An ecotoxicological assessment of a strigolactone mimic used as the active ingredient in a plant biostimulant formulation.

A risk assessment on the aquatic toxicity of the plant biostimulant strigolactone mimic (2-(4-methyl-5-oxo-2,5-dihydro-furan-2-yloxy)-benzo[de]isoquinoline-1,3-dione (SL-6) was performed using a suite of standardised bioassays representing different trophic groups and acute and chronic endpoints. In freshwater, three trophic groups of algae, crustacea and fish were used. Whilst in seawater, algae (unicellular and macroalgae), Crustacea and Mollusca were employed. In addition, the genotoxicity of SL-6 was determined with the comet assessment performed on unicellular marine algae, oysters, and fish embryos. This was the first time ecotoxicity tests have been performed on SL-6. In freshwater, the lowest LOEC was measured in the unicellular algae at 0.31 mg/L SL-6. Although, similar LOEC values were found for embryo malformations and impacts on hatching rate in zebrafish (LOEC 0.31-0.33 mg/L). Consistent malformations of pericardial and yolk sac oedemas were identified in the zebrafish embryos at 0.31 mg/L. In marine species, the lowest LOEC was found for both Tisbe battagliai mortality and microalgae growth at an SL-6 concentration of 1.0 mg/L. Significant genotoxicity was observed above control levels at 0.0031 mg/L SL-6 in the unicellular algae and 0.001 mg/L SL-6 in the oyster and zebrafish larvae. When applying the simple risk assessment, based on the lowest NOECs and appropriate assessment factors, the calculated predicted no effect concentration (PNEC), for the ecotoxicity and the genotoxicity tests were 1.0 µg/L and 0.01 µg/L respectively.

Growth and metabolism of pea (Pisum sativum) via biostimulants based on greener ZnO nanoparticles.

The purpose of this study was to identify the most important physiological and biological effects of green synthesis ZnO nanoparticles at a size of 65 nm, biostimulant (Folcare) and interaction biostimulant ZnO NPs on plant growth and metabolism. As our understanding of biostimulants' preventive and restorative modes of action has increased, it is critical to maintain the best crop output and quality possible. The reduction of fertilizers must be substituted by strategies that improve the nutrients uptake or their utilization by the plants. New processing methods are required as an efficient green process or an integrated (hybrid) process for different new technologies of interest. The effects of NPs, biostimulant, and combination ZnO NPs biostimulant on plant cell metabolism were examined in cytosol, chloroplast, and mitochondria of cells from the stems, roots, and leaves. The interaction NPs/biostimulant had a beneficial effect on the morphological and physiological indicators of plant health than when nanoparticles and biostimulant are applied separately. Folcare biostimulant coupled with zinc oxide nanoparticles improved pea crops growth. The improved of the quality of pea plants can be explained at least, in part, by increase in antioxidant activities during plant growth phenophase.

1. Highlighting the environmental effect the risk management of biostimulants based on ZnO bionanoparticles on the growth of pea- Identification of specific responses of plants to nano stresses: Analyzes of metabolitesAntioxidants enzymes2. Folcare coupled with zinc oxide bionanoparticles has a significant favorable influence on environmental conservation management by assisting plants in more effectively using nutrients.

Foliar application of biostimulants improves nutritional and bioactive quality of walnuts.

BACKGROUND: Owing to their health benefits, walnuts are attracting interest as a good option for nutritious meals, thereby promoting their production. Furthermore, the adoption of ecologically and environmentally friendly agriculture strengthens biostimulant use as a sustainable complement to traditional fertilizers. This study evaluated the effects of different foliar-applied biostimulants in walnut tree orchards, in northeastern Portugal, on walnuts' chemical composition and bioactivity. RESULTS: Walnut samples were rich in fat (particularly the polyunsaturated linoleic acid), dietary fiber and protein. Sucrose was the most prevalent soluble sugar, followed by glucose and fructose. Studied samples also showed an antioxidant activity comparable (or superior) to that of Trolox. Some plant biostimulants (e.g. Sprint Plus®) had a positive impact on the nutritional composition of walnuts, more specifically by boosting tocopherol levels, besides improving the bioactivity of walnut extracts against specific bacteria. CONCLUSION: Overall, this research demonstrated that important quality traits of walnuts can be improved using sustainable agricultural bioproducts and practices. © 2024 Society of Chemical Industry.

Transcriptome, Biochemical and Phenotypic Analysis of the Effects of a Precision Engineered Biostimulant for Inducing Salinity Stress Tolerance in Tomato.

Salinity stress is a major problem affecting plant growth and crop productivity. While plant biostimulants have been reported to be an effective solution to tackle salinity stress in different crops, the key genes and metabolic pathways involved in these tolerance processes remain unclear. This study focused on integrating phenotypic, physiological, biochemical and transcriptome data obtained from different tissues of Solanum lycopersicum L. plants (cv. Micro-Tom) subjected to a saline irrigation water program for 61 days (EC: 5.8 dS/m) and treated with a combination of protein hydrolysate and Ascophyllum nodosum-derived biostimulant, namely PSI-475. The biostimulant application was associated with the maintenance of higher K+/Na+ ratios in both young leaf and root tissue and the overexpression of transporter genes related to ion homeostasis (e.g., NHX4, HKT1;2). A more efficient osmotic adjustment was characterized by a significant increase in relative water content (RWC), which most likely was associated with osmolyte accumulation and upregulation of genes related to aquaporins (e.g., PIP2.1, TIP2.1). A higher content of photosynthetic pigments (+19.8% to +27.5%), increased expression of genes involved in photosynthetic efficiency and chlorophyll biosynthesis (e.g., LHC, PORC) and enhanced primary carbon and nitrogen metabolic mechanisms were observed, leading to a higher fruit yield and fruit number (47.5% and 32.5%, respectively). Overall, it can be concluded that the precision engineered PSI-475 biostimulant can provide long-term protective effects on salinity stressed tomato plants through a well-defined mode of action in different plant tissues.

Presence and future of plant phenotyping approaches in biostimulant research and development.

Commercial interest in biostimulants as a tool for sustainable green economics and agriculture concepts is on a steep rise, being followed by increasing demand to employ efficient scientific methods to develop new products and understand their mechanisms of action. Biostimulants represent a highly diverse group of agents derived from various natural sources. Regardless of their nutrition content and composition, they are classified by their ability to improve crop performance through enhanced nutrient use efficiency, abiotic stress tolerance, and quality of crops. Numerous reports have described modern, non-invasive sensor-based phenotyping methods in plant research. This review focuses on applying phenotyping approaches in biostimulant research and development, and maps the evolution of interaction of these two intensively growing domains. How phenotyping served to identify new biostimulants, the description of their biological activity, and the mechanism/mode of action are summarized. Special attention is dedicated to the indoor high-throughput methods using model plants suitable for biostimulant screening and developmental pipelines, and high-precision approaches used to determine biostimulant activity. The need for a complex method of testing biostimulants as multicomponent products through integrating other -omic approaches followed by advanced statistical/mathematical tools is emphasized.

Calcium Phosphate Particles Coated with Humic Substances: A Potential Plant Biostimulant from Circular Economy.

Nowadays, the use of biostimulants to reduce agrochemical input is a major trend in agriculture. In this work, we report on calcium phosphate particles (CaP) recovered from the circular economy, combined with natural humic substances (HSs), to produce a plant biostimulant. CaPs were obtained by the thermal treatment of Salmo salar bones and were subsequently functionalized with HSs by soaking in a HS water solution. The obtained materials were characterized, showing that the functionalization with HS did not sort any effect on the bulk physicochemical properties of CaP, with the exception of the surface charge that was found to get more negative. Finally, the effect of the materials on nutrient uptake and translocation in the early stages of development (up to 20 days) of two model species of interest for horticulture, Valerianella locusta and Diplotaxis tenuifolia, was assessed. Both species exhibited a similar tendency to accumulate Ca and P in hypogeal tissues, but showed different reactions to the treatments in terms of translocation to the leaves. CaP and CaP-HS treatments lead to an increase of P accumulation in the leaves of D. tenuifolia, while the treatment with HS was found to increase only the concentration of Ca in V. locusta leaves. A low biostimulating effect on both plants' growth was observed, and was mainly scribed to the low concentration of HS in the tested materials. In the end, the obtained material showed promising results in virtue of its potential to elicit phosphorous uptake and foliar translocation by plants.

An overview of the current plant biostimulant legislations in different European Member States.

Plant biostimulants are borderline substances that play an intermediate role between plant protection products and fertilisers. At present, such substances are regulated by national laws and have different names in the various European Member States. The aim of this paper is to provide an overview on the activity of these substances and on the national laws that regulate them, as they vary considerably from one Member State to another. The greatest difficulty in terms of the correct regulatory framework for these substances is related to their heterogeneity. This situation creates uncertainties for operators, control authorities and bodies that certify and control the organic production, and strongly limits the growth of these substances. This problem will be overcome with the amendment of Regulation EC No 2003/2003 which will shortly extend its scope to the category of plant biostimulants included within the fertilising additives.

Water-Soluble Lignins from Different Bioenergy Crops Stimulate the Early Development of Maize (Zea mays, L.).

The molecular composition of water-soluble lignins isolated from four non-food bioenergy crops (cardoon CAR, eucalyptus EUC, and two black poplars RIP and LIM) was characterized in detail, and their potential bioactivity towards maize germination and early growth evaluated. Lignins were found to not affect seed germination rates, but stimulated the maize seedling development, though to a different extent. RIP promoted root elongation, while CAR only stimulated the length of lateral seminal roots and coleoptile, and LIM improved only the coleoptile development. The most significant bioactivity of CAR was related to its large content of aliphatic OH groups, C-O carbons and lowest hydrophobicity, as assessed by (31)P-NMR and (13)C-CPMAS-NMR spectroscopies. Less bioactive RIP and LIM lignins were similar in composition, but their stimulation of maize seedling was different. This was accounted to their diverse content of aliphatic OH groups and S- and G-type molecules. The poorest bioactivity of the EUC lignin was attributed to its smallest content of aliphatic OH groups and largest hydrophobicity. Both these features may be conducive of a EUC conformational structure tight enough to prevent its alteration by organic acids exuded from vegetal tissues. Conversely the more labile conformational arrangements of the other more hydrophilic lignin extracts promoted their bioactivity by releasing biologically active molecules upon the action of exuded organic acids. Our findings indicate that water-soluble lignins from non-food crops may be effectively used as plant biostimulants, thus contributing to increase the economic and ecological liability of bio-based industries.

Next generation plant biostimulants & genome sequencing strategies for sustainable agriculture development.

The best environment for plant growth and development contains certain essential metabolites. A broad category of metabolites known as "plant biostimulants" (PBs) includes biomolecules such as proteins, carbohydrates, lipids, and other secondary metabolites related to groups of terpenes, specific nitrogen-containing compounds, and benzene ring-conjugated compounds. The formation of biomolecules depends on both biotic and abiotic factors, such as the release of PB by plants, animals, and microorganisms, or it can result from the control of temperature, humidity, and pressure in the atmosphere, in the case of humic substances (HSs). Understanding the genomic outputs of the concerned organism (may be plants or others than them) becomes crucial for identifying the underlying behaviors that lead to the synthesis of these complex compounds. For the purposes of achieving the objectives of sustainable agriculture, detailed research on PBs is essential because they aid in increasing yield and other growth patterns of agro-economic crops. The regulation of homeostasis in the plant-soil-microbe system for the survival of humans and other animals is mediated by the action of plant biostimulants, as considered essential for the growth of plants. The genomic size and gene operons for functional and regulation control have so far been revealed through technological implementations, but important gene annotations are still lacking, causing a delay in revealing the information. Next-generation sequencing techniques, such as nanopore, nanoball, and Illumina, are essential in troubleshooting the information gaps. These technical advancements have greatly expanded the candidate gene openings. The secondary metabolites being important precursors need to be studied in a much wider scale for accurate calculations of biochemical reactions, taking place inside and outside the synthesized living cell. The present review highlights the sequencing techniques to provide a foundation of opportunity generation for agricultural sustainability.

Higher Plant-Derived Biostimulants: Mechanisms of Action and Their Role in Mitigating Plant Abiotic Stress.

Modern agriculture is being challenged by deteriorating edaphoclimatic conditions and increasing anthropogenic pressure. This necessitates the development of innovative crop production systems that can sustainably meet the demands of a growing world population while minimizing the environmental impact. The use of plant biostimulants is gaining ground as a safe and ecologically sound approach to improving crop yields. In this review, biostimulants obtained from different higher plant sources are presented under the term higher plant-derived biostimulants (hPDBs). Their mechanisms of action regulate physiological processes in plants from germination to fructification, conditioned by responses induced in plant mineral nutrition and primary metabolism, specialized metabolism, photosynthetic processes, oxidative metabolism, and signaling-related processes. The aim of this review is to collect and unify the abundant information dispersed in the literature on the effects of these biostimulants, focusing on crops subjected to abiotic stress conditions and the underlying mechanisms of action.

Effect of Bacterial Extracellular Polymeric Substances from Enterobacter spp. on Rice Growth under Abiotic Stress and Transcriptomic Analysis.

Plant biostimulants have received attention as sustainable alternatives to chemical fertilizers. Extracellular polymeric substances (EPSs), among the compounds secreted by plant growth-promoting rhizobacteria (PGPRs), are assumed to alleviate abiotic stress. This study aims to investigate the effect of purified EPSs on rice under abiotic stress and analyze their mechanisms. A pot experiment was conducted to elucidate the effects of inoculating EPSs purified from PGPRs that increase biofilm production in the presence of sugar on rice growth in heat-stress conditions. Since all EPSs showed improvement in SPAD after the stress, Enterobacter ludwigii, which was not characterized as showing higher PGP bioactivities such as phytohormone production, nitrogen fixation, and phosphorus solubilization, was selected for further analysis. RNA extracted from the embryos of germinating seeds at 24 h post-treatment with EPSs or water was used for transcriptome analysis. The RNA-seq analysis revealed 215 differentially expressed genes (DEGs) identified in rice seeds, including 139 up-regulated and 76 down-regulated genes. A gene ontology (GO) enrichment analysis showed that the enriched GO terms are mainly associated with the ROS scavenging processes, detoxification pathways, and response to oxidative stress. For example, the expression of the gene encoding OsAAO5, which is known to function in detoxifying oxidative stress, was two times increased by EPS treatment. Moreover, EPS application improved SPAD and dry weights of shoot and root by 90%, 14%, and 27%, respectively, under drought stress and increased SPAD by 59% under salt stress. It indicates that bacterial EPSs improved plant growth under abiotic stresses. Based on our results, we consider that EPSs purified from Enterobacter ludwigii can be used to develop biostimulants for rice.

Management of Antracnosis with Electrochemically Activated Salt Solutions (EASSs) on Bean Culture.

Common bean (Phaseolus vulgaris L.) is an important crop for food security and for national economics for several countries worldwide. One of the most important factors of risk in common bean production is the fungal disease anthracnose caused by Colletotrichum lindemuthianum, which, in some cases, causes complete yield losses; this kind of plant disease is usually managed through the application of chemical products such as fungicides that are commonly not accepted by society. This rejection is based on the relationship of pesticides with health damage and environmental contamination. In order to help in solving these drawbacks, the present work proposes the use of electrochemically activated salt solutions (EASSs) as a safer pathogen control agent in crops, due to it having shown an elicitor and biostimulant effect on plants. With this background, this manuscript presents in vitro results of the evaluation of the inhibitory effect for multiple bean pathogens and in vivo results of EASS in the common bean-Colletotrichum pathosystem by evaluation of the infection severity and defense activation, such as secondary metabolite production and antioxidant activity. EASS presence in growth media had a strong inhibitory effect at the beginning of experiments for some of the evaluated fungi. EASSs showed an effect against the development of the disease when applied in specific doses to prevent distress in plants.

Application of Seaweed Generates Changes in the Substrate and Stimulates the Growth of Tomato Plants.

Ulva ohnoi is a cosmopolitan green seaweed with commercial potential given the biomass that may be generated. We evaluated the effects of substrate changes induced by U. ohnoi application on the vegetative response of tomato plants under greenhouse conditions. First, the decomposition dynamics and N release of the dry seaweed biomass were studied using the litterbag method. Subsequently, we evaluated the effect of seaweed powder (SP) or seaweed extract (SE) applications on substrate and plant growth. Additionally, the growth parameters responses evaluated were related to the changes in substrate properties associated with each treatment. The results showed that the dry seaweed biomass has a rapid rate of degradation (k = 0.07 day-1) and N release (k = 0.024 day-1). The SP application improved the physicochemical and biological characteristics of the substrate by increasing the availability of minerals, the fungi:bacteria ratio, and the growth morphophysiological parameters (length, area, dry and fresh weight), chlorophyll and mineral content. In contrast, SE treatment showed a positive effect on the root, mineral content, and soil microbes. This study highlights the agricultural potential of U. ohnoi powder as an alternative supplement that supports nutrition and promotes the vegetative growth of plants cultivated in soilless horticultural systems.

A plant's perception of growth-promoting bacteria and their metabolites.

Many recent studies have highlighted the importance of plant growth-promoting (rhizo)bacteria (PGPR) in supporting plant's development, particularly under biotic and abiotic stress. Most focus on the plant growth-promoting traits of selected strains and the latter's effect on plant biomass, root architecture, leaf area, and specific metabolite accumulation. Regarding energy balance, plant growth is the outcome of an input (photosynthesis) and several outputs (i.e., respiration, exudation, shedding, and herbivory), frequently neglected in classical studies on PGPR-plant interaction. Here, we discuss the primary evidence underlying the modifications triggered by PGPR and their metabolites on the plant ecophysiology. We propose to detect PGPR-induced variations in the photosynthetic activity using leaf gas exchange and recommend setting up the correct timing for monitoring plant responses according to the specific objectives of the experiment. This research identifies the challenges and tries to provide future directions to scientists working on PGPR-plant interactions to exploit the potential of microorganisms' application in improving plant value.

Effect of microbial plant biostimulants on fruit and vegetable quality: current research lines and future perspectives.

Fruit and vegetables hold a prominent place in dietary guidance worldwide and, following the increasing awareness of the importance of their consumption for health, their demand has been on the rise. Fruit and vegetable production needs to be reconsidered so that it can be productive and, meantime, sustainable, resilient, and can deliver healthy and nutritious diets. Microbial plant biostimulants (PBs) are a possible approach to pursuing global food security and agricultural sustainability, and their application emerged as a promising alternative or substitute to the use of agrochemicals (e.g., more efficient use of mineral and organic fertilizers or less demand and more efficient use of pesticides in integrated production systems) and as a new frontier of investigation. To the best of our knowledge, no comprehensive reviews are currently available on the effects that microbial plant biostimulants' application can have specifically on each horticultural crop. This study thus aimed to provide a state-of-the-art overview of the effects that PBs can have on the morpho-anatomical, biochemical, physiological, and functional traits of the most studied crops. It emerged that most experiments occurred under greenhouse conditions; only a few field trials were carried out. Tomato, lettuce, and basil crops have been primarily treated with Arbuscular Mycorrhizal Fungi (AMF), while plant grow-promoting rhizobacteria (PGPR) metabolites were used for crops, such as strawberries and cucumbers. The literature review also pointed out that crop response to PBs is never univocal. Complex mechanisms related to the PB type, the strain, and the crop botanical family, occur.

Combined physiological and metabolomic analysis reveals the effects of different biostimulants on maize production and reproduction.

Plant biostimulants (PBs) are a potential strategy to improve crop growth and grain quality. In the present study, 100 mg/L trehalose, chitosan, humic acid and gamma-aminobutyric acid treatments were applied to analyze the effects of maize production and reproductive characteristics. The contents of nitrogen, phosphorus and potassium and grain quality were significantly affected by the PBs, but not yield. The seed germination rate of all PB treatments was significantly reduced, but the drought resistance of progeny seedlings was significantly improved, with humic acid having the strongest effect. Liquid chromatography mass spectrometry analysis indicated that the disruption of the tricarboxylic acid cycle, probably due to the blockage of intermediate anabolism, reduced the supply of energy and nutrients in the early stages of germination, thus inhibiting seed germination, while the increased resistance of the offspring seedlings may be due to the up-regulation of the synthesis of unsaturated fatty acids and alkaloids by humic acid treatment. This study revealed the similarity and heterogeneity of the effects of different PBs on nutrient accumulation, yield characteristics and grain quality of maize, providing guidance for the application of PBs in intensive and sustainable agricultural production.

Oviposition Deterrent Activity of Fungicides and Low-Risk Substances for the Integrated Management of the Olive Fruit Fly Bactrocera oleae (Diptera, Tephritidae).

The control of Bactrocera oleae is fundamental to decreasing the significant production loss in olive cultivation. However, traditional containment based on the use of synthetic insecticides has been encountering serious limitations due to their negative effect on human health and the environment. Within the scope of integrated olive fly management, the use of products with repellency and oviposition deterrent activity might represent a more eco-friendly solution. In this study, we tested the oviposition deterrent activity of some commercial formulations already used in olive tree crops as fungicides (copper oxychloride, dodine, mancozeb, pyraclostrobin and difeconazole) and plant bio-stimulants (tannins, clay, flavonoids and a zinc-copper-citric acid biocomplex). The trials were conducted testing the oviposition behavior of mated olive fly females in both choice and no-choice assays. Our results showed that most of the substances have affected the ovipositional activity of the olive fly, except for difeconazole. Moreover, some products (copper oxychloride, flavonoids and tannins) have proven to differently influence the flies' oviposition comparing the two tests. The repellent effect of these commercial products should be further studied to prove whether the repellency was due either to the active ingredient or to the co-formulants, and to assess their effect in the open field.

Growth and metabolism of dark septate endophytes and their stimulatory effects on plant growth.

Dark septate endophytes (DSE) colonize plant roots extensively and increase host plant growth and nutrition. However, the development of DSE-produced metabolites as plant biostimulants has been largely ignored. DSE growth curves and extracellular metabolite components were analyzed and the growth-promoting effects of DSE extracellular metabolites on alfalfa (Medicago sativa L.) grown for 4, 8 12, 16 and 20 days were evaluated. The growth curve of the DSE strain Alternaria sp. shows days 0-8 in the growth phase, days 8-16 in the stable phase, and days 16-20 in the senescent phase. The extracellular metabolite components of DSE were significantly different at different growth stages. The biomass of alfalfa was increased significantly by DSE extracellular metabolites (P < 0.05). Biomass of alfalfa inoculated with DSE extracellular metabolites more than doubled after growth for 8 days and nutrient availability also increased significantly compared with the uninoculated control. Six DSE extracellular metabolites, calycosin 7-galactoside, 1-[(5-amino-5-carboxypentyl)amino]-1-deoxyfructose, N2-fructopyranosylarginine, 2-(4-methyl-5-thiazolyl)ethyl hexanoate, kenposide B, and medinoside E, were significantly positively correlated with alfalfa biomass (P < 0.01). This study combines the DSE extracellular metabolites with plant and soil traits to provide a theoretical basis for the use of DSE metabolites in the product development of plant biostimulants.

Diversity of Phosphate Chemical Forms in Soils and Their Contributions on Soil Microbial Community Structure Changes.

In many soils, the bioavailability of Phosphorus (P), an essential macronutrient is a limiting factor for crop production. Among the mechanisms developed to facilitate the absorption of phosphorus, the plant, as a holobiont, can rely on its rhizospheric microbial partners. Therefore, microbial P-solubilizing inoculants are proposed to improve soil P fertility in agriculture. However, a better understanding of the interactions of the soil-plant-microorganism continuum with the phosphorus cycle is needed to propose efficient inoculants. Before proposing further methods of research, we carried out a critical review of the literature in two parts. First, we focused on the diversity of P-chemical forms. After a review of P forms in soils, we describe multiple factors that shape these forms in soil and their turnover. Second, we provide an analysis of P as a driver of microbial community diversity in soil. Even if no rule enabling to explain the changes in the composition of microbial communities according to phosphorus has been shown, this element has been perfectly targeted as linked to the presence/absence and/or abundance of particular bacterial taxa. In conclusion, we point out the need to link soil phosphorus chemistry with soil microbiology in order to understand the variations in the composition of microbial communities as a function of P bioavailability. This knowledge will make it possible to propose advanced microbial-based inoculant engineering for the improvement of bioavailable P for plants in sustainable agriculture.

Selection of plant growth promoting rhizobacteria sharing suitable features to be commercially developed as biostimulant products.

Plant biostimulants (PBs) are an eco-friendly alternative to chemical fertilisers because of their minimal or null impact on human health and environment, while ensuring optimal nutrient uptake and increase of crop yield, quality and tolerance to abiotic stress. Although there is an increasing interest on microbial biostimulants, the optimal procedure to select and develop them as commercial products is still not well defined. This work proposes and validates a procedure to select the best plant growth promoting rhizobacteria (PGPR) as potential active ingredients of commercial PBs. The stepwise screening strategy was designed based on literature analysis and consists of six steps: (i) determination of the target crop and commercial strategy, (ii) selection of growth media for the isolation of microbial candidates, (iii) screening for traits giving major agronomical advantages, (iv) screening for traits related to product development, (v) characterisation of the mode of action of PGPR and (vi) assessment of plant growth efficacy. The strategy was validated using a case study: PGPR combined with humic acids to be applied on tomato plants. Among 200 bacterial strains isolated from tomato rhizosphere, 39 % were able to grow in presence of humic acids and shared the ability to solubilise phosphate. After the screening for traits related to product development, only 6 % of initial bacterial strains were sharing traits suitable for the further development as potential PBs. In fact, the selected bacterial strains were able to produce high cell mass and tolerated drought, aspects important for the mass production and formulation. These bacterial strains were not able to produce antibiotics, establish pathogenic interaction with plants and did not belong to bacterial species associated to human, animal and plant diseases. Most importantly, five of the selected bacterial strains were able to promote tomato seedling vigour in experiments carried out in vitro. These bacterial strains were furtherly characterised for their ability to colonize effectively tomato plant roots, produce phytohormones and solubilise soil minerals. This characterisation led to the selection of two candidates that showed the ability to promote tomato plant growth in experiments carried out in greenhouse conditions. Overall, this work provides a flow diagram for the selection of PGPR candidates to be successfully developed and commercialized as PBs. The validation of the flow diagram led to the selection of two bacterial strains belonging to Pantoea and Pseudomonas genera, potential active ingredients of new commercial PBs.