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Home > Potential of indole-3-acetic acid-producing rhizobacteria to resist Pb toxicity in polluted soil
Potential of indole-3-acetic acid-producing rhizobacteria to resist Pb toxicity in polluted soil
Bushra Rehman, Tamoor Ul Hassan & Asghari Bano
To cite this article: Bushra Rehman, Tamoor Ul Hassan & Asghari Bano (2019) Potential of indole-3-acetic acid-producing rhizobacteria to resist Pb toxicity in polluted soil, Soil and Sediment Contamination: An International Journal, 28:1, 101-121, DOI: 10.1080/15320383.2018.1539947
To link to this article: https://doi.org/10.1080/15320383.2018.1539947
Department of Plant Sciences, Quaid-i-Azam University, Islamabad, Pakistan; Department of Botany, Hazara University, Mansehra, Pakistan;
Department of BioSciences, University of Wah, Wah Cantt, Pakistan
Introduction
With the advent of modern civilization, industries are growing rapidly and uses of chemicals and heavy metals are increasing, which is adversely effecting the environment. Agricultural land bordering industrial areas are severely polluted with heavy metals (Guan et al., 2014). Human activities are also instigating serious changes in the environment. It has been observed that there are drastic changes in heavy metal concentrations resulting from mining, gas exhaust, metal smelting, and fuel production of power lines (Abdalla and El-Khoshiban 2012).
Oil refineries and drilling sites are often contaminated with heavy metals, and crops growing on the disposal sites of such areas face severe heavy metal stress. It has been observed that soils near oil refineries contain high contents of Pb, Cu, Cd, and Ni (Freije, 2015). Most plant species, including crops and weeds, cannot survive on oily polluted sites due to the toxic effects of heavy metals (Chibuike and Obiora, 2014). Remediation of heavy metal from such areas is necessary for the smooth functioning of the biogeochem- ical cycle (Wei and Yang, 2010). For petroleum hydrocarbon (PHC), which contaminates marine and terrestrial ecosystems, bioremediation is an effective tool (Scoma et al., 2016; Wang et al., 2016; Xue et al., 2015).
The plant-growth-promoting rhizobacteria (PGPR) are very effective to increase the growth of plants. They are associated with plant roots and may exert some beneficial effects on plant growth and nutrition through a number of mechanisms such as nitrogen fixation, production of siderophores, and transformation of nutrient elements when they are either applied to seeds or incorporated into the soil (Gopalakrishnan et al., 2015).
During rhizoremediation, exudates derived from the plant can help stimulate the survival and action of bacteria, which subsequently results in a more efficient degradation of pollutants. Rhizoremediation is the application of bacteria that can survive better in the root exudates of plants and contribute to sequester, detoxify, or degrade pollutants (Kuiper et al., 2004). Rhizobacteria not only mitigate heavy metal toxicity but also act as plant growth promoter (PGP) for crop. Rhizoremediation process has been reported to be an effective, low-cost, and socially accepted technology to remediate polluted soils (Chibuike and Obiora, 2014).
Indole-3-acetic acid (IAA) produced by microbes interferes a lot in the physiological processes of plants, and its level is significantly changed during stresses. Ahemad and Kibret (2014) documented that IAA not only affects plant cell division, differentiation, cell extension, stimulates growth and root development but also assists in water movement, controls vegetative growth, and initiates adventitious and lateral root formation. Physiologically, it mediates responses to light, florescence, and gravity; pigment formation; and affects photosynthesis and biosynthesis of metabolites.
The uptake, transport, and accumulation of Pb in plants are strongly dependent on soil type and plant species (Zaefarian et al., 2012). In maize, Pb mostly accumulates in roots, shoots, and seeds. Bashmakov et al. (2017) claimed that maize can help in the phytoex- traction of Pb and may remove 90 kg Pb per km2 annually.
Toxicity of Pb is distressing for both human and plants, but plant roots have the ability to tolerate a certain level of Pb. The permissible level of Pb in soil is less than 100 mg kg−1 and such soils are considered as non-polluted (Yilmaz et al., 2009). The tolerance level of plants to Pb varies, as crop plants are less immune. Higher accumulation of Pb in plants, especially in vegetables, is fatal when the Pb level is higher than 0.025 mg kg−1 per human body weight (Corley and Mutiti, 2017). Pb toxicity in soil stunts foliage, leads to wilting of leaves, generates reactive oxygen species (ROS), and inactivates enzymes (Fahr et al., 2013).
Growth and development of plants in response to environmental stresses involve multiple regulatory processes. Many studies have highlighted the effectiveness of rhizo- bacteria and their role in the bioremediation of PHC (Oliveira et al., 2015; Stroud et al., 2007; Wang et al., 2016); however, the role of IAA producers as a petroleum bioreme- diator has never been studied. Pb toxicity prevails in soil as a result of various anthro- pogenic activities, leaked gasoline, lead-based paints, coal combustion residues, and disposal of high metalliferous and petrochemicals (Zhang et al., 2010).
Application of IAA-producing bacteria for growth promotion of plants is a common practice as 90% culturable bacteria have IAA-producing potential (Idris et al., 2007). In such conditions, cross talk of IAA homeostasis and heavy metal toxicity is of particular interest, as this phytohormone mediates several attributes of plant growth and develop- ment (Lauro et al., 2017). Toxicity of Pb leads to the degradation of IAA, but exogenous application of IAA may compensate losses (Hac-Wydro et al., 2016).
In view of the serious hazardous effects of Pb on human health and ecosystem, the present investigation was aimed to isolate rhizobacteria from oil-contaminated soil, determine their tolerance level against applied Pb, and compared the role of IAA in the phyto-/bioremediation of Pb and on plant growth when used as a bio-inoculant on maize.
Materials and methods
Maize Zea mays L. plants were collected from a cultivated field at about 100 feet away from the Attock Oil Refinery waste disposal site (33° 32ʹ 0” North, 73° 5ʹ 0” East). Attock Oil Refinery is situated in Morgah, Rawalpindi, at a distance of 29 km from Islamabad, Pakistan. Plants were uprooted at vegetative stage (40 DAS), and from the top soil and the rhizosphere soil (7–10 cm depth), six bacterial strains were isolated. Among these, two bacteria E. aurantiacum strain MG228428 and B. firmus strain MG229068 were screened on the basis of Pb tolerance in culture media for application.
A greenhouse experiment was conducted to compare the nutrient content of soil and leaves with and without Pb and in combination with PGPR and a plant growth regulator IAA. Seeds of maize variety Kashmir Gold were collected from National Agricultural Research Center, Islamabad. Garden soil (EC = 0.3 decisiemen per meter, pH = 7.2, and field capacity = 55–60%) was mixed with sand at the ratio of 3:1 and autoclaved. The autoclaved soil/sand mixture was filled in earthen pots measuring 17 × 20 cm2, containing 8 kg soil/pot. No chemical or organic fertilizer was added in the soil. In each pot 7–10 seeds were sown and thinned to five plants/pot after germination.
Treatments comprised Pb = 500 ppm Pb, seed soaking with E. aurantiacum, seed soaking with B. firmus, E. aurantiacum + 500 ppm Pb, B. firmus + 500 ppm Pb, E. aurantiacum + 500 ppm + IAA (10−5 M), and B. firmus + 500 ppm Pb + IAA (10−5 M). Pb (NO3)2 at 500 ppm was applied to the soil with irrigation water prior to sowing (Zaefarian et al., 2012). Next, 50 g of seeds was soaked in 100 mL of IAA (10−5 M) for 12 h and re-dried under shade.
Seed sterilization was carried out by the method described by Almaghrabi et al. (2013) for maize seeds. Seeds were than incubated in 10 mL LB broth having bacterial isolates at the rate 1 × 108 CFU/mL at room temperature for 24 h. For each treatment, five replicates were made. Plants were harvested at two-leaf stage, after 80 days of sowing.
Isolation of rhizobacteria and seed inoculation
Soil (1 g) was taken at the depth of 6 cm from the top layer of maize rhizosphere of polluted areas mixed with 9 mL of autoclaved distilled water. The suspension was centrifuged at 3000 g for 10 min. The supernatant was used for making decimal dilutions. Next, 100 µL of decimal dilutions was used to spread on Luria Bertani (LB) agar media in petri plates from where a single colony was obtained. The LB broth was inoculated with fresh (24 h old) bacterial culture. The inoculated LB broth was incubated in a shaker for 48 h and centrifuged at 10000 g (10 min). Bacterial pellet was mixed with distilled water, and the optical density (at 660 nm) was adjusted to 1. E. aurantiacum and B. firmus had 12 × 10 8 and 11.6 × 10 8 CFU, respectively. The inoculated broth was used as a bio-inoculant on maize. Sterilized seeds of maize were soaked for 3 h in microbial culture of E. aurantiacum and B. firmus having 107cell/ mL. After shade drying of 45 min, seeds were sown in pots. Another set of seeds was sown in Pb solution and IAA solution prior to sowing, shade dried for 45 min, and sown.
Colony forming units
Viable cell counts were calculated as suggested by James (1978).
Viable Cell Count (C.F.U/mL) = (No. of colonies × dilution factor/volume of inoculum)
Quantification of IAA production in Salkowski reagent
Production of IAA in culture media was determined in Salkowski reagent using the Salkowski method (Ehmann, 1977). Bacterial strains were grown on LB broth and yeast malt dextrose broth (YMD broth) for 4 days at 28 °C. Bacterial cells were centrifuged at 5500 rpm for 10 min. One milliliter supernatant was mixed with 2 mL of Salkowski reagent containing 2% 0.5 FeCl3 in 35%
HClO4 solution. Absorbance was measured at 535 nm on a spectrophotometer, and IAA concentration was calculated with a standard curve.
Electrical conductivity and pH of soil
The pH of rhizospheric soil was measured by preparing 1:1 (soil:water) suspension (McKeague, 1978; McLean, 1982). Air-dried soil sample (10 g) was mixed in 10 mL distilled water and stirred for 1 h on a magnetic stirrer for homogenous mixing. Then the suspension was filtered with Whatmann No. 42 filter paper. The pH of the filtrate was determined with a pH meter, while the Electrical conductivity (EC) of the extract was recorded by an electrical conductivity meter. Readings were measured in microsiemens per centimeter (µS/cm).
Field capacity of rhizospheric soil
The field capacity of rhizospheric soil was determined by the method of Grewal et al. (1990).
Nutrient analysis of rhizospheric soil
The rhizospheric soil was analyzed for macro- and micronutrients (Na, Ca, Mg, K, Fe, Pb, Cd, Co, Zn, and Mn) following the Ammonium Bicarbonate-DTPA method developed by Soltanpour and Schwab (1977).
Extraction solution preparation
Extraction solution (0.005M) was determined by adding 1.97 g diethylenetriamine pen- taacetic acid (DTPA) to 800 mL distilled water. Ammonium hydroxide (2 mL) was added to facilitate dissolution. After dissolving DTPA, 79.06 g of ammonium bicarbonate (NH4HCO3) was added to the solution, and the pH was adjusted to 7.6 with ammonium hydroxide. The solution was diluted to 1 L with distilled water.
Extracting solution (10 mL) was added into 10 g air-dried rhizosphere soil. The mixture was shaken on a reciprocal shaker at 180 cycles/min for 15 min. Soil extract was filtered through Whatmann No. 42 filter paper. This rhizospheric soil extract was used to analyze different macro- and micronutrients on atomic absorption spectroscopy (Agilent, California 240 AA).
Nutrient analysis of plant tissues by the wet acid digestion method
Plants roots and leaves were analyzed for toxic heavy metals (Pb, Cd, Zn, Co, and Mn) uptake following the wet acid digestion method devised by Kingston and Haswell (1997). Plant tissue (1 g) was transferred to a 100 mL flask, and 10 mL of nitric-perchloric acid mixture (2:1) was added and allowed to stand overnight. After preliminary digestion, flasks were placed in a fume hood, and the temperature was raised to 150 °C for 1 h. The temperature was increased gradually to 235°C until all traces of nitric acid disappeared. Then it was allowed to cool for a few minutes, and then a few drops of distilled water were added carefully through a funnel. The extract was filtered by Whatmann No. 42 filter paper, and the volume of the filtrate was made up to 50 mL with distilled water. The extract was analyzed by atomic absorption spectroscopy (Agilent, California 240 AA).
Enrichment factor
The value of EF was calculated using the modified formula given by Loska et al. (2004) and the equation suggested by Buat-Menerd and Chesselt (1979):
EF = Cn (sample)/Cref (sample)
Bn (background)/Bref (background)
Cn (sample) = environmental content of the examined element.
Cref (sample) = content of the reference element in the examined environment.
Bn (background) = content of the examined element in the reference environment. Bref (background) = content of the reference element in the reference environment.
Geo-accumulation index (Igeo)
The Igeo was calculated by the Muller (1969) formula:
Igeo = log2 (Cn/1.5Bn)
Metal pollution index (MPI)
The MPI being a contamination index was calculated by the equation of Usero et al. (2000):
MPI = (Cf1× Cf2× Cf3×. . .Cfn) 1/n
Cf = contamination factor
Pollution load index (PLI)
PLI was calculated by the equation of Boszke et al. (2004):
PLI = (Cf1× Cf2× Cf3. . .Cfn)
Ecological risk assessment
According to Hakanson (1980), the potential ecological risk index (RI) is calculated by RI = ∑ (Ti × Ci/Co)
i = 1
BCF, biological accumulation coefficient (BAC), and TF
BCF was calculated as the metal concentration ratio of plant roots to soil given in the equation by Yoon et al. (2006). TF and BAC were calculated by the equations of Cui et al. (2007) and Li et al. (2007).
BCF = element content in root/element content in soil TF = element content in shoot/element content in root BAC = element content in shoot/element content in soils
DNA extraction
Extraction of genomic DNA of bacterial strain was carried out by using the Gen Elute Bacterial Genomic DNA Kit.
Heavy metal tolerance of PGPR
The bacterial strains were evaluated for their tolerance potential against different con- centrations of Pb (NO3)2 using the agar dilution method (Silambarasan and Jayanthi, 2010). The stock solution (100 ppm and 500 ppm) of Pb (NO3)2 was filtered through a millipore filter (0.22 µm, Corning® Germany). Two sets of petriplates were made contain- ing 100 and 500 ppm of Pb (NO3)2. Broth culture of bacteria (20 µl) having 108 cells of bacteria was spread on agar plates containing 100 and 500 ppm of Pb (NO3)2. Agar plates were than incubated for 24–48 h at 28°C until the colony appeared. For each set of plates, four replications were used. Agar plates lacking Pb (NO3)2 were taken as negative control. Appearance of bacterial colonies containing different concentrations of Pb indicated the survival efficiency of bacteria. The experiment was repeated in triplicate. Colony forming units were calculated as suggested by James (1978).
Determination of IAA in leaves
The extraction and purification for IAA were made following the method of Kettner and Doerffling (1995). Plant leaves (1 g) were ground in 80% methanol at 4°C with an antioxidant, butylated hydroxyl toluene (BHT) at 10 mg/l. The leaves and roots were extracted at 4°C in dark for 72 h with subsequent change of solvent. The extracted sample was centrifuged, and the supernatant was reduced to aqueous phase using rotary thin-film evaporator (RFE). The pH of the aqueous phase was adjusted to 2.5–3.0 and partitioned four times with half volume of ethyl acetate. The ethyl acetate was dried down completely using RFE. The dried sample was redissolved in 1 mL of methanol (100%) and analyzed on High performance Liquid Chromatography (HPLC) (Shimadzu, C-R4A Chromatopac; SCL-6B system controller, Japan) using a UV detector and a C-18 column (39x300 mm) for the identification of hormones. The sample (100 µl) was filtered through a 0.45 milli- pore filter and injected in column. Methanol, acetic acid, and water (29:1:70) were used, and the flow rate (0.5 mL/min) was adjusted for an average run of 15 min/sample. Pure IAA (Sigma Aldrich, USA) was used as standard, and IAA was identified on the basis of retention time and peak area. The detection of IAA was made at 280 nm (Sarwar et al., 1992).
Statistical analysis
The study was conducted using Complete Block Design with five replicates. The data were subjected to analysis of variance (ANOVA) using Statistix software 8.1 version to compare the effects of different treatments with control. The differences between the means were separated according to Steel and Torrie (1980) by least significant difference (LSD) at p = 0.05.
Results
Result from the 16s rRNA gene sequence with data nucleotide bank revealed that one of our isolates had 99% nucleotide similarity 1496/1508 with Exigobacterium auran- tiaca strain KJ722475. For the other strain, comparison of the nucleotide sequence with data nucleotide bank confirmed 99% similarity 1511/1520 nucleotides with Bacillus firmus strain MH071301. Nucleotide sequences were submitted to NCBI, and accession numbers MG228428 for E. aurantiacum strain and MG229068 for B. firmus were obtained.
Heavy metal tolerance potential of bacteria
Results revealed that survival efficiencies of E. aurantiacum and B. firmus declined by 12% and 15%, respectively, over the control at 100 ppm of Pb (Figure 1). At 500 ppm of Pb, CFU of E. aurantiacum and B. firmus decreased by 30% and 28%, respectively, as compared to the control.
Physicochemical analysis and pollution indices of soil
The rhizosphere soil analyses revealed that the polluted rhizosphere soil had electrical conductivity of 190 µS cm−1 and pH 6.85 (data not presented in table). PLI, MPI, and ecological risk assessment index (RI) of the oil-polluted field were 2.5, 0.3, and 27.3, respectively (Figure 2). The enrichment factor for Pb and Zn was 21 and 29, respec- tively, which was significantly higher than that for Cd and Mn (Figure 3). The Igeo of Pb was 1.2, which was higher than the Igeo of Cd, Zn, and Mn of the same soil (Figure 4).
IAA production in culture media and accumulation in treated leaves
E. aurantiacum and B. firmus exhibited IAA production in culture media. Addition of Pb at 500 ppm in culture media of E. aurantiacum and B. firmus significantly (40%–45%) inhibited IAA production in culture (Figure 5). Addition of IAA at 10−5 M in culture media inoculated with E. aurantiacum and B. firmus + 500 ppm Pb induced 30% and 20% more IAA over single inoculation of E. aurantiacum and B. firmus.
Inoculation of E. aurantiacum and B. firmus improved the IAA content of leaves by 27% and 34%, respectively, as compared to the control (Figure 6). Application of Pb with
E. aurantiacum and B. firmus reduced the IAA content by 25% in maize leaves over single inoculation. IAA application with E. aurantiacum and Pb accumulated 46% higher IAA in leaves over control. Similarly, 56% higher IAA was observed in maize leaves following the application of B. firmus + Pb + IAA.
Nutrient content in rhizosphere soil, roots, and leaves of maize collected from a polluted area
The Ca contents of maize growing in an oil-contaminated field were 450% higher in roots than in soil. Roots accumulated 16% higher Ca than leaves (Table 1). There were no significant differences between the Na contents of roots and soil, but 135% higher Na was observed in leaves as compared to roots and soil. Roots and leaves accumulated 14% greater Mg than soil. Roots accumulated 45% higher K and 30% less Fe than soil. Translocation of K was 31% higher from roots to leaves, but Fe was 50% less in leaves than in roots.
Heavy metal content in soil, roots, and leaves of maize collected from a polluted area
Maize growing in an oil-polluted field had 10% more Pb content in polluted roots than soil, while roots accumulated 50% higher Pb than leaves (Table 2). The Cd and Co contents were, respectively, 66% and 56% lower in roots than in soil. Leaves contained 33% less Cd and 17% Co than roots. Zn and Mn contents were, respectively, 192% and 610% higher in roots than soil, and translocation of Zn from roots to leaves was 40% less.
BCF, BAC, and TF
The values of BCF, BAC, and TF were more than one for Pb in oil-polluted soil (Table 3). TF value for Mn was also higher in all the tested metals.
Effects of different treatments on Pb accumulation in soil, roots, and leaves of
Zea mays in a greenhouse experiment
Inoculation of E. aurantiacum and B. firmus decreased 26% Pb accumulation in soil and 30% less Pb in leaves and roots over un-inoculated control (Table 4). E. aurantiacum and B. firmus application with 500 ppm Pb decreased 13% Pb in soil and 15% in roots and leaves over single inoculation of these strains. Addition of IAA with E. aurantiacum or B. firmus decreased 37% Pb in soil, 30% in roots, and 31% in leaves in Pb-stressed condition over control. No significant effects were observed on BCF and TF among the treatments. BAC was 11% higher over control in the plants treated with E. aurantiacum or B. firmus and supplemented with IAA under Pb-stressed condition.
Effects of different treatments on Pb accumulation on the growth characteristic of Zea mays grown in greenhouse
At 80 DAS, significantly higher (20%) plant height and shoot dry weight were observed in PGPR-inoculated plants (Table 5). Application of Pb with E. aurantiacum and B. firmus decreased plant height by 11% over single inoculation of these PGPR. Plants treated with IAA, Pb, and E. aurantiacum or B. firmus attained maximum height (172 cm) and shoot dry weight (107 g).
Chlorophyll content of maize leaves was increased by 18–24% when E. aurantiacum or B. firmus was applied as bio-inoculants (Table 5). Addition of Pb with PGPR decreased the chlorophyll content of treated maize leaves by 10%. The highest chlorophyll contents (37% greater than control) were observed when PGPR was inoculated with Pb and IAA.
Root length and root dry weight were, respectively, 30% and 32% higher over control in a single inoculation of E. aurantiacum and B. firmus (Table 5). Addition of Pb with
E. aurantiacum or B. firmus decreased root length and root dry weight (12–14%) over single inoculation. Maize plants attained 60% higher root length and root dry weight over control when E. aurantiacum and B. firmus were applied with Pb and IAA.
Discussion
In the present study, two bacteria viz. E. aurantiacum and B. firmus – were isolated from maize rhizosphere growing in an oil-polluted field and applied to check the bioremedia- tion potential of Pb. B. firmus has previously been isolated from a sugarcane field and was found to be helpful in the degradation of extensively used insecticide fipronil (Mandal et al. 2014). B. firmus has also been reported for the degradation of polcyclic aromatic hydrocarbons (Bayoumi, 2009). Similarly, Jeswani and Mukhergi (2012) documented the role of Exiguobacterium aurantiacumin in the degradation of polycyclic aromatic compounds.
The soil was polluted with Pb, Zn, Co, and Mn, whereas leaves accumulated Pb and Zn in much higher concentrations. Both BAC and BCFs were higher for Pb, whereas the TF value of Co was higher. Multiple factors like cation-exchange capacity, particle size, root architecture, and soil microflora are involved in Pb uptake (Tangahu et al., 2011). Plants face hostile environment under heavy metal stress soil because organic matter and nutrient availability are limited and soil acidification is higher (Becerra-Castro et al., 2012). The Igeo for the sampled soils was more than 1 and PLI values were more than 2 for Pb, which also indicates that soil was polluted with Pb according to the standards value (Omwene et al., 2018). MPI and R.I values were below as per standard risk indicator values. The Igeo, RI, and PLI are considered reliable sources for measuring the toxicity of environmentally sensitive elements (Shi et al., 2014; Zhao et al., 2014).
Heavy-metal-tolerant bacterial strains having plant-growth-promoting potential are valuable sources for reclaiming soil fertility and crop growth. In the present study, two bacterial strains E. aurantiacum and B. firmus isolated from oil-contaminated soil of Attock Oil Refinery were tolerant against Pb toxicity and IAA producer.
The bacterial strains E. aurantiacum and B. firmus were found tolerant to Pb applied in the culture medium up to 500 ppm as indicated by their growth pattern; this may be attributed to their IAA production in the culture medium. Both the strains showed higher production of IAA in culture media, but in the presence of Pb their IAA production potential decreased. Noteworthy, exogenous application of IAA either in the culture media stressed with Pb or to the plant augmented IAA production several fold higher than that of without IAA or without Pb. Yu et al. (2014) and Chen et al. (2016) showed the potential of IAA-producing bacteria to tolerate high concentrations of heavy metals in culture media. Escherichia, Enterobacter, and Serratia strains have been characterized for Pb tolerance and IAA production (Carlos et al., 2016). Previously, foliar application and seed soaking of IAA increased the phytoextraction of Pb in Zea mays and Picris divaricate (Du et al., 2011; Hadi et al., 2010).
The data revealed the ameliorative effects of PGPR and IAA on the nutrient contents of rhizosphere soil as well as roots of maize plants. While working on Pb and Cd stresses, Mojiri (2011) also found similar results in corn.
Pb treatment enhanced Pb uptake in roots and decreased its translocation to leaves, and inoculation of E. aurantiacum and B. firmus augmented this process. Oladejo et al. (2017) reported higher accumulation of Pb in shoots, leaves, and stems compared to roots of maize plants collected from a dumping site that receives commercial and municipal solid waste in Nigeria.
Seed soaking treatment with IAA significantly inhibited Pb accumulation in roots and more so in leaves. Fässler et al. (2010) demonstrated better phytoextraction of Pb in roots and shoots of sunflower following the application of different concentrations of IAA. Accumulation of heavy metals in edible parts of plants above the permissible limit is alarming because they provide basic diet to human and other animals (Zhao et al., 2014; Zhou et al., 2016). Corley and Mutiti (2017) documented that Pb translocation from soil to cabbage imparted serious consequences on human health.
Maize has the ability to reduce the Pb in soil because of its potential to accumulate heavy metals in roots and leaves. Sessitsch et al. (2013) demonstrated that microbial modification in heavy-metal-polluted soil improves absorptive properties of the roots by altering the root architecture and subsequent translocation. Salopek-Sondi et al. (2015) documented the contribution of IAA as a major factor in the shaping of root architecture under abiotic stresses. Mishra et al. (2016) revealed that application of Arbuscular mycorrhizal fungi Glomus, Acaulospora, and Scutellospora with plant-growth-promoting rhizobacteria belonging to genera Streptomyces, Azotobacter, Pseudomonas, and Paenibacillus reduced Fe toxicity in soil.
IAA-producing bacteria possibly help in the detoxification of Pb as evidenced by the root and shoot growth of the plant and other related parameters. It has been previously reported that exogenous application of IAA at 10 µM improved tolerance against heavy metal toxicity to Pisum sativum L. (Gangwar et al., 2012). Kumar et al. (2012) reported that exogenous application of IAA at 20 ppm reduced the accumulation of Pb in seeds of winter wheat (Triticum aestivum L.). Recently, Lyu et al. (2018) demonstrated that the addition of modified nanoscale carbon black (MBC) in Cd-contaminated soil decreases soil pH and Cd in bulk soil. Kaur et al. (2018) recommended that co-inoculation of Pseudomonas, Azotobacter, Azospirillum, Actinomyces, and Bacillus is better than single inoculation for bioremediation of As from wastewater of municipal systems. Similarly, Bacillus sp. SC2b, a Pb-resistant PGPR, was found to be effective in adsorbing heavy metals Cd, Pb, and Zn and improving plant growth (Ma et al., 2015). Toxicity of Pb and Zn was reduced by the application of Bacillus thuringiensis and Bacillus subtilis (Kumar et al., 2015a).
E. aurantiacum significantly decreased Pb content over that of the control. It was observed that the application of Acidithiobacillus thiooxidans as bio-inoculant assisted in the removal of toxic heavy metals from soil (Kumar and Nagendran, 2009). Upadhyay (2011) documented that rhizobacteria ameliorate the toxic effects of heavy metals by changing their valencies and bioavailability. Dary et al. (2010) demonstrated that the application of Bradyrhizobium sp., Pseudomonas sp., and Ochrobactrum cytisi alone as well as in consortium decreased Pb in soil, root, and shoot of Lupinus luteus after a mine spill.
Applications of bio-inoculants increased plant height of maize plant, shoot dry weight, number of leaves, chlorophyll, and IAA content of leaves in Pb-stressed soil. Application of Pb in culture media with bio-inoculant halted the growth of maize plant, but the addition of IAA with bio-inoculants ameliorated the adverse effects of Pb, demonstrating that these results suggest that IAA-producing bacteria induced tolerance of plants to Pb toxicity, and exogenously applied IAA assisted these bacteria and enhanced their efficacy so that minimum Pb was translocated to leaves. Auxin production is the function of 80% rhizosphere isolates (Idris et al., 2007).
Synthesis of IAA by rhizosphere bacteria in metalliferous environment seems to be the defense strategy of plants, which is vital for plant growth and development (Ahemad and Kibret, 2014). Mesa et al. (2015) reported that IAA production of Bacillus aryabhattai SMT48 was significantly increased in the presence of heavy metals. Similarly, Carlos et al. (2016) documented that IAA production was improved in four different bacteria in the presence of high Pb concentration. Kumar et al. (2015b) documented positive roles of plant growth promotion, IAA production, and Pb detoxification of Bacillus thuringiensis, Azotobacter chroococcum, Paenibacillus ehimensis, and Pseudomonas pseudo alcaligenes.
Among the growth parameters, control plants showed less shoot and root length, chlorophyll, and shoot and root dry weight. Ghani (2010) also observed decreases in maize growth and protein contents under Pb toxicity. Increases in root length, stem length, and chlorophyll of H. annuus were observed when Serratia K120 was applied as bio-inoculant under Pb and Zn stress (Marques et al., 2013). Pb-resistant and ACC deaminase-producing endophytic bacteria were reported to increase biomass Brassica napus (Zhang et al., 2011). Noteworthy, the combined effects of bio-inoculants and IAA were better than all other treatments. Growth improvements in different parameters of sunflower were observed when different concentrations of IAA were applied to Pb + Zn- stressed plants (Fässler et al., 2010). IAA plays a pivotal role in root structuring and brings out changes in water absorption and nutrient uptake/accumulation (Du et al., 2011).
Conclusions
The combined effects of exogenously applied IAA and IAA producing PGPR E. aurantiacum and B. firmus on the amelioration of Pb toxicity and maize growth are noteworthy and can be recommended for the bioremediation of other heavy metals to be tested. Supplementation of IAA with bacterial strains not only contributed significantly to the alleviation of Pb toxicity but also resulted in improved growth and physiology of maize. Application of these heavy- metal-tolerant bacteria with other plant-growth-promoting rhizobacteria in consortium may be helpful for the better reclamation of heavy metal toxicity.
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