# Source paper archive - Source title: Stress-adapted endophytic Pseudomonas strains isolated from unmanaged lupin promote potato growth and suppress verticillium wilt through complementary biocontrol and plant growth-promoting mechanisms - Author(s): Numan Ibne Asad, Lia Watson, Anab Mujtaba, Ievgen Lebedenko, Selvakumari Arunachalam, Hamind Reza Eisvand, Haleema Tariq, Donald L. Smith - Source landing URL: https://www.biorxiv.org/content/10.64898/2026.11.06.764942v1 - PDF URL: https://www.biorxiv.org/content/10.64898/2026.11.06.764942v1.full.pdf - PDF SHA-256: 0dd66859ea1c44c0886a3ce1e68a18cb42f5fc11c76fe11461f036f67932f0cd - Converter: pymupdf4llm 0.0.27 - Limits: Automated PDF text extraction; reading order, equations, tables and formatting may be incomplete or incorrect. Images are omitted, and no OCR was performed. Markup and links are neutralized for safe downloading. Consult the original PDF for the authoritative version. --- [bioRxiv preprint doi: https://doi.org/10.64898/2026.11.06.764942; this version posted November 9, 2026. The copyright holder for this preprint](https://doi.org/10.64898/2026.11.06.764942) (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made [available under a CC-BY-NC-ND 4.0 International license.](http://creativecommons.org/licenses/by-nc-nd/4.0/) 1 **Stress-adapted endophytic** _**Pseudomonas**_ **strains isolated from unmanaged lupin promote** 2 **potato growth and suppress verticillium wilt through complementary biocontrol and plant** 3 **growth-promoting mechanisms** 4 Numan Ibne Asad [1,2] - **,** Lia Watson¹ **,** Anab Mujtaba¹ **,** Ievgen Lebedenko¹ **,** Selvakumari 5 Arunachalam¹ **,** Hamid Reza Eisvand [1] **,** Haleema Tariq [1], Donald L. Smith¹ ***** 6 **¹** Department of Plant Science, Faculty of Agricultural and Environmental Sciences, McGill 7 University, Sainte-Anne-de-Bellevue, Québec, Canada 8 **²** School of the Environment, Florida A&M University, Tallahassee, Florida, USA 9 10 **Abstract** 11 Verticillium wilt, primarily caused by _Verticillium dahliae_, is among the most destructive soil 12 borne diseases impacting global potato production. Fungal propagules remain viable in soil for 13 extended periods, and conventional control measures often show limited efficacy. These 14 challenges underscore the necessity for sustainable disease-management strategies. We evaluated 15 two stress-adapted endophytic _Pseudomonas_ strains, B18 and B14, isolated from unmanaged wild 16 _Lupinus_ roots in Québec, Canada, for their plant growth–promoting and biocontrol potential 17 against Verticillium wilt in potato. 18 19 Both isolates exhibited multiple plant-beneficial traits. B18 demonstrated strong phosphate 20 solubilization, indole-3-acetic acid production, putative nitrogen fixation-related activity, 21 siderophore production, and hydrogen cyanide production. These traits correlated with enhanced 22 plant growth–promoting effects, including stimulation of seed germination and radicle growth. In 23 contrast, B14 primarily exhibited antagonistic activity against _Verticillium_ species through similar 24 plant-beneficial mechanisms, indicating complementary activities between the strains. The cell 25 free supernatants of B18 further supported its biostimulant activity, suggesting a significant role 26 for its extracellular metabolites. [bioRxiv preprint doi: https://doi.org/10.64898/2026.11.06.764942; this version posted November 9, 2026. The copyright holder for this preprint](https://doi.org/10.64898/2026.11.06.764942) (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made [available under a CC-BY-NC-ND 4.0 International license.](http://creativecommons.org/licenses/by-nc-nd/4.0/) 27 Greenhouse experiments using the _Verticillium_ -susceptible potato cultivar Russet Burbank, 28 inoculated with either strain, showed enhanced plant growth and reduced disease severity 29 compared with pathogen-only controls. Under pathogen challenge, plants treated with plant 30 growth–promoting rhizobacteria (PGPR) exhibited up to 62% greater shoot height and 31 approximately double the leaf production. Additionally, these plants produced 4.2 times more 32 tubers than pathogen-only plants. Disease progression, measured as the area under the disease 33 progress curve (AUDPC), was reduced by 84–92% following bacterial inoculation. While 34 individual strains effectively suppressed disease, the combined B18 plus B14 strains resulted in 35 the most substantial improvements in growth, biomass, root development, and tuber production. 36 37 These findings demonstrate that stress-adapted endophytic _Pseudomonas_ strains from wild 38 _Lupinus_ can contribute to sustainable potato production. The B18 plus B14 consortium, when 39 combined with two-strain inoculation, demonstrated the most significant effects. These findings 40 emphasize the potential of functionally complementary microbial inoculants for the integrated 41 management of _Verticillium_ wilt and other persistent soil-borne diseases in potatoes. 42 43 **Keywords:** _Pseudomonas_ ; endophytic bacteria; _Verticillium dahliae_ ; potato; plant growth 44 promotion; biological control; microbial consortium; _Lupinus_ . 45 46 47 48 49 50 51 [bioRxiv preprint doi: https://doi.org/10.64898/2026.11.06.764942; this version posted November 9, 2026. The copyright holder for this preprint](https://doi.org/10.64898/2026.11.06.764942) (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made [available under a CC-BY-NC-ND 4.0 International license.](http://creativecommons.org/licenses/by-nc-nd/4.0/) 52 **1.** **Introduction** 53 Potato ( _Solanum tuberosum_ L.) ranks among the most globally important food crops due to its 54 high nutritional value, adaptability, and economic significance. Despite its importance for food 55 security, potato production is continually threatened by soil-borne pathogens that reduce both yield 56 and tuber quality. _Verticillium_ species, in particular, are responsible for Verticillium wilt, a 57 destructive vascular disease prevalent in potato-growing regions worldwide. Infections by 58 _Verticillium dahliae_, _V. longisporum_, and related species result in chlorosis, wilting, vascular 59 discoloration, diminished tuber yield, and substantial economic losses, especially in intensive 60 production systems (Pegg and Brady, 2002; Klosterman et al., 2009). The persistence of 61 _Verticillium_ microsclerotia in soil and the broad host range of these pathogens present significant 62 challenges for effective disease management. 63 64 Current management strategies for Verticillium wilt primarily include crop rotation, the use of 65 resistant cultivars, soil fumigation, and direct application of chemical fungicides. These methods, 66 however, frequently yield inconsistent results, can be economically burdensome, and may 67 contribute to environmental concerns due to intensive chemical use (Fradin and Thomma, 2006; 68 Depotter et al., 2016). As a result, there is increasing interest in sustainable biological alternatives 69 that support plant health and suppress pathogen development. 70 71 Plant growth-promoting rhizobacteria (PGPR) have emerged as promising agents for sustainable 72 crop production and disease management. These beneficial microorganisms enhance plant 73 performance through mechanisms such as nutrient mobilization, phytohormone production, 74 biological nitrogen fixation, siderophore generation, pathogen suppression, and stress alleviation 75 (Lugtenberg and Kamilova, 2009; Glick, 2012). Within the PGPR group, _Pseudomonas_ species 76 are notable for their metabolic versatility, environmental adaptability, efficient root colonization, 77 and capacity to suppress a wide range of plant pathogens while promoting growth (Haas and 78 Défago, 2005; Weller, 2007). Additionally, many _Pseudomonas_ species produce diverse bioactive 79 metabolites that inhibit pathogens and enhance plant performance, making them valuable for 80 agricultural biotechnologies (Mercado-Blanco and Bakker, 2007; Pieterse et al., 2014). 81 [bioRxiv preprint doi: https://doi.org/10.64898/2026.11.06.764942; this version posted November 9, 2026. The copyright holder for this preprint](https://doi.org/10.64898/2026.11.06.764942) (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made [available under a CC-BY-NC-ND 4.0 International license.](http://creativecommons.org/licenses/by-nc-nd/4.0/) 82 Recent studies increasingly identify plant endophytes as reservoirs of agriculturally beneficial 83 microorganisms. Unlike free-living rhizosphere bacteria, endophytes establish close associations 84 within plant tissues, where they can directly enhance plant nutrition, stress tolerance, and disease 85 resistance (Compant et al., 2010; Hardoim et al., 2015). Wild and unmanaged plant populations in 86 challenging environments frequently harbor microbiomes with traits associated with adaptation 87 and plant fitness. These environments represent valuable yet underexplored sources of microbial 88 resources for sustainable agriculture (Rodríguez et al., 2009; Trivedi et al., 2020). 89 90 Accumulating evidence from previous literature indicates that microbial consortia frequently 91 outperform single-strain inoculants, as diverse microorganisms can provide complementary 92 ecological functions that enhance plant performance (Bashan et al., 2014; Niu et al., 2017; Berg et 93 al., 2021). Nevertheless, it remains uncertain whether functionally complementary endophytic 94 _Pseudomonas_ strains from stress-adapted wild hosts can simultaneously promote potato growth 95 and suppress Verticillium wilt. Furthermore, the potential of these isolates to serve as sources of 96 biologically active extracellular metabolites is not well understood. This study investigates the 97 effects of such _Pseudomonas_ consortia on potato performance and disease suppression, with a 98 specific focus on their dual functional potential. 99 100 In this study, we isolated endophytic _Pseudomonas_ strains from the roots of unmanaged wild lupin 101 plants growing in challenging environments and evaluated their growth-promoting and biocontrol 102 potential. We hypothesized that stress-adapted isolates would possess traits that enhance plant 103 growth and suppress Verticillium wilt, and that combining strains with complementary functions 104 would be more effective than using single strains. To test these hypotheses, we (i) characterized 105 growth-promoting traits and antifungal activity, (ii) evaluated the biological activity of cell-free 106 supernatants, (iii) assessed effects on seed germination and seedling development, and (iv) tested 107 the individual and combined ability of these strains to promote potato growth and reduce 108 _Verticillium wilt_ under greenhouse conditions. 109 110 111 112 [bioRxiv preprint doi: https://doi.org/10.64898/2026.11.06.764942; this version posted November 9, 2026. The copyright holder for this preprint](https://doi.org/10.64898/2026.11.06.764942) (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made [available under a CC-BY-NC-ND 4.0 International license.](http://creativecommons.org/licenses/by-nc-nd/4.0/) 113 **2. Materials and methods** 114 **2.1 Plant sampling and selection of candidate endophytes** 115 Healthy wild _Lupinus_ plants were collected from the lakefront area of the Macdonald Campus of 116 McGill University, Ste. Anne de Bellevue, Québec, Canada, in December 2024. Sampling focused 117 on plants from exposed field sites that persisted during seasonal low temperatures (below 0 °C) 118 and on nutrient-limited soils. Plants thriving under such environmental stress frequently harbor 119 endophytic microorganisms with enhanced adaptive and plant-beneficial traits. Therefore, the 120 sustained vigor of _Lupinus_ spp. under these conditions suggests that their root microbiome 121 represents a promising source of stress-adapted plant growth–promoting rhizobacteria (PGPR) for 122 subsequent greenhouse and field applications. 123 **2.2 Isolation and preservation of endophytic bacteria** 124 Root samples were thoroughly washed under running tap water to remove adhering soil particles 125 and surface-sterilized by sequential immersion in 75% ethanol for 10 min, 3% sodium hypochlorite 126 for 3–5 min, and sterile distilled water six times. To verify successful removal of epiphytic 127 microorganisms, aliquots of the final rinse water were plated onto King’s B medium (King et al., 128 1954), tryptic soy agar (TSA), and nutrient agar (NA), and incubated to confirm the absence of 129 microbial growth. Sterilized root tissues were aseptically macerated in sterile phosphate-buffered 130 saline (PBS), serially diluted (10 [-1] to 10 [-3] ), and spread onto the same media. Plates were incubated 131 at 28°C for 3–5 d. Colonies differing in morphology, pigmentation, size, or texture were purified 132 by repeated (3 times) streaking to obtain axenic cultures. 133 134 Pure isolates were subsequently grown in nutrient broth at 28°C with shaking (150 rpm) for 24– 135 48 h to reach a cell density of approximately 10 [8] to 10 [9] CFU mL [-1], estimated by OD₆₀₀ 136 measurements. Broth cultures were preserved in 50% glycerol at −80°C for long-term storage. 137 To prepare bacterial inoculum for downstream analyses, cells were harvested by centrifugation 138 (6000 × _g_, 10 min), washed twice, and resuspended in sterile 10 mM MgSO₄ solution. Cell density 139 was then adjusted to approximately 10 [8 ] CFU mL [-1] using OD₆₀₀ measurements. The MgSO₄ solution 140 was used as a low-nutrient carrier to maintain osmotic stability and bacterial cell viability during 141 inoculation procedures. 142 [bioRxiv preprint doi: https://doi.org/10.64898/2026.11.06.764942; this version posted November 9, 2026. The copyright holder for this preprint](https://doi.org/10.64898/2026.11.06.764942) (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made [available under a CC-BY-NC-ND 4.0 International license.](http://creativecommons.org/licenses/by-nc-nd/4.0/) 143 **2.5 Screening of biocontrol isolates by** _**in vitro**_ **antagonistic activity against Verticillium spp.** 144 A total of 24 distinct bacterial isolates recovered from lupin root endophytes were initially 145 screened for antagonistic activity against _Verticillium_ spp. using a dual-culture inhibition assay on 146 potato dextrose agar (PDA), a widely used preliminary method for identifying microbial 147 candidates with antifungal potential (Dennis and Webster, 1971; Weller, 2007). Sterile 90-mm 148 Petri dishes containing PDA were prepared, and a sterile filter paper disc (4.5 cm diameter) was 149 placed on the agar surface to standardize inoculation positions and ensure uniform spacing among 150 treatments. 151 152 A 5-mm mycelial plug excised from the actively growing margin of the fungal colony was 153 positioned at the center of each plate. Four equidistant inoculation points, approximately 2 cm 154 from the fungal plug, were marked around the center. At each point, 10 µL of bacterial suspension 155 (approximately 1 × 10 [8] CFU mL [-1] ) was applied. Control plates received the fungal plug alone 156 without bacterial inoculation. Plates were incubated at 28°C for 5 to 7 days and observed regularly 157 for fungal growth restriction, formation of inhibition zones, and visible changes in colony 158 morphology (Fig. S1). 159 160 At the end of the incubation period, fungal colony diameter was measured in millimetres using 161 two perpendicular axes across the colony through the center of the inoculation point, and the mean 162 value was used for analysis, as commonly applied in mycelial growth inhibition assays (Pandey _et_ 163 _al._, 1982; Dennis and Webster, 1971). Percentage inhibition of radial growth was calculated 164 according to the formula: 165 166 Inhibition (%) = [(fungal colony diameter in control (C) − fungal colony diameter in dual culture 167 (T) / fungal colony diameter in control (C)] × 100 168 169 Where **C** is the mean colony diameter of the fungal control plate, and **T** is the mean colony 170 diameter of the fungus in dual culture treatment (Vincent, 1947). 171 172 Each treatment was performed in triplicate using three independent biological replicates. Isolates 173 were comparatively ranked based on qualitative observations, such as clear suppression zones, [bioRxiv preprint doi: https://doi.org/10.64898/2026.11.06.764942; this version posted November 9, 2026. The copyright holder for this preprint](https://doi.org/10.64898/2026.11.06.764942) (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made [available under a CC-BY-NC-ND 4.0 International license.](http://creativecommons.org/licenses/by-nc-nd/4.0/) 174 restricted mycelial expansion, and altered fungal growth patterns, as well as quantitative inhibition 175 percentages. 176 177 Isolates that consistently exhibited the strongest and most reproducible antagonistic activity 178 against _Verticillium spp_ . were selected for further investigation. These isolates underwent 179 functional characterization of plant growth–promoting traits, seed bioassays, molecular 180 identification, and greenhouse evaluation of biological control efficacy in potato. 181 **2.4 Functional screening for plant growth–promoting traits** 182 The isolates identified in the dual culture assay as having the most antagonistic potential were 183 further evaluated for multiple plant growth–promoting (PGP) traits using established standard 184 biochemical assays. These assays were selected to assess functional mechanisms associated with 185 nutrient acquisition, phytohormone production, micronutrient mobilization, and pathogen 186 suppression. Appropriate positive and negative controls were included in each assay to ensure 187 reliability and consistency of interpretation (Fig. S2). 188 189 Indole-3-acetic acid (IAA) production was assessed qualitatively and quantitatively following the 190 method of Gordon and Weber (1951) with modifications. Isolates were grown in tryptic soy broth 191 (TSB) supplemented with L-tryptophan (0.5 to 1 g L [-1] ) at 28°C for 48 h. Cultures were then 192 centrifuged, and 1 mL of supernatant was mixed with 2 mL of Salkowski reagent. Development 193 of pink coloration indicated IAA production. Quantitative estimation was performed by measuring 194 absorbance at 530 nm and calculating IAA concentration from a standard curve (Patten and Glick, 195 2002). 196 197 Phosphate solubilization ability was evaluated on Pikovskaya’s agar containing insoluble 198 tricalcium phosphate (Pikovskaya, 1948). Bacterial cultures were spot-inoculated and incubated at 199 28°C for 5 to 7 days. The formation of clear halo zones around colonies indicated the solubilization 200 of inorganic phosphate. Solubilization efficiency was expressed as the phosphate solubilization 201 index (PSI), calculated as the ratio of total diameter (colony + halo zone) to colony diameter. 202 [bioRxiv preprint doi: https://doi.org/10.64898/2026.11.06.764942; this version posted November 9, 2026. The copyright holder for this preprint](https://doi.org/10.64898/2026.11.06.764942) (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made [available under a CC-BY-NC-ND 4.0 International license.](http://creativecommons.org/licenses/by-nc-nd/4.0/) 203 Nitrogen fixation-associated activity was evaluated qualitatively using a nitrogen-free 204 bromothymol blue (NFb) semi-solid medium, as described by Döbereiner (1995). Isolates were 205 inoculated into NFb medium and incubated at 28°C for 3 to 5 days. Development of bacterial 206 growth, accompanied by a colour change from green to blue, was interpreted as evidence of growth 207 under nitrogen-limited conditions and of ammonia production associated with potential nitrogen 208 fixing capability. 209 210 Because this assay provides only preliminary qualitative evidence, results are reported as nitrogen 211 fixation-associated activity rather than direct confirmation of biological nitrogen fixation. But 212 definitive confirmation of nitrogen fixation might require further assay like acetylene reduction, 213 15 N isotope incorporation experiments, or molecular characterization of nitrogenase-related genes. 214 215 Hydrogen cyanide (HCN) production, an important trait linked to suppression of soil-borne 216 pathogens, was assessed following Bakker and Schippers (1987). Isolates were grown on TSB agar 217 supplemented with glycine, and filter paper impregnated with picric acid solution was placed in 218 the lid of each plate. A colour change from yellow to brown after 2 to 4 days of incubation indicated 219 HCN production. 220 221 Siderophore production was evaluated using Chrome Azurol S (CAS) agar, as described by 222 Schwyn and Neilands (1987). The formation of orange halos surrounding colonies indicated the 223 secretion of iron-chelating siderophores. 224 225 These assays aimed to provide preliminary functional characterization of the isolates and to 226 identify traits commonly associated with plant growth promotion and biological control. The 227 assays were designed to demonstrate functional potential, not to quantify the contribution of 228 individual mechanisms under greenhouse conditions. 229 **2.5 Seed germination bioassays** 230 Tomato seeds served as a rapid and standardized model system to evaluate the effects of bacterial 231 inoculation on seed germination and early seedling development. Both tomato and potato are 232 members of the _Solanaceae_ family and share similar physiological characteristics during early [bioRxiv preprint doi: https://doi.org/10.64898/2026.11.06.764942; this version posted November 9, 2026. The copyright holder for this preprint](https://doi.org/10.64898/2026.11.06.764942) (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made [available under a CC-BY-NC-ND 4.0 International license.](http://creativecommons.org/licenses/by-nc-nd/4.0/) 233 development. Unlike potato, which is propagated from nutrient-rich tubers that may obscure 234 microbial effects during establishment, tomato seeds offer a sensitive and uniform system for 235 detecting growth-promoting responses. Additionally, tomato seeds germinate rapidly and 236 synchronously under controlled conditions, making them suitable for preliminary screening of 237 plant growth-promoting microorganisms prior to validation in the target crop. 238 239 Surface-sterilized seeds were placed on sterile filter paper in Petri dishes and treated with bacterial 240 suspensions adjusted to approximately 10 [5], 10 [6], or 10 [7] CFU mL [-1] . Sterile 10 mM MgSO 4 served 241 as the carrier control. Each treatment consisted of four biological replicates, with 25 seeds per Petri 242 dish. Each Petri dish was considered a single experimental unit. 243 244 Seeds were incubated under controlled growth conditions, and germination was recorded daily for 245 three days. A seed was considered germinated when visible radicle emergence occurred. 246 Germination percentage was calculated on a per-plate basis. Germination speed was assessed using 247 Maguire’s Germination Index (Maguire, 1962): 248 GRI = Σ(n i /t i ) 249 where n i represents the number of newly germinated seeds at time tᵢ. 250 **2.6 Cell-free supernatant assays** 251 To determine whether extracellular metabolites contributed to the observed plant responses 252 independently of bacterial colonization, cell-free supernatants (CFS) were prepared from liquid 253 cultures of the selected isolates. Cultures were grown at 28°C for 24 to 48 hours, centrifuged at 254 10,000 × g for 10 min, and filtered through sterile 0.22 μm membranes to remove bacterial cells. 255 256 Supernatants were diluted with sterile distilled water to final concentrations of 2.5, 5, and 10% 257 (v/v). Two controls were included: (i) sterile distilled water and (ii) uninoculated sterile culture 258 medium processed identically to the CFS preparations. These controls enabled discrimination 259 between the effects of bacterial metabolites and residual medium components. 260 261 262 [bioRxiv preprint doi: https://doi.org/10.64898/2026.11.06.764942; this version posted November 9, 2026. The copyright holder for this preprint](https://doi.org/10.64898/2026.11.06.764942) (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made [available under a CC-BY-NC-ND 4.0 International license.](http://creativecommons.org/licenses/by-nc-nd/4.0/) 263 **2.8 Molecular identification and phylogenetic analysis** 264 Forward and reverse partial 16S rRNA gene sequences of the selected lupin endophytic isolates 265 were obtained by Sanger sequencing at Genome Québec following submission of pure bacterial 266 culture samples. Raw reads were quality filtered, trimmed, and assembled into consensus 267 sequences. Closely related type and reference sequences were retrieved from NCBI for 268 comparative analysis. 269 270 Multiple sequence alignment was performed using the R package msa (Bodenhofer et al., 2015). 271 Phylogenetic relationships were inferred using maximum likelihood in phangorn (Schliep, 2011) 272 under the GTR+G+I substitution model. Branch support was assessed using 200 bootstrap 273 replicates. Trees were rooted using _Azotobacter vinelandii_ and visualized using ggtree (Yu et al., 274 2017) and iTOLV6 (Letunic and Bork 2024). 275 **2.9 Greenhouse evaluation of biocontrol efficacy against** _**Verticillium dahliae**_ 276 **2.9.1 Experimental design and treatments** 277 Greenhouse experiments were conducted using Agromix substrate in a randomized complete block 278 design (RCBD) to evaluate the preventive biocontrol efficacy of selected plant growth–promoting 279 rhizobacteria (PGPR) against _Verticillium dahliae_ in potato. The experiment consisted of eight 280 treatment combinations, each with eight biological replicates, resulting in a total of 64 281 experimental units (pots). Experimental units were arranged into blocks, with each block 282 containing all treatment combinations randomly assigned using a random number generator to 283 minimize positional effects and account for environmental variability within the greenhouse. 284 285 The potato cultivar Russet Burbank, known to be susceptible to _Verticillium_ wilt and commonly 286 used in disease screening studies, was selected to ensure consistent disease development and 287 maximize sensitivity for detecting treatment effects. 288 289 Based on the preliminary screening results, strain B18 was designated PGPR A and strain B14 was 290 designated PGPR B. The greenhouse experiment followed a 4 × 2 factorial arrangement, consisting 291 of four PGPR treatments [no PGPR (control), PGPR A (B18), PGPR B (B14), and PGPR A + B 292 (B18 + B14)] and two pathogen levels [absence or presence of _Verticillium dahliae_ ]. The [bioRxiv preprint doi: https://doi.org/10.64898/2026.11.06.764942; this version posted November 9, 2026. The copyright holder for this preprint](https://doi.org/10.64898/2026.11.06.764942) (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made [available under a CC-BY-NC-ND 4.0 International license.](http://creativecommons.org/licenses/by-nc-nd/4.0/) 293 combination of these factors generated eight treatment combinations: (i) healthy control, (ii) PGPR 294 A only, (iii) PGPR B only, (iv) PGPR A + B only, (v) pathogen only, (vi) PGPR A + pathogen, 295 (vii) PGPR B + pathogen, and (viii) PGPR A + B + pathogen. The healthy control received a sterile 296 10 mM MgSO 4 carrier solution supplemented with 0.25% carboxymethyl cellulose (CMC) and no 297 bacterial inoculum, whereas pathogen-free PGPR treatments received only a bacterial inoculum. 298 Pathogen treatments received _V. dahliae_ inoculation either alone or in combination with the 299 corresponding PGPR treatment. 300 301 This treatment structure allowed for the separation of direct plant growth–promoting effects under 302 healthy conditions from disease-protective effects under pathogen pressure. It also enabled 303 evaluation of potential additive or synergistic effects resulting from combined inoculation. 304 305 **2.9.2 Preparation and application of bacterial inoculum** 306 Healthy potato tubers were selected, cut into uniform cubes (~ 5g), surface-sterilized, and air-dried 307 prior to treatment to minimize background microbial interference. Bacterial inoculum was 308 prepared and adjusted to a cell concentration of approximately 10⁸ CFU mL⁻¹ in sterile 10 mM 309 MgSO 4 buffer, which served as a low-nutrient carrier to maintain osmotic stability and cell 310 viability without introducing excess nutrients (Bashan _et al._, 2014). The PGPR A+B consortium 311 was prepared by cultivating each strain (B18 and B14) separately, standardizing the resulting cell 312 suspensions to an equivalent cell density, and subsequently combining equal volumes of the two 313 suspensions immediately prior to inoculation. This method produced a dual-strain consortium of 314 B18 and B14 at a 1:1 ratio, aligning with established PGPR consortium inoculation protocols in 315 which individually prepared strains are standardized and combined before application (Hashmi et 316 al., 2019; Khan et al., 2022; Petrillo et al., 2022). 317 318 Tubers assigned to bacterial treatments were inoculated by immersion in the respective suspension 319 for 1 h. The inoculum suspension contained 0.25% carboxymethyl cellulose (CMC) as a sticker to 320 improve bacterial adhesion and facilitate early colonization of the tuber surface. 321 322 To enhance rhizosphere establishment, a second application of bacterial inoculum was delivered 323 as a soil drench on Day +3 after planting (10 mL pot [-1] at approximately 10 [8] CFU mL [-1] ). [bioRxiv preprint doi: https://doi.org/10.64898/2026.11.06.764942; this version posted November 9, 2026. The copyright holder for this preprint](https://doi.org/10.64898/2026.11.06.764942) (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made [available under a CC-BY-NC-ND 4.0 International license.](http://creativecommons.org/licenses/by-nc-nd/4.0/) 324 325 **2.9.3 Plant nutrition and irrigation management** 326 A minimal hydroponic nutrient input consisting of stock solutions A and B was applied during the 327 vegetative stage at approximately 0.0000625% (v/v; ~0.6 ppm) to maintain baseline plant nutrition 328 while minimizing interference with microbial interactions. Solution A primarily contained calcium 329 nitrate and iron chelate, whereas Solution B contained phosphate, magnesium sulphate, potassium 330 salts, and trace micronutrients (B, Mn, Zn, Cu, and Mo). 331 332 Prior to pathogen inoculation and throughout the experiment, all pots received autoclaved distilled 333 water as needed to maintain consistent soil moisture across treatments. 334 335 **2.9.4 Preparation and application of pathogen inoculum** 336 The pathogen inoculum consisted of _V. dahliae_ conidia harvested from potato dextrose agar (PDA) 337 cultures using sterile distilled water supplemented with Tween-20 to facilitate spore release. The 338 suspension was filtered to remove mycelial fragments and quantified using a hemocytometer under 339 light microscopy (Lievens _et al._, 2006). Spore concentration was adjusted to approximately 10⁶ 340 conidia mL⁻¹. 341 342 To improve infection consistency and better simulate natural inoculum pressure, actively growing 343 fungal mycelial plugs (5 mm diameter) were also introduced into the soil near the root zone. 344 Pathogen inoculation was performed approximately one week after plant emergence, when 345 seedlings were 1 to 2 cm tall and had reached a uniform, susceptible growth stage. This time point 346 was considered the baseline for Verticillium infection assessment. Inoculum was applied as a soil 347 drench (10 mL pot [-1] ) to allow direct contact between fungal propagules and the rhizosphere. 348 349 **2.9.5 Greenhouse Conditions** 350 Plants were maintained under controlled greenhouse conditions of 22/17°C Day/night temperature, 351 a 16 hours photoperiod, and 60–70% relative humidity. Irrigation was adjusted as required to 352 maintain uniform moisture among all treatments. 353 354 **2.9.6 Plant growth and disease assessments** [bioRxiv preprint doi: https://doi.org/10.64898/2026.11.06.764942; this version posted November 9, 2026. The copyright holder for this preprint](https://doi.org/10.64898/2026.11.06.764942) (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made [available under a CC-BY-NC-ND 4.0 International license.](http://creativecommons.org/licenses/by-nc-nd/4.0/) 355 Plant emergence was recorded during establishment and expressed as emergence percentage per 356 treatment using the formula: 357 Emergence (%) = (number of emerged pots / 8) × 100 358 359 Because each planted tuber piece was capable of producing multiple shoots, the total number of 360 shoots emerging per seed tuber was also recorded as an indicator of sprouting vigour and stem 361 multiplication. 362 Vegetative growth parameters, including shoot height (cm) and total leaf number per experimental 363 unit, were measured at emergence (0 dpi) and subsequently at 7, 14, 21, and 28 days post 364 inoculation (dpi). Shoot height was measured from the substrate surface to the apex of the tallest 365 stem using a ruler, while leaf number included all fully expanded leaves arising from each planted 366 tuber. 367 Leaf greenness, used as an indirect indicator of chlorophyll status and physiological performance, 368 was measured non-destructively using a SPAD chlorophyll meter (SPAD-502, Konica Minolta, 369 Japan or equivalent) on the youngest fully expanded leaves. Total leaf area was determined at 370 harvest using a portable leaf area meter (CI-202/CI-203 series, CID Bio-Science, USA), which 371 measures projected leaf surface area (cm [2] ) photometrically. 372 373 **2.9.7 Disease severity and final harvest measurements** 374 Disease severity was visually assessed at 7, 14, 21, and 28 days post-pathogen inoculation (dpi) 375 using a standardized 0–5 wilt severity scale based on foliar chlorosis, wilting, necrosis, and canopy 376 collapse, where: 0 = asymptomatic healthy plant; 1 = 1–25% foliage affected; 2 = 26–50% affected 377 with slight stunting; 3 = 51–75% affected with moderate wilting and necrosis; 4 = >75% affected 378 with severe wilt and chlorosis; and 5 = plant collapse or near death. To ensure scoring consistency, 379 all assessments were performed by the same evaluator. 380 381 Disease progression over time was summarized as the Area Under the Disease Progress Curve 382 (AUDPC), as described by Shaner and Finney (1977). At final harvest, plants were carefully 383 removed from the substrate and separated into shoots, roots, and tubers. Fresh biomass was 384 recorded immediately, then oven-dried to a constant weight for dry biomass determination. 385 Flowering incidence was determined by scoring each pot as flowering (1) or non-flowering (0) [bioRxiv preprint doi: https://doi.org/10.64898/2026.11.06.764942; this version posted November 9, 2026. The copyright holder for this preprint](https://doi.org/10.64898/2026.11.06.764942) (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made [available under a CC-BY-NC-ND 4.0 International license.](http://creativecommons.org/licenses/by-nc-nd/4.0/) 386 based on the presence of at least one visible inflorescence. Flowering incidence was expressed as 387 the percentage of pots that flowered within each treatment group. Tuber bulking response was 388 expressed as the number of tubers produced per seed tuber planted. Root system architecture, 389 including total root length, root surface area, root volume, and average root diameter, was 390 quantified using a flatbed scanner coupled with WinRHIZO image analysis software (Regent 391 Instruments Inc., Canada). 392 393 Collectively, these measurements quantified the effects of PGPR treatments on plant 394 establishment, growth promotion, physiological status, yield formation, root development, and 395 suppression of _Verticillium_ wilt. 396 397 **2.10 Statistical Analysis** 398 All statistical analyses were performed using R version 4.3.0 (R Core Team, 2023). Data were 399 visually inspected and evaluated for normality and homogeneity of variance using residual 400 diagnostic plots, Shapiro–Wilk tests, and Levene’s tests. When necessary, proportional data were 401 arcsine square-root transformed prior to analysis to satisfy model assumptions. 402 403 Greenhouse experiments were analyzed using analysis of variance (ANOVA) within a randomized 404 complete block design (RCBD), with treatment considered a fixed effect and block included as a 405 random factor. Variables measured at a single time point were analyzed using one-way ANOVA. 406 For variables measured repeatedly through time, observations were analyzed separately at each 407 sampling date to facilitate biological interpretation of treatment responses. Seed germination and 408 cell-free supernatant (CFS) experiments were analyzed using one-way ANOVA for each strain 409 and treatment group. 410 411 When significant treatment effects were detected ( _P_ < 0.05), pairwise comparisons among means 412 were performed using Tukey’s honestly significant difference (HSD) test at an experiment-wise 413 significance level of α = 0.05. Adjusted _P_ -values generated by Tukey’s multiple-comparison 414 procedure were used to control the family-wise error rate associated with multiple testing. 415 Treatment means sharing the same letter were not significantly different at _P_ ≥ 0.05, whereas [bioRxiv preprint doi: https://doi.org/10.64898/2026.11.06.764942; this version posted November 9, 2026. The copyright holder for this preprint](https://doi.org/10.64898/2026.11.06.764942) (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made [available under a CC-BY-NC-ND 4.0 International license.](http://creativecommons.org/licenses/by-nc-nd/4.0/) 416 means with different letters were considered significantly different at _P_ < 0.05. Estimated marginal 417 means (EMMeans) and corresponding 95% confidence intervals were calculated using the 418 emmeans package. Compact letter displays (CLD) were generated to summarize multiple 419 comparison results. 420 421 All figures were prepared using ggplot2 (Wickham, 2016). Results are presented as means ± 422 standard error (SE) or means with 95% confidence intervals (CI), as indicated in the respective 423 figures and tables. Statistical significance was declared at _P_ < 0.05. 424 425 The R scripts used for data processing, statistical analyses, and figure generation are publicly 426 [available on GitHub (https://github.com/numanibneasad/potato-pgpr-b18-b14), whereas the](https://github.com/numanibneasad/potato-pgpr-b18-b14) 427 experimental datasets, supplementary statistical summaries, and supporting files associated with 428 [this study are available through Zenodo ( https://zenodo.org/records/20542212?preview=1](https://zenodo.org/records/20542212?preview=1) ). 429 430 431 **Results** 432 433 **3.1 Isolation and selection of candidate endophytic bacteria** 434 Endophytic bacteria were isolated from surface-sterilized root tissues of wild _Lupinus_ plants 435 collected under cold-stress field conditions. The absence of microbial growth from the final rinse 436 water plated on both selective and non-selective media confirmed effective surface sterilization 437 and indicated the internal origin of the recovered isolates. Initial antagonistic screening against 438 _Verticillium_ spp. identified four candidate isolates with measurable inhibitory activity. Among 439 these, isolates B14 and B18 consistently demonstrated the most promising characteristics and were 440 selected for further investigation. Both isolates maintained stable growth during repeated 441 subculturing and exhibited strong plant growth–promoting and biocontrol potential, justifying 442 their selection for detailed characterization and greenhouse evaluation. 443 444 **3.3** _**In vitro**_ **suppression of** _**Verticillium**_ **spp.** [bioRxiv preprint doi: https://doi.org/10.64898/2026.11.06.764942; this version posted November 9, 2026. The copyright holder for this preprint](https://doi.org/10.64898/2026.11.06.764942) (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made [available under a CC-BY-NC-ND 4.0 International license.](http://creativecommons.org/licenses/by-nc-nd/4.0/) 445 Building on the observed pathogen suppression traits, both isolates were next evaluated for their 446 ability to inhibit mycelial growth of _Verticillium_ species (Table 2). 447 448 Both isolates significantly inhibited mycelial growth of all tested _Verticillium_ species (Table 2). 449 Against _V. dahliae,_ B14 caused 48.2% inhibition, compared with 37.4% for B18. Similar trends 450 were observed for _V. longisporum_, where B14 and B18 reduced fungal growth by 55.8% and 451 46.0%, respectively. 452 453 Against _V. albo-atrum_, B14 again produced greater suppression (43.2%) than B18 (32.9%). Across 454 all pathogens tested, B14 consistently outperformed B18 in direct antifungal activity. 455 These data indicate that both strains possess broad-spectrum antagonistic potential, with B14 456 showing stronger direct suppression of _Verticillium_ spp. under _in vitro_ conditions. This led to 457 further study of their effects on plant physiological processes. 458 459 **Table1:** _**In vitro**_ antagonistic activity of endophytic _Pseudomonas_ strains B18 and B14 against three _Verticillium_ pathogens in dual 460 culture assays. Values represent mean fungal radial growth (mm) from three biological replicates measured after 2 weeks of 461 incubation. Different letters indicate significant differences within each pathogen according to Tukey’s HSD test ( _P_ < 0.05). Dash 462 (—) indicates reference treatment or not applicable. **Control** **growth (mm)** **Inhibition** **(%)** **Significance vs** **group** **Growth** **reduction (mm)** **Treat** **Pathogen** **ment** **Radial** **growth (mm)** **Tukey** _**p**_ **-value** **control** **rou** _V. dahliae_ B18 23.74 ± SE 37.95 14.21 37.44 ** 0.0034 b _V. dahliae_ B14 19.66 ± SE 37.95 18.29 48.19 *** 0.0004 b _V. dahliae_ Control 37.95 ± SE 37.95 0.00 0.00 — — a _V. longisporum_ B18 22.00 ± SE 40.74 18.74 46.00 *** 0.0005 b _V. longisporum_ B14 18.00 ± SE 40.74 22.74 55.82 *** <0.0001 b _V. longisporum_ Control 40.74 ± SE 40.74 0.00 0.00 — — a _V. albo-atrum_ B18 26.00 ± SE 38.74 12.74 32.89 ** 0.0014 b _V. albo-atrum_ B14 22.00 ± SE 38.74 16.74 43.21 *** 0.0001 b _V. albo-atrum_ Control 38.74 ± SE 38.74 0.00 0.00 — — a 463 464 465 **3.2 Plant growth–promoting functional traits** 466 467 The two isolates, B14 and B18, were further tested for plant-beneficial traits, with each displaying 468 distinct functional profiles (Table 1). Strains B18 and B14 significantly solubilized inorganic [bioRxiv preprint doi: https://doi.org/10.64898/2026.11.06.764942; this version posted November 9, 2026. The copyright holder for this preprint](https://doi.org/10.64898/2026.11.06.764942) (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made [available under a CC-BY-NC-ND 4.0 International license.](http://creativecommons.org/licenses/by-nc-nd/4.0/) 469 phosphate, with phosphate solubilization index (PSI) values of 2.30 and 2.10, respectively, 470 compared with the non-inoculated control. No significant difference was detected between the two 471 strains. 472 473 **Table2.** Screening of plant growth–promoting (PGP) traits of bacterial isolates B18 and B14. Key plant growth–promoting traits 474 of B18 and B14 were assessed both qualitatively and quantitatively. Indole-3-acetic acid (IAA) production was quantified 475 spectrophotometrically (µg mL⁻¹). Phosphate solubilization efficiency is presented as the phosphate solubilization index (PSI), 476 based on halo development on Pikovskaya’s agar. Nitrogen fixation, hydrogen cyanide (HCN) production, and siderophore 477 production were evaluated qualitatively and are shown as either present (+) or absent (−). All measurements were under controlled 478 laboratory conditions. Values represent mean observations across (n=3) biological replicates. 479 Isolate PSI CLD IAA (µg mL [-1] ) CLD Nitrogen Fixation HCN CAS Strain B14 2.10 a 0.18 b +++ ++ ++ Strain B18 2.30 a 0.25 a +++ +++ +++ Control 1.00 b 0.06 c – – – 480 481 Quantitative evaluation revealed that IAA production differed significantly among treatments. 482 Strain B18 produced the highest IAA concentration (0.25 µg mL⁻¹), followed by B14 (0.18 µg 483 mL⁻¹), whereas the control showed only negligible background levels (0.06 µg mL [-1] ). 484 Both isolates showed strong nitrogen fixation (+++). Strain B18 showed higher HCN and 485 siderophore activity (+++) than B14 (++), suggesting greater potential for pathogen suppression 486 and micronutrient mobilization. 487 Overall, the findings demonstrate that B14 and B18 possess complementary functional 488 characteristics. B14 exhibits stronger antifungal activity, whereas B18 demonstrates greater plant 489 growth–promoting capacity. Therefore, these two isolates were selected for subsequent seed 490 germination, cell-free supernatant, and greenhouse experiments. 491 **3.2 Seed germination and seed vigour responses to bacterial inoculation** 492 Seed germination responded strongly to bacterial inoculation in a strain-dependent manner (Fig. 493 1). Overall, strain B18 exhibited substantially greater germination-promoting activity than strain [bioRxiv preprint doi: https://doi.org/10.64898/2026.11.06.764942; this version posted November 9, 2026. The copyright holder for this preprint](https://doi.org/10.64898/2026.11.06.764942) (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made [available under a CC-BY-NC-ND 4.0 International license.](http://creativecommons.org/licenses/by-nc-nd/4.0/) 494 B14, enhancing both germination speed and final germination percentage across inoculum 495 concentrations. 496 For strain B18, germination percentage was significantly influenced by inoculum concentration 497 during the early stages of germination (Day 1: F 3,12 = 15.49, _P_ < 0.001; Day 2: F 3,12 = 8.86, _P_ = 498 0.002). On Day 1, seeds treated with B18 reached germination levels ranging from approximately 499 70% to 92%, compared with approximately 60% in the MgSO 4 control. By Day 2, germination 500 increased to approximately 85 to 98% in inoculated treatments, whereas the control remained near 501 75%. At the final assessment (Day 3), all B18 treatments achieved near-complete germination (93– 502 100%), exceeding the control (approximately 92–94%). However, treatment differences were no 503 longer statistically significant at this stage (F 3,12 = 2.83, _P_ = 0.083), indicating convergence toward 504 maximum germination. Germination speed, estimated using Maguire’s Germination Index (MGI), 505 was significantly affected by B18 inoculation (F 3,12 = 17.21, _P_ < 0.001), with the highest MGI 506 observed at 10 [7] CFU mL [-1] . According to Tukey’s test, only the highest inoculum concentration 507 differed significantly from the control and lower concentrations, indicating a dose-dependent 508 enhancement of germination vigour. 509 In contrast, strain B14 produced a weaker but concentration-dependent stimulatory effect. 510 Germination percentage did not differ significantly among treatments on Day 1 (F 3,12 = 2.27, _P_ = 511 0.133), when little or no germination was observed in either the control or most inoculated 512 treatments. By Day 2, germination increased to approximately 24 to 38% for B14-treated seeds 513 compared with approximately 10% for the control, with treatment effects approaching significance 514 (F 3,12 = 3.48, _P_ = 0.050). At the final assessment (Day 3), germination reached approximately 36 515 to 55% across inoculum concentrations, with the highest response observed at 10 [6] CFU mL⁻¹ 516 (approximately 55%) compared with only 22% in the control. Treatment effects were significant 517 at this stage (F 3,12 = 4.58, _P_ = 0.023), demonstrating that B14 improved final germination despite 518 producing a weaker response than B18. Similarly, MGI differed significantly among B14 519 treatments (F 3,12 = 4.21, _P_ = 0.030), with the greatest increase occurring at 10 [6] CFU mL [-1], while 520 the remaining inoculum concentrations produced intermediate responses. 521 Collectively, these results demonstrate that both endophytic _Pseudomonas_ strains enhanced seed 522 vigour relative to the MgSO 4 control, but their effectiveness differed substantially. Strain B18 [bioRxiv preprint doi: https://doi.org/10.64898/2026.11.06.764942; this version posted November 9, 2026. The copyright holder for this preprint](https://doi.org/10.64898/2026.11.06.764942) (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made [available under a CC-BY-NC-ND 4.0 International license.](http://creativecommons.org/licenses/by-nc-nd/4.0/) 523 accelerated germination, increased final germination percentage, and produced the highest MGI 524 values, indicating superior stimulation of early seed physiological processes. In contrast, B14 525 generated a more moderate and concentration-dependent response. The stronger performance of 526 B18 during seed germination is consistent with its greater phosphate-solubilizing capacity and 527 higher indole-3-acetic acid production, suggesting a stronger ability to promote early seedling 528 establishment. To determine whether extracellular metabolites contributed to these responses, 529 additional assays were subsequently conducted using cell-free supernatants from each strain. 530 531 532 533 534 535 536 537 538 539 540 541 **Figure 1.** **Effect of endophytic** _**Pseudomonas**_ **strains on tomato seed germination and germination speed.** 542 Cumulative germination percentage and Maguire germination index (MGI) of tomato seeds treated with _P. sp._ 543 strain_B18 and _P. sp._ strain_B14 at Control, 10 [5], 10 [6], and 10 [7 ] CFU mL [-1] . The control treatment contained sterile 10 544 mM MgSO 4 only. Values represent mean ± 95% confidence intervals from four biological replicates, with 25 seeds 545 per replicate plate. Different letters indicate significant differences among inoculum levels within each strain and day 546 according to Tukey’s HSD test ( _P_ < 0.05). 547 548 549 550 [bioRxiv preprint doi: https://doi.org/10.64898/2026.11.06.764942; this version posted November 9, 2026. The copyright holder for this preprint](https://doi.org/10.64898/2026.11.06.764942) (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made [available under a CC-BY-NC-ND 4.0 International license.](http://creativecommons.org/licenses/by-nc-nd/4.0/) # 551 3.5 Effects of cell-free supernatants on seed germination and radicle development 552 To determine whether extracellular metabolites contributed to the observed seed responses, sterile 553 cell-free supernatants (CFS) obtained from strains B18 and B14 were evaluated in the absence of 554 live bacterial cells. Supernatants were tested at 2.5, 5, and 10% (v/v), corresponding to 25, 50, and 555 100 µL mL [-1], respectively. Water and uninoculated sterile growth medium controls were included 556 to distinguish metabolite-mediated effects from possible nutrient carryover. 557 The CFS derived from strain B18 significantly affected both germination percentage (F 6,22 = 3.68, 558 _P_ = 0.011) and radicle length (F 6,11 = 6.42, _P_ = 0.004) (Supplementary Fig. S3). Final germination 559 increased from approximately 86% in the water control to 92% following treatment with 2.5% 560 B18-CFS, representing the highest germination response observed among all treatments. 561 Germination remained high at 5 and 10% CFS (approximately 90 to 91%), indicating that maximal 562 stimulation occurred at the lowest concentration tested. Radicle growth exhibited a similar trend, 563 increasing from approximately 5.6 cm in the control to 6.5 cm at 2.5% B18-CFS. However, radicle 564 elongation declined at higher concentrations, reaching approximately 5.2 cm at 5% CFS and 565 approximately 4.5 cm at 10% CFS. According to Tukey’s multiple comparison test, the 2.5% B18 566 CFS treatment consistently produced the strongest stimulatory response, suggesting that 567 extracellular metabolites released by strain B18 directly promote seed germination and early root 568 development, with maximal activity occurring at relatively low concentrations. 569 In contrast, the CFS derived from strain B14 also significantly influenced germination percentage 570 (F 6,22 = 3.70, _P_ = 0.011) and radicle length (F 6,22 = 11.26, _P_ < 0.001), although the overall biological 571 response differed markedly from that of B18. Germination percentages ranged from 63 to 75% 572 across B14-CFS treatments, values that were comparable to or lower than the water control (77%). 573 Radicle responses were strongly concentration dependent. While 2.5% B14-CFS moderately 574 stimulated radicle elongation (approximately 5.6 cm), increasing the concentration substantially 575 reduced root growth, with radicle length declining to approximately 2.6 cm at 5% CFS and being 576 completely inhibited at 10% CFS. These findings indicate that metabolites present in B14-CFS 577 may exert inhibitory effects at elevated concentrations. [bioRxiv preprint doi: https://doi.org/10.64898/2026.11.06.764942; this version posted November 9, 2026. The copyright holder for this preprint](https://doi.org/10.64898/2026.11.06.764942) (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made [available under a CC-BY-NC-ND 4.0 International license.](http://creativecommons.org/licenses/by-nc-nd/4.0/) 578 The uninoculated medium controls did not consistently improve germination or radicle 579 development, confirming that the positive responses observed with B18-CFS were primarily 580 attributable to bacterial secreted metabolites rather than residual nutrients in the growth medium. 581 Collectively, these results demonstrate that strain B18 promotes seed germination and early root 582 growth through soluble extracellular compounds, whereas the beneficial effects associated with 583 strain B14 are likely dependent on live-cell colonization, rhizosphere interactions, or other indirect 584 plant–microbe processes that are not reproduced by cell-free supernatants alone. 585 586 **3.6 Phylogenetic identification of strains B14 and B18** 587 Maximum-likelihood analysis of partial 16S rRNA gene sequences placed both isolates within the 588 genus _Pseudomonas_ (Fig. 2). Strain B14 clustered most closely with _Pseudomonas marginalis_ and 589 _P. rhodesiae_ and was further associated with related environmental species including _P. arenae_ 590 and _P. grimontii_ . Bootstrap support within this clade ranged from moderate to strong (51–100%), 591 supporting a stable phylogenetic placement of B14 among plant-associated and environmental 592 _Pseudomonas_ taxa. 593 Strain B18 formed a separate lineage associated with _Pseudomonas reinekei_, _P. jessenii_, _P._ 594 _vancouverensis_, _P. uzonensis_, and closely related species. Bootstrap support within this cluster 595 ranged from low to strong (25 -100%), reflecting the close evolutionary relationships among these 596 taxa and the limited species-level resolution provided by partial 16S rRNA gene sequences. 597 Neither isolate clustered with the pathogenic reference species _Pseudomonas syringae_ or _P._ 598 _aeruginosa_, which formed distinct branches outside the isolate-associated clades. The 599 phylogenetic analysis therefore supports the classification of B14 and B18 as environmental or 600 plant-associated _Pseudomonas_ spp., consistent with their observed plant growth–promoting and 601 biocontrol characteristics. 602 603 [bioRxiv preprint doi: https://doi.org/10.64898/2026.11.06.764942; this version posted November 9, 2026. The copyright holder for this preprint](https://doi.org/10.64898/2026.11.06.764942) (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made [available under a CC-BY-NC-ND 4.0 International license.](http://creativecommons.org/licenses/by-nc-nd/4.0/) 604 605 606 607 608 . 609 610 611 612 613 614 615 616 617 618 619 620 621 **Figure 2 (2.1, 2.2).** Maximum-likelihood phylogenetic trees based on partial 16S rRNA gene sequences showing the 622 placement of strains B14 and B18 among closely related _Pseudomonas_ reference species. Trees were inferred under 623 the GTR+G+I model with bootstrap support indicated by node symbols. _Azotobacter vinelandii_ was used as the 624 outgroup. Strains B14 and B18 are highlighted in red. 625 **3.7 Greenhouse evaluation against** _**Verticillium dahliae**_ 626 **3.7.1 Plant establishment and vegetative growth** 627 Plant emergence remained consistently high in both the absence and presence of _Verticillium_ 628 _dahliae_, indicating successful tuber establishment across all treatments and demonstrating that 629 PGPR inoculation did not adversely affect early emergence (Fig. 3A). 630 Vegetative growth was significantly affected by treatment, particularly under pathogen challenge. 631 Final shoot height differed significantly among treatments in both pathogen-free (F 3,21 = 10.23, _P_ 632 < 0.001) and pathogen-challenged plants (F 3,21 = 8.90, _P_ < 0.001). In the presence of _V. dahliae_, [bioRxiv preprint doi: https://doi.org/10.64898/2026.11.06.764942; this version posted November 9, 2026. The copyright holder for this preprint](https://doi.org/10.64898/2026.11.06.764942) (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made [available under a CC-BY-NC-ND 4.0 International license.](http://creativecommons.org/licenses/by-nc-nd/4.0/) 633 pathogen-only plants exhibited severe growth suppression and reached only approximately 16.6 634 cm at the final assessment (Fig. 3B). Preventive PGPR application substantially alleviated 635 pathogen-induced growth reduction. The combined B18 plus B14 plus pathogen treatment 636 produced the tallest pathogen-challenged plants (27.0 cm), representing a 62.4% increase relative 637 to the pathogen-only treatment and achieving growth levels comparable to those observed under 638 pathogen-free conditions. Individual PGPR treatments also improved shoot growth compared with 639 pathogen-only plants; however, the consortium consistently produced the greatest response. 640 Leaf production followed a similar pattern and was significantly influenced by treatment under 641 both pathogen-free (F 3,21 = 21.10, _P_ < 0.001) and pathogen-challenged conditions (F 3,21 = 23.33, 642 _P_ < 0.001) (Fig. 3C). Plants inoculated with _V. dahliae_ alone developed the fewest leaves (39.4 643 leaves plant [-1] ), whereas the combined B18 plus B14 plus pathogen treatment produced the highest 644 leaf number (79.6 leaves plant⁻¹). This treatment approximately doubled leaf production relative 645 to pathogen-only plants and exceeded the healthy untreated control by approximately 33%, 646 demonstrating a strong recovery from pathogen-induced suppression and enhanced canopy 647 development. The individual PGPR treatments also significantly increased leaf production 648 compared with the pathogen-only control, although their effects were less pronounced than those 649 of the consortium. 650 Chlorophyll content was strongly affected by treatment (Fig. 3D). Significant treatment effects 651 were detected under both pathogen-free (F 3,21 = 18.00, _P_ < 0.001) and pathogen-challenged 652 conditions (F 3,21 = 34.57, _P_ < 0.001). In the presence of _V. dahliae_, chlorophyll content declined 653 markedly in pathogen-only plants, reaching approximately 26.2 SPAD units by the final 654 assessment. In contrast, PGPR-treated plants maintained significantly higher chlorophyll levels 655 throughout the experiment. The highest chlorophyll content under disease pressure was observed 656 in the B14 plus pathogen treatment (48.4 SPAD units), representing an 84.9% increase relative to 657 the pathogen-only control. Similarly, the B18 plus B14 plus pathogen treatment maintained 658 elevated chlorophyll content (47.6 SPAD units), indicating that PGPR inoculation effectively 659 reduced pathogen-associated physiological stress and preserved photosynthetic capacity. 660 In the absence of the pathogen, PGPR inoculation also enhanced plant performance relative to the 661 healthy untreated control. The combined B18 plus B14 treatment produced the greatest final shoot [bioRxiv preprint doi: https://doi.org/10.64898/2026.11.06.764942; this version posted November 9, 2026. The copyright holder for this preprint](https://doi.org/10.64898/2026.11.06.764942) (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made [available under a CC-BY-NC-ND 4.0 International license.](http://creativecommons.org/licenses/by-nc-nd/4.0/) 662 height (~29 cm), highest leaf number (~70 leaves plant⁻¹), and highest chlorophyll content (~50 663 SPAD units), demonstrating strong plant growth–promoting activity even under non-stress 664 conditions. 665 Collectively, these results demonstrate that PGPR inoculation significantly enhanced vegetative 666 growth and physiological performance under both healthy and pathogen-challenged conditions. 667 The combined B18 plus B14 consortium consistently produced the strongest response across all 668 measured parameters, indicating that the simultaneous application of functionally complementary 669 endophytic _Pseudomonas_ strains provided greater benefits than either strain alone. These findings 670 suggest that consortium-based inoculation strategies may offer an effective approach for 671 improving plant vigor while mitigating the detrimental effects of _V. dahliae_ infection. 672 [bioRxiv preprint doi: https://doi.org/10.64898/2026.11.06.764942; this version posted November 9, 2026. The copyright holder for this preprint](https://doi.org/10.64898/2026.11.06.764942) (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made [available under a CC-BY-NC-ND 4.0 International license.](http://creativecommons.org/licenses/by-nc-nd/4.0/) 673 **Figure 3.** Effects of PGPR treatments on potato establishment, growth, and physiological performance under non 674 pathogen and pathogen-challenged conditions. (A) Plant establishment response showing emergence (%) and shoot 675 no./tuber, (B) shoot height dynamics, (C) leaf number dynamics, and (D) chlorophyll index measured by SPAD over 676 the experimental period. Values represent mean ± standard error (SE) from biological replicates arranged in a 677 randomized complete block design (RCBD). Data were analyzed using RCBD ANOVA followed by Tukey’s HSD 678 multiple comparison test. Different letters indicate significant differences among treatments within each pathogen 679 group ( _P_ < 0.05). 680 681 **3.7.2 Flowering and tuber production** 682 PGPR inoculation significantly improved reproductive performance and daughter tuber production 683 under both pathogen-free and pathogen-challenged conditions (Fig. 4). Under _Verticillium dahliae_ 684 pressure, flowering was completely suppressed in the pathogen-only treatment throughout the 685 experiment, indicating severe impairment of reproductive development. In contrast, all PGPR 686 treated plants maintained flowering activity. By the final assessment, flowering incidence reached 687 approximately 75% in the B18 plus B14 plus pathogen treatment, compared with 0% in pathogen 688 only plants, demonstrating substantial recovery of reproductive capacity under disease stress (Fig. 689 4A). 690 A similar trend was observed under pathogen-free conditions, where PGPR inoculation enhanced 691 flowering relative to the healthy untreated control. The combined B18 plus B14 treatment 692 produced the highest flowering incidence (~75%), compared with ~25% in the healthy control, 693 indicating a positive effect of PGPR inoculation on reproductive development even in the absence 694 of disease (Fig. 4A). 695 Tuber production was significantly affected by treatment under both pathogen-free (F 3,21 = 16.06, 696 _P_ < 0.001) and pathogen-challenged conditions (F 3,21 = 5.90, _P_ = 0.004) (Fig. 4B). Under pathogen 697 pressure, the pathogen-only treatment produced the fewest daughter tubers (0.63 tubers plant⁻¹), 698 whereas the B18 + B14 + pathogen treatment produced the highest number (2.63 tubers plant⁻¹), 699 representing a 4.2-fold increase relative to the pathogen-only control. Similarly, under pathogen 700 free conditions, the combined B18 + B14 treatment produced the greatest tuber number (4.75 [bioRxiv preprint doi: https://doi.org/10.64898/2026.11.06.764942; this version posted November 9, 2026. The copyright holder for this preprint](https://doi.org/10.64898/2026.11.06.764942) (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made [available under a CC-BY-NC-ND 4.0 International license.](http://creativecommons.org/licenses/by-nc-nd/4.0/) 701 tubers plant⁻¹) compared with 1.75 tubers plant⁻¹ in the healthy control, corresponding to a 171% 702 increase (Fig. 4B). 703 Overall, PGPR inoculation mitigated disease-associated reductions in flowering and tuberization 704 while simultaneously enhancing reproductive performance under healthy conditions. The B18 + 705 B14 consortium consistently produced the strongest response, highlighting the benefits of 706 combining functionally complementary endophytic _Pseudomonas_ strains. 707 708 709 710 711 712 713 714 715 716 717 718 719 **Figure 4.** **Effects of PGPR treatments on flowering and tuber production in potato.** (A) Flowering incidence, 720 calculated as the percentage of pots showing visible inflorescence formation, and (B) tuber number per plant under 721 non-pathogen and pathogen-challenged conditions. For flowering incidence, each pot was scored as flowered or non 722 flowered based on the presence or absence of visible inflorescences, and the percentage of flowered pots was [bioRxiv preprint doi: https://doi.org/10.64898/2026.11.06.764942; this version posted November 9, 2026. The copyright holder for this preprint](https://doi.org/10.64898/2026.11.06.764942) (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made [available under a CC-BY-NC-ND 4.0 International license.](http://creativecommons.org/licenses/by-nc-nd/4.0/) 723 calculated within each treatment group. Values represent mean ± SE from biological replicates arranged in an RCBD. 724 Data were analyzed using RCBD ANOVA followed by Tukey’s HSD test. Different letters indicate significant 725 differences within each pathogen group ( _P_ < 0.05). 726 727 **3.7.3 Biomass accumulation and root system architecture** 728 PGPR inoculation significantly enhanced biomass accumulation and root development under both 729 pathogen-free and _Verticillium dahliae_ –challenged conditions (Fig. 5). Under pathogen pressure, 730 total fresh biomass differed significantly among treatments (F 3,17 = 4.55, _P_ = 0.016). The pathogen 731 only treatment produced the lowest biomass (11.4 g plant⁻¹), whereas the B18 + B14 + pathogen 732 treatment produced the highest value (16.0 g plant⁻¹), representing a 40.0% increase relative to the 733 pathogen-only control (Fig. 5A). Total dry biomass was similarly affected by treatment (F 3,16 = 734 3.45, _P_ = 0.042), increasing from 1.64 g plant⁻¹ in pathogen-only plants to 2.45 g plant⁻¹ in 735 consortium-treated plants, corresponding to a 49.0% improvement (Fig.5B). 736 Leaf area was significantly influenced by treatment under pathogen challenge (F 3,17 = 3.79, _P_ = 737 0.030). The pathogen-only treatment exhibited the smallest leaf area (136.7 cm [2] plant [-1] ), whereas 738 the B18 plus B14 plus pathogen treatment reached approximately 202.0 cm [2] plant [-1], representing 739 a 47.8% increase (Fig. 5D). Root dry weight also increased substantially in response to PGPR 740 inoculation and approached statistical significance under pathogen pressure (F 3,17 = 3.12, _P_ = 741 0.053). Consortium-treated (B18 plus B14) plants produced the greatest root biomass (0.77 g plant [-] 742 1 ), representing a 63.3% increase relative to pathogen-only plants (Fig. 5C). 743 Root architectural traits showed similar responses. Total root length differed significantly among 744 treatments under both pathogen-free (F 3,15 = 3.96, _P_ = 0.029) and pathogen-challenged conditions 745 (F 3,17 = 5.19, _P_ = 0.010). Under disease pressure, consortium-treated plants developed more 746 extensive root systems, reaching approximately 449 cm plant [-1] compared with 360 cm plant⁻¹ in 747 pathogen-only plants (Fig. 5E). Root surface area was also significantly affected under pathogen 748 challenge (F 3,17 = 3.92, _P_ = 0.027), indicating improved root system development despite disease 749 pressure (Fig. 5F). [bioRxiv preprint doi: https://doi.org/10.64898/2026.11.06.764942; this version posted November 9, 2026. The copyright holder for this preprint](https://doi.org/10.64898/2026.11.06.764942) (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made [available under a CC-BY-NC-ND 4.0 International license.](http://creativecommons.org/licenses/by-nc-nd/4.0/) 750 In the absence of the pathogen, PGPR treatments also promoted plant growth relative to the healthy 751 untreated control. The B18 and B14 consortium produced the greatest total fresh biomass (~16.8 752 g plant [-1] ), total dry biomass (~2.7 g plant [-1] ), leaf area (~225 cm [2] plant [-1] ), and root biomass (~0.97 753 g plant [-1] ), demonstrating strong plant growth–promoting activity even under non-stress conditions 754 (Fig. 5A, 5B, 5C). 755 Among the individual strains, B18 generally produced stronger effects on root development than 756 B14, particularly for total root length and root surface area. Nevertheless, the combined B18 and 757 B14 treatment consistently generated the greatest and most balanced improvements across both 758 aboveground and belowground traits. Similar trends were observed for leaf fresh weight, stem 759 fresh weight, leaf dry weight, stem dry weight, root volume, and average root diameter 760 (Supplementary Fig. S4). Collectively, these results demonstrate that endophytic _Pseudomonas_ 761 strains B18 and B14 substantially enhanced biomass accumulation and root system architecture 762 while mitigating the detrimental effects of _V. dahliae_ on potato growth. 763 764 765 766 767 768 769 [bioRxiv preprint doi: https://doi.org/10.64898/2026.11.06.764942; this version posted November 9, 2026. The copyright holder for this preprint](https://doi.org/10.64898/2026.11.06.764942) (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made [available under a CC-BY-NC-ND 4.0 International license.](http://creativecommons.org/licenses/by-nc-nd/4.0/) 770 771 **Figure 5** . **Effects of endophytic** _**Pseudomonas**_ **strains on potato biomass accumulation and root system** 772 **architecture under non-pathogen and** _**Verticillium dahliae**_ **–challenged conditions.** (A) Total fresh biomass, (B) 773 total dry biomass, (C) root dry weight, (D) leaf area, (E) total root length, and (F) root surface area measured at final 774 harvest. Values represent mean ± standard error (SE) from eight biological replicates arranged in a randomized 775 complete block design (RCBD). Treatments included Healthy control, PGPR A ( _Pseudomonas_ strain B18), PGPR B 776 ( _Pseudomonas_ strain B14), PGPR A + B consortium, Pathogen only ( _V. dahliae_ ), PGPR A + Pathogen, PGPR B + 777 Pathogen, and PGPR A + B + Pathogen. Data were analyzed using RCBD ANOVA with treatment as a fixed effect [bioRxiv preprint doi: https://doi.org/10.64898/2026.11.06.764942; this version posted November 9, 2026. The copyright holder for this preprint](https://doi.org/10.64898/2026.11.06.764942) (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made [available under a CC-BY-NC-ND 4.0 International license.](http://creativecommons.org/licenses/by-nc-nd/4.0/) 778 and block as a blocking factor, followed by Tukey’s honestly significant difference (HSD) test ( _P_ < 0.05). Different 779 letters indicate significant differences among treatments within each pathogen group. 780 **3.7.6 Disease suppression by PGPR treatments** 781 Disease progression, expressed as the area under the disease progress curve (AUDPC), was 782 significantly affected by PGPR treatment under _Verticillium dahliae_ challenge (F 3,21 = 14.59, _P_ < 783 0.001) (Fig. 6). The pathogen-only treatment exhibited the highest cumulative disease severity, 784 with a mean AUDPC value of 40.94, indicating severe wilt development in the absence of 785 biological protection. 786 Preventive PGPR inoculation substantially reduced disease progression relative to the pathogen 787 only control. AUDPC values were 3.44, 6.31, and 4.25 for the B18 plus pathogen, B14 plus 788 pathogen, and B18 plus B14 plus pathogen treatments, respectively. These values corresponded to 789 reductions in cumulative disease severity of 91.6, 84.6, and 89.6% relative to the pathogen-only 790 treatment. Although the B18 plus pathogen treatment produced the numerically lowest AUDPC 791 value, Tukey’s multiple comparison test detected no significant differences among the three PGPR 792 protected treatments ( _P_ - 0.05), indicating that all inoculation strategies provided similarly 793 effective suppression of Verticillium wilt. 794 No disease symptoms were observed in the non-inoculated treatments, and AUDPC values 795 remained at or near zero throughout the experiment. Consequently, statistical analysis was not 796 applicable for the non-pathogen treatments because disease severity did not develop under these 797 conditions. 798 Overall, the results demonstrate that both individual strains and their combined application 799 provided strong and consistent biological control of _V. dahliae_, reducing cumulative disease 800 severity by more than 84% compared with the pathogen-only control. While disease suppression 801 was statistically comparable among PGPR treatments, the B18 plus B14 consortium consistently 802 produced superior plant growth, biomass accumulation, root development, and tuber production 803 throughout the experiment. These findings suggest that the principal advantage of the consortium 804 lies not in greater disease suppression alone, but in its ability to simultaneously support plant 805 growth and maintain crop performance under pathogen pressure. [bioRxiv preprint doi: https://doi.org/10.64898/2026.11.06.764942; this version posted November 9, 2026. The copyright holder for this preprint](https://doi.org/10.64898/2026.11.06.764942) (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made [available under a CC-BY-NC-ND 4.0 International license.](http://creativecommons.org/licenses/by-nc-nd/4.0/) 806 807 808 809 810 811 812 813 814 815 816 817 818 819 820 821 822 823 824 825 826 **Figure 6.** **Effects of PGPR treatments on** _**Verticillium**_ **wilt progression in potato.** Disease progression was 827 summarized as the area under the disease progress curve (AUDPC) under non-pathogen and _Verticillium dahliae_ – 828 challenged conditions. Values represent mean ± SE from biological replicates arranged in an RCBD. Data were 829 analyzed using RCBD ANOVA followed by Tukey’s HSD test. Different letters indicate significant differences within 830 the pathogen-challenged group ( _P_ < 0.05). No statistical letters are shown for the non-pathogen group because AUDPC 831 values were zero across treatments. 832 833 **3.8 Integrated performance of single-strain and consortium treatments** 834 Across laboratory, growth chamber, and greenhouse experiments, the two endophytic 835 _Pseudomonas_ strains exhibited distinct but complementary functional profiles. Strain B18 836 consistently demonstrated stronger plant growth–promoting characteristics, including greater [bioRxiv preprint doi: https://doi.org/10.64898/2026.11.06.764942; this version posted November 9, 2026. The copyright holder for this preprint](https://doi.org/10.64898/2026.11.06.764942) (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made [available under a CC-BY-NC-ND 4.0 International license.](http://creativecommons.org/licenses/by-nc-nd/4.0/) 837 phosphate solubilization, higher indole-3-acetic acid production, stronger seed germination 838 stimulation, improved shoot and root growth, and enhanced physiological performance. In 839 contrast, strain B14 displayed stronger antagonistic activity against _Verticillium_ spp. _in vitro_ and 840 contributed substantially to the maintenance of chlorophyll content and plant health under 841 pathogen challenge. 842 Although both strains individually improved plant performance and reduced disease severity, their 843 combined application consistently produced the most balanced and comprehensive response across 844 measured agronomic traits. The B18 plus B14 consortium enhanced shoot growth, leaf production, 845 biomass accumulation, root development, flowering, tuber production, and disease tolerance under 846 both healthy and pathogen-challenged conditions. Importantly, while all PGPR treatments 847 provided statistically comparable suppression of Verticillium wilt, the consortium consistently 848 generated superior overall plant performance, indicating benefits beyond disease control alone. 849 Collectively, these findings support the concept of functional complementarity, whereby strains 850 possessing different biological capabilities contribute distinct but synergistic benefits to plant 851 performance. The results further suggest that carefully selected multi-strain PGPR consortia may 852 provide a more robust strategy for improving crop productivity and resilience than single-strain 853 inoculants. Consequently, the B18 plus B14 consortium represents a promising candidate for the 854 integrated management of Verticillium wilt and the development of sustainable potato production 855 systems. 856 857 858 859 860 861 [bioRxiv preprint doi: https://doi.org/10.64898/2026.11.06.764942; this version posted November 9, 2026. The copyright holder for this preprint](https://doi.org/10.64898/2026.11.06.764942) (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made [available under a CC-BY-NC-ND 4.0 International license.](http://creativecommons.org/licenses/by-nc-nd/4.0/) 862 **4. Discussion** 863 Our study shows that stress-adapted endophytic _Pseudomonas_ strains isolated from unmanaged 864 wild lupin roots have considerable potential to promote potato growth and suppress Verticillium 865 wilt under glasshouse conditions. Both isolates exhibited multiple plant growth–promoting (PGP) 866 traits, inhibited _Verticillium_ species _in vitro_, enhanced plant performance, and reduced disease 867 severity. The two strains displayed distinct functional profiles: B18 showed stronger growth 868 promoting characteristics, while B14 exhibited greater direct antifungal activity. Notably, the 869 combined application of both strains consistently led to the greatest improvements in plant growth, 870 physiological performance, biomass accumulation, tuber production, and disease suppression. 871 These data suggest that functionally complementary microbial inoculants may deliver broader 872 benefits than single-strain applications. 873 The antagonistic activity observed against _V. dahliae, V. longisporum_, and _V. albo-atrum_ is 874 consistent with previous reports identifying _Pseudomonas_ species as effective biological control 875 agents against soil-borne fungal pathogens (Haas and Défago, 2005; Weller, 2007). Of the two 876 isolates, B14 consistently manifested greater inhibition of fungal growth in dual-culture assays, 877 indicating stronger direct antagonistic potential. _Pseudomonas_ species are recognized for 878 producing a range of antimicrobial metabolites, volatile compounds, siderophores, and 879 extracellular enzymes that may contribute to fungal suppression (Mercado-Blanco and Bakker, 880 2007; Pieterse et al., 2014). However, as antimicrobial compounds were not directly characterized 881 in this study, the particular mechanisms underlying fungal inhibition remain undetermined. 882 Consequently, the noted suppression could be regarded as evidence of biocontrol potential rather 883 than confirmation of specific antimicrobial pathways. 884 Beyond pathogen suppression, both isolates exhibited multiple plant-beneficial functions, 885 including phosphate solubilization, indole-3-acetic acid (IAA) production, nitrogen fixation 886 associated activity, siderophore production, and hydrogen cyanide production. These traits are 887 typically linked to greater nutrient acquisition, root development, and plant vigour (Lugtenberg 888 and Kamilova, 2009; Glick, 2012). Strain B18 consistently surpassed B14 in phosphate 889 solubilization, IAA production, siderophore activity, and promotion of seed germination, showing 890 a stronger role in supporting plant growth and early seedling establishment. The observed 891 improvements in shoot growth, chlorophyll content, biomass accumulation, and root development [bioRxiv preprint doi: https://doi.org/10.64898/2026.11.06.764942; this version posted November 9, 2026. The copyright holder for this preprint](https://doi.org/10.64898/2026.11.06.764942) (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made [available under a CC-BY-NC-ND 4.0 International license.](http://creativecommons.org/licenses/by-nc-nd/4.0/) 892 in greenhouse experiments are consistent with previous studies that have demonstrated the positive 893 effects of multifunctional _Pseudomonas_ strains on plant growth (Weller, 2007; Mercado-Blanco 894 and Bakker, 2007). However, since nutrient uptake, phytohormone concentrations, and plant 895 physiological pathways were not directly measured, these mechanisms should be considered 896 plausible contributors rather than confirmed causes of the observed responses. 897 898 An important finding of this study is the better performance of the B18 plus B14 consortium. While 899 each strain improved plant performance and reduced disease severity, the combined treatment 900 consistently produced the strongest responses across nearly all measured parameters. This result 901 indicates that the consortium's enhanced performance results from functional complementarity 902 between the two isolates. The strain B18 exhibited stronger plant growth–promoting activity, 903 whereas B14 demonstrated greater antifungal capacity. Their combined application appears to 904 provide a wider range of beneficial functions than either strain alone. Similar results have been 905 reported for microbial consortia (Tao et al., 2020), where microorganisms with distinct ecological 906 functions outperform individual strains by simultaneously aiding nutrient acquisition, plant growth 907 promotion, pathogen suppression, and stress mitigation (Bashan et al., 2014; Niu et al., 2017; Berg 908 et al., 2021). 909 The concept of functional complementarity has received increasing attention in microbiome-based 910 agriculture. Instead of relying on a single primary mechanism, effective microbial consortia may 911 operate through the division of environmental roles, niche differentiation, and the integration of 912 multiple beneficial activities within the rhizosphere (Trivedi et al., 2020). Although direct 913 interactions between B18 and B14 were not investigated, the consistent superiority of the 914 consortium treatment under both healthy and pathogen-challenged conditions indicates that these 915 isolates occupy complementary functional niches. Such combinations may provide greater 916 stability and broader efficacy than single-strain inoculants, especially under shifting environmental 917 conditions. Therefore, the development of carefully selected microbial consortia may offer a 918 promising strategy to enhance both biological control and crop productivity. 919 920 An additional significant observation was the retention of biological activity in the cell-free 921 supernatant (CFS) of strain B18. Sterile CFS considerably enhanced seed germination and radicle [bioRxiv preprint doi: https://doi.org/10.64898/2026.11.06.764942; this version posted November 9, 2026. The copyright holder for this preprint](https://doi.org/10.64898/2026.11.06.764942) (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made [available under a CC-BY-NC-ND 4.0 International license.](http://creativecommons.org/licenses/by-nc-nd/4.0/) 922 elongation in the absence of live bacterial cells, indicating that extracellular metabolites 923 contributed to the observed plant responses. Comparable effects have been reported for bacterial 924 signaling molecules, phytohormones, volatile organic compounds, lipopeptides, and other low 925 molecular-weight metabolites produced by plant-associated microorganisms (Lee et al., 2009; 926 Subramanian and Smith, 2015; Raaijmakers and Mazzola, 2012). Although the specific active 927 compounds responsible for the stimulatory effects remain unidentified, these results suggest that 928 strain B18 may be a promising source of biologically active metabolites with agricultural potential. 929 The capacity of sterile bacterial culture filtrates to reproduce beneficial plant responses has 930 significant practical consequences. Metabolite-based biostimulants may offer several advantages 931 over live microbial inoculants, including improved formulation stability, extended shelf life, 932 simplified storage requirements, and reduced variability associated with microbial establishment 933 under field conditions (Backer et al., 2018). Subsequent research should therefore prioritize 934 bioassay-guided fractionation of B18 cell-free supernatants, combined with metabolomic and 935 chemical characterization, to identify the compounds accountable for growth promotion. These 936 studies could facilitate the advancement of next-generation biological products and improve 937 understanding of the molecular basis of plant–microbe interactions. 938 The origin of both isolates from unmanaged wild lupin plant populations that survive in cold, 939 environmentally challenging conditions may be relevant to their performance. Wild plants 940 frequently harbour microbiomes enriched in traits associated with nutrient acquisition, 941 environmental acclimation, and pathogen suppression (Compant et al., 2010; Hardoim et al., 942 2015). Those environments may serve as natural reservoirs for microorganisms with traits 943 advantageous for agricultural applications. The successful performance of B14 and B18 supports 944 the increasing recognition that stress-adapted natural ecosystems are valuable sources of elite 945 microbial inoculants for sustainable agriculture. 946 In summary, the findings show that stress-adapted endophytic _Pseudomonas_ strains isolated from 947 unmanaged wild lupin roots can significantly improve potato growth and reduce Verticillium wilt 948 severity under growth-chamber conditions. The better performance of the B18 plus B14 949 consortium demonstrates the importance of combining strains with complementary functional 950 traits and supports the wider concept that multifunctional microbial communities may offer more 951 consistent agricultural benefits than individual isolates. These results add to the expanding body [bioRxiv preprint doi: https://doi.org/10.64898/2026.11.06.764942; this version posted November 9, 2026. The copyright holder for this preprint](https://doi.org/10.64898/2026.11.06.764942) (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made [available under a CC-BY-NC-ND 4.0 International license.](http://creativecommons.org/licenses/by-nc-nd/4.0/) 952 of evidence supporting microbiome-based strategies for sustainable disease management and crop 953 production. 954 Some specific considerations should be taken into account when interpreting the results of this 955 study. First, all experiments were carried out under controlled environmental conditions; therefore, 956 field validation is necessary before any kind of agronomic recommendations can be made. 957 Environmental variation, soil characteristics, indigenous microbial communities, and agricultural 958 management practices may influence inoculant performance under commercial production 959 conditions. Second, taxonomic identification was based on partial 16S rRNA gene sequencing, 960 which provides limited species-level resolution within the genus _Pseudomonas_ . Whole-genome 961 sequencing would improve taxonomic resolution and facilitate the identification of genes 962 associated with plant growth promotion, secondary metabolite biosynthesis, biocontrol activity, 963 habitat adaptation, and biosafety. 964 Third, although multiple plant growth–promoting traits and strong antagonistic activity were 965 observed, the molecular and biochemical mechanisms underlying these responses were not directly 966 investigated. Future research needs to be conducted to integrate whole-genome sequencing, in 967 combination of plant metabolomic profiling, transcriptomics, and rhizosphere colonization 968 analyses to identify the genes, metabolites, and pathways accounting for disease suppression and 969 plant growth promotion. Specifically, characterization of the bioactive compounds present in B18 970 cell-free supernatants may detect novel metabolites suitable for development as microbial 971 biostimulants. Additionally, long-term persistence studies and investigations into interactions with 972 native microbial communities will be key to assessing the ecological stability, effectiveness, and 973 feasibility of deploying these strains under field conditions. 974 **5. Acknowledgments** 975 The authors gratefully acknowledge the financial support provided by McCain Foods and the 976 support of the Eastern Canada Oilseeds Development Alliance (ECODA) project. We thank all 977 members of the Donald Smith’s Laboratory, Department of Plant Science, McGill University, for 978 their assistance with greenhouse experiments, laboratory work, technical support, and valuable 979 scientific discussions throughout this study. The authors are especially grateful to Professor 980 Donald L. Smith for his guidance, mentorship, and continued support during the project. [bioRxiv preprint doi: https://doi.org/10.64898/2026.11.06.764942; this version posted November 9, 2026. The copyright holder for this preprint](https://doi.org/10.64898/2026.11.06.764942) (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made [available under a CC-BY-NC-ND 4.0 International license.](http://creativecommons.org/licenses/by-nc-nd/4.0/) 981 **6. Conflict of interest statement** 982 The authors declare that they have no known competing financial interests or personal 983 relationships that could have influenced the work reported in this paper. 984 **7. Funding** 985 This research was funded by McCain Foods and supported through the ECODA program within 986 the Donald Smith’s Laboratory, Department of Plant Science, McGill University. 987 988 989 990 991 992 993 994 995 996 997 998 999 1000 1001 1002 [bioRxiv preprint doi: https://doi.org/10.64898/2026.11.06.764942; this version posted November 9, 2026. The copyright holder for this preprint](https://doi.org/10.64898/2026.11.06.764942) (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made [available under a CC-BY-NC-ND 4.0 International license.](http://creativecommons.org/licenses/by-nc-nd/4.0/) 1003 **8.** **References** 1004 1. Backer R, Rokem JS, Ilangumaran G, Lamont J, Praslickova D, Ricci E, Subramanian S, 1005 Smith DL. 2018. Plant growth-promoting rhizobacteria: context, mechanisms of action, 1006 and roadmap to commercialization of biostimulants for sustainable agriculture. 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Yu G, Smith DK, Zhu H, Guan Y, Lam TTY. 2017. ggtree: an R package for 1139 visualization and annotation of phylogenetic trees with associated data. Methods Ecol 1140 Evol 8:28–36. 1141 57. Zar JH. 2010. Biostatistical Analysis, 5th edn. Upper Saddle River, NJ: Pearson Prentice 1142 Hall. 1143 1144 1145 1146 1147 1148 1149 [bioRxiv preprint doi: https://doi.org/10.64898/2026.11.06.764942; this version posted November 9, 2026. The copyright holder for this preprint](https://doi.org/10.64898/2026.11.06.764942) (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made [available under a CC-BY-NC-ND 4.0 International license.](http://creativecommons.org/licenses/by-nc-nd/4.0/) 1150 **Supplementary data:** 1151 1152 1153 **Supplementary Figure S1. Representative dual-culture antagonism assays of isolates B18 and B14 against three** 1154 _**Verticillium**_ **species after 4 weeks of incubation.** Colony diameter used for quantitative inhibition analysis was 1155 measured after 2 weeks of incubation, whereas the images shown here were captured after 4 weeks to better illustrate 1156 sustained fungal growth suppression, altered colony morphology, and long-term interaction patterns. Top row: 1157 _Verticillium dahliae_ ; middle row: _V. longisporum_ ; bottom row: _V. albo-atrum_ . In each row, the left plate represents the 1158 untreated fungal control, while the center and right plates show co-culture with isolates B18 and B14, respectively. 1159 1160 1161 [bioRxiv preprint doi: https://doi.org/10.64898/2026.11.06.764942; this version posted November 9, 2026. The copyright holder for this preprint](https://doi.org/10.64898/2026.11.06.764942) (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made [available under a CC-BY-NC-ND 4.0 International license.](http://creativecommons.org/licenses/by-nc-nd/4.0/) 1162 1163 1164 **Supplementary Figure S2. Phenotypic and biochemical characterization of endophytic** _**Pseudomonas**_ **isolates** 1165 **B14 and B18 used in this study.** Representative images showing preliminary plant growth–promoting and functional 1166 trait screening of the selected isolates. From left to right: (A) indole-3-acetic acid (IAA) production in broth culture 1167 indicated by development of pink to reddish coloration after Salkowski reagent reaction; (B) phosphate solubilization 1168 on Pikovskaya’s agar shown by halo formation surrounding colonies; (C) qualitative nitrogen fixation-associated 1169 growth in semi-solid NFb medium indicated by pellicle formation near the oxygen gradient; (D) siderophore 1170 production on chrome azurol S (CAS) medium indicated by color change around colonies; and (E) colony 1171 pigmentation and growth morphology on agar medium. Sterile uninoculated controls were included for comparison. 1172 These assays were used as preliminary screening tools to identify isolates with multiple plant growth–promoting and 1173 biocontrol-associated traits prior to greenhouse evaluation. 1174 1175 1176 1177 1178 1179 [bioRxiv preprint doi: https://doi.org/10.64898/2026.11.06.764942; this version posted November 9, 2026. The copyright holder for this preprint](https://doi.org/10.64898/2026.11.06.764942) (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made [available under a CC-BY-NC-ND 4.0 International license.](http://creativecommons.org/licenses/by-nc-nd/4.0/) 1180 1181 1182 1183 1184 1185 1186 1187 1188 **Supplementary Figure S3. Effects of cell-free supernatant (CFS) from bacterial isolates B18 and B14 on seed** 1189 **germination and radicle growth at different concentrations.** 1190 Four independent biological replicates were used for each treatment, with 25 seeds per replicate plate. Bars represent 1191 mean ± 95% confidence interval (CI). Different lowercase letters above bars indicate statistically significant 1192 differences among treatments within each panel according to one-way ANOVA followed by Tukey’s HSD post hoc 1193 test ( _P_ < 0.05). 1194 (A) Germination percentage of seeds treated with CFS from isolate B18 at 0, 2.5, 5, and 10% concentrations 1195 compared with the untreated control. 1196 (B) Radicle length of germinated seeds treated with B18 CFS at corresponding concentrations. 1197 (C) Germination percentage of seeds treated with CFS from isolate B14 at 0, 2.5, 5, and 10% concentrations 1198 compared with the untreated control. 1199 (D) Radicle length of germinated seeds treated with B14 CFS at corresponding concentrations. 1200 Overall, B18 CFS showed positive effects on germination and radicle elongation at moderate concentrations, whereas 1201 higher concentrations of B14 CFS reduced radicle growth, demonstrating concentration-dependent responses of 1202 seedling development to bacterial extracellular metabolites . 1203 1204 [bioRxiv preprint doi: https://doi.org/10.64898/2026.11.06.764942; this version posted November 9, 2026. The copyright holder for this preprint](https://doi.org/10.64898/2026.11.06.764942) (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made [available under a CC-BY-NC-ND 4.0 International license.](http://creativecommons.org/licenses/by-nc-nd/4.0/) 1205 1206 1207 1208 1209 1210 1211 1212 1213 1214 1215 1216 1217 1218 1219 1220 1221 1222 **Figure S4.** Effects of endophytic _Pseudomonas_ strains on additional biomass and root architecture traits of potato 1223 under non-pathogen and _Verticillium dahliae_ –challenged conditions. Panels show leaf fresh weight, stem fresh weight, 1224 leaf dry weight, stem dry weight, average root diameter, and root volume measured at final harvest. Values represent 1225 mean ± SE from eight biological replicates arranged in a randomized complete block design (RCBD). Data were 1226 analyzed using RCBD ANOVA followed by Tukey’s HSD test ( _P_ < 0.05). Different letters indicate significant 1227 differences among treatments within each pathogen group.