Unraveling the genome of Bacillus velezensis MEP218, a strain producing fengycin homologs with broad antibacterial activity: comprehensive comparative genome analysis.

Bacillus sp. MEP218, a soil bacterium with high potential as a source of bioactive molecules, produces mostly C16-C17 fengycin and other cyclic lipopeptides (CLP) when growing under previously optimized culture conditions. This work addressed the elucidation of the genome sequence of MEP218 and its taxonomic classification. The genome comprises 3,944,892 bp, with a total of 3474 coding sequences and a G + C content of 46.59%. Our phylogenetic analysis to determine the taxonomic position demonstrated that the assignment of the MEP218 strain to Bacillus velezensis species provides insights into its evolutionary context and potential functional attributes. The in silico genome analysis revealed eleven gene clusters involved in the synthesis of secondary metabolites, including non-ribosomal CLP (fengycins and surfactin), polyketides, terpenes, and bacteriocins. Furthermore, genes encoding phytase, involved in the release of phytic phosphate for plant and animal nutrition, or other enzymes such as cellulase, xylanase, and alpha 1-4 glucanase were detected. In vitro antagonistic assays against Salmonella typhimurium, Acinetobacter baumanii, Escherichia coli, among others, demonstrated a broad spectrum of C16-C17 fengycin produced by MEP218. MEP218 genome sequence analysis expanded our understanding of the diversity and genetic relationships within the Bacillus genus and updated the Bacillus databases with its unique trait to produce antibacterial fengycins and its potential as a resource of biotechnologically useful enzymes.

Mesorhizobium intechi sp. nov. isolated from nodules of Lotus tenuis in soils of the Flooding Pampa, Argentina.

Three symbiotic nitrogen-fixing bacteria (BD68T, BD66 and BD73) isolated from root nodules of Lotus tenuis in lowland soils of the Flooding Pampa (Argentina), previously classified as members of the Mesorhizobium genus, were characterized in this study. Phylogenetic analysis of their 16S rRNA gene sequences showed a close relationship to M. japonicum MAFF 303099T, M. erdmanii USDA 3471T, M. carmichaelinearum ICMP 18942T, M. opportunistum WSM 2975T and M. jarvisii ATCC 33699T, with sequence identities of 99.72%-100%. Multilocus sequence analysis of other housekeeping genes revealed that the three isolates belonged to a phylogenetically distinct clade within the genus Mesorhizobium. Strain BD68T was designated as the group representative and its genome was fully sequenced. The average nucleotide identity and in silico DNA-DNA hybridization comparisons between BD68T and the most related type strains showed values below the accepted threshold for species discrimination. Phenotypic and chemotaxonomic features were also studied. Based on these results, BD68T, BD66 and BD73 could be considered to represent a novel species of the genus Mesorhizobium, for which the name Mesorhizobium intechi sp. nov. is hereby proposed. The type strain of this species is BD68T (=CECT 9304T=LMG 30179T).

The Rhizobia-Lotus Symbioses: Deeply Specific and Widely Diverse.

The symbiosis between Lotus and rhizobia has been long considered very specific and only two bacterial species were recognized as the microsymbionts of Lotus: Mesorhizobium loti was considered the typical rhizobia for the L. corniculatus complex, whereas Bradyrhizobium sp. (Lotus) was the symbiont for L. uliginosus and related species. As discussed in this review, this situation has dramatically changed during the last 15 years, with the characterization of nodule bacteria from worldwide geographical locations and from previously unexplored Lotus spp. Current data support that the Lotus rhizobia are dispersed amongst nearly 20 species in five genera (Mesorhizobium, Bradyrhizobium, Rhizobium, Ensifer, and Aminobacter). As a consequence, M. loti could be regarded an infrequent symbiont of Lotus, and several plant-bacteria compatibility groups can be envisaged. Despite the great progress achieved with the model L. japonicus in understanding the establishment and functionality of the symbiosis, the genetic and biochemical bases governing the stringent host-bacteria compatibility pairships within the genus Lotus await to be uncovered. Several Lotus spp. are grown for forage, and inoculation with rhizobia is a common practice in various countries. However, the great diversity of the Lotus rhizobia is likely squandered, as only few bacterial strains are used as inoculants for Lotus pastures in very different geographical locations, with a great variety of edaphic and climatic conditions. The agroecological potential of the genus Lotus can not be fully harnessed without acknowledging the great diversity of rhizobia-Lotus interactions, along with a better understanding of the specific plant and bacterial requirements for optimal symbiotic nitrogen fixation under increasingly constrained environmental conditions.

Genome Sequence of the Symbiotic Type Strain Mesorhizobium helmanticense CSLC115N Isolated from Lotus corniculatus Nodules.

Mesorhizobium helmanticense is a novel species that was isolated from root nodules of Lotus corniculatus grown in an alfisol soil from Carbajosa de la Sagrada, a Mediterranean region in the province of Salamanca in northwest Spain. The whole-genome sequence of the type strain M. helmanticense CSLC115N is reported in this study.

Comparative symbiotic performance of native rhizobia of the Flooding Pampa and strains currently used for inoculating Lotus tenuis in this region.

The Flooding Pampa (FP) is the most important area for cattle breeding in Argentina. In this region, persistence and yield of typical forage legumes are strongly limited by soil salinity and alkalinity, which affect around 30% of the total area. Instead, naturalized Lotus tenuis is the main forage legume in this region. Rhizobial strains currently used for inoculating L. tenuis in the FP are exotic or native from non-saline soils of this region, their taxonomic identity being unknown. Assuming that rhizobia native from the most restrictive environments are well adapted to adverse conditions, the use of such isolates could improve the productivity of L. tenuis in the FP. Hence, the goal of this study was to evaluate the symbiotic efficiency of selected L. tenuis rhizobia native from the FP, as compared with strains currently used for field inoculation of this legume. Under non-stressing conditions, the symbiotic performance of native strains of FP exceeded those ones currently used for L. tenuis. Moreover, the symbiotic performance of the native strain ML103 was considerably high under salt stress, compared with strains currently used as inoculants. Analysis of 16S rRNA gene sequencing revealed that unclassified rhizobia currently used for field inoculation of L. tenuis and native strains grouped with the genus Mesorhizobium. As a whole, results obtained demonstrate that soils of the FP are a source of efficient and diverse rhizobia that could be used as a sustainable agronomic tool to formulate inoculants that improve forage yield of L. tenuis in this region.

Phenotypic plasticity with respect to salt stress response by Lotus glaber: the role of its AM fungal and rhizobial symbionts.

Our hypothesis is that Lotus glaber (a glycophytic species, highly tolerant to saline-alkaline soils) displays a plastic root phenotypic response to soil salinity that may be influenced by mycorrhizal and rhizobial microorganisms. Uninoculated plants and plants colonised by Glomus intraradices or Mesorhizobium loti were exposed to either 150 or 0 mM NaCl. General plant growth and root architectural parameters (morphology and topology) were measured and phenotypic plasticity determined at the end of the salt treatment period. Two genotypes differing in their salt tolerance capacity were used in this study. G. intraradices and M. loti reduced the total biomass of non-salinised, sensitive plants, but they did not affect that of corresponding tolerant ones. Root morphology of sensitive plants was greatly affected by salinity, whereas mycorrhiza establishment counteracted salinity effects. Under both saline conditions, the external link length and the internal link length of mycorrhizal salt-sensitive plants were higher than those of uninoculated control and rhizobial treatments. The topological trend (TT) was strongly influenced by genotype x symbiosis interaction. Under non-saline conditions, nodulated root systems of the sensitive plant genotype had a more herringbone architecture than corresponding uninoculated ones. At 150 mM NaCl, nodulated root systems of tolerant plants were more dichotomous and those of the corresponding sensitive genotype more herringbone in architecture. Notwithstanding the absence of a link between TTs and variations in plant growth, it is possible to predict a dissimilar adaptation of plants with different TTs. Root colonisation by either symbiotic microorganisms reduced the level of root phenotypic plasticity in the sensitive plant genotype. We conclude that root plasticity could be part of the general mechanism of L. glaber salt tolerance only in the case of non-symbiotic plants.

Modulation of polyamine balance in Lotus glaber by salinity and arbuscular mycorrhiza.

In this work we investigated the involvement of Glomus intraradices in the regulation of plant growth, polyamines and proline levels of two Lotus glaber genotypes differing in salt tolerance, after longterm exposure to saline stress. The experiment consisted of a randomized block design with three factors: (1) mycorrhizal treatments (with or without AM fungus); (2) two salinity levels of 0 and 200mM NaCl; and (3) L. glaber genotype. Experiments were performed using stem cuttings derived from L. glaber individuals representing a natural population from saline lowlands. One of the most relevant results was the higher content of total free polyamines in mycorrhized plants compared to non-AM ones. Since polyamines have been proposed as candidates for the regulation of root development under saline situations, it is possible that AM plants (which contained higher polyamine levels and showed improved root growth) were better shaped to cope with salt stress. Colonization by G. intraradices also increased (Spd+Spm)/Put ratio in L. glaber roots. Interestingly, such increment in salt stressed AM plants of the sensitive genotype, was even higher than that produced by salinization or AM symbiosis separately. On the other hand, salinity but not mycorrhizal colonization influenced proline levels in both L. glaber genotypes since high proline accumulation was observed in both genotypes under salt stress conditions. Our results suggest that modulation of polyamine pools can be one of the mechanisms used by AM fungi to improve L. glaber adaptation to saline soils. Proline accumulation in response to salt stress is a good indicator of stress perception and our results suggest that it could be used as such among L. glaber genotypes differing in salt stress tolerance.

Presence of different arbuscular mycorrhizal infection patterns in roots of Lotus glaber plants growing in the Salado River basin.

Morphological types of arbuscular mycorrhizal (AM) fungi associated with Lotus glaber in sodic soils of the Salado River basin were studied. At least eight colonization patterns (IP) of AM fungi in roots of L. glaber were observed after 30 plants were analyzed. Arum- and Paris-type infection were found in the same plant species. This result supports the idea that AM morphology is not solely under plant control, but is also influenced by fungal identity. One infection pattern, presumably corresponding to Glomus intraradices, and a second, possibly assignable to Glomus tenue, were the most commonly found. Our results reinforce previous suggestions that G. intraradices is well adapted to sodic-saline conditions and may play a role in the resistance of L. glaber to these soils.

Ornithine and arginine decarboxylase activities and effect of some polyamine biosynthesis inhibitors on Gigaspora rosea germinating spores.

The pathways for putrescine biosynthesis and the effects of polyamine biosynthesis inhibitors on the germination and hyphal development of Gigaspora rosea spores were investigated. Incubation of spores with different radioactive substrates demonstrated that both arginine and ornithine decarboxylase pathways participate in putrescine biosynthesis in G. rosea. Spermidine and spermine were the most abundant polyamines in this fungus. The putrescine biosynthesis inhibitors alpha-difluoromethylarginine and alpha-difluoromethylornithine, as well as the spermidine synthase inhibitor cyclohexylamine, slightly decreased polyamine levels. However, only the latter interfered with spore germination. The consequences of the use of putrescine biosynthesis inhibitors for the control of plant pathogenic fungi on the viability of G. rosea spores in soil are discussed.