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Environmental Sustainability

, Volume 1, Issue 4, pp 357–365 | Cite as

Iron biofortification in mungbean using siderophore producing plant growth promoting bacteria

  • Priyanka Patel
  • Goral Trivedi
  • Meenu Saraf
Original Article
  • 300 Downloads

Abstract

Iron is one of the essential elements for most organisms and for proper plant growth. Present study revealed that isolates Pantoea dispersa MPJ9 and Pseudomonas putida MPJ6 were potent iron chelating rhizobacteria which possess siderophore activity 89.9% and 85.3% respectively, under iron limited environment. Threshold level of iron for siderophore production was observed at 15 µM iron concentration. Isolates have potential of producing plant growth promoting attributes. Catecholate type siderophore were detected by HPLC showed peak at retention time of 2.9 and 4.6 min. Pot study results revealed at harvest time, bio-inoculum treated plants significantly increased vegetative parameters, iron content (100.3 ppm), protein (0.52 g/g), carbohydrates (0.67 g/g) as compared to un-inoculated plants, indicating that use of siderophore producing isolates could result in enhancement of iron content of the mungbean.

Keywords

Iron chelating rhizobacteria Plant growth promoting rhizobacteria Siderophore HPLC Biofortification Mungbean 

Introduction

Micronutrient deficiency is a significant problem to human health worldwide and people suffer from malnutrition. It may occur due to the lack of available micronutrients in soil and the edible parts of crops such as leaves or fruits. It may be reduced by enhancing the bio-available iron content through iron supplementation and food biofortification (Rana et al. 2012). Biofortification is a process of increasing the density of micronutrients in a crop through agronomic practices.

Mungbean is an important pulse crop having nutrients and antioxidants which may provide health benefits. It not only plays an important role in human diet but also in improving the soil fertility by fixing the atmospheric nitrogen. It is gaining attention as a short season crop that can tolerate dryland conditions and can serve as a multipurpose crop (Pataczek et al. 2018).

Iron bioavailability in soil habitats and plant surfaces is very low (Loper and Henkels 1997). To satisfy their iron necessity, microorganisms and plants have evolved particular mechanisms to chelate insoluble iron by the release of siderophores and uptake of iron siderophore complexes into cells through specific iron regulated outer membrane receptor proteins (Neilands 1984; Sharma and Johri 2003).

Plants live in intimate association with microorganisms that fulfil important functions in agricultural ecosystems (Trivedi et al. 2018). Plant growth promoting rhizobacteria (PGPR) are advantageous soil bacteria that inhabit plant roots and increase plant growth through various mechanisms in different ways (Patel et al. 2018a). Plant microbe interactions in the rhizosphere are responsible for plant health and soil fertility. Siderophores produced by rhizosphere bacteria may enhance plant growth by increasing the availability of iron near the root or by inhibiting the colonization of roots by the harmful microbes (Jha and Saraf 2015). Based on their structural features siderophores can be divided in two groups; hydroxymates and catecholates (Guan et al. 2001). High affinity iron transport systems in general are made up of certain components, which includes siderophores, outer membrane receptor proteins, periplasmic binding proteins, ATP-dependent ABC-type transporters, and the TonB-ExbB-ExbD protein complex, each is vital to the success of the transport system (Clark 2004).

Present study was carried out with the aim, to isolate the iron chelating bacteria and evaluate their effect on iron improvement and plant growth promotion in mungbean.

Materials and methods

Isolation of iron chelating bacteria

Soil sample collected from rhizospheric soil of mungbean plants were collected from Ahmedabad region (23.0225°N, 72.5714°E). Isolation of microorganisms was done on nutrient agar plates, yeast extract mannitol agar plates and King’s B medium by serial dilution (1:10) method and incubated at 30 °C for 24–48 h (Venkatkumar et al. 2017). Siderophore producing bacteria were detected on CAS-blue agar plates (Schwyn and Neilands 1987).

Detection of siderophore activity

Selected four isolates were assayed for quantitative estimation of siderophore production by CAS-shuttle siderophore assay using CAS reagent and absorbance was measured at 630 nm (Sayyed et al. 2005; Patel et al. 2018b). It was further confirmed by two specific siderophore production assays, viz., Arnow’s assay for catecholate type (Arnow 1937) and Csaky’s assay for hydroxymate type (Csaky 1948) by spectrophotometric method.

Effect of iron concentration

Effect of iron concentration on siderophore production by bacteria was checked using the succinic acid broth supplemented with different concentration of iron (FeCl3·6H2O) 1, 5, 10, 15, 20, 25, 50 and 100 µM. The flasks were incubated on shaker at 150 rpm at 30 °C for 24–48 h. Samples were withdrawn after 24 h intervals to check the growth curve and siderophore production (Sayyed et al. 2005; Bholay et al. 2012).

Plant growth promoting activity

Selected four iron chelating rhizobacteria were further checked for different plant growth promoting (PGP) traits like IAA production, phosphate solubilization, ammonia production, HCN production and EPS production. Production of IAA was studied by tryptone yeast broth with additional 1% tryptophan. After 72 h of incubation IAA production was measured using Salkowsky’s reagent at 536 nm by spectrophotometer (Sarwer and Kremer 1995). Phosphate solubilization was evaluated on liquid Pikovskyaya’s medium for 21 days. The concentration of the soluble phosphate in the supernatant was performed at every 7 days by SnCl2 method at 600 nm wavelength (Gaur 1990). Production of ammonia was checked in peptone water broth using Nessler’s reagent. After 10 days the absorbance was measured by spectrophotometer at wavelength of 540 nm (Dye 1962). HCN production was carried out by picrate assay on nutrient agar slant and filter paper impregnated strip with 0.5% picric acid, 2% sodium carbonate and incubate for 24–48 h (Castric 1975; Askeland and Morrison 1983). EPS production was studied on nutrient agar as basal medium supplemented with 5% sucrose as carbohydrate source. Plates were incubated for 24–48 h (Modi et al. 1989).

Characterization of catechol siderophore by HPLC

The bacterial culture was grown in 100 ml succinic acid broth for siderophore production. After 48 h of incubation culture was centrifuged at 10,000 rpm for 10 min and supernatant was acid hydrolysed to pH 2 with 1 N HCl. Extraction of siderophore from the culture supernatant was done twice with an equal volume of ethyl acetate. The ethyl acetate extract was dried through rotary evaporator and reconstitute it in methanol. 2,3-Dihydroxybenzoic acid (DHBA) was used as standard for catecholate type of siderophore. Type of siderophore was characterized by HPLC analysis (Shimadzu Prominence HPLC System) on a C18 reversed phase column (Phenomenex, 250 × 4.60 mm 5 micro), UV detector, using methanol/0.1% phosphoric acid (1:1) as solvent system (Berner et al. 1991). The compounds were detected with flow rate of 1 ml/min and at 220 nm wavelength.

Identification of isolates

The isolates were identified by series of morphological, biochemical and molecular identification. The isolates were studied for their morphological characteristics by performing Gram staining and motility test. Biochemical test of the isolates like IMViC tests, nitrate reduction, H2S production, catalase, oxidase, sugar tests, etc. were carried out to identify them according to the Bergey’s Manual of Systematic Bacteriology (Krieg and Holt 1984). Genetic characterization of selected bacteria was carried out based on 16S rRNA gene sequencing. Genomic DNA was isolated from bacterial sample using Chromous bacterial genomic DNA isolation kit. The 16S rRNA sequencing was performed through automated DNA sequencer and data analysed by sequencing analysis software v5.4 at Chromous Biotech Pvt. Ltd., Bangalore, India.

Effect of iron chelating rhizobacteria on iron enhancement of mungbean

Pot experiment was conducted to improve iron content and effect of four individual siderophore producing bacterial strains on mungbean. Seeds were surface sterilized using 0.1% HgCl2 and washed with sterile distilled water. Sterilized seeds were coated for 60 min in individual 48 h grown siderophore enriched culture broth (8 × 107 cells/ml) in succinic acid liquid medium and 1% carboxy methyl cellulose (CMC) as inoculum. Seeds were allowed to dry overnight under aseptic conditions after coating with CMC bacterial culture (Bhatia et al. 2008). Control was prepared by addition of 20 µM iron as FeCl3 in siderophore enriched succinic acid medium to eliminate siderophore (Sayyed et al. 2005). Coated seeds were sown in pots holding sterile soil. Seeded pots were irrigated with sterile distilled water after every 48 h to maintain the moisture which is required for the seed germination. At the stage of flowering, 10 ml of siderophore enriched culture broth was foliar sprayed individually to each respective pot as inoculum. All the studied treatments were performed three times and each with triplicates. Observations like seed germination percentage, seedling vigour index, increase in root, shoot length and weight were recorded. Seedling vigour index was also calculated by using the formula as per Abdul-Baki and Anderson (1973). Seedling vigour index = germination percentage × (average root length + average shoot length).

Iron content present in iron enriched mungbean

After 45 and 60 days after sowing (DAS) harvested mungbean were rinsed with distilled water and dried. Acid digestion of 1 g sample was carried out with 10 ml of concentrated nitric acid (HNO3) and perchloric acid (HClO4) with 2:1 ratio for 2 h at 70 °C and followed by diluting it to 100 ml with double distilled water. Acid digest was analysed for iron content present in mungbean by atomic absorption spectrophotometer (AAS) (Gupta 2006).

Protein and Carbohydrate analysis of iron enriched mungbean

Protein extraction from harvested mungbean was carried out with 0.1 M phosphate buffer and the extract was used for estimation of protein by Folin–Ciocalteu reagent method (Lowry et al. 1951). Extraction of carbohydrates from harvested mungbean was performed by ethanol homogenization. This extract was used for the estimation of total sugars through anthrone reagent method (Yemm 1954).

Statistical analysis

Data were analyzed by a one-way analysis of variance (ANOVA). All experiments were performed in triplicate and the values were presented as means ± standard errors. Differences were considered to be significant when the P value was ≤ 0.05.

Results

Isolation of iron chelating bacteria

Total 12 isolates were obtained from rhizospheric soil of leguminous plant. There were seven isolates on nutrient agar plates, two on yeast extract mannitol agar plate and one on King’s B medium. Out of those, 04 isolates showed orange yellow colour zone on CAS-blue agar plate, considered as siderophore positive. They were named as MPJ5, MPJ6, MPJ9 and MPJ11. The zone of siderophore production was observed in the range of 12–29 mm. Out of the four isolates, MPJ9 revealed the biggest zone of siderophore production of 29 mm, followed by MPJ6, MPJ11 and MPJ5 with zone of 25 mm, 18 mm and 12 mm, respectively (Table 1). These four isolates were selected for the further study.
Table 1

Zone and types of siderophore production

Isolates

Zone of siderophore (mm)

Hydroxymate

Catecholate

MPJ5

12

+ ve

− ve

MPJ6

25

+ ve

− ve

MPJ9

29

− ve

+ ve

MPJ11

18

+ ve

− ve

Detection of siderophore activity

Each of four isolates were grown on succinic acid liquid medium. After inoculation of liquid succinic acid broth, siderophore production was checked at every 24 h by CAS assay. All the four isolates were giving positive siderophore assay. MPJ9 revealed maximum (89.9%) siderophore production activity followed by MPJ6 (85.3% activity) at 48 h of incubation. Whereas, MPJ11 and MPJ5 showed 52.1% and 49.7% siderophore activity at 72 h of incubation respectively (Fig. 1). Significant production of siderophore 89.9% was occurred at the stationary phase of growth by MPJ9 isolate and production was decreased with the time increase. With time the pH also increased which was not favourable for siderophore production.
Fig. 1

Siderophore activity by different iron chelating bacteria

All the four isolates were also screened for hydroxymate and catecholate type of siderophore production. All the isolates showed positive results for hydroxymate type of siderophore except MPJ9. MPJ9 was observed positive for catecholate type of siderophore (Table 1).

Effect of iron concentration

Growth curve of bacteria at different iron concentrations (1–100 µM) at 48 h of incubation are shown in Fig. 2. Addition of ferric iron at different concentrations to the culture medium increased the growth of culture (Fig. 2), whereas it suppressed the production of the siderophore with increase in the iron concentration (Fig. 3). Highest siderophore activity was observed by MPJ9 followed by MPJ6, MPJ11 and MPJ5 and were 88.7%, 82.3%, 49.5% and 52.1% respectively, at 1 µM iron concentration, whereas no siderophore production was observed after 15 µM iron concentration which was considered as threshold level of iron (Fig. 3).
Fig. 2

Growth curve of bacteria at different iron concentrations (1–100 µM) at 48 h of incubation

Fig. 3

Effect of different iron concentrations on siderophore production

Plant growth promoting activity

Bacteria that colonize plant roots and promote plant growth are considered as PGPR. Siderophore producing iron chelating rhizobacteria are the PGPR. All the four isolates were capable of giving positive phosphate solubilization and IAA production. Moreover, MPJ9 isolate was the best phosphate solubilizer and IAA producer. MPJ6 and MPJ9 were able to produce ammonia and EPS. MPJ11 was observed to produce ammonia, whereas all the four isolates did not show HCN production (Table 2).
Table 2

Plant growth promoting characters of different iron chelating bacteria

Treatments

PO4 solubilization

IAA production

NH3 production

EPS production

HCN production

MPJ5

+

+

MPJ6

+++

+++

++

+

MPJ9

+++

+++

++

++

MPJ11

+

++

+

− no activity, + less activity, ++ moderate activity, +++ high activity

Extraction and HPLC analysis of siderophore

Siderophores were extracted with ethyl acetate from siderophore producing bacterial culture. Extract was reconstituted in methanol and used for HPLC analysis. HPLC analysis of extract obtained from culture of MPJ9 revealed a characteristic peak at retention time of 2.933 min. It was found similar to the peak of 2,3 DHBA a standard for catecholate type siderophore at retention time of 2.808 min (Fig. 4). Separation of the peak was obtained at retention time of 2.9 min, 4.6 min and 7.4 min. The comparative proportions of these peaks differ depending on the time of cultivation and the strain used; additional peaks have been observed in some strains under certain conditions.
Fig. 4

HPLC separation of catecholate siderophore from MPJ9 isolate. a Authentic 2,3-DHBA standard for catecholate siderophore. Retention time 2.808 min. b Catecholate produced by MPJ9 at retention time 2.933, 4.633 and 7.437 min

Identification of isolates

Results of biochemical tests showed that all the isolates were motile, Gram negative, catalase positive, oxidase negative and nitrate reduction positive (except MPJ6), H2S production negative, sucrose positive, mannitol positive, glucose positive, citrate utilization positive, MPJ9 showed IMVIC test positive, whereas MPJ6 showed indole positive, MPJ5 showed methyl red positive, MPJ5 and MPJ11 Voges Proskauer’s positive (Table 3).
Table 3

Biochemical tests of different iron chelating bacteria (+ for positive; − for negative)

 

MPJ5

MPJ6

MPJ9

MPJ11

Gram reaction

− ve

− ve

− ve

− ve

Motile

+ ve

+ ve

+ ve

+ ve

Indole

+

+

Methyl red

+

+

Voges Proskauer’s

+

+

+

Citrate utilization

+

+

+

+

Nitrate reduction

+

+

+

H2S production

Catalase

+

+

+

+

Oxidase

+

Sucrose

+

+

+

+

Mannitol

+

+

+

+

Glucose

+

+

+

+

Lactose

+

+

Trehalose

+

Identification of isolates through 16S rRNA sequencing was carried out using specific primers as below:
  • 16S Forward primer: 5′-AGAGTTTGATCMTGGCTCAG-3′.

  • 16S Reverse primer: 5′-TACGGYTACCTTGTTACGACTT-3′.

The size of PCR product was 1.5 kb, whereas sequence length of MPJ9 and MPJ6 were 903 bases and 859 bases, respectively. Identification through 16S rRNA sequencing identified that the strains MPJ9 and MPJ6 were from Pantoea and Pseudomonas genus as Pantoea dispersa and Pseudomonas putida respectively, having 99% similarity to the reported gene sequence and submitted to NCBI under the accession no. MG694540 and MG694539 respectively (Table 4).
Table 4

Phylogenetic similarity of isolates by 16S rRNA sequencing

Isolate

Genera

Strains

Similarity (%)

Accession no.

MPJ6

Pseudomonas sp.

Pseudomonas putida

99

MG694539

MPJ9

Pantoea sp.

Pantoea dispersa

99

MG694540

Effect of iron chelating rhizobacteria on iron enhancement of mungbean

Effect of different iron chelating bacteria on growth parameter of mungbean was studied by pot trials. Iron chelating rhizobacteria acted as beneficial bioinoculants for enhancement of iron concentration and vegetative growth parameter. Percentage of seed germination was significantly increase in comparison to control plants when iron chelating bacteria were applied as bioinoculants. MPJ9 had maximum percentage of seed germination 93.3%, followed by MPJ6, MPJ11 and MPJ5 at 90%, 80% and 76.6% respectively within 7 days (Table 5). Whereas seed vigour index was also calculated, and it was found 5010 followed by 4311, 3648 and 3263 pattern being same as seed germination rate, respectively. The root length and shoot length were also positively increase, MPJ9 showed highest shoot length of 43 cm, followed by 37.5 cm, 36.9 cm and 33 cm by MPJ11, MPJ6 and MPJ5, respectively. Whereas, maximum root length was observed by MPJ6 of 11 cm, followed by MPJ9, MPJ5 and MPJ11 of 10.7 cm, 9.5 cm and 8.1 cm, respectively (Table 5).
Table 5

Seed germination percentage and seed vigour index of mungbean

Treatments

Seed germination (%)

Seed vigour index

Shoot length (cm)

Root length (cm)

Control

60

1932

25.2

7

MPJ5

76.6

3263

33

9.5

MPJ6

90

4311

36.9

11

MPJ9

93.3

5010

43

10.7

MPJ11

80

3648

37.5

8.1

At the harvesting time, different bacterial strain treated mungbean plants were studied for their vegetative parameters. It revealed plant showed significant increase in fruit weight, fresh and dry weight of shoot and root in comparison to control plants. At the harvesting time, the best results for vegetative parameters of mungbean were recorded by MPJ9 and followed by MPJ6 isolate (Fig. 5). Appreciable fruit weight increase (3.92 g and 3.85 g per 100 seeds of mungbean plant) was observed by inoculation of MPJ9 and MPJ6 respectively. Highest fresh shoot weight of 3.02 g, dry shoot weight 1.43 g and dry root weight 0.62 g per plant was obtained by MPJ9, whereas maximum fresh root weight (1.61 g per plant) was showed by MPJ6.
Fig. 5

Influence of iron chelating bacteria on vegetative parameter of mungbean. *Significant difference (P ≤ 0.05)

Iron content enhancement in mungbean

This study was conducted to evaluate iron content enhancement by iron chelating bacteria. Iron was significantly enhanced by siderophore producing rhizobacteria in the range of 65.8–100.3 ppm in mungbean at the harvesting time. Study revealed maximum iron enhancement of 54.6 ppm in case of MPJ6 followed by 48.2 ppm, 40.4 ppm and 37.8 ppm in MPJ9, MPJ5 and MPJ11 treated plants respectively, after 45 days. Whereas, at harvesting time appreciable higher iron content was observed (100.3 ppm) in case of MPJ9 followed by 82.2 ppm, 74.4 ppm and 65.8 ppm in MPJ6, MPJ11 and MPJ5 treated plants respectively, after 60 days (Fig. 6). Finally, at harvesting time MPJ9 treated plant showed 3.4-fold increase and MPJ6 treated plant 2.8-fold increase in Fe content of mungbean compared with uninoculated plants 60 DAS (Fig. 6). Significant difference of p value was 0.05.
Fig. 6

Iron concentration enhance by iron chelating bacteria. *Significant difference (P ≤ 0.05)

Protein and carbohydrate analysis of iron enriched mungbean

When iron chelating bacteria were applied as bioinoculant they enhanced the iron content in the treated mungbean, as well as also improved the protein and carbohydrate concentrations. Maximum protein content was recorded to be 0.52 g/g by MPJ9 treatment followed by 0.43 g/g, 0.38 g/g and 0.33 g/g by MPJ6, MPJ11 and MPJ5 treated plants respectively (Fig. 7). Higher carbohydrate concentration was found (0.67 g/g) in MPJ9 treated plants followed by 0.63 g/g, 0.59 g/g and 0.44 g/g by MPJ6, MPJ5 and MPJ11 treatment which indicates MPJ6 showed carbohydrate concentration nearby the MPJ9 treated plant (Fig. 7). Finally, at harvesting time MPJ9 treated plant showed 2.5-fold increase and MPJ6 treated plant observed 2-fold increase in protein content in mungbean, whereas both the strains showed 1.5-fold increase in carbohydrate content compared with uninoculated plants 60 DAS (Fig. 7). Significant difference of p value was 0.05.
Fig. 7

Protein and carbohydrate concentration enhancement by iron chelating bacteria. *Significant difference (P ≤ 0.05)

Discussion

Iron chelating rhizobacteria can enhance plant growth by various direct and indirect mechanisms. Microbial siderophores are well known for their property to promote plant growth by contributing iron (Singh et al. 2014). Our study found P. dispersa MPJ9 and P. putida MPJ6 were the most potent PGPR and showed 89.9% and 85.3% siderophore activity, whereas, suppression of siderophore synthesis after 15 µM iron concentration. P. dispersa was capable to produce catecholate type of siderophore. Both P. dispersa and P. putida are earlier  reported as plant growth promoting iron chelating rhizobacteria (Shahzad et al. 2008; Weger et al. 1988), possessing ability to produce siderophore activity at 60.33% and 83% respectively (Panwar et al. 2016; Sayyed et al. 2005). Studies found P. dispersa was the best catecholate type of siderophore producing iron chelating microbe (Joshi et al. 2006). The reported threshold level of iron, which repressed siderophore synthesis in P. putida was found to be 20 µM iron concentration (Sayyed et al. 2005). P. dispersa reported to produce siderophore which can chelate iron from insoluble to soluble form and making it available to plants (Roy et al. 2016). Both the strains were capable of appreciable IAA production which can stimulate and facilitate plant growth. P. putida is also reported earlier to produce IAA and plant growth stimulation (Meliani et al. 2017).

Our study revealed the peak was shown in the culture extract sample at 2.9, 4.6 and 7.4 min which was very similar for 2,3-DHBA and enterobactin which indicate present of catecholate type of siderophore. Studies reported culture extracts of enterobacterial strain Erwinia herbicola produced enterobactin as catecholate type of siderophore. The peak for enterobactin was seen at 4.407 min whereas, peak obtained at 2.465 min for 2,3-DHBA as standard (Berner et al. 1991). Whereas, catecholate type siderophore produced by Rhizobium leguminosarum strain IARI 312 seemed similar to enterobactin and reported the peak for 2,3-DHBA at 2.9 min and a similar peak was found in the purified sample at 3.00 min (Clark 2004).

Studies also reported Pseudomonas strains can also improve vegetative parameters in maize plants under iron restricted conditions (Sharma and Johri 2003). Researchers reported that the Pseudomonas sp. as PGPR could better enhance the seed germination and vegetative growth of wheat plants (Kumar et al. 2018). Whereas, application of P. putida, strains improved iron translocation from root to grain through siderophore production in rice plants (Sharma et al. 2013). Siderophore produce by bacteria can improve iron to the iron-starved tomato plants (Radzki et al. 2013). Applications of different concentration of FeSO4 solution at different vegetative stages can improve mungbean iron and protein content in the range 78.50–146.43 ppm and 0.25–0.27 g/g (Ali et al. 2014). Whereas, our study revealed iron and protein content in the range of 65.8–100.3 ppm and 0.33–0.52 g/g respectively, which indicates the selected iron chelating PGPR strains work as potent bioinoculants which significantly can work without use of any added chemicals, can enhance iron content, protein content as well as carbohydrate content of the mungbean. Our findings highlight the role of PGPR strategy to progress towards biofortification with iron and its implementation in sustainable agriculture using mungbean as model crop.

Conclusion

P. dispersa followed by P. putida were the best iron chelating PGPR and show potent siderophore production activity. P. dispersa was capable of producing catecholate type of siderophore. At the harvesting time P. dispersa MPJ9 significantly increased mungbean iron content 3.4-fold times, protein content 2-fold and carbohydrate content 1.5-fold as compare to uninoculated plants. Study suggested both the strains can be used as biofertilizer for the purpose of iron biofortification in mungbean.

Notes

Acknowledgements

Authors are thankful to the Department of Microbiology and Biotechnology, Gujarat University for helpful to this research work.

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Copyright information

© Society for Environmental Sustainability 2018

Authors and Affiliations

  1. 1.Department of Microbiology and Biotechnology, University School of SciencesGujarat UniversityAhmedabadIndia

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