according to ITS sequences (Fig. 1). Morphologically, P. lunatus seeds from Los Tuxtlas resembled the most common type of P. lunatus seeds grown in Southern Mexico [J. Martínez-Castillo, personal communication]. By plant trap experiments with local P. lunatus and V. unguiculata seeds, nodule bacteria were sampled from milpa soils from a region of Los Tuxtlas where a large diversity of crops have been preserved [4]. P. lunatus and V. unguiculata isolates were slow growing bacteria. A total of 157 bradyrhizobia isolates were obtained, 98 from P. lunatus and 59 from Vigna (Supplementary Table S1). Isolates from Vigna were grouped into eight ERIC profiles, with two of them representing almost 80% of the profiles. From P. lunatus isolates, 52 ERIC profiles were identified and most of them were represented only by a single strain, and only three profiles had five or more isolates. Only one ERIC profile was found in common among isolates from both legumes. PCR-RFLP patterns of rpoB genes, previously used to characterize bradyrhizobial isolates from P. lunatus in Peru [27], showed four distinct patterns in Vigna isolates, and two of them included 95% of the isolates. The dominant ERIC profiles were included in these rpoB-based genotypes. On the other hand, P. lunatus isolates corresponded to eight patterns by rpoB PCR-RFLP analysis, one of which had 91% of the isolates and most ERIC profiles. The remaining rpoB patterns had one or two isolates. It is known that ERIC profiles may distinguish different strains within a single species that may be represented by single rpoB gene sequences. The bradyrhizobial populations of V. unguiculata and P. lunatus were different and only one rpoB PCR-RFLP pattern was shared. recA gene phylogenies have been used to describe bradyrhizobial diversity [38], and the current study showed that, on this basis, most P. lunatus symbionts were closely related to previously described isolates from Los Tuxtlas (Fig. 2). Bradyrhizobial isolates from P. lunatus corresponded to reported genospecies TUXTLAS-17, TUXTLAS-4, TUXTLAS-20 and TUXTLAS-2, to three novel genospecies (TUXTLAS-33, -34 and -35) and to Bradyrhizobium japonicum (Fig. 2). P. lunatus isolate Pop235 was closely related to Inga isolate Inga3-1a, while Pop363 was related to Inga32d. Inga species are native trees in Los Tuxtlas forest and their nodule bacteria were isolated and characterized previously [25]. Mexican isolates from P. lunatus were different to those from Peru (Fig. 2). Most isolates from Vigna grouped with Bradyrhizobium
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elkanii, whereas others grouped with genospecies TUXTLAS-17 and TUXTLAS-21. Representatives of all rpoB restriction patterns from Vigna and P. lunatus isolates were tested in the laboratory for nodulation of both legumes (Supplementary Table S3). All P. lunatus isolates could nodulate and fix nitrogen in Vigna and P. lunatus. Vigna isolates were capable of nodulating both legumes but only one (Pop352) could fix nitrogen in P. lunatus, which indicated that P. lunatus is a more restricted host while Vigna is considered promiscuous [14,28]. B. japonicum USDA 110 and B. elkanii USDA 76 nodulated and fixed nitrogen in Vigna but failed to nodulate in P. lunatus. B. elkanii USDA 76 formed only root bumps in P. lunatus. Additionally some bradyrhizobial strains previously described, representing native TUXTLAS genospecies [25] were tested in nodulation assays in the laboratory. All tested strains were found to nodulate P. lunatus with some of them fixing nitrogen (Supplementary Table S2). Based on nodZ sequences, most V. unguiculata isolates corresponded to nodZ gene group III (Fig. 3), according to a classification proposed earlier [33]. Group III is considered a pantropical ancient group of bradyrhizobial nodulation genes and most Vigna bradyrhizobia from Africa possess these genes [33]. Pop352, the only Vigna isolate able to fix nitrogen with P. lunatus, clustered in nodZ clade V together with Pop367 from P. lunatus and other bradyrhizobial isolates previously isolated from Los Tuxtlas (Fig. 3). Group V was proposed earlier based on a single sequence from a Brazilian Lupinus bradyrhizobia [33]. In this study, it was found that bradyrhizobia from Los Tuxtlas, as well as Brazilian strains isolated from different plants, had nodZ genes corresponding to group V, which seems to have an American origin. nifH gene phylogeny also separated isolates from groups III and V (Supplementary Fig. S1) and showed the same close relationship between Brazilian group V isolates and bradyrhizobia from Los Tuxtlas.
Discussion As polycultures in Mexico commonly include P. vulgaris it was surprising to find P. lunatus and V. unguiculata in milpas from Los Tuxtlas. In comparison to P. vulgaris and V. unguiculata, P. lunatusnodulated roots with Los Tuxtlas rhizobia exhibited the highest levels of acetylene reduction activity (not shown) and highest symbiont diversity. Of all Phaseolus species, P. lunatus seems to have the broadest geographical distribution. It is widely cultivated in the tropics and subtropics, possibly in relation to its capacity to form nodules with bradyrhizobia that are abundant in tropical soils [22,25]. Wild P. lunatus are found in Mexico [19,20,23] and have been described in Los Tuxtlas, but wild P. vulgaris have not been found in this area [Jaime Martínez-Castillo, personal communication]. Mexico, together with Peru, is a P. lunatus domestication site [10,23,27] but P. lunatus cultivars grown in Peru and Mexico are different and their bradyrhizobial symbionts seem to be specific for each domestication site ([27] and this work). Supposedly, there is a domestication origin of P. lunatus in Southern Mexico but it has not been located yet. Most P. lunatus isolates were found to correspond to genospecies TUXTLAS-17 that was reported as abundant in crop fields [25,26]. Genospecies TUXTLAS-20 and TUXTLAS-22 were less frequently isolated from P. lunatus, which corresponds to the observation in previous studies that they were less abundant in crop fields [25,26]. P. lunatus forms nodules with native bradyrhizobia in tropical Los Tuxtlas soils, whereas P. vulgaris symbionts are exotic and have been introduced to Los Tuxtlas [25,26]. Therefore, it is concluded that P. lunatus is a more adequate crop legume for Los Tuxtlas than P. vulgaris.
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Bradyrhizobium genosp. TUXTLAS-4 85v (FJ970368) Bradyrhizobium genosp. TUXTLAS-4 Inga3-1a (FJ970372) Pop235 [Phaseolus lunatus] (JQ966951) Bradyrhizobium genosp. TUXTLAS-5 1246v (FJ970345) Bradyrhizobium genosp. TUXTLAS-1 1681v (FJ970354) 99 Bradyrhizobium genosp. TUXTLAS-1 1041v (FJ970331) Bradyrhizobium genosp. TUXTLAS-8 35v (FJ970361) Bradyrhizobium genosp. TUXTLAS-8 42v (FJ970363) 99 Bradyrhizobium genosp. TUXTLAS-15 1028v (FJ970329) Bradyrhizobium genosp. TUXTLAS-10 1288v (FJ970346) 99 Bradyrhizobium genosp. TUXTLAS-10 1736m (FJ970357) Bradyrhizobium genosp. TUXTLAS-11 8v (FJ970369) 92 84 Bradyrhizobium genosp. TUXTLAS-11 44m (FJ970364) B. iriomotense EK05 (AB300996) Bradyrhizobium genosp. TUXTLAS-12 1648v (FJ970353) 72 Bradyrhizobium genosp. TUXTLAS-12 1784m (FJ970358) 98 Pop370 [Phaseolus lunatus] (JQ966967) Bradyrhizobium genosp. TUXTLAS-7 1018v (FJ970327) 99 Bradyrhizobium genosp. TUXTLAS-7 1595v (FJ970351) Bradyrhizobium genosp. TUXTLAS-6 1609v (FJ970352) 99 Bradyrhizobium genosp. TUXTLAS-6 1020v (FJ970328) B. yuanmingense LMTR 28 [Phaseolus lunatus] (AY591573) B. liaoningense LMG18230 (AY591564) Bradyrhizobium genosp. TUXTLAS-9 1031v (FJ970330) Bradyrhizobium genosp. TUXTLAS-14 1064v (FJ970333) B. canariense BTA-1 (AY591553) B. betae LMG21987 (AB353734) B. japonicum DSMZ30131 (AY591555) Pop363 [Phaseolus lunatus] (JQ966957) 99 95 B. japonicum Inga3-2d (FJ970374) 99 Pop231 [Phaseolus lunatus] (JQ966949) Pop271 [Phaseolus lunatus] (JQ966953) Pop306 [Vigna unguiculata] (JQ966955) 99 B. elkanii USDA76 (AY591568) 99 Pop255 [Phaseolus lunatus] (JQ966951) Pop253 [Phaseolus lunatus] (JQ966952) 93 Bradyrhizobium genosp. TUXTLAS-20 1017v (FJ970326) 99 Bradyrhizobium genosp. TUXTLAS-20 66v (FJ970367) Pop338 [Vigna unguiculata] (JQ966960) 95 82 Bradyrhizobium genosp. TUXTLAS-21 1325v (FJ970348) Bradyrhizobium genosp. TUXTLAS-18 1090v (FJ970335) 99 Pop234 [Phaseolus lunatus] (JQ966964) Bradyrhizobium genosp. TUXTLAS-22 1234v (FJ970344) 76 Pop321 [Vigna unguiculata] (JQ966956) 98 Pop304 [Vigna unguiculata] (JQ966959) Bradyrhizobium sp. Tpma1 -5 (EU099330 ) 71 Bradyrhizobium genosp. TUXTLAS-17 1110v (FJ970339) Bradyrhizobium genosp. TUXTLAS-17 109m (FJ970336) 91 78 Pop352 [Vigna unguiculata] (JQ966962) Bradyrhizobium sp. Dr3b-11 (EU099332) Bradyrhizobium sp. Cj3-3 (EU099333) Pop366 [Phaseolus lunatus] (JQ966958) Pop229 [Phaseolus lunatus] (JQ966948) Pop238 [Phaseolus lunatus] (JQ966950) Pop288 [Phaseolus lunatus] (JQ966954) Pop263 [Phaseolus lunatus] (JQ966966) Bradyrhizobium genosp. TUXTLAS-17 1730m (FJ970356) Pop218 [Phaseolus lunatus] (JQ966963) Pop342 [Vigna unguiculata] (JQ966961) Bradyrhizobium sp. LMTR 13 [ Phaseolus lunatus] (JX943615) Bradyrhizobium sp. LMTR 3 [ Phaseolus lunatus] (JX943616) Bradyrhizobium sp. LMTR 21 [ Phaseolus lunatus] (JX943617) Rhodopseudomomas palustris CGA009 (BX571963) Nitrobacter hamburgensis X14 (CP000319) 89 99
73
0.01
76
88
71
97
TUXTLAS-4
TUXTLAS-33
B. japonicum TUXTLAS-35 B. elkanii TUXTLAS-34
TUXTLAS-21 TUXTLAS-22
TUXTLAS-17
Fig. 2. recA gene phylogenetic relationships of Bradyrhizobium from Phaseolus lunatus and Vigna unguiculata isolated in this work (Pop strains). Geno(species) affiliations of Pop strains are indicated on the right-hand side. Novel genospecies are shown in bold. Bootstrap values >70% are indicated at the nodes. Bar, 1 nt substitution per 100 nt.
Vigna includes more than 200 species distributed throughout the tropics, some of them were domesticated and are widely used in tropical agriculture. The geographical origin of cowpea (V. unguiculata) is Africa and the bacteria associated with this legume have been reported as bradyrhizobia and some fast growing rhizobia. Cowpea bradyrhizobia isolated in Africa [33,39], China [40,41] and Brazil [42] were identified as B. elkanii, B. japonicum, Bradyrhizobium liaoningense, Bradyrhizobium yuanmingense and other unnamed Bradyrhizobium genospecies. In this work, the symbionts of Vigna were identified as B. elkanii and two previously reported genospecies (TUXTLAS-17 and TUXTLAS-21) [25,26]. Symbionts of V. unguiculata had already been studied using Vigna as
a trap plant from soils of another region in Los Tuxtlas [25]. B. elkanii and the three novel genospecies found here were not previously reported in that study [25,26]. This indicates that there is geographical heterogeneity of soil bacteria, which was not unexpected in view of the different landscape and environmental conditions therein [5,12]. However, it is remarkable that rhizobia similar to those found in Los Tuxtlas also exist in Central America and a microbiological corridor comprising such regions has been identified [16]. Approximately 40% of the forest cover has been lost in Los Tuxtlas [32] and soils are nutrient depleted [7]. Knowledge of the legume species grown in the Los Tuxtlas milpas is essential for
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37
99 ORS170 [Faidherbia albida, Senegal] (AJ431114) 98 SEMIA6160 [Albizia lebeck, Brazil] (HQ259524)
SEMIA6152 [Calopogonium sp., Brazil] (HQ259523) USDA3259 [Phaseolus lunatus, USA] (AM168393) R5 [Vigna unguiculata, Botswana] (EU364746) Pop321[Vigna sp.] (JX645470) 99 Pop304 [Vigna sp.] (JX645472)
99
81 USDA3139 [Astragalus canadensis, USA] (AJ431120)
SEMIA695 [Neonotonia wightii, Australia] (HQ259512) SEMIA6053 [Clitoria ternatea, Malaysia] (HQ259501) 99
SEMIA5026 [Glycine max, Thailand] (HQ259497) USDA76 [Glycine max, USA] (AM168395)
III.1
99 SEMIA5019 [Glycine max, Brazil] (HQ259496)
USDA135 [Glycine max, USA] (AJ431119) ORS130 [Faidherbia albida, Senegal] (AJ431111) SEMIA6028 [Desmodium uncinatum, Zimbabwe] (HQ259518)
98 82
SEMIA6146 [Centrosema sp., Brazil] (HQ259522)
93
SEMIA6148 [Neonotonia wightii, Brazil] (HQ259505) 98 Pop342 [Vigna sp., Mexico] (JX645473) SEMIA6069 [Leucaena leucocephala, Brazil] (HQ259502)
III.2
99
99
III.1 1234v [Vigna unguiculata, Mexico] (JX645477) SEMIA6101 [Dalbergia nigra, Brazil] (HQ259504)
99
CH2493 [Lupinus paraguariensis, Brazil] (AM168382) 96 SEMIA6099 [Dimorphandra exaltata, Brazil] (HQ259521)
SEMIA6440 [Arachis pintoi, Brazil] (HQ259509)
99
V
109m [Macroptilium atropurpureum, Mexico] (JX645478) 99
Pop352 [Vigna sp.] (JX645474) Pop367 [Phaseolus lunatus] (JX645471) 107m [Macroptilium atropurpureum, Mexico] (JX645476)
99 1017v [Vigna unguiculata, Mexico] (JX645475)
IV 99 88
II
VII A. caulinodans ORS571 (AP009384)
0.1 Fig. 3. Maximum likelihood phylogeny of nodZ gene sequences. Sequences of strains isolated from Los Tuxtlas are shown in bold. Those obtained in this study have the Pop prefix. nodZ clades are indicated with Roman numerals. Bootstrap values >70% are indicated at the nodes. Bar, 1 nt substitution per 10 nt.
determining which rhizobia could be used as inoculants for fulfilling our agro-ecological project aimed at maintaining soil fertility through symbiosis with native microorganisms. Acknowledgements To the memory of Jose Luis Blanco who provided seeds and contacts with farmers and was a wonderful friend. This work was part of the BioPop Project, FOMIX-CONACYT-Veracruz (Fondos Mixtos de Fomento a la Investigación Científica y Tecnológica – Consejo Nacional de Ciencia y Tecnología) 94427. We thank farmers in Ocotal Chico and Mazumeapan: Marcelino Gutiérrez Matias, Angel Gutiérrez Zacarías, Leopoldo González Mateo, Wenseslao Duarte González, Juan Santiago, Pedro González
Santiago, Roberto García Arias, Josafath Gutiérrez Matías, León Hernández Gutiérrez, Angel González Santiago, Isaías González Rodríguez, Diego González Mateo. To Luis E. Servín for providing primers, to Isis de la Rosa for technical assistance and to Michael Dunn for reading the paper. Funding was provided by UNAMPAPIIT (Universidad Nacional Autónoma de México – Programa de Apoyo a Proyectos de Investigación e Innovación Tecnológica) IN205412. Appendix A. Supplementary data Supplementary data associated with this article can be found, in the online version, at http://dx.doi.org/10.1016/j.syapm. 2012.10.006.
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References [1] Abbo, S., Saranga, Y., Peleg, Z., Kerem, Z., Lev-Yadun, S., Gopher, A. (2009) Reconsidering domestication of legumes versus cereals in the ancient near east. Q. Rev. Biol. 84, 29–50. [2] Aserse, A.A., Rasanen, L.A., Assefa, F., Hailemariam, A., Lindstrom, K. (2012) Phylogeny and genetic diversity of native rhizobia nodulating common bean (Phaseolus vulgaris L.) in Ethiopia. Syst. Appl. Microbiol. 35, 120–131. [3] Bitocchi, E., Nanni, L., Bellucci, E., Rossi, M., Giardini, A., Zeuli, P.S., Logozzo, G., Stougaard, J., McClean, P., Attene, G., Papa, R. (2012) Mesoamerican origin of the common bean (Phaseolus vulgaris L.) is revealed by sequence data. Proc. Natl. Acad. Sci. U.S.A. 109, E788–E796. [4] Blanco, J.L. (2006) Erosión de la agrodiversidad en la milpa zoque popoluca de Soteapan: Xutuchincon y Aktevet. Ph.D. Thesis, Universidad Iberoamericana, Mexico City. [5] Campos, C. (2004) El suelo. In: Guevara, S., Laborde, J., Sanchez-Ríos, G. (Eds.), Los Tuxtlas, El paisaje de la Sierra, Instituto de Ecología, A.C. & European Union, Xalapa, pp. 181–192. [6] Delgado-Salinas, A., Turley, T., Richman, A., Lavin, M. (1999) Phylogenetic analysis of the cultivated and wild species of Phaseolus (Fabaceae). Syst. Bot. 24, 438–460. [7] Eilittä, M., Arteaga, F., Diaz, M., Guerrero, C., Herrera, B., Narvaez, G., Paré, L., Robles, G., Triomphe, B. (2004) Cultivating maize with Mucuna in the Los Tuxtlas region of South-Eastern Veracruz, Mexico. In: Eilittä, M., Mureithi, J., Derpsch, R. (Eds.), Green Manure/Cover Crop Systems of Smallholder Farmers, Springer, Dordrecht, Netherlands, pp. 99–127. [8] Felsenstein, J. (1981) Evolutionary trees from DNA sequences: a maximum likelihood approach. J. Mol. Evol. 17, 368–376. [9] Goman, M., Byrne, R. (1998) A 5000-year record of agriculture and tropical forest clearance in the Tuxtlas, Veracruz, Mexico. Holocene 8, 83–89. [10] Kaplan, L. (1965) Archeology and domestication in American Phaseolus (Beans). Econ. Bot. 19, 358–368. [11] Kwak, M., Kami, J.A., Gepts, P. (2009) The putative mesoamerican domestication center of Phaseolus vulgaris is located in the Lerma-Santiago basin of Mexico. Crop Sci. 49, 554–563. [12] Laborde, J. (2004) Los Habitantes. In: Guevara, S., Laborde, J., Sanchez-Ríos, G. (Eds.), Los Tuxtlas, El paisaje de la Sierra, Instituto de Ecología, A.C. & European Union, Xalapa, pp. 61–84. [13] Larkin, M.A., Blackshields, G., Brown, N.P., Chenna, R., McGettigan, P.A., McWilliam, H., Valentin, F., Wallace, I.M., Wilm, A., Lopez, R., Thompson, J.D., Gibson, T.J., Higgins, D.G. (2007) Clustal W and Clustal X version 2.0. Bioinformatics 23, 2947–2948. [14] Lewin, A., Rosenberg, C., Meyer, H., Wong, C.H., Nelson, L., Manen, J.F., Stanley, J., Dowling, D.N., Denarie, J., Broughton, W.J. (1987) Multiple host-specificity loci of the broad host-range Rhizobium sp. NGR234 selected using the widely compatible legume Vigna unguiculata. Plant Mol. Biol. 8, 447–459. ˜ [15] López-Guerrero, M.G., Ormeno-Orrillo, E., Velázquez, E., Rogel, M.A., Acosta, J.L., Gónzalez, V., Martínez, J., Martínez-Romero, E. (2012) Rhizobium etli taxonomy revised with novel genomic data and analyses. Syst. Appl. Microbiol. 35, 353–358. [16] Lopez-Lopez, A., Rogel-Hernandez, M.A., Barois, I., Ortiz Ceballos, A.I., Martinez, ˜ J., Ormeno-Orrillo, E., Martínez-Romero, E. (2012) Rhizobium grahamii sp. nov., from nodules of Dalea leporina, Leucaena leucocephala and Clitoria ternatea, and Rhizobium mesoamericanum sp. nov., from nodules of Phaseolus vulgaris, siratro, cowpea and Mimosa pudica. Int. J. Syst. Evol. Microbiol. 62, 2264–2271. ˜ [17] Lloret, L., Ormeno-Orrillo, E., Rincón, R., Martínez-Romero, J., Rogel-Hernández, M.A., Martínez-Romero, E. (2007) Ensifer mexicanus sp. nov. a new species nodulating Acacia angustissima (Mill.) Kuntze in Mexico. Syst. Appl. Microbiol. 30, 280–290. [18] Maingi, J.M., Gitonga, N.M., Shisanya, C.A., Hornetz, B., Muluvi, G.M. (2006) Population levels of indigenous bradyrhizobia nodulating promiscuous soybean in two Kenyan soils of the semi-arid and semi-humid agroecological zones. J. Agric. Rural Dev. Trop. Subtrop. 107, 149–159. [19] Martinez-Castillo, J., Zizumbo-Villarreal, D., Gepts, P., Delgado-Valerio, P., Colunga-GarciaMarin, P. (2006) Structure and genetic diversity of wild populations of Lima bean (Phaseolus lunatus L.) from the Yucatan Peninsula, Mexico. Crop Sci. 46, 1071–1080. [20] Martínez-Castillo, J., Zizumbo-Villarreal, D., Perales-Rivera, H., ColungaGarcíamarin, P. (2004) Intraspecific diversity and morpho-phenological variation in Phaseolus lunatus L. from the Yucatan Peninsula, Mexico. Econ. Bot. 58, 354–380. [21] Martínez-Romero, E. (2003) Diversity of Rhizobium-Phaseolus vulgaris symbiosis: overview and perspectives. Plant Soil 252, 11–23. [22] Moreira, F.M., Haukka, K., Young, J.P. (1998) Biodiversity of rhizobia isolated from a wide range of forest legumes in Brazil. Mol. Ecol. 7, 889–895.
[23] Motta-Aldana, J.R., Serrano-Serrano, M.L., Hernandez-Torres, J., CastilloVillamizar, G., Debouck, D.G., Chacon, M.I. (2010) Multiple origins of Lima bean landraces in the Americas: evidence from chloroplast and nuclear DNA polymorphisms. Crop Sci. 50, 1773–1787. [24] Negrete-Yankelevich, S., Maldonado-Mendoza, I., Lázaro-Castellanos, J., Sangabriel-Conde, W., Martínez-Álvarez, J. Arbuscular mycorrhizal root colonization and soil P availability are positively related to agrodiversity in Mexican maize polycultures. Biol. Fertil. Soils, http://dx.doi.org/10.1007/s00374-0120710-5 ˜ [25] Ormeno-Orrillo, E., Rogel-Hernandez, M.A., Lloret, L., Lopez-Lopez, A., Martinez, J., Barois, I., Martínez-Romero, E. (2012) Change in land use alters the diversity and composition of Bradyrhizobium communities and led to the introduction of Rhizobium etli into the tropical rain forest of Los Tuxtlas (Mexico). Microb. Ecol. 63, 822–834. ˜ E., Rogel, M.A., Lloret, L., López-López, A., Martínez, J., Vin[26] Ormeno-Orrillo, uesa, P., Martinez Romero, E. (2009) Rhizobial diversity in different land use systems in the rain forest of Los Tuxtlas, Mexico. In: Barois, I., Huising, E.J., Ojosth, P., Trejo, D., De los Santos, M. (Eds.), Below-Ground Biodiversity in Sierra de Santa Marta, Los Tuxtlas, Veracruz, Mexico, Instituto de Ecología, Xalapa, pp. 65–84. ˜ ˜ [27] Ormeno-Orrillo, E., Vinuesa, P., Zúniga-Dávila, D., Martínez-Romero, E. (2006) Molecular diversity of native bradyrhizobia isolated from Lima bean (Phaseolus lunatus L.) in Peru. Syst. Appl. Microbiol. 29, 253–262. [28] Ren da, W., Chen, W.F., Sui, X.H., Wang, E.T., Chen, W.X. (2011) Rhizobium vignae sp. nov., a symbiotic bacterium isolated from multiple legume species. Int. J. Syst. Evol. Microbiol. 61, 580–586. [29] Rincón-Rosales, R., Lloret, L., Ponce, E., Martínez-Romero, E. (2009) Rhizobia with different symbiotic efficiencies nodulate Acaciella angustissima in Mexico, including Sinorhizobium chiapanecum sp. nov. which has common symbiotic genes with Sinorhizobium mexicanum. FEMS Microbiol. Ecol. 67, 103–117. [30] Saitou, N., Nei, M. (1987) The neighbor-joining method: a new method for reconstructing phylogenetic trees. Mol. Biol. Evol. 4, 406–425. [31] Silva, C., Vinuesa, P., Eguiarte, L.E., Martínez-Romero, E., Souza, V. (2003) Rhizobium etli and Rhizobium gallicum nodulate common bean (Phaseolus vulgaris) in a traditionally managed milpa plot in Mexico: population genetics and biogeographic implications. Appl. Environ. Microbiol. 69, 884–893. [32] Soto, M. (2004) El clima. In: Guevara, S., Laborde, J., Sanchez-Ríos, G. (Eds.), Los Tuxtlas., El paisaje de la Sierra, Instituto de Ecología, A.C. & European Union, Xalapa, pp. 195–199. [33] Steenkamp, E.T., Stepkowski, T., Przymusiak, A., Botha, W.J., Law, I.J. (2008) Cowpea and peanut in southern Africa are nodulated by diverse Bradyrhizobium strains harboring nodulation genes that belong to the large pantropical clade common in Africa. Mol. Phylogenet. Evol. 48, 1131–1144. [34] Tamura, K., Peterson, D., Peterson, N., Stecher, G., Nei, M., Kumar, S. (2011) MEGA5: molecular evolutionary genetics analysis using maximum likelihood, evolutionary distance, and maximum parsimony methods. Mol. Biol. Evol. 28, 2731–2739. [35] Thies, J.E., Bohlool, B.B., Singleton, P.W. (1991) Subgroups of the cowpea miscellany: symbiotic specificity within Bradyrhizobium spp. for Vigna unguiculata, Phaseolus lunatus, Arachis hypogaea, and Macroptilium atropurpureum. Appl. Environ. Microbiol. 57, 1540–1545. [36] Toledo, I., Lloret, L., Martínez-Romero, E. (2003) Sinorhizobium americanum sp. nov., a new Sinorhizobium species nodulating native Acacia spp. in Mexico. Syst. Appl. Microbiol. 26, 54–64. [37] Vincent, J.M. 1970 A Manual for the Practical Study of Root Nodule Bacteria, Blackwell Scientific Publications, Oxford. [38] Vinuesa, P., Silva, C., Werner, D., Martínez-Romero, E. (2005) Population genetics and phylogenetic inference in bacterial molecular systematics: the roles of migration and recombination in Bradyrhizobium species cohesion and delineation. Mol. Phylogenet. Evol. 34, 29–54. [39] Wolde-Meskel, E., Terefework, Z., Frostegard, A., Lindstrom, K. (2005) Genetic diversity and phylogeny of rhizobia isolated from agroforestry legume species in southern Ethiopia. Int. J. Syst. Evol. Microbiol. 55, 1439–1452. [40] Zhang, W.T., Yang, J.K., Yuan, T.Y., Zhou, J.C. (2007) Genetic diversity and phylogeny of indigenous rhizobia from cowpea [Vigna unguiculata (L.) Walp.]. Biol. Fertil. Soils 44, 201–210. [41] Zhang, Y.F., Wang, E.T., Tian, C.F., Wang, F.Q., Han, L.L., Chen, W.F., Chen, W.X. (2008) Bradyrhizobium elkanii, Bradyrhizobium yuanmingense and Bradyrhizobium japonicum are the main rhizobia associated with Vigna unguiculata and Vigna radiata in the subtropical region of China. FEMS Microbiol. Lett. 285, 146–154. [42] Zilli, J.E., Valisheski, R.R., Freire, F.R., Neves, M.C.P., Rumjanek, N.G. (2004) Assessment of cowpea rhizobium diversity in Cerrado areas of Northeastern Brazil. Braz. J. Microbiol. 35, 281–287.