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A simple agar plate assay for screening siderophore producer yeasts
Journal of Microbiological Methods 47 Ž2001. 273–279 www.elsevier.comrlocaterjmicmeth
A simple agar plate assay for screening siderophore producer yeasts Viviana Calvente, Marıa ´ Edith de Orellano, Gabriela Sansone, Delia Benuzzi, Maria Isabel Sanz de Tosetti ) ´ de Tecnologıa Area y Biotecnologıa, Facultad de Quımica, Bioquımica y Farmacia, ´ Quımica ´ ´ Departamento de Quımica, ´ ´ ´ UniÕersidad Nacional de San Luis, Chacabuco y Pedernera, 5700 San Luis, Argentina Received 10 September 2000; received in revised form 25 May 2001; accepted 25 May 2001
Abstract Yeasts produce hydroxamate-type siderophores Žiron-binding compounds. in response to Fe-stress conditions. Because these siderophores are important to the biocontrol of postharvest diseases of apple and pears, a method for screening siderophore producer yeast was developed. The screening method was carried out in special Petri dishes with eight or nine wells Ž25-mm diameter.. These wells were filled with siderophore production medium and seeded with yeasts isolated from epiphytic apple microflora. After yeasts grew Ž24–48 h., holes Ž2-mm diameter. were made in the agar of each well. Holes were filled with an acid solution of ferric perchlorate. After 10–15 min, reddish halos appeared in the bottom of the plate and their intensities were compared with standards. Standards were prepared in the same special dish with rhodotorulic acid solutions Žconcentrations between 0.05 and1 grl. plus 2% agar. When agar solidified into wells, holes were made and filled with ferric perchlorate solution. Color intensities of reddish halos were proportional to siderophore concentration and the detection limit was 0.1 grl. It was possible to correlate the production of siderophore in solid medium with the results obtained in liquid medium. The methodology was also a useful tool for making a preliminary assessment of the influence of different factors on the siderophore production. q 2001 Published by Elsevier Science B.V. Keywords: Ferric perchlorate; Agar plate assay; Siderophore
1. Introduction Microorganisms elaborate a variety of low-molecular-weight, high-affinity chelating agents, which solubilize ferric iron in the environment and transport it into the cell. These compounds are generically known as siderophores and they are typically found in iron-deficient cultures ŽNeilands, 1993.. )
Corresponding author. Fax: q54-652-30224. E-mail address: [email protected] ŽM.I. Sanz de Tosetti..
Siderophores exhibit considerable structural variability and affinity for iron, which determines the growth of a microorganism under competitive conditions when availability is a limiting factor ŽNeilands, 1989.. Most siderophores may be classified as either hydroxamates or phenolates–cathecolates. Yeasts produce only hydroxamate-type compound while bacteria produce hydroxamate as well as catecholate siderophores ŽRiquelme, 1996.. A variety of techniques has been developed to detect siderophores. To date, the most common de-
0167-7012r01r$ - see front matter q 2001 Published by Elsevier Science B.V. PII: S 0 1 6 7 - 7 0 1 2 Ž 0 1 . 0 0 3 3 3 - 5
274
V. CalÕente et al.r Journal of Microbiological Methods 47 (2001) 273–279
Fig. 1. Photography Ža. and scheme Žb. of the FP assay on solid medium for screening siderophore producer yeasts.
V. CalÕente et al.r Journal of Microbiological Methods 47 (2001) 273–279
tection method for siderophore production is the chrome azurol S ŽCAS. assay developed by Schwyn and Neilands Ž1987.. This assay is based on a competition for iron between the ferric complex of an indicator dye ŽCAS. and a chelator or siderophore produced by microorganisms. It depends only on the ability of the compound to bind iron with relatively high affinity but it does not provide information about the structure of the compound. Others, as the ferric perchlorate assay, are based on chemical properties of siderophores. The latter assay can be applied to detection of hydroxamate-type siderophores because these compounds are capable of forming stable iron complexes at low pH, while catechols do not react in this assay since the iron is dissociated from these compounds at low pH ŽPayne, 1994.. Recently, we have demonstrated that Rhodotorulic acid, a hydroxamate-type siderophore produced by Rhodotorula glutinis strains, improves the biological control of blue rot caused by Penicillium expansum in harvested apples ŽCalvente et al., 1999.. In view of this fact, we wanted to screen siderophore producer yeasts from epiphytic microflora of apple and pear fruits with the goal of applying them to biocontrol. Then, we developed a method of hydroxamate detection in solid medium based on the ferric perchlorate assay. This work describes the methodology and the results obtained.
2. Materials and methods 2.1. Microorganisms Yeasts isolated from apple ŽRed delicious. and pear ŽWilliam. fruits were isolated from the fruit by aseptic removal of the skin using a sterile scalpel. The material was transferred to a 500-ml flask with 250 ml of sterile medium Ž0.85% NaCl q 0.05% Tween 40.. The flask was shaken at 240 rpm for 1 h. Aliquots Ž200 ml. of the resulting suspension were spread on petri dishes. The fungal flora was isolated on GYP medium containing 2% glucose, 1% peptone, 0.5% yeast extract, 2% agar and 100 ppm chloramphenicol. Yeasts were identified by microscopic appearance and the Yeast Identification System API C 20 AUX ŽbioMerieux, France.. ´
275
2.2. Production of siderophores in liquid medium Culture medium for siderophore production consisted of: sucrose 25 grl, ŽNH 4 . 2 SO4 4 grl, PO4 H K 2 3 grl; citric acid 1 grl, MgSO4 0.08 grl, ZnSO4 0.002 grl, pH 6.8. Isolates were grown in 1000 ml flask cultures with 200 ml of production medium with shaking Ž200 rpm. for 72 h. The inoculum was grown in the same medium for 24 h at 30 8C with shaking Ž200 rpm.. The production medium was inoculated with 20 ml of this actively growing culture. 2.3. Siderophore detection in culture supernatants Siderophore was detected in culture medium supernatants according to the ferric perchlorate assay ŽAtkin et al., 1970.. The assay was carried out by mixing 0.5 ml of culture supernatant, or a suitable dilution thereof, with 2.5 ml 5 mM Fe 3 Cl–0.1 M HClO4 ŽpH 2. in a cuvette followed by the measurement of the maximum optical density ŽODmax . verTable 1 Ferric perchlorate ŽFP. assay for screening siderophore producer yeasts Isolated
Siderophore production in liquid medium Žgrl. a
FP assay on solid mediumb
SL1 Ž R. glutinis . SL14 Ž C. guillermondii) SL16 Ž C. guillermondii . SL19 Ž R. glutinis . SL27 Ž R. glutinis . SL29 Ž R. rubra. SL30 Ž R. glutinis . SL43 Ž Cryptococcus albidus . SL50 Ž R. rubra. SL55 Ž R. rubra. SL55a Ž R. glutinis . SL57 Ž R. glutinis . SL57c Ž R. glutinis . SL73 Ž R. glutinis . SL75 Ž R. glutinis .
0.09 0.03 0.03 0.42 0.33 0.38 0.02 0.03 0.23 0.58 0.55 0.40 0.53 0.11 0.14
y y y qq qq qq y y q qqq qqq qq qqq y y
a Values are the means of three experiments; standard deviations - 5%. b According to standards on solid medium: y: 0–0.1grl; q: 0.1–0.25 grl; qq: 0.25–0.5 grl; qqq: 0.5–0.75 grl; qqqq: 0.75–1 grl.
276
V. CalÕente et al.r Journal of Microbiological Methods 47 (2001) 273–279
Fig. 2. Photography Ža. and scheme Žb. of the FP assay on solid medium for assessing the effect to different compounds on siderophore production. ŽPM.: production medium, ŽAr.: arginine, ŽO.: ornithine, ŽU.: urea, ŽA.: ammonium acetate, ŽP.: proline, ŽL.: lysine, ŽGlu.: glutamine, ŽG Ac.: glutamic acid.
V. CalÕente et al.r Journal of Microbiological Methods 47 (2001) 273–279
277
sus a blank similarly prepared from sterile medium. The relationship of OD to concentration was assessed using the Lambert–Beer law. Patterns were made with Rhodotorulic acid, a hydroxamate-type siderophore, in concentration between 0.05 and 1 grl.
into wells. When agar solidified, 50 ml of a 5% solution of the compound to assay was put on the agar. Then, the yeast was seeded. After the microorganism grew, the above detection procedure was carried out.
2.4. Production and detection of siderophores in agar plates
3. Results
For production and detection of siderophores in solid medium, special dishes with wells Ž25 mm diameter. were used ŽFig. 1.. Production medium solidified with agar Ž2%. was used for screening siderophore producer yeasts. Wells were filled with the medium and seeded with different isolated, then plates were incubated at 30 8C. When the yeast grew Ž24–48 h., the detection reaction, with a little modification in the ferric perchlorate concentration was carried out. Little holes Ž2 mm diameter. were made in the agar with an appropriate hollow punch. These holes were filled with the acid solution of ferric perchlorate Ž20 mM Fe 3 Cl–0.1 M HClO4 .. After 10–15 min intensities of reddish halos in the bottom of the plate were compared with standards. Assays in solid and liquid media were carried out by triplicate. Experiments were repeated at least twice.
A preliminary assay for determining the sensitivity of the method was carried out. Different concentrations of ferric perchlorate Ž5, 20 and 40 mM. were used for assaying standards. With 20 mM ferric perchlorate, the detection limit in solid medium was 0.1 grl, while with a 5 mM solution, the detection limit was higher Ž0.25 grl.. On the other hand, with a 40 mM ferric perchlorate solution, it was not possible to observe any difference between standards of different concentrations, while with the 20 mM solution, color intensities of reddish halos were proportional to siderophore concentration ŽFig. 1.. Then, a little modification with respect to the original technique ŽAtkin et al., 1970. was introduced and a 20 mM Fe 3 Cl–0.1 M HClO4 solution was used for assay in solid medium. Results of the screening of siderophore producer yeasts in solid medium are shown in Fig. 1 and Table 1. As seen in the table, it was possible to correlate the production of siderophore in solid medium with the results obtained in liquid medium.
2.5. Standards preparation Rhodotorulic acid solutions of concentrations between 0.05 and 1 grl were prepared in distilled water. Dishes with wells were also used in this case. Wells were filled with Rhodotorulic solutions with 2% agar. When the agar solidified, holes Ž2 mm diameter. were made and were filled with the acid solution of ferric perchlorate. Color intensities of reddish halos were registered. 2.6. Application of the method for assessing influence of different compounds on siderophore production Assessment of different substances on the biosynthesis of siderophores was made by using production medium 2% agar. The medium was prepared and put
Table 2 Ferric perchlorate ŽFP. assay for assessing the effect of different compounds on siderophore production Compound added to production medium ŽPM.
FP assay on solid mediuma SL 29 SL 57c Ž R. rubra. Ž R. glutinis .
None ŽPM. Arginine ŽA. Ornithine ŽO. Urea ŽU. Ammonium acetate ŽA. Proline ŽP. Lysine ŽL. Glutamine ŽGlu. Glutamic acid ŽG Ac.
qq qq qq q qq qqq q qq qq
qqq qqq qqq qq qqq qqqq q qqq qqq
a According to standards on solid medium: y: 0–0.1grl; q: 0.1–0.25 grl; qq: 0.25–0.5 grl; qqq: 0.5–0.75 grl; qqqq: 0.75–1 grl.
278
V. CalÕente et al.r Journal of Microbiological Methods 47 (2001) 273–279
The results obtained in assessment of the influence of different substances on siderophore production are shown in Fig. 2 and Table 2. For these assays, isolated SL 29 and SL 57c were chosen. It was possible to assess the effect of each substance assayed on the siderophore production. In the presence of urea or lysine, siderophore production on solid medium decreased but it increased in the presence of proline. The effect was similar for both isolated substances.
4. Discussion Most of the methods to determine siderophores are carried out on the microorganism culture supernatant because siderophores are found there, with the exception of the cell wall-associated mycobactins. Cells must be removed by centrifugation and the supernatant assayed for the presence of the compounds. Thus, a screening of siderophore producer microorganisms in liquid medium can be arduous and expensive. With respect to the assays for determining siderophores, they can be either unspecific or based on chemical or biological properties of the siderophores ŽPayne, 1994.. The most used unspecific assay is the universal assay of chrome azurol S ŽCAS. developed by Schwyn and Neilands Ž1987.. This assay can be carried out in either liquid medium or solid one ŽMilagres et al., 1999. but only in liquid medium it can be used for estimating the quantity of siderophore present. Otherwise, phosphates interfere with the CAS assay, and in consequence simple growth media must be used in the study of siderophore production ŽSchwyn and Neilands, 1987.. Compounds other than phosphates Ži.e., proteins. also interfere with the CAS reaction and consequently give inaccurate results ŽRaaska and Mattila-Sandholm, 1995.. The ferric perchlorate assay is a method based in the chemical properties of siderophores. This reaction is carried out in liquid medium and is an adequate method for quantifying hydroxamates ŽAtkin et al., 1970.. Ferric hydroxamates are stable reddishorange colored complexes at low pH while ferric cathecols are reddish purple colored at neutral pH but the iron is dissociated and the color lost at acidic
pH ŽPayne, 1994.. Then, the ferric perchlorate assay provides not only a means of detection but also considerable information about the structure of the compound. The application of ferric perchlorate assay on solid medium, as it was demonstrated, not only gave us clues about the structure but it allowed us to know about the quantity of siderophore produced. As it was reported by Atkin et al. Ž1970. and verified by us, yeast strains of the same specie with some difference in their metabolic profiles have different ability for the rhodotorulic acid synthesis. Then, by means of the FP assay on solid medium it was possible select the best producer among yeasts of the same specie. Other application of this method was related with the improvement of the production of siderophore. The effect of different amino acids and nitrogen sources upon rhodotorulic acid biosynthesis in liquid medium was previously demonstrated by us ŽCalvente et al., in press.. In the present work it was demonstrated that it is possible to make a preliminary assessment of the influence of different factors on the siderophore production by means of FP assay in solid medium. In conclusion, the ferric perchlorate assay on solid medium can be a useful tool for screening hydroxamates producer microorganisms, because it allows to select not just the producer but the best producer and it also allows to evaluate the incidence of different factors when the goal is the improvement of the siderophore production.
Acknowledgements The authors are very grateful to Lidia Unger de Vacca for helping with the English text. We also thank Juan Carlos Soloa for his technical assistance. Financial support from Universidad Nacional de San Luis and Agencia de Promocion y ´ Cientıfica ´ Tecnologica, Argentina, is gratefully acknowledged. ´ References Atkin, C., Neilands, J., Phaff, H., 1970. Rhodotorulic acid from species of Leucosporidium, Rhodosporidium, Rhodotorula, Sporodiobolus, and Sporobolomyces and a new alanine-con-
V. CalÕente et al.r Journal of Microbiological Methods 47 (2001) 273–279 taining ferrichrome from Criptcoccus melibiosum. J. Bacteriol. 103, 722–733. Calvente, V., Benuzzi, D., Sanz de Tosetti, M.I., 1999. Antagonistic action of siderophores from Rhodotorula glutinis upon the post-harvest pathogen Penicillium expansum. Int. Biodeter. Biodegrad. 43, 167–172. Calvente, V., de Orellano, M.E., Sansone, G., Benuzzi, D., Sanz de Tosetti, M.I., 2001. Effect of nitrogen source and pH on siderophore production by Rhodotorula strains and their application to biocontrol of phytopathogenic moulds. J. Indust. Microbiol. Biotechnol., in press. Milagres, A., Machuca, A., Napoleao, D., 1999. Detection of siderophore production from several fungi and bacteria by a modification of chrome azurol S ŽCAS. agar plate assay. J. Microbiol. Methods 19, 223–234. Neilands, J.B., 1989. Siderophore systems of bacteria and fungi.
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In: Beveridge, T.J., Doyle, R.J. ŽEds.., Metals Ions and Bacteria. Wiley, New Jersey, pp. 141–164. Neilands, J.B., 1993. Siderophores. Arch. Biochem. Biophys. 302, 1–3. Payne, S., 1994. Detection, isolation and characterization of siderophores. Methods in Enzymology, 235, Academic Press, NY, pp. 329–344. Raaska, L., Mattila-Sandholm, T., 1995. Effects of iron level on the antagonistic action of siderophores from non-pathogenic Staphylococcus spp. J. Ind. Microbiol. 15, 480–485. Riquelme, M., 1996. Fungal siderophores in plant–microbe interactions. Microbiol. SEM 12, 537–546. Schwyn, B., Neilands, J.B., 1987. Universal chemical assay for the detection an determination of siderophores. Anal. Biochem. 160, 46–56.
A simple agar plate assay for screening siderophore producer yeasts Viviana Calvente, Marıa ´ Edith de Orellano, Gabriela Sansone, Delia Benuzzi, Maria Isabel Sanz de Tosetti ) ´ de Tecnologıa Area y Biotecnologıa, Facultad de Quımica, Bioquımica y Farmacia, ´ Quımica ´ ´ Departamento de Quımica, ´ ´ ´ UniÕersidad Nacional de San Luis, Chacabuco y Pedernera, 5700 San Luis, Argentina Received 10 September 2000; received in revised form 25 May 2001; accepted 25 May 2001
Abstract Yeasts produce hydroxamate-type siderophores Žiron-binding compounds. in response to Fe-stress conditions. Because these siderophores are important to the biocontrol of postharvest diseases of apple and pears, a method for screening siderophore producer yeast was developed. The screening method was carried out in special Petri dishes with eight or nine wells Ž25-mm diameter.. These wells were filled with siderophore production medium and seeded with yeasts isolated from epiphytic apple microflora. After yeasts grew Ž24–48 h., holes Ž2-mm diameter. were made in the agar of each well. Holes were filled with an acid solution of ferric perchlorate. After 10–15 min, reddish halos appeared in the bottom of the plate and their intensities were compared with standards. Standards were prepared in the same special dish with rhodotorulic acid solutions Žconcentrations between 0.05 and1 grl. plus 2% agar. When agar solidified into wells, holes were made and filled with ferric perchlorate solution. Color intensities of reddish halos were proportional to siderophore concentration and the detection limit was 0.1 grl. It was possible to correlate the production of siderophore in solid medium with the results obtained in liquid medium. The methodology was also a useful tool for making a preliminary assessment of the influence of different factors on the siderophore production. q 2001 Published by Elsevier Science B.V. Keywords: Ferric perchlorate; Agar plate assay; Siderophore
1. Introduction Microorganisms elaborate a variety of low-molecular-weight, high-affinity chelating agents, which solubilize ferric iron in the environment and transport it into the cell. These compounds are generically known as siderophores and they are typically found in iron-deficient cultures ŽNeilands, 1993.. )
Corresponding author. Fax: q54-652-30224. E-mail address: [email protected] ŽM.I. Sanz de Tosetti..
Siderophores exhibit considerable structural variability and affinity for iron, which determines the growth of a microorganism under competitive conditions when availability is a limiting factor ŽNeilands, 1989.. Most siderophores may be classified as either hydroxamates or phenolates–cathecolates. Yeasts produce only hydroxamate-type compound while bacteria produce hydroxamate as well as catecholate siderophores ŽRiquelme, 1996.. A variety of techniques has been developed to detect siderophores. To date, the most common de-
0167-7012r01r$ - see front matter q 2001 Published by Elsevier Science B.V. PII: S 0 1 6 7 - 7 0 1 2 Ž 0 1 . 0 0 3 3 3 - 5
274
V. CalÕente et al.r Journal of Microbiological Methods 47 (2001) 273–279
Fig. 1. Photography Ža. and scheme Žb. of the FP assay on solid medium for screening siderophore producer yeasts.
V. CalÕente et al.r Journal of Microbiological Methods 47 (2001) 273–279
tection method for siderophore production is the chrome azurol S ŽCAS. assay developed by Schwyn and Neilands Ž1987.. This assay is based on a competition for iron between the ferric complex of an indicator dye ŽCAS. and a chelator or siderophore produced by microorganisms. It depends only on the ability of the compound to bind iron with relatively high affinity but it does not provide information about the structure of the compound. Others, as the ferric perchlorate assay, are based on chemical properties of siderophores. The latter assay can be applied to detection of hydroxamate-type siderophores because these compounds are capable of forming stable iron complexes at low pH, while catechols do not react in this assay since the iron is dissociated from these compounds at low pH ŽPayne, 1994.. Recently, we have demonstrated that Rhodotorulic acid, a hydroxamate-type siderophore produced by Rhodotorula glutinis strains, improves the biological control of blue rot caused by Penicillium expansum in harvested apples ŽCalvente et al., 1999.. In view of this fact, we wanted to screen siderophore producer yeasts from epiphytic microflora of apple and pear fruits with the goal of applying them to biocontrol. Then, we developed a method of hydroxamate detection in solid medium based on the ferric perchlorate assay. This work describes the methodology and the results obtained.
2. Materials and methods 2.1. Microorganisms Yeasts isolated from apple ŽRed delicious. and pear ŽWilliam. fruits were isolated from the fruit by aseptic removal of the skin using a sterile scalpel. The material was transferred to a 500-ml flask with 250 ml of sterile medium Ž0.85% NaCl q 0.05% Tween 40.. The flask was shaken at 240 rpm for 1 h. Aliquots Ž200 ml. of the resulting suspension were spread on petri dishes. The fungal flora was isolated on GYP medium containing 2% glucose, 1% peptone, 0.5% yeast extract, 2% agar and 100 ppm chloramphenicol. Yeasts were identified by microscopic appearance and the Yeast Identification System API C 20 AUX ŽbioMerieux, France.. ´
275
2.2. Production of siderophores in liquid medium Culture medium for siderophore production consisted of: sucrose 25 grl, ŽNH 4 . 2 SO4 4 grl, PO4 H K 2 3 grl; citric acid 1 grl, MgSO4 0.08 grl, ZnSO4 0.002 grl, pH 6.8. Isolates were grown in 1000 ml flask cultures with 200 ml of production medium with shaking Ž200 rpm. for 72 h. The inoculum was grown in the same medium for 24 h at 30 8C with shaking Ž200 rpm.. The production medium was inoculated with 20 ml of this actively growing culture. 2.3. Siderophore detection in culture supernatants Siderophore was detected in culture medium supernatants according to the ferric perchlorate assay ŽAtkin et al., 1970.. The assay was carried out by mixing 0.5 ml of culture supernatant, or a suitable dilution thereof, with 2.5 ml 5 mM Fe 3 Cl–0.1 M HClO4 ŽpH 2. in a cuvette followed by the measurement of the maximum optical density ŽODmax . verTable 1 Ferric perchlorate ŽFP. assay for screening siderophore producer yeasts Isolated
Siderophore production in liquid medium Žgrl. a
FP assay on solid mediumb
SL1 Ž R. glutinis . SL14 Ž C. guillermondii) SL16 Ž C. guillermondii . SL19 Ž R. glutinis . SL27 Ž R. glutinis . SL29 Ž R. rubra. SL30 Ž R. glutinis . SL43 Ž Cryptococcus albidus . SL50 Ž R. rubra. SL55 Ž R. rubra. SL55a Ž R. glutinis . SL57 Ž R. glutinis . SL57c Ž R. glutinis . SL73 Ž R. glutinis . SL75 Ž R. glutinis .
0.09 0.03 0.03 0.42 0.33 0.38 0.02 0.03 0.23 0.58 0.55 0.40 0.53 0.11 0.14
y y y qq qq qq y y q qqq qqq qq qqq y y
a Values are the means of three experiments; standard deviations - 5%. b According to standards on solid medium: y: 0–0.1grl; q: 0.1–0.25 grl; qq: 0.25–0.5 grl; qqq: 0.5–0.75 grl; qqqq: 0.75–1 grl.
276
V. CalÕente et al.r Journal of Microbiological Methods 47 (2001) 273–279
Fig. 2. Photography Ža. and scheme Žb. of the FP assay on solid medium for assessing the effect to different compounds on siderophore production. ŽPM.: production medium, ŽAr.: arginine, ŽO.: ornithine, ŽU.: urea, ŽA.: ammonium acetate, ŽP.: proline, ŽL.: lysine, ŽGlu.: glutamine, ŽG Ac.: glutamic acid.
V. CalÕente et al.r Journal of Microbiological Methods 47 (2001) 273–279
277
sus a blank similarly prepared from sterile medium. The relationship of OD to concentration was assessed using the Lambert–Beer law. Patterns were made with Rhodotorulic acid, a hydroxamate-type siderophore, in concentration between 0.05 and 1 grl.
into wells. When agar solidified, 50 ml of a 5% solution of the compound to assay was put on the agar. Then, the yeast was seeded. After the microorganism grew, the above detection procedure was carried out.
2.4. Production and detection of siderophores in agar plates
3. Results
For production and detection of siderophores in solid medium, special dishes with wells Ž25 mm diameter. were used ŽFig. 1.. Production medium solidified with agar Ž2%. was used for screening siderophore producer yeasts. Wells were filled with the medium and seeded with different isolated, then plates were incubated at 30 8C. When the yeast grew Ž24–48 h., the detection reaction, with a little modification in the ferric perchlorate concentration was carried out. Little holes Ž2 mm diameter. were made in the agar with an appropriate hollow punch. These holes were filled with the acid solution of ferric perchlorate Ž20 mM Fe 3 Cl–0.1 M HClO4 .. After 10–15 min intensities of reddish halos in the bottom of the plate were compared with standards. Assays in solid and liquid media were carried out by triplicate. Experiments were repeated at least twice.
A preliminary assay for determining the sensitivity of the method was carried out. Different concentrations of ferric perchlorate Ž5, 20 and 40 mM. were used for assaying standards. With 20 mM ferric perchlorate, the detection limit in solid medium was 0.1 grl, while with a 5 mM solution, the detection limit was higher Ž0.25 grl.. On the other hand, with a 40 mM ferric perchlorate solution, it was not possible to observe any difference between standards of different concentrations, while with the 20 mM solution, color intensities of reddish halos were proportional to siderophore concentration ŽFig. 1.. Then, a little modification with respect to the original technique ŽAtkin et al., 1970. was introduced and a 20 mM Fe 3 Cl–0.1 M HClO4 solution was used for assay in solid medium. Results of the screening of siderophore producer yeasts in solid medium are shown in Fig. 1 and Table 1. As seen in the table, it was possible to correlate the production of siderophore in solid medium with the results obtained in liquid medium.
2.5. Standards preparation Rhodotorulic acid solutions of concentrations between 0.05 and 1 grl were prepared in distilled water. Dishes with wells were also used in this case. Wells were filled with Rhodotorulic solutions with 2% agar. When the agar solidified, holes Ž2 mm diameter. were made and were filled with the acid solution of ferric perchlorate. Color intensities of reddish halos were registered. 2.6. Application of the method for assessing influence of different compounds on siderophore production Assessment of different substances on the biosynthesis of siderophores was made by using production medium 2% agar. The medium was prepared and put
Table 2 Ferric perchlorate ŽFP. assay for assessing the effect of different compounds on siderophore production Compound added to production medium ŽPM.
FP assay on solid mediuma SL 29 SL 57c Ž R. rubra. Ž R. glutinis .
None ŽPM. Arginine ŽA. Ornithine ŽO. Urea ŽU. Ammonium acetate ŽA. Proline ŽP. Lysine ŽL. Glutamine ŽGlu. Glutamic acid ŽG Ac.
qq qq qq q qq qqq q qq qq
qqq qqq qqq qq qqq qqqq q qqq qqq
a According to standards on solid medium: y: 0–0.1grl; q: 0.1–0.25 grl; qq: 0.25–0.5 grl; qqq: 0.5–0.75 grl; qqqq: 0.75–1 grl.
278
V. CalÕente et al.r Journal of Microbiological Methods 47 (2001) 273–279
The results obtained in assessment of the influence of different substances on siderophore production are shown in Fig. 2 and Table 2. For these assays, isolated SL 29 and SL 57c were chosen. It was possible to assess the effect of each substance assayed on the siderophore production. In the presence of urea or lysine, siderophore production on solid medium decreased but it increased in the presence of proline. The effect was similar for both isolated substances.
4. Discussion Most of the methods to determine siderophores are carried out on the microorganism culture supernatant because siderophores are found there, with the exception of the cell wall-associated mycobactins. Cells must be removed by centrifugation and the supernatant assayed for the presence of the compounds. Thus, a screening of siderophore producer microorganisms in liquid medium can be arduous and expensive. With respect to the assays for determining siderophores, they can be either unspecific or based on chemical or biological properties of the siderophores ŽPayne, 1994.. The most used unspecific assay is the universal assay of chrome azurol S ŽCAS. developed by Schwyn and Neilands Ž1987.. This assay can be carried out in either liquid medium or solid one ŽMilagres et al., 1999. but only in liquid medium it can be used for estimating the quantity of siderophore present. Otherwise, phosphates interfere with the CAS assay, and in consequence simple growth media must be used in the study of siderophore production ŽSchwyn and Neilands, 1987.. Compounds other than phosphates Ži.e., proteins. also interfere with the CAS reaction and consequently give inaccurate results ŽRaaska and Mattila-Sandholm, 1995.. The ferric perchlorate assay is a method based in the chemical properties of siderophores. This reaction is carried out in liquid medium and is an adequate method for quantifying hydroxamates ŽAtkin et al., 1970.. Ferric hydroxamates are stable reddishorange colored complexes at low pH while ferric cathecols are reddish purple colored at neutral pH but the iron is dissociated and the color lost at acidic
pH ŽPayne, 1994.. Then, the ferric perchlorate assay provides not only a means of detection but also considerable information about the structure of the compound. The application of ferric perchlorate assay on solid medium, as it was demonstrated, not only gave us clues about the structure but it allowed us to know about the quantity of siderophore produced. As it was reported by Atkin et al. Ž1970. and verified by us, yeast strains of the same specie with some difference in their metabolic profiles have different ability for the rhodotorulic acid synthesis. Then, by means of the FP assay on solid medium it was possible select the best producer among yeasts of the same specie. Other application of this method was related with the improvement of the production of siderophore. The effect of different amino acids and nitrogen sources upon rhodotorulic acid biosynthesis in liquid medium was previously demonstrated by us ŽCalvente et al., in press.. In the present work it was demonstrated that it is possible to make a preliminary assessment of the influence of different factors on the siderophore production by means of FP assay in solid medium. In conclusion, the ferric perchlorate assay on solid medium can be a useful tool for screening hydroxamates producer microorganisms, because it allows to select not just the producer but the best producer and it also allows to evaluate the incidence of different factors when the goal is the improvement of the siderophore production.
Acknowledgements The authors are very grateful to Lidia Unger de Vacca for helping with the English text. We also thank Juan Carlos Soloa for his technical assistance. Financial support from Universidad Nacional de San Luis and Agencia de Promocion y ´ Cientıfica ´ Tecnologica, Argentina, is gratefully acknowledged. ´ References Atkin, C., Neilands, J., Phaff, H., 1970. Rhodotorulic acid from species of Leucosporidium, Rhodosporidium, Rhodotorula, Sporodiobolus, and Sporobolomyces and a new alanine-con-
V. CalÕente et al.r Journal of Microbiological Methods 47 (2001) 273–279 taining ferrichrome from Criptcoccus melibiosum. J. Bacteriol. 103, 722–733. Calvente, V., Benuzzi, D., Sanz de Tosetti, M.I., 1999. Antagonistic action of siderophores from Rhodotorula glutinis upon the post-harvest pathogen Penicillium expansum. Int. Biodeter. Biodegrad. 43, 167–172. Calvente, V., de Orellano, M.E., Sansone, G., Benuzzi, D., Sanz de Tosetti, M.I., 2001. Effect of nitrogen source and pH on siderophore production by Rhodotorula strains and their application to biocontrol of phytopathogenic moulds. J. Indust. Microbiol. Biotechnol., in press. Milagres, A., Machuca, A., Napoleao, D., 1999. Detection of siderophore production from several fungi and bacteria by a modification of chrome azurol S ŽCAS. agar plate assay. J. Microbiol. Methods 19, 223–234. Neilands, J.B., 1989. Siderophore systems of bacteria and fungi.
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