solubilization of metal phosphates by rhizoctonia solani

Solubilization of metal phosphates by Rhizoctonia solani

Helen JACOBS1, Graeme P. BOSWELL2, Frances A. HARPER1, Karl RITZ3#, Fordyce A. DAVIDSON2

and Geoffrey M. GADD1*

1 Division of Environmental and Applied Biology, Biological Sciences Institute, School of Life Sciences,University of Dundee, Dundee DD1 4HN, UK.2 Department of Mathematics, University of Dundee, Dundee DD1 4HN, UK.3 Soil-Plant Dynamics Group, Scottish Crop Research Institute, Invergowrie, Dundee DD2 5DA, UK.E-mail : [email protected]

Received 10 June 2002; accepted 11 October 2002.

The effects of different temperatures and pH on the growth and solubilization of insoluble calcium phosphate, cobaltphosphate, manganese phosphate, strontium hydrogen phosphate and zinc phosphate by Rhizoctonia solani on solidified

media were assessed. Solubilization of the metal phosphates was monitored by the production of a clear zone around orunderneath the fungus. R. solani efficiently solubilized all five metal phosphates except cobalt phosphate when grown onmedium at pH 7. Solubilization activity by R. solani decreased with increasing pH on medium containing calcium

phosphate but increased on strontium hydrogen phosphate-amended medium. The uptake of metals by the mycelia wasunaffected by the pH of the medium or the growth temperature. Small quantities of crystals were produced in the agarwhen R. solani was grown on calcium phosphate- and strontium hydrogen phosphate-amended media and these were

identified as calcium or strontium sulphates respectively: there appeared to be little or no production of insolubleoxalates although a role for oxalate in the overall solubilization process cannot be discounted. These results are discussedin relation to their physiological and environmental significance, and the important roles of fungi in effecting

transformations of insoluble metal-containing compounds in the environment.

INTRODUCTION

The ability of fungi to solubilize insoluble metal-con-taining minerals and other compounds is importantbecause of the resultant release of phosphates, othernutrients and metal cations into biogeochemical cycles(Sayer, Raggett & Gadd 1995, Gadd 1999, 2001, Gadd& Sayer 2000, Jacobs et al. 2002). While several mech-anisms may be involved (Burgstaller & Schinner1993), acidolysis mediated by H+ excretion and/or theproduction of organic acids appears to be the most sig-nificant. Many fungi can produce organic acids suchas citric acid, which supply both H+ and a metal-complexing anion (citratex), and mediate release ofmobile phosphate and metal species from insolublesources (Gadd 1999). In other instances, several fungican precipitate insoluble metal oxalates which alsohas resultant effects on release of, e.g. sulphate and

phosphate (Sayer & Gadd 1997, Gharieb & Gadd 1999,Sayer et al. 1999). A number of studies have examinedthe ability of soil fungi to solubilize insoluble min-eral phosphates. For example, the calcium phosphate-solubilizing ability of Penicillium bilaii has beendocumented (Kucey 1987) and this organism has beenproduced as a commercial preparation to increase theavailability of phosphate for cereal crops (Cunningham& Kuiack 1992). It appears that nutritional status canmarkedly affect the ability of fungi to solubilize insol-ublemetal compounds, includingphosphates (Altomareet al. 1999). An adequate source of carbon needs to bepresent (Dixon-Hardy et al. 1998, Jacobs et al. 2002),while solubilization of gypsum (CaSO4

. 2H2O) occurredin a medium containing nitrate but not in mediumcontaining ammonium as the nitrogen source : this cor-related with the production of substantial amounts ofoxalic acid in nitrate-containing medium (Gharieb &Gadd 1998, Gharieb 2000).

Rhizoctonia solani was chosen for this study as it isfound in a wide range of agricultural soils. It is aneconomically important plant pathogen that combines

* Corresponding author.# Permanent address: National Soil Resources Institute, Cranfield

University, Silsoe, Cranfield MK45 4DT, UK.

Mycol. Res. 106 (12): 1468–1479 (December 2002). f The British Mycological Society 1468

DOI: 10.1017/S0953756202006901 Printed in the United Kingdom.

strong saprophytic capabilities with facultative para-sitism of a wide range of plants (Thornton & Gilligan1999), although non-pathogenic binucleate strains arebeing investigated for biocontrol (Cartwright & Spurr1998). Some redistribution of nutrients through R.solani hyphae, when growing in nutritionally hetero-geneous conditions, has previously been reported byRitz (1995), although there is no information on themechanism of phosphate solubilization. Nevertheless,R. solani has been reported to produce calcium oxalatecrystals in vitro, and on the surface of infected hypo-cotyls of canola and mustard plants (Yang, Tewari &Verma 1993). Calcium oxalate is the most importantcrystalline metal oxalate in the environment and canbe associated with many species of free-living, plantsymbiotic and pathogenic fungi (Gadd 1999, 2001). Theobjective of this work was to examine the ability ofR. solani to solubilize a variety of metal phosphates,some containing potentially toxic metals, and attemptto understand what mechanisms may be employed. Theinfluence of pH and temperature on growth and solu-bilization ability was also examined, together with as-sessment of the amounts of solubilized metals taken upby the mycelia. The results are discussed in relation tofungal physiology, mechanisms of fungal transform-ations of insoluble metal-containing compounds andtheir possible environmental significance.

MATERIALS AND METHODS

Organism, medium and culture conditions

Rhizoctonia solani (anastomosis Group 4 (R3), IMI385768) was routinely maintained on potato dextroseagar (PDA, Oxoid CM139) in 90 mm diam Petri dishesat 25 xC. For all experiments a defined medium (Dixon-Hardy et al. 1998)was used of the following composition(g lx1 distilled water) : (NH4)2SO4, 5; KH2PO4, 0.5;MgSO4

. 7H2O, 0.2; CaCl2 . 6H2O, 0.05; NaCl, 0.1;FeCl3 . 6H2O, 0.0025; ZnSO4

. 7H2O, 0.004; MnSO4.

4H2O, 0.004; CuSO4. 5H2O, 0.0004; D-glucose, 10;

Noble agar (Difco), 10. Mineral salt solutions wereautoclaved separately before being added to glucose-containing molten agar at approximately 55–60 x. Agarmedium and mineral salt solutions were sterilized byautoclaving at 121 x for 20 min.

Assessment of growth and solubilization

To assess the ability of Rhizoctonia solani to solubilizeinsolublemetal phosphates, commercial preparations ofCo3(PO4)2 . 8H2O and Zn3(PO4)2 . 2H2O (Alfa, JohnsonMatthey, Royston, Herts.), Mn2(PO4)2 . 3H2O (VWRInternational, Poole, Dorset) and Ca3(PO4)2 (VWR In-ternational) were used. Strontium phosphate was pre-pared by mixing aliquots of Sr(NO3)2 (10 g in 100 mldistilled H2O) with excess amounts of a solution ofNa2HPO4 (10 g in 100 ml distilled H2O). The precipitatewas allowed to settle and was washed five times with

distilled de-ionized water prior to drying at 60 x. Theprecipitate was identified as strontium hydrogen phos-phate (SrHPO4) by powder X-ray diffraction with aPhillips X-ray powder diffractometer using Cu Ka

radiation. SrHPO4 was added to media at a concen-tration of 0.1% (w/v) : the other insoluble metalphosphates were added to the medium to a final con-centration w5 mM (Sayer et al. 1995). The initial pHof control medium was pH 4.6: the addition of themetal phosphates increased the pH of the medium topH 5.8, Ca3(PO4)2 ; pH 5.7, Co3(PO4)2 . 8H2O; pH 5.3,Mn3(PO4)2 . 3H2O; pH 5.5, SrHPO4 and Zn3(PO4)3 .

2H2O. The medium was also adjusted to pH 6 and 7 bythe addition of 1 MNaOHafter autoclaving. The funguswas grown at three temperatures, 15 x, 25 x and 30 x.

To facilitate recovery of the fungus from the agar,sterilized Cellophane discs (P 00, UCB Cellophane,Bridgewater, Somerset) were placed aseptically on thesurface of the agar. The Cellophane discs (85 mm diam)were prepared by boiling in distilled water before beingautoclaved (Gray, Wilding & Markham 1991). Plateswere inoculated centrally with 4 mm diam plugs ofR. solani cut from the edge of a growing colony. Eachcombination of treatment was replicated five times.Colony radius and clear zones of solubilization weremeasured as the mean of two perpendicular radii untilthe colony reached 40 mm diam, with a minimum ofeight recordings. Solubilization of the metal phosphateswas clearly observed by using a light source underneaththe plates.

The pH of the agar under the growing colony and inabiotic controls was measured before and after incu-bation using a double junction flat tip pH electrode(BDH). Biomass dry weight was determined by remov-ing the mycelial mat from the Cellophane and dryingin tared foil cups to a constant weight at 80 x. Afterdry weight measurements were taken, mycelia weredigested in 2 ml concentrated HNO3 at 25 x overnight,then heated to 90 x for 8 h. After the samples werediluted appropriately with double-distilled H2O, sol-utions were analysed for metal ion content using a PyeUnicam SP9 atomic absorption spectrophotometer(AAS) with respect to standard solutions in acidifiedddH2O. Lanthanum chloride (0.1%of the final volume)was added to the calcium and strontium standardsolutions and the samples.

Isolation and characterization of crystals formed inagar medium

Fungal colonies were removed from the agar by liftingthe Cellophane membrane. Crystals that had formedin the agar under Rhizoctonia solani colonies were ex-tracted bymelting the agar iny100 ml of distilled waterusing a microwave oven (PROline Microchef ST44) seton maximum power for approximately 2 min. Crystalswere recovered from the solution and were placed ondouble-sided carbon adhesive tape on 10 mm diamaluminium stubs and dried overnight. The crystals were

H. Jacobs and others 1469

examined using a JEOL JSM-35 scanning electronmicroscope (SEM) coupled with energy dispersiveX-ray micro-analysis (EDXA, link interface p1449).For EDXA, uncoated samples were analysed at a countrate of 1500 counts sx1 for at least 100 s at a voltage of25 kV. For SEM, the samples were sputter-coated for5 min using a Polaron E5100 series II ‘cool ’ sputter-coater fitted with a Au/Pd target. Crystals were alsoexamined using powder X-ray diffraction as describedpreviously.

Identification of crystals by high performance liquidchromatography (HPLC)

Crystals derived from growth of Rhizoctonia solanion strontium hydrogen phosphate- and calcium phos-phate-amendedmedium,were dissolved in concentratedhydrochloric acid. Analysis of free organic acids wasdetermined by injecting a 20 ml sample into a Waters(Watford), HPLC system comprising a 600E systemcontroller, a 600 pump and a 717plus autosampler. Theorganic acids were separated with a BioRad AminexHPX-87H (BioRad Laboratories, Richmond, CA) ion-exclusion column (300 mmr7.8 mm) at room tempera-ture. Prior to injection, samples were diluted 100-fold inMilliQH2O,mixedwith cation-exchange resin (BioRad,AG 50W-X4), and filtered through 0.45 mm pore sizemembrane filters (Whatman, Maidstone). The mobilephase was 4 mM H2SO4 at a flow rate of 0.6 ml minx1.Detection was carried out at 210 nm with a Waters 486tuneable wavelength detector for a run of 30 min.

Data analysis

Colony radial growth rate (Kr ; Pirt 1967) and rate ofsolubilization were calculated by linear regression ofcolony radius or clear zone of solubilization with time.Growth rates were used to calculate the colony exten-sion rate of Rhizoctonia solani in the presence of the

metal phosphates compared to the control. The valueswere expressed as a ratio of the colony growth rate ofR. solani in the presence of a metal phosphate (Rm), tothat of the control (Rc). Likewise, the rate ofmetal phos-phate solubilization (Rs) was calculated using the rate ofextension of the clear zone compared to the growth rateof the fungus in the presence of the metal phosphate(Rm). Analysis of variance (ANOVA) was used to de-termine the effects of temperature and initial pH ofthe media on the growth and solubilization ability ofR. solani. Correlation coefficients were used for com-parisons betweengrowth rate, rate of solubilization, bio-mass dry weight, final pH of the medium, metal uptake,incubation temperature and initial pH of themedium byR. solani onmetal phosphate-amendedmedia. Student’st-test (two samples, variances not assumed equal) wasused to assess pairwise comparisons of metal uptakebetween the control and metal-amended media. Allstatistics were calculated usingMINITABRelease 12.1.Statistical significance was assessed at the 95% level ofconfidence.

RESULTS

Effect of temperature and pH on growth

The growth rate of Rhizoctonia solani was significantlyincreased when grown at 25 x and 30 x compared to 15 x

(P<0.05, ANOVA) (Fig. 1). The addition of calciumphosphate had the greatest positive effect on the growthof R. solani, followed by manganese phosphate andstrontium hydrogen phosphate (Figs 1–2). Zinc phos-phate inhibited the growth of R. solani at all tempera-tures and pH values compared to the control. Cobaltphosphate increased the growth of R. solani in mediumwith an initial pH of 6.0 (P<0.05), but inhibited thegrowth of R. solani in medium adjusted to pH 7.0compared to the control.

Fig. 1. Colony radial growth rate (mm hx1) of Rhizoctonia solani on media containing metal phosphates. Colonies were

grown at (%) 15 xC, ( ) 25 x and (&) 30 x over 7 d. Results are means of five replicates ¡S.E.M.

Fungal solubilization of metal phosphates 1470

Colony extension rates in the presence of the metalphosphateswere compared and the values expressed as aratio of the colony growth rate of R. solani in the pres-ence of a metal phosphate (Rm), to that of the control(Rc) (Fig. 2). A ratio of above 1.0 indicates that themetalphosphate had increased the fungal growth rate com-pared to the control whereas a ratio of less than 1.0indicated the metal phosphate inhibited growth. Theratio of growth on metal-amended media compared tocontrol medium (Rm/Rc) decreased with an increase inmedium pH for calcium phosphate, manganese phos-phate and strontiumhydrogen phosphate (Fig. 2). Therewas a strong positive correlation between the initialpH of the medium and the growth rate of R. solani onthe control medium (P<0.05) and on the zinc phos-phate-amended medium (P<0.05) (Table 1). Theincrease in temperature significantly correlated with theincrease in the rate of growth by R. solani on zincphosphate-amended medium, but not with the othermetal phosphates. The growth rate of R. solani alsopositively correlated with the biomass dry weight whengrown on the control medium, and calcium-, manga-nese phosphate- and strontium hydrogen phosphate-amended media (Table 1). However, when R. solaniwasgrown on cobalt- and zinc phosphate-amended media,the morphology was sparse, resulting in a low pro-duction of biomass (Fig. 3).

Effect of temperature and pH on solubilization ofmetal phosphates

The rate of solubilization of the metal phosphates byRhizoctonia solani, i.e. the rate of extension of the clearzone of solubilization in the agar, was greater at 25 x and30 x than at 15 x (Fig. 4). However, this was not truefor manganese phosphate and strontium hydrogen

phosphate at pH 5.3 and 5.5 respectively. Calciumphosphate and manganese phosphate were both solu-bilized at a slower ratewhen the initial pHof themediumwas pH 7 compared to medium with an initial pH of 6.The solubilization rate of strontium hydrogen phos-phate increased with increasing initial pH of the media(Table 1). A negative correlation for the solubilizationof cobalt phosphate with the initial pH of the mediumwas observed due to the cobalt phosphate not beingsolubilized at pH 7 at any of the temperatures tested.

In Fig. 5, the rate of metal phosphate solubilization(Rs), i.e. the rate of extension of the clear zone, is com-pared to the growth rate of the fungus in the presence ofthe metal phosphate (Rm). A ratio of above 1.0 indicatesthat the rate of solubilization is greater than the growthof the fungus, and the clear zone is therefore greater thanthe size of the colony. The rate of solubilization ofmanganese phosphate by R. solani was between 1.4 and2.6 times greater than the growth rate ofR. solani at 15 x,compared to between 0.1 and 1.4 times greater at 25 x

and 30 x (Fig. 5). R. solani grown on medium with aninitial pH of 5.5 and 6 solubilized zinc phosphate up tofour times greater than the rate of growth, and this wasvisuallymanifest as a small colony surroundedby a largeclear zone: on media adjusted to pH 7 the rate of solu-bilization was approximately equal to the growth rate.Calcium phosphate and cobalt phosphate were bothsolubilized by R. solani at approximately the same rateas growth, except for cobalt phosphatemediumadjustedto pH 7 where no visible solubilization occurred.

Change in pH of solid medium during growth

The pH of the media decreased after 7 d growth ofRhizoctonia solani regardless of the presence or absenceof metal phosphates (Table 2). At 15 x, the pH of the

Fig. 2. Rate of extension of the colony radial growth rate (mm hx1) of Rhizoctonia solani on metal-amended media (Rm),

in relation to growth on control media (Rc), expressed as a ratio (Rm/Rc). Colonies were grown at (%) 15 xC, ( ) 25 x and(&) 30 x over 7 d. Results are means of five replicates ¡S.E.M.


control mediumwas reduced to pH 3.0–3.3 regardless ofthe initial pH of the medium.WhenR. solaniwas grownat 25 x and 30 x the pH of the medium decreased fur-ther to between 2.3 and 2.7 (Table 2). Of the metalphosphates tested, the greatest decrease in the pH ofthe medium was observed on strontium hydrogenphosphate-containing medium although this was notsignificantly different to the pH change recorded forcontrol medium, calcium phosphate- or manganesephosphate-amendedmedia (P<0.05, ANOVA). At 15 x

and 25 x, there was no significant decrease in the pH ofthe media on addition of zinc phosphate and cobaltphosphate, with the exception of zinc phosphate in pH 7adjusted medium at 25 x (P<0.05). The pH of zincphosphate- and cobalt phosphate-amended medium

decreased during growth of R. solani at 30 x (Table 2).There was a significant (P<0.05) negative correlationbetween the initial and the final pH of the media forcalcium- and manganese phosphate-amended media,and this was also the trend for the other metal phos-phates (Table 1). There was no significant difference(P<0.05, ANOVA) between the pH of the mediumbefore inoculation and the abiotic controls after 7 dincubation.

Crystal formation

The sparse production of crystals of different shapesand sizes were only observed in calcium phosphate-and strontium hydrogen phosphate-amended media.

Table 1. Correlation coefficients for comparisons between growth rate, rate of solubilization, biomass dry weight, final pH of the medium,

metal uptake, incubation temperature and initial pH of the medium by Rhizoctonia solani grown on medium amended with metal

phosphates. Significant correlations are shown in bold (P<0.05).

Metal phosphate

Correlation coefficient between measurements

Growth Solubilization Biomass Final pH Metal uptake

Control

Solubilization NA – – – –

Biomass 0.699 NA – – –

Final pH x0.398 NA x0.788 – –

Metal uptake NA NA NA NA –

Incubation temperature 0.662 NA 0.251 0.113 NA

Initial pH 0.706 NA 0.269 0.113 NA

Ca3(PO4)2Solubilization 0.858 – – – –

Biomass 0.943 0.752 – – –

Final pH x0.800 x0.561 x0.753 – –

Metal uptake x0.410 x0.443 x0.600 0.294 –

Incubation temperature x0.295 0.683 0.858 x0.797 x0.368

Initial pH x0.234 x0.582 0.533 x0.684 x0.108

Co2(PO4)2 . 8H2O

Solubilization 0.389 – – – –

Biomass 0.324 x0.472 – – –

Final pH x0.275 x0.523 0.374 – –

Metal uptake 0.148 0.530 x0.667 x0.809 –

Incubation temperature 0.193 0.382 0.259 x0.500 x0.174

Initial pH 0.153 x0.751 0.858 x0.538 x0.065

Mn3(PO4) . 3H2O


Biomass 0.948 0.771 – – –

Final pH x0.949 x0.821 x0.879 – –

Metal uptake x0.209 x0.362 x0.176 0.210 –

Incubation temperature 0.071 0.490 0.852 x0.893 x0.04

Initial pH 0.107 x0.109 0.259 x0.760 0.069

SrHPO4


Biomass 0.770 0.361 – – –

Final pH x0.700 x0.358 x0.906 – –

Metal uptake 0.212 0.530 0.286 x0.253 –

Incubation temperature 0.292 0.288 0.708 x0.807 0.380

Initial pH 0.505 0.873 0.327 x0.509 0.250

Zn3(PO4)2 . 2H2O


Biomass 0.503 0.426 – – –

Final pH 0.268 x0.372 x0.236 – –

Metal uptake x0.064 x0.444 x0.534 0.366 –

Incubation temperature 0.719 0.874 0.419 x0.558 0.491

Initial pH 0.832 0.182 0.649 x0.649 0.561


Spherical crystals, approximately 200–600 mm in length(Fig. 6a,c), star-shaped crystals with three protrudingarms 300 mm in length (Fig. 6b) and rectangular crystals350 mm in length (Fig. 6a), were observed using scan-ning electron microscopy. The crystals formed byRhizoctonia solani when grown in strontium hydrogenphosphate-amended medium had a regular octahedralshape approximately 25 mm in length with some crystalsappearing to be fused together (Fig. 6d). X-ray micro-analysis of the crystals showed that the correspondingmetals were present in the samples, i.e. calcium in crys-tals from the Ca3(PO4)2-amended medium and stron-tium in crystals from the SrHPO4-amended medium.Sulphur was also detected in both samples (Fig. 7).Further examination of the crystals using powder X-ray

diffraction analysis revealed the presence of CaSO4 andSrSO4 respectively (Fig. 8). HPLC analysis of the dis-solved crystals resulted in small identical peaks occur-ring at 7.1 min, which corresponded with oxalic acid(results not shown) but this appeared to be present innegligible quantities. Furthermore, oxalate was not de-tected using staining with silver nitrate and rubeanicacid (Yasue 1969), while ion chromatography revealedthe presence of sulphate (results not shown).

Effect of temperature and pH on metal uptake

Trace amounts of metals were detected in Rhizoctoniasolani grown in the absence of metal phosphates(Table 3). Significantly greater amounts (P<0.05, t-test)

Fig. 3. Biomass dry weight of Rhizoctonia solani on media containing metal phosphates. Colonies were grown at (%) 15 xC,( ) 25 x and (&) 30 x over 7 d. Results are means of three replicates ¡S.E.M.

Fig. 4. Rate of extension of the clear zone of solubilization of metal phosphates (mm hx1) by Rhizoctonia solani. Colonies weregrown at (%) 15 xC, ( ) 25 x and (&) 30 x. Results are means of five replicates ¡S.E.M.


were found in R. solani grown on metal phosphate-amended media. Overall the metal content in R. solanimycelium was greatest for manganese followed by co-balt (Table 3). No significant linear correlation betweenthe metal content of R. solani mycelia and the pH ortemperature of the medium for any of the metals wasobserved (Table 1).

Correlations between growth, biomass dry weight,metal phosphate solubilization, metal uptake and finalpH of the media

Table 1 shows the correlation coefficients betweeneach of the measurements taken for each of the metalphosphates and the control. The results for calciumand manganese phosphate, and strontium hydrogenphosphate-amended media were similar, i.e. the rate ofgrowth strongly correlated with the rate of solubiliz-ation and the biomass dryweight, and the rate of growthnegatively correlated with the final pH of the media asdid the biomass dry weight. No correlation was detectedbetween the results for zinc phosphate. The addition ofcobalt phosphate to the medium was the only treatmentto display any correlation for metal uptake. Biomassdry weight and the final pH of the medium correlatednegatively with metal uptake by the colony.

DISCUSSION

Metal phosphate solubilization by fungi is an importantbiological process in the soil environment, makingphosphates and metal cations available to plants andother microorganisms. It is a widespread phenomenonand Sayer et al. (1995) found that one third ofmore than50 fungal soil isolates tested were able to solubilizeat least one metal compound out of zinc oxide, zincphosphate and cobalt phosphate. In themajority of con-ditions tested in this study, Rhizoctonia solani efficientlysolubilized all fivemetal phosphates. This contrasts withthe findings of Agnihotri (1970) who reported that R.solani failed to solubilize three phosphatic compounds,tricalcium phosphate, hydroxyapatite and fluorapatite,in liquid culture and also reported that the growth of

Fig. 5.Rate of extension of the clear zone of solubilization (Rs) in relation to the rate of growth onmetal-amended medium (Rm)

expressed as a ratio (Rs/Rm), for Rhizoctonia solani in metal phosphate amended media. Colonies were grown at (%) 15 xC,( ) 25 x and (&) 30 x. Results are means of five replicates ¡S.E.M.

Table 2. Change in medium pH after 7 d growth of Rhizoctonia

solani on medium of differing initial pH and at different

temperatures. A single reading was taken in the centre of three

replicate plates using a flat tip pH electrode. There was no

significant difference between the initial pH of the medium and

abiotic controls after 7 d incubation at each temperature

(P<0.05, ANOVA).

Metal phosphate Initial pH

pH of medium after 7 d

15 xC 25 xC 30 xC

Control 4.6¡0.0 3.1¡0.02 2.3¡0.01 2.4¡0.01

6.1¡0.04 3.0¡0.15 2.7¡0.06 2.3¡0.0

7.2¡0.04 3.3¡0.03 2.3¡0.01 2.5¡0.03

Ca3(PO4)2 5.8¡0.0 4.4¡0.08 3.4¡0.01 3.2¡0.12

6.1¡0.04 4.0¡0.15 3.9¡0.16 3.7¡0.04

7.2¡0.04 3.9¡0.25 3.7¡0.05 3.4¡0.05

Co3(PO4)2 . 8H2O 5.7¡0.0 5.5¡0.16 5.7¡0.04 4.5¡0.06

6.1¡0.04 6.3¡0.03 6.3¡0.04 4.3¡0.32

7.2¡0.04 6.6¡0.05 5.4¡0.61 6.5¡0.07

Mn2(PO4)2 . 3H2O 5.3¡0.0 4.5¡0.08 2.8¡0.02 2.8¡0.02

6.1¡0.04 4.8¡0.04 3.3¡0.19 2.9¡0.1

7.2¡0.04 4.2¡0.08 2.8¡0.08 3.3¡0.03

SrHPO4 5.5¡0.0 3.7¡0.04 2.3¡0.03 2.4¡0.09

6.1¡0.04 3.7¡0.12 2.3¡0.02 2.3¡0.01

7.2¡0.04 2.7¡0.26 2.3¡0.0 2.4¡0.01

Zn3(PO4)2 . 2H2O 5.5¡0.0 5.5¡0.1 5.5¡0.06 4.2¡0.03

6.1¡0.04 5.6¡0.04 5.8¡0.01 4.1¡0

7.2¡0.04 6.2¡0.03 5.5¡0.06 5.9¡0.03


R. solani did not change the pH of the culture medium.These discrepancies may partly be due to media andstrain differences as high phosphate concentrations inliquid media would increase buffering capacity and per-haps negate fungal acid production. It is known that dif-ferent isolates ofR. solani can differ considerably in theirpathogenicity to crops (Jeger et al. 1996), and it seemsunlikely that a soil fungus would not possess any ef-fective mechanism for inorganic phosphate acquisition.

Incorporating metal phosphates into a solid mediumprovides an easy screening method for assessing thesolubilization ability of fungi, provided that clear zonesof solubilization can be observed which indicates thefungus is actively producing metabolites required forsolubilization to occur. Although this method cannotreadily be used in a quantitative manner, it can be usefulfor comparing toxic effects of different metal com-pounds toward a single fungal isolate, and toleranceof different fungal isolates towards metal compounds.In related studies using this technique, the screeningprocedure is generally limited to a single incubationtemperature and pH of the medium (Sayer et al. 1995).However, it is evident that these factors can have a sig-

nificant effect on the solubilizing ability of R. solani andthis should be considered when other fungi are screened.

In the present study, small quantities of crystals wereonly formedwhenR. solaniwas grown in the presence ofstrontium hydrogen phosphate and calcium phosphate,and this was independent of temperature or the pH ofthemedium.Thecrystals isolated fromthecalciumphos-phate- and strontium hydrogen phosphate-amendedmedium were not homogeneous in morphology and asignificant proportion of the crystals were found to beCaSO4 or SrSO4, respectively. Calcium and strontiumwere the onlymetals that form insoluble sulphates of themetals tested. While this represents a novel biogeo-chemical cycle, although perhaps of minor importancein the system tested, it may also indicate the involvementof an additional mechanism of phosphate solubilizationbesides oxalic acid production, since only small amountsof oxalate were detected in concentrated extracts of thecrystals. However, a possible role for oxalate should notbe completely discounted since total oxalate was notmeasured in the system. The production of insolublecalcium oxalate is a common phenomenon in manyfungi (Gharieb, Sayer & Gadd 1998, Gadd 1999,

Fig. 6. Scanning electron micrographs of crystals purified from agar under colonies of Rhizoctonia solani growing on agar

amended with (a–c) 5 mM Ca3(PO4)2, (d) 0.1% (w/v) SrHPO4. Bars=(a–c) 100 mm and (d) 10 mm.


Gharieb & Gadd 1999, Ramsay, Sayer & Gadd 1999,Gharieb 2000), while Yang et al. (1993) reported theproduction of calcium oxalate by R. solani in vitro andon the surface of infected hypocotyls of Brassica rapa,B. napa and Camelina sativa. However, in this study,insoluble crystals were not formed when R. solaniwas grown on zinc phosphate-, cobalt phosphate- ormanganese phosphate-amended media despite solu-bilization occurring. This also indicates that there areadditional mechanisms of solubilization occurringbesides oxalate production such as acidolysis and/orthe production of organic acids that form soluble com-plexes. Nutrition can also be an important factor inmetal solubilization and immobilization (Dixon-Hardyet al. 1998, Gharieb 2000) with, for example, citric acidproduction by P. bilaii being stimulated under carbon-limiting conditions (Cunningham & Kuiack 1992).Changes in the amount of carbon source available toR. solani, as well as physico-chemical changes in themedium, may therefore alter the quantity and type ofacid produced.

It was clear that the presence of an insoluble metalcompound was not necessary for acidification of themediumbyR. solani as the decrease in pHof themediumunder growing colonies of R. solani was similar in the

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5 10 15 20 25 30 35 40 45 50 55 60

(a) 10

8

6

4

2

0

Cou

nts

per

seco

nd

10 20 30 40 50 60

(b)

2θ°

0.8

0.7

0.6

0.5

0.4

0.3

0.2

0.1

0

Cou

nts

per

seco

nd

5 10 15 20 25 30 35 40 45 50 55 60

(c)80

70

60

50

40

30

20

10

0

Cou

nts

per

seco

nd

10 20 30 40 50 60

(d)

2θ°

Fig. 8. Typical powder X-ray diffraction patterns for crystals extracted from agar after growth of Rhizoctonia solani on(a) calcium phosphate-amended medium and (c) strontium phosphate-amended medium. (b) and (d) are powder X-ray

diffraction patterns for CaSO4 and SrSO4, respectively. Typical spectra are shown from one of several determinations.

Fig. 7. Typical spectra obtained by energy dispersive X-raymicro-analysis of purified crystals obtained under colonies ofRhizoctonia solani grown on agar amended with (a) 5 mM

Ca3(PO4)2 and (b) 0.1% (w/v) SrHPO4.


presence andabsence of themetal phosphates.However,although Aspergillus niger produces organic acids asa result of normal metabolism (Burgstaller & Schinner1993, Sayer & Gadd 1997), their production may bemarkedly affected by the presence of insoluble metalcompounds (Gadd & Sayer 2000). Citric acid pro-duction by Penicillium simplicissimum was enhanced byadsorption of zinc oxide by themycelium, this substanceprobably acting as a buffering agent (Franz, Burgstaller& Schinner 1991). In this study, we found a strongnegative correlation between the final pH of the mediaand the rate of growth and biomass dry weight of R.solani grown on calcium phosphate-, manganese phos-phate- and strontium hydrogen phosphate-amendedmedium. Growth of R. solani in the presence of zincphosphate and cobalt phosphate did not lead to such amarked reduction in the final pH of the media as theother metal phosphates. This was possibly due to inhi-bition of the fungus by these compounds and/or releasedmetal species resulting in a decrease in growth, biomassand acid production.

The initial pH of the medium did not influence thegrowth rate of R. solani except on control and zincphosphate-amended media. It is known that the passive(and active) uptake of metal cations is influenced byexternal pH and adsorption of many cationic speciesdecreases with external pH as protons compete morestrongly with metal ions for sorbing sites (Morley &Gadd 1995, Morley et al. 1996). In the absence of metalphosphates, R. solani may be more susceptible tochanges in pH due to a reduction in medium bufferingcapacity. The amount of R. solani biomass producedwhen grown on cobalt phosphate-amended mediumincreased with the initial pH of the medium and this wasnot observed for any of the othermetal phosphates. Thissuggests that the pH of the medium is altering cobaltspeciation and/or reducing cobalt uptake byR. solani, toan extent which would otherwise have an inhibitoryeffect on growth. Previous studies on the effects of pHon the availability of phosphate have shown contrastingresults (Barrow 1984, Whitelaw 2000). However, pHeffects on the solubility and speciation of metals are welldocumented (Gadd & Griffiths 1978, Morley & Gadd1995, Gadd 1999, 2001, Gadd& Sayer 2000). In soil, themobile concentrations of metals such as Zn, Ni and Cdwere increased two-fold by a unit decrease in pH (Giller,Witter & McGrath 1998).

Our work also shows that the metal component of thephosphate may influence the availability of the phos-phate, as biomass and growth rate varied dependingupon themetal phosphate present, although toxic effectsmay also contribute to this observation. In liquid cul-tures, the initial pHof themedium can affect the amountof phosphate solubilization that occurs with an opti-mum pH range for growth resulting in higher levels ofsolubilization (Ahmed & Robson 1968). Burgstaller &Schinner (1993) found that the production of organicacids by A. niger was also influenced by the pH of themedium in which it is grown; citric, gluconic and oxalicT

able3.ResidualmetalcontentofRhizoctonia

solanimycelium

after

7dgrowth

onmedium

unamended

withmetalphosphates(control)andamended

withmetalphosphates(5

mMCa3(PO

4)2,

5m

MCo3(PO

4)2. 8H

2O,5m

MMn2(PO

4)2. 3H

2O,5m

MZn3(PO

4)2. 2H

2O,0.1%

SrH

PO

4).Coloniesweredigestedin

concentratedHNO

3andthemetalioncontentwasanalysedwithrespectto

thecalcium,

cobalt,manganese,strontium

andzincstandard

solutions.Resultsare

meansofthreereplicates¡

S.E.M.

InitialpH

of

medium/

temperature

(xC)

Metalcontent,nmol(m

gdry

wt)x

1

Ca

Co

Mn

Sr

Zn

Control

Amended

Control

Amended

Control

Amended

Control

Amended

Control

Amended

pH

4.6¡

0.0

(5.8¡0.0)

(5.7¡0.0)

(5.3¡

0.0)

(5.5¡0.0)

(5.5¡0.0)

15

0¡0

4.01¡0.28

0.12¡0.01

6.15¡1.86

0¡0

7.85¡

1.11

0.72¡

0.02

1.49¡0.12

0.14¡0.02

7.14¡4.98

25

0.29¡0.02

4.01¡0.28

0.08¡0.01

5.56¡0.32

0.05¡0.01

11.85¡

0.25

0.21¡

0.03

1.47¡0.19

0.09¡0.01

3.09¡1.57

30

0.23¡0.13

3.29¡0.16

0.06¡0.002

5.45¡0.76

0.4¡0.01

4.42¡

2.26

0.20¡

0.03

2.01¡0.18

0.08¡0.003

2.00¡0.43

pH

6.1¡

0.04

15

0.34¡0.17

3.54¡0.45

0.07¡0.04

5.01¡1.46

0.02¡0.02

8.81¡

1.42

0.27¡

0.05

1.26¡0.07

0.09¡0.04

2.03¡0.37

25

0.61¡0.01

2.87¡0.18

0.05¡0.05

3.13¡0.15

0.08¡0.01

4.98¡

0.27

0.29¡

0.02

1.95¡0.07

0.16¡0.01

3.66¡0.59

30

0.46¡0.23

3.55¡0.58

0.1¡0.02

7.30¡0.06

0.05¡0.01

3.75¡

0.18

0.27¡

0.02

1.67¡0.05

0.12¡0.02

2.38¡0.36

pH

7.2¡

0.04

15

0.29¡0.15

6.02¡0.24

0.08¡0.01

3.15¡0.76

0¡0

6.74¡

1.12

0.26¡

0.03

1.78¡0.06

0.13¡0.01

4.40¡1.06

25

0.35¡0.01

3.88¡0.57

0.1¡0.01

4.78¡0.96

0.06¡0.01

7.59¡

1.09

0.17¡

0.02

1.62¡0.06

0.11¡0.02

4.94¡1.55

30

0.43¡0.02

4.16¡0.3

0.1¡0.01

4.06¡0.24

0.04¡0.01

7.22¡

1.62

0.21¡

0.01

1.89¡0.04

0.1¡0.01

2.33¡0.72


acids were produced in that order with an increase inmedium pH.

The amount of metal taken up into the mycelium waslower when grown on strontium hydrogen phosphate-amended medium than on the other metal phosphates,which may be due to greater immobilization of releasedstrontium in the agar. The biomass dry weight andgrowth rate of R. solani when grown on strontiumhydrogen phosphate-amended medium was almostas great as when grown on calcium phosphate- andmanganese phosphate-amended media indicating themetal phosphatewas readily solubilized but themajorityof the strontium remained in the agar, where some wasassociated with sulphate. Strontium has no apparentbiological function (Huh et al. 1991, Anderson &Appanna 1994) although it is chemically similar to cal-cium and tends to accumulate in areas rich in calcium(Anderson&Appanna 1994). In the absence of calcium,strontium can be substituted and this can be of signifi-cance if the strontium is in the form of radioactive90Sr, which is produced during nuclear fission (Paulus,Komarneni &Roy 1992). Strontium-containing oxalatecrystals have also been reported by Connolly, Shortle &Jellison (1999) where the majority of the crystals werefound inside themycelial cord of thewood-decay fungusResinicium bicolor.

This study has demonstrated the ability of R. solanito solubilize insoluble calcium phosphate, cobalt phos-phate,manganese phosphate, strontiumhydrogenphos-phate and zinc phosphate, and the marked influence ofcertain physico-chemical factors on this phenomenon.The ability of R. solani to solubilize calcium (and other)phosphate minerals is an important attribute as thisinfluences nutrient cycling and may enable the break-down of fertilizers in soils while the fungus is growingsaprophytically.Wealso provide evidence for secondarymineral formation by those metals forming insolublesulphates (calcium and strontium).While this representsa novel fungal-mediated biogeochemical cycle, the sig-nificance of this process in the environment remains tobe fully assessed. Other questions are also raised re-garding the possible mechanisms of solubilization andcrystallization, particularly the relationship betweenproton- and ligand-mediated dissolution. Fungi mayexhibit a spectrum of mechanisms by which insolublemetal compounds may be dissolved, and such mechan-isms may exhibit species specificity.

ACKNOWLEDGEMENTS

F.A.D., K.R. and G.M.G. gratefully acknowledge financial support

from the Biotechnology and Biological Sciences Research Council

(94/MAF12243): G.M.G. also gratefully acknowledges research sup-

port from British Nuclear Fuels plc. K.R. further acknowledges

support from the Scottish Executive Environment and Rural Affairs

Department. Martin Kierans (University of Dundee Centre for High

Resolution Imaging and Processing) is thanked for assistance with

scanning electron microscopy, Ewan Starke for other technical as-

sistance and John Barnes (Department of Chemistry, University of

Dundee) is thanked for assistance with powderX-ray diffraction of the

crystals.

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Corresponding Editor: N. P. Money


solubilization of metal phosphates by rhizoctonia solani

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