Accepted Manuscript Siderophore production by Pseudomonas aeruginosa FP6, a biocontrol strain for Rhizoctonia solani and Colletotrichum gloeosporioides causing diseases in chilli Bhaktavatchalu Sasirekha, Srividya Shivakumar PII:
S2452-316X(16)30101-6
DOI:
10.1016/j.anres.2016.02.003
Reference:
ANRES 39
To appear in:
Agriculture and Natural Resources
Received Date: 28 August 2015 Accepted Date: 4 February 2016
Please cite this article as: Sasirekha B, Shivakumar S, Siderophore production by Pseudomonas aeruginosa FP6, a biocontrol strain for Rhizoctonia solani and Colletotrichum gloeosporioides causing diseases in chilli, Agriculture and Natural Resources (2016), doi: 10.1016/j.anres.2016.02.003. This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
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Agriculture and Natural Resources. 2016. 50(4): xx–xx.
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Agr. Nat. Resour. 2016. 50(4): xx–xx.
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Siderophore production by Pseudomonas aeruginosa FP6, a biocontrol strain for
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Rhizoctonia solani and Colletotrichum gloeosporioides causing diseases in chilli
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Bhaktavatchalu Sasirekhaa and Srividya Shivakumarb,*
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a
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b
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Bangalore-560 011, Karnataka, India.
Received 28 August 2015
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Accepted 4 February 2016
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Keywords:
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Biocontrol,
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Chilli,
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Optimization,
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P. aeruginosa FP6,
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Siderophore
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*Corresponding author.
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E–mail address:
[email protected]
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Department of Microbiology, Centre for Post Graduate Studies, Jain University, Jayanagar,
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Department of Microbiology, Acharya Bangalore B School, Bangalore-560 091, India.
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Abstract
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Siderophores are compounds secreted under low iron stress, which act as specific
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ferric iron chelating agents. Owing to their potential in the biological control of fungal
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phytopathogens, they may be used as an alternative strategy to chemical control.
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Pseudomonas aeruginosa FP6, previously isolated from rhizospheric soil samples was
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screened for its siderophore production on a chrome-azurol S agar plate. Change in the color
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of the chrome-azurol S agar from blue to orange red confirmed the siderophore producing
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ability of P. aeruginosa FP6. The effects of various physicochemical parameters on
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siderophore production were studied. The maximum siderophore production was obtained in
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succinate medium (125 µM) followed by King’s B medium (105 µM). The presence of
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sucrose and mannitol increased the siderophore production. Yeast extract proved to be the
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most suitable nitrogen source. Media supplemented with Pb2+, Mn2+ and Mg2+ showed
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appreciable siderophore production as well as growth of cultures. An increase in the iron
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concentration favored growth but substantially reduced siderophore production. The strain
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when tested for its in-vitro antagonistic activity against Rhizoctonia solani and
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Colletotrichum gloeosporioides on King’s B media, with and without FeCl3, showed a
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significant reduction in R. solani growth with FeCl3 supplementation compared to the control
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(without FeCl3), suggesting the role of siderophore mediated antagonism of R. solani.
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Antifungal activity was not influenced by FeCl3 in the case of C. gloeosporioides, suggesting
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the presence of other antagonistic mechanisms.
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Introduction
Chilli (Capsicum annuum L.) belongs to the family Solanaceae and is one of the
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important vegetable-cum-spice crops cultivated in India, which, after China, is not only
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largest producer but also the largest exporter of chilli in the world, with a cultivated area of
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0.81 million ha in 2010–2011 producing 1.22 million t of green chilli (Anonymous, 2011).
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Andhra Pradesh is the largest chilli-producing region in India, contributing about 26% to the
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total area, followed by Maharashtra (15%), Karnataka (11%), Orissa (11%) and Madhya
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Pradesh (7%) with other states contributing nearly 22% to the total area under chilli
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cultivation (Jagtap et al., 2012). Chilli is known to suffer from as many as 83 different diseases, of which more than 40
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are caused by fungi (Rangasami, 1988). Anthracnose (fruit rot) and damping off are the two
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major fungal diseases incited by Colletotrichum gloeosporioides and Rhizoctonia solani
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which often cause high yield loss (Than et al., 2008). With the ever-increasing awareness of
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the negative health-implications and environmental concerns regarding prolonged chemical
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usage there is a burgeoning demand for the development of alternative and safer methods of
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disease management.
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Biological control has emerged as a very popular alternative because it offers a way of
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controlling pathogens that does not involve the use of chemicals. ‘Siderophores’ (derived
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from the Greek meaning “iron carriers”) are low molecular weight (below 1000 Da),
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ferric-ion-specific chelating agents produced by bacteria and fungi to combat low iron stress
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(Ngamau et al., 2014).
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For several fluorescent Pseudomonades, it has been suggested that the
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siderophore-mediated competition for iron with soil borne pathogens is an important
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mechanism for biological control as most of these plants are able to use bacterial iron
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siderophore complexes as a source of iron from the soil with a competitive advantage under
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iron stress and thereby restrict proliferation and root colonization by phytopathogens
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(Decheng et al., 2005). Siderophore-producing rhizobacteria are also known to impart
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induced systemic resistance to plants and suppressiveness to the soil and have been
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implicated in the biocontrol of several plant diseases (Akhtar et al., 2011).
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The presence of heavy metals—even in traces—is toxic and detrimental to both flora
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and fauna (Priyadarshini and Rath, 2012). Plant growth-promoting rhizobacteria capable of
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growing in the presence of a variety of heavy metals are seen as potent bioinoculants
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(Nilanjana et al., 2008). Siderophores are also found to complex with heavy metals like
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cadmium, lead, nickel, arsenic (III, V), aluminum, magnesium zinc, copper, cobalt and
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strontium as well as iron (Sayyed and Chincholkar, 2010).
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Siderophore-based biological control agents are gaining commercial significance as
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they are safer, do not lead to biomagnification and also provide iron nutrition to the crops
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thereby promoting plant growth (Sayyed et al., 2007). Therefore, the present study was
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undertaken to study the effects of culturing conditions on the siderophore production of P.
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aeruginosa FP6 and its effect on biocontrol activity against Colletotrichum gloeosporioides
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and Rhizoctonia solani.
Materials and Methods
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Iron decontamination
All glass ware was soaked overnight in 6M HCl and rinsed with distilled water several times to remove any traces of iron.
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Bacterial strain
The P. aeruginosa FP6 strain used in this study was isolated from a rhizospheric soil
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sample (Bangalore) and was identified as Pseudomonas aeruginosa (Sasirekha et al., 2013a).
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The nucleotide sequence of the 16S rRNA of Pseudomonas aeruginosa FP6 has been
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deposited in the GenBank database under the accession number JN861778. The organism was
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maintained on King’s B agar at 4°C.
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Screening for siderophore production
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Siderophore production was qualitatively assayed as described by Schwyn and
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Neilands (1987). Briefly, overnight culture of P. aeruginosa FP6 was spot inoculated onto a
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chrome azurol S (CAS) agar plate and incubated for 18 to 24 hr at 28°C. The basic principle
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underlying the test is that when a strong ligand L (for example, siderophore) is added to a
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highly coloured dye-Fe3+ complex, the iron-ligand complex is formed and the release of free
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dye is accompanied by a colour change.
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Siderophore estimation
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An amount of 100 µL of 24 hr broth culture (1 × 108 colony forming units (cfu) per
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mL) was inoculated into 100 mL of iron-free succinic acid broth medium. The flask was
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incubated at 30°C for 24 hr in a rotary shaker (120 revolutions per minute ;rpm). Then, the
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broth culture was centrifuged for 10 min at 10,000 rpm. The amount of siderophore produced
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was determined by measuring the absorbance of the supernatant at 400 nm. The concentration
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was calculated using the absorption maximum (λ = 400 nm) and the molar extinction
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coefficient (ε = 20,000/M/cm) according to Rachid and Bensoltane (2005).
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Spectrophotometric characteristics of siderophore
P. aeruginosa FP6 was inoculated into 100 mL of iron-free succinic acid broth
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medium. The flask was incubated at 30°C for 24 hr in a rotary shaker (120 rpm). Then, the
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broth culture was centrifuged for 10 min at 10,000 rpm. After centrifugation, the supernatant
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was scanned for peaks in ultraviolet-visible spectrum range (420–450 nm for hydroxamate
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and 490–515 nm for catecholate) using a spectrophotometer (UV-2450; Shimadzu; Tokyo,
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Japan. Siderophore quenching was studied by adding 10 µL of Fe3+ solution to 3 mL of crude
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culture supernatant to achieve a final concentration of 3.3 mΜ.
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Siderophore production in different media
Siderophore production occurs only under iron-deficient conditions; hence, different
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iron-deficient media like King’s B, succinate, glucose and glutamate medium (Table 1) were
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separately inoculated. Samples were withdrawn every 6 hr and assayed for their siderophore
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content as described above. The media supporting the maximum siderophore production was
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used for further studies (Sayyed et al., 2005).
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Optimization of physiochemical parameters for siderophore production
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Various physico-chemical parameters were optimized for siderophore production
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using succinate media. The parameters tested were: carbon source, nitrogen source, iron and
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heavy metal.
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Effect of different carbon and nitrogen sources on siderophore production
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In order to examine the effect of different carbon sources on siderophore production,
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100mL of succinate broth was supplemented with 1 g/L of different carbon sources—glucose,
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sucrose, lactose, maltose, mannitol and starch. Each flask was incubated at 37°C for 24 hr in a
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rotary shaker (120 rpm). Growth and siderophore production were estimated. In order to
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determine the effect of different nitrogen sources on siderophore production, 100 µL of 24 hr
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bacterial broth (1 × 108 cfu/mL) was inoculated into 100 mL of succniate broth containing 1
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g/L of different nitrogen sources—yeast extract, peptone, sodium nitrate, potassium nitrate,
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ammonium nitrate, ammonium chloride and urea (replaced with ammonium sulphate in
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succinate medium). Each flask was incubated at 37°C for 24 hr in a rotary shaker (120 rpm).
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Growth and siderophore production in these media were compared with those of succinate
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medium containing ammonium sulphate (Tailor and Joshi, 2012).
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Influence of iron and heavy metals on siderophore production
The threshold level of iron which represses siderophore biosynthesis was determined
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by externally supplementing succinic media with 0–250 µM of iron (FeCl3.H2O). Following
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incubation at 37°C and rotary shaking at 120 rpm, growth and siderophore production were
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estimated. To determine the influence of different heavy metals on siderophore production,
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each 100 mL of succinate broth was separately supplemented with 10 µM of different heavy
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metals—cobalt (CoCl2), magnesium (MgCl2), manganese (MnCl2), mercury (HgCl2) and lead
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(PbCH3CHOO). Following the incubation at 30°C for 24–48 hr, the siderophore content was
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estimated.
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Siderophore-mediated antifungal activity Potato dextrose agar supplemented with FeCl3 (100 mg/mL) was seeded with an agar
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plug (4 mm diameter) of actively growing fungal culture of C. gloeosporioides and R. solani.
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Simultaneously, P. aeruginosa FP6 was streaked 3 cm away from the agar plug on the side
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towards the edge of the Petri plates. The plates were incubated at 30°C for 24–48 hr. The
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same experiment was repeated in the absence of FeCl3 and was used as the control (Ramesh
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Kumar et al., 2002).
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Plasmid curing
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Curing of the plasmid from the P. aeruginosa FP6 strain was done by exposing the
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overnight grown culture of P. aeruginosa to ethidium bromide (500 µg/mL). The derivatives
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of P. aeruginosa which were cured of the plasmid were selected on the basis of their inability
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to grow on culture media supplemented with kanamycin (1,000 µg/mL) unlike the parent
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strain, which is kanamycin resistant. The treated culture of P. aeruginosa was initially plated
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on a complete medium without kanamycin. The colonies of P. aeruginosa obtained were
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picked and spotted on medium containing kanamycin. Colonies that were unable to grow on
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the medium containing kanamycin were derivatives of P. aeruginosa that were cured of the
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plasmid and were designated as P. aeruginosa PC 1 to PC 100. The plasmid-cured strains
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were screened for siderophore production (Naidu and Yadav, 1997).
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Plasmid DNA isolation
The presence of plasmid in the plasmid-cured strains (P. aeruginosa PC 1 to PC 100)
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and the parent strain (P. aeruginosa FP6) was detected using the alkaline lysis method (Davis
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et al., 1986). The purified plasmid DNA was visualized by resolving on 0.8% agarose gel
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electrophoresis.
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Results and Discussion
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The siderophore production detected using CAS agar showed orange-colored colonies
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after 24 hr of incubation due to the siderophoral removal of Fe from the dye which primarily
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indicated the ability of P. aeruginosa FP6 to produce siderophore (Figure 1). Several
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researchers (Sayyed et al., 2005; Omidvari et al., 2010) have reported the production of
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siderophore by various microorganisms. P. aeruginosa FP6 produced 85.7 µM siderophore in
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succinate medium. Spectrophotometric analysis of the culture in standard succinate medium
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showed a peak between 420 nm and 450 nm (Figure 2) indicating the presence of siderophore
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of a ferric hydroxamate nature in P. aeruginosa FP6 (Ali and Vidhale, 2011). The amount of
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hydroxamate type of siderophore produced was 18.18 mg/L whereas Gull and Hafeez (2012)
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reported 15.5 µg/mL of hydroxamate siderophore in P. fluorescens Mst 8.2. Chandra et al.,
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(2007) reported 32 µg/mL of the hydroxamate type of siderophore by Mesorhizobium loti
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after 48 hr of incubation.
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Siderophore production in different media Among the different iron deficient media tested, the glucose and glutamate media
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supported the growth of FP6 but showed low siderophore production. The maximum
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siderophore production was obtained on succinate medium (125 µM) followed by King’s B
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medium (105 µM). Increased siderophore production in succinate media can be substantiated
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by the structure of pyoverdins in which the three amino moiety of the chromophore is
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substituted with various acyl groups derived from succinate, malate, α-ketoglutarate (Linget
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et al., 1992).
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The time course study revealed that siderophore production commenced after a lag
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period of 6 hr after inoculation. Maximum siderophore production was observed at 36 hr of
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incubation, after which, it started to decrease as the the culture had reached its stationary
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phase in the succinate medium (Figure 3A). King’s B medium supported maximum
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siderophore production from 24 hr of incubation (Figure 3B). Statistically, the results showed
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a positive correlation between siderophore production and time in both succinate (coefficient
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of determination, r2 = 0.91) and King’s B (r2 = 0.84) medium. This was in contrast to the
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work by Tailor and Joshi (2012) who reported maximum siderophore production at 24 hr, and
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Sayyed et al., (2005) who reported maximum siderophore production between 24 hr and 30
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hr in modified succinate medium using fluorescent Pseudomonas spp. Prashant et al. (2009)
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reported that A. calcoaceticus produced optimum siderophore at 36 hr of incubation, similar
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to the results in the current study.
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Effect of carbon and nitrogen sources on siderophore production
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The carbon source and its availability play a secondary but important role in
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regulating siderophore production. Siderophore production varied with the type of carbon
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source in Pseudomonas sp. (Sayyed et al., 2005). Along similar lines, FP6 showed a varied
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response to the presence of different sugars, with mannitol and sucrose supporting increased
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growth and siderophore production (Figure 4A).
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Of the various nitrogen sources tested, the optimum siderophore yields of 104.8 µM
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and 92.9 µM were obtained in yeast extract and urea supplemented media, respectively
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(Figure 4B). Urea was used with the view that the unutilized part of it serves as N fertilizer
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when the post-fermentation broth is used for field application. Utilization of urea by the
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isolate in the current study also suggested its possible exploitation for bioremediation of
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alkaline soils by reducing the excess amount of urea present in the soil (Sayyed et al., 2010).
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Influence of iron and heavy metal on siderophore production
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As shown in Figure 5A, the growth of FP6 increased with an increasing concentration
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of iron tested (1–250 µM). However, an Fe concentration ranging from 5 µM upward
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repressed siderophore production. An amount of 100 µM Fe in the media completely
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repressed siderophore synthesis by P. aeruginosa FP6. Statistically, the results showed a
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positive correlation (r2= 0.94) between the iron concentration and siderophore production
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(Figure 5B). The increase in growth with an increase in the iron concentration reflected the
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iron requirement by the strain for cellular processes. It has been reported that the siderophore
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transport system contains several gene products, all of which are negatively regulated by
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Fe-binding protein (Fur) for ferric uptake regulation (Klaus, 2002). Similar observations have
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been made by Sayyed et al., (2005) who reported that an increase in the iron concentration up
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to 100 µM favored growth but affected the siderophore production of P. fluorescens NCIM
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5096 and P. putida NCIM 2847. Prashant et al. (2009) have shown that the biosynthesis of
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siderophores in Acinetobacter calcoaceticus was suppressed in the presence of 20 µM free Fe,
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emphasizing the role of iron in siderophore production.
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Baysse et al. (2000) reported that siderophore can form complexes with metals with
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lower affinity other than Fe3+. In the current study, media supplemented with lead, manganese
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and magnesium salts supported siderophore production but this was reduced compared to the
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control (Table 2). Metals like Cd2+, Hg2+ and Co2+ showed an inhibitory effect on both growth
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and siderophore production which may have been due to the competitive binding of the
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metals, thereby elevating oxidative stress in the bacterial cells (Dimkpa et al., 2009).
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Chelation of metal ions may reduce their toxicity and hence allow pigment production. It has
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been reported that Mn2+ may substitute for Fe2+ in the intracellular control of siderophore
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synthesis (Williams, 1982).
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Siderophore mediated antifungal activity
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P. aeruginosa FP6 inhibited R. solani in the absence of FeCl3 (72.25%), which was
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reduced to 12% in the presence of FeCl3, suggesting that siderophore mediated antagonism
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whereas the strain showed an inhibitory effect against C. gloeosporioides in either the
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presence or absence of FeCl3, which may have been due to the production of other antifungal
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metabolites (Table 3).
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Plasmid curing
Plasmid curing was carried out to determine whether the siderophore genes are
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located on plasmid or chromosomal DNA. The antibiotic susceptibility test of the wild strain
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showed sensitivity to most of the antibiotics except kanamycin, (1,000 µg/mL) and
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rifampicin (500 µg/mL); therefore kanamycin was used as a marker (Van den Broek et al.,
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2003; Sasirekha et al., 2013b). In total, 100 colonies were obtained, of which 88 colonies
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were able to grow on Luria Bertanni broth (LB) medium but not on LB medium
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supplemented with kanamycin. Thus, those colonies which grew on LB medium were
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selected for further studies. The plasmid curing efficiency was 88%.
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Plasmid analysis of the wild strain showed the presence of four bands suggesting the
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presence of one or more plasmids in this strain (Figure 6, lane 1). All cured clones showed
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the loss of plasmid bands (Figure 6, lane 2) which were present in the original wild strain,
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which clearly showed the relationship between the loss of plasmid and the loss of kanamycin
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resistance but no loss in siderophore production, indicating the chromosomal location of the
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gene.
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Siderophore production by Pseudomonas aeruginosa FP6 was high under the iron-stressed
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conditions, suggesting that siderophore chelates Fe from soil and there is a lignand exchange
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with plants and thereby negatively affecting the growth of several fungal pathogens—an
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important feature in plant disease suppression. The appreciable siderophore production
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observed in the presence of lead, magnesium and manganese can be attributed to the ability
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of rhizobacterial strains to mitigate the toxic effects of metals besides their role in providing
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plants with the sufficient amounts of growth promoting substances.
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Acknowledgements The authors sincerely thank the management of Jain University, Karnataka, India for providing the necessary infrastructural and financial support to carry out this work.
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Sasirekha, B., Srividya, S., Sullia, S.B. 2013b. Molecular detection of antibiotic related genes
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from Pseudomonas aeruginosa FP6, an antagonist towards Rhizoctonia solani and
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Colletotrichum gloeosporioides. Turk. J. Biol. 37: 289–295.
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Sayyed, R.Z., Chincholkar, S.B. 2010. Growth and siderophore production Alcaligenes
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faecalis is influenced by heavy metals. Indian J. Microbiol. 50 :179–182.
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Sayyed, R.Z., Gangurde, N.S., Patel, P.R., Joshi, S.A., Chincholkar, S.B. 2010. Siderophore
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production by Alcaligenes faecalis and its application for growth promotion in
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Arachis hypogaea. Indian J. Biotechnol. 9: 302–307.
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Sayyed, R.Z., Naphade, B.S., Chincholklar, S.B. 2007. Siderophore producing A. feacalis promoted the growth of Safed musali and Ashwagandha. J. Med. Arom. Pl. 29: 1–5.
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Sayyed, R.Z., Badgujar, M.D., Sonawane, H.M., Mhaske, M.M., Chincholkar, S.B. 2005. Production of microbial iron chelators (siderophores) by fluorescent pseudomonads.
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Indian J. Biotechnol. 4: 484–490.
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Schwyn, R., Neilands, J.B. 1987. Universal chemical assay for detection and determination of siderophores. Anal. Biochem. 160: 47–56. Tailor, A.J., Joshi, H.B. 2012. Characterization and optimization of siderophore production
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from Pseudomonas fluorescens strain isolated from sugarcane rhizosphere. J. Env. Res. Dev. 6 :688–694.
Than, P.P., Jeewon, R., Hyde, K.D., Pongsupasamit, S., Mongkolporn, O., Taylor, P.W.J. 2008.
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Characterization and pathogenicity of Colletotrichum species associated with
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anthracnose disease on chilli (Capsicum spp.) in Thailand. Plant Pathol. 57: 562–572.
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Van den Broek, D., Chin-A-Woeng, T.F., Eijkemans, K., Mulders, I.H., Bloemberg, G.V.,
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Lugtenberg, B.J.J. 2003. Biocontrol traits of Pseudomonas spp. are regulated by phase
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variation. Mol. Plant Microbe In. 16: 1003–1012.
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Williams, R.J.P. 1982. Free manganese (II) and iron (II) cations can act as intracellular cell controls. FEBS Lett. 140: 3–10.
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Table 1 Composition of various culture media used for siderophore production
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Medium Component (g/L) Glutamic acid -
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0.56
KH2PO4
-
3
-
(NH4)2 SO4
-
1
-
MgSO4.7H20
-
0.2
-
Succinic acid
-
4
-
Glycerin
10
-
Peptone
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-
Glucose
-
Urea
-
Glutamic acid
-
(NH4)2 NO3
-
-
-
-
-
-
-
-
-
-
10
-
-
0.85
-
-
-
1
-
-
1
-
-
-
0.02
-
-
-
0.02
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-
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-
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NaCl
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Glucose
K2HPO4
Na2SO4
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Succinate
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Table 2 Influence of heavy metals on siderophore production
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Siderophore (µM)*
Heavy metal (10µM)
5.75 ± 0.05
Pb
88.21 ± 0.04
Co
10.35 ± 0.12
Mn
78.80 ± 0.019
Cd
7.69 ± 0.037
Mg
79.20 ± 0.026
*
values are mean ± SE of triplicates.
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Hg
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105.00 ± 0.02
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Table 3 Inhibition of Colletotrichum gloeosporioides and Rhizoctonia solani by
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Pseudomonas aeruginosa FP6 in either the presence or absence of FeCl3
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Inhibition zone (%)*
Fungal pathogen
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C. gloeosporoides
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*
With FeCl3
72.25 ± 0.065
12.22 ± 0.078
72.12 ± 0.057
61.40 ± 0.086
values are mean ± SE of triplicates.
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Without FeCl3
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Figure 1 Siderophore production on chrome azurol S medium
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Figure 2 Absorption characteristics of Pseudomonas aeruginosa FP6 culture filtrate
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confirming the hydroxamate nature of siderophores
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(B)
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120 Siderophore production (µM)
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40
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y = -0.1313x + 7.9973x - 38.088
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r = 0.8469
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6
12
18
24
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48
54
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Time (hr)
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Figure 3 Correlation and equation (r2 is the coefficient of determination) between
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siderophore production and time on: (A) succinate medium; (B) King’s B medium
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(A)
a
b 2
b
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(B) e
d
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c
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Figure 4 Effect of source on siderophore production: (A) carbon; (B) nitrogen. Different
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lowercase letters above each column indicate significant differences within sources,
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according to least significant difference (p < 0.05). Error bars show mean ± SE
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(B)
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(A) 90 80 70 60 50 40 30 20 10 0
Siderophore production
Siderophore production (µM)
Growth
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Figure 5 Graphs of (A) influence of iron level on siderophorogenesis and growth by P.
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aeruginosa FP6; (B) correlation between iron concentration and siderphore production and
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equation (r2 is the coefficient of determination)
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Figure 6 Photograph showing absence of plasmid in cured strain (lane2) compared to wild
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strain (lane1) and 1,000–5,000 bp ladder (lane M)
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