Redalyc.Efects of Metal Ions (Cd2+, Cu2+, Zn2+) on the Growth And

Interciencia ISSN: 0378-1844 [email protected] Asociación Interciencia Venezuela

Machuca, Ángela; Navias, David; Milagres, Adriane M. F.; Chávez, Daniel; Guillén, Yudith Efects of metal ions (Cd2+, Cu2+, Zn2+) on the growth and chelating-compound production of three ectomycorrhizal fungi Interciencia, vol. 39, núm. 4, abril, 2014, pp. 221-227 Asociación Interciencia Caracas, Venezuela

Available in: http://www.redalyc.org/articulo.oa?id=33930412002

How to cite Complete issue Scientific Information System More information about this article Network of Scientific Journals from Latin America, the Caribbean, Spain and Portugal Journal's homepage in redalyc.org Non-profit academic project, developed under the open access initiative Effects of metal ions (Cd2+, Cu2+, Zn2+) on the growth and chelating-compound production of three ectomycorrhizal fungi Ángela Machuca, David Navias, Adriane M. F. Milagres, Daniel Chávez and Yudith Guillén

SUMMARY

Several studies have shown that ecotypes of ectomycorrhizal inter- and intraspecific variation in the fungal responses at low fungi from metal-contaminated sites present a high metal toler- and high metal concentrations. Some ecotypes of Rhizopogon ro- ance under in vitro conditions, but it is not clear whether fungi seolus and Suillus luteus were the most tolerant at 1mM Cu and from non-contaminated sites can also develop ecotypes with high 10mM Zn. S. luteus SRS showed tolerance to the three metals. metal tolerance. Tolerance to Cd2+, Cu2+ and Zn2+ ions, and pro- The addition of Cu and Cd stimulated CAS-detected metal-che- duction of chelating compounds as a detoxification mechanism lating compounds and dark pigmentation production in all iso- were evaluated in ectomycorrhizal fungi collected from three un- lates. In the presence of Cd the lowest pH values of the culture contaminated sites (SIS, SRS and ESC). The fungi were grown in media were detected. Hydroxamates and catecholates were de- solid medium with 5 and 20μmol·l‑1 Cd, 0.1 and 1mmol·l‑1 Cu, and tected only in some isolates, and the catecholates were stimulat- 1 and 10mmol·l‑1 Zn, and the tolerance index was determined. ed by Cd in S. luteus and S. bellinii. Among fungi collected from The metal-chelating compounds were determined by Chrome uncontaminated sites it was possible to found ecotypes tolerant Azurol S (CAS) assay, and the chemical nature (hydroxamate or to high metal concentrations and also producers of siderophore- catecholate) of the compounds was analyzed. There was a clear type chelating compounds.

tudies have demonstrated dez et al., 2000a; Jentschke and Goldbold, portant that the screening of native ecotypes the importance of ectomy- 2000; Urban, 2011). Metal-tolerant ecotypes of ectomycorrhizal fungi be site/host specif- corrhizal fungi for the may play a crucial role in the establishment ic, ecologically adapted to particular tree plant species with which they form symbi- of mycorrhizal tree seedlings in contaminat- species and edaphoclimatic conditions of the otic associations, since under certain condi- ed soils for the restoration or remediation of region where the mycorrhizal seedlings will tions these fungi may develop ecotypes industrial and metalliferous sites (Godbold be introduced (Ray et al., 2005; Carrillo- with tolerance or resistance to high metal et al., 1998; Schützendübel and Polle, 2002; González and González-Chávez, 2012). concentrations and thus can protect their Urban 2011). Nevertheless, when the selec- The mechanisms of metal host plants when they grow in metal con- tion of fungal ecotypes is done for the tolerance described in fungi include extra- taminated soils (Hartley et al., 1997; Blau- aforementioned applications, it is very im- cellular chelation through organic com-

Keywords / Catecholates / Fungi / Metals / Radial Growth Rate / Rhizopogon roseolus / Suillus bellinii / Suillus luteus / Received: 01/29/2014. Modified: 03/31/2014. Accepted: 03/04/2014.

Ángela Machuca. Biochemist, Universidad de Concepción (UdeC), Chile. M.S. and Ph.D. in Biochemistry, Universidade Estadual de Campinas (Unicamp), Brasil. Professor, Universidad de Concepción, Chile. Address: Labo- ratorio de Biotecnología de Hongos, Departamento de Ciencias y Tecnología Vegetal, Escuela de Ciencias y Tecnología, UdeC, Campus Los Ángeles, Chile. e-mail: [email protected] David Navias. Forestry Excecution Engineer, UdeC, Chile. Functionary, Servicio Agrícola Ga- nadero (SAG), Chile. e-mail: [email protected] Adriane M. F. Milagres. Food Engineer, Universidade de Viçosa, Brazil, Doctor en Ciencia con mención en Bioquímica, Unicamp, Brasil. Professor, Universidade de São Paulo, Brasil. e-mail: [email protected] Daniel Chávez. Forestry Excecution Engineer, UdeC, Chile. Forestry Engineer, Universidad Arturo Prat, Chile. Doctoral Candidate, UdeC, Chile. e-mail: [email protected] Yudith Guillén. Forestry Engineer and M.S. in Forest Products Technology, Universidad de Los Andes, Venezuela. Doctor in Forestry Sciences, UEDC, Chile. Professor, Universidad Nacional Experimental de Guayana, Ve nezuela. e-mail: [email protected]

APR 2014, VOL. 39 Nº 4 0378-1844/14/04/221-07 $ 3.00/0 221 pounds released by the fungal hyphae, such Table I as organic acids and siderophores. These Location and soil characteristics of the ectomycorrhizal metal-chelating compounds play a very im- fungi collection sites portant role in metal acquisition by plants, Characteristics fungi and bacteria from their environments Origin Sites Metal content* (mg·kg‑1 dry soil) Isolates (Guerinot, 1994; Winkelmann, 2002) and their participation in processes of metal de- San Isidro (SIS) Rhizopogon roseolus SIS Bio Bio Province Sandy loam soil, pH 5.9 Suillus luteus SIS toxification has been also demonstrated 36º55’0”S; 72º0’21”O Fe: 9.4, Zn: 0.1, Cu: 0.2 Suillus bellinii SIS (White et al., 1997; Jentschke and Godbold, Santa Rosa (SRS) Rhizopogon roseolus SRS 2000; Machuca, 2011). Organic acids such Bio Bio Province Sandy soil, pH 6.1 Suillus luteus SRS as oxalic and citric acids are excreted by 37º14’19”S; 72º24’46”O Fe: 4.8, Zn: 0.2, Cu: 0.1 Suillus bellinii SRS mycorrhizal fungi or plant roots into the Escuadron (ESC)** rhizosphere, where they may both mobilize Concepcion Province Clay soil, pH 4.1 Rhizopogon roseolus ESC metal ions from insoluble sources or immo- 73º09’12.76”S; 36º58’55.21”O Fe: 27.2, Zn: 1.1, Cu: 4.1 Suillus luteus ESC bilize them through precipitation (Meharg, 2003; Bellion et al., 2006). Siderophores are *Metal content as total concentration. **During the period when the fruiting bodies were collect- highly specific ferric iron (Fe3+) chelators ed, the S. bellinii species was not found at the ESC site. produced by many fungi and bacteria in re- sponse to Fe deficiency, but they are also ecotypes to be applied in future investiga- 1987; Santiago-Martínez et al., 2003). capable of forming stable complexes with tions on the mycorrhization of forest spe- Each fungal isolate was inoculated in trip- many other metal ions in addition to Fe. cies, assessing the effect of the fungi when licate, with and without metal, and the These compounds can be classed into two the plants are introduced in disturbed soils, means of the values reported. The toler- major groups according to their chemical poor in essential nutrients and/or contami- ance of each isolate was expressed as the structure: hydroxamates and catecholates nated with metals. Tolerance Index (% TI) calculated com- (Hider, 1984; Renshaw et al., 2002). Al- pared to the growth of control culture though siderophores have been associated Material and Methods (without metal). Some morphological with metal detoxification and tolerance in changes in the fungal colonies were visu- bacteria and fungi (Rogers et al., 2001; Il- Fungal isolates and collection sites ally evaluated during the incubation peri- lmer and Buttinger, 2006; Braud et al., od. At the end of the incubation period the 2010; Rajkumar et al. 2010), production of Fruiting bodies of ecto- changes in pH were evaluated in the solid siderophores by ectomycorrhizal fungi mycorrhizal fungi Rhizopogon roseolus Fr., media, with and without metal ions, using growing in the presence of metal ions other Suillus bellinii (Inz.) Kuntze and Suillus lu- a surface pH electrode. than Fe and their possible relation with the teus L. Gray associated with young Pinus tolerance has not been described in the lit- radiata D. Don plantations were collected Detection of metal-chelating compounds in erature to date. from three forest sites located in the central- fungal extracts Several studies have southern region of Chile, where the soils shown that fungal ecotypes from contami- had different chemical and physical charac- At the end of the incuba- nated sites present a higher metal tolerance teristics (Table I). Pure cultures of different tion period (40 days), the content of each than isolates from non-contaminated sites, isolates were obtained from carpophores dish, including mycelium plus MMN me- and in some cases the tolerance displayed and identified at the Fungi Biotechnology dium, with and without metals (Cd, Cu or by the ecotypes is metal-specific to the site Laboratory, Universidad de Concepcion, Zn), was extracted with 20ml of deionised (Adriaensen et al., 2005; Colpaert, 2008; Chile. The stock of fungal cultures was cold water for 20min under agitation. The Colpaert et al., 2011; Urban, 2011). Howev- maintained and cultivated regularly on extracts were filtered through a 0.45μm er, in many cases metal-sensitive ecotypes plates containing solid modified Melin- Millipore membrane and the presence of have also been isolated from contaminated Norkrans(MMN) medium with pH adjusted metal-chelating compounds in the filtrates sites and it is not clear whether fungi found to 5.8 (Marx, 1969) and incubated in the was determined. To do this, 1ml of the fil- in uncontaminated sites can also present dark at 24 ±1°C for 25-30 days. tered fungal extract was mixed with 1ml tolerant ecotypes under in vitro conditions. of Chrome Azurol S (CAS; Schwyn and The aim of this work was to evaluate the Effects of metal ions on fungal growth Neilands 1987). This assay is based on the effect of cadmium (Cd2+), copper (Cu2+) and ability of siderophore-type metal-chelating zinc (Zn2+) ions on the rate of growth and Petri dishes (90mm di- compounds to bind and remove Fe3+ pres- metal-chelating compound production in ameter), containing 20ml of MMN medi- ent in the CAS reagent, producing a solid medium by ecotypes of ectomycorrhi- um, pH adjusted to 5.8 before autoclaving, change in the reagent color from blue to zal fungi collected from different uncon- were prepared with and without the addi- orange or pink. After 1h of incubation at taminated soils. The ectomycorrhizal fungi tion of Cd2+, Cu2+ and Zn2+ions, which room temperature, the absorbance of the Rhizopogon roseolus, Suillus luteus and were added as sulphate salts to the medi- mixture at 630nm (A630) was measured. A Suillus bellinii used in this study were iso- um to give final concentrations of 5 and reference was prepared with extracts ob- lated from fruiting bodies collected from 20μmol·l‑1 Cd; 0.1 and 1mmol·l‑1 Cu; and 1 tained from dishes containing non-inocu- Pinus radiata plantations in different sites and 10mmol·l‑1 Zn. The dishes were inoculated MMN solid medium (with and with- in the central-southern region of Chile. lated in the centre with a mycelium-agar out metals). The percent metal-chelating These fungal species were chosen as they disk (5mm diameter) obtained from the compound units in the extracts were cal- are found with high frequency in young stock plates and incubated at 24 ±1°C in culated by subtracting the sample (As) pine forests and primary plantations as pio- the dark. The growth of the colonies was from the reference (Ar), so that %A630 neer species. The selection of metal-tolerant evaluated at regular intervals for 40 days metal-chelating compound units= (Ar-As/ ecotypes and efficient producers of metal- and the results were expressed as average Ar)×100 (Payne, 1994; Machuca and Mila- chelating compounds will also allow these radial growth rate in mm/day (Harnett, gres, 2003).

222 APR 2014, VOL. 39 Nº 4 Detection of hydroxamates and catecholates Table II Radial growth rate (mm/day) of ectomycorrhizal fungi The presence of the hy- growing in solid MMN medium at two metal concentrations droxamate and catecholate ligands in the Cd2+ (mmol·L‑1) Cu2+ (mmol·L‑1) Zn2+ (mmol·L‑1) filtrate extracts was determined by the col- Control 5 20 0.1 1 1 10 orimetric Csáky and Arnow assays, respec- R. roseolus tively. The Csáky assay (Csáky, 1948) con- SIS 0.80 0.46a* (58) 0.15bc*(19) 0.48b*(60) 0.00d* 0.70a (87) 0.00b* sisted of the digestion of samples contain- SRS 0.79 0.39a* (49) 0.12c* (15) 0.37c*(47) 0.00d* 0.58a (73) 0.00b* ing hydroxamic acids in sulphuric acid to ESC 0.76 0.23b* (30) 0.15bc*(20) 0.64a (84) 0.39c*(51) 0.69a (91) 0.00b* detect bound hydroxylamine. A pink color S. luteus indicates a positive reaction for the pres- SIS 0.59 0.26a*(44) 0.12b*(20) 0.59b(100) 0.00e* 0.72b(122) 0.75a*(127) ence of hydroxamate structures and the ab- SRS 0.45 0.25a*(56) 0.23a*(51) 0.48c(107) 0.40c*(89) 0.61c*(136) 0.63c*(140) sorbance of the solution was measured at ESC 1.59 0.20a*(13) 0.21a*(13) 0.71a*(45) 0.31d*(19) 0.85a*(53) 0.00d* 526nm. In the Arnow assay (Arnow, 1937), S. bellinii catecholate structures give a yellow color SIS 0.38 0.13a*(34) 0.07b*(18) 0.52a*(137) 0.00c* 0.49b(129) 0.00c* when they react with nitrite-molybdate in SRS 0.58 0.04b*(7) 0.04b*(7) 0.36b*(62) 0.00c* 0.63a (109) 0.00c* acid medium and change to an intense or- Values of growth rate followed by (*) are significantly different (p< 0.05) compared to the con- ange-red when the medium is made strong- trol (without metal) for each isolate. ly basic. The solution has a maximum ab- a‑eAt different concentrations of each metal, for isolates from each species, values followed by the same letter are not significantly different (p< 0.05). Values in parentheses corresponding to sorbance at 510nm. Hydroxylamine hydro- Tolerance Index (%) calculated compared to the control. Values are the means of three replicates chloride and 2,3-dihydroxybenzoic acid ± standard errors. (2,3-DHBA) were used as standards for the calibration curves of the Csáky and Arnow assays, respectively, and results expressed rate of some isolates were significantly re- um, when grown in the absence of metal as µmoles·l‑1. duced, while in others the growth was not ions. In the presence of Cd the pH values affected and in S. bellinii SIS the growth were lower than those obtained with Zn and Statistical analysis was significantly stimulated (TI>100%). Cu, attaining values in the range 2.9-3.8, in- However, when the Cu was increased to dependently of the species/isolate and Cd A one-way ANOVA 1mmol·l‑1, the growth of most isolates was concentration. No pH changes were detected (p<0.05) was applied to determine the dif- completely inhibited and only R. roseolus compared to the controls in the presence of ferences between growth of the control ESC and S. luteus SRS and ESC showed Cu or Zn, except for S. luteus ESC, which (without metal) and metal treatment for each tolerance. In no case was the growth of S. decreased the pH to 3 in the presence of isolate. To compare the growth of the iso- luteus SRS affected, showing at 1mmol·l‑1 both metal ions (Table III). lates from the same species at two different the highest TI (89%) among all the isolates metal concentrations, a factorial design was (Table II). Unlike Cd and Cu, at the lowest Effect of metal ions on metal-chelating used (ANOVA, p<0.05) and the statistical Zn concentration (1mmol·l‑1) all the isolates compound production differences between these treatments were displayed tolerance to the metal (TI>70%), identified using Tukey`s multiple comparison except S. luteus ESC (TI 53%). However, All extracts from fungal test. All statistical analyses were performed when the concentration increased to cultures displayed a positive CAS reaction, with three replicates, using STATISTICA 10mmol·l‑1 the radial growth rate of most of but the percentages of metal-chelating com- software (v. 6.0). the isolates was completely inhibited and pound units were different depending on only S. luteus SIS and SRS showed toler- species/isolates and type and concentration Results ance (TI>00%). The radial growth rate of of metal ion. The color changes produced these isolates was significantly higher in the during the CAS assay were also different Effects of metal ions on fungal growth presence of 1 and 10mmol·l‑1 Zn than in its among species, and whereas the extracts of absence (Table II). R. roseolus produced a change from blue to When the isolates of ecto- The metal ions caused purple, with the S. luteus and S. bellinii ex- mycorrhizal fungi grew in solid MMN me- modifications in some morphological char- tracts these changes were from blue to or- dium without metal ions (control cultures) acteristics of the mycelia of all the isolates ange-reddish. In the absence of metal, the different radial growth rates were observed. (data not shown). The isolates that grew at presence of the metal-chelating compounds S. luteus ESC showed the highest growth the highest Cu and Zn concentrations dis- was detected in all the extracts, with the rate, followed by the three isolates of R. ro- played abundant aerial mycelium and thick highest percentage of metal-chelating com- seolus (Table II). In the presence of metal mycelial cord formation. In addition, a clear pounds (highest CAS reaction) produced by ions differential responses in the growth influence of the metal ions on the dark pig- the SIS isolates of R. roseolus and S. belli- rate and tolerance index (TI) were observed, mentation production was also observed at nii (Figures 1a and c). In the presence of depending on the metal ion and its concen- the highest Cd and Cu concentrations in all metal ions R. roseolus and S. luteus dis- tration, and on the species/isolate (Table II). the isolates. Nevertheless, a very marked played different CAS reactions, while S. Two concentrations of Cd significantly in- difference was observed between the spe- bellinii showed a reduction in the metal-che- hibited the radial growth rate of all isolates cies R. roseolus, which produced pigmented lating compounds (Figure 1c).The presence compared to the controls, mainly at mycelium, and S. luteus and S. bellinii, of Cu, but not Cd or Zn, stimulated the pro- 20μmol·l‑1, and the TI were lower than those which secreted the pigments to the medium, duction of the metal-chelating compounds in obtained with Cu and Zn. S. luteus ESC producing an intense dark coloration on the R. roseolus SIS and SRS, and in the ESC and S. bellinii SRS were the most sensitive agar, more so in the presence of Cd. All isolate the three metal ions produced a re- to Cd, whereas S. luteus SRS displayed the fungal isolates decreased the pH of the me- duction in these compounds (Figure 1a). highest TI at 20μmol·l‑1(Table II). At a low dium to a range from 4 to 4.7 compared Similarly, the production of compounds by Cu concentration (0.1mmol·l‑1) the growth with the initial pH 5.8 of the MMN medi- S. luteus ESC also was reduced by metal

APR 2014, VOL. 39 Nº 4 223 Table III production compared to the control. In addi- Changes in the pH of solid MMN medium during the growth tion, the catecholate production by R. roseo- of ectomycorrhizal fungi at two metal concentrations lus and S. luteus was also stimulated by Cd Control Cd2+ (mmol·L‑1) Cu2+ (mmol·L‑1) Zn2+ (mmol·L‑1) and Cu, respectively, but not by Zn. The hydroxamates were detected in R. roseolus in 0 5 20 0.1 1 1 10 the presence of Cu and Zn, and in the pres- R. roseolus SIS 4.0 ±0.6 3.4 ±0.1 3.6 ±0.2 4.2 ±0.0 n.d. 4.2 ±0.1 n.d. ence of the three metal ions in the case of S. R. roseolus SRS 4.3 ±0.2 3.8 ±0.0 3.4 ±0.1 4.2 ±0.1 n.d. 4.0 ±0.2 n.d. bellinii. However, in S. luteus grown in the ± ± ± ± ± ± R. roseolus ESC 4.2 0.4 3.4 0.0 3.5 0.1 4.8 0.1 4.1 0.1 4.1 0.1 n.d. presence of the three metal ions hydroxamate S. luteus SIS 4.0 ±0.1 3.2 ±0.1 3.4 ±0.0 4.5 ±0.1 n.d. 4.2 ±0.1 5.4 ±0.1 structures were not detected (Table IV). S. luteus SRS 4.0 ±0.2 3.2 ±0.1 3.1 ±0.0 4.6 ±0.2 4.0 ±0.1 4.0 ±0.1 5.3 ±0.0 S. luteus ESC 4.5 ±0.2 3.2 ±0.1 3.2 ±0.0 3.2 ±0.2 3.0 ±0.1 3.2 ±0.1 n.d. S. belliniiSIS 4.7 ±0.4 3.0 ±0.1 3.1 ±0.0 4.2 ±0.1 n.d 4.8 ±0.2 n.d. Discussion S. bellinii SRS 4.1 ±0.1 3.0 ±0.0 2.9 ±0.1 4.0 ±0.0 n.d. 3.9 ±0.1 n.d. Some studies have dem- Initial pH of solid MMN medium was adjusted to 5.8 before autoclaving. Values are the means onstrated that species of ectomycorrhizal of three replicates ± standard errors. fungi that grow on sites strongly contami- n.d.: not determined because these isolates were not grown under these conditions. nated with metal ions can exhibit high tolerance under laboratory controlled conditions ions, mainly by Cu, which was responsible ions, the catecholate concentrations detected in the absence of mycorrhizal association for a complete absence of CAS reaction. In by the Arnow assay were higher than hy- (Hartley et al.1997; Meharg, 2003; Colpaert the SIS and SRS isolates the addition of Cd droxamates detected by the Csáky assay. In et al., 2004, 2011). Nevertheless, it has not and Cu respectively, but not Zn, stimulated the absence of metal, only the S. luteus SRS always been possible to establish a direct re- the production of metal-chelating com- and S. bellinii extracts displayed the pres- lationship between the presence and concen- pounds (Figure 1b). Those isolates that grew ence of both type of structures(Table IV). tration of metal in the soils and the toler- at 1mmol·l‑1 Cu and 10mmol·l‑1 Zn presented The addition of metal ions stimulated both ance that fungal species collected from no CAS reaction. catecholate and hydroxamate production, but those sites can develop in vitro. This study The hydroxamate and cat- it was dependent on species/isolate and the demonstrated that the ectomycorrhizal spe- echolate structures were also determined in type and concentration of metal. High cate- cies R. roseolus, S. luteus and S. bellinii as- the fungal extracts that showed a positive cholate concentrations were detected in S. sociated with pine plantations from three CAS reaction. Independently of species/iso- bellinii in the presence of the three metal different uncontaminated sites, presented late and of the presence or absence of metal ions, but only Cd stimulated the catecholate metal tolerant isolates when cultivated in vi-

Figure 1. Metal-chelating compounds (%) siderophore-type detected by CAS assay in the extracts of cultures of ectomycorrhizal fungi isolates growing in solid MMN medium at different metal concentrations. SIS, SRS and ESC correspond to the different fungi collection sites (see Table I). Values are the means of three replicates ±standard errors.

Table IV Detection of catecholate and hydroxamate structures (mmol·l‑1) in extracts of ectomycorrhizal fungi growing in solid MMN medium at two metal concentrations Cd2+ (µmol·l‑1) Cu2+ (mmol·l‑1) Zn2+ (mmol·l‑1) Control 5 20 0.1 1 Catechol - Hydroxam Catechol - Hydroxam Catechol - Hydroxam Catechol - Hydroxam Catechol - Hydroxam R. roseolus SIS 0.0 0.0 12.3 ±0.1 0.0 3.6 ±0.1 0.0 0.0 5.3 ±0.1 0.0 5.8 ±0.2 R. roseolus SRS 0.0 0.0 0.0 0.0 4.5 ±0.0 0.0 0.0 8.9 ±0.2 0.0 4.3 ±0.2 R. roseolus ESC 3.7 ±0.1 0.0 7.5 ±0.2 0.0 19.6 ±0.3 0.0 0.0 5.6 ±0.0 0.0 6.5 ±1.0 S. luteus SIS 0.0 0.0 10.5 ±0.2 0.0 34.5 ±0.5 0.0 12.8 ±0.3 0.0 0.0 0.0 S. luteus SRS 26.7 ±0.4 4.0 ±0.1 46.7 ±2.2 0.0 71.2 ±3.3 0.0 30.0 ±1.2 0.0 0.0 0.0 S. luteus ESC 0.0 5.6 ±0.1 32.5 ±1.0 0.0 22.0 ±2.0 0.0 15.8 ±1.0 0.0 0.0 0.0 S. bellinii SIS 98.7 ±1.2 2.8 ±0.0 287.0 ±4.1 0.0 320.0 ±3.1 0.0 89.0 ±2.7 0.0 85.6 ±2.0 3.7 ±0.2 S. bellinii SRS 118.5 ±3.2 4.9 ±0.1 344.9 ±3.8 2.6 ±0.1 283.0 ±2.1 4.5 ±0.8 54.0 ±2.4 3.9 ±0.5 64.5 ±1.1 4.9 ±0.3 Catecholate were detected by Arnow test; hydroxamate were detected by Csáky test. Values are the means of three replicates ± standard errors.

224 APR 2014, VOL. 39 Nº 4 tro in solid MMN medium with Cd2+, Cu2+ Cd than Paxillus involutus when grown in It has been suggested that organic acids and Zn2+ ions. The effect of each metal ion in vitro conditions, but the contrary was ob- such as oxalic and citric acid can alleviate on the growth rate was evaluated at a low served for Ni. On the other hand, an intra- metal toxicity through immobilization, concentration and at another ten times high- specific variation has been also observed for forming insoluble metal-complexes outside er, except Cd, a non-essential metal, which Al among P. tinctorius strains (Egerton- the cells of several organisms (Bellion et al., was increased only four times due to its Warburton and Griffin, 1995) and for Zn 2006; Machuca, 2011). In a previous study, high toxic potential on living organisms, among S. luteus strains (Blaudez et al., isolates of S. luteus, R. luteolus and Sclero- even at very low concentrations (Trevors et 2000a). In the present study, the three R. derma verrucosum produced several organic al. 1986; Blaudez et al., 2000b). This was roseolus isolates displayed a similar sensi- acids (oxalic, citric, succinic and malonic) evident when, in spite of the low concentra- tivity at high concentrations of metal ions, and metal siderophore type chelating-com- tions of Cd used the TI values were lower except the ESC isolate, which was the only pounds when grown in liquid MMN in the than those obtained with Cu and Zn with one that grew at 1mmol·l‑1 Cu (51% TI). For absence and presence of low concentrations all the isolates (Table II). S. luteus, the SIS and SRS isolates were the of Fe (35μmol·l‑1; Machuca et al., 2007). On Several studies have dem- most tolerant at two Zn concentrations the other hand, depending on their concen- onstrated that fungal ecotypes from contam- (>100% TI) and SRS showed the highest tration, oxalic and citric acids can react pos- inated sites have a higher metal tolerance tolerance to Cd (51% TI) and Cu (89% TI) itively with CAS in the same form as sider- (TI) than isolates from non-contaminated as well; however, in this species the isolate ophore-type iron-chelating compounds do sites, and in some cases the tolerance dis- ESC was also found to be most sensitive to (Oberegger et al., 2001). Thus, the produc- played by the ecotypes is metal-specific to Cd, Cu and Zn, even though at the site of tion of organic acids by ectomycorrhizal the site. For instance, ecotypes of ectomy- origin of this isolate the highest Cu and Zn fungi could be contributing to the acidifica- corrhizal fungi from Cu-contaminated sites concentrations were detected (Table I). By tion of the media as well as to the positive showed tolerance in vitro to Cu but not to contrast, for R. roseolus ESC there seemed CAS reaction. Zn (Colpaert et al., 2004; Adriaensen et al., to be a relationship among the tolerance and Recently, the role of sider- 2005; Colpaert, 2008; Urban, 2011). On the the level of metal at its site of origin, at ophores in the increase of bacterial metal other hand, the metal tolerance that a fungal least for Cu. tolerance has been highlighted, particularly species can display is dependent on the spe- Although the literature in Pseudomona ssp. (Rajkumar et al., 2010; cies and isolate or ecotype, as well as on mentions that tolerance to one metal does Schalk et al., 2011; Cao et al., 2012). Brau- the metal ion involved and its concentration not necessarily imply tolerance to other det al. (2010) demonstrated that in P. aeru- (Gadd, 1993; Galli et al., 1994).The soils of metals (Adriaensen et al., 2005; Ray et al., ginosa the presence of siderophores reduced the forest sites from where the R. roseolus, 2005; Colpaert, 2008), the present results the toxicity of metals (Cu, Zn, Al and Pb S. luteus and S. bellinii isolates were col- show that S. luteus SRS from an uncontam- among others) by extracellular chelation in lected presented low and similar levels of inated site is remarkable among the various media supplemented or not with Fe. More- metals, except for the ESC site, which isolates studied because it showed a multiple over, Cu at 100μmol·l‑1markedly increased showed a slightly higher level of Fe, Zn and metal tolerance for Cd, Cu and Zn in solid the siderophore production, but it was de- Cu (Cd contents were not determined) than medium. This isolate displayed only a slight pendent on the type of siderophore. To date the SIS and SRS sites, in addition to a tex- or no growth inhibition at the higher metal the stimulation or repression of the produc- ture and acidic pH different from the other concentrations assayed, mainly in the pres- tion of siderophores by many metals other sites (Table I).Although the collection sites ence of 10mmol·l‑1 Zn. This is an important than Fe has been extensively described for chosen for this study were not contaminated characteristic if the fungal isolate is re- bacteria, but this issue has been scarcely with metal ions, some isolates were capable quired to be applied in association with studied in fungi such as ectomycorrhizal of growing and tolerating in vitro the con- their host plant for reforestation of metal- fungi (Illmer and Buttinger, 2006; Sinha centrations of metals assayed, which were contaminated sites, since in most cases a and Mukherjee, 2008; Schalk et al., 2011). higher than those at their sites of origin. contaminated soil contains toxic levels of In the present study, all the isolates of ecto- The R. roseolus, S. luteus and S. bellinii metal ion mixtures. Among the fungal spe- mycorrhizal fungi showed different levels of isolates displayed a different TI under the cies studied herein, S. luteus has been the iron-chelating compounds as detected by the conditions assayed, depending on the type most frequently reported in the literature CAS assay, when grown in MMN solid me- and concentration of the metal ions. The tol- with regard to metal tolerance. Populations dium supplemented with 18μmol·l‑1 Fe, in erance was evaluated by measuring the ra- of this specie from metal contaminated eco- the absence or presence of Cd, Cu and Zn dial growth rate (mm/day) in solid medium, systems have been studied for adaptive tol- (Figure 1). Differences in the colors of posi- which is considered a good parameter for erance to Cu, Zn and Cd, as well as in re- tive CAS reaction were observed among R. assessing the ability of fungal mycelia to gard to some mechanisms and genes impli- roseolus and two Suillus species, which explore or colonize soils. Among the ecto- cated in their tolerance and the protective could be related to chelating compounds mycorrhizal fungi there was a clear inter- effect on seedlings exposed to metal (Col- with different chemical structures. In the and intra-specific variation in the responses paert et al., 2004, 2011; Muller et al., 2007; presence of metals the production of these at two concentrations of metals. According Krznaric et al., 2009; Ruytinx et al., 2011). compounds was reduced, stimulated or not to the TI values found, S. luteus and S. bell- The changes observed in affected, depending on the species/isolate. inii were the most tolerant and sensitive the pH of the media and in the pigmenta- Zn reduced the production of chelating species, respectively, principally at high tion both of mycelia and media, mainly at compounds for the three species and a re- concentrations of the three ions. Several high metal concentrations, may be related to duction was also observed in R. roseolus in studies carried out in axenic cultures have different metal tolerance strategies devel- the presence of Cd, but not Cu, which in- demonstrated interspecific variations among oped by fungi (Gadd 1993; Jentschke and creased these compounds in the SIS and ectomycorrhizal fungi (Colpaert and Van Goldbold, 2000; Martino et al., 2000; Bel- SRS isolates. For S. luteus, Cd and Cu stim- Assche, 1987; Blaudez et al., 2000a; Ray et lion et al., 2006). The decrease in pH ob- ulated the production of these compounds in al., 2005). Blaudez et al. (2000a) observed served in all fungal cultures in the absence the SIS and SRS isolates, respectively. In that S. luteus, S. variegatus and Pisolithus or presence of metal ions, mainly with Cd, contrast, in S. bellinii the three metal ions tinctorius were more tolerant to Zn, Cu and is likely related to organic acid production. reduced the chelating compounds produc-

APR 2014, VOL. 39 Nº 4 225 tion. Although it was not possible to estab- ACKNOWLEDGEMENTS Gadd GM (1993) Interactions of fungi with toxic lish any relationship between the metal tol- metals. New Phytol. 124: 25-60. erance and chelating compound production This study was support- Galli U, Schüepp H, Brunold C (1994) Heavy for the different species/isolates, it is impor- ed by the DIUC project No.211.418.002- metal binding by mycorrhizal fungi. Physiol. Plant 92: 364-368. tant to note that the chelating compounds 1.0, University of Concepcion, Chile. Godbold DL, Jentschke G, Winter S, Marschner P were determined at the end of the incuba- (1998) Ectomycorrhizae and amelioration of tion period (40 days) and a peak of produc- REFERENCES metal stress in forest trees. Chemosphere 36: tion may have occurred before that time. 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Efecto de iones metálicos (Cd2+, Cu2+, Zn2+) en el crecimiento y producción de compuestos quelantes de metal en tres hongos ectomicorrícicos Ángela Machuca, David Navias, Adriane M. F. Milagres, Daniel Chávez y Yudith Guillén RESUMEN

Diversos estudios han demostrado que hongos aislados de si- ca en las respuestas a bajas y altas concentraciones de metales. tios contaminados con metales presentan alta tolerancia in vitro, Algunos ecotipos de Rhizopogon roseolus y Suillus luteus fueron pero no está claro si hongos provenientes de sitios no contami- los más tolerantes a 1mM Cu y 10mM Zn. S. luteus SRS presentó nados también pueden presentar ecotipos tolerantes. La toleran- tolerancia a los tres metales. La adición de Cu y Cd estimuló cia a Cd2+, Cu2+ y Zn2+ y la producción de compuestos quelantes la producción de compuestos quelantes detectados con CAS y la como posible mecanismo de destoxificación, fue evaluada en tres producción de pigmentos oscuros en todos los ecotipos. Los me- hongos ectomicorrícicos recolectados en tres sitios no contami- nores valores de pH de los medios de cultivo fueron detectados nados (SIS, SRS y ESC). Los hongos fueron cultivados en medio en presencia de Cd. Hidroxamatos y catecolatos fueron detecta- sólido con 5 y 20μmol·l‑1 Cd; 0,1 y 1mmol·l‑1 Cu; y 1 y 10mmol·l‑1 dos sólo en algunos ecotipos, y los catecolatos fueron estimula- Zn, y se determinó el índice de tolerancia. Los compuestos que- dos por Cd en S. luteus y S. bellinii. Entre los hongos recolecta- lantes fueron determinados con Chrome Azurol S (CAS), y su dos en sitios no contaminados fue posible encontrar ecotipos to- naturaleza química (hidroxamatos o catecolatos) fue analizada. lerantes a altas concentraciones de metal y además productores Entre los hongos existió una clara variación inter e intraespecífi- de compuestos quelantes del tipo sideróforos.

EfeIto de Íons metálicos (Cd2+, Cu2+, Zn2+) NO creScimento E produÇÃO de compOstos quelantes de metal eM trÊs FUngos ectomicorríZicos. Ángela Machuca, David Navias, Adriane M. F. Milagres, Daniel Chávez e Yudith Guillén RESUMO

Diversos estudos têm demonstrado que fungos isolados de traespecífica nas respostas em baixas e altas concentrações de locais contaminados com metais apresentam alta tolerância in metais. Alguns ecótipos de Rhizopogon roseolus e Suillus luteus vitro, mas não está claro si fungos provenientes de locais não foram os mais tolerantes a 1mM Cu e 10mM Zn. S. luteus SRS contaminados também podem apresentar ecótipos tolerantes. A apresentou tolerância aos três metais. A adição de Cu e Cd esti- tolerância a Cd2+, Cu2+ e Zn2+ e a produção de compostos mulou a produção de compostos quelantes detectados com CAS e quelantes como possível mecanismo de detoxificação, foi ava- a produção de pigmentos escuros em todos os ecótipos. Os me- liada em três fungos ectomicorrízicos recolhidos em três locais nores valores de pH dos meios de cultivo foram detectados em não contaminados (SIS, SRS e ESC). Os fungos foram cultivados presença de Cd. Hidroxamatos e catecolatos foram detectados em meio sólido com 5 e 20μmol·l-1 Cd; 0,1 e 1mmol·l-1 Cu; y somente em alguns ecótipos, e os catecolatos foram estimulados 1 y 10mmol·l-1 Zn, e se determinou o índice de tolerância. Os por Cd em S. luteus e S. bellinii. Entre os fungos recolhidos em compostos quelantes foram determinados com Cromo Azurol S locais não contaminados foi possível encontrar ecótipos toleran- (CAS), e sua natureza química (hidroxamatos ou catecolatos) foi tes a altas concentrações de metal e além disso produtores de analisada. Entre os fungos existiu uma clara variação inter e in- compostos quelantes do tipo sideróforos.

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