کود زیستی چند منظوره از Pseudomonas putida PT: رویکردی برای تحریک همزمان رشد ذرت و زیست‌پالایی خاک‌های آلوده به کادمیوم

Introduction

Pollution of agricultural soils with heavy metals, due to the disposal of untreated toxic waste of industries such as mining, leatherworking, metallurgy and electric appliance manufacturing, has become a serious problem in the world (1-6). On the other hand, increasing world’s population has dramatically increased the demand for high yield crops (7). This matter has caused to overuse chemical fertilizers, leading to a large-scale release of heavy metals into the environment, particularly Cd(8), and decline of soil quality and fertility (9). Soluble heavy metals in the soil solution or those that are easily solubilized by root exudates can be uptaken by plant (10). The reduction of plants growth and crops yield are the results of heavy metal accumulation in plant tissues (1, 11). On the other hand, plants contaminated with heavy metals (vegetables, fruits, and grains) are the most important metal entrance routes to the human food chain especially in the case of cadmium (12-15). Cadmium seriously threatens human health with its carcinogenic, mutagenic and teratogenic properties (16). Since heavy metals, unlike some other pollutants, cannot be decomposed to compounds with low toxicity, their toxic effects increase gradually in the environment (17). So, the remediation of metal-contaminated soils is necessary to keep sustainable agriculture and subsequently a healthy life. High costs, incomplete metal removal, forming secondary pollution, destruction of soil texture and decreasing soil fertility are the disadvantages of physicochemical methods used for remediating polluted soils. In recent years, biological methods (bioremediation) have gained more prominence as an alternative technology due to simplicity, efficiency and low costs (18). Plant growth promoting bacteria (PGPB) are a heterogeneous group of bacteria that can enhance plant growth by synthesizing or facilitating the uptake of nutrients from the soil. Also, they can protect plants against phytopathogenic microorganisms (19-26). Using heavy metal resistant microbes promoting plant growth is a low-cost and eco-friendly approach to remove metals from the soil and increase plant growth without effects on soil structure (27). The soil is an unpredictable environment, so finding a suitable niche for survival amongst the competitors, predators and harsh environmental conditions like toxic compounds for inoculated bacteria is often difficult. In general, after the inoculation of bacterial cells without a suitable formulation into the soil, the bacterial population decreases rapidly. A major function of inoculant formulation is to provide a proper microenvironment for improving the survival and retention of bacterial cells (28). Also, an appropriate formulation protects the stability of bacterial cells during storage and transportation (29). Formulation of inoculant is an industrial art which converts a laboratory proven bacteria to a commercial product. The aim of this study is to produce a multipurpose biofertilizer which can remove cadmium from polluted agricultural soils as well as enhancing plant growth. To achieve this objective: (1) the effects of some organic compounds present in plant root exudate on growth, chemotactic responses and biofilm formation of P. putida PT as a plant growth promoting bacteria were evaluated; (2) biofertilizers were produced by two different methods (seed immersion and bacterial cells immobilization on rice bran); and (3) the effect of each biofertilizer on growthof Zea maize and the amount of Cd²⁺ adsorption from the soil by this plant was compared.

Materials and Methods

Source of Bacterial Strain: P. putida PT was isolated previously from the rhizosphere of plants cultivated in agricultural soil polluted by heavy metals. The selected bacterial strain was characterized by using standard morphological, physiological and biochemical tests according to Bergey’s Manual of Determinative Bacteriology, followed by molecular identification based on 16S-rRNA gene sequence analysis (GenBank accession NO. KX963368).

Chemicals and Media Preparation: A synthetic minimal salt medium (MSM) containing (g L^-1): KH₂PO₄, 2.25; K₂HPO₄, 2.25; (NH₄)2SO₄, 1.0; MgCl₂.6H₂O, 0.2; NaCl, 0.1; FeCl₃.6H₂O was used as basal medium for chemotaxis, growth and biofilm formation tests. Some amino acids (i.e. alanine, histidine and tryptophan), sugars (i.e. sucrose, mannitol and glucose) and organic acids (i.e. succinic acids, malic acid and citric acid) which had been reported in previous studies as the most important compounds present in plant root exudate (30) were selected for further investigation in mentioned experiments.  Stock solutions of these compounds were prepared in MSM. The metal stock solution was prepared in double-distilled water and sterilized by a syringe filter (0.45 µM). All chemicals were purchased from Sigma (Sigma Chemical Company, Germany) or Merck (Germany).

Chemotaxis Assay: Swim plates for the qualitative analysis of chemotaxis were prepared in MSM which was solidified by adding 0.2% agar (31). A colony of P. putida PT (grown on tryptic soy agar (TSA) medium (Typical composition (g/litre): pancreatic digest of casein 15.0; papaic digest of soya bean 5.0; sodium chloride 5.0; agar-agar 15.0.)) was inoculated into the agar at the center of each plate. Then, crystals of amino acids, organic acids and sugars were placed separately on the right side of each plate. After 24 h incubation at 30˚C, the halos of bacterial movement toward chemoattractants were investigated. The plate containing one drop of sterile distilled water instead of organic compounds was considered as negative control.

Evaluation of P. Putida PT Growth: P. putida PT growth in the presence of some organic compounds present in plant root exudates was evaluated using 96-well microtiter plate based on the protocol described previously (32) with minor modifications. Briefly, each well of the microtiter plate was loaded with 150 µL of different concentrations of organic acids (25, 50 and 100 µM), sugars (10, 50 and 100 mM) and amino acids (5 and 10 mM). Then, 50 µL of diluted bacterial culture in MSM (OD6_00nm= 0.1) was added to each well. Control wells contained 150 µL of MSM and 50 µL of bacterial culture. All experiments were done in triplicate. After 24 h incubation at 30˚C, the OD (optical density) was measured by an ELISA reader at 630nm.

Biofilm Formation Assay: Biofilm formation was assayed by following the protocol described previously with some modifications (32). Flat-bottomed microtiter plates were loaded according to conditions mentioned in the previous section and incubated at 30˚C for 24h.  The growth phase medium was removed carefully by pipetting, and each well was rinsed with 250μL of saline (0.09% NaCl). The remaining attached bacterial cells were fixed by adding 200 μL of ethanol to each well. After 15 min, the wells were emptied and left to dry at room temperature. Then, each well was stained with 200 μL of 0.1% (w/v) crystal violet solution for 10 min. Excess dye was rinsed off by normal saline slowly. Finally, 100 ml of 30% (v/v) acetic acid was added to each well, and the OD was read by ELISA reader at 630 nm after 15 min.

Biofertilizer Preparation: Biofertilizers were prepared in two different types of formulation: bacterial inoculation using the seed immersion method and immobilization on rice bran. P. putida PT was cultured in conical flasks containing 50 ml Tryptic Soy Broth (TSB) medium at 30°C on an orbital shaker at 120 rpm until reaching exponential growth phase. The bacterial suspension was adjusted to the approximate concentration of 2.5_˟10⁹ CFU/ml.

In seed immersion method, maize (Z. mays) seeds provided by the Soil and Water Research Institute of Isfahan were transferred to the bacterial suspension (1 seed per 1 ml of bacterial inoculants) and allowed to incubate for 30 min at room temperature. To prepare immobilized cells on rice bran, the bacterial suspension was added to the sterilized rice bran (1 ml of bacterial suspension per 1 gram of rice bran) and incubated at room temperature for 5 days (33).

Pot Experiments: Pot experiments for studying the effect of the biofertilizers on the plant growth and heavy metal uptake in Z. mays were conducted in greenhouse condition. Physicochemical properties of the soil used in the experiments were as follows: 55% sand; 18.4% silt; 24% clay; cation exchange capacity (CEC) of 7.5 cmol/kg; 10000 mg/kg Fe; 330 mg/kg Mn; 83 mg/kg Zn; 25 mg/kg Cu and 30 mg/kg Pb. Soil samples were dried at room temperature and passed through a sieve with a diameter of 2 mm. The metal salt was dissolved in distilled water and mixed thoroughly with the soil (~ 40000 ppb Cd in each kg of soil). The experiment involved three separate treatments with three replications: (I) plants growing in the inoculated soil with the immobilized bacterial cells on rice bran; (II) inoculated plants by seed immersion method (III) control treatment (plants growing in soil in the absence of bacterial cells). After 70 days of incubation, plants were removed from the pots carefully and were rinsed with distilled water. After measuring the fresh weight, dry weight of each plant was determined after drying at 70 ˚C for 48h. The cadmium concentration in Z. mays aerial tissues was determined using Atomic Absorption Spectrometry (AAS). All experiments were performed triplicates.

Statistical Analysis: One-factor analysis of variance (ANOVA) followed by Scheffe’s post hoc analysis were performed to assess the difference between different treatments. Level of significance unless specified is P < 0.05.

Results

Chemotaxis Assay: P. putida PT migration from the inoculation point to organic compounds added to swim plates exhibited positive chemotactic responses of this bacterium to all amino acids, sugars and organic acids tested in this study. No chemotactic response was detected in the negative control plate which was contained one drop of sterile distilled water instead of organic compounds (Figure 1).

Evaluation of P. Putida PT Growth: To evaluate the influence of plant root exudates on P. putida PT growth, different amino acids, sugars and organic acids at various concentrations were added to the microorganism culture medium. Citric acid at concentrations from 25 and 50 µM did not produce a significant effect on the microorganism growth. Malic acid at concentrations from 50 to 100 µM and succinic acid at the concentration of 100 µM significantly decreased P. putida PT growth. Succinic acid at concentrations from 25 to 100 µM and malic acid at the concentration of 25 µM significantly enhanced the growth compared to control (Fig. 2a). Alanine, tryptophan, and histidine revealed no significant effects on growth (Fig. 2b). Also, the results indicated that P. putida PT growth is independent regarding the variation in the concentration of glucose. The growth of microorganism significantly decreased by mannitol and sucrose at the concentration of 10 mM but, these sugars at concentrations from 50 to 100 mM enhanced the growth significantly (Fig. 2c).

Fig. 1- P. putida PT chemotactic response to (a) tryptophan, (b) alanine, (c) histidine, (d) malic acid, (e) citric acid, (f) succinic acid, (g) mannitol, (h) glucose and (i) sucrose in qualitative swim plate assays. A positive chemotactic response is indicated by the formation of a swimming ring toward the chemoattractant (arrows represent P. putida PT movement in the direction of the chemoattractants). No response was detected when only a drop of sterile distilled water was added (plate (j) as negative control).

Fig. 2- Effects of different types and concentrations of (a) organic acids, (b) amino acids and (c) sugars which are present in root exudates on P. putida PT growth. Each value is the mean of triplicates. Bars represent standard error. Data of columns indexed by the same letter are not significantly different (P < 0.05) according to the Scheffe test (p < 0.05). 

Biofilm Formation Assay: Biofilm formation in P. putida PT was measured following incubation in the presence of different types of amino acids, sugars, and organic acids which are present in plant root exudate. Twenty four hours after incubation, succinic acid at the concentration of 50 μM could significantly enhance the biofilm formation of P. putida PT. Malic acid and citric acid revealed no effects on the biofilm formation (Fig. 3a). Histidine at concentrations from 5 to 10 mM and tryptophan at the concentration of 10 mM significantly stimulated the P. putida PT biofilm formation compared to the control. Alanine represented no significant effect on the biofilm formation (Fig. 3b). All of the sugars tested in this study induced biofilm formation and this stimulatory effect was concentration-dependent. Glucose and sucrose at concentrations from 50 to 100 mM and mannitol at the concentration of 10 mM significantly increased biofilm formation (Fig. 3c).

Fig. 3- Effects of different types and concentrations of (a) organic acids, (b) amino acids and (c) sugars which are present in root exudates on biofilm formation in P. putida PT. Each value is the mean of triplicates. Bars represent standard error. Data of columns indexed by the same letter are not significantly different according to the Scheffe test (p < 0.05).