I. Cakmak Sabanci University Istanbul, Turkey

cakmak@sabanciuniv.edu

Detrimental Effects of Glyphosate Detrimental Effects of Glyphosate on Nutritional Status of Plants on Nutritional Status of Plants

with Micronutrientswith Micronutrients

Glyphosate:Glyphosate: extensively applied herbicide

• Increasing usage in minimum- and no-tillage agricultural practices

• Extensive application on RR crops

.

In regions with extensive use of glyphosate there are increasing reports on:

• reductions in growth and yield, • increases in disease problems,• increased use of insecticides and fungicides,• inhibition of N fixing bacteria• increased use of foliar micronutrient fertilizers• micronutrient deficiencies

Particular micronutrient deficiencies induced by

Glyphosate: Manganese and Iron Deficiencies

Repeated use of glyphosate induces Fe and Mn deficiencies in soybeans

in USA

(Photo: Prof. Don Huber)

(areas which are not treated with glyphosate)

+ gyphosate

+ seed Fe treatment

+ gyphosate

– seed Fe treatment

- gyphosate

+ seed Fe treatment

Photo: N.C. Hansen, Fort Collins, USA

Glyphosate-induced Fe-deficiency chlorosisGlyphosate-induced Fe-deficiency chlorosis

(Jolley et al., Soil Sci Plant Nutr. 50, 793-981, 2004)

visual chlorosis scrore grain yield Treatment (1=green to 5 =severe) (t/ha)

– Fe + Fe* – Fe + Fe*

Control 3.1 2.8 1.01 1.70 (no herbicide)

Glyphosate 3.7 3.3 0.27 0.61

* 50g Fe/ha as FeEDDHA applied to seeds

Interaction of seed applied Fe and glyphosate application on Fe deficiency chlorosis in soybeans; Minnesota, USA

(Jolley et al., Soil Sci Plant Nutr. 50, 793-981, 2004)

Foliar and Soluble Micronutrient* Sales in USA

0

500

1000

1500

2000

2500

3000

3500

4000

4500

5000

B Cu Fe Mn Zn

20002001200220032004

(*represents approx 30% of US Sales)

(Courtesy of Prof. P. Brown, UC Davis)

RoundUp Ready Corn, Soy

Glyphosate is toxic to plants whether from a drift or residues in soil

http://www.uky.edu/Ag/Tobacco/Pages/RoundupFloat.html

A significant amount of glyphosate applied to target plants reaches the soil as a result of direct contact, wash off from leaves and exudation from roots of the treated weeds.

entweb.clemson.edu.

Up to 10 % of the applied glyphosate move to non-target plants

no Glyphosate

withGlyphosate

Application of glyphosate on target plants (weeds)/glyphosate resistant cultivars; very quick uptake by leaves.

Rapid translocation of glyphosate from shoot to root

REMEMBER

Glyphosate is highly phloem mobile

Release of glyphosate into therhizosphere

Marschner and Römheld, 1995; Plant and Soil

Rhizosphere/SoilPlasma

MembraneApoplasm Cytoplasm

H+

RFeIII ChelateChelator

Reductants

(Chelators)

H+

FeII

FeIII

FeIII

FeIII

FeIII

TR

PUMP

Adaptive Root Responses of Strategy-I Plants to Fe Deficiency

R: Ferric ReductaseR

Effect of iron deficiency in cucumber on pH decreasing and reducing capacity of the roots

+Fe +Fe-Fe-Fe

Romheld et al., 1981

Time dependent inhibition of ferric reductase activity in Fe-deficient sunflower plants (+Glyp: 1.89 mM glyphosate

or 6% of the recommended rate for weed control)

0

20

40

60

80

-Glyp. +Glyp.6h

+Glyp.12h

+Glyp.24h

-Glyp.+Fe

Fe

redu

ctio

n ca

paci

ty

(µm

ol F

e(II

) g

- 1 r

oot

dry

wt

3 h

- 1)

LSD0.05

Fe deficient

Control (+Fe)

Ozturk et al., 2007

Effect of Glyphosate on Ferric Reductase

Dose dependent inhibition of ferric reductase activity by glyphosate (+Glyp: 0.32, 0.95 and 1.89 mM

0

20

40

60

80

-Glyp. +Glyp.0.32 mM

+Glyp. 0.95 mM

+Glyp. 1.89 mM

-Glyp. +Fe

Fe

redu

ctio

n ca

paci

ty

(µm

ol F

e(II

) g

- 1 r

oot

dry

wt

3 h

- 1)

LSD0.05

Fe deficient

Control (+Fe)

Ozturk et al., 2007

Foliar applications of glyphosate (Glyp) at 0.32 mM, 0.95 mM and 1.89 mM for 24h.

Ozturk et al., 2007

Staining of Ferric Reductase Activity

GLYPHOSATE

Fe Deficient Plants

-Fe -Fe +Glyp (1.89 mM)

Effect of foliar applied of glyphosate (1.89 mM ) for 24hon ferric reductase activity.

Ozturk et al., 2007

Shikimate accumulation(µmol g -1 FW)

0

3

6

9

12

15

ShootShoot

Do

se D

epen

den

t S

hik

imat

e A

ccu

mu

lati

on

Ozturk et al., 2007

0

2

4

6

8

Shikimate accumulation(µmol g -1 FW)

RootRoot

Ozturk et al., 2007

Do

se D

epen

den

t S

hik

imat

e A

ccu

mu

lati

on

Shoot Root

Shikimate accumulation(µmol g -1 FW)

0

3

6

9

12

15

0

2

4

6

8

Time Dependent Shikimate Accumulation

Ozturk et al., 2007

ROOT UPTAKE TRANSLOCATION TO SHOOT

0

5

10

15

20

25 59Fe

LSD0.05=7.0

0

200

400

600

800 59Fe

LSD0.05=108

0

25

50

75

100

12554Mn

LSD0.05=3.6

0

300

600

900

120054Mn

LSD0.05=22.5

0

25

50

75

10065Zn

LSD0.05= ns

0

750

1500

2250

300065Zn

LSD0.05= ns

Ro

ot

up

tak

e r

ate

(

nm

ol

g-1 r

oo

t d

ry w

t 4

h-1)

Sh

oo

t tr

an

slo

ca

tio

n r

ate

(

nm

ol

g-1 r

oo

t d

ry w

t 4

h-1)

Control Glyphosate Control Glyphosate

Effect of 1.89 mM glyphosate (equivalent to 6 % of the recommended dosage for weed control in the field) on root uptake and shoot translocation of 59Fe, 54Mn and 65Zn in sunflower plants.

Eker et al., 2006: J. Agric. Food Chem. 26, 10019 -10025

Short-Term Experiments:Glyphosate inhibits root uptake and

root-to-shoot transport of micronutrients

59Fe

54Mn

65Zn

Root Uptake Translocation

Rhizosphere/Soil PlasmaMembrane

PHYTOSIDEROPHORES

TRFeIII-PS

TR: Transporter Protein

ZnII-PS

Apoplasm Cytoplasm

FeIII

FeIII

ZnII

ZnIIFeIII-PSZnII-PS

Adaptive Root Responses to Iron and Zinc Deficiencies in Cereals

Marschner and Römheld, 1995; Plant and Soil

Biosynthetic Pathway of Mugineic Acid Family Phytosiderophores

H3CS NH2

COOH

NH NH2

COOH COOHCOOH

N

NH O

COOH COOHCOOH

N NH OH

COOH COOHCOOH

N

OH

NH OH

COOH COOHCOOH

N

3"-oxo acid

NH OH

COOH COOHCOOH

NHHO

NH OH

COOH COOHCOOH

NHO

OH

NH OH

COOH COOHCOOH

NHO

S-adenosyl-methionine

NAAT

NAS

NH OH

COOH COOHCOOH

NHO

OH

Nicotianamine

methionine

DMA

mugineic acid family phytosiderophores

AVA

MA

HMA epiHMA

epiHDMA

IDS3

IDS3

IDS2

IDS2

DMAS

SAMS

Mugineic Acid Family

Mori et al., 1990

Fe OH FeNH2

Fe3+-Deoxymugineic acid Fe3+-Nicotianamine

Similar Stereochemical Structure between Fe3+-DMA and Fe3+-NA

Mori, et al, 1990

PhytosiderophorePhytosiderophoreReleaseRelease fromfrom RootsRoots

0

40

80

120

160

Ph

yto

sid

ero

ph

ore

re

lea

se

(nm

ol P

S p

lan

t-1 3

h-1)

+Fe

-Fe, 0% Glyp

-Fe, 1% Glyp

-Fe, 3% Glyp

-Fe, 9% Glyp

+Fe -Fe Ozturk et al., 2008

Phytosiderophore Release from Wheat Roots

0

40

80

120

160

Ph

yto

sid

ero

ph

ore

re

lea

se

(nm

ol P

S p

lan

t-1 3

h-1)

+Fe

-Fe, 0% Glyp

-Fe, 1% Glyp

-Fe, 3% Glyp

-Fe, 9% Glyp

Glyphosate ApplicatonOzturk et al., 2008

Control (+Fe, -Glyp)

-Fe, -Glyp -Fe, 1% Glyp

-Fe, 3% Glyp -Fe, 9% Glyp

Control (+Fe, -Glyp)

-Fe, -Glyp -Fe, 1% Glyp

-Fe, 3% Glyp -Fe, 9% Glyp

0.0

0.1

0.2

0.3

0.4

Mob

ilize

d Z

n (µ

g g-1

soi

l)

Mob

ilize

d Z

n, µ

g -1

soil

Mobilization of Zn from Calcareous Soil by Phytosiderophores

Glyphosate ApplicationOzturk et al., 2008

0.0

0.1

0.2

Mob

ilize

d M

n, µ

g -1

soil

Control (+Fe, -Glyp)

-Fe, -Glyp -Fe, 1% Glyp

-Fe, 3% Glyp -Fe, 9% Glyp

Control (+Fe, -Glyp)

-Fe, -Glyp -Fe, 1% Glyp

-Fe, 3% Glyp -Fe, 9% Glyp

Mobilization of Mn from Calcareous Soil by Phytosiderophores

Glyphosate ApplicationOzturk et al., 2008

Application of glyphosate on target plants (weeds)/glyphosate resistant cultivars; very quick uptake by leaves.

Rapid translocation of glyphosate from shoot to root

REMEMBER

Glyphosate is highly phloem mobile

Release of glyphosate into therhizosphere

Induction of Fe deficiency chlorosis in non-target plants (sunflower)

Glyphosate application to target plants (soybean)

Development of Fe deficiency symptoms in non-target plants …

Glyphosate applied to target plants

is released into the rhizosphere

SoybeanTarget

SoybeanTarget

SoybeanTarget

SoybeanTarget

SunflowerIndicator

SunflowerIndicator

(Neumann et al., 2005, J. Plant Dis. Prot.)

Inhibited Mn acquisition by non-target plants (sunflower) grown in nutrient solution together with glyphosate treated target plants (soybean) for 2, 4 and 6 days.

54Mn in shoots

Glyphosate released in the rhizosphere reduces Mn uptake

Neumann et al., 2005, J. Plant Dis. Prot.

KS 4202 RR

KS 4202

Significantly lower Mn uptake/accumulation in RR-soybeans

(B.Gordon, 2006; Kansas State Univ.)

Inhibition of MnO2 Reduction in Soil-filled Rhizoboxes after Glyphosate Treatment to Target Plants (soybean)

(Neumann et al., 2005)

Control + Glyphosate

MnO2 Reduction in Soil-filled Rhizobox Culture of sunflower (non-target) and RR-Soybean with and without foliar Glyphosate spray

Glyphosate Reduces Grain Mn, Mg and Ca

Glyphosate Application, %Glyphosate Application, %

0 0.3 0.6 0.9

0.0

2.0

4.0

6.0

8.0

control 0.3 0.6 0.90

2

4

6

8

10

12

control 0.3 0.6 0.90

4

8

12

16

20

control 0.3 0.6 0.9

Dry

mat

ter

yiel

d, g

pla

nt-1

Glyphosate treatment, % of recommendedGlyphosate treatment, % of recommended

Grain Straw Whole plant

GGrain, straw and whole plant rain, straw and whole plant ddryry matter yield as matter yield as affected by drift rates of glyphosate in soybeanaffected by drift rates of glyphosate in soybean

Glyphosate Applied: 3 Times; one before flowering and 2 times after flowering

Control 0.3 0.6 0.9 Control 0.3 0.6 0.9 Control 0.3 0.6 0.9

Yazici et al., 2008

Glyphosate applied 3 times before flowering

Gra

in c

onc

ent

ratio

n,

%

Glyphosate treatment, % of recommended

K Mg Ca

Grain concentration of K, Mg and Ca as affected by drift rates of glyphosate in soybean

0.6

0.9

1.2

1.5

1.8

control 0.3 0.6 0.90.10

0.15

0.20

0.25

0.30

control 0.3 0.6 0.90.2

0.3

0.4

0.5

control 0.3 0.6 0.90 0.3 0.6 0.90 0.3 0.6 0.90 0.3 0.6 0.9

GlyphosateGlyphosate treatment, % of recommendedtreatment, % of recommended

K Mg Ca

Yazici et al., 2008

Gra

in c

once

ntra

tion,

m

g kg

-1

Grain concentration of Grain concentration of FeFe, M, Mnn and and ZnZn as as affected by drift rates of glyphosate inaffected by drift rates of glyphosate in soybeansoybean

Fe Mn Zn

30

40

50

60

70

80

90

100

control 0.3 0.6 0.930

40

50

60

70

80

control 0.3 0.6 0.920

30

40

50

60

control 0.3 0.6 0.9

Glyphosate treatment, % of recommendedGlyphosate treatment, % of recommended

0 0.3 0.6 0.9 0 0.3 0.6 0.90 0.3 0.6 0.9

Fe Mn Zn

Yazici et al., 2008

Seed Mn and Ca very importantfor seed viability and seedling vigour

0

20

40

60

80

100

120

140

control 0.3 0.6 0.90

200

400

600

800

control 0.3 0.6 0.9

Dry matter yield of young Dry matter yield of young andand old leaves old leaves of of soybeansoybeanplantsplants as affected by drift rates of glyphosateas affected by drift rates of glyphosate

Young leaves Old leaves

Dry

mat

ter

yiel

d,

g pl

ant-1

GlyphosateGlyphosate treatment, % of recommendedtreatment, % of recommended

0 0.3 0.6 0.90 0.3 0.6 0.9

Yazici et al., 2008

K Mg Ca

Concentration of K, Mg and Ca in soybean oncentration of K, Mg and Ca in soybean leaves as affected by drift rates of leaves as affected by drift rates of glyphosateglyphosate

Leaf

conc

entr

atio

n, %

1.5

2.0

2.5

3.0

3.5

control 0.3 0.6 0.9

0.2

0.3

0.4

0.5

0.6

0.7

control 0.3 0.6 0.9

0.6

0.9

1.2

1.5

1.8

control 0.3 0.6 0.9

Glyphosate treatment, % of recommendedGlyphosate treatment, % of recommended

0 0.3 0.6 0.9 0 0.3 0.6 0.9 0 0.3 0.6 0.9

Hande et al., 2008

Hande et al., 2008

Plants with low Mg are highly Plants with low Mg are highly sensitive to glyphosatesensitive to glyphosate

Hande et al., 2008

Plants with low Mg are highly Plants with low Mg are highly sensitive to glyphosatesensitive to glyphosate

What is the effect of Glyphosate??

Glyphosate binds with the cations to form a strong complex which is not bio-available.

Only unbound glyphosate act as a herbicide. www.loveland.co.uk/ Gifs/X-Change-du-pont.gif

Control GlyphosateGlyphosate+ Calcium

Effect of Glyphosate with and without calcium in the tank

Effectiveness of Glyphosate

GlyphosateGlyphosate binds with the cations to form a strong complex which is not bio-available. Only unbound glyphosate act as a herbicide.

Conclusions

• Routine glyphosate use in agricultural systems results in considerable side effects on plant growth and mineral nutrition of plants

• Glyphosate is antagonistic to the uptake, transport and accumulation (tissue concentration) of Fe,Mn, Ca and Mg possibly due to the formation of poorly soluble glyphosate-metal complexes (??)

Conclusions-cont

• Glyphosate impairs genetic adaptation mechanisms of plants to Fe deficiency

• Plants grown under low Mg are very sensitive to glyphosate

• A new risk assessment for glyphosate is urgently needed,

Root Tips:Co-localization of Ferric

Reductase and Glyphosate Accumulation

Leading 10 Health Risk Factors in Developing Countries, % Cause of Disease Burden

Underweight 14.9%

Unsafe sex 10.2%

Unsafe water 5.5%

Indoor smoke 3.7%

Zinc DeficiencyZinc Deficiency 3.2%3.2%Iron deficiencyIron deficiency 3.1%3.1%Vitamin A def. 3.0%

Blood pressure 2.5%

Tobacco 2.0%

Cholesterol 1.9%WHO, 2002

Copenhagen Consensus-2004Copenhagen Consensus-2004Worldwide Panel of Distinguished Economists

including Nobel Prize-Winners

Top Four Global Challenges

Source:: http://www.copenhagenconsensus.com)

• Control of HIV/AIDS• Providing micronutrients (Fe, Providing micronutrients (Fe, Zn..) Zn..) to human populationsto human populations• Trade Liberalization• Control of Malaria

Interference of glyphosate with root uptake and transport ofmicronutrients may also represent a potential threat to human nutrition and human health

T. Tesfamariam Fanghua Ye C. Weishaar

K. Stock-de Oliveira Souza E. Landsberg S. Kohls University Hohenheim (U.H.)V. Römheld T. Yamada I. Cakmak

(S. Bott: Uni.Hohenheim)

L. Ozturk Sabanci UniversityG. Neumann

Sabanci Sabanci UniversityUniversity

Obrigado…

-Glyp. 208 ± 16 58 ± 8 21.2 ± 1.0

+Glyp. 0.32 mM 202 ± 16 55 ± 8 21.3 ± 0.7

+Glyp. 0.95 mM 208 ± 15 56 ± 11 21.7 ± 1.4

+Glyp. 1.89 mM 194 ± 23 53 ± 9 21.8 ± 2.3

+Fe -Glyp. 226 ± 30 61 ± 13 39.2 ± 1.9

LSD0.05

-Fe

Chloropyll

(mg plant-1) (SPAD)

24 12 2.3

Dry matter productionTreatments Shoot Root

Shoot and root dry matter production and chlorophyll level as influenced from glyphosate application for 24 h

Ozturk et al., 2007

0

3

6

9

12

15

Tot

al c

arbo

hydr

ates

(mg

gluc

ose

equi

v. g

-1 F

W)

Carbohydrates in Apical Parts of Roots

Ozturk et al., 2007

+ Glyphosate

Marschner and Römheld, 1995; Plant and Soil

RhizospherePlasma

MembraneApoplasm Cytoplasm

H+

RFeIII ChelateChelator

Reductants

(Chelators)

H+

FeII

FeIII

FeIII

FeIII

FeIII

TR

PUMP

Root Responses to Fe Deficiency in Strategy-I Plants

NAD(P)H

NAD(P)+

R: Ferric ReductaseR

0

10

20

30

40N

AD

PH

oxi

datio

n(n

mol

min

-1 g

-1 F

W)

Capacity of Roots to Oxidize NADPH

Ozturk et al., 2007

+ Glyphosate

Close Relationship between Fe Deficiency Tolerance and Ferric Reductase Activity in Soybean

Marschner and Romheld, 1995

Soybean Genotypes

Rel

ease

of

chel

atin

g c

om

po

un

ds

(µm

ol

Fe

equ

iv.

g-1 f

resh

wt.

h-1)

0

10

20

30

40

50

60

+Fe

-Fe

Release of Phytosiderophores from Roots Correlate with Tolerance Fe Deficiency Chlorosis

rice sorghum maize oat Wheat

(Römheld & Marschner (1986): Adv. in Plant Nutr 2, 155-204)

Chlorosis resistance