Efficacy of talc bio-based formulation of Bacillus amyloliquefaciens and Rhizobium gallicum for Phaseolus vulgaris L. seeds coating

Efficacy of talc bio-based formulation of Bacillus amyloliquefaciens and Rhizobium gallicum for Phaseolus vulgaris L. seeds coating | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article Efficacy of talc bio-based formulation of Bacillus amyloliquefaciens and Rhizobium gallicum for Phaseolus vulgaris L. seeds coating Yosra SENDI, Khouloud BEN RZOUGA, Sana LAMLOUM, Sabrine JEDER, and 2 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-5489726/v1 This work is licensed under a CC BY 4.0 License Status: Published Journal Publication published 28 Jul, 2025 Read the published version in Journal of Plant Diseases and Protection → Version 1 posted 5 You are reading this latest preprint version Abstract The aim of this study is to evaluate the effectiveness of Bacillus amyloliquefaciens / Rhizobium gallicum - talc formulations to control fungal diseases and to promote plant growth in the common bean ( Phaseolus vulgaris L.). Bacillus amyloliquefaciens strain PVB17 and R. gallicum strain Ma1.12 inhibited Fusarium oxysporum , Alternaria alternata and Macrophomina phaseolina growth by up to 70% and 50%, respectively. Both bacterial strains are able to produce siderophores, indole-3-acetic acids (IAA), and exopolysaccharides. Interestingly, R. gallicum strain Ma1.12 IAA production reached 910 µg. mL − 1 , a value to be reported for the first time and has never been reported in various bacterial species. Talc was sampled from the region of Grombalia and powder was prepared. Gum Arabic was used as an adhesive in the bacterial formulation. The shelf life assessment- during two months of incubation- showed that B. amyloliquefaciens and R. gallicum strains are able to survive at more than 10 8 cfu. g − 1 of talc formulation. Whether used separately or combined, coating common bean seeds with Ma1.12 and PVB17 strains and growing them in a peat/sand mixture resulted in a significant increase in shoot and root dry weights up to 200% and 400%, respectively. Interestingly, the biocontrol assay of a pathogenic F. oxysporum with the talc bacterial formulations is associated with a decrease in fungal incidence in plant growth. A Field trial conducted in the region of Boucharray showed that seed coating with the formulations of R. gallicum strain Ma1.12 and B. amyloliquefaciens strain PVB17 significantly increased plant growth, nodule number, and pod weight. Combined seeds-coating using both bacterial strains gave the highest increases in shoot dry weight (by 108%), nodules number (by 861%), and pods weight (by 209%) in comparison to the controls. Seeds coating with the bacterial formulation are associated with decreased disease symptoms. Biocontrol Bacterial inoculum Common bean Plant growth promotion Seed treatment Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Introduction There is increasing interest in the use of beneficial microorganisms as alternatives to chemical pesticides and synthetic fertilizers in agricultural production (Gupta et al. 2022 ; Ansabayeva et al. 2024). Plant growth promoting microorganisms (PGPMs) provide essential agrosystems services that support plant growth, such as crop nutrient improvement, phytostimulation, plant tolerance to biotic and abiotic stresses, biocontrol of pests and dieseases, and water uptake enhancement (Koskey et al. 2021; Ansabayeva et al. 2024). While several scientific research papers revealed potentially hyge positive effects of PGPMs on plants, only a handful concentrate on delivery systems or formulation (Ma et al. 2021 ; Khan et al. 2023 ). The ‘formulation’ refers to the laboratory or industrial process of unifying the carrier with beneficial microbial strains for their productive use in various sectors, including agriculture (Balla et al. 2022 ). This technology harnesses the power of potential microbial strains possessing specific properties, such as nitrogen (N) fixation, Phosphorus (P) solubilization, siderophores and phytohormones production, and pathogen protection (Pirtilla et al. 2021 ; Khan et al. 2023 ). A number of commercial formulations of PGPMs are being developed recently, however their use is limited which may be due to some constraints such as reliability of the formulation, cost effectiveness, compatibility with soil conditions and mode timing of application (O’Callaghan et al. 2022 ; Khan et al. 2023 ). The review of Khan et al. ( 2023 ) gave a resume on formulation samples of beneficial microorganisms developed from 2000 to 2019. Accordingly, Bacillus sp., Pseudomonas sp., Azospirillum sp., and rhizobia strains are the main formulated microorganisms. Bioformulations are commonly applied as soil inoculation (direct soil inoculation, plant treatment (seedling/ root dipping, foliar spray), and seed coating (Khan et al. 2023 ). Pre-coated seed treatment with bacterial formulations is considered the most efficient method at field scales (O’Callaghan 2022). In order to increase the stabilization of microbes into the formulation and enhance their adhesion potential, some adjuvants/adhesives such as xanthan gum, methylcellulose, and gum arabic have been used (Khan et al. 2023 ). Despite the technological advancements in microbial bioformulations, there are some key factors that can affect the efficiency of microbial formulations (Rojas-Sánchez et al. 2022 ). This includes the selection of appropriate strain, the carrier type, the storage carrier, the microbial shelf-life, the environmental competition, and the application method. For these reasons, much more research efforts should be directed toward identifying the appropriate bioformulation of microbial strains regarding these considerations. In this context, this study was undertaken to develop and assess the efficacy of talc-biobased formulations of Bacillus amyloliquefaciens - as biocontrol agent- and Rhizobium gallicum as a N-fixing bacterium- for P. vulgaris L. seed coating under various experimental conditions including greenhouse and field trials. Material and Methods Bacterial strains and growth conditions The B. amyloliquefaciens strains PVB17 (Genbank accession number NR 112685.1) and R. gallicum strain Ma1.12 (Mrabet et al. 2006) were used in this study. Strain PVB17 is a P. vulgaris L. rhizospheric bacteria and Ma1.12 is a root symbiotic rhizobia isolated from P. vulgaris L. -root nodules in Tunisia. Bacillus amyloliquefaciens is maintained on Luria-Bertani (LB) and R. gallicum on Yeast Extract Mannitol Agar (YEMA) media and grown at 28 °C. Fungal strains Five fungal strains previously isolated from infested field-grown P. vulgaris L. plants in Tunisia and representing the main abundant pathogenic fungal species according to Sendi et al. (2019) were used in the present study. Those strains are assigned respectively to Alternaria alternata strain PVF4 (KU831493), Fusarium cerealis strain PVF10 (KU831499), F. oxysporum strain PVF26 (KU831515), F. tricinctum strain PVF30 (KU8131515), and Macrophomina phaseolina strain PVF32 (KU8131521). Fungal strains were grown on potato-dextrose agar (PDA) medium and stored at 4 °C. Antibiosis test of B. amyloliquefaciens PVB17 and R. gallicum Ma1.12 against the fungal strains The antibiosis test was performed on PDA medium for B. amyloliquefaciens strain PVB17 and on GN (Glucose nitrate) medium for R. gallicum strain Ma1.12 –for the reason of growth medium compatibility- and according to the protocol of Mrabet et al. (2015). The percentage of growth inhibition of each fungal strain was calculated considering the control fungal growth using the following formula (Mrabet et al. 2015): Growth inhibition (%) = [A − B/A] x 100; Where A is the diameter of fungal growth in the control and B is the distance of fungal growth in front of each of the two bacterial streaks. Preparation of fungal inoculums Twenty fungal discs (0.7 mm of diam.) from a fungal fresh culture were placed into an Erlenmeyer flask containing 200 g of sterilized sorghum seeds imbibed with 70 mL of sterile distilled water. The fungal preparation was incubated at 24 °C for five days in the darkness for growing mycelium. The density of each fungal strain in Sorghum bicolor L. was measured and adjusted to 10 6 CFU (colony forming units) per gram of Sorghum bicolor L., the density to be used for the inoculation of plants. Research for PGPR activities in B. amyloliquefaciens and R. gallicum strains Indole-3-acetic acids (IAA) production was measured according to the protocol of Bano and Musarrat (2003) and siderophores production was assessed using the chromeazurol (CAS) agar assay as described by Schwyn and Neilands (1987). Wells were made on the CAS medium and 100 μl of each bacterial suspension was incubated in the dark for two days. Positive results were indicated by the formation of a light halo around the colonies, showing a visual change in color from dark blue to yellow. HCN production using the method of Feigl and Anger (1966). The bacterial isolates were streaked on a medium Luria Bertani (LB). A Whatman filter paper was placed at the top of the plate. The plates were sealed with parafilm and incubated for four days at 30 o C. Production of HCN was indicated by the development of blue color, and EPS production was assessed according to the protocol of ShivaKumar and Vijayendra (2006). Thirty days after bacterial incubation in a medium saturated with saccharose (20 g. L -1 ) at 30°C, strains with a mucous appearance are EPS-productive. Phosphate solubilization activity was assessed on Pikovskaya’s agar medium (g. L -1 ; yeast extract 0.5, dextrose 10, calcium phosphate 5, ammonium sulfate 0.5, potassium chloride 0.2, magnesium sulfate 0.1, manganese sulfate 0.0001, ferrous sulfate 0.0001, agar 15) was used for isolation of phosphate-solubilizing microorganisms (Pikovskaya, 1948). Bacterial strains able to solubilize precipitated calcium phosphate to produce clear zones around colonies were assessed and measured for both bacterial strains B. amyloliquefaciens strain PVB17 and R. gallicum strain Ma1.12. Each test was performed in triplicate. Development of talc bio-based formulations for B. amyloliquefaciens and R. gallicum strains Preparation of mineral carriers Clay samples were collected from the formation of Saouaf located in Grombalia location at the northeast of Tunisia which is characterized by clays intercalated with sandstone (Ghrasalli and Mzali 2017). The mineral composition of the Saouaf argillaceous formation is kaolinite (20-35%), smectite (40-60%), and illite (10-20%) and the average clay fraction is of 57% according to Gharsalli and Mzali (2017). Clay samples were crushed using an electric Retsch shredder for 10 min. Clay powder was therefore three times autoclaved for 30 min at 120 °C. Then, dried up to 20% humidity and kept in sterile plastic bags. Preparation of bacterial suspensions Bacillus amyloliquafciens strain PVB17 was cultivated in LB broth medium and optical density (OD) at 620 nm was adjusted to the value 0.6 giving 10 8 CFU. mL -1 . Rhizobium gallicum strain Ma1.12 was cultivated in YEM (Yeast Extract Mannitol) broth medium at 28 °C for 48 h and OD 620 nm adjusted to the value 0.8 corresponding to 10 8 CFU. mL -1 . A Sterile solution of Gum arabic (Lot 55H1455) was used as an additive to the bacterial preparation, mixing 50 mL of gum arabic solution (10%) with 100 mL of bacterial suspension and kept at room temperature for 30 min before use. Development of talc bio-based formulations and seed coating Twenty mL of bacterial suspension/gum arabic preparation was mixed with 80 g of autoclaved talc powder in sterile Petri dishes. P. vulgaris L. seeds, cv. Coco blanc, were surface disinfected with mercury chloride solution (0.2%) for 2 min and then rinsed with sterile distilled water. Then, the seeds were soaked in a double volume of sterile distilled water containing the mentioned formulations (10 g/1 Kg of seeds). One hour later, the bacterial suspension was drained off and the seeds were dried at room temperature under sterile conditions for 30 min and planted as described by Vidhyasekaran et al. (1997). Shelflife assessment From each talc bacterial preparation, 1 g was added to 9 mL of sterile distilled water and a series of dilutions from 10 -1 to 10 -10 were prepared. A volume of 100 µl of each dilution was suspended on solid growth medium (LB or YEMA) and incubated at 28 °C and growing colonies were counted (CFU. mL -1 ) at 0, 15, 30, 45, and 60 days of incubation. The talc-bacterial formulations were kept in sterile plastic bags at room temperature during the performed experiment. Effectiveness of talc bio-based formulations on host plant bioferilization and bioprotection under greenhouse conditions P. vulgaris L. seed plantation and treatments P. vulgaris L. cv. Coco blanc seeds were planted in plastic pots of 1 Kg containing a mix of sterile peat/sand at a ratio of 1:2. In case of fungal treatments, 15 g of infected sorghum seeds were added and mixed with peat/sand mixture in each pot 24 h before plantation step. Plantlets were grown in a growth chamber under controlled conditions (28 °C, HR 60%, Photoperiod 16/8). The following treatments were considered : (i) Control plants, (ii) pots infected with the fungal inoculum, (iii) Coated-seeds with the R. gallicum strain Ma1.12, (iv) Coated-seeds with the B. amyloliquefaciens strain PVB17, (v) Coated-seeds with both formulations of R. gallicum strain Ma1.12 and B. amyloliquefaciens strain PVB17, (vi) plant co-treated F. oxysporum strain PVF26 and B. amyloliquefaciens strain PVB17 formulation, and (vii) plant treated with F. oxysporum strain PVF26, B. amyloliquefaciens strain PVB17, R. gallicum strain Ma1.12. In each pot, three P. vulgaris L. seeds were planted and five pots were considered for each treatment. The culture was grown for 40 days before harvest. Recorded parameters Both growth and disease parameters were recorded at harvesting time which consist of shoot dry weight, root dry weight, nodules number, and plant health status according to Sendi et al. (2020). Bacterial effectiveness of talc formulation in field plots A field trial was performed in the region of Boucharray (North-eastern Tunisia) and three experimental blocks were considered. Each block is designed as 10 mL long and three lines were considered for each treatment in each block. Lines were separated by a distance of 30 cm. P. vulgaris L. seeds were sown in order of eight seeds per 20 cm 2 and each seeds mass is distant of 20 cm from the previous one. In each block, the following treatments were included : Control, Coated seeds with R. gallicum strain Ma1.12 formulation, Coated seeds with B. amyloliquefaciens strain PVB17 formulation, and Coated seeds with combined formulations of R. gallicum strain Ma1.12 and B. amyloliquefaciens strain PVB17. During cultivation, plants were irrigated with well water when needed. P. vulgaris L. plants were harvested two months post plantation in order of 24 randomized plants per treatment. Shoot and root dry weights, pod number and weight, and the health state of grown plants were recorded. Statistical analyses Different parameters were analyzed by comparing means and variances using the SPSS version 20 software. Means were compared with the test Duncan at p = 0.05. Results Antifungal effectiveness of B. amyloliquefaciens strain PVB14 and R. gallicum strain Ma1.12 Antibiosis tests showed that B. amyloliquefaciens strain PBV17 is able to inhibit the in vitro growth of A. alternata PVF4, F. cerealis PVF10, F. oxysporum PVF26, F. tricinctum PVF30, and M. phaseolina PVF32 by 55% to 73% (Fig. 1). Rhizobium gallicum strain Ma1.12 inhibited the mycelial growth of different fungal strains from 50% to 64% (Fig. 1). In Fig. 2, an illustration of the inhibition of F. oxysporum strain PVF26 by B. amyloliquefaciens strain PVB17 is shown. Characterization of PGPR activities in B. amyloliquefaciens strain PVB17 and R. gallicum strain Ma1.12 The PGPR traits characterization showed that both bacterial strains are able to produce siderophores (Fig. 3). Strain Ma1.12 of R. gallicum is able to produce 910 µg. mL -1 of auxins whereas B. amyloliquefaciens strain PVB17 is producing only 25.19 µg. mL -1 (Table 1). Both bacterial strains produced a high amount of exopolysaccharides (EPS) in a growth medium saturated with saccharose. However, phosphates solubilization is only detected for R. gallicum strain Ma1.12 (Table 1). Viability of B. amyloliquefaciens strain PVB17 and R. gallicum strain Ma1.12 in the talc bio-formulation The assessment of cell viability of bacterial strains showed that B. amyloliquefaciens strain PVB17 and R. gallicum strain Ma1.17 are able to survive into the talc formulation at up to 10 9 and 10 8 CFU. g -1 of talc formulation, respectively, during two months of incubation (Table 2). Incidence of talc bio-based formulations of B. amyloliquefaciens PVB17 and R. gallicum Ma1.12 on P. vulgaris L. growth P. vulgaris L. cv. Coco blanc plants of talc-coated seeds showed similar root and shoot growth capacity as the talc non-coated seeds (Fig. 4). Plants from seeds coated with R. gallicum Ma1.12 talc formulation showed a significant increase of 5.3 folds and 2 folds of root and shoot growth, respectively (Fig. 4). Root and shoot dry weights increased by about 3 folds and 1.3 folds, respectively, when seeds are coated with B. amyloliquefaciens PVB17 talc formulation. The simultaneous application of Ma1.12 and PVB17 talc formulations to P. vulgaris L. seeds is associated with an increase of root dry weight by 5 folds and of shoot dry weight by 2 folds in comparison to the non-coated seeds. Biocontrol effectiveness of the bacterial talc formulations of P. vulgaris L. against F. oxysporum attacks Root and shoot growth of P. vulgaris L. plants grown in peat/sand mixture inoculated with F. oxysporum strain PVF26 is significantly increased for seeds coated with R. gallicum strain Ma1.12 and B. amyloliquefaciens strain PVB17 applied separately or combined (Fig. 5). The root and shoot dry weights are increased up to 5.64 folds compared to control plants only infected with the fungal strain. Severe leaf chlorosis and root rot symptoms in P. vulgaris plants inoculated with F. oxysporum strain PVF26 was observed. However, the leaf chlorosis and root rot symptoms were significantly reduced, particularly under PVB17/Ma1.12 combined seed-treatment (data not shown). When inoculated with F. oxysporum strain PVF26, The treatment involving R. gallicum strain Ma1.12 is associated with root nodulation of up to 207 nodules per plant (data not shown). Field effectiveness of the talc bio-based formulation of B. amyloliquefaciens strain PVB17 and R. gallicum strain Ma1.12 The analysis of the variance of shoot and root dry weights, pod number, and weight showed that there are no differences between various blocks for each treatment (P > 0.05). Accordingly, results were averaged for each treatment and presented in table 3. P. vulgaris L. cv. Coco blanc seeds coated with R. gallicum strain Ma1.12 is associated with a significant increase of nodules number up to 186 nodules/plant (Table 3). This increase was associated with a significant enhancement of shoot dry weight, pod number, and weight. When coated with the B. amyloliquefaciens strain PVB17, common bean plant growth, and pod production are also significantly increased, however less than results given by R. gallicum strain Ma1.12 (Table 3). Nodules number is significantly increased also. Interestingly, when coated with combined formulations of B. amyloliquefaciens PVB17 and R. gallicum Ma1.12, P. vulgaris L. plants showed an increase of 10 folds in nodules number, 2 folds in shoot dry weight, 2 folds in pods number, and 3 folds in pods weight in comparison to the control non coated plants (Table 3). Seed coating with B. amyloliquefaciens strain PVB17 and R. gallicum strain Ma1.12 is associated with a clear improvement in the health status of grown P. vulgaris L. plants compared to the control ones (Data not shown). This was observed both for the aerial and root parts. Discussion The anti-fungal activity of B. amyloliquefaciens strain PVB17 is demonstrated on F. oxysporum , F. cerealis , F. tricinctum , A. alternata and M. phaseolina . These fungal species are reported to be predominant in different agricultural soils and to exhibit severe diseases on various beans (Darvishnia et al. 2023 ; Wu et al. 2024 ) and even on other legumes and non-legume plants (Ekwomadu et al. 2023, a review). Using B. amyloliquefaciens inoculants in the biocontrol of plant pathogens is recognized as an important feature for more sustainable agriculture (Luo et al. 2022 , a review). The antifungal efficacy of B. amyloliquefaciens is proven to be linked to its ability to produce secondary metabolites as lipopeptides (surfactin, fengycin, bacillomycin, iturin), lytic enzymes, siderophores, and microbial volatile organic compounds (Ji et al. 2021 ; Luo et al. 2022 , a review). The second bacterial strain used in this study is R. gallicum strain Ma1.12 characterized by its high symbiotic effectiveness with P. vulgaris L. plants (Mrabet et al. 2006 ). Results showed that both strains of B. amyloliquefaciens PVB17 and R. gallicum Ma1.12 were able to produce remarkably siderophores and indole-3-acetic acid (IAA). The IAA production by R. gallicum strain Ma1.12 is about 910 µg. mL − 1 . This value has never been reported in different bacterial species. In fact, Küçük and Cevheri ( 2016 ) in a study involving many Rhizobium sp. strains reported that the maximum IAA production was about 165 µg. mL − 1 . Auxins control several stages of plant growth such as cell division, tissue differentiation, and apical dominance in plant roots (Seo et al. 2021 ). The treatment of IAA-producing rhizobacteria increases plant growth (Khozo et al. 2024 , a review). Bacillus amyloliquefaciens strain PVB17 and R. gallicum strain Ma1.12 are shown to produce great amounts of siderophores. Siderophore-producing rhizobacteria improve plant growth and protection since they improve iron nutrition and hinder the growth of pathogens by limiting the iron available for the pathogen, generally, fungi, which are unable to absorb the iron-siderophore complex (Deb and Tatung 2024 ). After N, P is the most limiting nutrient for plants. The ability of R. gallicum strain Ma1.12 to solubilize inorganic phosphates may explain- partially- the PGPR effectiveness of this bacterial strain, besides N fixation property. It was recognized that microorganisms that are able to solubilize complex-structured phosphates, providing it in a form suitable for plant uptake, are among PGPR groups that increase the P availability to plants (Elhaissoufi et al. 2024). Despite the antifungal activity of B. amyloliquefaciens strain PVB17 and the symbiotic effectiveness of R. gallicum strain Ma1.12, the challenge is how to process for their use at field scales. In this regard, talc was used as a mineral carrier to formulate both bacterial strains and to assess their plant growth-promoting traits on P. vulgaris L. under greenhouse and field trials. Interestingly, the self-life study of both bacterial strains in talc formulation showed that B. amyloliquefaciens strain PVB17 and R. gallicum strain Ma1.12 survive at high frequency during a period of two months of assessment. This ability to survive into talc formulation is necessary but not determinant for the success of developed formulations. In fact, it was reported that the success of microbial seed treatments depends not only on formulations that ensure the survival of the inoculant during seed treatment and storage but also on its ability to multiply in the spermosphere, colonize root, and in some cases the surrounding soil, where it then perform its required function (Khan et al. 2023 ). Therefore, confirmation of talc bioformulation efficacy on P. vulgaris L. plants was necessary to provide much more evidence of its effectiveness. This study showed that P. vulgaris L. seeds coating with B. amyloliquefaciens strain PVB17 and R. gallicum strain Ma1.12, combined or separately, was associated with significant improvement of shoot and root dry weights. This result indicates the effectiveness of talc formulations of both bacterial strains. On another side, P. vulgaris L. seeds coated only with talc gave similar shoot and root growth as in control non-coated seeds underlying that there’s no harmful effect of talc on P. vulgaris L. seed germination and plant growth. Another challenge for us was to confirm if the talc-biobased formulation of B. amyloliquefaciens strain PVB17 and R. gallicum strain Ma1.12 was able to protect P. vulgaris L. plants against the pathogen F. oxysporum . The biocontrol assay confirmed the efficacy of talc formulation of both bacterial strains applied separately or combined. The shoot and root dry weights increased by about 5 fold. Moreover, nodulation by R. gallicum was more than 200 nodules per plant. These findings confirmed the efficacy of talc-formulations of both bacterial strains in protecting P. vulgaris L. plants from F. oxysporum strain PVF26 and therefore in enhancing plant growth. Field assays showed the effectiveness of the talc formulation of both bacterial strains in enhancing nodulation of P. vulgaris L. induced by R. gallicum strain Ma1.12 and in increasing plant growth and pods number and weights, particularly when both bacterial formulations are combined. This fact demonstrated the field efficacy of the developed formulations of both bacterial strains. The increase in nodule number induced by the R. gallicum strain Ma1.12 under field conditions is a key fact of the enhancement of plant growth and pod production. In fact, nodulation by rhizobia was shown to be improved when rhizobia were combined with plant growth promoting rhizobacteria in legumes, and plant growth was shown to be increased (Kaschuk et al. 2022 ). It was recognized that commercial application of PGPR for control of soil-borne diseases depends upon the development of commercial formulations in which bacteria can survive for a considerable length of time, on the development of a suitable method of application to control pathogen establishment and disease development, and assessment of their efficacy under field conditions (O’Callaghan 2022 ; Khan et al. 2023 ). Hence, this study demonstrated the efficacy of talc-formulation of B. amyloliquefaciens strain PVB17 and R. gallicum strain Ma1.12 as seed coating inoculant. Further investigations on the usefulness of the developed formulations in other cultivated crops will be tested and agro-economical efficacy assessed. Declarations Acknowledgment The authors wish to thank Dr. Houcem Mzali, Geologist Researcher at the Centre for Research and Water Technologies of Borj-Cedria (CERTE) for technical assistance during the sampling talc from the region of Grombalia, and for a sufficient description of the geological formation of this region. Funding This study was supported by the Ministry of Higher Education and Scientific Research of Tunisia, [Grant Laboratory of Legumes and Sustaible Agrosystems 2018-2023]. Data availability statement The research data can be available to reader upon the acceptance of the paper according to the springer regulation. Conflict of interest The authors declare that they have no any potential financial or non-financial conflict of interest. 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Plant Stress 11 :100341. https://doi.org/10.1016/j.stress.2023.100341 Küçük Ç, Cevheri, C (2016) Indole acetic acid production by Rhizobium sp. isolated from pea ( Pisum sativum L. ssp. arvense). Turk J Life Sc 1:43‒45 Luo L, Zhao C, Wang E, Raza A, Yin C (2022) Bacillus amyloliquefaciens as an excellent agent for biofertilizer and biocontrol in agriculture: An overview for its mechanisms. Microbiological Research 259 (2022) 127016. https://doi.org/10.1016/j.micres.2022.127016 Ma Y, Vosátka M, Rensing G, Freitas H (2021) Editorial : Advanced microbial biotechnologies for sustainable agriculture. Front Microbiol 12 :634891. https://doi.org/10.3389/fmicb.2021.634891 Mrabet M,Mnasri B,Ben Romdhane S, Laguerre G, Aouani ME, Mhamdi R (2006) Agrobacterium strains isolated from root nodules of common bean specifically reduce nodulation by Rhizobium gallicum . FEMS Microbiol Ecol 56:304‒309. https://doi.org/10.1111/j.1574-6941.2006.00069.x Mrabet M, Elkahoui S, Tarhouni B, Djebali N (2015) Potato seed dressing with Pseudomonas aeruginosa strain RZ9 enhances yield and reduces black scurf. Phytopathol Mediterr 54:265‒74. https://doi.org/10.14601/Phytopathol_Mediterr-15171 O’Callaghan M, Ballard RA, Wright D (2022) Soil microbial inoculants for sustainable agriculture: limitations and opportunities. Soil Use Manage 38 :1340-1369. DOI:10.1111/sum.12811 Pikovskaya RI (1948) Mobilization of phosphorus in soil connection with the vital activity of some microbial species. Microbiol 17:362‒370 Pirttila AM, Mohammad P, Tabas H, Baruah N, Koskimäki JJ (2021) Biofertilizers and biocontrol agents for agriculture: how to identify and develop new potent microbial strains and traits. Microorganisms 9 (4), 817. doi: 10.3390/microorganisms9040817 Rojas-Sánchez B, Guzmán-Guzmán P, Morales-Cedeño LR, Orozco-Mosqueda MDC, Saucedo-Martı́nez BC, Sánchez-Yáñez JM, et al. (2022) Bioencapsulation of microbial inoculants: mechanisms, formulation types and application techniques. App Biosci 1 (2): 198–220. doi: 10.3390/applbiosci1020013 Schwyn B, Neilands JB (1987) Universal chemical assay for the detection and determination of siderophores. Analy Biochem 160:47‒56. https://doi.org/10.1016/0003-2697(87)90612-9 Sendi Y, Ben Romdhane S, Mhamdi R, Mrabet M (2019) Diversity and geographic distribution of fungal strains infecting field-grown common bean ( Phaseolus vulgaris L.) in Tunisia. Eur J Plant Pathol (2019) 153:947–955. https://doi.org/10.1007/s10658-018-01612-y Sendi Y, Pfeiffer T, Koch E, Mhadhbi H, Mrabet M (2020) Potential of common bean ( Phaseolus vulgaris L.) root microbiome in the biocontrol of root rot disease and traits of performance. J Plant Dis Protec 127:453-462. https://doi.org/10.1007/s41348-020-00338-6 Seo DH, Jeong H, Choix YD, Jang G (2021) Auxin controls the division of root endodermal cells. Plant Physiol 187:1577-1586. https://doi.org/10.1093/plphys/kiab341 ShivaKumar S, Vijayendra SVN (2006) Production of exopolysaccharides by Agrobacterium sp. CFR‐24 using coconut water – a byproduct of food industry. Lett App Microbiol 42: 477‒482. https://doi.org/10.1111/j.1472-765X.2006.01881.x Vidhyasekaran P, Rabindran R, Muthamilan M, Nayar K, Rajappan K, Subramanian N, Vasumathi K (1997) Development of a powder formulation of Pseudomonas fluorescens for control of rice blast. Plant Pathol 46:291‒297. https://doi.org/10.1046/j.1365-3059.1997.d01-27.x Wu L, Hwang SF, Strelkov SE, Fredua-Agyeman R, Oh SH, Bélanger RR, Wally O, Kim YM (2024) Pathogenicity, host resistance, and genetic diversity of Fusarium species under controlled conditions from soybean in Canada. J Fungi 10 : 303. https://doi.org/10.3390/jof1005030 Tables Table 1. PGPR traits characterization of B. amyloliquefaciens strain PVB17 and R. gallicum strain Ma1.12. IAA/indole-3-acetic acids, P/Phosphates, EPS/exopolysaccharides. Siderophore Production (Halo diam. in cm) IAA production (µg. mL -1 ) P. solubilisation (Halo diam. in cm) EPS production B. amyloliquefaciens strain PVB17 4.3 (± 0.2) 25.19 (± 5.21) ND +++ R. gallicum strain Ma1.12 3.1 (± 0.13) 910.32 (± 35.05) 3.3 (± 0.57) +++ +++ : High production level. ND: Non detected. P: phosphates. Standard error values for each parameter are indicated in parentheses. Table 2. Viability of B. amyloliquefaciens strain PVB17 and R. gallicum strain Ma1.12 into the talc bio-based formulation during two months. Bacterial colonies were counted every 15 days using the dilution method and spreading on a growth medium. Bacterial strains Time of incubation (Day) Colonies counting (CFU. g -1 of talc formulation) B. amyloliquefaciens strain PVB17 0 4x10 10 15 3.8x10 9 30 2.2x10 9 45 2.7x10 9 60 2.3x10 9 R. gallicum strain Ma1.12 0 3x10 9 15 5.4x10 8 30 3.7x10 8 45 2.1x10 8 60 1.8x10 8 Table 3. Field effectiveness of talc formulations of B. amyloliquefaciens strain PVB17 and R. gallicum Ma1.12 on P. vulgaris L. cv. Coco blanc in the region of Boucharray. The harvest was done 60 days post plantation. Treatment Nodules number SDW (g. plant -1 ) Pods number Pods weight (g. plant -1 ) Control 21 c (± 5) 4.2 d (± 0.43) 4.02 d (± 0.5) 16.5 d (± 3.4) R. gallicum Ma1.12 186 a (± 21) 7.38 b (± 0.84) 6.3 b (± 1) 39.97 b (± 3.2) B. amyloliquefaciens PVB17 47 b (± 12) 5.34 c (± 0.54) 5.47 c (± 0.73) 30.11 c (± 5.6) R. gallicum Ma1.12 + B. amyloliquefaciens PVB17 202 a (± 33) 8.76 a (± 1.39) 8.72 a (± 1.33) 51.03 a (± 8.1) * Each recorded parameter corresponds to an average of a three experimental blocks. Values between parentheses represent standard errors. SDW/Shoot dry weight. Cite Share Download PDF Status: Published Journal Publication published 28 Jul, 2025 Read the published version in Journal of Plant Diseases and Protection → Version 1 posted Editorial decision: Accept 04 Jul, 2025 Reviewers agreed at journal 02 May, 2025 Reviewers invited by journal 25 Apr, 2025 Editor assigned by journal 09 Apr, 2025 First submitted to journal 08 Apr, 2025 You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. We do this by developing innovative software and high quality services for the global research community. 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Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-5489726","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":448096529,"identity":"756ec02f-7aeb-4fe6-9b4d-553ef1074b1f","order_by":0,"name":"Yosra SENDI","email":"","orcid":"","institution":"","correspondingAuthor":false,"prefix":"","firstName":"Yosra","middleName":"","lastName":"SENDI","suffix":""},{"id":448096530,"identity":"7679c534-723b-40cb-afa2-5732119a3ea4","order_by":1,"name":"Khouloud BEN RZOUGA","email":"","orcid":"","institution":"","correspondingAuthor":false,"prefix":"","firstName":"Khouloud","middleName":"BEN","lastName":"RZOUGA","suffix":""},{"id":448096531,"identity":"cfb250fe-3314-484c-89b0-9a2ec973ae39","order_by":2,"name":"Sana LAMLOUM","email":"","orcid":"","institution":"","correspondingAuthor":false,"prefix":"","firstName":"Sana","middleName":"","lastName":"LAMLOUM","suffix":""},{"id":448096532,"identity":"a115562d-4eaf-4eaa-8588-681e57183bbe","order_by":3,"name":"Sabrine JEDER","email":"","orcid":"","institution":"","correspondingAuthor":false,"prefix":"","firstName":"Sabrine","middleName":"","lastName":"JEDER","suffix":""},{"id":448096533,"identity":"5f17d009-0902-4f9e-91e4-d75a9275daec","order_by":4,"name":"Fethi BARHOUMI","email":"","orcid":"","institution":"","correspondingAuthor":false,"prefix":"","firstName":"Fethi","middleName":"","lastName":"BARHOUMI","suffix":""},{"id":448096534,"identity":"8b3429a4-ae08-4d4b-be0b-47ebf4ce2a77","order_by":5,"name":"Moncef MRABET","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAA5ElEQVRIiWNgGAWjYNACGxDBA8QVDAwGDBCaAEiDajlwBqrlDNFaDrYRoUW3vffghw8JDNHm7WcPPv4477C8OXvzAYaDe3BrMTtzLllyRgJD7pwzeckGB7cdNtzZcyyB4cAzPFpu5Jgx8/5gyJ3BkGMmAdTCuOFGjgHzhwMEtPwB2jKD/435j4NzDtuDtDAcIKSFAaRFIseM4WDD4UTCWs6cMZbsAWt5lyxx5lh68oYzxxIO4NVyvMfwww+ww3IPfqiosbbdcLz54AN8WqDgP4zRDCYJa0ACdaQoHgWjYBSMghECAGkZXtPajbHMAAAAAElFTkSuQmCC","orcid":"","institution":"Centre of Biotechnology of Borj-Cédria","correspondingAuthor":true,"prefix":"","firstName":"Moncef","middleName":"","lastName":"MRABET","suffix":""}],"badges":[],"createdAt":"2024-11-20 09:46:12","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-5489726/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-5489726/v1","draftVersion":[],"editorialEvents":[{"content":"https://doi.org/10.1007/s41348-025-01128-8","type":"published","date":"2025-07-28T16:13:31+00:00"}],"editorialNote":"","failedWorkflow":false,"files":[{"id":81527022,"identity":"4bf5029d-fb19-4dc9-92e4-3bd73484b054","added_by":"auto","created_at":"2025-04-28 09:00:14","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":31997,"visible":true,"origin":"","legend":"\u003cp\u003ePercentages of inhibition of fungal strains \u003cem\u003eA. alternata\u003c/em\u003ePVF4, \u003cem\u003eF. cerealis\u003c/em\u003e PVF10, \u003cem\u003eF. oxysporum\u003c/em\u003e PVF26, \u003cem\u003eF. tricinctum\u003c/em\u003ePVF30, and \u003cem\u003eM. phaseolina\u003c/em\u003e PVF32 by \u003cem\u003eB. amyloliquefaciens\u003c/em\u003e strain PVB17 and \u003cem\u003eR. gallicum\u003c/em\u003e strain Ma1.12. Each percentage is an average of three replicates and values were recorded when the mycelia of the control treatments invade the whole Petri dishes surface which depends on the fungal strain. Bars represent standard errors.\u003c/p\u003e","description":"","filename":"1.png","url":"https://assets-eu.researchsquare.com/files/rs-5489726/v1/a5a017ad78c5142585cc044c.png"},{"id":81527026,"identity":"a5279de5-ac4d-4bdf-a67f-4b1b25a89623","added_by":"auto","created_at":"2025-04-28 09:00:15","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":392357,"visible":true,"origin":"","legend":"\u003cp\u003eInhibition of \u003cem\u003eF. oxysporum\u003c/em\u003e strain PVF26 growth by the \u003cem\u003eB. amyloliquefaciens\u003c/em\u003e strain PVB17 in a dual culture test on PDA growth medium. a, Control growth; b, antibiosis effect. The result is recorded five days after the antibiosis test initiation.\u003c/p\u003e","description":"","filename":"2.png","url":"https://assets-eu.researchsquare.com/files/rs-5489726/v1/7519cf23ba7d8cf0f14d7c51.png"},{"id":81527024,"identity":"88cc5e87-f414-4502-9318-78fc1c7d3da5","added_by":"auto","created_at":"2025-04-28 09:00:15","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":233778,"visible":true,"origin":"","legend":"\u003cp\u003eSiderophores production by \u003cem\u003eB. amyloliquefaciens\u003c/em\u003e strain PVB17 and \u003cem\u003eR. gallicum\u003c/em\u003e strain Ma1.12 in chromeazurol (CAS) agar assay according to Schwyn and Neilands (1987).\u003c/p\u003e","description":"","filename":"3.png","url":"https://assets-eu.researchsquare.com/files/rs-5489726/v1/60eab70cdce7b41766a8421b.png"},{"id":81527023,"identity":"b45718af-b884-49df-9bad-4fd584d79561","added_by":"auto","created_at":"2025-04-28 09:00:15","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":107932,"visible":true,"origin":"","legend":"\u003cp\u003eEffect of talc bio-based formulation of \u003cem\u003eR. gallicum\u003c/em\u003e strain Ma1.12 and\u003cstrong\u003e \u003c/strong\u003e\u003cem\u003eB. amyloliquefaciens\u003c/em\u003e strain PVB17 on \u003cem\u003eP. vulgaris\u003c/em\u003e L. plants root and shoot dry weights. \u003cem\u003eP. vulgaris\u003c/em\u003e L. plants were grown in a peat/sand mixture for 40 days under controlled conditions. Bars represent standard errors.\u003c/p\u003e","description":"","filename":"4.png","url":"https://assets-eu.researchsquare.com/files/rs-5489726/v1/f3b86f0e8e20ba3e26b80d70.png"},{"id":81527029,"identity":"2c00dffa-b27f-408d-ad8c-1f55522a1e9a","added_by":"auto","created_at":"2025-04-28 09:00:15","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":105217,"visible":true,"origin":"","legend":"\u003cp\u003eBiocontrol of \u003cem\u003eF. oxysporum\u003c/em\u003e strain PVF26 with the talc bio-based formulation of \u003cem\u003eR. gallicum\u003c/em\u003e strain Ma1.12 and\u003cstrong\u003e \u003c/strong\u003e\u003cem\u003eB. amyloliquefaciens\u003c/em\u003e strain PVB17 on \u003cem\u003eP. vulgaris\u003c/em\u003e L. plants root and shoot dry weights. \u003cem\u003eP. vulgaris\u003c/em\u003e L. plants were grown in a peat/sand mixture for 40 days under controlled conditions. Bars represent standard errors.\u003c/p\u003e","description":"","filename":"5.png","url":"https://assets-eu.researchsquare.com/files/rs-5489726/v1/fd64121576b51ba4c7fbc57f.png"},{"id":88268403,"identity":"04101bdb-460f-47be-b0fe-309d2f116b25","added_by":"auto","created_at":"2025-08-04 16:51:30","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":2277368,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-5489726/v1/4936b592-56e2-45ad-bfcc-919c20dbdbbd.pdf"}],"financialInterests":"","formattedTitle":"Efficacy of talc bio-based formulation of Bacillus amyloliquefaciens and Rhizobium gallicum for Phaseolus vulgaris L. seeds coating","fulltext":[{"header":"Introduction","content":"\u003cp\u003eThere is increasing interest in the use of beneficial microorganisms as alternatives to chemical pesticides and synthetic fertilizers in agricultural production (Gupta et al. \u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e2022\u003c/span\u003e ; Ansabayeva et al. 2024). Plant growth promoting microorganisms (PGPMs) provide essential agrosystems services that support plant growth, such as crop nutrient improvement, phytostimulation, plant tolerance to biotic and abiotic stresses, biocontrol of pests and dieseases, and water uptake enhancement (Koskey et al. 2021; Ansabayeva et al. 2024). While several scientific research papers revealed potentially hyge positive effects of PGPMs on plants, only a handful concentrate on delivery systems or formulation (Ma et al. \u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e2021\u003c/span\u003e ; Khan et al. \u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e2023\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eThe \u0026lsquo;formulation\u0026rsquo; refers to the laboratory or industrial process of unifying the carrier with beneficial microbial strains for their productive use in various sectors, including agriculture (Balla et al. \u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2022\u003c/span\u003e). This technology harnesses the power of potential microbial strains possessing specific properties, such as nitrogen (N) fixation, Phosphorus (P) solubilization, siderophores and phytohormones production, and pathogen protection (Pirtilla et al. 2021 ; Khan et al. \u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e2023\u003c/span\u003e). A number of commercial formulations of PGPMs are being developed recently, however their use is limited which may be due to some constraints such as reliability of the formulation, cost effectiveness, compatibility with soil conditions and mode timing of application (O\u0026rsquo;Callaghan et al. \u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e2022\u003c/span\u003e ; Khan et al. \u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e2023\u003c/span\u003e). The review of Khan et al. (\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e2023\u003c/span\u003e) gave a resume on formulation samples of beneficial microorganisms developed from 2000 to 2019. Accordingly, \u003cem\u003eBacillus\u003c/em\u003e sp., \u003cem\u003ePseudomonas\u003c/em\u003e sp., \u003cem\u003eAzospirillum\u003c/em\u003e sp., and rhizobia strains are the main formulated microorganisms. Bioformulations are commonly applied as soil inoculation (direct soil inoculation, plant treatment (seedling/ root dipping, foliar spray), and seed coating (Khan et al. \u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e2023\u003c/span\u003e). Pre-coated seed treatment with bacterial formulations is considered the most efficient method at field scales (O\u0026rsquo;Callaghan 2022). In order to increase the stabilization of microbes into the formulation and enhance their adhesion potential, some adjuvants/adhesives such as xanthan gum, methylcellulose, and gum arabic have been used (Khan et al. \u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e2023\u003c/span\u003e). Despite the technological advancements in microbial bioformulations, there are some key factors that can affect the efficiency of microbial formulations (Rojas-S\u0026aacute;nchez et al. \u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e2022\u003c/span\u003e). This includes the selection of appropriate strain, the carrier type, the storage carrier, the microbial shelf-life, the environmental competition, and the application method. For these reasons, much more research efforts should be directed toward identifying the appropriate bioformulation of microbial strains regarding these considerations.\u003c/p\u003e \u003cp\u003eIn this context, this study was undertaken to develop and assess the efficacy of talc-biobased formulations of \u003cem\u003eBacillus amyloliquefaciens\u003c/em\u003e- as biocontrol agent- and \u003cem\u003eRhizobium gallicum\u003c/em\u003e as a N-fixing bacterium- for \u003cem\u003eP. vulgaris\u003c/em\u003e L. seed coating under various experimental conditions including greenhouse and field trials.\u003c/p\u003e"},{"header":"Material and Methods","content":"\u003cp\u003e\u003cstrong\u003eBacterial strains and growth conditions\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe \u003cem\u003eB. amyloliquefaciens\u003c/em\u003e strains PVB17 (Genbank accession number NR 112685.1) and \u003cem\u003eR.\u003c/em\u003e \u003cem\u003egallicum\u003c/em\u003e strain Ma1.12 (Mrabet et al. 2006) were used in this study. Strain PVB17 is a \u003cem\u003eP.\u003c/em\u003e \u003cem\u003evulgaris\u003c/em\u003e L. rhizospheric bacteria and Ma1.12 is a root symbiotic rhizobia isolated from \u003cem\u003eP. vulgaris \u003c/em\u003eL. -root nodules in Tunisia. \u003cem\u003eBacillus amyloliquefaciens\u003c/em\u003e is maintained on Luria-Bertani (LB) and \u003cem\u003eR. gallicum\u003c/em\u003e on Yeast Extract Mannitol Agar (YEMA) media and grown at 28 \u0026deg;C.\u003c/p\u003e\n\n\u003cp\u003e\u003cstrong\u003eFungal strains\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eFive fungal strains previously isolated from infested field-grown \u003cem\u003eP.\u003c/em\u003e \u003cem\u003evulgaris\u003c/em\u003e L. plants in Tunisia and representing the main abundant pathogenic fungal species according to Sendi et al. (2019) were used in the present study. Those strains are assigned respectively to \u003cem\u003eAlternaria alternata\u003c/em\u003e strain PVF4 (KU831493), \u003cem\u003eFusarium cerealis\u003c/em\u003e strain PVF10 (KU831499), \u003cem\u003eF. oxysporum\u003c/em\u003e strain PVF26 (KU831515), \u003cem\u003eF. tricinctum\u003c/em\u003e strain PVF30 (KU8131515), and \u003cem\u003eMacrophomina\u003c/em\u003e \u003cem\u003ephaseolina\u003c/em\u003e strain PVF32 (KU8131521). Fungal strains were grown on potato-dextrose agar (PDA) medium and stored at 4 \u0026deg;C.\u003c/p\u003e\n\n\u003cp\u003e\u003cstrong\u003eAntibiosis test of \u003cem\u003eB. amyloliquefaciens\u003c/em\u003e PVB17 and \u003cem\u003eR. gallicum\u003c/em\u003e Ma1.12 against the fungal strains\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe antibiosis test was performed on PDA medium for \u003cem\u003eB. amyloliquefaciens\u003c/em\u003e strain PVB17 and on GN (Glucose nitrate) medium for \u003cem\u003eR. gallicum\u003c/em\u003e strain Ma1.12 \u0026ndash;for the reason of growth medium compatibility- and according to the protocol of Mrabet et al. (2015). The percentage of growth inhibition of each fungal strain was calculated considering the control fungal growth using the following formula (Mrabet et al. 2015):\u003c/p\u003e\n\u003cp\u003eGrowth inhibition (%) = [A \u0026minus; B/A] x 100;\u003c/p\u003e\n\u003cp\u003eWhere A is the diameter of fungal growth in the control and B is the distance of fungal growth in front of each of the two bacterial streaks. \u003c/p\u003e\n\n\u003cp\u003e\u003cstrong\u003ePreparation of fungal inoculums\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eTwenty fungal discs (0.7 mm of diam.) from a fungal fresh culture were placed into an Erlenmeyer flask containing 200 g of sterilized sorghum seeds imbibed with 70 mL of sterile distilled water. The fungal preparation was incubated at 24 \u0026deg;C for five days in the darkness for growing mycelium. The density of each fungal strain in \u003cem\u003eSorghum bicolor\u003c/em\u003e L. was measured and adjusted to 10\u003csup\u003e6\u003c/sup\u003e CFU (colony forming units) per gram of \u003cem\u003eSorghum bicolor\u003c/em\u003e L., the density to be used for the inoculation of plants.\u003c/p\u003e\n\n\u003cp\u003e\u003cstrong\u003eResearch for PGPR activities in\u003c/strong\u003e\u003cem\u003e \u003cstrong\u003eB. amyloliquefaciens\u003c/strong\u003e \u003c/em\u003e\u003cstrong\u003eand\u003c/strong\u003e\u003cem\u003e \u003cstrong\u003eR. gallicum\u003c/strong\u003e \u003c/em\u003e\u003cstrong\u003estrains\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eIndole-3-acetic acids (IAA) production was measured according to the protocol of Bano and Musarrat (2003) and siderophores production was assessed using the chromeazurol (CAS) agar assay as described by Schwyn and Neilands (1987). Wells were made on the CAS medium and 100 \u0026mu;l of each bacterial suspension was incubated in the dark for two days. Positive results were indicated by the formation of a light halo around the colonies, showing a visual change in color from dark blue to yellow. HCN production using the method of Feigl and Anger (1966). The bacterial isolates were streaked on a medium Luria Bertani (LB). A Whatman filter paper was placed at the top of the plate. The plates were sealed with parafilm and incubated for four days at 30 \u003csup\u003eo\u003c/sup\u003eC. Production of HCN was indicated by the development of blue color, and EPS production was assessed according to the protocol of ShivaKumar and Vijayendra (2006). Thirty days after bacterial incubation in a medium saturated with saccharose (20 g. L\u003csup\u003e-1\u003c/sup\u003e) at 30\u0026deg;C, strains with a mucous appearance are EPS-productive. \u003c/p\u003e\n\u003cp\u003ePhosphate solubilization activity was assessed on Pikovskaya\u0026rsquo;s agar medium (g. L\u003csup\u003e-1\u003c/sup\u003e; yeast extract 0.5, dextrose 10, calcium phosphate 5, ammonium sulfate 0.5, potassium chloride 0.2, magnesium sulfate 0.1, manganese sulfate 0.0001, ferrous sulfate 0.0001, agar 15) was used for isolation of phosphate-solubilizing microorganisms (Pikovskaya, 1948). Bacterial strains able to solubilize precipitated calcium phosphate to produce clear zones around colonies were assessed and measured for both bacterial strains \u003cem\u003eB. amyloliquefaciens\u003c/em\u003e strain PVB17 and \u003cem\u003eR. gallicum\u003c/em\u003e strain Ma1.12. Each test was performed in triplicate.\u003c/p\u003e\n\n\u003cp\u003e\u003cstrong\u003eDevelopment of talc bio-based formulations for\u003cem\u003e B. amyloliquefaciens \u003c/em\u003eand\u003cem\u003e R. gallicum \u003c/em\u003estrains\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003ePreparation of mineral carriers\u003c/p\u003e\n\u003cp\u003eClay samples were collected from the formation of Saouaf located in Grombalia location at the northeast of Tunisia which is characterized by clays intercalated with sandstone (Ghrasalli and Mzali 2017). The mineral composition of the Saouaf argillaceous formation is kaolinite (20-35%), smectite (40-60%), and illite (10-20%) and the average clay fraction is of 57% according to Gharsalli and Mzali (2017). Clay samples were crushed using an electric Retsch shredder for 10 min. Clay powder was therefore three times autoclaved for 30 min at 120 \u0026deg;C. Then, dried up to 20% humidity and kept in sterile plastic bags.\u003c/p\u003e\n\n\u003cp\u003ePreparation of bacterial suspensions\u003c/p\u003e\n\u003cp\u003e\u003cem\u003eBacillus amyloliquafciens\u003c/em\u003e strain PVB17 was cultivated in LB broth medium and optical density (OD) at 620 nm was adjusted to the value 0.6 giving 10\u003csup\u003e8 \u003c/sup\u003eCFU. mL\u003csup\u003e-1\u003c/sup\u003e. \u003cem\u003eRhizobium gallicum\u003c/em\u003e strain Ma1.12 was cultivated in YEM (Yeast Extract Mannitol) broth medium at 28 \u0026deg;C for 48 h and OD 620 nm adjusted to the value 0.8 corresponding to 10\u003csup\u003e8\u003c/sup\u003e CFU. mL\u003csup\u003e-1\u003c/sup\u003e. A Sterile solution of Gum arabic (Lot 55H1455) was used as an additive to the bacterial preparation, mixing 50 mL of gum arabic solution (10%) with 100 mL of bacterial suspension and kept at room temperature for 30 min before use.\u003c/p\u003e\n\n\u003cp\u003eDevelopment of talc bio-based formulations and seed coating\u003c/p\u003e\n\u003cp\u003eTwenty mL of bacterial suspension/gum arabic preparation was mixed with 80 g of autoclaved talc powder in sterile Petri dishes. \u003cem\u003eP. vulgaris\u003c/em\u003e L. seeds, cv. Coco blanc, were surface disinfected with mercury chloride solution (0.2%) for 2 min and then rinsed with sterile distilled water. Then, the seeds were soaked in a double volume of sterile distilled water containing the mentioned formulations (10 g/1 Kg of seeds). One hour later, the bacterial suspension was drained off and the seeds were dried at room temperature under sterile conditions for 30 min and planted as described by Vidhyasekaran et al. (1997).\u003c/p\u003e\n\n\u003cp\u003eShelflife assessment\u003c/p\u003e\n\u003cp\u003eFrom each talc bacterial preparation, 1 g was added to 9 mL of sterile distilled water and a series of dilutions from 10\u003csup\u003e-1\u003c/sup\u003e to 10\u003csup\u003e-10\u003c/sup\u003e were prepared. A volume of 100 \u0026micro;l of each dilution was suspended on solid growth medium (LB or YEMA) and incubated at 28 \u0026deg;C and growing colonies were counted (CFU. mL\u003csup\u003e-1\u003c/sup\u003e) at 0, 15, 30, 45, and 60 days of incubation. The talc-bacterial formulations were kept in sterile plastic bags at room temperature during the performed experiment.\u003c/p\u003e\n\n\u003cp\u003e\u003cstrong\u003eEffectiveness of talc bio-based formulations on host plant bioferilization and bioprotection under greenhouse conditions\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cem\u003eP. vulgaris\u003c/em\u003e L. seed plantation and treatments\u003c/p\u003e\n\u003cp\u003e\u003cem\u003eP. vulgaris\u003c/em\u003e L. cv. Coco blanc seeds were planted in plastic pots of 1 Kg containing a mix of sterile peat/sand at a ratio of 1:2. In case of fungal treatments, 15 g of infected sorghum seeds were added and mixed with peat/sand mixture in each pot 24 h before plantation step. Plantlets were grown in a growth chamber under controlled conditions (28 \u0026deg;C, HR 60%, Photoperiod 16/8). The following treatments were considered : (i) Control plants, (ii) pots infected with the fungal inoculum, (iii) Coated-seeds with the \u003cem\u003eR. gallicum\u003c/em\u003e strain Ma1.12, (iv) Coated-seeds with the \u003cem\u003eB. amyloliquefaciens\u003c/em\u003e strain PVB17, (v) Coated-seeds with both formulations of \u003cem\u003eR. gallicum\u003c/em\u003e strain Ma1.12 and \u003cem\u003eB. amyloliquefaciens\u003c/em\u003e strain PVB17, (vi) plant co-treated \u003cem\u003eF. oxysporum\u003c/em\u003e strain PVF26 and \u003cem\u003eB. amyloliquefaciens\u003c/em\u003e strain PVB17 formulation, and (vii) plant treated with \u003cem\u003eF. oxysporum\u003c/em\u003e strain PVF26, \u003cem\u003eB. amyloliquefaciens\u003c/em\u003e strain PVB17, \u003cem\u003eR. gallicum\u003c/em\u003e strain Ma1.12. In each pot, three \u003cem\u003eP. vulgaris\u003c/em\u003e L. seeds were planted and five pots were considered for each treatment. The culture was grown for 40 days before harvest.\u003c/p\u003e\n\n\u003cp\u003eRecorded parameters\u003c/p\u003e\n\u003cp\u003eBoth growth and disease parameters were recorded at harvesting time which consist of shoot dry weight, root dry weight, nodules number, and plant health status according to Sendi et al. (2020).\u003c/p\u003e\n\n\u003cp\u003e\u003cstrong\u003eBacterial effectiveness of talc formulation in field plots\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eA field trial was performed in the region of Boucharray (North-eastern Tunisia) and three experimental blocks were considered. Each block is designed as 10 mL long and three lines were considered for each treatment in each block. Lines were separated by a distance of 30 cm. \u003cem\u003eP. vulgaris\u003c/em\u003e L. seeds were sown in order of eight seeds per 20 cm\u003csup\u003e2\u003c/sup\u003e and each seeds mass is distant of 20 cm from the previous one. In each block, the following treatments were included : Control, Coated seeds with \u003cem\u003eR. gallicum\u003c/em\u003e strain Ma1.12 formulation, Coated seeds with \u003cem\u003eB. amyloliquefaciens\u003c/em\u003e strain PVB17 formulation, and Coated seeds with combined formulations of \u003cem\u003eR. gallicum\u003c/em\u003e strain Ma1.12 and \u003cem\u003eB. amyloliquefaciens\u003c/em\u003e strain PVB17. During cultivation, plants were irrigated with well water when needed.\u003c/p\u003e\n\u003cp\u003e\u003cem\u003eP. vulgaris\u003c/em\u003e L. plants were harvested two months post plantation in order of 24 randomized plants per treatment. Shoot and root dry weights, pod number and weight, and the health state of grown plants were recorded. \u003c/p\u003e\n\u003cp\u003e\u003cem\u003e \u003c/em\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eStatistical analyses\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eDifferent parameters were analyzed by comparing means and variances using the SPSS version 20 software. Means were compared with the test Duncan at p = 0.05.\u003c/p\u003e"},{"header":"Results","content":"\u003cp\u003e\u003cstrong\u003eAntifungal effectiveness of\u003cem\u003e B. amyloliquefaciens \u003c/em\u003estrain PVB14 and\u003cem\u003e R. gallicum \u003c/em\u003estrain Ma1.12\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAntibiosis tests showed that \u003cem\u003eB. amyloliquefaciens\u003c/em\u003e strain PBV17 is able to inhibit the \u003cem\u003ein vitro\u003c/em\u003e growth of \u003cem\u003eA. alternata\u003c/em\u003e PVF4, \u003cem\u003eF. cerealis\u003c/em\u003e PVF10, \u003cem\u003eF. oxysporum\u003c/em\u003e PVF26, \u003cem\u003eF. tricinctum\u003c/em\u003e PVF30, and \u003cem\u003eM. phaseolina\u003c/em\u003e PVF32 by 55% to 73% (Fig. 1). \u003cem\u003eRhizobium gallicum\u003c/em\u003e strain Ma1.12 inhibited the mycelial growth of different fungal strains from 50% to 64% (Fig. 1). In Fig. 2, an illustration of the inhibition of \u003cem\u003eF. oxysporum\u003c/em\u003e strain PVF26 by \u003cem\u003eB. amyloliquefaciens\u003c/em\u003e strain PVB17 is shown.\u003c/p\u003e\n\n\u003cp\u003e\u003cstrong\u003eCharacterization of PGPR activities in \u003cem\u003eB. amyloliquefaciens\u003c/em\u003e strain PVB17 and \u003cem\u003eR. gallicum\u003c/em\u003e strain Ma1.12\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe PGPR traits characterization showed that both bacterial strains are able to produce siderophores (Fig. 3). Strain Ma1.12 of \u003cem\u003eR. gallicum\u003c/em\u003e is able to produce 910 \u0026micro;g. mL\u003csup\u003e-1\u003c/sup\u003e of auxins whereas \u003cem\u003eB. amyloliquefaciens\u003c/em\u003e strain PVB17 is producing only 25.19 \u0026micro;g. mL\u003csup\u003e-1\u003c/sup\u003e (Table 1). Both bacterial strains produced a high amount of exopolysaccharides (EPS) in a growth medium saturated with saccharose. However, phosphates solubilization is only detected for \u003cem\u003eR. gallicum\u003c/em\u003e strain Ma1.12 (Table 1).\u003c/p\u003e\n\n\u003cp\u003e\u003cstrong\u003eViability of \u003cem\u003eB. amyloliquefaciens\u003c/em\u003e strain PVB17 and \u003cem\u003eR. gallicum\u003c/em\u003e strain Ma1.12 in the talc bio-formulation\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe assessment of cell viability of bacterial strains showed that \u003cem\u003eB. amyloliquefaciens\u003c/em\u003e strain PVB17 and \u003cem\u003eR. gallicum\u003c/em\u003e strain Ma1.17 are able to survive into the talc formulation at up to 10\u003csup\u003e9\u003c/sup\u003e and 10\u003csup\u003e8\u003c/sup\u003e CFU. g\u003csup\u003e-1\u003c/sup\u003e of talc formulation, respectively, during two months of incubation (Table 2).\u003c/p\u003e\n\n\u003cp\u003e\u003cstrong\u003eIncidence of talc bio-based formulations of \u003cem\u003eB. amyloliquefaciens\u003c/em\u003e PVB17 and \u003cem\u003eR. gallicum\u003c/em\u003e Ma1.12 on \u003cem\u003eP. vulgaris\u003c/em\u003e L. growth\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cem\u003eP. vulgaris\u003c/em\u003e L.\u003cem\u003e \u003c/em\u003ecv. Coco blanc plants of talc-coated seeds showed similar root and shoot growth capacity as the talc non-coated seeds (Fig. 4). Plants from seeds coated with \u003cem\u003eR. gallicum\u003c/em\u003e Ma1.12 talc formulation showed a significant increase of 5.3 folds and 2 folds of root and shoot growth, respectively (Fig. 4). Root and shoot dry weights increased by about 3 folds and 1.3 folds, respectively, when seeds are coated with \u003cem\u003eB. amyloliquefaciens\u003c/em\u003e PVB17 talc formulation. The simultaneous application of Ma1.12 and PVB17 talc formulations to \u003cem\u003eP. vulgaris\u003c/em\u003e L.\u003cem\u003e \u003c/em\u003eseeds is associated with an increase of root dry weight by 5 folds and of shoot dry weight by 2 folds in comparison to the non-coated seeds.\u003c/p\u003e\n\u003cp\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eBiocontrol effectiveness of the bacterial talc formulations of \u003cem\u003eP. vulgaris\u003c/em\u003e L.\u003c/strong\u003e \u003cstrong\u003eagainst \u003cem\u003eF. oxysporum\u003c/em\u003e attacks\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eRoot and shoot growth of \u003cem\u003eP. vulgaris\u003c/em\u003e L.\u003cem\u003e \u003c/em\u003eplants grown in peat/sand mixture inoculated with \u003cem\u003eF. oxysporum\u003c/em\u003e strain PVF26 is significantly increased for seeds coated with \u003cem\u003eR. gallicum\u003c/em\u003e strain Ma1.12 and \u003cem\u003eB. amyloliquefaciens\u003c/em\u003e strain PVB17 applied separately or combined (Fig. 5). The root and shoot dry weights are increased up to 5.64 folds compared to control plants only infected with the fungal strain. Severe leaf chlorosis and root rot symptoms in \u003cem\u003eP. vulgaris\u003c/em\u003e plants inoculated with \u003cem\u003eF. oxysporum\u003c/em\u003e strain PVF26 was observed. However, the leaf chlorosis and root rot symptoms were significantly reduced, particularly under PVB17/Ma1.12 combined seed-treatment (data not shown). When inoculated with \u003cem\u003eF. oxysporum\u003c/em\u003e strain PVF26, The treatment involving \u003cem\u003eR. gallicum\u003c/em\u003e strain Ma1.12 is associated with root nodulation of up to 207 nodules per plant (data not shown).\u003c/p\u003e\n\n\u003cp\u003e\u003cstrong\u003eField effectiveness of the talc bio-based formulation of \u003cem\u003eB. amyloliquefaciens\u003c/em\u003e strain PVB17 and \u003cem\u003eR. gallicum \u003c/em\u003estrain Ma1.12\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe analysis of the variance of shoot and root dry weights, pod number, and weight showed that there are no differences between various blocks for each treatment (P \u0026gt; 0.05). Accordingly, results were averaged for each treatment and presented in table 3. \u003cem\u003eP. vulgaris\u003c/em\u003e L.\u003cem\u003e \u003c/em\u003ecv. Coco blanc seeds coated with \u003cem\u003eR. gallicum\u003c/em\u003e strain Ma1.12 is associated with a significant increase of nodules number up to 186 nodules/plant (Table 3). This increase was associated with a significant enhancement of shoot dry weight, pod number, and weight. When coated with the \u003cem\u003eB. amyloliquefaciens\u003c/em\u003e strain PVB17, common bean plant growth, and pod production are also significantly increased, however less than results given by \u003cem\u003eR. gallicum\u003c/em\u003e strain Ma1.12 (Table 3). Nodules number is significantly increased also. Interestingly, when coated with combined formulations of \u003cem\u003eB. amyloliquefaciens\u003c/em\u003e PVB17 and \u003cem\u003eR. gallicum\u003c/em\u003e Ma1.12, \u003cem\u003eP. vulgaris\u003c/em\u003e L.\u003cem\u003e \u003c/em\u003eplants showed an increase of 10 folds in nodules number, 2 folds in shoot dry weight, 2 folds in pods number, and 3 folds in pods weight in comparison to the control non coated plants (Table 3). Seed coating with \u003cem\u003eB. amyloliquefaciens\u003c/em\u003e strain PVB17 and \u003cem\u003eR. gallicum\u003c/em\u003e strain Ma1.12 is associated with a clear improvement in the health status of grown \u003cem\u003eP. vulgaris\u003c/em\u003e L.\u003cem\u003e \u003c/em\u003eplants compared to the control ones (Data not shown). This was observed both for the aerial and root parts.\u003c/p\u003e"},{"header":"Discussion","content":"\u003cp\u003eThe anti-fungal activity of \u003cem\u003eB. amyloliquefaciens\u003c/em\u003e strain PVB17 is demonstrated on \u003cem\u003eF. oxysporum\u003c/em\u003e, \u003cem\u003eF. cerealis\u003c/em\u003e, \u003cem\u003eF. tricinctum\u003c/em\u003e, \u003cem\u003eA. alternata\u003c/em\u003e and \u003cem\u003eM. phaseolina\u003c/em\u003e. These fungal species are reported to be predominant in different agricultural soils and to exhibit severe diseases on various beans (Darvishnia et al. \u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e2023\u003c/span\u003e ; Wu et al. \u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e2024\u003c/span\u003e) and even on other legumes and non-legume plants (Ekwomadu et al. 2023, a review). Using \u003cem\u003eB. amyloliquefaciens\u003c/em\u003e inoculants in the biocontrol of plant pathogens is recognized as an important feature for more sustainable agriculture (Luo et al. \u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e2022\u003c/span\u003e, a review). The antifungal efficacy of \u003cem\u003eB. amyloliquefaciens\u003c/em\u003e is proven to be linked to its ability to produce secondary metabolites as lipopeptides (surfactin, fengycin, bacillomycin, iturin), lytic enzymes, siderophores, and microbial volatile organic compounds (Ji et al. \u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e2021\u003c/span\u003e ; Luo et al. \u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e2022\u003c/span\u003e, a review). The second bacterial strain used in this study is \u003cem\u003eR. gallicum\u003c/em\u003e strain Ma1.12 characterized by its high symbiotic effectiveness with \u003cem\u003eP. vulgaris\u003c/em\u003e L. plants (Mrabet et al. \u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e2006\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eResults showed that both strains of \u003cem\u003eB. amyloliquefaciens\u003c/em\u003e PVB17 and \u003cem\u003eR. gallicum\u003c/em\u003e Ma1.12 were able to produce remarkably siderophores and indole-3-acetic acid (IAA). The IAA production by \u003cem\u003eR. gallicum\u003c/em\u003e strain Ma1.12 is about 910 \u0026micro;g. mL\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e. This value has never been reported in different bacterial species. In fact, K\u0026uuml;\u0026ccedil;\u0026uuml;k and Cevheri (\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e2016\u003c/span\u003e) in a study involving many \u003cem\u003eRhizobium\u003c/em\u003e sp. strains reported that the maximum IAA production was about 165 \u0026micro;g. mL\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e. Auxins control several stages of plant growth such as cell division, tissue differentiation, and apical dominance in plant roots (Seo et al. \u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e2021\u003c/span\u003e). The treatment of IAA-producing rhizobacteria increases plant growth (Khozo et al. \u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e2024\u003c/span\u003e, a review). \u003cem\u003eBacillus amyloliquefaciens\u003c/em\u003e strain PVB17 and \u003cem\u003eR. gallicum\u003c/em\u003e strain Ma1.12 are shown to produce great amounts of siderophores. Siderophore-producing rhizobacteria improve plant growth and protection since they improve iron nutrition and hinder the growth of pathogens by limiting the iron available for the pathogen, generally, fungi, which are unable to absorb the iron-siderophore complex (Deb and Tatung \u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e2024\u003c/span\u003e). After N, P is the most limiting nutrient for plants. The ability of \u003cem\u003eR. gallicum\u003c/em\u003e strain Ma1.12 to solubilize inorganic phosphates may explain- partially- the PGPR effectiveness of this bacterial strain, besides N fixation property. It was recognized that microorganisms that are able to solubilize complex-structured phosphates, providing it in a form suitable for plant uptake, are among PGPR groups that increase the P availability to plants (Elhaissoufi et al. 2024).\u003c/p\u003e \u003cp\u003eDespite the antifungal activity of \u003cem\u003eB. amyloliquefaciens\u003c/em\u003e strain PVB17 and the symbiotic effectiveness of \u003cem\u003eR. gallicum\u003c/em\u003e strain Ma1.12, the challenge is how to process for their use at field scales. In this regard, talc was used as a mineral carrier to formulate both bacterial strains and to assess their plant growth-promoting traits on \u003cem\u003eP. vulgaris\u003c/em\u003e L. under greenhouse and field trials.\u003c/p\u003e \u003cp\u003eInterestingly, the self-life study of both bacterial strains in talc formulation showed that \u003cem\u003eB. amyloliquefaciens\u003c/em\u003e strain PVB17 and \u003cem\u003eR. gallicum\u003c/em\u003e strain Ma1.12 survive at high frequency during a period of two months of assessment. This ability to survive into talc formulation is necessary but not determinant for the success of developed formulations. In fact, it was reported that the success of microbial seed treatments depends not only on formulations that ensure the survival of the inoculant during seed treatment and storage but also on its ability to multiply in the spermosphere, colonize root, and in some cases the surrounding soil, where it then perform its required function (Khan et al. \u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e2023\u003c/span\u003e). Therefore, confirmation of talc bioformulation efficacy on \u003cem\u003eP. vulgaris\u003c/em\u003e L. plants was necessary to provide much more evidence of its effectiveness. This study showed that \u003cem\u003eP. vulgaris\u003c/em\u003e L. seeds coating with \u003cem\u003eB. amyloliquefaciens\u003c/em\u003e strain PVB17 and \u003cem\u003eR. gallicum\u003c/em\u003e strain Ma1.12, combined or separately, was associated with significant improvement of shoot and root dry weights. This result indicates the effectiveness of talc formulations of both bacterial strains. On another side, \u003cem\u003eP. vulgaris\u003c/em\u003e L. seeds coated only with talc gave similar shoot and root growth as in control non-coated seeds underlying that there\u0026rsquo;s no harmful effect of talc on \u003cem\u003eP. vulgaris\u003c/em\u003e L. seed germination and plant growth.\u003c/p\u003e \u003cp\u003eAnother challenge for us was to confirm if the talc-biobased formulation of \u003cem\u003eB. amyloliquefaciens\u003c/em\u003e strain PVB17 and \u003cem\u003eR. gallicum\u003c/em\u003e strain Ma1.12 was able to protect \u003cem\u003eP. vulgaris\u003c/em\u003e L. plants against the pathogen \u003cem\u003eF. oxysporum\u003c/em\u003e. The biocontrol assay confirmed the efficacy of talc formulation of both bacterial strains applied separately or combined. The shoot and root dry weights increased by about 5 fold. Moreover, nodulation by \u003cem\u003eR. gallicum\u003c/em\u003e was more than 200 nodules per plant. These findings confirmed the efficacy of talc-formulations of both bacterial strains in protecting \u003cem\u003eP. vulgaris\u003c/em\u003e L. plants from \u003cem\u003eF. oxysporum\u003c/em\u003e strain PVF26 and therefore in enhancing plant growth.\u003c/p\u003e \u003cp\u003eField assays showed the effectiveness of the talc formulation of both bacterial strains in enhancing nodulation of \u003cem\u003eP. vulgaris\u003c/em\u003e L. induced by \u003cem\u003eR. gallicum\u003c/em\u003e strain Ma1.12 and in increasing plant growth and pods number and weights, particularly when both bacterial formulations are combined. This fact demonstrated the field efficacy of the developed formulations of both bacterial strains. The increase in nodule number induced by the \u003cem\u003eR. gallicum\u003c/em\u003e strain Ma1.12 under field conditions is a key fact of the enhancement of plant growth and pod production. In fact, nodulation by rhizobia was shown to be improved when rhizobia were combined with plant growth promoting rhizobacteria in legumes, and plant growth was shown to be increased (Kaschuk et al. \u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e2022\u003c/span\u003e). It was recognized that commercial application of PGPR for control of soil-borne diseases depends upon the development of commercial formulations in which bacteria can survive for a considerable length of time, on the development of a suitable method of application to control pathogen establishment and disease development, and assessment of their efficacy under field conditions (O\u0026rsquo;Callaghan 2022 ; Khan et al. \u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e2023\u003c/span\u003e). Hence, this study demonstrated the efficacy of talc-formulation of \u003cem\u003eB. amyloliquefaciens\u003c/em\u003e strain PVB17 and \u003cem\u003eR. gallicum\u003c/em\u003e strain Ma1.12 as seed coating inoculant. Further investigations on the usefulness of the developed formulations in other cultivated crops will be tested and agro-economical efficacy assessed.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eAcknowledgment\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors wish to thank Dr. Houcem Mzali, Geologist Researcher at the Centre for Research and Water Technologies of Borj-Cedria (CERTE) for technical assistance during the sampling talc from the region of Grombalia, and for a sufficient description of the geological formation of this region.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFunding\u003c/strong\u003e\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eThis study was supported by the Ministry of Higher Education and Scientific Research of Tunisia, [Grant Laboratory of Legumes and Sustaible Agrosystems 2018-2023].\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eData availability statement\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe research data can be available to reader upon the acceptance of the paper according to the springer regulation.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConflict of interest\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors declare that they have no any potential financial or non-financial conflict of interest.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n \u003cli\u003eAnsabayeva A, Makhambetov M, Rebouh NY, Abdelkader M, Saudy HS, Hassan KM, Nasser MA, Ali MAA, Ebrahim M (2025) Plant growth-promoting microbes for resilient farming systems: mitigating environmental stressors and boosting crops productivity\u0026mdash;a review. 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Plant Physiol 187:1577-1586. https://doi.org/10.1093/plphys/kiab341\u003c/li\u003e\n \u003cli\u003eShivaKumar S, Vijayendra SVN (2006) Production of exopolysaccharides by \u003cem\u003eAgrobacterium\u003c/em\u003e sp. CFR‐24 using coconut water \u0026ndash; a byproduct of food industry. Lett App Microbiol 42: 477‒482. https://doi.org/10.1111/j.1472-765X.2006.01881.x\u003c/li\u003e\n \u003cli\u003eVidhyasekaran P, Rabindran R, Muthamilan M, Nayar K, Rajappan K, Subramanian N, Vasumathi K (1997) Development of a powder formulation of \u003cem\u003ePseudomonas\u003c/em\u003e \u003cem\u003efluorescens\u003c/em\u003e for control of rice blast. Plant Pathol 46:291‒297. https://doi.org/10.1046/j.1365-3059.1997.d01-27.x\u003c/li\u003e\n \u003cli\u003eWu L, Hwang SF, Strelkov SE, Fredua-Agyeman R, Oh SH, B\u0026eacute;langer RR, Wally O, Kim YM (2024) Pathogenicity, host resistance, and genetic diversity of \u003cem\u003eFusarium\u003c/em\u003e species under controlled conditions from soybean in Canada. J Fungi 10 : 303. https://doi.org/10.3390/jof1005030\u003c/li\u003e\n\u003c/ol\u003e"},{"header":"Tables","content":"\u003cp\u003e\u003cstrong\u003eTable 1.\u003c/strong\u003e PGPR traits characterization of \u003cem\u003eB. amyloliquefaciens\u003c/em\u003e strain PVB17 and \u003cem\u003eR. gallicum\u003c/em\u003e strain Ma1.12. IAA/indole-3-acetic acids, P/Phosphates, EPS/exopolysaccharides.\u003c/p\u003e\n\u003cdiv align=\"\"\u003e\n \u003ctable border=\"0\" cellspacing=\"0\" cellpadding=\"0\"\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 141px;\"\u003e\u003cbr\u003e\u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 142px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eSiderophore Production\u003c/strong\u003e\u003c/p\u003e\n \u003cp\u003e\u003cstrong\u003e(Halo diam. in cm)\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 113px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eIAA production (\u0026micro;g. mL\u003csup\u003e-1\u003c/sup\u003e)\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 123px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eP. solubilisation\u003c/strong\u003e\u003c/p\u003e\n \u003cp\u003e\u003cstrong\u003e(Halo diam. in cm)\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 85px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eEPS production\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 141px;\"\u003e\n \u003cp\u003e\u003cstrong\u003e\u003cem\u003eB. amyloliquefaciens\u003c/em\u003e\u003c/strong\u003e\u003cstrong\u003e\u0026nbsp;strain PVB17\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 142px;\"\u003e\n \u003cp\u003e4.3 (\u0026plusmn; 0.2)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 113px;\"\u003e\n \u003cp\u003e25.19 (\u0026plusmn; 5.21)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 123px;\"\u003e\n \u003cp\u003eND\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 85px;\"\u003e\n \u003cp\u003e+++\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 141px;\"\u003e\n \u003cp\u003e\u003cstrong\u003e\u003cem\u003eR. gallicum\u003c/em\u003e\u003c/strong\u003e\u003cstrong\u003e\u0026nbsp;strain Ma1.12\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 142px;\"\u003e\n \u003cp\u003e3.1 (\u0026plusmn; 0.13)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 113px;\"\u003e\n \u003cp\u003e910.32 (\u0026plusmn; 35.05)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 123px;\"\u003e\n \u003cp\u003e3.3 (\u0026plusmn; 0.57)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 85px;\"\u003e\n \u003cp\u003e+++\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n \u003c/table\u003e\n\u003c/div\u003e\n\u003cp\u003e\u003csup\u003e+++\u003c/sup\u003e: High production level. ND: Non detected. P: phosphates. Standard error values for each parameter are indicated in parentheses.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eTable 2.\u0026nbsp;\u003c/strong\u003eViability of \u003cem\u003eB. amyloliquefaciens\u003c/em\u003e strain PVB17 and \u003cem\u003eR. gallicum\u003c/em\u003e strain Ma1.12 into the talc bio-based formulation during two months. Bacterial colonies were counted every 15 days using the dilution method and spreading on a growth medium.\u003c/p\u003e\n\u003ctable border=\"0\" cellspacing=\"0\" cellpadding=\"0\"\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 173px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eBacterial strains\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 217px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eTime of incubation (Day)\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 214px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eColonies counting (CFU. g\u003csup\u003e-1\u003c/sup\u003e of talc formulation)\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd rowspan=\"5\" valign=\"top\" style=\"width: 173px;\"\u003e\n \u003cp\u003e\u003cem\u003e\u0026nbsp;\u003c/em\u003e\u003c/p\u003e\n \u003cp\u003e\u003cem\u003eB. amyloliquefaciens\u003c/em\u003e strain PVB17\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 217px;\"\u003e\n \u003cp\u003e0\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 214px;\"\u003e\n \u003cp\u003e4x10\u003csup\u003e10\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 217px;\"\u003e\n \u003cp\u003e15\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 214px;\"\u003e\n \u003cp\u003e3.8x10\u003csup\u003e9\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 217px;\"\u003e\n \u003cp\u003e30\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 214px;\"\u003e\n \u003cp\u003e2.2x10\u003csup\u003e9\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 217px;\"\u003e\n \u003cp\u003e45\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 214px;\"\u003e\n \u003cp\u003e2.7x10\u003csup\u003e9\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 217px;\"\u003e\n \u003cp\u003e60\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 214px;\"\u003e\n \u003cp\u003e2.3x10\u003csup\u003e9\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd rowspan=\"5\" valign=\"top\" style=\"width: 173px;\"\u003e\n \u003cp\u003e\u003cem\u003e\u0026nbsp;\u003c/em\u003e\u003c/p\u003e\n \u003cp\u003e\u003cem\u003eR. gallicum\u003c/em\u003e strain Ma1.12\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 217px;\"\u003e\n \u003cp\u003e0\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 214px;\"\u003e\n \u003cp\u003e3x10\u003csup\u003e9\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 217px;\"\u003e\n \u003cp\u003e15\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 214px;\"\u003e\n \u003cp\u003e5.4x10\u003csup\u003e8\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 217px;\"\u003e\n \u003cp\u003e30\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 214px;\"\u003e\n \u003cp\u003e3.7x10\u003csup\u003e8\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 217px;\"\u003e\n \u003cp\u003e45\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 214px;\"\u003e\n \u003cp\u003e2.1x10\u003csup\u003e8\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 217px;\"\u003e\n \u003cp\u003e60\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 214px;\"\u003e\n \u003cp\u003e1.8x10\u003csup\u003e8\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n\u003c/table\u003e\n\u003cp\u003e\u0026nbsp;\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eTable 3.\u0026nbsp;\u003c/strong\u003eField effectiveness of talc formulations of \u003cem\u003eB. amyloliquefaciens\u003c/em\u003e strain PVB17 and \u003cem\u003eR. gallicum\u003c/em\u003e Ma1.12 on \u003cem\u003eP. vulgaris\u003c/em\u003e L. cv. Coco blanc in the region of Boucharray. The harvest was done 60 days post plantation.\u0026nbsp;\u003c/p\u003e\n\u003ctable border=\"0\" cellspacing=\"0\" cellpadding=\"0\"\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 198px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eTreatment\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 95px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eNodules number\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 94px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eSDW (g. plant\u003csup\u003e-1\u003c/sup\u003e)\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 98px;\"\u003e\n \u003cp\u003e\u003cstrong\u003ePods number\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 119px;\"\u003e\n \u003cp\u003e\u003cstrong\u003ePods weight (g. plant\u003csup\u003e-1\u003c/sup\u003e)\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 198px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eControl\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 95px;\"\u003e\n \u003cp\u003e21\u003csup\u003ec\u003c/sup\u003e (\u0026plusmn; 5)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 94px;\"\u003e\n \u003cp\u003e4.2\u003csup\u003ed\u0026nbsp;\u003c/sup\u003e(\u0026plusmn; 0.43)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 98px;\"\u003e\n \u003cp\u003e4.02\u003csup\u003ed\u003c/sup\u003e (\u0026plusmn; 0.5)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 119px;\"\u003e\n \u003cp\u003e16.5\u003csup\u003ed\u003c/sup\u003e (\u0026plusmn; 3.4)\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 198px;\"\u003e\n \u003cp\u003e\u003cstrong\u003e\u003cem\u003eR. gallicum\u003c/em\u003e\u003c/strong\u003e\u003cstrong\u003e\u0026nbsp;Ma1.12\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 95px;\"\u003e\n \u003cp\u003e186\u003csup\u003ea\u003c/sup\u003e (\u0026plusmn; 21)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 94px;\"\u003e\n \u003cp\u003e7.38\u003csup\u003eb\u003c/sup\u003e (\u0026plusmn; 0.84)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 98px;\"\u003e\n \u003cp\u003e6.3\u003csup\u003eb\u003c/sup\u003e (\u0026plusmn; 1)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 119px;\"\u003e\n \u003cp\u003e39.97\u003csup\u003eb\u003c/sup\u003e (\u0026plusmn; 3.2)\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 198px;\"\u003e\n \u003cp\u003e\u003cstrong\u003e\u003cem\u003eB. amyloliquefaciens\u003c/em\u003e\u003c/strong\u003e\u003cstrong\u003e\u0026nbsp;PVB17\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 95px;\"\u003e\n \u003cp\u003e47\u003csup\u003eb\u003c/sup\u003e (\u0026plusmn; 12)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 94px;\"\u003e\n \u003cp\u003e5.34\u003csup\u003ec\u003c/sup\u003e (\u0026plusmn; 0.54)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 98px;\"\u003e\n \u003cp\u003e5.47\u003csup\u003ec\u003c/sup\u003e (\u0026plusmn; 0.73)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 119px;\"\u003e\n \u003cp\u003e30.11\u003csup\u003ec\u003c/sup\u003e (\u0026plusmn; 5.6)\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 198px;\"\u003e\n \u003cp\u003e\u003cstrong\u003e\u003cem\u003eR. gallicum\u003c/em\u003e\u003c/strong\u003e\u003cstrong\u003e\u0026nbsp;Ma1.12 + \u003cem\u003eB. amyloliquefaciens\u003c/em\u003e PVB17\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 95px;\"\u003e\n \u003cp\u003e202\u003csup\u003ea\u003c/sup\u003e (\u0026plusmn; 33)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 94px;\"\u003e\n \u003cp\u003e8.76\u003csup\u003ea\u003c/sup\u003e (\u0026plusmn; 1.39)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 98px;\"\u003e\n \u003cp\u003e8.72\u003csup\u003ea\u003c/sup\u003e (\u0026plusmn; 1.33)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 119px;\"\u003e\n \u003cp\u003e51.03\u003csup\u003ea\u003c/sup\u003e (\u0026plusmn; 8.1)\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n\u003c/table\u003e\n\u003cp\u003e* Each recorded parameter corresponds to an average of a three experimental blocks. Values between parentheses represent standard errors. SDW/Shoot dry weight.\u003c/p\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":true,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"journal-of-plant-diseases-and-protection","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"jpdp","sideBox":"Learn more about [Journal of Plant Diseases and Protection](https://www.springer.com/journal/41348)","snPcode":"41348","submissionUrl":"https://www.editorialmanager.com/jpdp","title":"Journal of Plant Diseases and Protection","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"Springer Hybrid","inReviewEnabled":true,"inReviewRevisionsEnabled":false},"keywords":"Biocontrol, Bacterial inoculum, Common bean, Plant growth promotion, Seed treatment","lastPublishedDoi":"10.21203/rs.3.rs-5489726/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-5489726/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eThe aim of this study is to evaluate the effectiveness of \u003cem\u003eBacillus amyloliquefaciens\u003c/em\u003e/ \u003cem\u003eRhizobium gallicum\u003c/em\u003e- talc formulations to control fungal diseases and to promote plant growth in the common bean (\u003cem\u003ePhaseolus vulgaris\u003c/em\u003e L.). \u003cem\u003eBacillus amyloliquefaciens\u003c/em\u003e strain PVB17 and \u003cem\u003eR. gallicum\u003c/em\u003e strain Ma1.12 inhibited \u003cem\u003eFusarium oxysporum\u003c/em\u003e, \u003cem\u003eAlternaria alternata\u003c/em\u003e and \u003cem\u003eMacrophomina phaseolina\u003c/em\u003e growth by up to 70% and 50%, respectively. Both bacterial strains are able to produce siderophores, indole-3-acetic acids (IAA), and exopolysaccharides. Interestingly, \u003cem\u003eR. gallicum\u003c/em\u003e strain Ma1.12 IAA production reached 910 \u0026micro;g. mL\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e, a value to be reported for the first time and has never been reported in various bacterial species. Talc was sampled from the region of Grombalia and powder was prepared. Gum Arabic was used as an adhesive in the bacterial formulation. The shelf life assessment- during two months of incubation- showed that \u003cem\u003eB. amyloliquefaciens\u003c/em\u003e and \u003cem\u003eR. gallicum\u003c/em\u003e strains are able to survive at more than 10\u003csup\u003e8\u003c/sup\u003e cfu. g\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e of talc formulation. Whether used separately or combined, coating common bean seeds with Ma1.12 and PVB17 strains and growing them in a peat/sand mixture resulted in a significant increase in shoot and root dry weights up to 200% and 400%, respectively. Interestingly, the biocontrol assay of a pathogenic \u003cem\u003eF. oxysporum\u003c/em\u003e with the talc bacterial formulations is associated with a decrease in fungal incidence in plant growth. A Field trial conducted in the region of Boucharray showed that seed coating with the formulations of \u003cem\u003eR. gallicum\u003c/em\u003e strain Ma1.12 and \u003cem\u003eB. amyloliquefaciens\u003c/em\u003e strain PVB17 significantly increased plant growth, nodule number, and pod weight. Combined seeds-coating using both bacterial strains gave the highest increases in shoot dry weight (by 108%), nodules number (by 861%), and pods weight (by 209%) in comparison to the controls. Seeds coating with the bacterial formulation are associated with decreased disease symptoms.\u003c/p\u003e","manuscriptTitle":"Efficacy of talc bio-based formulation of Bacillus amyloliquefaciens and Rhizobium gallicum for Phaseolus vulgaris L. seeds coating","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-04-28 09:00:10","doi":"10.21203/rs.3.rs-5489726/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"decision","content":"Accept","date":"2025-07-04T09:37:25+00:00","index":"","fulltext":""},{"type":"reviewerAgreed","content":"","date":"2025-05-02T05:03:34+00:00","index":0,"fulltext":""},{"type":"reviewersInvited","content":"","date":"2025-04-25T15:18:34+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2025-04-09T07:04:53+00:00","index":"","fulltext":""},{"type":"submitted","content":"Journal of Plant Diseases and Protection","date":"2025-04-08T09:45:25+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"journal-of-plant-diseases-and-protection","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"jpdp","sideBox":"Learn more about [Journal of Plant Diseases and Protection](https://www.springer.com/journal/41348)","snPcode":"41348","submissionUrl":"https://www.editorialmanager.com/jpdp","title":"Journal of Plant Diseases and Protection","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"Springer Hybrid","inReviewEnabled":true,"inReviewRevisionsEnabled":false}}],"origin":"","ownerIdentity":"d573d062-fe36-4198-9aca-e9e167739998","owner":[],"postedDate":"April 28th, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"published-in-journal","subjectAreas":[],"tags":[],"updatedAt":"2025-08-04T16:44:43+00:00","versionOfRecord":{"articleIdentity":"rs-5489726","link":"https://doi.org/10.1007/s41348-025-01128-8","journal":{"identity":"journal-of-plant-diseases-and-protection","isVorOnly":false,"title":"Journal of Plant Diseases and Protection"},"publishedOn":"2025-07-28 16:13:31","publishedOnDateReadable":"July 28th, 2025"},"versionCreatedAt":"2025-04-28 09:00:10","video":"","vorDoi":"10.1007/s41348-025-01128-8","vorDoiUrl":"https://doi.org/10.1007/s41348-025-01128-8","workflowStages":[]},"version":"v1","identity":"rs-5489726","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-5489726","identity":"rs-5489726","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}