new proximal sensors of vegetation: towards a non

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1Ebernburg, October 2012

New proximal sensors of vegetation:

towards a non destructive quantitative

estimation of plant constituents

Zoran G. Cerovic

CNRS, Univ. Paris-Sud, Orsay, France

[email protected]

"to see the invisible"

VIS UV

Ebernburg-Workshop "Leaf Optics" 10-12 October 2012

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Plant pigments as indicators of crop status, leaves & fruits

Chlorophyll a & b

+OHO

OCH3

OH

OGlu

OH

OCH3

Oenin

Quercitrin

OHO

OH

OH

O

OGlu

OH

Chlorophylls in leaves & (grapes)

! Nitrogen & (maturity)

Anthocyanins (leaves) & grape skin

! Colour ! Phen

Flavonols leaves & grapes ! Nitrogen! Phen

! Light

UV IR

Flav Car Anth Chl

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Analytical optical methodsNon-destructive

Destructive

Extraction &

Assay

Sugar

Malic acid

Anthocyanins

Total Phenolics

Refractrometry

Absorbance

Spectral bands

Perkin Elmer

Varian

Agilent…

FT-NIRS

All constituents

Reflection

Spectra

Foss

Colorimetry

Colour

Hue

(CIELab)

(CIRG)

Reflection

(Absorptance)

Spectral bands

Spectra

Diostem (imaging)

Minolta CM-2600d

Agilent

Vis

Near infrared &

Chemometics

All constituents

Reflection

Transmission

Spectra

Luminar 5030

Nirvana

(Spectron)

NIR

Fluorescence

Anthocyanins

Flavonols

Chlorophyll

BGF

Fluorescence

Absorbance

(screening)

Spectral bands

Dualex 3

Dualex 4

Multiplex 3

Mounted Mx 3

VisUV

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Advantages of optical sensor solutions

• non-destructive

• non-contact (remote)

• rapid (light)

• high frequency

• large surfaces

• large sampling

• mapping possible

Optical sensing

• more sensitive

• more precise

• established protocols

• limited sampling

• labour intensive

• delayed results

chemical analysis

Field and production monitoring

compared to

Qualitative Quantitative

Field & Laboratory Laboratory

Quantitative

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Leaf optical properties in the UV-VIS - Ebernburg 2006

In the range 220 - 360 nm

Transmittance = 0.24 - 0.32 %

Reflectance = 4.2 - 5.8 %

For 10 crops

Gausman et al. (1975)

Rodriguez & Gausman (1977)

Grant et al. (2003)

UV-B

T = 0 %

R = 5 %

A B C

100

80

60

40

20

0

absorptance (%)

700600500400300wavelength (nm)

400

300

200

100

0

fluorescence (QSEU)

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Leaf and grape absorbers - grape quality factors - Ebernburg 2009

FLAV = log(FRF_R/FRF_UV)

FlavonolsCerovic et al. (2002) PCE, 25, 1663

SFR_G = FRF_G/RF_G

ChlorophyllsAgati et al. (1993) JPPBB, 17, 163

ANTH = log(FRF_R/FRF_G)

ANTH_GR = log(FRF_G/FRF_R)

FERARI = log(5000/FRF_R)

BRR_FRF = YF_UV/FRF_UV

AnthocyaninsCerovic et al. (2008) JAFC, 55, 1053

Cerovic et al. (2007) 6thECPA

NBI_G = FRF_UV/RF_G

Nitrogen deficiency

Meyer et al. (2005) ECPA, 55, 1053

Cartelat et al. (2003) 4thECPA

Cerovic et al. (2005) 5thECPA

Multiplex indices

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Optical sensors

Dualex:

Chlorophylls

Flavonols

Multiplex:

Chlorophylls

Flavonols

Anthocyanins

(Stilbenes)

leaf-clip

proximal sensor : leaves and grapes

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The Dualex 4 leaf-clip

Chlorophylls

(Epidermal) Flavonols

Data logger

GPS

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Dualex 4 - Pigments as indicators of nitrogen nutrition

Chlorophyll a & b

Chlorophylls

! Nitrogen

Quercitrin

OHO

OH

OH

O

OGlu

OH

Flavonols

! Light (LMA)

1G Lab 1999 & Field

2000

2 G 2003 4G, 20093G, 2005/06

DUALEX: from 1G to 4 Generation

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UV NIRChlorophyll in vivo

Chlorophyll a in solution

Chlorophyll fluorescence screening basis of the ABC method

Cerovic et al. (1999) Agronomie, 19: 543

upper

epidermis

lower

epidermis

mesophyll

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Flavonoids are present in theepidermis. They absorb UV radiationand screen the mesophyll.

Chlorophyll from the mesophyll emitsnear-IR fluorescence measurable onboth sides of the leaf.

Dualex leaf-clip based on the ABC method

Goulas et al. (2004) Applied Optics 43, 4488-4496

Red

Ultra-

violet

LED

Infra-red fluorescence

Photodiode

Leaf

section

under UV

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Dualex flavonols - quantitative linear responseCerovic et al. (2012) Physiol Plant, 146: 251

Dualex 3 calibration with

absorbance standards

Dualex 4 to Dualex 3 comparison

Louis et al. (2009) Funct Plant Biol, 36: 732

Dualex 3 vs. oak-leaf extracts

Dualex and UV-A PAM

calibration and comparison

Pfündel et al. (2007) Photosynth Res 93: 205

Barthod et al. (2007) J Exp Bot, 36: 1753

Dualex units

0 1 2 3 4

Maple and ash leaf

extracts vs. Dualex 2

Goulas et al. (2004) App Optics 43: 4488

Wheat leaf extracts

vs. Dualex 1

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Dualex 4 chlorophyll: quantitative linear response

Cerovic et al. (2010) Physiol Plant, 146: 251

repeatability

< 1%

reproducibility

< 3%

accuracy

16%

(single species)

(7%)

k[(I(850)/Io(850)/I(710)/Io(710))-1] + c

k[log(I(940)/Io(940)) - log(I(650)/Io(650))] + c

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1. The opposite dependence on nitrogenincreases the dynamic range

2. The parallel dependence

on leaf age decreasesleaf position influence

Three Beneficial effects

3. The ratio of two

surface-based measurements

avoids the influence of LMA

60

50

40

30

20

Chl/Phen

2520151050

azote (kg/ha) 240

160

60

0

The Chl/Flav ratio: Nitrogen Balance Index (NBI)

N%

PHEAllocation

Prot Phen

Protm

Chlm

Chla

Phenm

Flavm

Flava

SPAD Dualex 3

LMA

Prot, Phen Allocation

Dry mass

Dualex 4

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The Multiplex proximal sensor for leaves and fruits

Chlorophylls

(epidermal) Flavonols

Anthocyanins

Stilbenes

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Multiplex proximal sensor - a multiparametric sensor

1.5 - 2005 2G - 2007 3G – 2008 / 2009

4G

MULTIPLEX: towards 4G

COOH

OH

OH

Caffeic acid

Chlorophyll a & b

Quercitrin

OHO

OH

OH

O

OGlu

OH

+OHO

OCH3

OH

OGlu

OH

OCH3

Oenin

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20 !s flashes4 excitations3 emission channelsRepeated 500 times6000 measurements0.5 s per sampleIn situUnder daylight

The Multiplex sensor

Emission (nm) Excitation

UV Blue (B) Green (G) Red-Orange (R) YF (590) YF_UV YF_B = R YF_G = R YF_R = R

RF (685) RF_UV RF_B RF_G RF_R

FRF (735) FRF_UV FRF_B FRF_G FRF_R

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Chlorophyll estimation from fluorescence reabsorption

Rabinowitch (1951) Book100

80

60

40

20

0

absorptance (%)

800700600500400wavelength (nm)

400

300

200

100

0

fluorescence (QSEU)

685RF

735FRF

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Multiplex chlorophyll-related signals and ratios

fruits leaves

(grapevine) (kiwi)

fruits leaves

SFR

signals ratios

Babani et al. (1996) JPP, 148: 471

Betemps et al. (2012) J Sci Food Agric 92: 1855 Tremblay et al. (2012) Agron Sustain Dev 32: 451

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Multiplex FLAV index - leaf epidermal flavonols

integrated irradiance meter disease susceptibility

Agati et al. (2008) Funct. Plant Biol. 35: 77

Agati et al. (2011) Environ. Exp. Bot. 73: 3

Dalla Marta et al. (2008) Sci. Agric. (Piracicaba, Braz.) 65: 65

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Mapping and zoning of leaves in viticulture

LD Chl Flav Chl/Flav LD*Chl/Flav

Martin et al. (2007)

Meggio et al. (2010)

Mabrouk et al. (1998)

Stamatiadis et al. (2010)

Bavaresco & Eibach (1987)

Agati et al. (2008)

Cerovic et al. (2007) patent

Cerovic et al. (2009)

Debuisson et al. (2012)

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Chlorophyll fluorescence excitation screening in berries

Skin (grape berry exocarp):

Single layer of clear epidermal cells

Six hypodermal layers

Agati et al. (2007) JAFC, 55, 1053-1061

A B

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Multiplex berry anthocyanins - quantitative non-linear response

ANTH_RG = A[exp(-aR Anth) - exp(-aG Anth)] + log(!RG)

Ben Ghozlen et al. (2010) Sensors, 10:10040 signals ratios

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Multiplex-330: Stilbenoid fluorescence

UV-exited

"blue"

fluorescence

HCA

Fungi

stilbenesResveratrol

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Disease detection: Downy mildew in grapevine

Bellow et al. (2012) submitted to JXB

Bellow et al. (2012) J Exp Bot 63:3697

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Plant Biospectroscopy team

ZoranCerovic

SylvieMeyer

GwendalLatouche

Plant Ecophysiology DepartmentEcology, Systematics and Evolution Laboratory

University Paris-Sud XI - CNRS UMR 8079

ConstanceLaureau

SebastienBellow

PeterStreb

Jean-MarcDucruet

Thanks to:Jean-Luc Ayral

Nicolae Moise

Naïma Ben Ghozlen

Marine Le Moigne

Sophie Lejealle

Guillaume

Masdoumier

Aurélie Cartelat (Paris)

Juliette Louis (Paris)

Erwin Dreyer (INRA-Nancy)

Yves Goulas (Palaiseau)

Ismaël Moya (Palaiseau)

Eric Serrano (Toulouse)

Sébastien Debuisson (CIVIC-Epernay)

Giovanni Agati (Firenze)

Erhard Pfündel (Würzburg)

Fermin Morales (Zaragosa)

Guy Samson (Trois-Rivières)

Nicolas Tremblay (Montreal)

Kathrin Bürling (Bonn)

France

FORCE-AInternational

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Drawing logos using light

David Alan Walker

(1928-2012)

Botanisches InstitutAbt. Ökophysiologie der PflanzenProf. Dr. W. BilgerAm Botanischen Garten 3 - 924118 Kiel

VISUV-A

new proximal sensors of vegetation: towards a non

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