Violets of the section Melanium, their colonization by arbuscular mycorrhizal fungi and their occurrence on heavy metal heaps

Page 1

This article appeared in a journal published by Elsevier. The attachedcopy is furnished to the author for internal non-commercial researchand education use, including for instruction at the authors institution

and sharing with colleagues.

Other uses, including reproduction and distribution, or selling orlicensing copies, or posting to personal, institutional or third party

websites are prohibited.

In most cases authors are permitted to post their version of thearticle (e.g. in Word or Tex form) to their personal website orinstitutional repository. Authors requiring further information

regarding Elsevier’s archiving and manuscript policies areencouraged to visit:

http://www.elsevier.com/copyright

Page 2

Author's personal copy

Journal of Plant Physiology 168 (2011) 1191–1199

Contents lists available at ScienceDirect

Journal of Plant Physiology

journa l homepage: www.e lsev ier .de / jp lph

Violets of the section Melanium, their colonization by arbuscular mycorrhizalfungi and their occurrence on heavy metal heaps

A. Słomkaa, E. Kutaa, G. Szarek-Łukaszewskab, B. Godzikb, P. Kapustab, G. Tylkoc, H. Bothed,∗

a Institute of Botany, Jagiellonian University, 52 Grodzka str., 31-044 Cracow, Polandb W. Szafer Institute of Botany, Polish Academy of Sciences, 46 Lubicz str., 31-512 Cracow, Polandc Institute of Zoology, Jagiellonian University, 6 Ingardena str., 30-060 Cracow, Polandd Botanical Institute, The University of Cologne, Zülpicherstr. 47b, 50923 Cologne, Germany

a r t i c l e i n f o

Article history:Received 21 September 2010Received in revised form24 November 2010Accepted 13 January 2011

Keywords:AMF-colonization of violetsDithizone stainingHeavy metals in violetsMetalophytesViolaZinc violets

a b s t r a c t

Violets of the sections Melanium were examined for their colonization by arbuscular mycorrhizal fungi(AMF). Heartsease (Viola tricolor) from several heavy metal soils was AMF-positive at many sites but not atextreme biomes. The zinc violets Viola lutea ssp. westfalica (blue zinc violet) and ssp. calaminaria (yellowzinc violet) were always AMF-positive on heavy metal soils as their natural habitats. As shown for theblue form, zinc violets germinate independently of AMF and can be grown in non-polluted garden soils.Thus the zinc violets are obligatorily neither mycotrophs nor metalophytes. The alpine V. lutea, likelyancestor of the zinc violets, was at best poorly colonized by AMF. As determined by atomic absorptionspectrometry, the contents of Zn and Pb were lower in AMF colonized plants than in the heavy metal soilsfrom where the samples had been taken. AMF might prevent the uptake of toxic levels of heavy metalsinto the plant organs. Dithizone staining indicated a differential deposition of heavy metals in tissues ofheartsease. Leaf hairs were particularly rich in heavy metals, indicating that part of the excess of heavymetals is sequestered into these cells.

© 2011 Elsevier GmbH. All rights reserved.

Introduction

Members of the Violaceae family have developed the tendencyto occupy heavy metal soils. Metalophytes from the three generaHybanthus, Rinorea and Viola have been reported to accumulateNi, Zn, Cd, and Pb (Ernst, 1998; Psaras et al., 2000; Prasad andde Oliveira Freitas, 2003). In Central Europe, heavy metal heapshost remarkable violets of the section Melanium Ging. within thegenus Viola. Two endemic violets of heavy metal soils have a veryrestricted local distribution in the western part of Central Europe.The yellow zinc violet occurs in the area between Aachen/Germanyand Liège/Belgium. The blue form is found only on a ditch andits neighboring meadow at the village Blankenrode in the vicinityof Paderborn in eastern Westfalia. Both violets are of top prior-ity among the endangered taxa of Europe (Schmeil and Fitschen,2009). Morphological criteria let Nauenburg (1986) to concludethat the blue and the yellow zinc violets are separate species. Hetermed the yellow violet Viola calaminaria and the blue violet asViola guestphalica. More recent molecular studies (DNA sequenc-ing of the ITS1-5.8S rDNA-ITS2-regions), however, indicated thatthe blue and the yellow zinc violets originate from the alpine

∗ Corresponding author. Tel.: +49 221 4702760; fax: +49 221 4705039.E-mail address: [email protected] (H. Bothe).

Viola lutea, are closely related, and should be termed V. lutea ssp.calaminaria and V. lutea ssp. westfalica, respectively (Hildebrandtet al., 2006). Both zinc violets are strongly colonized by arbuscularmycorrhizal fungi, which might prevent uptake of heavy met-als into the plant tissues (Hildebrandt et al., 1999; Kaldorf et al.,1999). Earlier measurements (Ernst, 1982) indicated that leavesof V. lutea ssp. calaminaria contain as low amounts of heavy met-als as non-metalophytes, also occupying the heavy metal heap ofBreinigerberg close to Aachen. Others reported higher concentra-tions of heavy metals in the aboveground parts of the zinc violetsunder laboratory conditions (Jedrzejczyk et al., 2002). In a fieldstudy with V. lutea ssp. calaminaria performed in Belgium, high con-centration of Zn in leaves were found to retard the development oflarvae of the butterfly Issoria lathonia, which did not happen whenthe larvae fed on the non-accumulating Viola tricolor (Noret et al.,2007). Thus the concentration of heavy metals in the zinc violetsrequires reassessment.

V. tricolor (heartsease; wild pansy) thrives on both heavy metalheaps and on non-polluted soils, particularly on sand and gravel,and is abundant especially in Eastern European countries. More-over, this species is indigenous to or naturalized in large parts ofEurope, the Middle East, Central Asia, the U.S.A. (Rimkiene et al.,2003) and even Korea (Choi et al., 1996). V. tricolor has been usedin folk medicine against respiratory problems such as bronchi-tis, asthma and cold symptoms (Rimkiene et al., 2003), exhibits

0176-1617/$ – see front matter © 2011 Elsevier GmbH. All rights reserved.doi:10.1016/j.jplph.2011.01.033

Page 3

Author's personal copy

1192 A. Słomka et al. / Journal of Plant Physiology 168 (2011) 1191–1199

antioxidative activities (Vukics et al., 2008), has cytotoxic effects onhuman cancer cell lines (Svangård et al., 2004) and antimicrobialactivity (Witkowska-Banaszczak et al., 2005).

Members of the Violaceae family are reported to be AMF-positive in general (Harley and Harley, 1987; Newman and Reddell,1987). To our knowledge, heavy metal accumulation by heavymetal tolerant violets has not yet been assessed in relation to AMFcolonization. The mycorrhizal colonization of the zinc violets, oftheir putative parents V. lutea, collected either from the Vosgesmountains in France or the Sudeten mountains in Poland, and ofV. tricolor from different habitats were examined with unexpectedresults. The heavy metals concentration in soils as well as in rootsand aboveground parts of plants was determined in order to assessany impact of AMF on the levels of heavy metals in these plants inboth polluted and non-affected soils.

Materials and methods

Plant material

The origin of the plant material and site descriptions are sum-marized in Table 1.

Determination of root colonization by AMF

Following the standard protocol of Phillips and Hayman (1970),roots were rinsed with tap water and cleared in 10% KOH for20–25 min at 90 ◦C, then thoroughly washed with water and stained(30 min at room temperature) with a solution of 0.05% (w/v)of trypan blue (C. I. 23850 direct Blue 14, Sigma) dissolved inlactate/glycerol/H2O = 1/1/1. Destaining of the roots was done in50% glycerol/H2O (v/v) overnight. The fungal structures, but notthe plant cells, retained the blue color. Microscopic determinationof the AMF colonization was performed at 125-fold magnificationon at least 300 roots per sample using the gridline intersect method,slightly modified (Schmitz et al., 1991).

Heavy metal concentrations in plant organs and in soils measuredby atomic absorption spectrometry (AAS)

Soils and plants of 5 Viola taxa (Viola lutea, V. lutea ssp. sudetica, V.lutea ssp. westfalica, V. lutea ssp. calaminaria, and V. tricolor) from 9populations (Table 4) were collected in the field between 2006 and2009. Three plants taken from the immediate vicinity of each sam-pling area were mixed and separated into roots, stems, leaves andflowers with peduncles for the determinations. Dependent on pop-

Table 1Sites of the analyzed plant material.

Taxon Locality Geographic coordinates Collection date

Viola lutea ssp. westfalica D-Blankenrode N 51◦11′45′′ 05.06.2006E 08◦03′15′′ 17.07.2008

D-private garden in Erftstadt near Cologne(plants grown from seeds collected inBlankenrode)

N 50◦46′44′′ 10.06.2006

E 06◦49′41′′ 21.08.2008PL-private garden in Ostrowy Górnicze inSosnowiec (plants grown from seeds collectedin Blankenrode)

N 50◦17′24′′ June 2004

E 19◦26′21′′

V. lutea ssp. calaminaria D-Breinigerberg, meadow at the sport fields N 50◦43′32′′ 15.06.200625.08.2008

E 06◦13′22′′

V. lutea, blue and yellow morph F-Hohneck, Vosges mountains N 48◦02′28′′ 19.07.2007E 06◦58′80′′

V. lutea ssp. sudetica PL-Snieznik, Sudeten mountains N 50◦12′25′′ 30.06.2009E 16◦49′51′′

A-ArnoldsteinSite A N 46◦33′14′′ 04.09.2008

E 13◦34′23′′

Site B N 46◦33′36′′ 04.09.2008E 13◦41′22′′

I-Cave del Predil N 46◦26′44′′ 04.09.2008E 13◦34′23′′

HU-Nagyszénás near Budapest N 47◦34′30′′ 27.05.2009E 18◦51′43′′

PL-Trzebionka heap in Trzebinia N 50◦09′27′′ 27.08.2008E 19◦25′13′′ 27.06.2009

PL-Saturn heap in Olkusz N 50◦17′12′′ 27.08.2008E 19◦32′96′′

PL-Warpie heap in Chrzanów N 50◦09′00′′ 27.08.2008E 19◦25′96′′

PL-Bukowno heap near Olkusz, calamine heaps N 50◦17′30′′ 21.05.200727.08.200827.06.2009

E 19◦28′20′′

PL-Zakopane along the railroad N49◦20′09′′ 21.05.2007E 20◦00′05′′

PL-Central Zakopane (Chełbonski Museumvicinity)

N 49◦18′55′′ 21.05.2007

E 19◦56′51′′

PL-Botanical Garden Cracow N 50◦03′44′′ 27.08.2008E 19◦57′30′′

Page 4

Author's personal copy

A. Słomka et al. / Journal of Plant Physiology 168 (2011) 1191–1199 1193

Tab

le2

Myc

orrh

izal

colo

niz

atio

nof

viol

ets

coll

ecte

dfr

omd

iffe

ren

tsi

tes.

Taxo

nSi

teSi

ted

escr

ipti

onD

ate

ofco

llec

tion

Roo

tsn

on-c

olon

ized

(%)

Intr

arad

ical

hyp

hae

(%)

Arb

usc

ule

s(%

)V

esic

les

(%)

No.

ofp

lan

tsan

alyz

ed

Vio

latr

icol

orB

uko

wn

oh

eap

ASm

allh

ill,

flat

wit

hD

iant

hus

cart

husi

anor

um,S

cabi

osa

ochr

oleu

ca20

0882

.9±

8.8

17.1

±8.

612

.6±

7.9

1.1

±1.

35

2009

47.4

±15

.252

.5±

15.5

44.2

±19

.06.

0±

3.6

3

BM

ead

oww

ith

Card

amin

opsi

sar

enos

a,D

iant

hus

cart

husi

anor

um,C

arex

hirt

a,A

rmer

iam

arit

ima

ssp

.hal

leri

2008

68.0

±12

.431

.3±

12.0

27.8

±10

.20.

9±

1.6

3

2009

46.4

±25

.553

.6±

25.5

40.4

±28

.26.

2±

4.6

3

CM

ead

owcl

ose

tofo

rest

wit

hA

rmer

iam

arit

ima

ssp

.hal

leri

,Equ

setu

mar

vens

e,Ca

rdam

inop

sis

aren

osa,

Rum

exac

etos

a

2008

69.6

±3.

130

.0±

3.4

22.5

±6.

14.

2±

6.0

3

2009

36.3

±12

.274

.8±

12.2

63.6

±19

.39.

6±

3.0

3

DG

rave

lare

a,m

inin

gd

itch

esw

ith

crip

ple

dp

ines

,Car

lina

acau

lis,

Bisc

utel

lala

evig

ata,

Sile

nevu

lgar

isss

p.

hum

ilis,

Arm

eria

mar

itim

ass

p.h

alle

ri

2007

95.7

±3.

74.

2±

3.6

4.2

±3.

51.

2±

2.1

3

2008

94.1

±4.

07.

0±

3.7

3.9

±1.

10

±0

320

0989

.9±

7.2

10.1

±7.

28.

7±

8.4

0.4

±0.

83

Trze

bion

kah

eap

Ayo

un

g(3

0ye

ars)

,dry

,ste

epar

tifi

cial

slop

ew

ith

pos

t-fl

otat

ion

was

tes

2008

96.3

±0.

63.

7±

0.6

3.7

±6.

20

±0

4

2009

95.4

±1.

74.

3±

2.2

1.1

±1.

80.

3±

0.5

3Sa

turn

hea

pin

Olk

usz

On

eof

the

old

est

(800

year

s)Po

lish

hea

vym

etal

hea

p;

chal

k20

0854

.2±

8.6

45.6

±8.

645

.3±

8.9

14.7

±0.

73

War

pie

hea

pin

Ch

rzan

ówM

ead

owcl

ose

tofo

rest

;ch

alk

2008

51.3

±12

.848

.8±

12.7

48.8

±12

.72.

7±

2.5

3

Cen

tral

Zako

pan

eG

ard

enin

the

vici

nit

yof

Ch

ełbo

nsk

iM

use

um

2007

52.4

±7.

147

.4±

7.1

47.4

±7.

13.

4±

3.5

3

Zako

pan

eIn

the

grav

elal

ong

the

rail

road

2007

57.5

±3.

942

.3±

3.8

42.3

±3.

80

±0

3C

raco

wB

otan

ical

Gar

den

2008

91.0

±8.

214

.9±

3.3

12.3

±3.

00

±0

3A

rnol

dst

ein

AIn

the

rive

rbed

Gai

litz

2008

45.2

±9.

954

.2±

9.9

34.9

±7.

613

.0±

5.1

4B

Sam

esi

te,∼

300

map

art

2008

51.6

±14

.248

.5±

14.4

28.0

±12

.717

.8±

10.2

5C

ave

del

Pred

ilG

rave

lalo

ng

the

Gai

litz

2008

62.5

±27

.437

.6±

27.0

18.9

±16

.512

.6±

10.6

5N

agy-

szén

ásn

ear

Bu

dap

est

Gyp

sum

rock

2009

22.0

±13

.7(7

3.6

±7.

5)a

(2.2

±0.

6)a

(1.8

±.1

)a2

V.l

utea

ssp

.cal

amin

aria

Bre

inig

erbe

rgA

Zn-m

ead

owat

the

spor

tfi

eld

s20

0651

.5±

9.6

47.5

±8.

624

.9±

5.0

4.2

±3.

83

B20

0830

.7±

7.7

68.7

±7.

054

.9±

12.0

46.1

±4.

03

V.l

utea

ssp

.wes

tfal

ica

Bla

nke

nro

de

Pban

dZn

dit

ch20

0627

.5±

8.8

79.2

±6.

270

.5±

11.7

28.8

±9.

73

Priv

ate

gard

enin

Erft

stad

tR

iver

sed

imen

t20

0661

.1±

11.5

38.9

±11

.536

.8±

12.0

3.4

±2.

33

V.l

utea

Vos

ges

mou

nta

ins

Hoh

nec

k,on

gran

ite

2007

97.9

±2.

01.

9±

1.8

1.9

±1.

70

±0

3V

.lut

eass

p.s

udet

ica

Sud

eten

mou

nta

ins

Snie

znik

,on

gran

ite

2009

85.1

±8.

615

.2±

8.4

8.2

±4.

43.

3±

2.5

3

aTh

etw

osa

mp

les

con

tain

edex

clu

sive

lyd

ark

sep

tate

end

oph

ytes

wit

hm

icro

scle

roti

a,d

ata

ther

efor

ein

brac

kets

.

Page 5

Author's personal copy

1194 A. Słomka et al. / Journal of Plant Physiology 168 (2011) 1191–1199

ulation size, 4–16 samples/population were analyzed. In the caseof the extreme rare V. lutea ssp. westfalica and V. lutea ssp. calami-naria, only three specimens were analyzed. Few roots, stems, leavesand flowers were taken from these V. lutea ssp. westfalica plants sothat each individual survived sampling. For the determinations ofheavy metal concentrations, roots and other organs were rinsedwith deionized water (3×), sonicated for 5 min and mineralizedusing Tecator (Sweden) Kjeldahl digestion system in mixture ofconcentrated nitric acid (HNO3, MERCK suprapure) and perchloricacid (HClO3, MERCK suprapure) 4/1 (w/v). The obtained digestivewas concentrated by evaporation to about 0.5 ml and then dilutedwith deionised water to a final vol of 10 ml. In this solution theelements Zn, Cd, and Pb, were determined by the atomic absorp-tion spectrometry with a Varian 220 FS model. The accuracy of thedeterminations was ascertained by analyzing one blank and tworeference samples after every 10th measurement. Concentrationsof elements were referred to two standard reference materials:SRM 1575, SRM 1570a and international inter-laboratory mosssamples M2 and M3.

Soil samples of the upper 10 cm (with litter removed) were col-lected simultaneously with the plants on the sampling dates. SoilpH was measured as described in ISO10390. A suspension of theair-dried soil passed through a 2 mm aperture sieve was made upto five times its vol by water. Suspension pH was measured usinga Mettler Toledo MP125 pH-meter. For available elements in H2Oextracts, 5 g of the air-dried soil with 50 ml H2O was shaken for 1 hon a reciprocal shaker at slow speed. The soil extract was filteredthrough membrane filters (0.2 �m mesh) and directly analyzedfor Zn, using atomic absorption spectrometry (AAS) Varian 220 FS(flame method), and for Cd and Pb, using graphite tube GTA 110.Concentrations of available elements were determined in standardmaterials CPI P/N4400-010118.

Determinations by AAS were made in the Department of Ecologyof the Institute of Botany, the Polish Academy of Sciences in Cracow.

Germination experiment with the blue zinc violet

Pots filled with 1 kg of fertile garden soil (originating fromdeposits of the river Erft at D-Erftstadt-Bliesheim) were eachplanted with approximately 100 seeds of V. lutea ssp. westfalicaon September 2nd, 2008. More than 90% of all seeds germinatedin darkness after 2 weeks. Seedlings were grown outdoors inthe light over winter. Where applicable (see Table 3), soil wassteam-sterilized in an autoclave for 1 h to destroy endogenousmicroorganisms including mycorrhizal fungi. Glomus intraradicesSy167 (Hildebrandt et al., 1999) was used as mycorrhizal fungus.This isolate colonized several plants in many different experimentsperformed in the Cologne laboratory over years. Their mycorrhizalcolonization was assessed immediately after harvesting on March20th, 2009. Three independent counts in each case on at least100 roots were made for each trail (see Table 3). On March 20th,2009, there were no visually observable differences in plant vigourbetween the three treatments.

Staining of heavy metals in plant tissues and organs by dithizone

Roots, leaves and stem fragments of plants from control sitecentral Zakopane (Chełbonski museum vicinity) and Bukowno

heap (site C) were rinsed in tap water and then 3–4 times indistilled water. Transverse and longitudinal sections of stemsand roots were made and the abaxial epidermis of leaves wasremoved to facilitate analysis of epidermal hairs. Heavy met-als were detected in whole organs and in sections (in tissuesand cells) after staining with dithizone (diphenylthiocarbazone,Aldrich; 30 mg dissolved in 60 ml acetone and 20 ml distilled water)as described by Seregin and Ivanov (1997) and slightly modifiedby Wierzbicka and Pielichowska (2004). Samples were stained indarkness for 1.5 h, then rinsed in water and analyzed under a stere-oscope microscope. Metals (Zn, Pb, Cd)-dithizonate complexes inorgans and tissues stained red at different intensities dependingon the metal.

Data analysis

Both one-way ANOVA followed by Tukey’s test post hoc forheavy metal concentrations in plants and Kruskal–Wallis’ test forheavy metals concentration in soils were performed using STATIS-TICA ver.7.0.

Results

Colonization of the violets collected from field sites by arbuscularmycorrhizal fungi

Most samples of heartsease (V. tricolor) were strongly AMF-colonized regardless of their origin from heavy metal heaps ornon-polluted areas in Poland and the Alps (Arnoldstein, Cave delPredil; Table 2). A high percentage of the stained roots of all thesesamples showed both intraradical hyphae, arbuscules and vesicles.There were, however, remarkable exceptions. Whereas heartseasefrom the three other sites of Bukowno heap was strongly colo-nized, this was not the case at site D where the colonization leveland the arbuscules formation was at most only 10% in all threesampling years (Table 2). Site D is characterized by coarse graveloccupied by only few drought-resistant plants such as the two met-alophytes Silene vulgaris ssp. humilis (with curved shoots, unlike tothe specimens occurring on non-polluted sites) and Armeria mar-itima ssp. halleri as well as Biscutella laevigata, Potentilla rupestris,Gypsophila repens, the beauty Carlina acaulis and few others besidesV. tricolor. Heartsease on the steep slope of Trzebionka heap wasfound also not to be colonized (or rather poorly), both in 2008 and2009, thus on an area which was similarly dry as the gypsum areaof Nagy-szénás close to Budapest. Here the two samples of heart-sease were colonized not by AMF but by another, non-mycorrhizalfungus which showed blackish septae within the stained hyphaeand therefore likely was a dark septate endophyte (Table 2). Thethree samples from the Cracow Botanical Garden also showed a lowdegree of colonization (around 15%); there is no obvious explana-tion for that.

Plants of the mountain pansy (V. lutea) group showed a non-uniform pattern of AMF colonization (Table 2). V. lutea from theFrench Vosges mountains with 2n = 48 (Hildebrandt et al., 2006)was poorly colonized (2.1%), whereas the Sudeten pansy (V. luteassp. sudetica) with 2n = 50 (Krahulcová et al., 1996) was slightly(4.9%) colonized. A colonization of less than 5% is generally regardedas meaningless. The endemic and rare zinc violets which are likely

Table 3Non-dependence of the germination of V. lutea ssp. westfalica on mycorrhizal fungi.

Intraradical hyphae Arbuscules Vesicles

Non-sterilised soil 0.008 ± 0.005 0.003 ± 0.003 0.003 ± 0.003Sterilised soil 0.013 ± 0.002 0.005 ± 0.005 0.000 ± 0.000Sterilised soil + Glomus 0.043 ± 0.039 0.020 ± 0.020 0.000 ± 0.000

Data given in %. The number of roots showing any sign of colonization is identical with that for intraradical hyphae.

Page 6

Author's personal copy

A. Słomka et al. / Journal of Plant Physiology 168 (2011) 1191–1199 1195

Tab

le4

Tota

lcon

cen

trat

ion

ofh

eavy

met

als

inso

ilan

din

dif

fere

nt

par

tsof

viol

ets

from

seve

rall

ocat

ion

sd

eter

min

edby

atom

icab

sorp

tion

spec

trom

etry

.

Vio

lata

xon

;ye

arco

llec

ted

Soil

Roo

tSt

emLe

afFl

ower

wit

hp

edu

ncl

e

ZnPb

Cd

pH

ZnPb

Cd

ZnPb

Cd

ZnPb

Cd

ZnPb

Cd

V.l

utea

ssp

.w

estf

alic

aB

lan

ken

-ro

de

2008

90,2

00a

2800

an

.d.

∼4.5

1819

±35

7a

135

±97

c9

±6

d14

87±

445

a37

24b

7±

5cd

3719

±89

a3

±2

d3

±0

f86

2±

299

a2

±1

e2

±2

c

V.l

utea

ssp

.w

estf

alic

aG

ard

enin

Ost

row

yG

órn

icze

2004

183d

53d

n.d

.n

.d.

1931

±58

0a

24±

18d

4±

1d

e10

22±

25b

4±

2e

1±

0f

616

±85

2c

2±

2d

1±

0g

697

±23

c2

±2

e0

±0

d

V.l

utea

ssp

.ca

lam

inar

iaB

rein

iger

-be

rg20

08

4500

–42,

200b

10,2

69c

n.d

.∼6

.023

75a

19,3

45a

318

a14

51a

5704

a18

9a

1881

b25

18a

104

a95

6ab

2723

a2

c

41,2

75c

V.l

utea

Hoh

nec

k,V

osge

sM

ts20

07

n.d

.n

.d.

n.d

.∼4

.092

9±

306

b13

2±

57c

8±

5d

252

d0

f1

f29

2e

0d

e0

g19

4d

0ef

0d

V.t

rico

lor

Bu

kow

no

hea

psi

teD

2008

7271

±45

1314

91±

1035

85±

746.

819

76±

553

a38

4±

93d

51±

15b

1720

±48

4a

25±

18d

38±

15b

2331

±42

1ab

74±

67b

31±

16b

1311

±30

4a

132

±84

b25

±8

a

Satu

rnh

eap

inO

lku

sz20

0818

67±

7630

26±

560

76±

287.

430

9±

35c

65±

33cd

11±

3d

209

±36

d12

±10

de

9±

2cd

232

±50

e4

±6

d7

±2

d14

8±

21e

21±

8d

5±

2c

War

pie

hea

pin

Ch

rzan

ów20

08

11,3

43±

2194

5180

±21

910

2±

86.

346

8±

67c

302

±17

0b

25±

8c

316

±29

c35

±5

c16

±3

c48

6±

62d

35±

6c

22±

4c

165

±28

de

62±

19c

13±

2b

Cen

tral

Zako

pan

e20

07

83±

5730

±7

1±

17.

281

±6

d1

±2

e3

±1

e85

±4

e6

±2

e3

±1

e14

0±

11f

0±

0d

e4

±0

e80

±5

f0

±0

ef3

±1

c

Zako

pan

eal

ong

rail

road

2007

794

±20

468

±89

10±

37.

114

1±

24d

0±

0e

2±

1e

147

±73

de

0±

0f

2±

1e

132

±24

f0

±0

de

1±

1g

100

±22

f0

±0

ef1

±1

d

Stat

isti

csH

=16

.58;

0.00

1<

P<

0.01

H=

16.1

5;0.

001

<P

<0.

01H

=16

.18;

0.00

1<

P<

0.01–

F=

21.1

6;P

<0.

001

F=

66.2

6;P

<0.

001

F=

3.2;

0.00

1<

P<

0.01F

=22

.01;

P<

0.00

1F

=16

2.0;

P<

0.00

1F

=54

.01;

P<

0.00

1F

=16

.82;

P<

0.00

1F

=19

2.21

P<

0.00

1F

=19

.80;

P<

0.00

1F

=33

.70;

P<

0.00

1F

=67

.05;

P<

0.00

1F

=25

.93;

P<

0.00

1

n.d

.—n

otd

etec

ted

.Mea

ns

wit

hd

iffe

ren

tle

tter

sin

one

colu

mn

are

sign

ifica

ntl

yd

iffe

ren

tby

AN

OV

Afo

llow

edby

Tuke

y’s

test

.Dat

agi

ven

inm

g/kg

.a

Thes

ed

ata

wer

eta

ken

from

Ern

st(1

974)

.b

Thes

ed

ata

wer

eta

ken

from

Sch

wic

kera

th(1

944)

.c

Thes

ed

ata

wer

eta

ken

from

Sim

on(1

975,

1978

).d

Thes

ed

ata

wer

eta

ken

from

Jed

rzej

czyk

etal

.(20

02).

Page 7

Author's personal copy

1196 A. Słomka et al. / Journal of Plant Physiology 168 (2011) 1191–1199

descendents of the mountain pansy (Hildebrandt et al., 2006) werealways strongly colonized (38.9–72.5%). This was seen both for theyellow zinc violet (V. lutea ssp. calaminaria) of the Aachen-Liègearea and for the blue form (V. lutea ssp. westfalica) of the Blanken-rode ditch, in accordance with earlier determinations (Hildebrandtet al., 1999). The blue zinc violet was also strongly colonized (38.9%)when grown for years in a private allotment in fertile and non-polluted garden soil (Table 2). Despite this, zinc violets are notentirely dependent on symbiosis with AMF, as documented for thegermination and the juvenile stage of the blue zinc violet (Table 3).Roots of the plants that had germinated in autumn and had beengrown over winter showed almost nil intraradical hyphae, arbus-cule or vesicle formation when grown in a fertile garden soil,regardless of whether the soil had been sterilized, not sterilizedor sterilized but supplemented with Glomus intraradices SY167propagules.

Determination of heavy metals by atomic absorptionspectrometry (AAS)

The heavy metal soils in which both blue (V. lutea ssp. west-falica) and the yellow (V. lutea ssp. calaminaria) zinc violets thrivecontain high concentrations of both Zn and Pb (Schwickerath, 1944;Ernst, 1974; Simon, 1975, 1978). As Table 4 shows, Zn and Pb levelsin all the organs of both zinc violets collected from heavy met-als soils were considerably lower than the soils. Nevertheless, theblue zinc violet from the heavy metals soil (Blankenrode) appar-ently accumulated fairly high concentrations of Zn and Pb in itscells; the levels of these heavy metals were drastically higher insamples from Blankenrode than in plants originating from the non-polluted soil of a private allotment at Sosnowiec, Poland (comparelines 1 and 2 of Table 4). As regard to heartsease, the concentra-tions of Zn and Pb were significantly lower in all organs than in thesoils of all heavy metal polluted areas (Saturn heap, Warpie heap,Bukowno heap—site D, moderately polluted Zakopane railroad bed;Table 4). Plants from all V. tricolor populations deposited Zn alsointo leaves, since the Zn concentrations were comparable in rootsand leaves, regardless of the soil pollution level. In the reproduc-tive organs (flowers with peduncles), the Zn concentrations werelower than in the other organs. Pb and Cd were retained mainly inroots (Table 4). Heartsease samples from Bukowno, site D which –as just mentioned were non-AMF – showed extremely high levelsof Zn, Pb and Cd, often 5–20 times the levels in the organs of theplants from the other heavy metal soils (Table 4). Unlike the bluezinc violet, heartsease did not accumulate heavy metals in non-polluted soils, as shown for the specimens collected from centralZakopane.

Anova followed by the Tukey’s test indicated that the between-site differences in heavy metal content in soil and in plantswere highly significant (Table 4) but there was no direct cor-relation (P > 0.05) between the concentration of heavy metals inplants (roots, stems) and soils and the degree of AMF coloniza-tion.

Heavy metals in heartsease followed a similar but not so distinctpattern as in Table 4 when the concentrations of water extractablePb, Zn and Cd were determined (Table 5). Once more, concentra-tions of Zn and Pb were highest in both in roots and abovegroundparts in the non-AMF-colonized samples from Bukowno site D. TheAMF-colonized samples from Bukowno site A, B and C showedhigher amounts of Zn, Pb and Cd than the specimens from Trze-bionka (Table 5). The Trzebionka soil contained higher levels ofZn than the Snieznik soil of Sudeten Mts. from which V. luteassp. sudetica was collected. However, the concentration of water-extractable Zn at Trzebionka was distinctly lower than at the foursites at Bukowno, indicating that Trzebionka was only moderatelycontaminated by heavy metals. Ta

ble

5C

once

ntr

atio

nof

wat

er-e

xtra

ctab

lem

etal

sin

soil

and

tota

lam

oun

tof

met

als

inro

ots

and

abov

egro

un

dp

arts

ofV

iola

lute

ass

p.s

udet

ica

and

V.t

rico

lor.

Soil

Roo

tA

bove

grou

nd

par

t

ZnPb

Cd

pH

ZnPb

Cd

ZnPb

Cd

V.l

utea

ssp

.sud

etic

a1

±0

0±

00

±0

5.0

225

±7.

718

.2±

7.1

1.4

±0.

491

.8±

206.

2±

0.4

0.5

±0.

1V

.tri

colo

rTr

zebi

onka

hea

p21

±9

0±

00

±0

6.8

504.

8±

158.

511

8±

388.

3±

0.5

206.

6±

201.

115

.4±

7.4

4.3

±2.

6B

uko

wn

oh

eap

,sit

eA

36±

180

±0

3±

17.

025

27.1

±29

40.9

278.

2±

204.

754

.4±

65.7

500.

7±

223.

529

.3±

19.2

7.6

±2.

4B

53±

250

±0

4±

17.

014

31.9

±11

3.5

262.

8±

46.1

44.1

±15

686.

9±

314.

346

.6±

20.1

20.0

±9.

9C

87±

61

±0

3±

06.

818

25.2

±71

0.4

258.

4±

298.

628

.6±

11.7

1227

.6±

716.

536

.9±

20.8

17.1

±7.

2D

285

±97

4±

33

±2

6.8

2576

.1±

311.

641

8.5

±10

.339

.8±

1113

10.7

±98

7.3

55.8

±34

.132

.5±

16.3

Dat

agi

ven

inm

g/kg

.All

the

pla

nt

and

soil

mat

eria

lwas

coll

ecte

din

2009

.

Page 8

Author's personal copy

A. Słomka et al. / Journal of Plant Physiology 168 (2011) 1191–1199 1197

Fig. 1. Heavy metal deposits in roots, stems and leaves of Viola tricolor as indicated by dithizone staining. Plants grown on a heavy metals heap were from Bukowno site Cand control plants from a non-polluted site from central Zakopane. (A) Not stained roots from the control site; (B) stained root from Bukowno (left) and not colored rootafter staining from the control site (right); (C) intensely colored root from Bukowno, longitudinally sectioned stem parts (D) parts of the stem from Bukowno plants (the twostems at right) and non-colored sample of a control plant (left stem); (E and F) transverse section of stems, from the control site (E) and from Bukowno (F); (G) petiole andpart of the blade of a leaf of a Bukowno plant; (H and I) hairs of the abaxial leaf epidermis of a Bukowno plant.

Localization of heavy metals in plant tissues by dithizone staining

Dithizone forms colored complexes with Zn and other heavymetals and was used to map levels of heavy metals in heartseasetissues from site C of Bukowno heavy metals heap and from thenon-polluted central Zakopane control site. Total roots and stemsof heartsease from the heavy metal heap stained dark red whereasplants from the control site hardly changed their color by staining(Fig. 1A–D). Transverse sections of the stems stained intensively,particularly in the xylem and phloem vessels and in the innermostcortical cells in plants from the heavy metal soil, but not from thecontrol site (Fig. 1E and F). In leaves, particularly the petiole was

strongly colored in specimens from the heavy metal soil (Fig. 1G).Hairs of the abaxial leaf epidermis of the plants from the heavymetal heap showed crystal deposits in addition to an intense darkred/violet staining (Fig. 1H and I). Thus, heavy metals are differ-entially deposited in the tissues of heartsease originating from theheavy metal soil.

Discussion

Heartsease (V. tricolor) generally is said to be AMF positive, butreviews (Harley and Harley, 1987; Wang and Qui, 2006) indicatethat the reports are not uniform. In Norway, for example, there are

Page 9

Author's personal copy

1198 A. Słomka et al. / Journal of Plant Physiology 168 (2011) 1191–1199

habitats where plants are strongly colonized and others where theyare not (Eriksen et al., 2002). In previous studies (Pawłowska et al.,1996; Zubek et al., 2003), heartsease samples from the metal heapsin the vicinity of Olkusz, Southern Poland were consistently foundto be highly AMF-positive. In the present investigation this was con-firmed for most of plants collected from the different heavy metalsoils in Southern Poland and elsewhere, and also from non-pollutedsoils, but not everywhere. Bukowno heap site D had the highestmetal content, but the plants from there were not AMF-positive.Other sites where heartsease was not found to be AMF-positivewere the Trzebionka steep heap and the gypsum rock close toBudapest. These sites are commonly very dry. Lack of moisture mayhave prevented the growth of the AM fungi. On the other hand,in extremely water stressed habitats, in salt marshes, AMF havebeen reported to alleviate water stress (Füzy et al., 2008; and oth-ers). It has been suggested that dark septate endophytes becomemore important than AMF in extremely stressed environments(Read and Haselwandter, 1981). Indeed, the samples from the gyp-sum rock close to Budapest were strongly infected by these fungi,but this was not detected at the Bukowno site D or at Trzebionka.The degree of mycorrhizal colonization is known to undergo sea-sonal fluctuations. However, specimens from the two Polish sites,Bukowno D and Trzebionka, repeatedly collected in different years,were always AMF negative. Several years of field observations indi-cated that they were not substantially impaired in their growth ascompared to the samples from the other habitats. One might beinclined to say that such variations in the AMF colonization aredue to subtle fluctuations of soil conditions within short distances.However, the occurrence of heartsease at these extreme habitatswithout AMF colonization is, admittedly, not understood by us.The plants collected were perennial. Zabłocka (1936) suggestedthat only the annual but not the perennial V. tricolor plants arenon-mycorrhizal.

The zinc violets (V. lutea ssp. calaminaria and V. lutea ssp. west-falica) have been regarded as obligate metalophytes (meaningthey can live only on heavy metal soils; Ernst, 1974; Nauenburg,1986), and as always AMF-positive (Hildebrandt et al., 1999; Toninet al., 2001). Those statements have to be modified. Both zincviolets are descendent of the alpine V. lutea (Hildebrandt et al.,2006) and survive in lowlands only on heavy metal soils probablybecause they cannot compete with other plants on non-pollutedsoils (Hildebrandt et al., 2006). Both the blue and the zinc vio-let can be grown in fertile garden soils provided overgrowth byother plants is prevented, as now verified at several locations for(unpublished results cited in Bothe et al., 2010; Jedrzejczyk et al.,2002). At their natural stands and when grown in garden soils, theyare strongly AMF-positive (Hildebrandt et al., 1999; this publica-tion). However, their germination is not AMF-dependent duringtheir early growth, as shown in the present study for V. lutea ssp.westfalica. Thus, caution is called in describing a plant as an obligatemetalophyte or a strict mycotroph.

Mycorrhizal colonization of plants is high in alpine grasslandsabove the timberline (Read and Haselwandter, 1981). AMF-positiveplants should be more abundant in ecosystems with nutrient defi-ciencies and lower availability of organic nutrients in soils (Read,1981). Treseder and Cross (2006), however, argued that there isnot necessarily an inverse correlation between AMF colonizationof plants and organic nutrient content in soils. Nevertheless, bothalpine to montane habitats in the present study (Hohneck in Vos-ges mountains and Snieznik in Sudeten mountains) are nutrientpoor, both in minerals and organic matter, and they are stronglyacidic. These soil conditions should favor colonization of plantsby mycorrhizal fungi. However, all V. lutea plants (the yellow andblue morphs of Vosges mountains and V. lutea ssp. sudetica ofSnieznik) were not or at best poorly AMF-colonized whereas theirpresumed descendants, the zinc violets of the heavy metal soils,

were strongly AMF-positive. There is no obvious explanation forthe non-colonization of the alpine V. lutea plants. A future charac-terization of these alpine meadows should reveal whether otherplants are also not AMF-positive and whether the AMF spores con-tent of the soils is low there. Zabłocka (1936) reported mycorrhizalcolonization of V. lutea ssp. sudetica, but did not provide informationabout the degree of colonization or the sampling site.

Heartsease specimens from heavy metal soils had the highestZn and Pb contents when not colonized by AMF (Bukowno siteD, Trzebionka; see Tables 4 and 5). The levels of both Zn and Pbin the organs of AMF colonized heartsease and of the blue zincviolet plants were lower than in the soils (Tables 4 and 5). Suchfindings are in line with earlier data showing that AMF coloniza-tion causes a reduction of the uptake of heavy metals into plants(reviewed by Hildebrandt et al., 2007). Using specific transporterproteins, the fungi deposit heavy metals into their cell walls andsequester them into their vacuoles or out of the cells across theplasmalemma particularly in the extraradical hyphae. This resultsin a reduction of the plants’ heavy metal content upon mycorrhizalcolonization. Those heavy metals inevitably reaching the cytoplasmdespite of the action of transporters might exert toxicity, primarilyby generating reactive radicals. This is counteracted by the cells byenhanced expression of oxidative stress responsive fungal genes(Ouziad et al., 2005). Słomka et al. (2008) showed that the activi-ties of enzymes involved in oxidative stress relief and H2O2 contentdiffered in plants collected from the same sites as for the presentstudy, indicating that heartsease is a species well adapted to heavymetal contaminated soils.

When the violets were AMF-colonized, AMF counts betweendifferent specimens taken from one site showed large variations.This was the case for samples from both metalliferous and non-metalliferous sites as it is typical for samples of all plants fromdiverse biomes. As recently stated (Feldmann et al., 2009; Botheet al., 2010) the critical threshold value for an effective AMF sym-biosis might be at a degree between 20 and 30% colonization,determined by the gridline intersect method (Giovannetti andMosse, 1980). Higher colonization values of the plant roots maynot yield more effective symbioses. Moreover, fungal structures,in particular arbuscules, undergo seasonal fluctuations. Thus, nodirect correlation can be made between the degree of mycorrhizalcolonization and the heavy metal content of soils; the present studybears this out. Simple counting of the degree of root colonizationcannot give a proper assessment of the effectiveness of the plant-mycorrhiza symbiosis.

Dithizone staining showed distinct differences between heart-sease plants from a metalliferous habitat (Bukowno, site C) andfrom a non-polluted soil (central Zakopane). This method alsorevealed differential element deposition in heartsease tissues.Dithizone complexes with Zn and is often used to document higherZn concentrations in tissues, e.g. in human brain (Frederickson,1989). However, also other elements besides Zn stain with dithi-zone, for example Cd in the Brassicaceae Biscutella laevigata(Pielichowska and Wierzbicka, 2004). The procedure can be alsoused to localize heavy metals in plant organs, especially in sam-ples from polluted sites (Baranowska-Morek and Wierzbicka, 2004;Olko et al., 2008). Dithizone staining only shows qualitative differ-ences and cannot be exploited to quantify heavy metal deposits. Theparticularly intensive staining of leaf hairs was obvious in heart-sease from the heavy metal soil. This could indicate that heavymetals taken up by the plants are sequestered into hairs while stillliving. In a similar way, NaCl is deposited into hairs of halophyticspecies of the genus Atriplex, and the hairs fall off when overloaded(Lüttge, 1975). Such an adaptation may be important for metalo-phytes that can cope with high concentrations of heavy metals suchas heartsease, the zinc violets and also other non-Violaceae such asB. laevigata (Pielichowska and Wierzbicka, 2004).

Page 10

Author's personal copy

A. Słomka et al. / Journal of Plant Physiology 168 (2011) 1191–1199 1199

Acknowledgement

The authors are indebted to Zuzanna Banach, Institute of Zool-ogy, Cracow, for her help in some experiments.

References

Baranowska-Morek A, Wierzbicka M. Localization of lead in root tips of Dianthuscarthusianorum. Acta Biol Cracoviensa Ser Bot 2004;46:45–56.

Bothe H, Turnau K, Regvar M. The potential role of arbuscular mycorrhizal fungi inprotecting endangered plants and habitats. Mycorrhiza 2010;20:445–57.

Choi HW, Kim JS, Bang JW. Chromosome numbers and DNA polymorphism of Violain Korea. Korean J Genetics 1996;18:241–8.

Eriksen M, Bjureke KE, Dhillion SS. Mycorrhizal plants of traditionally managedboreal grassland in Norway. Mycorrhiza 2002;12:117–23.

Ernst WHO. Evolution of plants on soils anthropogenically contaminated by heavymetals. In: Van Raamsdonk LWD, den Nijs JCM, editors. Plant evolution in man-made habitats. Proceedings of VIIth symposium of IOPB. Amsterdam: Elsevier;1998.

Ernst WHO. Schwermetallvegetation der Erde. Stuttgart: Gustav Fischer; 1974. p.171–188.

Ernst WHO. Schwermetallpflanzen. In: Kinzel H, editor. Pflanzenökologie und Min-eralstoffwechsel. Stuttgart: E. Ulmer; 1982. p. 474–506.

Feldmann F, Gillesen M, Hutter I, Schneider C. Should we breed for effective mycor-rhiza symbioses? In: Feldmann F, Alford DV, Furk C, editors. Crop plant resistanceto biotic and abiotic factors, current potential and future demands. Braun-schweig: DPG Selbstverlag; 2009, ISBN 978-3-941261-05-1 p. 507–22.

Frederickson C. Neurobiology of zinc and zinc-containing neurons. Int Rev Neurobiol1989;31:145–238.

Füzy A, Tóth T, Hildebrandt U, Biró B, Bothe H. Drought, but not salt-load determinesthe apparent effectiveness of halophytes colonized by arbuscular mycorrhizalfungi. J Plant Physiol 2008;165:1181–92.

Giovannetti M, Mosse B. Evaluation of techniques for measuring vesicular arbuscularmycorrhizal infection in roots. New Phytol 1980;84:489–500.

Harley JL, Harley EL. A check-list of mycorrhiza in the British flora. New Phytol1987;150 SO:1–102.

Hildebrandt U, Hoef-Emden K, Backhausen S, Bothe H, Bozek M, Siuta A, et al. Therare endemic zinc violets of Central Europe originate from Viola lutea Huds. PlantSyst Evol 2006;257:205–22.

Hildebrandt U, Kaldorf M, Bothe H. The zinc violet and its colonisation by arbuscularmycorrhizal fungi. J Plant Physiol 1999;154:709–17.

Hildebrandt U, Regvar M, Bothe H. Arbuscular mycorrhiza and heavy metal toler-ance. Phytochemistry 2007;68:139–46.

Jedrzejczyk M, Rostanski A, Malkowski E. Accumulation of zinc and lead in selectedtaxa from genus Viola L. Acta Biol Cracoviensa Ser Bot 2002;44:49–55.

Kaldorf MO, Kuhn AJ, Schröder WH, Hildebrandt U, Bothe H. Selective elementdeposits in maize colonized by a heavy metal tolerance conferring arbuscularmycorrhizal fungus. J Plant Physiol 1999;154:718–28.

Krahulcová A, Krahulec F, Kirschner J. Introgressive hybridization between a native.and an introduced species: Viola lutea subsp. sudetica versus V. tricolor. FoliaGeobot et Phytotaxon 1996;31:219–44.

Lüttge U. Salt glands. In: Baker DA, Hall JL, editors. Ion transport in plant cells andtissues. Amsterdam, Oxford: North-Holland Publishing Comp; 1975. p. 335–76.

Nauenburg JD. Untersuchungen zur Variabilität, Ökologie und Systematik der Violatricolor-Gruppe in Mitteleuropa. Thesis, The University of Göttingen, Germany,1986, 126 pp.

Newman EI, Reddell P. The distribution of mycorrhizas among families of vascularplants. New Phytol 1987;106:745–51.

Noret N, Josens G, Escarré J, Lefébvre C, Panichelli S, Meerts P. Development of Issorialathonia (Lepidoptera: Nymphalidae) on zinc-accumulating and nonaccumulat-ing Viola species (Violaceae). Environ Toxicol Chem 2007;26:565–71.

Olko A, Abratowska A, Zylkowska J, Wierzbicka M, Tukiendorf A. Armeria maritimafrom a calamine heap – initial studies on physiologic – metabolic adaptationsto metal-enriched soils. Ecotoxicol Environ Saf 2008;69:209–18.

Ouziad F, Hildebrandt U, Schmelzer E, Bothe H. Differential gene expressions inarbuscular mycorrhizal-colonized tomato grown under heavy metal stress. JPlant Physiol 2005;162:634–49.

Pawłowska TE, Blaszkowski J, Rühling A. The mycorrhizal status of plants col-onizing a calamine spoil mound in Southern Poland. Mycorrhiza 1996;6:499–505.

Pielichowska M, Wierzbicka M. Uptake and localisation of Cadmium by Bis-cutella laevigata, a Cadmium hyperaccumulator. Acta Biologica Cracoviensia2004;46:57–63.

Phillips JM, Hayman DS. Improved procedures for clearing roots and stainingparasitic and vesicular arbuscular mycorrhizal fungi for rapid assessment ofinfection. Trans Br Mycol Soc 1970;55:158–68.

Prasad MNV, de Oliveira Freitas HM. Metal hyperaccumulation inplants—biodiversity prospecting for phytoremediation technology. ElectrJ Biotechnol 2003;6:285–321.

Psaras GK, Constantinidis T, Cotsopoulos B, Manetas Y. Relative abundance ofnickel in the leaf epidermis of eight hyperaccumulators: evidence that themetal is excluded from both guard cells and trichomes. Ann Bot 2000;86:73–8.

Read DJ. Mycorrhizas in ecosystems. Experientia 1981;47:376–91.Read DJ, Haselwandter K. Observations on the mycorrhizal status of some alpine

plant communities. New Phytol 1981;88:341–52.Rimkiene S, Ragazinskiene O, Savickiene N. The cumulation of Wild pansy (Viola tri-

color L.) accessions: the possibility of species preservation and usage in medicine.Medicina (Mex) 2003;39:411–4.

Schmeil O, Fitschen J. Flora von Deutschland und Angrenzenden Ländern. 94th ed.Wiebelsheim, Germany: Quelle and Meyer; 2009.

Schmitz O, Danneberg G, Hundeshagen B, Klingner A, Bothe H. Quantification ofvesicular-arbuscular mycorrhiza by biochemical parameters. J Plant Physiol1991;139:106–14.

Schwickerath M. Das Hohe Venn und seine Randgebiete. Jena: Gustav Fischer; 1944.Seregin IV, Ivanov VB. Histochemical investigation of cadmium and lead distribution

in plants. Russian J Plant Physiol 1997;44:791–6.Simon E. La dinamique de la vegetation de quelques sites metalliferés dans

les facteurs édaphiques. Bulletin Societé Royale Botan Belgique 1975;108:237–86.

Simon E. Heavy metals in soils, vegetation development and heavy metal toler-ance in plants populations from metalliferous areas. New Phytol 1978;812:175–88.

Słomka A, Libik-Konieczny M, Kuta E, Mizalski Z. Metalliferous and non-metalliferous populations of Viola tricolor represent similar mode ofantioxidative response. J Plant Physiol 2008;165:1610–9.

Svangård E, Göransson U, Hocaoglu Z, Gullbo J, Larsson R, Claeson P, et al. Cytotoxiccyclotides from Viola tricolor. J Nat Prod 2004;67:144–7.

Tonin C, Vandenkoornhuyse P, Joner EJ, Strczek J, Leyval C. Assessment of arbuscularmycorrhizal fungi diversity in the rhizosphere of Viola calaminaria and effect ofthese fungi on heavy metal uptake by clover. Mycorrhiza 2001;10:161–8.

Treseder KK, Cross A. Global distributions of arbuscular mycorrhizal fungi. Ecosys-tems 2006;9:305–16.

Vukics V, Kery A, Bonn GK, Guttman A. Major flavonoid components of heart-sease (Viola tricolor L.) and their antioxidant activities. Anal Bioanal Chem2008;390:1917–25.

Wang B, Qui Y-L. Phylogenetic distribution and evolution of mycorrhizas in landplants. Mycorrhiza 2006;16:299–363.

Wierzbicka M, Pielichowska M. Adaptation of Biscutella laevigata L., a metal hyper-accumulator, to growth on a zinc-lead waste heap in southern Poland. I.Differences between waste-heap and mountain populations. Chemosphere2004;54:1663–74.

Witkowska-Banaszczak E, Bylka W, Matławska I, Goslinska O, Muszynski Z. Antimi-crobial activity of Viola tricolor herb. Fitoterapia 2005;76:458–61.

Zabłocka W. Untersuchungen über die Mykorrhiza bei der Gattung Viola. BullIntern L’acad Polonaise Sciences Lettres Ser B: Sciences Naturelles Annee 1936:93–103.

Zubek S, Orlowska E, Turnau K. Mycorrhiza of Viola tricolor L. and Plantago lanceolataL. as indicator of succession tendencies. In: Abstract of the fourth internationalconference on Mycorrhia; 2003.