Effects of a Hydroxamate Siderophore on Iron Absorption by ... - NCBI

Plant Physiol. (1984) 76, 36-39 0032-0889/84/76/0036/04/$0 1.00/0

Effects of a Hydroxamate Siderophore on Iron Absorption by Sunflower and Sorghum' Received for publication January 6, 1984 and in revised form May 2, 1984

GARY R. CLINE2, C. P. PATRICK REID*, PAUL E. POWELL, AND PAUL J. SZANISZLO Department of Forest and Wood Sciences, Colorado State University, Fort Collins, Colorado 80523 (G. R. C., C. P. P. R); and, Department of Microbiology, University of Texas, Austin, Texas 78712 (P. E. P., P. J. S.) other natural chelating agents. A limited number of previous studies have indicated that plants can utilize HS in their absorption of Fe (12, 15). The purpose of this study was to confirm these earlier studies, examine the mechanisms involved, and determine if Fe deficiency among some plant species might be related to their ability to acquire Fe from HS. Hydroxamate siderophores have been strongly implicated in the Fe nutrition of fungi. The Fe uptake mechanism involves absorption of the intact Fe-HS chelate, reduction and subsequent dissociation within the cells, and release of HS to the environment (8, 18, 19). If some plant species have evolved similar mechanisms to utilize Fe-HS, they may be more efficient in uptake of Fe in natural environments than those not possessing such mechanisms. An Fe-inefficient species, sorghum, and an Fe-efficient species, sunflower, were grown hydroponically with various levels of both Fe and DFOB, a naturally occurring HS. The objective was to establish varying levels of total Fe, Fe chelated with DFOB, and of solubilized, nonchelated Fe to determine which forms of solubilized Fe were directly available to each plant species. The interactions among these parameters are discussed below. The terms Fe-inefficient and Fe-efficient are used to characterize plant species which tend to exhibit or not exhibit, respectively, Fe deficiency symptoms when grown in media low in available Fe (1).

ABSTRACr When Fe was supplied at 100 micromolar in nutrient solution of pH 7.5, 10 and I micromolar levels of the siderophore desferroxamine B (DFOB), a microbial iron transport compound, significantly (a = 0.05) enhanced growth and reduced chlorosis of an Fe-inefficient variety of sorghum (Sorghum bicolor L.). Although significantly adverse effects resulted when both Fe and desferrioxamine B (DFOB) were added at 100 micromolar as FeDFOB, the plants were relatively healthy when grown with 100 micromolar DFOB plus 200 micromolar Fe. It was concluded that sorghum absorbed Fe from the pool of nonchelated, solubilized Fe, and utilized DFOB as a shuttle agent, in equilibrium with this pool, to transport Fe from fmely suspended solid phase Fe particles to the membrane of absorbing root cells. In contrast to sorghum, absorption of Fe by the Fe-efficient species sunflower (Helianthus aIMMus L.) was related to the level of FeDFOB and independent of the level of solubilized, nonchelated Fe. The latter was decreased whenever the concentration of DFOB was equal to or greater than the concentration of total Fe. For an Fe concentration of 10 micromolar, significantly larger and greener plants were obtained when DFOB was present at 1, 10, or 100 micromolar than in the absence of DFOB. When grown with 100 micromolar FeDFOB, sunflower plnts appeared larger and less chlorotic than those supplied with 100 micromolar Fe and no DFOB. Sunflower apparently was able to utlilie FeDFOB more directly than was sorghum. It is suggested that sunflower acquires Fe after binding FeDFOB at membrane sites and/or by producing sufficient reductants in the rhizosphere to reduce biologically significant levels of Fe(III)DFOB to the less stable Fe(II)DFOB.

MATERIALS AND METHODS Seeds of sorghum (SC 102), Sorghum bicolor L., and sunflower, Helianthus annuus L., were germinated for 6 and 5 d, respectively, on moistened blotter paper or vermiculite. Seedlings with shoots approximately 2 cm in height were washed with distilled H20 and transferred to containers (one plant/container) containing 500 ml of nutrient solution. Sorghum was grown in onethird strength modified Hoagland nutrient solution (7), whereas full strength nutrient solution was used for the more rapidly growing sunflower. Treatments consisted of varying the levels of Fe and DFOB as described below for each species. The nutrient solutions were adjusted to pH 7.5 with KOH, and 1.0 g/l CaC03 was added as a buffering agent. The sunflower and sorghum were grown for I I and 14 d, respectively, with continuous aeration of the nutrient solution. A photoperiod of 16 h with a photon flux density of 300 ME m 2 s-' was provided by a metal halide lamp. After the growth period, the plants were freeze-dried and weighed. The final solution pH values varied randomly among replicates from pH 7.0 to 7.5. Five successive experiments were conducted with sorghum; each experiment contained four or six replications of either six or four treatments, respectively. All experiments involved appropriate control treatments which included Fe but not DFOB. Dry

Hydroxamate siderophores (HS)3 are Fe chelating agents produced by many microorganisms when subjected to Fe stress (5, 1 1, 16). Using the Arthrobacter pavescens JG-9 bioassay, Powell et al. (13, 14) found that HS occur in soils at levels sufficiently high to potentially affect the absorption of Fe by plants. Cline et al. (4) demonstrated that HS are able to form stable Fe chelates over the normal pH range of soils, in contrast to a number of 'Supported by National Science Foundation grant DEB-79-11276. From a dissertation by the senior author submitted to the Academic Faculty of Colorado State University in partial fulfillment of the requirements for the Ph.D. degree. 2 Present address: Caesar Kleberg Wildlife Research Institute, College of Agriculture, Texas A & I University, Kingsville, TX 78363.

'Abbreviations: HS, hydroxamate siderophore; DFOB, desferrioxamine B obtained from Ciba-Giegy as Desferal, the methanesulfonate salt; XDFOB, free ligand of DFOB.

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SIDEROPHORE EFFECT ON PLANT IRON UPTAKE weights of plants from treatments containing DFOB were statistically compared with dry weights of plants from control treatments using Student's t test (a = 0.05). Treatment selection in

successive experiments was based

results obtained in previous ones and consisted of combinations of 10, 100, or 200 Mm Fe and 0, 1, 10, or 100 Mm DFOB (see Table I). Mean dry weights and chlorosis ratings of all replications of each treatment are reported, but only plants from individual experiments containing both treatments being compared were included in the statistical analysis to avoid any confounding by possible differences in growing conditions among the five experiments. Sunflower was grown in three successive and identical experiments; each experiment contained four replications of the same six treatments. Data from the three experiments were pooled within each treatment for the statistical analysis as described above. Treatments consisted of 10 or 100 Mm Fe and 0, 5, or 100 Mm DFOB in a 2 x 3 factorial design. When Fe and DFOB were added at equimolar concentrations, FeDFOB was prepared separately and then added to the nutrient solution. Equimolar FeDFOB was prepared by adding 1.5 times the required amount of DFOB to 21 ml of nutrient solution containing I mm Fe plus an amount of additional Fe equal to that needed to saturate DFOB with Fe. It was necessary to upon

repeatedly adjust the pH to 7.5 with KOH during the 1st h to precipitate nonchelated Fe and to offset the extreme acidity produced as Fe displaced H from DFOB during the formation of FeDFOB. The solution was then shaken for 18 to 24 h to allow for complete saturation of DFOB with Fe. Solid phase Fe (i.e. nonchelated Fe) was then removed by centrifugation at 7000g for 10 min, after which a volume of supematant containing the required amount of FeDFOB was removed and added to nutrient solution treatments. At neutral pH, the saturation level of nonchelated Fe in equilibrium with solid phase Fe (i.e. Fe(OH)3 (amorph.)) is only 0.1 nm (calculated for Fe(OH)3(amorph.) at pH 7 [9]) and is negligible relative to the level of FeDFOB in all experimental treatments containing DFOB. Experimental treatments were based on the following rationale: when the concentration of added Fe exceeds that of DFOB, the level of solubilized Fe is determined by its equilibrium with solid phase Fe(OH)3(amorph.), and essentially all DFOB is chelated with Fe (3). A small portion of DFOB will always exist as the free ligand or chelate with other metals (M). Since the term DFOB is commonly used to indicate that Fe is not chelated to the compound, the term XDFOB indicates the free ligand (i.e. the most anionic form of DFOB which is not complexed with any positive ion). The entire system can be described by the following equilibria (equation 1): Total solubilized nonchelated Fe

it

Fe(OH)3(amorph.) + 3H+ = 3H20 + Fe3+

(1)

Fe(OH)3(amorph.).

Fe3+

=

Fe3+, Log Ko7ol 10-31'36 x FeDFOB/XDFOB

FeDFOB

=

XDFOB +

When total Fe exceeds the sum of all Fe solution species, the Fe should exist as solid phase Fe(OH)3(amorph.). Since the equilibrium (i.e saturation) level of total solubilized, nonchelated Fe, including Fe3+, in equilibrium with Fe(OH)3(amorph.) is only 0.1 nm at pH 7, essentially all Fe in excess of chelated Fe excess

exists as Fe(OH)3(amorph.). When the concentration of total added DFOB is greater that that of total Fe, Fe(OH)3(amorph.) cannot form and precipitate, and therefore all Fe is in solution, existing primarily as FeDFOB. In this case, the level of Fe3+ is determined by its equilibrium with FeDFOB (equation 2) and is less than that maintained by equilibrium with

(2)

RESULTS AND DISCUSSION Experiments designed to examine Fe absorption by sorghum revealed that all plants were severely chlorotic and stunted (dry weight near 0.1 g) when total Fe was 10 AM, irrespective of the level of DFOB (Table I). In these experiments a healthy plant of 1.0 g biomass would require about 100 Mg Fe assuming 100 ,gg/ g tissue concentration (17). At least 1350 Mg Fe were always supplied to each plant; thus, even at 10 AM Fe, there was an adequate supply of total Fe. In the 10 Mm Fe treatments, absorption of Fe appeared to have been limited by diffusion of Fe from solid phase Fe(OH)3(amorph.) through the solution phase (only 0.1 nm at pH 7 in the absence of chelating agent [9]) to plant roots. It was somewhat surprising that the addition of DFOB did not increase yields or decrease chlorosis when total Fe was 10 AM. This might be explained if Fe was removed from FeDFOB in the rhizosphere, and rechelation of DFOB with Fe was limited by diffusion of Fe from dissolving solid phase. When total Fe was raised to 100 AM and no DFOB was added, plants were much larger (mean dry weight of 0.239 g) and less chlorotic, although they still exhibited Fe stress symptoms. Since the level of total Fe exceeded the saturation level of the precipitated Fe(OH)3(amorph.) solid phase, the level of solubilized Fe was identical (0.1 nM) for both 10 and 100 AM total Fe in the absence of DFOB. Conditions should have been similar in the 10 and 100 jAM Fe treatments except that the latter contained 10 times as much solid phase Fe as the former. We believe the beneficial effects derived when total Fe was raised from 10 to 100 AM were Table I. Effects of Various Molar Ratios of DFOB/Fe on the Growth of Sorghum (SC 102) in One-Third Strength Modified Hoagland Nutrient Solution Buffered at pH 7 to 7.5 The data result from five combined experiments. DFOB

Replications

Oto 100 0

36 22

Fe

XDFOB + M = MDFOB

FeDFOB

-31.36

(3) Total solubilized nonchelated Fe Thus, the levels of Fe3+ and total solubilized, nonchelated Fe are proportional to the ratio of the levels of FeDFOB/XDFOB (equation 3) when the level of added DFOB exceeds that of total Fe. In the experiments, this ratio was varied by adding different amounts of total Fe and total DFOB to nutrient solution. After monitoring the growth of sorghum and sunflower plants in these solutions, conclusions were drawn regarding the abilities of the two plant types to utilize FeDFOB as an Fe source and the mechanisms involved.

+

it

=

Total Plant Dry Wt.

Severity Chlorosis8of

g± SE

AM

0.1 9.4 0.239 ± 0.02 5.1 1 7 0.310 ± 0.05* 3.9 100 10 4 0.291 ± 0.04* 3.6 100 100 22 0.178 ± 0.02* 6.1 200 100 6 0.431 ± 0.04** 2.2 a Mean of all plants per treatment which were individually rated as: 0 = dark green, I = green, 3 = light green with interveinal yellowing, 5 = equal green and yellow areas, 7 = yellow with green veins, 9 = completely yellow, 10 = yellow with brown areas. *, Significantly (a = 0.05) different from 100 Mm Fe and 0 M DFOB treatment as determined by Student's t test. **, Significantly (a = 0.05) different from both the 100 Mm Fe, 100 Mm DFOB and the 100 Mm Fe, 0 M DFOB treatments. 10 100 100

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CLINE ET AL.

due to a more rapid rate of diffusion caused by more solid phase particles being in suspension. We believe Fe diffusion would be enhanced if more solid phase Fe particles were in suspension from which Fe could dissolve and diffuse to absorbing root cells as they remove Fe from solution. No other explanation would appear logical. Although the nutrient solutions were agitated by aeration, Fe concentration gradients still probably occurred in the intermediate rhizosphere, including the cortical free space and between the cell wall and plasmalemma of absorbing root cells as acknowledged by others (6). At 100 Mm Fe, the addition of DFOB at 1 and 10 Mm significantly (a = 0.05) increased yields and decreased chlorosis of sorghum. This indicated that the plants could utilize FeDFOB as a source of Fe when sufficient solid phase Fe was present and when the concentration of Fe exceeded that of DFOB. The above 0, 1, and 10 Mm DFOB treatments with 100 Mm Fe contained the same levels of solubilized, nonchelated Fe (i.e. 0.1 nM) since the level of total Fe exceeded the level of DFOB (see "Materials and Methods"). However, when Fe and DFOB were added in equimolar quantities as FeDFOB (see "Materials and Methods"), the yields were significantly less (a = 0.05), and chlorosis was more severe than when DFOB was deleted. This indicated that the sorghum probably absorbed Fe from the solubilized, nonchelated Fe pool, causing FeDFOB to dissociate to XDFOB (i.e. free ligand) near the absorption site to replace the absorbed Fe in accordance with equation 2. Some XDFOB would have rechelated with other ions, mainly H+ (3). When solid phase Fe was present (i.e. total Fe exceeded added DFOB), XDFOB could rechelate with Fe in accordance with equation 1. However, when no solid phase was present (e.g. when Fe and DFOB were equimolar), XDFOB could not become rechelated with Fe, and the solution level of the directly available, nonchelated Fe pool was lowered as XDFOB accumulated and the ratio of FeDFOB/ XDFOB decreased (equation 3). In this instance, XDFOB probably competed with the plant for Fe. When the level of Fe was increased from 100 to 200 Mm, the plant dry weight of sorghum more than doubled and chlorosis was markedly reduced (Table 1). With added Fe, XDFOB could become rechelated with Fe and the level of the solubilized, inorganic Fe pool could not be suppressed by a large accumulation of XDFOB. Thus, we postulate that when the amount of Fe exceeded that of added DFOB, allowing precipitation of solid phase Fe, FeDFOB was beneficial to the growth of sorghum because it acted as a shuttle agent and enhanced the diffusion of Fe from the relatively abundant solid phase to absorbing root cells. As with sorghum, sunflower plants were stunted and chlorotic when grown with 10 ,M Fe in the absence of DFOB (Table II). However, when DFOB was added at 5 gM, the Fe-efficient sunflower responded with significantly (a = 0.05) increased growth and reduced chlorosis. It is particularly interesting that the plants grown in nutrient solution with 10 ,uM Fe and 100 Mm DFOB were significantly larger and less chlorotic than those with 10 ;M Fe and no DFOB. When the concentration of DFOB exceeded that of Fe, it would be expected that the level of solubilized, nonchelated Fe was lowered far below the level maintained by equilibrium with Fe(OH)3(amorph.) (see "Materials and Methods."). Calculation indicated that both the levels of Fe3+ and total solubilized, nonchelated Fe were 109 times less in the 10 ,M Fe plus 100 $LM DFOB treatment than in the 10 Mm Fe treatment without DFOB. This was accomplished by using the computer modeling program of Cline et al. (3) to predict the levels of FeDFOB and XDFOB (10 Mm and 0.068 nm, respectively), and then substituting these values into equation 3. The fact that sunflower plants were larger and healthier in the 10 UM Fe plus 100 Mm DFOB treatment suggested FeDFOB may have been bound at membrane sites as reported for fungi (8, 18, 19). In

grown

Plant Physiol. Vol. 76, 1984

Table II. Effects of Various Molar Ratios ofDFOB/Fe on the Growth ofSunflower in a Full Strength Modified Hoagland Nutrient Solution Buffered at pH 7 to 7.S The data result from three combined identical experiments with 12 replications per treatment. DFOB

Fe JM

Dry Wt g

Severity Chlorosie8of

SE

10 0 0.340 0.05 7.1 10 5 0.498 ± 0.06* 3.7 10 100 0.591 0.10* 3.5 100 0 0.587 0.06 1.3 5 100 0.579 0.07 1.3 100 100 0.759 0.11 0.3 a Mean of all plants per treatment which were individually rated as described in Table I. *, Significantly (a = 0.05) different from the 10 pM Fe and 0 M DFOB treatment as determined by Student's t test.

contrast to sorghum, Fe absorption by sunflower was directly related to the level of FeDFOB and apparently independent of the level of solubilized, nonchelated Fe. Sunflower probably absorbed Fe directly from FeDFOB via a reduction process (2) at specific membrane sites or in the rhizosphere, or FeDFOB may have been absorbed intact across the absorbing membrane as has been reported for duckweed using ferrichrome as a representative HS (12). If sunflower plants were absorbing Fe from the solubilized, nonchelated pool as is supposed for sorghum, adverse effects would have been expected in the case where the molarity of DFOB was 10 times that of Fe. In contrast with sorghum, the direct use of FeDFOB by sunflower was also supported by the observation that plants grown with 100 gM FeDFOB were apparently healthier and larger than those grown with 100 MM Fe without DFOB. However, sunflower plants grown with DFOB and Fe were not significantly (a = 0.05) larger than control plants grown with 100 pM Fe without DFOB (Table II) and may be a consequence of the control plants being relatively more healthy in this comparison, as evidenced by their chlorosis rating ofonly 1.3. Although the growth ofsorghum was apparently limited by diffusion of Fe in the 100 Mm Fe treatment without DFOB (Table I), the growth of Fe-efficient sunflower plants was much less affected under these conditions. The fact that solubilized, nonchelated Fe was calculated to be suppressed by a factor of 109 in the 100 Mm DFOB plus 10 Mm Fe treatment, relative to controls without DFOB, was somewhat offset by the increase in total solubilized Fe (i.e., diffusible Fe) by a factor of I0O, from 0.1 nm without DFOB to 10 juM as FeDFOB. Thus, it could be argued that sunflower absorbed nonchelated Fe similarly to sorghum, and the benefits derived by sunflower in the 100 gM DFOB plus 10 Mm Fe treatment relative to the control were the result of increased diffusion of Fe. We believe this explanation to be less likely than a more

direct utilization of FeDFOB by sunflower since solubilized, nonchelated Fe was suppressed by much more than diffusible Fe was increased. Furthermore, if the diffusion explanation were true for sunflower, release of Fe by FeDFOB at the membrane of absorbing root cells would drastically reduce the ratio of FeDFOB/XDFOB in the above treatment and further suppress the level of solubilized, nonchelated Fe (equation 3), making a favorable plant response even less likely. The greening of plants in all treatments after foliar application of FeSO4 indicated that all observed chlorosis was the result of a Fe deficiency related to Fe content and that DFOB did not appear to reduce the availability of any other metals below critical levels. It may be important to note that a disappearance of some FeDFOB (specifically the DFOB portion of the molecule) and/

SIDEROPHORE EFFECT ON PLANT IRON UPTAKE or DFOB sometimes occurred randomly in any rootbathing solution containing DFOB as evidenced by the loss of the characteristic color of the solutions. When observed, the disappearance was only after 11 and 14 d of growth by sunflower and sorghum, respectively (the final day of growth for each species). However, treatment effects were obvious 3 to 6 d prior to any observed DFOB color disappearance and occurred in treatment replications where no disappearance of chelating agent was apparent. This is especially relevant in the 100 pM DFOB plus 10 jM Fe treatments in which sunflower plants were significantly larger and greener than in the absence of DFOB. Degradation of over 90% of the DFOB would have had to occur for the molarity of Fe to exceed that of DFOB and to have prevented the treatment from suppressing the level of nonchelated Fe (see "Materials and Methods"). The validity of the results is also supported by the adverse effect observed with sorghum grown with 100 AM FeDFOB. Ifdegradation or disappearance of DFOB was a significant factor, Fe(OH)3(amorph.) would have precipitated and the level of solubilized, nonchelated.Fe could not have been reduced below that present in the absence of DFOB and reduced the growth of sorghum.

SUMMARY The growth and degree of chlorosis of sorghum, an Fe-inefficient species, appeared to be directly related to both the concentration and diffusion rate of solubilized, nonchelated Fe. The observed effects of DFOB upon sorghum growth and chlorosis were dependent upon the level of DFOB and how it affected the concentration of nonchelated Fe. It is concluded that sorghum absorbed Fe from the solubilized, nonchelated pool of Fe and that FeDFOB could be of benefit to sorghum in the presence of solid phase Fe (i.e. when the molarity of DFOB was less than that of Fe) by dissociating and increasing the rate of diffusion of Fe from small suspended solid phase particles to the root surface. Thus, FeDFOB at nonexcessive levels would be expected to be beneficial to sorghum grown in soil since solid phase Fe is always abundant. Sunflower, an Fe-efficient plant species, appeared to be able to absorb Fe directly from FeDFOB since absorption of Fe was directly related to the level of FeDFOB and not related to the concentration of solubilized nonchelated Fe, which was substantially lowered whenever the concentration of total Fe was less than or equal to the concentration DFOB. Sunflower may acquire Fe after binding FeDFOB to membrane sites and/or producing sufficient reductants to reduce Fe(III)DFOB to the much less stable Fe(II)DFOB. Because plants have evolved in the presence of HS in soil, they may possess different mechanisms for acquiring Fe from Fe-HS than from synthetically chelated

39

Fe. It appears that Fe-efficient and Fe-inefficient species may be differentiated based on their ability to use HS in the absorption of Fe. However, these experiments were conducted using one specific HS compound, and it is cautioned that Fe absorption rates and mechanisms may vary with different types of sidero-

phores.

Acknowledgmenis-The authors wish to thank Mary McClintock for her able

technical assistance.

LITERATURE CliTD 1. BROWN JC 1978 Mechanism of iron uptake by plants. Plant Cell Environ 1: 249-257 2. CHANEY RL, JC BROWN, LO TIFFIN 1972 Obligatory reduction of ferric chelates in iron uptake by soybeans. Plant Physiol 50: 208-213 3. CLINE GR, PE POWELL, PJ SZANIszLO, CPP REID 1982 Comparison of the abilities of hydroxamic, synthetic, and other organic acids to chelate iron and other ions in nutrient solution. Soil Sci Soc Am J 46: 1158-1164 4. CLINE GR, PE POWELL, PJ SZANSLO, CPP REID 1983 Comparison of the abilities of hydroxamic and other organic acids to chelate iron and other ions in soils. Soil Sci 136: 145-157 5. EMERY T 1977 The storage and transport of iron. In Helmet Sigel, ed, Metal Ions in Biological Systems, Vol 7, Iron in Model and Natural Compounds. Marcel Dekker, New York, pp 77-125 6. HALVORSON AD, WL LINDSAY 1977 The critical Zn2 concentration for corn and the nonabsorption of chelated zinc. Soil Sci Soc Am J 41: 531-534 7. HALVORSON AD, WL LINDSAY 1972 Equilibrium relationships of metal chelates in hydroponic solutions. Soil Sci Soc Am Proc 36: 755-761 8. LEONG J, JB NEILANDS, KN RAYMOND 1974 Coordination of isomers of biological iron transport compounds III. (1) Transport of A-cis chromic desferriferrichrome by Ustilago sphaerogena. Biochem Biophys Res Commun 60: 1066-1071 9. LINDSAY WL 1979 Chemical Equilibria in Soils. John Wiley and Sons, New York 10. MARTELL AE, RM SMITH 1977 Critical Stability Constants, Vol 3, Other Organic Ligands. Plenum, New York I 1. NEILANDSJB 1979 Biomedical and environmental significance of siderophores. In N Kharasch, ed, Trace Metals in Health and Disease. Raven Press, New York, pp 27-41 12. ORLANDO JA, JB NEILANDS 1982 Ferrichrome compounds as a source of iron for higher plants. In H Kehl, ed, Chemistry and Biology of Hydroxamic Acids. S. Karger, Basel, pp 123-129 13. POWELL PE, GR CLINE, CPP REID, PJ SZANISZLO 1980 Occurrence of hydroxamate siderophore iron chelators in soils. Nature 287: 833-834 14. POWELL PE, PJ SZANIszLO, CPP REID 1983 Confirmation of occurrence of hydroxamate siderophores in soil by a novel Escherichia coli bioassay. Appl Environ Micro 46: 1080-1083 15. STurz E 1964 Aufnahme von Ferrioxamin B durch Tomatenpflanzen. Experientia 20: 430-431 16. WAID JS 1975 Hydroxamic acids in soil systems. In EA Paul, AD McLaren, eds. Soil Biochemistry, Vol 4. Marcel Dekker, New York, pp 65-101 17. WALLIHAN EF 1966 Iron. In HD Chapman, ed, Diagnostic Criteria for Plants and Soils. University of California, Division of Agricultural Sciences, Riverside, pp 203-212 18. WIEBE C, G WINKELMANN 1975 Kinetic studies on the specificity of chelateiron uptake in Aspergillus. J Bacteriol 123: 837-842 19. WINKELMANN G 1974 Uptake of iron by Neurospora crassa. III. Iron transport studies with ferrichrome-type compounds. Arch Microbiol 98: 39-50