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Lupin is a high-protein legume crop that grows in a wide range of edaphoclimatic conditions where other crops are not viable. Its unique seed nutrient profile can promote health benefits, and it has been proposed as a phytoremediation plant. Most rhizobia nodulating Lupinus species belong to the genus Bradyrhizobium, comprising strains that are phylogenetically related to B. cytisi, B. hipponenese, B. rifense, B. iriomotense/B. stylosanthis, B. diazoefficiens, B. japonicum, B. canariense/B. lupini, and B. retamae/B. valentinum. Lupins are also nodulated by fast-growing bacteria within the genera Microvirga, Ochrobactrum, Devosia, Phyllobacterium, Agrobacterium, Rhizobium, and Neorhizobium. Phylogenetic analyses of the nod and nif genes, involved in microbial colonization and symbiotic nitrogen fixation, respectively, suggest that fast-growing lupin-nodulating bacteria have acquired their symbiotic genes from rhizobial genera other than Bradyrhizobium. Horizontal transfer represents a key mechanism allowing lupin to form symbioses with bacteria that were previously considered as non-symbiotic or unable to nodulate lupin, which might favor lupin’s adaptation to specific habitats. The characterization of yet-unstudied Lupinus species, including microsymbiont whole genome analyses, will most likely expand and modify the current lupin microsymbiont taxonomy, and provide additional knowledge that might help to further increase lupin’s adaptability to marginal soils and climates.

Nodule endophytes and associated bacteria are non-symbiotic bacteria that colonize legume nodules. They accompany nodulating rhizobia and can form beneficial associations, as some of them are plant growth-promoting rhizobacteria (PGPR) that are able to promote germination and plant growth and increase tolerance to biotic and abiotic stress. White lupin (Lupinus albus) is a legume crop that is gaining relevance as a suitable alternative to soybean as a plant protein source. Eleven nodule-associated bacteria were isolated from white lupin nodules grown in a Tunisian soil. They belonged to the genera Rhizobium, Ensifer, Pseudomonas and Bacillus. Their plant growth-promoting (PGP) and enzymatic activities were tested in vitro. Strains Pseudomonas sp., L1 and L12, displayed most PGP activities tested, and were selected for in planta assays. Inoculation with strains L1 or L12 increased seed germination and had the same positive effects on all plant growth parameters as did inoculation with symbiotic Bradyrhizobium canariense, with no significant differences among treatments. Inoculation with efficient nitrogen-fixing rhizobia must compete with rhizobia present in the soil that sometimes nodulate efficiently but fix nitrogen poorly, leading to a low response to inoculation. In such cases, inoculation with highly effective PGPR might represent a feasible alternative to boost crop productivity.

Thirty-two bacterial isolates were obtained from root nodules of Lupinus angustifolius growing in Northern Tunisia. Phylogenetic analyses based on recA and gyrB partial gene sequences grouped the strains into six clusters: four clusters belonged to the genus Bradyrhizobium (22 isolates), one to Microvirga (8 isolates) and one to Devosia (2 isolates), a genus that has not been previously reported to nodulate lupin. Representative strains of each group were further characterized. MultiLocus Sequence Analysis (MLSA) based on recA and glnII gene sequences separated the strains within the genus Bradyrhizobium into four divergent clusters related to B. canariense, B. liaoningense, B. lupini, and B. algeriense, respectively. The latter might constitute a new Bradyrhizobium species. The strains in the Microvirga cluster showed high identity with M. tunisiensis. The Devosia isolates might also represent a new species within this genus. An additional phylogenetic analysis based on the symbiotic gene nodC affiliated the strains to symbiovars genistearum, mediterranense, and to a possibly new symbiovar. These results altogether contributed to the existing knowledge on the genetic diversity of lupin-nodulating microsymbionts and revealed a likely new, fast-growing, salt-tolerant rhizobial species within the genus Devosia as a potentially useful inoculant in agricultural practices or landscape restoration.

Calicotome villosa is a spontaneous Mediterranean legume that can be a good candidate as pioneer plants to limit regression of vegetation cover and loss of biodiversity in Tunisian arid soils. In order to grow legumes in such soils, pairing rhizobia and nodule associated bacteria (NAB) might provide numerous advantages. In this work, cultivable biodiversity of rhizobial symbionts and NAB in nodules of C. villosa plants growing in five arid regions of south Tunisia was characterized. Phylogenetic analysis using 16S rDNA gene, dnak, recA and nodD sequences separated nodule-forming bacteria in six clades associated to genera Ensifer, Neorhizobium, Phyllobacterium and Rhizobium. Among NAB, the strain Variovorax sp. CT7.15 was selected due to its capacity to solubilise phosphate and, more interestingly, its high level of aminocyclopropane-1-carboxylate deaminase (ACC deaminase) activity. C. villosa plants were inoculated with representative rhizobia of each phylogenetic group and co-inoculated with the same rhizobia and strain CT7.15. Compared with single rhizobia inoculation, co-inoculation significantly improved plant growth and nodulation, ameliorated plant physiological state and increased nitrogen content in the plants, independently of the rhizobia used. These results support the benefits of pairing rhizobia and selected NAB to promote legume growth in arid or degraded soils

Calicotome villosa is a spontaneous Mediterranean legume that can be a good candidate as pioneer plants to limit regression of vegetation cover and loss of biodiversity in Tunisian arid soils. In order to grow legumes in such soils, pairing rhizobia and nodule associated bacteria (NAB) might provide numerous advantages. In this work, cultivable biodiversity of rhizobial symbionts and NAB in nodules of C. villosa plants growing in five arid regions of south Tunisia was characterized. Phylogenetic analysis using 16S rDNA gene, dnak, recA and nodD sequences separated nodule-forming bacteria in six clades associated to genera Ensifer, Neorhizobium, Phyllobacterium and Rhizobium. Among NAB, the strain Variovorax sp. CT7.15 was selected due to its capacity to solubilise phosphate and, more interestingly, its high level of aminocyclopropane-1-carboxylate deaminase (ACC deaminase) activity. C. villosa plants were inoculated with representative rhizobia of each phylogenetic group and co-inoculated with the same rhizobia and strain CT7.15. Compared with single rhizobia inoculation, co-inoculation significantly improved plant growth and nodulation, ameliorated plant physiological state and increased nitrogen content in the plants, independently of the rhizobia used. These results support the benefits of pairing rhizobia and selected NAB to promote legume growth in arid or degraded soils

Calicotome villosa is a spontaneous Mediterranean legume that can be a good candidate as pioneer plants to limit regression of vegetation cover and loss of biodiversity in Tunisian arid soils. In order to grow legumes in such soils, pairing rhizobia and nodule associated bacteria (NAB) might provide numerous advantages. In this work, cultivable biodiversity of rhizobial symbionts and NAB in nodules of C. villosa plants growing in five arid regions of south Tunisia was characterized. Phylogenetic analysis using 16S rDNA gene, dnak, recA and nodD sequences separated nodule-forming bacteria in six clades associated to genera Ensifer, Neorhizobium, Phyllobacterium and Rhizobium. Among NAB, the strain Variovorax sp. CT7.15 was selected due to its capacity to solubilise phosphate and, more interestingly, its high level of aminocyclopropane-1-carboxylate deaminase (ACC deaminase) activity. C. villosa plants were inoculated with representative rhizobia of each phylogenetic group and co-inoculated with the same rhizobia and strain CT7.15. Compared with single rhizobia inoculation, co-inoculation significantly improved plant growth and nodulation, ameliorated plant physiological state and increased nitrogen content in the plants, independently of the rhizobia used. These results support the benefits of pairing rhizobia and selected NAB to promote legume growth in arid or degraded soils

Calicotome villosa is a spontaneous Mediterranean legume that can be a good candidate as pioneer plants to limit regression of vegetation cover and loss of biodiversity in Tunisian arid soils. In order to grow legumes in such soils, pairing rhizobia and nodule associated bacteria (NAB) might provide numerous advantages. In this work, cultivable biodiversity of rhizobial symbionts and NAB in nodules of C. villosa plants growing in five arid regions of south Tunisia was characterized. Phylogenetic analysis using 16S rDNA gene, dnak, recA and nodD sequences separated nodule-forming bacteria in six clades associated to genera Ensifer, Neorhizobium, Phyllobacterium and Rhizobium. Among NAB, the strain Variovorax sp. CT7.15 was selected due to its capacity to solubilise phosphate and, more interestingly, its high level of aminocyclopropane-1-carboxylate deaminase (ACC deaminase) activity. C. villosa plants were inoculated with representative rhizobia of each phylogenetic group and co-inoculated with the same rhizobia and strain CT7.15. Compared with single rhizobia inoculation, co-inoculation significantly improved plant growth and nodulation, ameliorated plant physiological state and increased nitrogen content in the plants, independently of the rhizobia used. These results support the benefits of pairing rhizobia and selected NAB to promote legume growth in arid or degraded soils

[EN] Water shortage throughout the world, especially in arid regions in the later decades has led to search for alternatives to save potable fresh water. Treated wastewater (TWW) appears to be an opportunity for irrigation. However, it could represent a stress factor for plants, and influence their metabolism, changing their secondary metabolites and, consequently, their biological properties. Eucalyptus camaldulensis essential oil (EO) had been reported to possess phytotoxic activity. The main objective of this work was to compare the chemical composition and herbicidal activity of E. camaldulensis EO obtained from leaves of young plants and old trees irrigated with well water (WW) and TWW. Germination tests were performed in vitro against Amaranthus hybridus, Chenopodium album, Echinochloa crus-galli and Lolium perenne. The EOs composition was analyzed by gas chromatography and gas chromatography/mass spectrometry. A high percentage of oxygenated monoterpenes, with 1,8-cineole as main compound, was found in the EOs from leaves of young plants irrigated with both types of water. The EO from leaves of old trees irrigated with WW contained a main fraction of monoterpene hydrocarbons (45.17%) with p-cymene as principal compound. The highest herbicidal potential was shown by the EO from young plants irrigated with TWW. It completely inhibited A. hybridus and L. perenne germination, and nearly blocked the others at all concentrations assayed. It also showed strong phytotoxic activity on seedling length. The results suggest the possible use of TWW to irrigate Eucalyptus crops as it enhances the EOs herbicidal potential that could be used as natural herbicides.

[EN] Water shortage throughout the world, especially in arid regions in the later decades has led to search for alternatives to save potable fresh water. Treated wastewater (TWW) appears to be an opportunity for irrigation. However, it could represent a stress factor for plants, and influence their metabolism, changing their secondary metabolites and, consequently, their biological properties. Eucalyptus camaldulensis essential oil (EO) had been reported to possess phytotoxic activity. The main objective of this work was to compare the chemical composition and herbicidal activity of E. camaldulensis EO obtained from leaves of young plants and old trees irrigated with well water (WW) and TWW. Germination tests were performed in vitro against Amaranthus hybridus, Chenopodium album, Echinochloa crus-galli and Lolium perenne. The EOs composition was analyzed by gas chromatography and gas chromatography/mass spectrometry. A high percentage of oxygenated monoterpenes, with 1,8-cineole as main compound, was found in the EOs from leaves of young plants irrigated with both types of water. The EO from leaves of old trees irrigated with WW contained a main fraction of monoterpene hydrocarbons (45.17%) with p-cymene as principal compound. The highest herbicidal potential was shown by the EO from young plants irrigated with TWW. It completely inhibited A. hybridus and L. perenne germination, and nearly blocked the others at all concentrations assayed. It also showed strong phytotoxic activity on seedling length. The results suggest the possible use of TWW to irrigate Eucalyptus crops as it enhances the EOs herbicidal potential that could be used as natural herbicides.

The genetic diversity of bacterial populations nodulating Lupinus micranthus in five geographical sites from northern Tunisia was examined. Phylogenetic analyses of 50 isolates based on partial sequences of recA and gyrB grouped strains into seven clusters, five of which belong to the genus Bradyrhizobium (28 isolates), one to Phyllobacterium (2 isolates), and one, remarkably, to Microvirga (20 isolates). The largest Bradyrhizobium cluster (17 isolates) grouped with the B. lupini species, and the other five clusters were close to different recently defined Bradyrhizobium species. Isolates close to Microvirga were obtained from nodules of plants from four of the five sites sampled. We carried out an in-depth phylogenetic study with representatives of the seven clusters using sequences from housekeeping genes (rrs, recA, glnII, gyrB, and dnaK) and obtained consistent results. A phylogeny based on the sequence of the symbiotic gene nodC identified four groups, three formed by Bradyrhizobium isolates and one by the Microvirga and Phyllobacterium isolates. Symbiotic behaviors of the representative strains were tested, and some congruence between symbiovars and symbiotic performance was observed. These data indicate a remarkable diversity of L. micranthus root nodule symbionts in northern Tunisia, including strains from the Bradyrhizobiaceae, Methylobacteriaceae, and Phyllobacteriaceae families, in contrast with those of the rhizobial populations nodulating lupines in the Old World, including L. micranthus from other Mediterranean areas, which are nodulated mostly by Bradyrhizobium strains.

The genetic diversity of bacterial populations nodulating Lupinus micranthus in five geographical sites from northern Tunisia was examined. Phylogenetic analyses of 50 isolates based on partial sequences of recA and gyrB grouped strains into seven clusters, five of which belong to the genus Bradyrhizobium (28 isolates), one to Phyllobacterium (2 isolates), and one, remarkably, to Microvirga (20 isolates). The largest Bradyrhizobium cluster (17 isolates) grouped with the B. lupini species, and the other five clusters were close to different recently defined Bradyrhizobium species. Isolates close to Microvirga were obtained from nodules of plants from four of the five sites sampled. We carried out an in-depth phylogenetic study with representatives of the seven clusters using sequences from housekeeping genes (rrs, recA, glnII, gyrB, and dnaK) and obtained consistent results. A phylogeny based on the sequence of the symbiotic gene nodC identified four groups, three formed by Bradyrhizobium isolates and one by the Microvirga and Phyllobacterium isolates. Symbiotic behaviors of the representative strains were tested, and some congruence between symbiovars and symbiotic performance was observed. These data indicate a remarkable diversity of L. micranthus root nodule symbionts in northern Tunisia, including strains from the Bradyrhizobiaceae, Methylobacteriaceae, and Phyllobacteriaceae families, in contrast with those of the rhizobial populations nodulating lupines in the Old World, including L. micranthus from other Mediterranean areas, which are nodulated mostly by Bradyrhizobium strains.