Fig 1.
A graphical representation of isolation, screening of halotolerant bacteria and their effect on alfalfa plant growth under salt stress.
Table 1.
Source of bacterial strains.
Fig 2.
Plant Growth promoting traits of halotolerant bacterial isolates.
(A) Pikovskaya’s medium and (B) NBRIP medium for phosphate solubilization. (C) mineral salts medium for zinc solubilization. (D) IAA production assay in a 96-well plate. Halos around bacteria in A-C indicate positive activity. Results are summarized in Fig 3.
Fig 3.
Heat map of putative plant growth promoting bacterial properties.
The data was normalized to scale different property values uniformly, ensuring comparability across bacterial strains. Each parameter was transformed to a range between 0 and 1 using min-max normalization, where the minimum observed value was set to 0 and the maximum to 1. The qualitative data indicators (e.g., “-” “+”, “++”, “+++”) were converted into numerical values based on an arbitrary scale before applying min-max normalization, where the minimum observed value was set to 0 and the maximum to 1.
Fig 4.
Testing of halotolerant strains on LB agar with 0, 1, 2, 3 & 4 M NaCl.
No growth was observed on LB with 0 salt.
Fig 5.
Effect of Kushneria strains on alfalfa plant growth in greenhouse open pot trials.
(A) photographs of plants after harvesting from each treatment (No salt control without bacteria, Salt control without bacteria, A3 inoculation, B5 inoculation). (B) the total plant fresh weight is shown. Data points indicate means ± SEM of at least 10 biological replicates. The statistically significant difference is marked with asterisks (Tukey’s HSD, P < 0.05). Significance: P < 0.001 ‘***’. (C) Plants in open pots before harvesting.
Fig 6.
Alfalfa plant growth trial with Kushneria strains under salt stress conditions.
Harvested alfalfa plants are shown at the bottom. Each pot was inoculated with the indicated bacterial isolate. See Fig 7 for the data analysis.
Fig 7.
Effect of Kushneria strain inoculation on alfalfa in growth chamber trials.
(A) plant height, (B) root length, (C) plant fresh weight, and D) plant dry weight. Data points show the means ± SEM of at least 15 biological replicates. Statistically significant differences are marked with asterisks (Tukey’s HSD, P < 0.05). Significance codes: 0.001 ‘***’; 0.01 ‘**’; 0.05 ‘*’.
Fig 8.
Alfalfa plant growth trial with Kushneria strains under salt stress conditions (top panel).
Harvested alfalfa plants (bottom panel). All plants were grown in closed pots with a single watering upon planting and inoculated with the indicated bacterial isolate except for the controls (uninoculated No salt & salt). See Fig 9 for analysis of plant length and weight measurements.
Fig 9.
Effect of Kushneria strain on alfalfa plants.
(A) shoot length, (B) root length, (C) shoot fresh weight, and (D) root fresh weight. Data points show the means ± SEM of at least 13 biological replicates except for E4 which has 10 replicates and the unstressed control which has 4 replicates. Statistically significant differences are marked with asterisks (Tukey’s HSD, P < 0.05). Significance codes: 0.001 ‘***’; 0.01 ‘**’; 0.05 ‘*’.
Fig 10.
Survival determination of inoculated bacteria recovered from alfalfa grown in pots.
Bacteria from crushed root tissue after harvest were grown on LB medium containing 1M NaCl with 10-6 dilution after harvesting. (A) No inoculation (B) A3 inoculation (C) B5 inoculation.
Fig 11.
Visualization of B5 strain expressing GFP in alfalfa root tissue.
(A & B) No inoculation, (C & D) B5 + GFP inoculation. Root sample from alfalfa inoculated with B5 strain, constitutively expressing the GFP gene as compared to control, visualized using the Echo Revolve microscope. Fluorescent GFP signal highlights bacterial colonization within the root tissue.
Fig 12.
Potential mechanisms for salt-tolerant plant growth promoting rhizobacterial mitigation of salt stress in plants.
Concept from [26].