Análisis comparativo de bradyrhizobia aislados de Lupinus angustifolius y Lupinus mariae-josephi.

Los altramuces (Lupinus sp.) son leguminosas con alto contenido proteico y alto valor en alimentación animal. Todas las especies de Lupinus que crecen en la Península Ibérica, incluida L. angustifolius, requieren suelos ácidos, excepto L- mariae-josephi, una especie recientemente descrita, que excepcíonalmente crece en suelos alcalinos. Se han analizado las relaciones filogenéticas entre bacterias endosimbióticas (rizobios) de L. mariae-josephi y de L. angustifolius y su posición evolutiva respecto a rizobios aislados de otras especies de Lupinus y de otras leguminosas. Con este fin se comparó la secuencia aminoacídica de proteínas esenciales ("housekeeping"), RecA, AtpD, Glnl l, y de la proteína simbiótica NodC, así como también la secuencia nucleotídica intergénica 16S-23S (ITS). La caracterización simbiótica de L. angustifolius y L. mariae-josephi implicó la realización de ensayos de inoculación cruzada, empleando plantas cuyos endosimbiontes están filogenéticamente próximos. Además, se estudió por microscopía de fluorescencia la distribución de bacteroides en los nodulos, utilizando rizobios que expresan GFP. Se espera identificar factores de la simbiosis con rizobios que capacitan a L .mariae-josephi para crecer en suelos básicos

Endangered Lupinus mariae-josephae species: conservation efforts

A lupin endemism, Lupinus mariae-josephae (Lmj), singularly has been identified in the Valencia province, in Eastern Spain. This lupin thrives in alkaline-limed soils with high pH, a unique habitat for lupins, from a small area in Valencia region. In these soils, Lmj grows in just a few small, defined patches, and previous conservation efforts directed towards controlled plant reproduction have been unsuccessful. This lupin was thought to be extinct in Valencia until 2007, when it was discovered in a limestone patch. The reasons behind Lmj endangered status are presently unknown. This study will focus on the symbiosis between Lmj and rhizobia, and how this relationship might impact the population size of Lmj. We have previously shown that Lmj plants establish a specific root nodule symbiosis with bradyrhizobia present in those soils, and we reasoned that the paucity of these bacteria in soils might contribute to the lack of success in reproducing plants for conservation purposes

Phenotypic and molecular diversity of Lupinus mariae-josephi endosymbiotic bacteria.

Lupinus mariae-josephi is a new Lupinus species recently described in a Southeastern area of Spain (Valencia) in soils of singularly high pH and active lime content. Bacteria from L. mariaejosephi have been isolated and correspond to extra-slow growing bacteria symbiotically and phylogenetically distant to endosymbiotic strains nodulating other Lupinus sp. native of the Iberian Peninsula and adapted to growth in acid soils. Cross-inoculation experiments revealed that the L. mariae-josephi endosymbiotic bacteria are unable to nodulate or efficiently fix nitrogen with well-known Lupinus spp. Their species affiliation was examined by a multilocus sequence analysis of four housekeeping genes (16S rDNA, glnII, recA, atpD) and the symbiotic nodC gene. Single and concatenated phylogenetic analyses of these genes consistently revealed that L. mariae-josephi endosymbiotic bacteria belong to a clade, within the Bradyrhizobium genus, highly differentiated from the Bradyrhizobium clade that includes currently named Bradyrhizobium species as well as the endosymbiotic bacteria from Lupinus species tested in this study. Within this new clade the L. mariae-josephi bacteria nested in several subgroups that may correspond to novel sister species. The phylogenetic analysis based on the nodC gene showed that L. mariae-josephi endosymbiotic bacteria define a novel branch in the nodC Bradyrhizobium tree and likely have a common unique ancestor for the symbiotic genes with nodule isolates from Retama spp. At this moment two draft genome sequences belonging to a Bradyrhizobium isolated from L. angustifolius (ISLU101) and to a Bradyrhizobium isolated from L. mariae-josephi (LmjC) have been obtained. The first analysis showed that both genomes correspond to very large chromosomes (>8000 genes) with a high number of unique proteins. nod genes organization are highly conserved among ISLU101, LmjC and B. japonicum USDA110. LmjC presents a single cluster with nod genes from diverse origins. ISLU101 fix genes are found in a single cluster homologous to that of USDA110. LmjC possess a complete copy of fix genes homologous to that from USDA110 and an incomplete one similar to S. meliloti. LmjC possess TypeIII and TypeIV secretion systems. ISLU101 has a Type IV homologous to that of photosynthetic Bradyrhizobium BTAi1, and two copies of Type VI, one homologous to that of USDA110 and the other to R. leguminosarum. Preliminary data indicate that ISLU101 contains a potential second replicon (~100 genes) with high homology to Bradyrhizobium BTai1 plasmid sequenc

Symbiosis of the endangered Lupinus mariae-josephae lupin especies: Successful "in situ" propagation with rhizobial inoculation

Region, in Eastern Spain. This lupine thrives in alkaline soils with high pH, a unique habitat for lupines. In these soils, Lmj grows in just a few defined patches, and previous conservation efforts directed towards controlled plant reproduction have been unsuccessful. A legislative decree (70/2009, page 20156 Anex I) published in the el 'Diario Oficial de la Comunitat Valenciana' shows Lmj in a category corresponding, in the latest version of the Red List of IUCN (IUCN, 2012) (International Union for Conservation of Nature and Nature Resources), to an ?Endangered? legume species not extinct in the wild. Most current IUCN criteria used to define rare, small-range legume species, are based on history of reproductive traits such as number of pods and seeds. We have previously shown that Lmj plants establish a specific root nodule symbiosis with bradyrhizobia present in those soils, and we reasoned that the paucity of these bacteria in soils might contribute to the lack of success in reproducing plants for conservation purposes. Greenhouse experiments using Lmj trap-plants showed an absence, or very low concentration, of Lmj-nodulating bacteria in ?terra rossa? soils of Valencia outside of Lmj plant patches. No Lmj endosymbiotic bacteria were found in ?terra rossa? or alkaline red soils outside the Valencia Lmj endemism region in the Iberian Peninsula or Balearic Islands. Among the rhizobia able to establish an efficient symbiosis with L. mariae-josephae plants, two Bradyrhizobium sp. strains, LmjC and LmjM3, were selected as inocula for seed coating. Two planting experiments were carried out in consecutive years under natural conditions in areas with edapho-climatic characteristics identical to those sustaining natural Lmj populations, and successful reproduction of the plant was achieved. Interestingly, the successful reproductive cycle was absolutely dependent on seedling inoculation with effective bradyrhizobia, and optimal performance was observed in plants inoculated with LmjC, a strain that had previously shown the most efficient behavior under controlled conditions. These results define conditions for L. mariae-josephae conservation and for extension to alkaline-limed soil habitats, where no other known lupine can thrive. Broadly speaking, the work singularly identified the rhizobial symbiosis as a factor affecting the conservation of legumes and often being exceedingly vulnerable to threats. Our results also indicate that seed inoculation with N2-fixing, efficient Rhizobium strains is a strategy to consider in the conservation of endangered legume specie

Nitrogen fixation by native Bradyrhizobia in symbiosis with Lupinus mariae-josephae requires a T3SS encoding a NopE-like effector

Several bradyrhizobial isolates from L. mariae-josephae root nodules [1] contain a type III secretion system (T3SS) within a cluster of about 30 genes. Among those genes, ttsI codes for the transcriptional activator of the system. Mutation of ttsI resulted in the formation of white, non-fixing nodules with the natural legume host, L. mariae-josephae. The T3SS cluster also contains a gene coding for a NopE-like protein. NopE proteins have been demonstrated to be effectors in the Bradyrhizobium-soybean symbiosis [2] and belong to a small group of poorly characterized proteins from plant-associated bacteria that contain one or two autocleavage motifs known as DUF1521 (Schirrmeister et al. 2011). The amino acid sequence of a NopE-like protein in the L. mariae-josephae strain LmjC contains just one autocatalytic motif. This is unlike NopE1 and NopE2 proteins secreted by the T3SS of B. japonicum, that contain two motifs [3]. The autocleavage of LmjC NopE protein was analyzed after expression in E. coli and purification. Two protein fragments of the predicted sizes appeared in the presence of Ca2+, Cu2+, Cd2+, Zn2+ and Mn2+ cations. In contrast, autocleavage did not take place in the presence of Ni2+, Co2+ or Mg2+. Site-directed mutagenesis of the DUF1521 motif in LmjC NopE abolished self-cleavage in vitro. Symbiotic competence of a NopE- mutant with the L. mariae-josephae host was not affected. Possible roles of NopE are discussed

Lupinus mariae-josphi, a new lupin endemic of soils with active lime and high pH in South Eastern Spain, is nodulated by a new bacterial lineage within Bradyrhizobium genus

Lupinus mariae-josephi is a recently described species (Pascual, 2004) able to grow in soils with high pH and active lime content in the Valencia province (Spain). L. mariae-josephi endosymbionts are extremely slowgrowing bacteria with genetic and symbiotic characteristics that differentiate them from Bradyrhizobium strains nodulating Lupinus spp. native of the Iberian Peninsula and adapted to grow in acid soils. Cross-inoculation experiments revealed that all the endosymbiotic isolates from L. mariae-josephi tested are legume-host selective and are unable to nodulate species such as L. angustifolius, and L. luteus. In contrast, Bradyrhizobium strains from Lupinus spp. tested were able to nodulate L. mariae-josephi, although the nodules fixed nitrogen inefficiently. Phylogenetic analysis was performed with housekeeping genes (rrn, glnII, recA, atpD) and nodulation gene nodC. Housekeeping gene phylogeny revealed that L. mariae-josephi rhizobia form a strongly supported monophyletic group within Bradyrhizobium genus. This cluster also includes B. jicamae and certain strains of B. elkanii. Contrarily, isolates from other Lupinus spp. native of the Iberian Peninsula were grouped mainly within B. canariense and two B. japonicum lineages. Phylogenetic analysis of L. mariae-josephi isolates based on the nodC symbiotic gene defined a solid clade close to isolates from Algerian Retama spp. and to fast-growing rhizobia

Relevance of bacterial secretion systems Type III and Type VI in the Bradyrhizobium-Lupinus simbiosis

One of the most studied models in plant-microbe interaction is the symbiosis Rhizobium-legume. This symbiosis is highly specific and depends on several molecular signals produced by both partners. Some of these signals are bacterial proteins named effectors that are translocated into the plant cells by secretion systems similar to contractile nanomachines (injectisomes). The injectosomes puncture and deliver the effectors into the target cell. The two main injectiosomes are the secretion system type III (T3SS) and the secretion system type VI (T6SS). The genome of many rhizobia encodes T3SS and/or T6SS but their role in symbiosis is mostly unknown. The aim of this work is to study the symbiotic relevance of T3SS and T6SS of Bradyrhizobia that nodulate lupins that thrive in alkaline (Lupinus mariae-josephae) and acid soils (L. angustifolius) in the Iberian Peninsul

Native bradyrhizobia isolated from Lupinus mariae-josephae possess an essential T3SS for symbiosis

Analysis of the genome sequence of bradyrhizobia strains isolated from root nodules of Lupinus mariae-josephae revealed the presence of a type III secretion system (T3SS). Mutagenesis of ttsI gene that codes for the transcriptional activator (TtsI) resulted in the formation of white, non-fixing nodules in L. mariae-josephae. The T3SS cluster includes a gene coding for a NopE-like protein with an autocleavage motif. The NopE protein is an effector in the Bradyrhizobium-soybean symbiosis (Wenzel et al., 2010). The autocatalytic properties of the purified NopE-like protein have been studied

Endosymbiotic bacteria nodulating a new endemic lupine Lupinus mariae-josephi from alkaline soils in Eastern Spain represent a new lineage within the Bradyrhizobium genus

Lupinus mariae-josephi is a recently described endemic Lupinus species from a small area in Eastern Spain where it thrives in soils with active lime and high pH. The L. mariae-josephi root symbionts were shown to be very slow-growing bacteria with different phenotypic and symbiotic characteristics from those of Bradyrhizobium strains nodulating other Lupinus. Their phylogenetic status was examined by multilocus sequence analyses of four housekeeping genes (16S rRNA, glnII, recA, and atpD) and showed the existence of a distinct evolutionary lineage for L. mariae-josephi that also included Bradyrhizobium jicamae. Within this lineage, the tested isolates clustered in three different sub-groups that might correspond to novel sister Bradyrhizobium species. These core gene analyses consistently showed that all the endosymbiotic bacteria isolated from other Lupinus species of the Iberian Peninsula were related to strains of the B. canariense or B. japonicum lineages and were separate from the L. mariae-josephi isolates. Phylogenetic analysis based on nodC symbiotic gene sequences showed that L. mariae-josephi bacteria also constituted a new symbiotic lineage distant from those previously defined in the genus Bradyrhizobium. In contrast, the nodC genes of isolates from other Lupinus spp. from the Iberian Peninsula were again clearly related to the B. canariense and B. japonicum bv. genistearum lineages. Speciation of L. mariae-josephi bradyrhizobia may result from the colonization of a singular habitat by their unique legume host

Bradyrhizobium sp endosymbiont bacteria nodulate several species of spontaneous leguminous plants of Genisteae tribe from northeastern of Algeria.

218 bacterial isolates obtained from different legume species belonging to the Gensisteae tribe (Lupinus micruntus, Retama sphaerocarpa, Retama raetam and Cytisus vilosus) from different sampling sites in Algeria were studied. Cultivated on YMA medium these strains show a slow growth. The phylogenetic analysis based on 16S rDNA and the household genes of the representatives of these strains indicates that they belong to the genus Bradyrhizobium. A variety of Bradyrhizobium sp. (1, 2, 3) is present in this collection with the presence of strains that can lead to new species. (1) Boulila Farida et al., (2009). Syst. Appl. Microbiol. 32:245?255. (2) Ahnia, et al (2014). Antonie van Leeuwenhoek, J.Microbiol. ISSN 0003-6072, Volume 105 [6] 1121?1129. (3) Bourebaba and al., (2016). Syst. Appl. Microbiol. 39 266?274. This study was supported by the AECID project A1/038234/11. Bourebaba acknowledges support of the National Exceptional. Program (PNE), a fellowship from the Ministry of Higher Education and Scientific research of Algeria at CBGP, Madrid