Optimization of Siderophore Production by Bacillus Subtilis DR2 and its Effect on Growth of Coriandrum Sativum

Under scarce iron conditions, several bacteria, fungi and plants, secrete ferric iron-specic ligands, generically termed as siderophores that are able to bind with insoluble ferric ion thereby making them available to the host organisms. Siderophore producing bacteria was isolated from the rhizospheric soil of Eragrostis cynosuroides by CAS agar screening and CAS-shuttle assay method. Among ve positive isolates, DR2 produced a relatively high level of siderophore (69.81 SU%) and was identied as catecholate type. Further, it was identied as Bacillus subtilis DR2 (KP455653) based on 16S rRNA gene sequencing and phylogenetic analysis. Media optimization revealed that the strain B. subtilis DR2 showed maximum siderophore yield (80.60 SU%) under optimized condition of 72 h incubation at 35 °C in succinate media at pH 8, supplemented with sucrose as carbon and NaNO 3 as nitrogen sources. It was further tested as seed inoculants under pot culture conditions and was found to be very ecient in seed germination and growth promotion of Coriandrum sativum. Thus, the present study signies that B. subtilis DR2 may be a promising candidate with potential of plant growth promotion to be used as biofertilizer for various crops.


Introduction
Rhizosphere is a dynamic environment, harbouring diverse array of microorganims. It is a rich repository of plant growth promoting rhizobacteria that enhances crop productivity and maintains soil health in a sustainable way 1 . Bacteria that stimulate plant growth either directly or indirectly, have been regarded as plant growth promoting rhizobacteria (PGPR). They promote plant growth via different mechanisms to make the respective elements available, like nitrogen xation (N 2 ), siderophore production (Fe), IAA production, phosphate solubilisation (PO 4 ), etc. All of them have their own vital roles in plant growth promotion.
Iron is a vital trace element for living organisms. But due to very low solubility of Fe 3+ in the earth's crust, its availability is limited and it cannot be utilized by them. The element is predominantly present as oxide and hydroxide forms with their characteristic very low solubility. In response to this, microorganisms have evolved a strategy for acquiring iron through siderophore production 2, 3 .
A siderophore is a low-molecular weight, iron chelating compound having high a nity for ferric iron, converting them into ferrous form, which is to be further utilized in the metabolic processes 4 . Rhizospheric bacteria capable of producing siderophores increase the bioavailability of iron near the root to promote plant growth 5 . Based on the ligands used to chelate ferric iron, bacterial siderophores are categorized into four types: catecholate, hydroxamate, carboxylate and salicylate 6 . Various bacterial species of Bacillus, Aeromonas, Aerobacter, Enterobacter, Escherichia, Mycobacterium, Klebsiella, Vibrio, Salmonella and Yersinia are reported to produce siderophores. Bacillus spp. is known to be excellent siderophore producers and stimulate plant growth through enhanced phosphate nutrition, iron, potassium and nitrogen uptake. Inoculation of crop plant with different strains of PGPR increased not only the yield but also the quality of various medicinal plants as well as spice crops 7,8 . Several PGPR have been isolated all over the world with only few being commercialized including the species of Bacillus, Pseudomonas, Azobacter, Enterobacter, Azosprillum, Klebsiella, Serratia and Variovorax 9,10 . Among them, Bacillus spp. are reported to secrete metabolites that provide considerable tolerance property in any adverse condition and prevent from pathogen infection, hence they are better for several crops [11][12][13] .
Various PGPR attributes of Bacillus species involved in increased productivity of rice, maize, wheat, cucumber, soybean, potato, apple, tomato and ornamental plants has been validated through green house as well as eld trials 14 . In the present scenario, the natal approaches are generally used as an alternative to chemical fertilizers for enhancing crop productivity apart from including plant nutrient management systems. It is opined that a potent PGPR is one that must be able to colonize plant rhizosphere, promote growth with its multi-spectrum mode of action, good survivality, ecofriendly, and tolerant to temperature, ultraviolet (UV) radiation, oxidizing agents and various other unfavourable conditions 15 .
Eragrostis cynosuroides known as kusha or dharbham grass is a medicinal plant and has extensive properties of acting as antimicrobial, antioxidant and anticancerous agents. This plant has also an ability to absorb ultraviolet radiation 16 . In the view of aforesaid bene ts this grass has been chosen because its growth is independent of any exogenous supply of chemical fertilizer and it grows luxuriantly attaining huge biomass in short period of time.
Coriandrum sativum (Coriander), a member of family Apiaceae is an important herbal spice crop, generally grown in winter season of India. Its different parts are valued for its culinary and medicinal properties. The green herb being sink of various vitamins like, vitamin A, vitamin C, and vitamin B 2 17,18 are used as avouring agents in preparation of delicacies (sauces, soup, breads, cakes, confectionery and meat products), while the seeds act as tonic, stomachic, diuretic, carminative and aphrodisiac.
So keeping in mind, the immersing importance of coriander crop, the present study is aimed for production and estimation of siderophores produced by B. subtilis and their role in vegetative growth promotion of Coriandrum sativum seedlings under in vitro pot culture experiment.

Iron Decontamination
All glasswares used in the present study were soaked overnight in 6 M hydrochloric acid (HCl) and thoroughly rinsed with distilled water (DW) to remove any traces of iron.

Sample collection and isolation of rhizospheric bacteria
Rhizospheric soil sample of Eragrostis cynosuroides plant was collected from road side (devoid of any fertilizer) of Danapur, Patna, Bihar, India (25º 34' 56.2" N, 85º 2' 37.06" E). The plant was uprooted with the help of trowel for the collection of rhizospheric soil. The soil sample was collected and transferred in sterile ziplock polythene bags to the laboratory for further study and processed within three hours. The soil suspension was prepared by adding 1 g of soil sample to 10 mL sterile DW and diluted up to 10 -6 dilution and spread on to nutrient agar media (NAM) and incubated at 30±2 °C for 24 h. Bacterial colonies appear on plates were puri ed; sub cultured repeatedly to get axenic culture and preserved at 4°C in NAM for further use. The isolates were designated as DR1, DR2, DR3, DR4, DR5, DR6 and DR7. All the experiments were carried out in triplicates.

Siderophore production
Preparation of Chrome Azurol S medium The Modi ed Chrome Azurol Sulfonate (CAS) agar plate 19 was prepared by mixing two solutions given below: Solution 1: It was prepared by dissolving 60.5 mg CAS in 50 ml de-ionized water and mixing it with a 10 ml Fe 3+ solution (containing 1 mmol L -1 FeCl 3 .6H 2 O in 10 mmol L -1 HCl). Solution 2: It was prepared by dissolving 72.9 mg of hexa decyl trimethyl ammonium bromide (HDTMA) in 40 ml de-ionized water resulting in a dark blue solution.
Both solutions (solution 1 and solution 2) were autoclaved separately and mixed slowly. The nal mixture of 100 ml volume was added to 900 ml of succinate agar medium (pH 7). After solidi cation of the media on petri plates, 24 h old bacterial isolates were inoculated and incubated at 30±2 °C for 24-72 h. Formation of orange halo zone from dark blue color around the colonies was indicative positive result.
Quantitative Assay of siderophore production (CAS-Shuttle Assay) It was carried out by CAS shuttle assay 19 . The isolates were cultured in succinate medium and incubated in shaker incubator at 120 rpm and 30±2 °C for 24 h. The fermented broth was centrifuged at 10,000 rpm for 10 minutes at 4 ºC. An aliquot of 0.5 ml of supernatant (cell-free extract) was mixed with 0.5 ml of CAS solution. The resulting color obtained was measured after 20 min of incubation at the wave length of 630 nm, using the UV-VIS spectrophotometer (Systronics, Ahmedabad, India), referring the uninoculated CAS solution as blank. The percentage of siderophore units (SU) was estimated as the proportion of CAS color shifted using the formula: Where, Ar -absorbance of reference (CAS assay solution + uninoculated media) and As -absorbance of the sample (CAS assay solution + cell-free supernatant).

Phenotypic and genotypic characterization of isolates
The most e cient isolate was further characterized on the basis of its morphological, cultural and biochemical characteristics as per the Bergey's Manual of Systematic Bacteriology. The potential strain was identi ed by 16S rRNA gene sequence analysis. The sequence will be submitted to National Center for Biotechnology Information (NCBI) for accession number. PCR based 16S rRNA gene ampli cation and sequencing of the isolated bacterium was carried out using universal primers at Xcelris lab Ltd, Ahmedabad, Gujarat, India.

Characterization of siderophores
The characterization of the siderophore as catechol or hydroxamate types was carried out as follows: Hydroxamate type of Siderophore (Tetrazolium salt test): A pinch of tetrazolium salt and 1-2 drops of 2 N NaOH was added to 0.1 ml supernatant of the test culture. Instant appearance of a red to deep-red color was indicative of presence of hydroxamate siderophores 2 .
Catecholate type of Siderophore (Arnow's Test): In this assay 1 ml of cell-free supernatant was mixed with 1 ml of 0.5 M HCl and 1 ml of nitrate molybdate to turn the mixture solution yellow. Further, 1 ml of 1 M NaOH was added, mixed and incubated for 5 min at room temperature, resulting in red color formation. The color was stable for 1 hour and the absorbance was measured at 510 nm using a UV-VIS spectrophotometer 20 .

Detection of siderophores by Thin Layer Chromatography (TLC)
The culture supernatant of siderophore producer strain was spotted on 10×20 mm silica gel plates and allowed to dry. The plates were run in an n-butanol: acetic acid: distilled water (12:3:5) solvent system until the solvent front reached the top. Thereafter it was dried and 0.1 M FeCl 3 (prepared in 0.1 N HCl) was sprayed. Appearance of a wine-colored spot indicated a hydroxamate-type siderophore, while that of a dark gray spot indicated catechol-type siderophore 21 .
Optimization of physicochemical parameters for siderophore production The biological production of siderophores is governed by several environmental factors like growth medium, temperature, pH, incubation time, carbon sources, nitrogen sources etc. In the present study, the optimization experiments were initiated by evaluating the optimum nutrient medium for siderophore production. The three different nutrient media tested in the current study were Nutrient broth, JNFbbroth and Succinate broth. The siderophore production was monitored by using 50 ml medium each, separately inoculated with 0.25 ml of 24 h old culture, incubated at 37 °C in shaker incubator (120 rpm for 24 h).
The optimization of other physicochemical parameters for production of siderophores was studied by varying one parameter at a time, while keeping the others constant. These varying parameters included, incubation time (24 h, 48 h, 72 h, 96 h, 120 h), temperature (25 °C, 30 °C, 35 °C, 40 °C), and pH (5,6,7,8,9,10). In addition, the effect of 0.1 % solution of different carbon sources (glucose, sucrose, fructose, lactose, mannitol) and nitrogen sources (urea, sodium nitrate, ammonium sulphate) were also studied on siderophore production. The bacterial isolates were inoculated in the succinate medium and the estimation was done on the above mentioned quantitative assay.

Pot experiment
In order to evaluate the potential of selected isolate in coriander plant, a pot experiment was conducted in a growth chamber at the Department of Botany, Patna University. The coriander seeds purchased from local market (Bakarganj, Patna, Bihar, India) were surface sterilized by exposing to 2-3 % of NaOCl followed by 70 % ethanol solution for 3 min followed by rinsing with autoclaved DW, at least for three times 10 . Sterilized seeds were soaked in autoclaved DW for 24 h at room temperature inside closed petri dishes. Further seeds were transferred in bacterial suspension (10 8 cfu ml -1 ) at 30 º C for 6 h and sown in the pot having sterile soil (by autoclaving at 15 lbs/121 °C for 3 h) to a depth of 5 mm as a test (inoculated seeds) and control (uninoculated seeds) 10 . Sterile water was used for maintaining moisture in the pots as per requirements and observed for seed germination, root length and shoot length with respect to control and after two weeks, plants were harvested, roots were washed free of soil and shoot and root lengths were measured. The germination percentage was calculated. After one week, seedling vigour was recorded in terms of root and shoot length with the help of a measuring scale. Each treatment was carried out in three replications. Also number of normal and abnormal seedlings and dead seeds were counted. Germination percentage was determined by the following formula-Germination (%) = Normal seedlings / Total number of seed taken × 100 The entire plant was dried in an oven at 72 ºC for 48 h and fresh weight and dry weight were recorded as seedling growth parameter. Total biomass was calculated after deducting the dry weight from wet weight. The plants involved in our study comply with institutional guidelines.

Statistical Analysis
The data obtained were statistically analyzed by using software (SPSS 16.0), and graphically represented as the mean ± standard deviation (n=3).

Results
Sample collection and isolation of rhizospheric bacteria Choice for soil sample was guided by the luxuriant growth of Eragrostis cynosuroides on roadside, which are practically zero chemical fertilizer zones. Seven isolates (DR1-DR7) appeared on solid NAM, which upon repeated sub culturing retained their growth and preserved at 4 °C in NA medium.
Qualitative screening of siderophore production Siderophore production is one of the important traits of PGPR. Since last few decades, they are attaining much attention because of their application in various other elds, apart from agriculture. In this context, the bacterial isolates were qualitatively screened for siderophore production trait. It was found that 5 out of 7 isolates gave positive test for the siderophore production with formation of varying intensity of orange zones (Fig.1) and were used for the further study. The zone of coloration was 13 mm, 31 mm, 16 mm, 8 mm and 22 mm in DR1, DR2, DR4, DR6 and DR7 respectively.
Quantitative assay for siderophore production The CAS forms a tight complex with the ferric ion resulting in blue coloration of medium, and upon addition of siderophores to the medium, iron are chelated from the dye complex, eventually changing the color of medium from blue to orange (Fig. 2). The zone sizes produced by these isolates and SU% of siderophore production are represented in g. 3. Among these 5 isolates, DR2 showed maximum siderophore production i.e. 69.81 SU% in comparison to DR1 (27.51 SU%), DR4 (31.95 SU%), DR6 (15.67 SU%) and DR7 (53.49 SU%) and thus selected for further study.

Phenotypic and genotypic characterization of isolates
Morphological, physiological and biochemical characteristics of the potential isolate DR2 appeared as Gram positive, rod having motility and the colony with creamy white, round, irregular margin. The isolate was also foundd positive for oxidase, citrate, ammonia, Voges-Proskauer reaction and starch hydrolysis. Based on nucleotide homology and phylogenetic analysis of the 16S rRNA gene sequence showed the highest similarity with B. subtilis strain ZJHD1-70 (GenBank Accession Number: KF585036.1) (Fig. 4). So the isolate DR2 has been identi ed as B. subtilis .The nucleotide sequence analysis of the isolate DR2 was compared with available data base using BlastN tool on NCBI (http://www.ncbi.nlm.nih.gov/ BLAST). The nucleotide sequence of identi ed isolate was deposited as B. subtillis DR2 at NCBI with an accession number of KP455653 (Fig.4).

Characterization of siderophores
The selected strain B. subtilis showed a strong positive reaction with formation of red color in the Arnow's test and negative reaction in tetrazonium salt, infers the presence of catechol-type siderophore (Fig.5).
Optimization of physicochemical parameters for siderophore production Optimization of culture media Optimization of nutrient media prior to physicochemical parameters is essential for ensuring maximum microbial growth and hence, maximum siderophore production. For this three different media viz., Nutrient broth, JNFband Succinate medium were individually inoculated with the test strain B. subtilis DR2. Among the three different nutrient media tested, highest siderophore production was observed in succinate medium (71.51 SU%) followed by nutrient media (67.23 SU%) and JNFb -(59.31 SU%) (Fig. 6).

Optimization of incubation time
There is variation in siderophore production at various time intervals and was found to be optimum after 72 h incubation (72.48 SU%). The isolate B. subtilis DR2 showed a gradual increase in siderophore production till 72 h, after which the production declined from 50.38 to 32.36 at 96 h to 120 h, respectively of incubation as noted in our study (Fig.7).

Optimization of temperature
The siderophore production varied with variation in temperatures and thus 35 °C under shaking condition of 120 rpm (74.54 SU%) was observed for maximum production (Fig.8).
Optimization of pH pH plays a vital role in the solubility of iron in production media and thereby, siderophore production. In our study the maximum siderophore production (75.80 %) was found at pH 8.0 (Fig. 9).

Optimization of carbon source
It was carried out with different sources of carbon, such as sucrose, glucose, fructose, mannitol and lactose. Supplementation of growth media with various carbon sources increases the growth capacity of bacteria and therefore enhances siderophore production. In our investigation, presence of sucrose as Csource in the medium produced maximum (78.06 SU%) siderophore as compared to other carbon sources (Fig.10).

Optimization of nitrogen source
During the evaluation of different suitable nitrogen sources in culture media, sodium nitrate was appeared to be the best suited for siderophore production as 80.60 SU% by B. subtilis DR2. The other nitrogen sources, such as ammonium sulphate (60.32 SU%), potassium nitrate (43.86 SU%) and urea (21.67 SU%) gave lesser amount of siderophore production (Fig.11).

Pot studies
Pot culture complements to the eld measurement in having experimental microbial status under controlled conditions. Application of this process favours, transferability of the experimental results to natural conditions. These experiments are cost e cient, easy to conduct and broadly applicable. Such pot culture is a simple and fast method to demonstrate that inoculation of rhizobacteria can increase the biomass and yield of any test plant. In our pot study, the results revealed that inoculation of Coriandrum sativum seeds with bacterial strain B. subtilis DR2 had a positive stimulatory effect on all the growth parameters, as compared to the control (Table 1). In the tested plant material % enhancements in seed germination, root length, shoot length and biomass were recorded as 64, 51.85, 47.65 and 70.94, respectively (Fig.12).

Discussion
In the present study, 7 rhizospheric bacteria were isolated from the rhizosphere of grass Eragrostis cynosuroides (Kusha). Among them, 5 isolates (DR1, DR2, DR4, DR6 and DR7) were siderophore positive, of which the most promising isolate, namely DR2 (the highest Siderophore producing strain) was identi ed as B. subtilis DR2 based on its morphological, biochemical, and genotypic characters. Genus Bacillus is the most commonly reported PGPB with its well documented abilities of PGP properties like siderophore production, IAA production, nitrogen xation, ACC deaminase synthesis and P-solubilization by numerous researchers [22][23][24] .
All the ve siderophore producers showed more than 8 mm zone on CAS agar plate, where B. subtilis DR2 gave maximum siderophore zone of 31 mm. There are similar reports of maximum siderophore zone by salinity tolerant B. subtilis 1 (15.8 mm), impacting on plant growth of wheat under saline soil 25
Upon quantitative assessment also, DR2 appeared as the best siderophore producer among all the isolates i.e., 69.81 SU%, resembling with the reports on Bacillus sp. VITVK5 and Enterobacter sp. VITVK6 showing 60.06 % and 61.79 % siderophore production respectively 4 .
In another study, similar results were reported in B. subtilis CTS-G24 which produced 59 % and 64 % siderophore units in nutrient and succinate media, respectively 27 . On the basis of highest siderophore producing abilities, the DR2 was selected for further optimization and pot experiment studies.
Several workers across the globe reported several plant growth promoting rhizobacteria having ability to produce catecholates and hydroxamate type, siderophore 28-30 . In our investigation, B. subtilis DR2 showed a catechol-type siderophore, which is in accordance with the ndings of other workers 31 . Another studies also con rmed the production of catecholate type of siderophore by Bacillus spp. under iron limiting condition 32 and catecholic siderophore 2, 3-dihydroxybenzoate-glycine-threonine trimeric ester bacillibactin by B. subtilis strain CAS15 33 . In the similar way, it has been stated that when the culture ltrates of the siderophore producers were analysed, there was revelation of presence of catechol type siderophores in B. subtilis and R. radiobacter, while hydroxamate-type siderophores for P. allii and B. megaterium 29 .
In the present work, the in uence of B. subtilis physiology and the impact of various physical and chemical culture conditions on production of siderophore have been studied. Siderophores are iron-speci c compounds, which are secreted under low iron stress conditions. B. subtilis DR2 showed maximum siderophore production in succinate medium, without the addition of iron, while its productivity may be repressed, due to presence of iron traces in nutrient broth and JNFb − broth media. Similar study has also been reported, in which succinate media supported maximum siderophore production (64 SU%) by B. subtilis CTS-G24 27 .
There are several reports on siderophore production by Bacillus spp., under the range of 24 h to 120 h of incubation 34,29 . In the present investigation, B. subtilis DR2 achieved highest siderophore production (72.48 SU%) at 72 h of incubation, which declined, thereafter. In accordance with this, optimum siderophore production at 72 h of incubation has been reported in Bacillus sp. IFM22 35 . Other workers have reported highest production of siderophore (80 SU%) by Bacillus sp. at an incubation period of 36 h 36 .
Like incubation, microorganisms are also profoundly affected by temperature of their habitat, as it in uences their growth and metabolite secretion. At low temperature, as the growth rate is slow, the siderophore production is also low. However, as the temperature increases up to optimal limit, the growth rate enhances, leading to production of more biomass, consequently higher amount of siderophore production takes place. In this study, 35°C was found to be optimum for siderophore production by B. subtilis DR2 (74.54 SU%). Optimum production of siderophores have also been reported over a wide range of temperatures i.e., 25-45°C by Bacillus VITVK5, Bacillus VITVK6 and Enterobacter sp. 37,38,4 . However, similar to our ndings, the temperature range of 37°C has been cited as optimum for siderophore production in B. anthracis 39 .
A change in pH of culture medium affects both microbial growth and bioavailability of iron 40 . In the present work, experiments investigating the effect of pH, clearly re ected that the pH, close to ambient condition (8.0) supported maximum growth and siderophore production. In Streptomyces fulvissimus also the highest siderophore production (93% unit) was re ected at pH 8.0 41 . As per the report of Agro services international at a high pH value, insolubility of iron increases, which is in accordance with our nding 4 . During the optimization process, siderophore production showed an increase at pH 7.5 and ranged between 18-30%, while at pH of 8.5, siderophore production was reported at peak, ranging from 30 to 60% by ve bacteria (Bacillus cereus, Pseudoalteromonas tetraodonis, Micrococcus aloeverae, Psychrobacter pocilloporae, and Pseudomonas weihenstephanensis) 31 .
Carbon source provides energy for growth and various metabolic activities of microorganism. Siderophore production of B. subtilis is in uenced by the nature of the carbon source. The present study on analysis of the effect of various carbon sources on siderophore production revealed that sucrose has most profound effect, acting as best inducer of B. subtilis DR2. Other workers have also reported sucrose, as best inducer of siderophore production in different species of Bacillus viz., Bacillus sp. VITVK5 as 83.17 SU% 4 . Nitrogen, as one of the most important nutritional factors, serves as the building block material of organisms, so is used as the basal component of medium. Hence, various organic and inorganic compounds were tested in media as a source of nitrogen for siderophore production and sodium nitrate appeared to be the best suited for siderophore production as 80.60 SU% by B. subtilis DR2. Similar results were reported in the bacterial strains of Bacillus sp. VITVK5 (61.94%) and Enterobacter sp. VITVK6 (61.32%), where sodium nitrate was used as nitrogen source 4 .
In order to evaluate the PGP potential of B. subtilis DR2, its effect on seed germination of Coriandrum sativum has been investigated. The bene cial effects of the B. subtilis DR2 on the C. sativum were observed and evidenced by the increase in the % germination, root length, plant fresh and dry biomass of the inoculated plants as compared to the non-inoculated controls (Table 1) 42 . The enhancement in growth parameters after inoculation with B. subtilis DR2 may be, due to total nutrient uptake, different PGP activities, such as siderophore production, IAA production, phosphate solubilization, nitrogen xation, etc 43,23 . These results corroborate with the previous nding, which reported that B. cereus ALT1 diminished Cd stress, strengthened antioxidant system and boosted growth in Soybean 44 . In a recent report, an endophytic bacteria B. subtilis has been observed to enhance drought tolerance and growth in wheat plant, due to its siderophore producing property 45 . Such sort of extensive research work becomes necessary as it re ects that replacement of chemical fertilizers with PGPR as inoculants is a much effective and sustainable approach for plant growth promotion.

Conclusion
Iron is a vital element required by all living organisms for their numerous cellular activities. Under ironde cit condition, microorganisms (PGPRs) produce low molecular weight siderophores to chelate iron (Fe 3+ ) molecules from the environment for their survival and support for overall crop improvement. The isolate B. subtillis DR2 was found to be quite effective in promoting seed germination and seedling growth of Coriandrum sativum, in terms of enhanced root, shoot length and biomass production. Therefore, it is suggested that the use of this potential strain as a potent biofertilizer can be bene cial for coriander cultivation and other crops also. Its application in pot experiment favours integration of biological management for plant improvement. The reproducibility of the result needs to be further standardized, so that the bacterium could be recommended as biofertilizers.

Declarations
Availability of data and material The authors declare that all the data supporting the ndings of this study are available within the article and from the corresponding authors on reasonable request.

Plant
Treatments Germination %  Figure 1 Qualitative and quantitative assessment of siderophore production by potential isolates Figure 2 The appearance of orange color and zone formation by DR2 indicating siderophore production on CAS agar plate Figure 3 Quantitative estimation of siderophore production by DR2. Instant appearance of red colour in Arnow's test