identification and characterisation of siderophore

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IDENTIFICATION AND CHARACTERISATION OF SIDEROPHORE POSITIVE

PSEUDOMONAS FROM NORTH INDIAN ROSEWOOD (DALBERGIA SISSOO) Roxb.

FOREST ECOSYSTEM

PRAGATI SRIVASTAVA, VANDANA JAGGI, HEMANT DASILA & MANVIKA SAHGAL

Department of Microbiology, G. B. Pant University of Agriculture & Technology, Pantnagar, Uttarakhand, India

ABSTRACT

Dalbergia sissoo Roxb., common name shisham, natural as well as plantation forests, are facing the large scale

mortality induced by poor soil fertility. Iron scarcity in Dalbergia sissoo leads to iron deficiency-induced chlorosis

(IDIC) which makes this tree susceptible to fungal pathogens and lepidopteron attack. Since Dalbergia sissoo provides

valuable timber as well as enriches soil nitrogen, its large scale decline incurs a huge economic loss. Several

rhizosphere dwelling bacteria secrete ferric iron-chelating agents, siderophores, under iron-deficient conditions. The

siderophore positive bacteria are significant as plant growth promoting and biocontrol agents. Therefore this paper

deals with the isolation, identification, and characterization of siderophore positive bacteria from the Dalbergia sissoo

plantation forest from the Tarai region of western Himalayas. The siderophore production in twenty shisham

rhizosphere bacteria was assayed qualitatively and quantitatively through chrome azurol ‘S’ assay. In all 10 isolates

were siderophore positive and identified as Pseudomonas, Streptomyces, and Burkholderia. Out of which, the five

strains, viz., R2, R4, B3 B6, and B9, that showed production of higher siderophore units (65- 90 % SU) were identified

as belonging to Genus Pseudomonas. Further characterization and optimization studies revealed that all five strains

produced siderophores in the range of 80-100 % SU under pH 7-9 and without the addition of FeCl3 in the growth

medium. These Pseudomonas strains are promising candidates for siderophore production and hold promise as iron

biofertilizers for use in plantation forestry.

KEYPOINTS: Seasonal microbial diversity in Dalbergia sissoo, Optimization of siderophore production & molecular

identification of selected isolates

Received: Sep 22, 2020; Accepted: Oct 11, 2020; Published: Oct 31, 2020; Paper Id.: IJASRAUG202032

INTRODUCTION

Dalbergia sissoo (shisham) is prominent timber species of India. It is widely distributed all along Sub-Himalayan

Tract generally up to an altitude of 900 m and occasionally to 1500 mabsl. Natural and plantation sissoo forests are

common in Bihar, Haryana, Punjab, and Uttar-Pradesh. It is suitable for agroforestry systems and can be grown

successfully in combination with fodder grasses, fruit trees, and crops. Because of its strength, elasticity, durability,

and colour grain attractive surface, the shisham wood is a highly valuable timber. Besides, it is an important tree of

social forestry and fixes nitrogen (Lal and Singh, 2012; Ahmad et al, 2013; Rashid et al 2019). In recent years,

the decline of shisham tree (Dalbergia sissoo Roxb.) plantations and natural forests have been observed in foothills

of Himalayas from eastern Afghanistan through Pakistan to India and Nepal (Sagta and Nautiyal, 2001; Ashraf et

al., 2010). Fungal dieback is a major threat to this multipurpose tree (Ahmad et al., 2016, 2017) and has affected

millions of trees in Southern Asia (Voget et al., 2011).

Orig

ina

l Article

International Journal of Agriculture

Science and Research (IJASR)

ISSN (P): 2250–0057; ISSN (E): 2321–0087

Vol. 10, Issue 4, Aug 2020, 239-256

© TJPRC Pvt. Ltd.

240 Pragati Srivastava, Vandana Jaggi, Hemant Dasila & Manvika Sahgal

Impact Factor (JCC): 8.3083 NAAS Rating: 4.13

Nutrient elements, such as phosphorous and iron are known to limit crop and forest plantation yields

(Schulze and Mooney, 2012; Kumar 2015). Iron has an innumerable function in the plant system. It is present as a

prosthetic group in cytochrome b and c, it regulates the structure and function of stomata. Iron is required for the

functioning of Photosystem I and II and is present as Rieske iron-sulphur protein in the electron transport chain,

during photosynthesis. Unlike phosphorous, iron is abundantly present in the soil but unavailable to plants due to low

solubility of Fe3+ form in alkaline pH and aerobic environment. Iron limitation in Dalbergia sissoo leads to iron

deficiency-induced chlorosis which makes this perennial tree vulnerable to attack by fungal pathogens and

lepidopterans. Mortality is more common in monoculture Sissoo plantation forests where if a single tree is affected;

the whole plantation is adversely affected. Micro-organisms possess a mechanism to overcome the iron limitation by

the production of “siderophore” (Kleoper et al., 1980; Neilands, 1995; Ahmed and Holmstorm, 2014). This is a

low molecular weight Fe3+ metal-chelating molecule. According to the co-ordinating group that chelates Fe3+ ion,

four major siderophore types are (i) catecholate (ii) hydroxymate (iii) carboxylate (iv) mixed ligands (Ali and

Vidhale, 2013). Catecholate type of siderophores contains phenolate or 2, 3 dihydroxy benzoate as a co-ordinating

group. Some known examples of catecholate type are azotochelin and aminochelin (Wittman et al., 2001) and

Enterobactin, a common siderophore produced by members of family Enterobacteriacae: E. coli, Salmonella

typhimurium, Aerobacter aerogenes (Ward et al., 1999). The second most common type of siderophore is

hydroxymate. It contains a C (=O) N-(OH) R group, where R group can be an amino acid or its derivative (Řezanka

et al., 2019). Hydroxymate siderophore has a strong affinity for Fe3+ ion with binding constant in the range 1022 -

1032 per moles. Some examples of hydroxymate siderophores include ferrichrome produced by soil fungi (Zahnerat

et al, 1963; O’Sulvian & O’Gara 1992; Schalk et al., 2011). They are used as therapeutics. For example

ferrioxamines, the linear tri-hydroxamates produced by Streptomyces and Nocardia, are used for the treatment of

thalassemia (WHO, 2013). Carboxylate type of siderophore contains hydroxyl carboxylate/carboxylate as a

coordinating moiety (Schwyn and Neilands, 1987). A common example is Rhizoferrin (Munzinger et al., 1999) and

Rhizobactin (Kim et al., 2016). Mostly fungus produces mixed ligand type siderophores. Similarly, siderophores

produced by Pseudomonas mainly pyoverdine, pseudobactins, pyoverdine are also examples of mixed ligand type

siderophores (Abddalah 1991; Meyer 2000; Meneely & Lamb). Siderophores produced by Pseudomonas are

categorized as, low (primary) and high affinity (secondary) siderophores. Pyoverdine and pseudobactin are high-

affinity whereas pyochelin, pseudomonine, quinolobactin, thioquinolobactin, pyridine-2, 6-dithiocarboxylic acid

(PDTC) are low-affinity siderophores. The genus Pseudomonas is one of the most diversified genera and well known

for plant growth promoting and disease management properties. They are cosmopolitan and inhabit diverse

environments such as an ocean, hydrothermal vents, or soil. Pseudomonas, Agrobacterium, Arthrobacter,

Azotobacter, Azospirillum, Bacillus, Burkholderia, Caulobacter, Chromobacterium, Erwinia, Flavobacterium,

Micrococcus, Allorhizobium, Azorhizobium, Bradyrhizobium, Mesorhizobium and Rhizobium are known to enhance

mineral nutrition, phytohormones synthesis, and suppress the growth of soil-borne pathogens via siderophore

production (Bhattacharya and Jha, 2012). Therefore, siderophore producing microorganisms have a colonizing

advantage over other microorganisms in the rhizosphere (Haas and Défago, 2005). Siderophore positive

Pseudomonas fluorescens is known to control bacterial pathogens causing bacterial soft rot of potato, tomato

bacterial wilt, the bacterial canker of tomato (David et al 2018, Shamigarah et al 2015). Heavy metal resistant

Pseudomonas aeruginosa RZS3 show antagonism against Aspergillus niger NCIM 1025, A. flavus NCIM 650,

https://www.frontiersin.org/articles/10.3389/fmicb.2018.02213/full#B52

Identification and Characterisation of Siderophore Positive Pseudomonas from North 241

Indian Rosewood (Dalbergia Sissoo) Forest Ecosystem


Fusarium oxysporum NCIM 1281, Alternaria alternata ARI 715, Cercospora arachichola, Metarhizium anisopliae

NCIM 1311 and P. solanacerum NCIM 5103 (Sayyed and Patel 2011). Abo Zaid et al. (2020) screened twenty

Pseudomonas strains against six plant pathogenic fungi and observed that two putative biocontrol strains P. aeruginosa

F2 and P. fluorescens JY3 were also producing siderophore. In these strains, siderophore production was highest in

batch and exponential fed-batch fermentation. These examples indicate that siderophore positive Pseudomonas species

can serve as multifunctional microbial inoculants. Their beneficial effects include both plant growth enhancement and

biological control of phytopathogenic fungi (Kumar et al., 2017). Although siderophore positive rhizobacteria from

crop cultivation systems have been extensively characterized. But very little is known about the siderophore producing

bacteria from forest ecosystems specifically from Dalbergia sissoo forests and plantations. Therefore, the present study

was planned with the following specific objectives i) Analysis of physico-chemical properties and microbial diversity in

rhizospheric and bulk soil samples from Dalbergia sissoo forests ecosystem in two different seasons “cold dry” and

"monsoon” season using culture-dependent approach, ii) isolation and screening of siderophore producing bacteria and

optimization of physicochemical parameters for enhanced siderophore production. The selected strains can serve as iron

bio inoculants for commercial agriculture in iron scarce agroforestry ecosystems where the bio-availability of iron is

compromised.

MATERIAL AND METHODS

Soil Sampling

Soil samples (Bulk, B and Rhizospheric, R) were collected from a ten year old shisham plantation, in two different seasons,

cold dry season (Oct- Dec) and monsoon season (June-Aug) maintained at Agroforestry Research Centre, G.B Pant

University of Agriculture & Technology, Pantnagar (28°58′N 79°25′E / 28.97°N 79.41°E). Soil was immediately

transferred in polyethylene bags and transported to laboratory and refrigerated at 40 C. The samples were serially diluted

and plated on nutrient agar plates. The plates were incubated at 28±20C in BOD incubator for 3 to 5 days. The colonies

were distinguished and pure cultures were maintained in separate plates.

Physicochemical Characteristics of Shisham Rhizospheric Soil

The soil pH was determined by the slurry method wherein soil and distilled deionized water were mixed in the ratio

1:5 and measured with a glass electrode of microprocessor based pH meter, century CP 931 (Miller and Donochue,

1992). For measurement of electrical conductivity (EC), soil and water were mixed in ratio 1:25, and reading

recorded with digital microprocessor based conductivity meter (Systronic Model 306). Total organic carbon (OC)

was determined by Kjeldahl digestion method (TKN). Available phosphorous (AP) content was measured

colourimetrically after extraction with 0.5 mol l-1 NaHCO3 (pH8.5) for 30 minute (Olsen et al., 1954). Available

potassium (AK) content was measured with a flame photometer after extraction with 1 mol l-1 NH4Ac (pH 7.0) for 30

minutes (Yuan et al., 1983). Minor trace element like iron and zinc were measured using an atomic absorption

spectrometry (Yao et al., 2003). The correlation between soil factors within two seasons was analysed statistically by

two-way ANOVA (P<0.05).

Quantitative and Qualitative Assay for Siderophore Production

All twenty bacterial isolates were grown in LB Broth at 30oC and 120 rpm for 48-72 hrs. and spectrophotometrically

screened for siderophore production. The production of siderophores was further confirmed by CAS Agar test



developed by Schwyn and Neilands (1987) and modified by Alexendar and Zuberer (1991). For 100 ml CAS

Solution, 60.5 mg of CAS dye was diffused into 50 ml of deionized water to which 10 ml of FeCl3.6H20 solution was

added. 72.9 mg Hexa Decyl Trimethyl Ammonium Bromide (HDTMA) was separately dissolved in 40 ml of deionized

water and added to CAS solution to make up 100 ml volume. The resulting solution was autoclaved for 30 min at 1210C

at 15 psi. For CAS agar test, 0.5 ml of CAS solution was added to 0.5 ml of culture supernatant and incubated for 5

minutes. The absorbance of solution was measured at 630 nm and the amount of siderophore was calculated and

represented as % siderophore units using the formula% of Siderophore= Ar-As/Ar*100 (Set et al, 2017) Where, Ar

=Absorbance of the reference (CAS Reagent); As= absorbance of the sample at 630 nm. Confirmation was done by

qualitative CAS agar test. The log phase bacterial culture was spotted on nutrient agar plates amended with CAS

solution. The plates were incubated at 28°C under dark for 3-5 days. The appearance of yellow to orange zone confirms

the siderophore production. All the assays were carried out in triplicates.

Characterisation of Siderophore

Detection of Hydroxamate Type of Siderophore

Cskay method (1948) was used for the detection of hydroxymate type siderophores. Culture filtrate (1ml) and 6N H2SO4

(1ml) were boil together for 10minutes to release the bound hydroxymate. Then 10 ml of an indicator solution (sodium

arsenite 1ml + alpha naphthylamine 1ml, volume made up to 10 ml with DW) was added to check the colour change

reaction from orange to pink and absorbance of resulting solution was read at 526 nm taking hydroxylamine HCL was

taken as a standard.

Detection of Catechol Type of Siderophore

Catecholate type of siderophore was assayed by Arnow’s test (Arnow, 1937). To 1 ml bacterial supernatant, 1 ml 0.5N

HCL was added, followed by 1ml Nitrate Molybdate reagent (10 gm. sodium Nitrite+ 10 gm. sodium molybdate in 100

ml double distilled water). The resulting solution was incubated at 28 ± 1 ° C for 10 -15 minute. The absorbance was

measured at 510 nm taking catechol as a standard. Water and catechol were taken as negative and positive control

respectively.

PCR Amplification, Phylogenetic Analysis and Sequencing of 16S r DNA

Bacterial genomic DNA of all the 10 isolates recovered from Dalbergia sissoo plantation forest ecosystem was extracted

(Bazzicalupo and Fani, 1995) and 16S rDNA was amplified using Primers GM3f (5’TACCTTGTTGTTACGACTT3’) and

GM4r (5’TACCTTGTTACGACTT3’) (Muyzer et al., 1995). The amplified 1492 bp 16S rDNA region of all 10 isolates

was sequenced on 3730 DNA sequencer using ABI big dye terminator technology (Central Instrumental facility, Biotech

Centre UDSC, New Delhi) using same set of primers as used for 16S rRNA gene amplification.

The strains were identified using nearly complete sequence of 16S rDNA gene on EzTaxon server

(http://eztaxon_e.ezbiocloud.net) and blast search on NCBI server. The phylogenetic and molecular analysis was

performed with all the closely related taxa according to procedure described previously using MEGA version 7.0 (Tamura

et al., 2011, Roohi et al., 2012). Amplified PCR products of the selected strains were submitted to NCBI Data Bank.

Optimization of Physicochemical Parameters for Siderophore Production:

Varying sources of carbon and nitrogen, pH and concentration of iron and heavy metals were optimized for enhanced

http://eztaxon_e.ezbiocloud.net/




siderophore production.

(i) Effect of pH

The effect of varying pH (3-11) on siderophore production was studied. The succinate broth with different pH was

inoculated with log phase of each bacterial culture separately and incubated at 370C for 48-72 h at 120 rpm. Thereafter, 1

ml culture filtrate was added to 1 ml CAS solution and absorbance measured at 630 nm and % siderophore units calculated.

(ii) Effect of Iron Concentration

The succinic acid medium was supplemented with varying concentration of iron to determine, threshold level of iron which

repressed siderophore production. Log phase bacterial culture was inoculated separately in succinate broth amended with

varying FeCl3.6H2O concentration (0, 25, 50, 100, 150 µM) and incubated at 370C for 48-72 h at 120 rpm. Thereafter, 1ml

culture filtrate was added to CAS solution (1 ml) and absorbance was measured at 630 nm. Siderophore yield was

calculated as % siderophore unit.

(iii) Effect of Carbon and Nitrogen Source on Siderophore Production

The succinate broth (100 ml) was supplemented with 1g l-1 of four different carbon sources. Each bacterial isolate was

inoculated separately in succinate broth supplemented with sucrose, glucose, starch and mannitol and incubated at 370C for

48-72 h at 120 rpm. After 72 h, 1 ml culture filtrate was added to 1 ml CAS solution (1:1). The absorbance was measured

at 630 nm and % siderophore units calculated.

Similarly, loopful of log phase bacterial culture was inoculated into succinate broth supplemented with ammonium nitrate,

yeast extract, proteose peptone and potassium nitrate separately. The flasks were incubated at 37oC, 120 rpm for 48-72 h

after which 1ml culture filtrate was added to 1 ml of CAS solution and the absorbance measured at 630 nm and %

siderophore units were calculated.

v) Effect of Heavy Metals on Siderophore Production

To evaluate the influence of heavy metals on siderophore production, succinate broth (100 ml) was supplemented with

10µm of each, HgCl2, MnCl2, CdCl2 and NiCl2, separately followed by incubation at 37oC for 48-72 hrs. The %

siderophore unit was estimated.

Statistical Analysis

Data was analysed using two way Anova under Completely Randomized Design. Each of the parameters tested,

significantly affects the siderophore production. Moreover the interaction of each parameter with the isolates also is highly

significant.

RESULTS

Soil Physicochemical Analysis

The soil texture at the experimental site was sandy loam. During the cold season, the pH of both the rhizospheric and

bulk soil was near neutral. It was 6.72 and 7.0 respectively. In contrast during monsoon season, pH for rhizospheric

and bulk soil was alkaline and 8 and 8.5 respectively. During the cold dry season, electrical conductivity was 21.86

dsm-1 for (RS) and 21.14 dsm-1 for (BS) whereas in monsoon season 52.03dsm-1 and 43.58 dsm-1 for RS and BS

respectively. In contrast, Total organic carbon (TOC) in the monsoon season was higher than the cold dry season.



During the monsoon season, TOC in (RS) and (BS) was 45909 and 43768 kg ha-1 whereas in cold dry season 25900

and 25000 kg ha-1respectively. Available phosphorous (AP) in the monsoon season was 360.161 kg ha in (RS) and

382.67 kg ha-1 in (BS) whereas in cold dry season 661.79kg ha-1 in (RS) rhizospheric soil and 54.04 kg ha-1 in (BS)

respectively. Total Kjeldahl nitrogen (TKN) in monsoon season was 815.356kg ha-1 in (RS) and 301.05kg ha-1 (BS)

whereas 200.70 kg ha-1 and 112.896 kg ha-1 in RS and BS respectively during the cold dry season. In contrast,

potassium content in the cold dry season was 352 kg ha-1 in RS and 246 Kg ha-1 in BS whereas 168.87 kg ha-1 in RS

and 125.98 kg ha-1 in BS during the monsoon season. Iron content was higher in monsoon season 19.53 kg ha-1 in RS

and 17.46 kg ha-1 BS and in the cold dry season, it was 17.45kg ha-1 in RS and 12.58 kg ha-1 in bulk soil respectively.

Zinc content was 1.34 kg ha-1 in RS and 1.008 kg ha-1 in BS in monsoon season and 0.947 kg ha-1 in RS and 1.064 kg

ha-1 in BS during the cold dry season. Figure 1(a) and (b)

0500

1000

Qu

an

tity

in K

g/h

act

are

Physicochemical parameters of

Rhizospheric soil from Dalbergia forest

ecosystem

1st Season 2nd Season

(a) (b)

Figure 1: Graphical Representation of Soil Physicochemical Parameters from Dalbergia sissoo Forest Ecosystem in

Two Different Seasons a) Rhizospheric and b) Bulk Soil

Quantitative and Qualitative Estimation of siderophore

All the 10 bacterial isolates depicted yellow to orange halo zone on CAS medium indicating positive for siderophore

production (Figure2). Each of the 10 isolates were producing 100-90 % siderophore units (Table1)

Figure 2: Siderophore production by bacterial isolates from Dalbergia sissoo Roxb. plantation forest ecosystem in a

CAS agar Plate Assay

Table1: Identification of the Siderophore Producing Bacteria through 16S rDNA Sequencing and Qualitative and

Quantitative Estimation of Siderophore Production in Bacterial Strains Recovered from Dalbergia sissoo

Plantation Forest Ecosystem




Isolate Identified Strains

Quantitative

Analysis

( % SU)

Qualitative

Analysis

%

Similarity

Accessions

No

R3 Pseudomonas constantinni 72.23±0.05 ++ 92.75 MN759444

R2 Pseudomonas benzenevorans 73.33±0.5 +++ 90.95 MN759445

B9 Pseudomonas chlororaphis 86.32±0.005 +++ 98.41 MN759442

B6 Pseudomonas lini 70.79±0.01 ++ 97.61 MN759443

B8 Pseudomonas monteilli 80.36±0.005 +++ 93.13 MN759447

B3 Pseudomonas azotoformans 82.66±0.05 +++ 97.68 MN759446

B2 Pseudomonas cedrina sub spcedrina 71.91±0.005 ++ 83.94 -*

R4 Pseudomonas paralactis 68.33±0.01 ++ 86.15 -*

R5 Streptomyces lavendulae 88.33±0.57 +++ 90.62 MN759448

R10 Burkholderia territorii 91.33±0,5 +++ 86.73 -* Data are represented by the means of three replicates± standard deviation,(+++), high production;(++), medium production;(+)low.

*for three isolates (R4,B6,R10) accession number could not be retrieved in NCBI due low% similarity.

Identification of Siderophore Producing Isolates

The siderophore positive, 10 bacterial isolates were identified through 16S rDNA sequencing analysis (Table 1). The majority

of eight isolates were identified as Pseudomonas and one each as Burkholderia and Streptomyces. Their evolutionary

relationship was also derived (Figure 3). All the five best siderophore producing strains based on quantitative and qualitative

CAS assay were from the genus Pseudomonas. These five Pseudomonas strains were selected for optimization studies.

Figure 3: Evolutionary relationships of bacteria from Dalbergia sissoo plantation forest ecosystems. The three bacterial strains

B9, B6 and R4 are showing a close relationship with Pseudomonas fluorescens sp. chlororaphis, lini, paralactis and R10 showing a

similarity with Burkholderia territorri whereas R3, R2, B3, B2, B8 show similarity with Pseudomonas sp. constantini,

benzenevorans, azotoformans, cedrina sub sp cedrina ,monteilli and R5 showing similarity with Streptomyces lavendulae.

Type of Siderophore Produced



Only one strain produced hydroxymate type of siderophore and none catecholate type of siderophore. The hydoxymate

type of siderophore was produced Streptomyces lavendulae strain R5.

Siderophore Production in Pseudomonas benzenivorans Strain R2

Maximum siderophore production was achieved at pH 7.0 (93.13% SU) and 9.0 (90.47% SU). Amongst the nitrogen

sources, NH4NO3 was the best utilizable nitrogen source yielding 80.51% siderophore unit. 50μM FeCl3 was best suited for

Pseudomonas benzenivorans yielding up to 48.98 % siderophore unit. Substitution of succinic acid broth with MnCl2

favoured siderophore production in the range 41.56 %SU. After optimization of the factors, the % SU increased with

respect to pH and NH4NO3 whereas decreased with respect to other factors such as carbon source, heavy metals salts and

Fe (Figure 4).

Siderophore Production in Pseudomonas paralactis Strain (R4)

The maximum siderophore production in Pseudomonas paralactis strain R4 was achieved at pH 7(98.57 % SU). Peptone

was the best utilizable N source yielding 54.27% SU. At 50μMFeCl3, 73.26% SU was obtained. Substitution of SSM broth

with CdCl2 yielded 31.34% SU. Upon optimization of the factors % SU increased with pH and Fe concentration. On the

contrary addition of peptone and CdCl2 resulted into siderophore yields lower than obtained in quantitative CAS assay

(<68.33% SU). The siderophore production positively correlated with pH and Fe concentration whereas negatively with

the other factors (Figure 4).

Siderophore Production in Pseudomonas lini Strain B6

The highest siderophore production in Pseudomonas lini strain B6 was attained at pH 11 (92.56% SU). Amongst four N

sources, the addition of peptone gave the highest yield (42.33% SU). The addition of 50μM FeCl3 resulted in the higher

% SU (42.49%SU). The substitution of SSM broth with MnCl2 yielded 41.83%SU. Only the single factor pH was

positively correlated with siderophore production. The other factors were negatively regulated with siderophore

production Figure 4.

Siderophore Production in Pseudomonas azotoformans Strain B3

The maximum siderophore production in Pseudomonas azotoformans strain B3 was attained at pH 11 (89.05% SU). Yeast

extract was the best utilizable N source resulting in 39.33 %SU yields. An addition of 50μM FeCl3 resulted in a

siderophore yield of 61.66 %SU. Moreover, the substitution of SSM broth with MnCl2 resulted in a siderophore yield of

45.45%SU. The pH of the medium alone was the major factor in increasing the amount of siderophore produced. The

remaining factors negatively regulated siderophore production and % SU was lower than 82.66%SU Figure 4.

Siderophore Production in Pseudomonas chlororaphis Strain B9

Maximum siderophore production in Pseudomonas chlororaphis strain B9 was attained at pH 7 (82.98% SU). Peptone was

the best utilizable N source yielding 45.40%SU. The addition of 50μM FeCl3 resulted in siderophore yield of 41.02%SU.

The substitution of SSM broth with MnCl2 resulted in 42.03%SU. The single major factor enhancing the siderophore yield

was pH. The other factors negatively regulated the siderophore production as % SU achieved lower than in quantitative

CAS assay(<86.32%SU) Figure 4.

DISCUSSIONS




Out of the 10 strains majority are within genus Pseudomonas. The reason for the dominance of Pseudomonas species

among the cultivable diversity in the Dalbergia sissoo ecosystem could be that Pseudomonas are fast growers, can utilize

various carbon sources for energy production, and have remarkable physiological and genetic adaptability (Spiere et al.,

2000). Various studies have reported that soil harbours the higher percentage of gamma Proteobacteria, amongst which

Pseudomonas have higher population density (Joshi et al., 2019). Moreover, Pseudomonas species are dominant in

unexplored soil of the Himalayan region (Shah et al. 2016). The Pseudomonas strains from this study were identified as

(R2) Pseudomonas benzenivorans, (R4) Pseudomonas paralactis (B6) Pseudomonas lini, (B3) Pseudomonas

azotoformans, and (B9) Pseudomonas chlororaphis based on their 16SrDNA sequences. The similarity level of these

strains with reference standards was low, 86-98%. Hence, if these strains are represented to housekeeping gene analysis,

could be identified as new species (Hofmann et al., 2020). These five strains are siderophore positive with production of

siderophore in range 65-90 % SU Besides, these possess other PGP traits also. Plant beneficial properties of the identified

Pseudomonas species have been proved earlier also. Previously, P. paralactis sp nov. as a biofertilizer for promoting

runner bean growth (Mihalache et al., 2016), P. lini, as a PGPR (Kiranpreet et al., 2018, 2019), P. azotoformans ASS1

protecting the plant against biotic stresses (Ma et al., 2017) and P. chlororaphis is a major soil bioinoculant for

horticultural crops and plantation trees and acts as a bio-control agent against phytopathogenic fungi due to phenazine

production (Arrebola et al., 2019; Woeng et al., 2000; Kim et al., 2000) and P. benzenivorans sp nov. has been reported

to be a remarkable xenobiotic degrader (Lang et al., 2010) .

The below-ground microbial community composition is affected by above-ground vegetation and soil

physiochemical properties. The soil of forestry ecosystems with co-cultivation of a leguminous tree Acacia mangium with

Eucalyptus urophylla or Eucalyptus grandis dominates in Firmicutes and Proteobacteria (Rachid et al., 2013). Moreover,

the cultivation of legumes favoured the growth of beneficial microbes especially Pseudomonas species in the rhizosphere

(Baker et al., 2013). In Dalbergia sissoo Roxb. plantation forests, the concentration of N and P is always higher and

shows seasonal variation (Vitousek 1984). The soil phosphorous is high because it is directly interlinked with the

regulation of nitrogen in biological nitrogen fixation (Sharma et al., 2013). Other physiological factors such as pH,

electrical conductivity, the total organic matter, micronutrients (Fe and Zn) depend on soil type. Sandy loam texture of the

soil allows good propagation of the shisham trees. However, during rains, the nutrient rich topsoil from the sub-Himalayan

tract is carried away to the agriculture fields leading to the increment of clay and nutrient content (Fe, Zn, K P, and N).

These soil conditions make the tree more vulnerable to fungus and insect-pest attack. Phytopathogenic fungi Rhizoctonia

solani, Fusarium oxysporum, Fusarium solani, Ganoderma sp., Fomes lucidium, Phellinus gilvus infect the trees (Bakshi

1974; Mukherjee et al., 1997; Ariful Islam et al., 2018).

The abundance of Pseudomonas influences soil Fe availability by releasing siderophores, organic acids, and mobilization

of Fe oxides. Soil Fe content in Dalbergia sissoo plantation forest was between 19.53 kg ha-1 (RS) and 17.46 kg ha-1(BS)

in monsoon season and 17.45 kg ha-1 (RS) and 12.58 kg ha-1 (BS) in the cold dry season. An estimated value of 40,000 kg

ha-1 of Fe is the standard value present in the agricultural land of 50 cm depth (Shenker and Chen, 2004). Therefore, the

value obtained in our study was below the threshold value. Thus its bioavailability for plant optimal growth was restricted.

Among the various abiotic factors, pH affects the availability of iron. During the cold dry season, pH was between 6.72

and 7 in RS and BS respectively whereas upon the onset of rains, it increased up to 8 in RS and 8.5 in BS. It is already

known that the increase in pH lowers the bioavailability of iron. (Rengel et al., 2015). A single unit increment in the soil

pH above neutral leads to a 95% decline in Fe accessibility to plants. At pH 7 or above, under aerobic conditions, the



concentration of inorganic Fe in solution is 10 -10 M, which is many-fold lower than the amount required for optimal plant

growth (Romheld and Marschner 1986). The composition of soil microbial population also affects the solubility and

accessibility of Fe to plants (Becker and Asch, 2005).

Quantitatively, siderophore produced by five selected Pseudomonas strains in succinate broth was between 65-

90% SU, P. benzenevorans R2 (73.33%), P. paralactis R4 (68.33%), P. lini B6 (70.79), P. azotoformans B3 (82.66%),

and P. chlororaphis B9 (86.32%). The siderophore production was enhancing when the pH of medium was 7-11. The

probable reason is that the pH of medium regulates the dissolution and precipitation of Fe and its availability to the

growing bacteria.(Mengel 1994; Lindsay 1986; Columbo et al.,2016). The amount of siderophore produced in P.

benzenivorans strain R2 at pH 7 and pH 9 was 93.13% and 90.47% SU respectively. The siderophore yield in P. paralactis

strain R4 was 98.57% SU and P chlororaphis (B9) was 82.98 % SU. The siderophore yield in P. lini (B6) at pH 9 and

pH11 was 68.67 % and 92.56 % SU respectively. Alkaline pH of the medium decreases the solubility of iron thus making

it unavailable to the growing bacteria creating an iron-depleted environment suitable for siderophore production. P.

aeruginosa strain JAS-25 was grown best at pH 7 in King’s B medium, with the production of 130 μM of siderophore

(Sulochna et al., 2014).Similarly (Carlos et al., 2019) studied the iron chelating ability of five siderophore positive strains

in alkaline pH. Bacillus subtilis had the highest chelating capacity at pH 9. The optimum siderophore production in

Pseudomonas azotoformans strain B3 siderophore was achieved at pH 11. The addition of FeCl3 in the medium affected

the siderophore production. The addition of 25 μM FeCl3 resulted in a siderophore in the range of 10-30 %SU. Upon

increasing iron concentration, there was a steep decline in the % SU. This could be because once iron concentration in the

medium reached above the threshold value required for siderophore production, it negatively regulates iron acquisition

genes (Crichton., 2012; Tailor and Joshi.,2012; Ganesapilli and Sinha.,2015). For example, siderophore production by

two P. aeruginosa strains RSP5 and RSP8 in SSM medium without added iron was 134 μg/ml, and 210 μg/ml respectively

and with 20 μM Fe, 10 μg/ml, and 75 μg/ml respectively (Sah et al., 2017). In contrast, the production of siderophore by

Pseudomonas fluorescence SSM medium is independent of iron in the medium (Nair et al 2007; Bholay et al., 2012;

Sinha et al., 2018). Of the four nitrogen sources amended in succinate medium, the addition of ammonium nitrate

enhanced siderophore production in strains.

All the five, selected Pseudomonas strains from this study were able to synthesize siderophore efficiently in the

presence of heavy metal, MnCl2. Apart from MnCl2 other metals were negatively correlated with siderophore production in

all the strains. Previously Berraho et al. (1997) studied the effect of heavy metals on siderophore production and reported

that the addition of 100 μM of Mo and Mn concentration enhanced the siderophore production by up to 45 % and 100%

respectively. The possible reason for enhanced siderophore production in the presence of heavy metals is the ability of

Mn2+ to substitute for Fe2+ in the intracellular spaces inside the cell to control siderophore synthesis (Williams, 1982).

Baysee et al 2000 described that fluorescent pseudomonas can form complexes with other metals at a lower affinity than

Fe’ hence these strains can be suitable for bioremediation of metal contaminated soils. Major findings of the study include

that these five strains have major nutritional requirements which can be utilized for commercialization as bio-inoculants.

Major factors like pH, Fe concentration and Ammonium nitrate influencing siderophore production can be further

optimized using statistical software for scaling up siderophore production using batch fermenters.




(a) (b)

Figure 4(a): Effect of pH on Siderophore Production Figure 4(b): Effect of Iron Concentration on Siderophore

Production

(c) (d)

(e)

Figure 4(c): Effect of Carbon Sources 4(d) Nitrogen Source 4(e) Heavy Metals Concentration on Siderophore

Production

CONCLUSIONS

The present study elucidates that different species belonging to the genus Pseudomonas have specific optimal conditions

for siderophore production. The siderophore production by strains was higher in iron-deficient SSM broth as revealed in

higher % SU. During optimization studies, Among the various abiotic factors tested, pH and Fe concentration were

influencing siderophore production. Upon maintaining the pH of SSM broth between7-9, a marked increment in % SU was

observed, minimal or no iron was required to initiate siderophore production. Ammonium sulphate itself in the SSM broth



served as an efficient utilizable N source rather than other amended N sources. These optimization results could be utilized

at large scale production of siderophore and can be effectively used as an iron inoculant in iron deprived soil. The

Pseudomonas strains from Dalbergia sissoo Roxb. plantation ecosystem can be used as an effective bio-inoculant to

protect this natural perennial heritage sissoo. The availability of siderophore-producing bacteria in the rhizosphere region is

worthy of importance in agriculture, providing iron to the plants and preventing the growth of phytopathogens which are

iron-dependent. To our knowledge, this is the first report on siderophore-producing bacteria from Dalbergia sissoo forest

ecosystem. Since the application of siderophore-producing bacteria as bio-inoculant is of immense importance in a agro-

ecosystem as well as tree based agroforestry ecosystem to improve yield and maintain soil fertility level, the findings of

this study are highly significant.

Conflict of Interest: The authors declare that they have no conflict of interest.

Acknowledgement: Authors thanks Dr Laksmi Tewari (Head of Department), Department of Microbiology GBPUA&T,

Pantnagar Uttarakhand for providing facilities and working environment. We would like to express our gratitude to the

expert of Agroforestry Dr Salil Tewari (Head of Department), Department of Genetics & Plant Breeding, GBPUA&T,

Pantnagar Uttarakhand .

REFERENCES

1. 1-Lal, H. S., & Sanjay, S. (2012). Ethnomedicinal uses of Dalbergia sissoo Roxb in Jharkhand. International Journal of

Ayurvedic and Herbal Medicine, 2(1), 198-201.

2. Ahmad, B. I., Khan, R. A., & Siddiqui, M. T. (2013). Incidence of dieback disease following fungal inoculations of sexually and

asexually propagated shisham (D albergia sissoo). Forest Pathology, 43(1), 77-82.

3. Lal, H. S., Singh, S. (2012): Ethnomedicinal uses of Dalbergia sissoo Roxb in Jharkhand. – International Journal of Ayurvedic

& Herbal Medicine 2(1): 198-201.

4. Ashraf, M. U. H. A. M. M. A. D., Mumtaz, A. S., Riasat, R., & Tabassum, S. (2010). A molecular study of genetic diversity in

shisham (dalbergia sissoo) plantation of NWFP, Pakistan. Pak. J. Bot, 42(1), 79-88.

5. Sagta HC and Nautiyal S (2001). Growth performance and genetic divergence of various provenances of Dalbergia sissoo

Roxb. at nursery stage. Silvae Genet. 50: 93-99

6. Ahmad, I., Atiq, M., Gul, S., Hannan, A., Siddiqui, M. T., Nawaz, M. F., & Ahmed, S. (2016). Dieback disease predictive model

for sexually and asexually propagated Dalbergia sissoo(Shisham). Pak J Bot, 48(4), 1645-1650.

7. Ahmad, I., Hannan, A., Ahmad, S., Asif, M., Nawaz, M. F., Tanvir, M. A., & Azhar, M. F. (2017). Fungi associated with

decline of Kikar (Acacia nilotica) and Red River Gum (Eucalyptus camaldulensis) in Faisalabad. International Journal of

Biological, Biomolecular, Agricultural, Food and Biotechnological Engineering, 11(2), 171-174.

8. Vogel, S., Tantau, H., Mielke-Ehret, N., Hoque, M. I., Sarker, R. H., Saha, M. L., ... & Mühlbach, H. P. (2011). Detection of

virus particles and double-stranded RNA in dieback affected dalbergia sissoo Roxb. from Bangladesh. Bangladesh Journal of

Botany, 40(1), 57-65.

9. Kloepper, J. W., Leong, J., Teintze, M., & Schroth, M. N. (1980). Pseudomonas siderophores: a mechanism explaining

disease-suppressive soils. Current microbiology, 4(5), 317-320.

10. Neilands, J. B. (1995). Siderophores: structure and function of microbial iron transport compounds. Journal of Biological

Chemistry, 270(45), 26723-26726.




11. Ahmed, E., & Holmström, S. J. (2014). Siderophores in environmental research: roles and applications. Microbial

biotechnology, 7(3), 196-208.

12. Ali, S. S., & Vidhale, N. N. (2013). Bacterial siderophore and their application: a review. Int J Curr Microbiol Appl Sci, 2(12),

303-312.

13. Corbin, J. L., & Bulen, W. A. (1969). Isolation and identification of 2, 3-dihydroxybenzoic acid and. N2, N6-di (2, 3-

dihydroxybenzoyl)-l-lysine formed by iron-deficient Azotobacter vinelandii. Biochemistry, 8(3), 757-762.

14. Hishe, H. A. D. G. U., et al. "The influence of socioeconomic factors on deforestation: A case study of the dry Afromontane forest

of Desa’a in Tigray Region, Northern Ethiopia." International Journal of Agricultural Science and Research 5.3 (2015): 339-348.

15. PAGE, W. J., & VON TIGERSTROM, M. A. R. G. A. R. E. T. (1988). Aminochelin, a catecholamine siderophore produced by

Azotobacter vinelandii. Microbiology, 134(2), 453-460.

16. Cornish, A. S., & Page, W. J. (1995). Production of the triacetecholate siderophore protochelin by Azotobacter vinelandii.

BioMetals, 8(4), 332-338.

17. Wittmann, S., Heinisch, L., Scherlitz-Hofmann, I., Stoiber, T., Ankel-Fuchs, D., & Möllmann, U. (2004). Catecholates and

mixed catecholate hydroxamates as artificial siderophores for mycobacteria. Biometals, 17(1), 53-64.

18. Řezanka, T., Palyzová, A., Faltýsková, H., & Sigler, K. (2019). Siderophores: Amazing Metabolites of Microorganisms. In

Studies in Natural Products Chemistry (Vol. 60, pp. 157-188).Elsevier.

19. ZAHNER, H. – KELLER-SCHIERLEIN, W. – HÜTTER,R. – HESS-LESINGER, K. – DEER, A. 1963. Stoffwechselprodukte von

Mikroorganismen. Sideramineaus Aspergillacaeen. In Archives of Microbiology, vol.45, vol. 119–135.

20. O'sullivan, D. J., & O'Gara, F. (1992). Traits of fluorescent Pseudomonas spp. involved in suppression of plant root

pathogens. Microbiology and Molecular Biology Reviews, 56(4), 662-676.

21. Schalk, I. J., Hannauer, M., & Braud, A. (2011). New roles for bacterial siderophores in metal transport and tolerance.

Environmental microbiology, 13(11), 2844-2854.

22. WHO Model List of Essential Medicines. World Health Organization. October, 2013. Retrieved 22 April 2014.

23. Münzinger, M., Taraz, K., Budzikiewicz, H., Drechsel, H., Heymann, P., Winkelmann, G., & Meyer, J. M. (1999). S, S-

rhizoferrin (enantio-rhizoferrin)–a siderophore of Ralstonia (Pseudomonas) pickettii DSM 6297–the optical antipode of R, R-

rhizoferrin isolated from fungi. Biometals, 12(2), 189-193.

24. Kim, M. J., Kim, J. H., & Nam, S. W. (2011). Constitutive overexpression of Pseudoalteromonas carrageenovora arylsulfatase

in E. coli fed-batch culture. Korean Journal of Chemical Engineering, 28(4), 1101-1104.

25. ABDALLAH, M.A. 1991. Pyoverdines and pseudobactins. In WILKELMANN,G. (Ed) CRC handbook of microbial iron

chelates. Boca Raton, Florida : CRC Press. pp 139–152.

26. 24- Meyer, J. M. (2000). Pyoverdines: pigments, siderophores and potential taxonomic markers of fluorescent Pseudomonas

species. Archives of microbiology, 174(3), 135-142.

27. HOUDA, DEROUECHE, and CHAKALI GAHDAB. "SPATIAL AND TEMPORAL ANALYSIS OF DECLINE IN DJELFA

PINE FOREST (ALGERIA)." International Journal of Agricultural Science and Research (IJASR) 4.3 (2014): 155-164.

28. Meneely, K. M., & Lamb, A. L. (2007). Biochemical characterization of a flavin adenine dinculeotide-dependent

monooxygenase, ornithine hydroxylase from Pseudomonas aeruginosa, suggests a novel reaction mechanism. Biochemistry,

46(42), 11930-11937.



29. Sridevi, V., et al. "Weed management in lowland rice (Oryza sativa L.) ecosystem a review." International Journal of

Agricultural Science and Research (IJASR) 3.3 (2013): 13-21.

30. Bhattacharyya, P. N., & Jha, D. K. (2012). Plant growth-promoting rhizobacteria (PGPR): emergence in agriculture. World

Journal of Microbiology and Biotechnology, 28(4), 1327-1350.

31. Haas, D., & Défago, G. (2005). Biological control of soil-borne pathogens by fluorescent pseudomonads. Nature reviews

microbiology, 3(4), 307-319.

32. Kumar, V., Menon, S., Agarwal, H., & Gopalakrishnan, D. (2017). Characterization and optimization of bacterium isolated

from soil samples for the production of siderophores. Resource- Efficient Technologies, 3(4), 434-439.

33. SEBTI, SAFIA, and GAHDAB CHAKALI. "DISTRIBUTION AND IMPORTANCE OF THE PINE PROCESSIONARY MOTH

WINTER NESTS THAUMETOPOEA PITYOCAMPA (DENIS & SCHIFFERMÜLLER)(LEPIDOPTERA: NOTODONTIDAE)

IN THE FORESTS CEDAR OF THE NATIONAL PARK OF CHRÉA (ALGERIA)." International Journal of Agricultural

Science and Research (IJASR) 4.5 (2014): 77-84.

34. Sah, S., & Singh, R. (2015). Siderophore: structural and functional characterisation–a comprehensive review. Agriculture

(Polnohospodárstvo), 61(3), 97-114.

35. Spiers, A. J., Buckling, A., & Rainey, P. B. (2000). The causes of Pseudomonas diversity. Microbiology, 146(10), 2345-2350.

36. Sah, S., & Singh, R. (2016). Phylogenetical coherence of Pseudomonas in unexplored soils of Himalayan region. 3 Biotech,

6(2), 170.

37. Rachid, C. T. C. C., Balieiro, F. C., Peixoto, R. S., Pinheiro, Y. A. S., Piccolo, M. C., Chaer, G. M., & Rosado, A. S. (2013).

Mixed plantations can promote microbial integration and soil nitrate increases with changes in the N cycling genes. Soil

Biology and Biochemistry, 66, 146-153.

38. Bakker, M. G., Otto-Hanson, L., Lange, A. J., Bradeen, J. M., & Kinkel, L. L. (2013). Plant monocultures produce more

antagonistic soil Streptomyces communities than high-diversity plant communities. Soil biology and biochemistry, 65, 304-312.

39. Andriesse, J. P. (1988). Nature and management of tropical peat soils (No. 59). Food & Agriculture Org..

40. Vitousek, P. M., & Sanford Jr, R. L. (1986). Nutrient cycling in moist tropical forest. Annual review of Ecology and

Systematics, 17(1), 137-167.

41. Sharma, S. B., Sayyed, R. Z., Trivedi, M. H., & Gobi, T. A. (2013). Phosphate solubilizing microbes: sustainable approach for

managing phosphorus deficiency in agricultural soils. SpringerPlus, 2(1), 587.

42. Naqvi, S. A. H., Mushtaq, S., Malik, M. T., ur Rehman, A., Fareed, S., & Zulfiqar, M. A. (2019). Factors leading towards

Dalbergia sissoo decline (Syndrome) in Indian sub-continent: A critical review and future research agenda. Pakistan Journal

of Agricultural Research, 32(2), 302.

43. Azaizeh, H., Fulder, S., Khalil, K., & Said, O. (2003). Ethnobotanical knowledge of local Arab practitioners in the Middle

Eastern region. Fitoterapia, 74(1-2), 98-108.

44. Simpson, P. (1993). The cabbage trees (Cordyline australis) are dying: investigations of Sudden Decline in New Zealand. In

Forest Decline in the Atlantic and Pacific region (pp. 280-292). Springer, Berlin, Heidelberg.

45. Negi, J. D. S., Pokhriyal, T. C., Sharma, S. D., & Bhandari, R. S. (1999). Shisham mortality in India–A case study.

Unpublished Report.

46. Mukerjee, S. K., Saroja, T., & Seshadri, T. R. (1971). Dalbergichromene: A new neoflavonoid from stem-bark and heartwood




of Dalbergia sissoo. Tetrahedron, 27(4), 799-803.

47. Troup, R. S. (1921). The Silviculture of Indian Trees 1 (Rev. Edition 1980).

48. Bakshi, B. K. (1974). Control of root disease in plantations in reforested stands (with special reference to Khair, Sissoo,

Eucalyptus etc.). Indian Forester, 100(1).

49. Sah, S., Singh, N., & Singh, R. (2017). Iron acquisition in maize (Zea mays L.) using Pseudomonas siderophore. 3 Biotech,

7(2), 121.

50. Dave, B. P., & Dube, H. C. (2000). Detection and chemical characterisation of siderophores of rhizobacterial fluorescent

Pseudomonas. Indian Phytopathology, 53(1), 97-98.

51. Sulochana, M. B., Jayachandra, S. Y., Kumar, S. A., Parameshwar, A. B., Reddy, K. M., & Dayanand, A. (2014). Siderophore

as a potential plant growth-promoting agent produced by Pseudomonas aeruginosa JAS-25. Applied biochemistry and

biotechnology, 174(1), 297-308.

52. Kumar, V., Menon, S., Agarwal, H., & Gopalakrishnan, D. (2017). Characterization and optimization of bacterium isolated

from soil samples for the production of siderophores. Resource- Efficient Technologies, 3(4), 434-439.

53. Tailor, A. J., & Joshi, B. H. (2012). Characterization and optimization of siderophore production from Pseudomonas

fluorescens strain isolated from sugarcane rhizosphere. Journal of Environmental Research and Development, 6(3A), 688-

694.

54. Sharma, T., Kumar, N., & Rai, N. (2016). Production and optimization of siderophore producing Pseudomonas species

isolated from Tarai region of Uttarakhand. Int J Pharm Biol Sci, 7(1), 306-314.

55. Ferreira, C. M., Vilas-Boas, Â., Sousa, C. A., Soares, H. M., & Soares, E. V. (2019). Comparison of five bacterial strains

producing siderophores with ability to chelate iron under alkaline conditions. AMB Express, 9(1), 78.

56. Gascoyne, D. J., Connor, J. A., & Bull, A. T. (1991). Isolation of bacteria producing siderophores under alkaline conditions.

Applied microbiology and biotechnology, 36(1), 130-135.

57. Murugappan, R. M., Aravinth, A., Rajaroobia, R., Karthikeyan, M., & Alamelu, M. R. (2012).Optimization of MM9 medium

constituents for enhancement of siderophoregenesis in marine Pseudomonas putida using response surface methodology.

Indian journal of microbiology, 52(3), 433-441.

58. Wafaa M, H., & Mostafa, A. E. S. (2012). Production and optimization of Pseudomonas fluorescens biomass and metabolites

for biocontrol of strawberry grey mould. American Journal of Plant Sciences, 2012.

59. Biniarz, P., Coutte, F., Gancel, F., & Łukaszewicz, M. (2018). High-throughput optimization of medium components and

culture conditions for the efficient production of a lipopeptidepseudofactin by Pseudomonas fluorescens BD5. Microbial cell

factories, 17(1), 121.

60. King, E. O., Ward, M. K., & Raney, D. E. (1954). Two simple media for the demonstration of pyocyanin and fluorescin. The

Journal of laboratory and clinical medicine, 44(2), 301-307.

61. Berraho, E. L., Lesueur, D., Diem, H. G., & Sasson, A. (1997). Iron requirement andsiderophore production in Rhizobium

ciceri during growth on an iron-deficient medium. World Journal of Microbiology and Biotechnology, 13(5), 501-510.

62. Williams, R. J. P. (1982). Free manganese (II) and iron (II) cations can act as intracellular cellcontrols. Febs Letters, 140(1),

3-10.

63. Shi, P., Xing, Z., Zhang, Y., & Chai, T. (2017, January). Effect of heavy-metal on synthesisof siderophores by Pseudomonas



aeruginosa ZGKD3. In IOP Conference Series: Earth and Environmental Science (Vol. 52, No. 1, p. 012103). IOP Publishing.

64. Baysse, C., De Vos, D., Naudet, Y., Vandermonde, A., Ochsner, U., Meyer, J. M., ... &Cornelis, P. (2000). Vanadium interferes

with siderophore-mediated iron uptake in Pseudomonasaeruginosa. Microbiology, 146(10), 2425-2434.

65. Sinha, S., & Mukherjee, S. K. (2008). Cadmium–induced siderophore production by a high Cdresistant bacterial strain

relieved Cd toxicity in plants through root colonization. Current microbiology, 56(1), 55-60.

66. Dao, K. H. T., Hamer, K. E., Clark, C. L., & Harshman, L. G. (1999). Pyoverdine productionby Pseudomonas aeruginosa

exposed to metals or an oxidative stress agent. Ecological applications, 9(2), 441-448.

67. Audenaert, K., De Meyer, G. B., & Höfte, M. M. (2002). Abscisic acid determines basal susceptibility of tomato toBotrytis

cinerea and suppresses salicylic acid-dependent signalingmechanisms. Plant physiology, 128(2), 491-501.

68. Buysens, S., Heungens, K., Poppe, J., & Hofte, M. (1996). Involvement of pyochelin andpyoverdin in suppression of Pythium-

induced damping-off of tomato by Pseudomonas aeruginosa 7NSK2. Appl. Environ. Microbiol., 62(3), 865-871.

69. Lee, C. H., Lewis, T. A., Paszczynski, A., & Crawford, R. L. (1999). Identification of an extracellular catalyst of carbon

tetrachloride dehalogenation from Pseudomonas stutzeri strain KC as pyridine-2, 6-bis (thiocarboxylate). Biochemical and

biophysical research communications, 261(3), 562-566.

70. Bisht, R., Chaturvedi, S., Srivastava, R., Sharma, A. K., & Johri, B. N. (2009). Effect ofarbuscular mycorrhizal fungi,

Pseudomonas fluorescens and Rhizobium leguminosarum on the growth and nutrient status of Dalbergia sissoo Roxb.

Tropical Ecology, 50(2), 231.

71. Boer, W. D., Folman, L. B., Summerbell, R. C., & Boddy, L. (2005). Living in a fungal world: impact of fungi on soil bacterial

niche development. FEMS microbiology reviews, 29(4), 795-811.

72. Lang, E., Burghartz, M., Spring, S., Swiderski, J., & Spröer, C. (2010). Pseudomonas benzenivorans sp. nov. and

Pseudomonas saponiphila sp. nov., represented by xenobiotics degrading type strains. Current microbiology, 60(2), 85-91.

73. von Neubeck, M., Huptas, C., Glück, C., Krewinkel, M., Stoeckel, M., Stressler, T., & Wenning, M. (2017). Pseudomonas lactis

sp. nov. and Pseudomonas paralactis sp. nov., isolated from bovine raw milk. International journal of systematic and

evolutionary microbiology, 67(6), 1656-1664.

74. Mihalache, G., Zamfirache, M. M., Hamburda, S., Stoleru, V., Munteanu, N., & Stefan, M. (2016). SYNERGISTIC EFFECT

OF Pseudomonas lini AND Bacillus pumilus ON RUNNERBEAN GROWTH ENHANCEMENT. Environmental Engineering &

Management Journal (EEMJ), 51(8).

75. Padda, K. P., Puri, A., & Chanway, C. (2019). Endophytic nitrogen fixation–a possible ‘hidden’source of nitrogen for

lodgepole pine trees growing at unreclaimed gravel miningsites. FEMS microbiology ecology, 95(11), fiz172.

76. Delorme, S., Lemanceau, P., Christen, R., Corberand, T., Meyer, J. M., & Gardan, L. (2002).

77. Pseudomonas lini sp. nov., a novel species from bulk and rhizospheric soils. International journal of systematic and

evolutionary microbiology, 52(2), 513-523.

78. 72-Ma, Y., Rajkumar, M., Moreno, A., Zhang, C., & Freitas, H. (2017). Serpentine endophytic bacterium Pseudomonas

azotoformans ASS1 accelerates phytoremediation of soil metals under drought stress. Chemosphere, 185, 75-85.

79. Arrebola, E., Tienda, S., Vida, C., De Vicente, A., & Cazorla, F. M. (2019). Fitness features involved in the biocontrol

interaction of pseudomonas chlororaphis with host plants: the case study of PcPCL1606. Frontiers in microbiology, 10.

80. Chin-A-Woeng, T. F., Bloemberg, G. V., Mulders, I. H., Dekkers, L. C., & Lugtenberg, B. J.(2000). Root colonization by




phenazine-1-carboxamide-producing bacterium Pseudomonaschlororaphis PCL1391 is essential for biocontrol of tomato foot

and root rot. Molecular plantmicrobe interactions, 13(12), 1340-1345.

81. Anzai, Y., Kim, H., Park, J. Y., Wakabayashi, H., & Oyaizu, H. (2000). Phylogeneticaffiliation of the pseudomonads based on

16S rRNA sequence. International journal of systematic and evolutionary microbiology, 50(4), 1563-1589.

82. Islam, M. A., Nain, Z., Alam, M. K., Banu, N. A., & Islam, M. R. (2018). In vitro study ofbiocontrol potential of rhizospheric

Pseudomonas aeruginosa against Fusarium oxysporum f. sp. cucumerinum. Egyptian Journal of Biological Pest Control,

28(1), 90.

83. Guez, J. S., Chenikher, S., Cassar, J. P., & Jacques, P. (2007). Setting up and modelling ofoverflowing fed-batch cultures of

Bacillus subtilis for the production and continuous removal oflipopeptides. Journal of biotechnology, 131(1), 67-75.

84. Kumar, V. (2015). Nursery and plantation practices in forestry. Scientific Publishers.

85. Crichton, R. R. (2012). Transport, Storage, and Homeostasis of Metal Ions Elsevier.

86. Ganesapillai, M., & Simha, A. S. P. (2016). Separation processes and technologies as the mainstay in chemical, biochemical,

petroleum and environmental engineering: a special issue. Resource-Efficient Technologies, 2, 12.

87. Joshi, S., Jaggi, V., Tiwari, S., Sah, V. K., & Sahgal, M. (2019). Multitrate phosphate solubilizing bacteria from Dalbergia

sissoo Roxb. Rhizosphere in natural forests of Indian Central Himalayas. Environ Ecol, 37(3A), 894-908.

88. Shenker, M., & Chen, Y. (2005). Increasing iron availability to crops: fertilizers, organo‐fertilizers, and biological

approaches. Soil Science & Plant Nutrition, 51(1), 1-17.

89. Römheld, V., & Marschner, H. (1986). Evidence for a specific uptake system for iron phytosiderophores in roots of grasses.

Plant physiology, 80(1), 175-180.

90. Hofmann, M., Heine, T., Schulz, V., Hofmann, S., & Tischler, D. (2020). Draft genomes and initial characterization of

siderophore producing pseudomonads isolated from mine dump and mine drainage. Biotechnology Reports, 25, e00403.

identification and characterisation of siderophore

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