Appl Biochem Biotechnol (2014) 174:297–308 DOI 10.1007/s12010-014-1039-3
Siderophore as a Potential Plant Growth-Promoting Agent Produced by Pseudomonas aeruginosa JAS-25 M. B. Sulochana & S. Y. Jayachandra & S. Anil Kumar & A. B. Parameshwar & K. Mohan Reddy & A. Dayanand
Received: 28 June 2013 / Accepted: 25 June 2014 / Published online: 26 July 2014 # Springer Science+Business Media New York 2014
Abstract Siderophores scavenges Fe+3 from the vicinity of the roots of plants, and thus limit the amount of iron required for the growth of pathogens such as Fusarium oxysporum, Pythium ultimum, and Fusarium udum, which cause wilt and root rot disease in crops. The ability of Pseudomonas to grow and to produce siderophore depends upon the iron content, pH, and temperature. Maximum yield of siderophore of 130 μM was observed at pH 7.0±0.2 and temperature of 30 °C at 30 h. The threshold level of iron was 50 μM, which increases up to 150 μM, favoring growth but drastically affecting the production of siderophore by Pseudomonas aeruginosa JAS-25. The seeds of agricultural crops like Cicer arietinum (chick pea), Cajanus cajan (pigeon pea), and Arachis hypogaea (ground nut) were treated with P. aeruginosa JAS-25, which enhanced the seed germination, root length, shoot length, and dry weight of chick pea, pigeon pea, and ground nut plants under pot studies. The efficient growth of the plants was not only due to the biocontrol activity of the siderophore produced by P. aeruginosa JAS-25 but also may be by the production of indole acetic acid (IAA), which influences the growth of the plants as phytohormones. Keywords Siderophore . Agricultural plants . Growth promotion . Pseudomonas sp . Indole . Acetic acid
Introduction Severe global economic losses to agricultural crops are encountered due to plant diseases caused by more than 60 pathogens leading to the loss of 30 % crop yield about US$416 million [1]. Rhizosphere is a dynamic environment, which harbors diverse group of microbes. Some of the bacteria, which directly or indirectly stimulate plant growth, have been referred to as plant M. B. Sulochana (*) : S. Y. Jayachandra : S. A. Kumar : A. B. Parameshwar : K. M. Reddy Department of PG Studies and Research in Biotechnology, Gulbarga University, Gulbarga 585106 Karnataka, India e-mail:
[email protected] A. Dayanand Department of PG Studies and Research in Microbiology, Gulbarga University, Gulbarga 585106, India
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growth-promoting rhizobacter (PGPR) [2]. Siderophores are low-molecular mass compounds (1,500 Da) with high iron affinity [3, 4], which allows soil microorganisms to sequester and solubilize ferric ion in poor environments of iron. They are useful in health care as a drug and in agriculture for plant disease management and also in plant growth improvement [5, 6]. Public concern about chemical pesticides has fostered an interest in the application of bacteria for the biological control of pathogenic fungi affecting agricultural crops [7]. The biological control of plant diseases with bacterial antagonism is a potential alternative to expensive chemical control [8]. Biocontrol through siderophore-mediated competition for iron has emerged as a sustainable approach for integrated plant disease management [9–13]. Bacterial plant growth promotion can result from direct or indirect mechanisms, including siderophores production. Siderophore production was also postulated to be an important mechanism for the biocontrol activity of PGPR [14]. Recently, there has been an increasing interest in the use of biological control agents and siderophores produced by several fluorescent Pseudomonas, as an alternative to take into account, due to the fact that they reduce the rhizospheric population of phytopathogenic fungi and bacteria [15]. Pseudomonas sp.-producing siderophores play a vital role in stimulating plant growth and in controlling several plant diseases [16]. Siderophores are thought to facilitate the biocontrol activity by sequestering iron from pathogens, thus limiting their growth [17]. Any factor influencing either the growth or siderophore production by a bacterial antagonist would greatly influence the efficacy of that antagonist in plant growth promotion and disease suppression [18]. In the present investigation, the effect of siderophore produced by Pseudomonas aeruginosa JAS-25 on the plant growth has been studied. Significant growth of the agricultural plants was observed proportionally in the increasing order of chick pea followed by groundnut and pigeon pea.
Materials and Methods P. aeruginosa JAS-25 was isolated from the saprophytic soil sample in our laboratory and was used for the present study. The bacterial culture was maintained on King B medium at 4 °C by periodic subculturing. Type strain (MTCC 2581) was used as a positive control strain. 16S rRNA Identification of the Strain P. aeruginosa JAS-25 The genomic DNA was isolated as described by Ausubel [19], and PCR assay was performed using Applied Biosystems, model 9800 with 50 ng of DNA extract. The PCR master mixture contained 2.5 μL of 10× PCR reaction buffer (with 1.5 M MgCl2), 2.5 μL of 2 mM dNTPs, 1.25 μL of 10 pm/μL of each oligonucleotide primer 16S_8F (5′-AGAGTTTGATCCTGGC TCAG-3′) and 16S_1391R (5′-GACGGGCGGTGTGTRCA-3′), 0.2 μL of 3 U/μL Taq DNA polymerase and 15.76 μL of glass-distilled PCR water. Initially, denaturation was accomplished at 94 °C for 3 min and 32 cycles of amplification consisting of denaturation at 94 °C for 30 s, annealing at 55 °C for 30 s, and extension at 72 °C for 1.30 min. A final extension phase at 72 °C for 10 min was performed, and the PCR product was purified by PEG-NaCl method. The sample was mixed with 0.6 times volume of PEGNaCl, 20 % (PEG (MW 6,000) and 2.5 M NaCl) and incubated for 20 min at 37 °C. The precipitate was collected by centrifugation at 3,800 rpm for 20 min, and the pellet was washed with 70 % ethanol, air dried, and dissolved in 12 μL sterile distilled water. The sample was sequenced using a 96-well sequencing plate and the thermocycling for the sequencing reactions began with an initial denaturation at 94 °C for 2 min. Thirty-five cycles
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of PCR consisting of denaturation at 94 °C for 10 s, annealing at 50 °C for 10 s, and extension at 60 °C for 4 min using primers 704 F (5′-GTAGCGGTGAAATGCGTAGA-3′) and 907R (5′-CCGTCAATTCMTTTGAGTTT-3′), and to this, 10 μL of Hi-Di formamide was added and vortexed briefly. The DNA was denatured by incubating at 95 °C for 3 min, kept on ice for 5–10 min and was sequenced in a 3730 DNA analyzer. The sequences obtained were analyzed using Sequence Scanner Software, and the rDNA sequence contigs were generated using Chromas Pro, and then they were analyzed using online databases NCBI-BLAST to find the closest match of the contig sequence [20, 21]. Phylogenetic analysis was performed using the software packages PHYLIP [22] and MEGA version 4 [23] after obtaining multiple alignment data available from databases by CLUSTAL_X [24]. Pairwise evolutionary distances were computed using the correction method [25], and clustering was performed using the neighbor-joining method [26]. Bootstrap analysis was used to evaluate the tree topology of the neighbor-joining data by performing 1,000 resampling [27]. Production and Assay of Siderophore The production of siderophore was carried out in succinate medium [28] with pH 7.0. The medium was inoculated with 1 % (v/v) inoculum of 24-h-old culture of P. aeruginosa JAS-25 and incubated at 30 °C for 30 h with constant shaking at 120 rpm. The amount of siderophore secreted into the culture medium was determined by measuring the absorbance of the supernatant at 400 nm. Chrome Azurol Sulfonate Liquid Assay Quantitative detection of siderophore was assayed spectrophotometrically as described by Karuna Gokarn et al. [29]. To determine the presence of siderophore, the supernatant (24 h) was poured into the dark blue CAS broth in equal proportion (0.5–0.5 mL). To this shuttling solution, sulfosalicylic acid (10 μL) was added and the color obtained was determined using the spectrophotometer at 630 nm after 20 min of incubation. Necessary blank (minimal medium) and reference solution (minimal medium+CAS dye + shuttle solution) were used as a negative control. Csaky’s Assay Qualitative assay was performed in order to determine the type (hydroxymate or catechol) of siderophore produced by the strain. To determine the presence of siderophore, the supernatant from 24-h grown culture was poured into the dark blue CAS broth in equal proportion (0.5– 0.5 mL). To this, 3 mL of Na-acetate, 1 mL of sulfanilic acid, and 0.5 mL iodine solution was added. After 3–5 min, excess of iodine was removed with 1 mL of Na-arsenate solution. One milliliter of alpha naphthylamine was added, and water was used to make up the volume to 10 mL, and the color developed after 20–30 min was measured as the absorbance with the help of ultraviolet (UV)–visible spectrophotometer at 526 nm using hydroxylamine as a standard [30]. Partial Purification of Siderophore To 100 mL of succinate media, 1 mL culture of P. aeruginosa JAS-25 was inoculated and incubated for 48 h at 30 °C in a shaker. Cells were harvested by centrifugation at 10,000 rpm
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for 5 min, and the cells obtained were resuspended in 100 mL of distilled water and recentrifuged, and then transferred to 50 mL methanol. The flasks containing the bacterial suspension were kept in boiling water for 5–10 min and intermittently swirled to facilitate the release of bacterial pigments. The suspension was cooled and centrifuged to remove the cells. Supernatant was transferred to a flask, and equal volumes of 6 % KOH in methanol was added and warmed at 40 °C for 10 min by swirling the content intermittently. The contents were transferred to the separating flask and 2 volumes of diethyl ether and water was added to separate the layer. The flasks were gently shaken to release the bacterial pigments into the ether phase. It was allowed to stand for a few minutes for the separation, and the ether phase was collected. If the water layer was still colored, re-extraction was carried out once again with diethyl ether, washed with water, and dehydrated by adding anhydrous solid sodium sulfate. The ether was then transferred to the flask evaporator and dried at 30 °C. Subsequently, the material was redissolved in 10 mL of distilled water and filtered using Whatman No. 1 filter paper. The absorbance of the filtrate was read at 350 nm using a spectrophotometer. Antifungal Activity of Siderophore by Well Plate Assay The antifungal activity of the partially purified siderophore produced by P. aeruginosa JAS-25 was studied against three important phytopathogens, Fusarium oxysporum f. sp. ciceri (NCIM 1008; wilt of chickpea), Fusarium udum (MTCC 3829; wilt of pigeonpea), and Aspergillus niger (MTCC 872; wilt of groundnut) by well plate assay. Spores of the test fungi (105 cfu/mL) were inoculated on potato dextrose agar plates. Small wells of (4×2 mm2) were made in the agar plates with one as control and 0.1 mL of the partially purified siderophore was added and incubated at 30 °C for 4 days. The activity of partially purified siderophore was assessed on the basis of the zone of inhibition produced by the Pseudomonas sp. JAS-25 against the phytopathogens [31]. Inhibition of Spore Germination The partially purified siderophore was inoculated along with the test fungus and inhibition of spore germination by siderophore was observed using microphotograph microscope (Nikon Fx-35Dx) 10×20 magnification. The mycelial samples were picked up from the periphery of the zone of inhibition, and control plates were mounted directly onto the microscopic slide and observed for the inhibition of the spores and the mycelial deformation under the microphotograph microscope [31]. Effect of pH King B medium was prepared with different pH ranging from 5 to 11 with a difference of pH 1 for the study of pH. These flasks were inoculated with the test isolates and incubated at 30 °C for 30 h, and the production of siderophore was estimated spectrophotometrically at 435 nm. Effect of Temperature Effect of temperature was studied by using the test isolates, and these were inoculated in the King B medium with optimum pH 7 and incubated for 30 h at different temperatures ranging from 25 to 55 °C at a difference of 5 °C. Siderophore produced was estimated and recorded.
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Effect of Iron Concentration Different concentrations of FeCl3 ranging from 0 to 300 μg/L were used at a difference of 50 μg in King B media to study its effect. The test isolates were inoculated and incubated with optimum pH 7 and temperature of 30 °C for 30 h on a shaker at 120 rpm. The growth and production of siderophore by P. aeruginosa was recorded. Detection of Indole Acetic Acid The production of indole acetic acid (IAA) was determined according to the standard method [32]. P. aeruginosa JAS-25 was inoculated in the nutrient broth with 500 μg/mL of tryptophan and without tryptophan and were incubated at 30 °C for 24 h. Five milliliters of culture was taken from each tube and centrifuged at 10,000 rpm for 15 min. An aliquot of 2 mL supernatant was transferred to fresh tubes to which 100 μL of 10 mM orthophosphoric acid and 4 mL of Salkowski’s reagent (1 ml of 0.5 M FeCl3 in 50 mL of 35 % perchloric acid) were added. The mixture was incubated at room temperature for 30 min, and the absorbance of pink color developed was read at 530 nm. The IAA concentration was determined by using a standard calibration curve of standard IAA. Effect of Siderophore on Plants The seeds of Cicer arietinum (Chickpea), Cajanus cajan (Pigeon pea), and Arachis hypogaea (Ground nut) were surface sterilized with 1 % (w/v) mercuric chloride followed by three washings with sterile water. These sterilized seeds were mixed with the siderophore-rich broth of P. aeruginosa JAS-25 for 10 min. These seeds were sown ten seeds per pot containing infested soil and control soils in the pots. These seeded pots were watered regularly and observed for the growth of the plant and also the rate of germination of seeds after 15 days of sowing along with their respective controls.
Results and Discussion Identification of the Strain, Antifungal Activity The bacterial strain P. aeruginosa JAS-25 was characterized by biochemical and microscopic features as P. aeruginosa, which was further confirmed by 16S rRNA sequence. Relevant sequences were obtained from the BLASTn sequence alignment tool, and the tree was constructed using MEGA version 4 using neighbor-joining method [23] as shown in Fig. 1. 16S rRNA sequences of P. aeruginosa JAS-25 was submitted to NCBI GenBank, and the accession number JX104229 was obtained. Antifungal activity and inhibition of spore germination was studied against the phytopathogens like F. oxysporum f. sp. ciceri, F. udum, and A. niger by well plate assay and microphotographic method. Zone of inhibition of 3.0 cm was observed for F. udum and A. niger, proving it as potential biocontrol agent against phytopathogens [31]. CAS Liquid Assay The amount of siderophore produced by P. aeruginosa JAS-25 was observed using CAS liquid method; the strain showed good pigmentation and color change of CAS broth from dark blue
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Fig. 1 Phylogenetic tree of the Pseudomonas sp. JAS-25 constructed by MEGA version 4. Relevant sequences were obtained by BLASTn analysis
to yellowish orange compared with the reference solution. The reference solution showed greatest absorbance (blue color) as all the blue color is measured (Ar). Samples (i.e., culture supernatant taken after growing the cultures at 37 °C/24 h under static and shaker conditions) showed lower readings as siderophore removes the iron from the dye complex (As). The values of the siderophore excreted were determined using the formula: Ar − As 100 where Ar gives the percentage of the siderophore units. Type of Siderophore The type of siderophore produced by JAS-25 was determined as a potent producer of hydroxamate type of siderophore, which was determined by following the assay methods of Csaky [30] and Arnow [33]. The change in the color from yellow to orange (red) on addition of NaOH indicated it as positive for the production of a hydroxamate type of siderophore. Partial Purification of Siderophore Siderophore was partially purified using organic solvents like methanol and diethyl ether which were water soluble. The partially purified siderophore showed good fluorescence under UV and also indicated high antifungal activity [31] against the plant pathogens. Effect of pH The highest production of siderophore and growth of P. aeruginosa JAS-25 was studied in King B medium, with pH ranging from 5 to 9 and a difference of pH 1. P. aeruginosa JAS-25 showed no production of siderophore up to pH 6 and thereafter showed rapid increase in the production of siderophore up to pH 7 with the highest production of 130 μM, and then decreased gradually. pH plays an important role in the solubility of iron, thereby making its availability to the organism for its growth. At neutral pH, maximum yield of siderophore was
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obtained due to good growth of bacteria in the presence of iron in insoluble form. This stress of iron induces siderophore production; with increase in pH toward alkalinity, the production of siderophore ceased, due to the fact that alkaline pH helps in excess solubalization of iron, which in turn increases the iron content of the medium as shown in Fig. 2. The effect of pH on the siderophore production and growth of P. aeruginosa PSS was studied in succinate media, and maximum amount of siderophore of 60 μM was produced at optimum pH 7, as reported by Diaz de Villegas [34]. Maximum yield of 87 and 83 % was obtained by P. fluorescens and P. putida respectively in succinate media at optimum pH 7, as confirmed by Sayyed et al. [9]. Effect of Temperature Effect of temperature on the production of siderophore by JAS 25 in King B medium is as shown in Fig. 3. The production of siderophore was carried out for 30 h with the optimum pH 7. The production of siderophore gradually increased with the increase in temperature from 25 °C. Maximum production of siderophore of 130 μM was observed at 30 °C, and then gradually decreased as temperature increased (35 °C). As per Digat and Mattar [35], at temperature of 30 °C, most of the strains from temperate countries grow slowly or not at all and they did not produce any fluorescent siderophores (with a few exceptions), while tropical strains produced siderophores. Temperature is a key factor influencing both colonization by affecting the predisposing of pathogens to microbial antagonism. It also helps in the regulation of the growth and the production of metabolites, such as antibiotics and siderophores. The optimum temperature for the growth of P. fluorescens RGAF 19 and P. fluorescens RG26 in King B broth and maximum log (mol cell−1) of 18 was observed at 25 °C, which was confirmed by Landa et al. [36]. Effect of Iron on Siderophore Production Effect of iron on growth and siderophore production by JAS 25 in King B media is as shown in Fig. 4. King B media was prepared with different concentration of iron ranging from 0 to
Fig. 2 Effect of pH on the production of siderophore by Pseudomonas aeruginosa JAS-25 in KB medium
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Fig. 3 Effect of temperature on the production of siderophore by Pseudomonas aeruginosa JAS-25 in KB medium
300 μg/L. The growth of P. aeruginosa JAS-25 increased as concentration of iron increased from 0 to 150 μg/L and highest production of siderophore of 74.12 % was observed by P. aeruginosa JAS-25 in 150 μM of iron and decreased rapidly as the concentration of iron increased further. Siderophores are iron specific, which are secreted under low iron stress and captures iron from the environment. Conversely, the biosynthesis and recreation of siderophore are strictly regulated by environmental factors of which iron concentration is the most
Fig. 4 Effect of iron on growth and siderophore production by Pseudomonas aeruginosa JAS-25 in KB medium
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important one. The production of siderophore in the media was inversely proportional to the iron concentration. The growth of the culture increased with the increasing concentration of iron, whereas siderophore production repressed at higher concentration of iron. The excretion of siderophore continued till the end of the log phase and ceased as the cultures entered into the stationary phase. The threshold level of iron, which repressed the siderophore production, was found to be 20 μg. Maximum production of siderophore by P. fluorescens and P. putida was 87 and 83 %, respectively, which were obtained at 1 μM of iron and was confirmed [9]. The maximum amount of siderophore produced in succinate media was 34.7 μM at zero concentrations of iron and remained almost constant up to 10 μg/L of iron with 34.61 μM of siderophore production and decreased rapidly as the concentration of iron increased, was reported by Djibaoui and Bensoltane [37]. Pot Study Method The effects of the siderophore on the growth of the agricultural crops are indicated in Table 1. The siderophore produced by P. aeruginosa JAS-25 was used as a growth-promoting agent on C. arietinum (Chickpea), C. cajan (pigeon pea), and A. hypogaea (Ground nut) is shown in Fig. 5 along with their controls. The treated seeds with and without siderophore were used for the study and observed for the growth of shoot length and root length in centimeters, shoot and root dry weight in grams, and rate of seed germination was recorded in percentage. The plants showed good efficient growth with seeds treated with siderophore produced by P. aeruginosa JAS-25 when compared with their corresponding controls. The rate of seed germination observed for chickpea, followed by groundnut and pigeon pea were 30, 20, and 20 %, respectively. Accordingly, there was a significant increase in shoot length, root length, and their dry weights. Ten percent increase in seed germination, 20 % increase in root length, and 31 % increase in shoot length was evident by P. fluorescens NCIM 5096, which was observed and proved by Sayyed et al. [38]. Twenty percent increase in shoot length, 10 % increase in shoot dry weight, and 10 % increase in root dry weight, was proved and confirmed by Landa et al. [36]. The efficient growth of the plants was not only due to the biocontrol activity of the siderophore produced by P. aeruginosa JAS-25 but may also be by the production of IAA, which influences the growth of the plants as an phytohormone. The release of this phytohormone Table 1 Effect of siderophore produced by Pseudomonas aeruginosa JAS-25 on the growth of Cicer arietinum (chikpea), Cajanus cajan (pigeon pea) and Arachis hypogaea (ground nut) Test
Plants
% germination Growth variables Shoot length Root length Shoot dry weight Root dry weight (cm) (cm) (gm) (gm)
Control Cicer arietinum
10 %
18±1.21
6.30±1.09
0.49±0.2
0.06±0.02
JAS-25 C. arietinum
30 %
22±2.02
8.33±1.25
0.65±0.1
0.08±0.01
Control Cajanus cajan
10 %
15±2.13
3.5±0.82
0.35±0.4
0.04±0.03
JAS-25 C. cajan
20 %
4.6±0.97
0.58±0.2
0.05±0.01
17.16±2.0
Control Arachis hypogaea 10 %
10.16±1.03
4.89±1.16
0.53±0.13
0.09±0.05
JAS-25 A. hypogaea
12.51±1.24
5.99±1.19
0.7±0.11
0.15±0.04
“±” standard error
20 %
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Fig. 5 Effect of siderophore on growth of chick pea (a), pigeon pea (b), and ground nut plant (c), respectively
IAA in the stationary phase has been evidently proved as the plant growth-promoting ability of the test strain P. areuginosa NJ-15 was reported by Bano and Musarrat [39].
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The current study is evident that the fluorescent pseudomonads investigated is capable of producing plant growth-promoting substances and antifungal substances which act as biocontrol agents [31]. The bio-inoculant (P. aeruginosa JAS-25) applied significantly improved the plant nutrition and also promoted the plant growth by stimulating plant growth hormone (IAA) production. Hence, P. aeruginosa JAS-25 isolated from saprophytic soil is a potential candidate for the production of siderophore, which helps in the development of bio-inoculants for the agricultural crops. Acknowledgments Financial support (UGC no. F. no. 37-162/2009(SR)) by UGC, Government of India, Ministry of Science and Technology, New Delhi is highly acknowledged for granting UGC Major Research Project. Our sincere thanks to Dr. Yogesh S. Shouche, Scientist, National Center for Cell Sciences, Pune, Maharashtra, India for his kind help in the identification of the strain.
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