Ana Bejarano

Soil salinity is a major problem affecting crop production worldwide. Lately, there have been great research efforts in increasing the salt tolerance of plants through the inoculation of plant growth-promoting endophytic bacteria. However, their ability to promote plant growth under no-stress and salinity-stress conditions remains largely uncertain. Here, we carried out a global meta-analysis to quantify the plant growth-promoting effects (improvement of morphological attributes, photosynthetic capacity, antioxidative ability, and ion homeostasis) of endophytic bacteria in plants under no-stress and salinity-stress conditions. In addition, we elucidated the underlying mechanisms of growth promotion in salt-sensitive (SS) and salt-tolerant (ST) plants derived from the interaction with endophytic bacteria under no-stress and salinity-stress conditions. Specifically, this work encompassed 42 peer-reviewed articles, a total of 77 experiments, and 24 different bacterial genera. On average, endophytic bacterial inoculation increased morphological parameters. Moreover, the effect of endophytic bacteria on the total dry biomass, number of leaves, root length, shoot length, and germination rate was generally greater under salinity-stress conditions than no-stress conditions. On a physiological level, the relative better performance of the bacterial inoculants under the salinity-stress condition was associated with the increase in total chlorophyll and chlorophyll-b, as well as with the decrease of 1-aminocylopropane-1-carboxylate concentration. Moreover, under the salinity-stress condition, bacterial inoculation conferred a significantly higher increase in root K⁺ concentration and decrease in leaf Na⁺ concentration than under the no-stress condition. In SS plants, bacterial inoculation induced a higher increase in chlorophyll-b and superoxide dismutase activity, as well as a higher decrease in abscisic acid content, than in ST plants. Under salinity-stress, endophytic bacterial inoculation increased root K⁺ concentration in both SS and ST plants but decreased root Na⁺ concentration only in ST plants. Overall, this meta-analysis suggests that endophytic bacterial inoculation is beneficial under both no salinity-stress and salinity-stress conditions, but the magnitude of benefit is definitely higher under salinity-stress conditions and varies with the salt tolerance level of plants.

The rhizosphere is a dynamic region governed by complex microbial interactions where diffusible communication signals produced by bacteria continuously shape the gene expression patterns of individual species and regulate fundamental traits for adaptation to the rhizosphere environment. Lysobacter spp. are common bacterial inhabitants of the rhizosphere and have been frequently associated with soil disease suppressiveness. However, little is known about their ecology and how diffusible communication signals might affect their behavior in the rhizosphere. To shed light on the aspects determining rhizosphere competence and functioning of Lysobacter spp., we carried out a functional and transcriptome analysis on the plant beneficial bacterium Lysobacter capsici AZ78 (AZ78) grown in the presence of the most common diffusible communication signals released by rhizosphere bacteria. Mining the genome of AZ78 and other Lysobacter spp. showed that Lysobacter spp. share genes involved in the production and perception of diffusible signal factors, indole, diffusible factors, and N-acyl-homoserine lactones. Most of the tested diffusible communication signals (i.e., indole and glyoxylic acid) influenced the ability of AZ78 to inhibit the growth of the phytopathogenic oomycete Pythium ultimum and the Gram-positive bacterium Rhodococcus fascians. Moreover, RNA-Seq analysis revealed that nearly 21% of all genes in AZ78 genome were modulated by diffusible communication signals. 13-Methyltetradecanoic acid, glyoxylic acid, and 2,3-butanedione positively influenced the expression of genes related to type IV pilus, which might enable AZ78 to rapidly colonize the rhizosphere. Moreover, glyoxylic acid and 2,3-butanedione downregulated tRNA genes, possibly as a result of the elicitation of biological stress responses. On its behalf, indole downregulated genes related to type IV pilus and the heat-stable antifungal factor, which might result in impairment of twitching motility and antibiotic production in AZ78. These results show that diffusible communication signals may affect the ecology of Lysobacter spp. in the rhizosphere and suggest that diffusible communication signals might be used to foster rhizosphere colonization and functioning of plant beneficial bacteria belonging to the genus Lysobacter.

Determining the mode of action of microbial biocontrol agents plays a key role in their development and registration as commercial biopesticides. The biocontrol rhizobacterium Lysobacter capsici AZ78 (AZ78) is able to inhibit a vast array of plant pathogenic oomycetes and Gram-positive bacteria due to the release of antimicrobial secondary metabolites. A combination of MALDI-qTOF-MSI and UHPLC-HRMS/M was applied to finely dissect the AZ78 metabolome and identify the main secondary metabolites involved in the inhibition of plant pathogenic microorganisms. Under nutritionally limited conditions, MALDI-qTOF-MSI revealed that AZ78 is able to release a relevant number of antimicrobial secondary metabolites belonging to the families of 2,5-diketopiperazines, cyclic lipodepsipeptides, macrolactones and macrolides. In vitro tests confirmed the presence of secondary metabolites toxic against Pythium ultimum and Rhodococcus fascians in AZ78 cell-free extracts. Subsequently, UHPLC-HRMS/MS was used to confirm the results achieved with MALDI-qTOF-MSI and investigate for further putative antimicrobial secondary metabolites known to be produced by Lysobacter spp. This technique confirmed the presence of several 2,5-diketopiperazines in AZ78 cell-free extracts and provided the first evidence of the production of the cyclic depsipeptide WAP-8294A2 in a member of L. capsici species. Moreover, UHPLC-HRMS/MS confirmed the presence of dihydromaltophilin/Heat Stable Antifungal Factor (HSAF) in AZ78 cell-free extracts. Due to the production of HSAF by AZ78, cell-free supernatants were effective in controlling Plasmopara viticola on grapevine leaf disks after exposure to high temperatures. Overall, our work determined the main secondary metabolites involved in the biocontrol activity of AZ78 against plant pathogenic oomycetes and Gram-positive bacteria. These results might be useful for the future development of this bacterial strain as the active ingredient of a microbial biopesticide that might contribute to a reduction in the chemical input in agriculture.

Intraspecies, interspecies and interkingdom signalling occurring via diffusible communication signals (DCSs) continuously shapes the gene expression patterns of individual bacterial species in the rhizosphere, affecting bacterial functions within the rhizosphere microbial community. To unravel how DCSs influence rhizosphere competence of plant beneficial rhizobacteria, we carried out a functional and transcriptome analysis on the plant beneficial bacterium Lysobacter capsici AZ78 (AZ78). Results reveal that 13-methyltetradecanoic acid and indole, glyoxylic acid and 2,3-butanedione play a relevant role in the interaction between L. capsici members and other soil-living (micro)organisms. DCSs regulated mechanisms of multistress and multidrug tolerance and persistence, including detoxification, motility, antibiotic production, and expression of secretion systems. In particular, 13-methyltetradecanoic acid, glyoxylic acid and 2,3-butanedione might enable AZ78 to rapidly colonize the rhizosphere. Moreover, glyoxylic acid and 2,3-butanedione elicit biological responses to outcompete other (micro)organisms. In contrast, indole inactivates twitching motility and antibiotic production. These results demonstrate that DCSs influence the functioning of plant beneficial rhizobacteria and suggest that plant beneficial L. capsici strains could use them to foster rhizosphere colonization and enhance in vivo activities to increase soil health and plant fitness.

Inhibitory activity of the biocontrol bacterial strain Lysobacter capsici AZ78 is related to the production of cyclo(l-Pro-l-Tyr), a 2,5-diketopiperazine with in vitro and in vivo toxic activity against Phytophthora infestans and Plasmopara viticola. Further investigation of culture filtrate organic extracts showed its ability to produce other 2,5-diketopiperazines. They were isolated and identified by spectroscopic (¹H NMR and ESIMS) and physic (specific optical rotation) methods as cyclo(l-Pro-l-Val), cyclo(d-Pro-d-Phe), cyclo(l-Pro-l-Leu), and cyclo(d-Pro-l-Tyr). When tested against the phytopathogenic Gram-positive bacterium Rhodococcus fascians LMG 3605, cyclo(l-Pro-l-Val) showed a toxic activity similar to chloramphenicol at a comparable concentration. Overall, these data suggest that 2,5-diketopiperazines represent a class of metabolites characterizing the metabolome of L. capsici AZ78. Furthermore, the toxic activity showed by cyclo(l-Pro-l-Val) against R. fascians LMG 3605 broaden the spectrum activity of 2,5-diketopiperazines against phytopathogenic microorganisms enforcing their potential development as biopesticides.

Damages of the (agro)ecosystem by extensive use of chemical fertilizers and pesticides, the global dying of bee populations possibly linked to pesticide spraying, and stricter regulations for pesticide use together with successful use of microbials in IPM programs are pushing on the development and commercialization of new microbial products and a large and growing biostimulants and biocontrol market. This review focuses on microbial inoculants including bacteria, fungi, and viruses used as biostimulant or biocontrol agent for foliar application and covers all important steps from inoculant development to successful field application. Topics presented comprise typical spraying equipment including the importance of the spraying process and relating effects, furthermore formulation development including classification and adjuvants, and thirdly regulatory aspects as currently applied or under discussion. Microbial inoculants for foliar spray reported in scientific literature are summarized and contrasted with selected commercial products. Special attention is given to factors most important in microbial spray: (a) type of active ingredient (bacteria, fungi, viruses), (b) mode of action (ingestion, contact, competition), (c) interaction with the plant leaf surface, (d) droplet size in terms of microbe concentration and leaf coverage, and (e) environmental conditions during spraying. Finally, we want to emphasize that timely administration is of utmost importance for successful spraying and maximum efficacy. This might be supported by weather stations and disease/pest models as an important step towards precision farming.

Summary: The application of beneficial, plant‐associated microorganisms is a sustainable approach to improving crop performance in agriculture. However, microbial inoculants are often susceptible to prolonged periods of storage and deleterious environmental factors, which negatively impact their viability and ultimately limit efficacy in the field. This particularly concerns non‐sporulating bacteria. To overcome this challenge, the availability of protective formulations is crucial. Numerous parameters influence the viability of microbial cells, with drying procedures generally being among the most critical ones. Thus, technological advances to attenuate the desiccation stress imposed on living cells are key to successful formulation development. In this review, we discuss the core aspects important to consider when aiming at high cell viability of non‐sporulating bacteria to be applied as microbial inoculants in agriculture. We elaborate the suitability of commonly applied drying methods (freeze‐drying, vacuum‐drying, spray‐drying, fluidized bed‐drying, air‐drying) and potential measures to prevent cell damage from desiccation (externally applied protectants, stress pre‐conditioning, triggering of exopolysaccharide secretion, ‘helper’ strains). Furthermore, we point out methods for assessing bacterial viability, such as colony counting, spectrophotometry, microcalorimetry, flow cytometry and viability qPCR . Choosing appropriate technologies for maintenance of cell viability and evaluation thereof will render formulation development more efficient. This in turn will aid in utilizing the vast potential of promising, plant beneficial bacteria as sustainable alternatives to standard agrochemicals.

Herein, we present a workflow for the development of talc-based microbial inoculants for foliar spray consisting of four steps. These include together with decision-making criteria (1) the selection of additives based on their capability to wet juvenile maize leaves, (2) their adhesion on the plant, (3) their interaction with the biological systems, and (4) the choice of thickener for good dispersion stability. In total, 29 additives including polysaccharides and proteins, polyols, glycosides, oils, waxes, and surfactants (e.g., chitosan, gelatin, glycerol, saponin, castor oil, polyethylene, rhamnolipid) were evaluated. Contact angle and spreading index measurements revealed that the use of 5% Geloil, 1% rhamnolipid, or suitable combinations of Geloil + rhamnolipid and Nurture Yield S 2002 + rhamnolipid enhanced wetting of hydrophobic maize leaves and adherence, similarly to the commercial wetting agents recommended for plant protection 1% Prev B2 and 1% Trifolio S Forte. Interaction of additives with biological systems was based on biocompatibility and phytotoxicity assays, and cell viability monitoring using the endophytic Gram-negative bacterium Paraburkholderia phytofirmans PsJN. Results from biocompatibility assays indicated that in contrast to rhamnolipid and Prev B2 Geloil, Nurture Yield S 2002 and Trifolio S Forte fully supported bacterial growth within a concentration range of 1 to 5%. Dose-dependent phytotoxicity was observed in plants treated with rhamnolipid. Most efficient formulation was composed of PsJN, talc, xanthan, and Geloil. Beyond that, the proposed workflow is expected to generally provide guidance for the development of spray formulations and help other researchers to optimize their choices in this area.