Siderophore mediated antagonism of fluorescent Pseudomonads against soil borne plant pathogenic fungi in West Bengal , India

The rhizobacterial isolates from different agro ecological region of West Bengal, used in this study, were identified as fluorescent pseudomonads based on biochemical and molecular characterization. Six effective antagonist isolates were selected for further studies of secondary metabolite (siderophore) production with respect to bacterial growth at different time intervals (6 to 96 h), the nature of siderophore production (hydroxamate and/or catecholate type), effect of iron supplementation at its different concentration on siderophore production and antagonistic potentiality. Observations of higher level of growth and siderophore production, for most of the isolates, were recorded after 72 h of incubation. The maximum zone of inhibition and siderophore production was recorded at 0.5 μM and the least at 10 μM iron concentration. With the gradual increase in iron supplementation in king’s B medium, the siderophore production as well as in vitro antagonistic potentiality decreased in terms of enhancement of diameter of fungal mycelium and reduction of percent inhibition of pathogen by fluorescent pseudomonads in dual culture assay method.


INTRODUCTION
Many genera of plant root-inhabiting bacteria have potential as biological control agents (Weller, 1988).The genus Pseudomonas belongs to the γ-subclass of the Proteobacteria and includes mostly fluorescent Pseudomonads as well as a few non-fluorescent species.Particularly fluorescent Pseudomonas spp.has received much attention because of their suggested involvement in natural disease suppressiveness of certain soils (Weller et al., 2002).Iron-regulated metabolites are mainly referred to as siderophores.The siderophores are highaffinity iron chelators in bacteria, enabling uptake of Fe (Braun, 2001).Iron is essential for the growth of almost all organisms but, in many environments, the amount of free iron is below 10 -7 M, which is the required concentration by most bacteria for growth (Ratledge and Dover, 2000).Bacteria have developed several strategies for the acquisition, solubilization and transportation of iron (Guérinot, 1994).The most efficient mechanism of iron acquisition is the secretion of high affinity iron-chelating compounds, so-called siderophores.The siderophores chelate iron in the extracellular environment and the resulting ferric siderophore complex is recognized by *Corresponding authors.E-mail: subrata_mithu@yahoo.co.in, kanish.sarker@gmail.com.
# Authors contributed equally to this work.siderophore-specific membrane receptors, enabling uptake of Fe by the bacterial cells (Braun, 2001).Many bacteria produce more than one type of siderophore, as shown for Enterobacter cloacae, Mycobacterium smegmatis and Pseudomonas spp.(Adilakshmi et al., 2000;Mercado-Blanco et al., 2001).There are several reports where the effective role of siderophore on antagonistic potentiality under in vitro and in vivo cinditions, has clearly been discussed (Pal and Gokarn, 2010;Adhikari et al., 2013;Subashri et al., 2013).
Although considerable structural variation exists among the several dozen siderophores chemically characterized at the present time, most can be classified as hydroxamates or catechols (Neiland, 1981).The present study on siderophore producing fluorescent pseudomonads was concentrated on the screening for their siderophoregenesis, siderotyping and siderophore mediated antibiosis under in-vitro conditions against pathogenic fungi with different concentration of iron supplement.

Isolation of bacteria
Soil samples were collected from different crop rhizospheres from different agro-ecological region of West Bengal (Table 1).Roots were shaken vigorously to remove loosely adhering rhizospheric soil.To isolate rhizospheric bacteria, 10 g of rhizospheric soil were shaken in 90 ml sterile phosphate buffer saline (PBS) and were four fold diluted and plated on King's B (KB) medium.A total of 100 bacterial isolates were isolated and fluorescent colonies, screened through UV light (k = 365 nm), with different morphological characteristics were subcultured by repeated streaking on KB medium.On the basis of siderophore production property five highly and one moderately siderophore producing isolates were selected for further studies.

Characterization of siderophore producing bacteria
Bacterial identification was done by biochemical analysis on the basis of the tests of oxidase, arginine dihydrolase, denitrificacation, gelatine hydrolysis, levan production, and acid production from trehalose, utilization of tryptophane and L-tartrate coupled with amplification of the Pseudomonas genus specific 618 bp fragment of 16S rDNA genes by PCR using the primers PA-GS-F and PA-GS-R corresponding to the 5' (5'-GACGGGTGAGTAATGCCTA-3') end and 3' end (5'-CACTGGTGTTCCTTCCTATA-3') of the 16S rDNA gene (Spilker et al., 2004).Amplification was confirmed by analyzing 5uL of each PCR reaction mixture on a 1% agarose gel (Sambrook et al., 1989).

Qualitative assay
Siderophore production by all the isolates was tested qualitatively by Chrome Azural S (CAS) plate assay (Schwyn and Neilands, 1987).The isolates were spotted over cetrimide agar and incubated for 48 h at 28°C.After incubation, a thin layer of CAS reagent in 0.7% agar was spread on the bacterial growth and plates were again incubated for 24 h at 28°C, formation of yellow orange color zone around the colonies in plate assay indicated the siderophore production.

Quantitative assay
All the isolates were grown at 28±2°C on a rotary shaker in King's B broth for three days and centrifuged at 10,000 rpm for 10 min and the supernatant was collected.The pH of the supernatant was adjusted to 2.0 with 1 N HCl and equal quantity of ethyl acetate was added in a separating funnel, mixed well and ethyl acetate fraction was collected.5 mL of ethyl acetate fraction was mixed with 5 mL of Hathway's reagent (The reagent was prepared by adding 1 mL of 0.1 M ferric chloride in 0.1 N HCl to 100 mL of distilled water, and to this 1 mL of 0.1 M potassium ferricyanide was added).The absorbance for dihydroxy phenols was read at 700 nm in a Spectrophotometer (Reeves et al., 1983).A standard curve was prepared using different concentration of dihydroxy benzoic acid at 700 nm.The quantity of siderophore synthesized was expressed as µ mol benzoic acid mL -1 of culture filtrate.

Hydroxamate type of siderophore
Csaky's test: Hydroxamate type of siderophore was determined by hydrolyzing 1 ml supernatant of overnight grown culture with 1 ml of 6 N H2SO4 in boiling water bath for 6 h or 130°C for 30 min.Further, this hydrolyzed sample was buffered by adding 3 ml of sodium acetate solution.To this, 0.5 ml iodine was added and allowed to react for 3-5 min.After completion of reaction, the excess iodine was destroyed with 1 ml of sodium arsenate solution.Finally 1 ml of α -Naphthylamine solution was added and allowed to develop the colour.Wine red colour formation indicates production of hydroxamate type of siderophore (Gillan et al., 1981).

Neilands spectrophotometric assay
The hydroxamate nature of siderophore was detected by Neilands spectrophotometric assay (Jalal and Vander Helm, 1990;(Neilands, 1981) where a peak between 420-450 nm on addition of 2% aqueous solution of FeCl3 to 1 mL of supernatant indicated presence of Ferrate hydroxamate.

Catecholate type of siderophore Arnow's Test
To 1 ml of cell-free supernatant, 1 ml of nitrite-molybdate reagent with 1 ml NaoH solution was added.Finally, 1 ml of 0.5 N HCl was added and allowed to develop colour.Yellow colour formation indicates production of catecholate type siderophore (Arnow, 1937).

Spectrophotometric assay
Catecholate nature of siderophore was detected by the method of Jalal and Vander Helm (1990) using spectrophotometric assay where a peak at 495 nm on addition of 2% aqueous solution of FeCl3 to 1 mL of supernatant indicated the presence of siderophores of catecholate nature.

Temporal variation of bacterial growth and siderophore production
All the six fluorescent pseudomonads were grown in king's B medium by submerged culture method in 50 mL conical flasks and incubated at 28±2°C on a rotary shaker at 150 rpm.Samples were withdrawn in duplicate at definite time intervals from 6 h up to 96 h.One set was used for growth measurement at 600 nm and the other for estimation of siderophore at 700 nm (Reeves et al., 1983).

Influence of iron on production of siderophore
In order to determine the concentration of iron at which siderophore biosynthesis is minimized in fluorescent pseudomonads under study, all the cultures were grown in king's B media, externally supplemented with different concentrations of iron (FeCl3.6H2O) viz., 0.5, 1, 1.5, 2.5, 5 and 10 µM.Following the incubation at 28±2°C for 72 h at 120 rpm, siderophore contents were estimated at 700 nm.To remove traces of iron, glasswares were cleaned with 6 M HCl and with double distilled water.

In vitro antagonistic effect on mycelial growth of phytopathogen under different iron limiting conditions
An agar plug (7 mm diameter) taken from actively growing fungal culture such as Rhizoctonia solani and Sclerotium rolfsii were placed on the surface of the plate-enhancing medium supplemented with FeCl3 at different concentration that is 0.5, 1, 1.5, 2.5, 5, 10 µM.Simultaneously, fluorescent pseudomonas isolates were streaked 3 cm away from the agar plug at both sides towards the edge of Petri plates (Kumar et al., 2000).Plates inoculated with fungal agar plugs alone were used as control.The plates were incubated at 28±2°C until fungal mycelia completely covered the agar surface in control plate.Observations on mycelial growth of test pathogens were recorded and percent inhibition of pathogen growth were calculated: %I= 100(C-T)/C Adhikari et al. 4691 Where, I = inhibition of mycelial growth, C = growth of pathogen in the control plate (cm) and T = growth of pathogen in dual cultures (cm).

Statistical analysis
All the isolates were clustered with respect to their siderophoregenesis at different ferric ion concentration, zone of inhibition, inhibition percentage, temporal growth measurement and siderophore production at different time intervals by critical difference at 5% probability level using DMRT by SPSS v.10.

RESULTS AND DISCUSSION
Characterization of siderophore producing bacteria

Biochemical characterization
Among the bacterial isolates, five highly and one moderately siderophore producing isolates were confirmed as fluorescent pseudomonads based on biochemical tests such as gram reaction, oxidase, arginine dihydrolase, denitrificacation, gelatine hydrolysis, levan production, and acid production from trehalose, utilization of tryptophane and L-tartrate.Microscopic observation showed that all the six isolates were rod shaped, motile, gram negative, and fluorescent in the presence of UV light (Rao et al., 1999;Srivastava et al., 1999).All the isolates exhibited positive reaction in catalase, oxidase, arginine di hydrolase and negative in TABAC hypersensitivity test (Bossis et al., 2000).All the five isolates except FPK 10 were positive in gelatin liquefaction test; isolates MFP9 and FPK7 showed positive in trehalose test; MFP9 was positive in levan production and FPK7, FPK8 showed denitrification activity; whereas, the isolate TFP16 exhibited positive reaction in all the cases except TABAC hypersensitivity (Table 2).

Molecular characterization
PCR assays employing genus specific primer pair PA-GS-F/PA-GS-R produced 618 bp DNA fragments of the predicted size for all the six isolates (Figure 1).With the presence of the 618 bp amplicon the six putative siderophore producing isolates were confirmed as Pseudomonas sp.(Spilker et al., 2004).All the isolates produced diffusible fluorescent pigment which was confirmed in the presence of UV light, which indicated that all the isolates belonged to fluorescent pseudomonads.

Qualitative assay for siderophore production
All the six isolates were subjected to screening for side-  rophore production.Isolates CHFP23, FPK8, FPK10 produced a strong reaction with the CAS reagent (Table 3).After incubating for a period of 24 h in the dark at 28°C, a change in color from blue to orange was observed in the agar plate (Figure 2).The color change from blue to orange resulted by siderophoretic removal of Fe from the dye.
The siderophores have very high affinity for ferric iron and form ferric-siderophores complex making it unavailable to other organisms but the producer organism can utilize these complexes via a specific receptor in their outer cell membrane (Buyer and Leong, 1986).Fluorescent pseudomonads produce several siderophores such as pyoverdine, pyochelin (Dave and Dube, 2000) and are important to agriculture because they can reduce crop yield losses caused by bacteria and fungi in the root environment.The protective activity is related to the production and excretion of siderophores which efficiently chelate the iron in the root environment.This iron deficiency leads to an impaired growth of the deleterious micro-organisms.13.9 ± 0.30

Assay for siderophore typing
The type of siderophore was determined by Csaky and Arnow assay, where isolates TFP16 and FPK8 has shown hydroxamate type of siderophore production that is formation of wine red colour in supernatant.Further spectrophotometric analysis of the culture, TFP16 and FPK8 in King's B medium showed a sharp peak between 420 to 450 nm (Figure 3a) and clearly indicated the presence of siderophores of ferrate hydroxamate nature.Catecholate type of siderophore was determined by Arnow's test where the formation of yellow colour was observed in the supernatant of the isolates MFP9, CHFP23, FPK7, FPK10.A peak seen between 490 to 505 nm confirmed the presence of siderophores of catecholate nature (Figure 3b and 3c) (Ali and Vidhale, 2011).Evidence for the production of catecholate or hydroxamate siderophores by different bacterial species has been shown previously (Hohlneicher et al., 1995;Howard et al., 2000;Gull and Hafeez, 2012).
Siderophores usually form a stable, hexadentate, octahedral complex with Fe 3+ preferentially compared to other naturally occurring abundant metal ions.Siderophores are usually classified by the ligands used to  chelate the ferric iron.The majors groups of siderophores include the catecholates (phenolates), hydroxamates and carboxylates (ederivatives of citric acid) (Hofte, 1993).

Temporal variation of bacterial growth and siderophoregenesis
In the growth and siderophore production, as depicted in Figure 4a to 4f, an exponential phase from 6 to 48 h was observed regarding the isolates MFP9, CHFP23, TFP16 and FPK8; stationary phase was initiated after 48 h.In the case of the isolates FPK7 and FPK10, growth was ceased after 72 h.Threshold level of siderophore production was observed after 12 h of incubation.Syeed et al. ( 2005) also found similar result that siderophore production started after 12 h of incubation, while working  with two fluorescent pseudomonads.For all the five isolates except MFP9, production of siderophore was increased up to 72 h and declined thereafter, for MFP9 it was after 48 h.Thus, maximum siderophore yield was observed at the end of log phase growth (Sharma and Johri, 2003).

Influence of iron on siderophoregenesis
In order to assess the influence of iron concentration on siderophore release of six different isolates, King's B media supplemented with different concentration of ferric ion was used.After 72 h of incubation, maximum side-  rophore productions for all the isolates under study were obtainable at 0.5 μM Fe 3+ concentration and the lowest siderophore level was recorded at 10.0 μM Fe 3+ (Table 5).Siderophores are iron-specific compounds which are synthesized and secreted under iron stress condition and for each isolate under study siderophore production was  gradually repressed with the increasing concentration of iron (0.5, 1, 1.5, 2.5, 5, 10µM FeCl 3 ) (Budzikiewicz, 1993;Rachid and Ahmed, 2005;Sayyed et al., 2005).
The universal repressor 'Fur' acts together with iron under iron rich conditions.When the intra-cellular environment is iron rich, the Fe 2+-Fur complex bind to promoter containing a 'Fur box' and represses transcription of the genes involving in iron uptake.Moreover,    there are several reports of Pseudomonas aeruginosa and Vibrio anguillarum regarding positive regulatory mechanisms of siderophore producing genes and iron uptake systems by a combination of positive regulatory proteins and cognate siderophore but the details are under study (Chen et al., 1996;Crosa, 1997;Heinrichs and Poole, 1996;Brandel et al., 2012).

Invitro antagonistic effect on mycelial growth of phytopathogen under different iron limiting conditions
All the isolates were tested for in vitro siderophore mediated antagonism at different iron (Fe 3+ ) concentration against R. solani and S. rolfsii.All the six isolates showed percent inhibition, to some extent, at all the different iron concentrations.Although, the present study argued that percent inhibition and or zone of inhibition decreased with increasing iron concentration when all the other parameters such as growth temperature, incubation period, defined media compositions are constant (Kumar et al., 2002;Sharma and Johri, 2003).
At 0.5 µM iron concentration, the percent inhibition of mycelial growth of S .rolfsiiand R. solani for FPK8 were 71.1 and 73.3% respectively, which are the maximum among all the isolates, whereas, the same was 52.2% at 10 µM concentration of iron.The similar phenomenon is true with regard to the other isolates (Table 6a and b).It is apparent from the data that the siderophore mediated      antagonism may be one of the important characters for the fluorescent pseudomonas under study.

Conclusion
Based on these results, it can be concluded that the production of different and potent antifungal hydroxymate and catecholate siderophore by fluorescent pseudomonads against R. solani and S. rolfsii are being reported and could be effective bio-control agent against these phytopathogens.Reduction of antagonism by the addition of iron indicated that siderophores were inhibitory to fungal growth and showed a significant effect.Though different mechanisms might be responsible for the inhibition of R. solani and S. rolfsii but siderophore pro-duction might be the main bio-control mechanism associated with the antagonistic potentiality of the rhizobacterial isolates of West Bengal.

Figure 1 .
Figure 1.Agarose gel electrophoresis of PCR amplified genus specific 618 bp 16S rDNA amplicons; Lanes are named with respective isolates; L: 100 bp molecular marker.

Figure 2 .
Figure 2. Siderophore production by bacterial isolates in CAS plate assay.

Figure 3a .
Figure 3a.Absorption characteristics FPK8 and TFP16 extract confirming hydroxamate nature of siderophores.Scanning of absorption characteristics were between 400-550 nm; a sharp peak between 420 and 450 nm confirmed hydroxymate nature of siderophore of FPK8 and TFP16.

Figure 3b .
Figure 3b.Absorption characteristics FPK7 and FPK10 extract confirming catecholate nature of siderophores.Scanning of absorption characteristics were between 400-550 nm; a sharp peak between 490 and 510 nm confirmed catecholate nature of siderophore of FPK7 and FPK10.

Figure 3c .
Figure 3c.Absorption characteristics MFP9 and CHFP23 extract confirming catecholate nature of siderophores.Scanning of absorption characteristics were between 400-550 nm; a sharp peak between 490 and 510 nm confirmed catecholate nature of siderophore of MFP9 and CHFP23.

Figure 4a .
Figure 4a.Temporal growth measurement and siderophore production of MFP9 as a function of time.Vertical bar shows critical difference values.Values are mean of three replications.A common letter (letters on the bars) means they are not significantly different (p= 0.05) by DMRT.

Figure 4b .
Figure 4b.Temporal growth measurement and siderophore production of CHFP23 as a function of time.Vertical bar shows critical difference values.Values are mean of three replications .A common letter (letters on the bars) means they are not significantly different (p= 0.05) by DMRT.

Figure 4c .
Figure 4c.Temporal growth measurement and siderophore production of TFP16 as a function of time.Vertical bar shows critical difference values.Values are mean of three replications .A common letter (letters on the bars) means they are not significantly different (p= 0.05) by DMRT.

Figure 4d .
Figure 4d.Temporal growth measurement and siderophore production of FPK7 as a function of time.Vertical bar shows critical difference values.Values are mean of three replications .A common letter (letters on the bars) means they are not significantly different (p= 0.05) by DMRT.

Figure 4e .
Figure 4e.Temporal growth measurement and siderophore production of FPK8 as a function of time.Vertical bar shows critical difference values.Values are mean of three replications .A common letter (letters on the bars) means they are not significantly different (p= 0.05) by DMRT.

Figure 4f .
Figure 4f.Temporal growth measurement and siderophore production of FPK10 as a function of time.Vertical bar shows critical difference values.Values are mean of three replications .A common letter (letters on the bars) means they are not significantly different (p= 0.05) by DMRT.
of three replications.A common letter (on the superscript of each value) means they are not significantly different (p= 0.05) by DMRT.

Table 1 .
Origins and different crop rhizospheres of the isolates.

Table 2 .
Different biochemical tests performed to identify the isolates.

Table 3 .
Sideropore production under in vitro condition by six different fluorescent pseudomonads.

Table 4 .
Sideropore production under in vitro condition by six different fluorescent pseudomonads.

Table 6a .
Siderophore mediated in vitro antagonism against S. rolfsii at different iron concentration.

Table 6b .
Siderophore mediated in vitro antagonism against R. solani at different iron concentration.
Values are mean of three replications.A common letter (on the superscript of each value) means they are not significantly different (p= 0.05) by DMRT.