Rhizosphere bacteria help in enhance the tomato plants growth -

Rhizosphere bacteria help in enhance the tomato plants growth

The inoculation of beneficial bacteria is known to enhance plant growth under these stresses, such as phosphorus starvation or salt stress. The efficiency of selected beneficial bacterial strains in improving tomato plant growth to better cope with double stresses in salty and P-deficient soil conditions. Six strains of Arthrobacter and Bacillus with different reservoirs of plant growth-promoting traits were tested in vitro for their abilities to tolerate 2–16% (w/v) NaCl concentrations, and shown to retain their motility and phosphate-solubilizing capacity under salt stress conditions. Bacterial isolates from Cameroonian soils mobilized P from different phosphate sources in shaking culture under both non-saline and saline conditions. They also enhanced plant growth in P-deficient and salt-affected soils by 47–115%, and their PGP effect was even increased in higher salt stress conditions. The results provide valuable information for prospective production of effective bio-fertilizers based on the combined application of local rock phosphate and halotolerant phosphate-solubilizing bacteria. This constitutes a promising strategy to improve plant growth in P-deficient and salt-affected soils.

Microorganism Selection and Storage

Three Arthrobacter strains (V54, V64, and V84) and three Bacillus strains (V62, V39, and V1) isolated from maize rhizosphere were used in this study. Strains were stored at −80 ◦C in standard nutrient broth medium with glycerol (50% v/v).

Bacterial Inoculum Preparation

To prepare an inoculum from each bacterial strain, a single pure bacterial colony was transferred into a 100-mL Erlenmeyer flask containing 50 mL of nutrient broth and grown on a rotary shaker at 28 ◦C for 24 h. Bacterial cells were harvested and washed three times in 0.05 M sterilized NaCl solution after centrifugation (10,000× g) for 15 min at 4 ◦C and finally suspended in 0.05 M sterilized NaCl solution. The optical density (OD) of the suspension was adjusted using a spectrophotometer to 0.2 at 620 nm wavelength, which meant that the population reached 108 colony forming units (CFU) mL⁻¹, to be used in greenhouse experiments.

Characterization of Bacterial Strains under Normal Condition

To assess the swarming ability of the bacterial strains, 10 µL of each bacterial suspension adjusted to OD 0.2 at 620 nm were spotted at the center of a plate containing 25 mL of a semisolid nutrient broth medium (NB; per liter: 5 g peptone, 3 g yeast extract, and 5 g of Agar-Agar). For the swimming motility assay, the plate containing the nutrient broth medium (NB; per liter: 5 g peptone, 3 g yeast extract, and 3 g of Agar-Agar) were inoculated with 10 µL of each bacterial suspension on the surface of the plate. The plates in triplicate were incubated at 28 ◦C for 48 h and motility was measured from the center towards the periphery of the plate.

Qualitative Characterizations of Bacteria for Phosphate Solubilization on Plates

The efficacy of the different strains to solubilize sparingly soluble phosphate sources was first assessed on plates. The ability of the bacterial strains to solubilize all seven different inorganic phosphate sources: tricalcium phosphate (TCP), hydroxyapatite, and five rock phosphates (RPs) of different origins was assessed on plates filled with the National Botanical Research Institute’s Phosphate growth medium. In total, 10 µL of each bacterial suspension were transferred onto a single point of compartmented Petri dishes. Plates in triplicate for each treatment and each phosphate source were incubated at 28 ◦C for 5 days. The halo (yellow) zone surrounding the bacterial colony indicated phosphate solubilization. The solubilization index (SI) was used as an indicator for the isolate´s efficiency, and was calculated in the following way:

SI = (colony diameter + diameter of halo zone)/colony diameter

Quantitative Estimation of Phosphate Solubilization in Liquid Broth

Bacterial strains were tested in liquid NBRIP media to assess their capability to release P from TCP and Cameroonian RP (CRP). The local RP source (CRP) as well as TCP, the most used inorganic phosphate, were chosen for screening phosphate-solubilizing microorganisms. In all cases, 50 mL of NBRIP medium were distributed into 100-mL Erlenmeyer flasks. After sterilization and cooling, 1 mL of the bacterial suspensions was used to inoculate flasks. Each treatment was replicated three times and non-inoculated flasks supplemented with different phosphate sources and 1 mL of 0.05 M sterile NaCl served as controls. Incubation was performed at 28 ◦C, 180 rpm for 5 days. At the end of the incubation time, the cultures were transferred into sterile falcon tubes, centrifuged at 10,000× g for 10 min at 4 ◦C, and the supernatants filtered through 0.2-µm filters. The pH of the filtrate was measured in each case using a pH 110 meter (VWR, Darmstadt, Germany) and the available P was determined following the colorimetric molybdate blue method.

Bacterial Tolerance to Salt

Salt tolerance of bacterial strains was tested on Standard I Nutrient agar plates amended with various concentrations of NaCl (2–16%; w/v).

Motility Test under Salt Stress

The swarming and swimming ability of bacterial strains under salt stress was performed, on the same media supplemented with different concentrations of NaCl (2–16%; w/v), and motility was measured as before.

Phosphate Solubilization under Salt Stress

The influence of salt on the phosphate-solubilizing ability of the bacterial strains was performed essentially on NBRIP medium supplemented with different concentrations of NaCl (2 and 4%, w/v).

Plant Inoculation Experiment

A greenhouse experiment using tomato plant was conducted to evaluate the inoculation effect of the six bacterial strains: (Arthrobacter strains V54, V64, and V84; Bacillus strains V62, V39, and V1) on plant growth and P uptake under different growth conditions in the greenhouse. Phosphorus stress was triggered by fertilization with poorly soluble Cameroonian RP (CRP). The growth conditions included three treatments: P stress without salt stress (CRP + EC = 0 ds m⁻¹), P stress + low salt stress (CRP + low S; CRP + EC = 8 ds m⁻¹), and P stress + high salt stress (CRP + high S; CRP + EC = 12 ds m⁻¹). The inoculation comprised eight treatments, including six bacterial treatments (inoculated plants with each one of the bacterial strains), a non-inoculated control (negative control), and a non-inoculated treatment fertilized with KH₂PO₄ (without phosphorus stress). Surface-sterilized tomato seeds were inoculated by immersion in 1 mL of each bacterial suspension (OD = 0.2 at 620 nm) (microbial treatments) or 1 mL 0.05 M sterilized NaCl solution (control treatments) for 15 min, then sown in quartz sand and maintained in a phyto-chamber (25/20 ◦C day/night temperature) for 14 days. Afterwards, seedlings were potted in pots containing 1 L of mixed quartz sand and vermiculite (1/1). All pots (CRP, CRP + low S, and CRP + high S) were mixed with Cameroonian RP 350 mg P g⁻¹ soil (equivalent to P fertilization of 80 kg P ha⁻¹). The positive control pots were supplemented with the same amount of P applied as soluble phosphate (KH₂PO₄). In all inoculated pots, seedlings were transferred to a pit and finely covered with soil and re-inoculated the following day with 2.5 mL (OD = 0.2 at 620 nm) of the respective bacterial suspension or with 0.05 M sterilized NaCl solution for the control treatments (negative and positive). Pots were watered with 30 mL of the corresponding modified, lacking P, Hoagland solution five times per week and with 30 mL of osmose water twice per week. Salt stress was imposed by adding 20% NaCl to the Hoagland solution until reaching the targeted concentration. Plant growth was documented over six weeks after transplanting in a greenhouse at a day/night temperature 25/23 ◦C and 75% air humidity

At the harvest, the plant height, number of leaves, and stem diameter were recorded. Then, the aerial part was separated from the root part and plant roots were thoroughly washed in tap water and deionized water. Shoot, root, and total fresh biomass were documented. The dry biomass of shoots and roots was determined after they were oven dried at 60 ◦C for 72 h. Shoot/root ratio was calculated and the dry matter content was expressed as the percentage of dry weight in fresh weight. Oven-dried tissues were used to determine the shoot and root P concentration by the phospho-vanadomolybdate colorimetric method.

Results

Phosphate Source and Strain-Dependent Phosphate-Solubilizing Efficacy on Agar Plates

Among the six tested bacteria, all strains, except Bacillus strain V1, were able to solubilize at least one inorganic phosphate source on the agar plates. The phosphate-solubilizing ability across all P sources: tricalcium phosphate, hydroxyapatite, and Algerian, Cameroonian, Malian, Mexican, and Moroccan rock phosphate (RP) differed depending on the bacterial strain. Arthrobacter strain V54 and Bacillus strain V62 were the only strains able to solubilize all the seven phosphate sources. Bacillus strains V62 and V39 showed the highest solubilization index (SI = 5.1) for tricalcium phosphate (TCP). Hydroxyapatite was best solubilized by V62 and V64 (SI = 4.0).

Phosphate Source and Strain-Dependent Phosphate-Solubilizing Efficiency in Liquid Culture under Non-Stress Conditions

To quantify the specific bacterial phosphate-solubilizing activity in liquid culture, used the most easily solubilized TCP and the local insoluble phosphate, Cameroonian RP (CRP). The amount of soluble P and changes in pH were monitored for five days in NBRIP medium. In the liquid culture supplemented with TCP or CRP, all bacterial strains solubilized phosphate, but at different rates, depending on the strain and the phosphate source. Bacterial inoculation significantly increased the TCP and CRP solubilization compared to the non-inoculated control. Bacterial-induced solved P amounts, estimated in the NBRIP supernatant, varied from 36.3 to 179.9 mg L⁻¹ in TCP, with the highest solubilization recorded in Arthrobacter strains V84 (179.9 mg L⁻¹) and V54 (169.6 mg L⁻¹). In contrast, with CRP, the estimated amount of P solubilized varied from 36.4 to 54.5 mg L⁻¹ with the maximum concentration in Bacillus strain V62 (54.5 mg L⁻¹), followed by Arthrobacter strains V84 (44.2 mg L⁻¹). Bacillus strain V1, which was unable to show any phosphate-solubilizing activity on solid media supplied with the different phosphate sources, could mobilize phosphate from TCP and CRP in liquid culture. However, compared to all strains, it recorded the lowest P concentration of 36 mg L⁻¹ with TCP and CRP. Solubilization of phosphates was associated with a pH decrease in the NBRIP medium.

To determine their phosphate-solubilizing activity under saline conditions, bacterial strains were also tested for their ability to solubilize tricalcium phosphate (TCP) and Cameroonian RP (CRP) in the presence of different concentrations (2% and 4%) of NaCl in NBRIP broth. In contrast with CRP, the greatest amount of solubilized P at the same concentration of NaCl was found in Arthrobacter V54 (81.5 mg P L⁻¹) and Bacillus strain V62 (71.9 mg P L⁻¹). In the presence of 4% NaCl, the highest amount of solubilized P was recorded for Bacillus strain V39 (193.1 mg P L⁻¹) in TCP, followed by Arthrobacter strain V54 (164.9 mg P L⁻¹). Bacillus strains V62 (61.04 mg P L⁻¹) and V39 (47.5 mg P L⁻¹) showed the highest capability to release P from CRP. Under saline conditions (2% and 4% NaCl), the solubilization of the two phosphate sources in liquid medium corresponded to a decrease in the medium pH.

Bacillus strain V39 showed an even higher phosphate-solubilizing activity when the NaCl concentration increased to 4%. The salt concentration effect varied specifically with the bacterial strain; however, all selected bacterial strains solubilized at least the same amount of P from both phosphate sources (TCP and CRP) under 2% and even 4% NaCl as under normal growth conditions.

All six selected bacterial strains caused a significant increase in shoot dry weight compared to the non-inoculated control plant under P-deficient and high salt stress conditions (CRP + high S). Among the six bacterial inoculations, two bacterial strains, V54 (Arthrobacter sp.) and V39 (Bacillus sp.), significantly enhanced plant height under CRP and CRP + high S; V54 alone induced a significant increase in plant height under CRP + low S. Strain V54 exhibited the greatest effect on plant height under CRP (24.1%) and CRP + low S (26.1%), whereas V39 promoted the highest plant height under CRP + high S (27.8%).

Effect of Bacteria on P uptake of the tomato plant phosphorus shoot and root contents as a direct measure of PGP activities were clearly affected by different bacterial inoculants. Three strains (V39, V54, and V62) under CRP, two strains (V54 and V62) under CRP + low S, and all the strains under CRP + high S resulted in significant increases in P shoot content of tomato plants compared to the negative control. The maximum increase was recorded for plants inoculated by Bacillus strain V39 in CRP (1.8 mg shoot−1 ), while Arthrobacter strain V54 induced the highest P shoot content under CRP + low S (1.1 mg shoot⁻¹ ) and CRP + high S (1.0 mg shoot⁻¹ ). The result of bacterial inoculation revealed that the highest amount of total P uptake was obtained in tomato plants inoculated with Bacillus strain V39 (2.0 mg plant⁻¹ ) and Arthrobacter strain V54 (2.0 mg plant⁻¹ ), with an increase of 122% and 118%, respectively, over the non-inoculated control plants under CRP. The maximum value of the total P uptake was achieved by inoculation with Arthrobacter strain V54 under CRP + low S (1.3 mg plant⁻¹ ) and CRP + high S (1.2 mg plant⁻¹ ), which induced an increase of 65% and 117%, respectively, over the non-inoculated control. Only the Arthrobacter strain V54 additionally induced a significant higher P concentration (mg P per g plant dry matter) in the tomato shoot under all salt conditions  while the Bacillus strain V39 inoculation yielded in significant higher shoot dry matter content under all salt conditions compared to the respective non inoculated control.

Conclusions

In this study Arthrobacter and Bacillus strains help tomato plants cope better with double stresses of salinity and P deficiency under controlled glasshouse conditions. This study highlights the capacity of the selected bacteria to solubilize phosphate in the presence of high salt concentrations and to promote tomato plant growth under P deficiency and even under combined P and salt stresses. This provides valuable information for producing effective bio-fertilizers based on the combined application of rock phosphate and halotolerant phosphate-solubilizing bacteria, and offers promising potential to improve plant growth in P-deficient and salt-affected soils.

Reference:

Tchakounté, G.V.T., Berger, B., Patz, S., Becker, M., Fankem, H., Taffouo, V.D. and Ruppel, S., 2020. Selected rhizosphere bacteria help tomato plants cope with combined phosphorus and salt stresses. Microorganisms, 8(11), p.1844.