Lotus spp. are widely used as a forage to improve pastures, and inoculation with elite rhizobial strains is a common practice in many countries. However, only a few Lotus species have been studied in the context of plant-rhizobia interactions. In this study, forty highly diverse bacterial strains were isolated from root nodules of wild Lotus parviflorus plants growing in two field locations in Portugal. However, only 10% of these isolates could nodulate one or more legume hosts tested, whereas 90% were thought to be opportunistic nodule associated bacteria. Phylogenetic studies place the nodulating isolates within the Bradyrhizobium genus, which is closely related to B. canariense and other Bradyrhizobium sp. strains isolated from genistoid legumes and Ornithopus spp. Symbiotic nodC and nifH gene phylogenies were fully consistent with the taxonomic assignment and host range. The non-nodulating bacteria isolated were alpha- (Rhizobium/Agrobacterium), beta- (Massilia) and gamma-proteobacteria (Pseudomonas, Lysobacter, Luteibacter, Stenotrophomonas and Rahnella), as well as some bacteroidetes from genera Sphingobacterium and Mucilaginibacter. Some of these nodule-associated bacteria expressed plant growth promotion (PGP) traits, such as production of lytic enzymes, antagonistic activity against phytopathogens, phosphate solubilization, or siderophore production. This argues for a potential beneficial role of these L. parviflorus nodule-associated bacteria.

There is growing interest in using plant-beneficial microorganisms to partially replace chemicals and help reduce the environmental impact of agriculture. Formulated microbial products or inoculants for agriculture contain single strains or a consortium of live microbes, well characterized and biosafe, which can contribute to the growth, health, and development of a plant host. This concept conforms to the definition of probiotics. However, some plant-growth-promoting microorganisms (PGPMs) have been considered a category of biostimulants since some years ago, despite the traditional concept of biostimulants involves substances or materials with no fertilizer value, which in minute amounts promote plant growth. The inclusion of PGPMs together with substances has also involved a significant distortion of the classical concept of biostimulants. Regulations such as the recent EU Fertilizing Products Regulation (EU No. 2019/1009) have incorporated the new definition of biostimulants and included microbials as a subcategory of biostimulants. We discuss that this regulation and the forthcoming European harmonized standards disregard some key features of microbial products, such as the live, true biological nature of their active principles. The factors that determine the complex functional compatibility of plant–microbe associations, and important biosafety issues that concern the intentional release of microbes into the environment, seem to be also ignored. We anticipate that by equating microbials to chemicals, the biological nature of microbial products and their specific requirements will be underestimated, with pernicious consequences for their future development and success.

Bacterial exopolysaccharides (EPS) have been implicated in a variety of functions that assist in bacterial survival, colonization, and host–microbe interactions. Among them, bacterial linear β-glucans are polysaccharides formed by D-glucose units linked by β-glycosidic bonds, which include curdlan, cellulose, and the new described Mixed Linkage β-Glucan (MLG). Bis-(3′,5′)-cyclic dimeric guanosine monophosphate (c-di-GMP) is a universal bacterial second messenger that usually promote EPS production. Here, we report Rhizobium etli as the first bacterium capable of producing cellulose and MLG. Significant amounts of these two β-glucans are not produced under free-living laboratory conditions, but their production is triggered upon elevation of intracellular c-di-GMP levels, both contributing to Congo red (CR⁺) and Calcofluor (CF⁺) phenotypes. Cellulose turned out to be more relevant for free-living phenotypes promoting flocculation and biofilm formation under high c-di-GMP conditions. None of these two EPS are essential for attachment to roots of Phaseolus vulgaris, neither for nodulation nor for symbiotic nitrogen fixation. However, both β-glucans separately contribute to the fitness of interaction between R. etli and its host. Overproduction of these β-glucans, particularly cellulose, appears detrimental for symbiosis. This indicates that their activation by c-di-GMP must be strictly regulated in time and space and should be controlled by different, yet unknown, regulatory pathways.

Extracellular matrix components of bacterial biofilms include biopolymers such as polysaccharides, nucleic acids and proteins. Similar to polysaccharides, the secretion of adhesins and other matrix proteins can be regulated by the second messenger cyclic diguanylate (cdG). We have performed quantitative proteomics to determine the extracellular protein contents of a Rhizobium etli strain expressing high cdG intracellular levels. cdG promoted the exportation of proteins that likely participate in adhesion and biofilm formation: the rhizobial adhesion protein RapA and two previously undescribed likely adhesins, along with flagellins. Unexpectedly, cdG also promoted the selective exportation of cytoplasmic proteins. Nearly 50% of these cytoplasmic proteins have been previously described as moonlighting or candidate moonlighting proteins in other organisms, often found extracellularly. Western blot assays confirmed cdG-promoted export of two of these cytoplasmic proteins, the translation elongation factor (EF-Tu) and glyceraldehyde 3-phosphate dehydrogenase (Gap). Transmission Electron Microscopy immunolabeling located the Gap protein in the cytoplasm but was also associated with cell membranes and extracellularly, indicative of an active process of exportation that would be enhanced by cdG. We also obtained evidence that cdG increases the number of extracellular Gap proteoforms, suggesting a link between cdG, the post-translational modification and the export of cytoplasmic proteins.