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.

IDENTIFICATION AND CHARACTERISATION OF SIDEROPHORE POSITIVE 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 dwelling 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 Original 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

Article 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).

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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,

Impact Factor (JCC): 8.3083 NAAS Rating: 4.13 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 -1 colourimetrically after extraction with 0.5 mol l NaHCO3 (pH8.5) for 30 minute (Olsen et al., 1954). Available -1 potassium (AK) content was measured with a flame photometer after extraction with 1 mol l 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

www.tjprc.org [email protected] 242 Pragati Srivastava, Vandana Jaggi, Hemant Dasila & Manvika Sahgal 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

Impact Factor (JCC): 8.3083 NAAS Rating: 4.13 Identification and Characterisation of Siderophore Positive Pseudomonas from North 243 Indian Rosewood (Dalbergia Sissoo) Forest Ecosystem 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 0 varying FeCl3.6H2O concentration (0, 25, 50, 100, 150 µM) and incubated at 37 C 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 o 10µm of each, HgCl2, MnCl2, CdCl2 and NiCl2, separately followed by incubation at 37 C 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. www.tjprc.org [email protected] 244 Pragati Srivastava, Vandana Jaggi, Hemant Dasila & Manvika Sahgal

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)

Physicochemical parameters of Rhizospheric soil from Dalbergia forest ecosystem 1000 500

0 Quantity in Kg/hactarein Quantity 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

Impact Factor (JCC): 8.3083 NAAS Rating: 4.13 Identification and Characterisation of Siderophore Positive Pseudomonas from North 245 Indian Rosewood (Dalbergia Sissoo) Forest Ecosystem Quantitative Qualitative % Accessions Isolate Identified Strains Analysis Analysis Similarity No ( % SU) 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

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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

Impact Factor (JCC): 8.3083 NAAS Rating: 4.13 Identification and Characterisation of Siderophore Positive Pseudomonas from North 247 Indian Rosewood (Dalbergia Sissoo) Forest Ecosystem 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 , 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 www.tjprc.org [email protected] 248 Pragati Srivastava, Vandana Jaggi, Hemant Dasila & Manvika Sahgal 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.

Impact Factor (JCC): 8.3083 NAAS Rating: 4.13 Identification and Characterisation of Siderophore Positive Pseudomonas from North 249 Indian Rosewood (Dalbergia Sissoo) Forest Ecosystem

(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

www.tjprc.org [email protected] 250 Pragati Srivastava, Vandana Jaggi, Hemant Dasila & Manvika Sahgal 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 .

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