Microbials for Agriculture: Why Do They Call Them Biostimulants When They Mean Probiotics?

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.

The Role of Two Linear ß-Glucans Activated by c-di-GMP in Rhizobium etli CFN42.

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 ß-glucans are polysaccharides formed by D-glucose units linked by ß-glycosidic bonds, which include curdlan, cellulose, and the new described Mixed Linkage ß-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 ß-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 ß-glucans separately contribute to the fitness of interaction between R. etli and its host. Overproduction of these ß-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.

Selection and characterization of Spanish Trifolium-nodulating rhizobia for pasture inoculation.

Identification of elite nitrogen-fixing rhizobia strains is a continuous and never ending effort, since new legume species can be cultivated in different agro systems or are introduced into new areas. This current study reports on the taxonomic affiliation and symbiotic proficiency of nine strains of Trifolium-nodulating rhizobia isolated from different pasture areas in Spain, as well as three Rhizobium leguminosarum bv. trifolii reference strains, on eleven Trifolium species. Based on 16S rRNA gene sequences the strains belonged to the R. leguminosarum species complex. Additional phylogenetic analyses of the housekeeping genes recA, atpD and rpoB showed the strains were closely related to the species R. leguminosarum, R. laguerreae, R. indicum, R. ruizarguesonis or R. acidisoli. In addition, three strains had no clear affiliation and could represent putative new species, although two of the reference strains were positioned close to R. ruizarguesonis. nodC gene phylogeny allowed the discrimination between strains isolated from annual or perennial Trifolium species and placed all of them in the symbiovar trifolii. Neither geographic origin nor host-plant species could be correlated with the taxonomic affiliation of the strains and a high degree of phenotypic diversity was found among this set of strains. The strong interaction of plant species with the rhizobial strains found for biological nitrogen fixation (BNF) was noteworthy, and allowed the identification of rhizobial strains with a maximum proficiency for certain trefoil species. Several strains showed high BNF potential with a wide range of clover species, which made them valuable strains for inoculant manufacturers and they would be particularly useful for inoculation of seed mixtures in natural or cultivated pastures.

Impact of c-di-GMP on the Extracellular Proteome of Rhizobium etli.

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.

Diversity, Phylogeny and Plant Growth Promotion Traits of Nodule Associated Bacteria Isolated from Lotus parviflorus.

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.

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.

Mesorhizobium olivaresii sp. nov. isolated from Lotus corniculatus nodules.

In this study four Mesorhizobium strains isolated from Lotus corniculatus nodules in Granada (Spain) were characterized. Their 16S rRNA gene sequences were closely related to those of M. albiziae LMG 23507T and M. chacoense Pr5T showing 99.4 and 99.2% similarity values, respectively. The analysis of concatenated rpoB, recA, atpD and glnII genes showed they formed a cluster with internal similarities higher than 97%. The closest species also were M. albiziae LMG 23507T and M. chacoense Pr5T showing similarity values lower than 92% in rpoB, recA and glnII genes and lower than 96.5% in the atpD gene. These results indicated that the L. corniculatus strains belong to a new species of genus Mesorhizobium which was confirmed by DNA-DNA hybridization and phenotypic characterization. Therefore a new species with the name Mesorhizobium olivaresii sp. nov. is proposed, and the type strain is CPS13T (LMG 29295T=CECT 9099T).

Phyllobacterium loti sp. nov. isolated from nodules of Lotus corniculatus.

Strain S658(T) was isolated from a Lotus corniculatus nodule in a soil sample obtained in Uruguay. Phylogenetic analysis of the 16S rRNA gene and atpD gene showed that this strain clustered within the genus Phyllobacterium. The closest related species was, in both cases, Phyllobacterium trifolii PETP02(T) with 99.8 % sequence similarity in the 16S rRNA gene and 96.1 % in the atpD gene. The 16S rRNA gene contains an insert at the beginning of the sequence that has no similarities with other inserts present in the same gene in described rhizobial species. Ubiquinone Q-10 was the only quinone detected. Strain S658(T) differed from its closest relatives through its growth in diverse culture conditions and in the assimilation of several carbon sources. It was not able to reproduce nodules in Lotus corniculatus. The results of DNA-DNA hybridization, phenotypic tests and fatty acid analyses confirmed that this strain should be classified as a representative of a novel species of the genus Phyllobacterium, for which the name Phyllobacterium loti sp. nov. is proposed. The type strain is S658(T)( = LMG 27289(T) = CECT 8230(T)).

Phylogenetic relationship of Lotus uliginosus symbionts with bradyrhizobia nodulating genistoid legumes.

Lotus species are legumes with potential for pastures in soils with low-fertility and environmental constraints. The aim of this work was to characterize bacteria that establish efficient nitrogen-fixing symbiosis with the forage species Lotus uliginosus. A total of 39 isolates were obtained from nodules of L. uliginosus naturally growing in two different locations of Portugal. Molecular identification of the isolates plus the commercial inoculant strain NZP2039 was performed by REP-PCR, 16S rRNA RFLP, and 16S rRNA, glnII and recA sequence analyses. Limited genetic diversity was found among the L. uliginosus symbionts, which showed a close phylogenetic relationship with the species Bradyrhizobium japonicum. The symbiotic nifH, nodA and nodC gene sequences were closely related with the corresponding genes of various Bradyrhizobium strains isolated from Lupinus and other genistoid legumes and therefore were phylogenetically separated from other Lotus spp. rhizobia. The L. uliginosus bradyrhizobia were able to nodulate and fix nitrogen in association with L. uliginosus, could nodulate Lotus corniculatus with generally poor nitrogen-fixing efficiency, formed nonfixing nodules in Lotus tenuis and Lupinus luteus roots and were unable to nodulate Glycine soja or Glycine max. Thus, L. uliginosus rhizobia seem closely related to B. japonicum biovar genistearum strains.

Characterization of strains unlike Mesorhizobium loti that nodulate lotus spp. in saline soils of Granada, Spain.

Lotus species are forage legumes with potential as pastures in low-fertility and environmentally constrained soils, owing to their high persistence and yield under those conditions. The aim of this work was the characterization of phenetic and genetic diversity of salt-tolerant bacteria able to establish efficient symbiosis with Lotus spp. A total of 180 isolates able to nodulate Lotus corniculatus and Lotus tenuis from two locations in Granada, Spain, were characterized. Molecular identification of the isolates was performed by repetitive extragenic palindromic PCR (REP-PCR) and 16S rRNA, atpD, and recA gene sequence analyses, showing the presence of bacteria related to different species of the genus Mesorhizobium: Mesorhizobium tarimense/Mesorhizobium tianshanense, Mesorhizobium chacoense/Mesorhizobium albiziae, and the recently described species, Mesorhizobium alhagi. No Mesorhizobium loti-like bacteria were found, although most isolates carried nodC and nifH symbiotic genes closely related to those of M. loti, considered the type species of bacteria nodulating Lotus, and other Lotus rhizobia. A significant portion of the isolates showed both high salt tolerance and good symbiotic performance with L. corniculatus, and many behaved like salt-dependent bacteria, showing faster growth and better symbiotic performance when media were supplemented with Na or Ca salts.