ACADIAN SEAPLANTS LIMITEDACADIAN SEAPLANTS LIMITEDACADIAN SEAPLANTS LIMITEDACADIAN SEAPLANTS LIMITED

ADVANCES IN THE USE OF ADVANCES IN THE USE OF ASCOPHYLLUM ASCOPHYLLUM NODOSUMNODOSUM SEAPLANT EXTRACTS FOR CROP SEAPLANT EXTRACTS FOR CROP

PRODUCTIONPRODUCTIONPRODUCTIONPRODUCTIONLINKING LABORATORY AND FIELD RESEARCHLINKING LABORATORY AND FIELD RESEARCH

Jeffrey Norrie, Ph.D., P.Ag. CPHJeffrey Norrie, Ph.D., P.Ag. CPHAcadian Seaplants Ltd., Dartmouth, Nova Scotia, CanadaAcadian Seaplants Ltd., Dartmouth, Nova Scotia, Canada

Major Processing FacilitiesMajor Processing FacilitiesPrince Edward IslandPrince Edward Island

New BrunswickNew Brunswick

Nova ScotiaNova ScotiaPennfield, NBPennfield, NB

Head Office Head Office Dartmouth, NSDartmouth, NS

Cornwallis, NSCornwallis, NSYarmouth, NSYarmouth, NS

M i USAM i USACharlesville, NSCharlesville, NS

Maine, USAMaine, USA

www.acadianseaplants.com

HanaHana--norinori™™

Alimentation animaleAlimentation animale

Ascophyllum nodosumAscophyllum nodosum “Norwegian Kelp” or “Knotted wrack”“Norwegian Kelp” or “Knotted wrack”

Bay of FundyBay of FundyBay of FundyBay of FundyBay of FundyBay of FundyBay of FundyBay of Fundy

R&D Challenges� Main product for agriculture:

� Ascophyllum nodosum algal extracts

� Research from lab to field on achieving maximum benefit from seaweed extracts

� Technology Transfer of information to Sales and MarketingSales and Marketing

� Get best-use information into the hands of growers and market partnersgrowers and market partners

R&D Challenges� Different countries, different crops, different

varieties, different growing conditions (soil, t t h )water, atmosphere, crop programs)

� Research field trials, coop trials, joint trials

� Draw on expertise from greater scientific community to examine seaweed products y pto understand best use strategies

Ascophyllum nodosum

Stimulate proteins and growth promoting

Growth response (cell division and differentiation)

Cause-effect bioassays

Biostimulant-Nutrient activity

growth promoting compounds

Enzymes productionMediate cellular processes

differentiation) bioassays

Foliar nutritional component

Mediate cellular processes Defensive proteinsStructural proteinsAnti-oxidants

Fruit fillingCell filling

Protection against biotic and abiotic stress Better yields

Bud retention and fruit setLonger shelf lifeHigher fruit numberBetter fruit quality

Different fractions

Different active ingredients

Different growth stages Better fruit qualityDifferent crops

Interactions with other crop inputs

ChallengeChallenge

• Sensitivity¾How do we show differences between treatments?¾Remove experimental error

• Cause and effect• Provide scientific explanations• Data Data DataData Data Data

ASL R&D Center for I nnovationASL R&D Center for I nnovation

Cornwallis Business Park, Clementsport, Nova ScotiaCornwallis Business Park, Clementsport, Nova Scotia

Benefits of Ascophyllum extracts� Many benefits are attributed to

seaweed extracts� How are these identified?

� How do we best use seaweed extracts in Agriculture?

� Starts with examination of our species, extraction technology and base constituents.

What isWhat is AscophyllumAscophyllum SeaweedSeaweed

A C l i f

What is What is AscophyllumAscophyllum Seaweed Seaweed Extract?Extract?

� A Complex mix of:• Plant growth regulators

¾ A i Gibb lli C t ki i B t i¾ Auxins, Gibberellins, Cytokinins, Betaines, • Amino acids

Organic acids• Organic acids• Carbohydrates

Vitamins• Vitamins• Macro- and micro-nutrients

Plant Growth Hormones___________________

AUXINSCYTOKININSTrans-zeatin

AUXINSIndole acetic acid

a s eatdihydro-zeatin dihydro-zeatin riboside trans zeatin riboside ABSCISSIC ACIDtrans-zeatin riboside Isopentyladenosineisopentyladenoside

ABSCISSIC ACID

GIBBERELLINSGA33GA4

Other Bioactives of Interest

Oligosaccharides Polyphenolics

Betaine Levels Betaine Levels (ppm)(ppm)

SampleSample GB GB ABABABAB AVABAVABA. nodosumA. nodosum M1M1 1515--3030 150150--350350 5050--6060SSEPSSEP 3030--6060 130130--260260 100100--200200

Acadian Seaplants R&D

� What kinds of projects are we looking at?

Acadian Seaplants R&D

p j g� Active ingredient identification:

� HPLC technologies� HPLC technologies� Carbohydrate profiling� Plant Hormones

� Gas Chromatography/Mass spectrometry� Specific molecules

� NMR technologies� Comparative profiles of molecular constituents

� In vivo testing

NMR Spectra of ASL ExtractsNMR Spectra of ASL ExtractsLocalities and SeasonsLocalities and Seasons

Ingomar NS Sep 2004

Barrington NS Jun 2004

Bear Point NS Mar 2004

Glenwood NS Jan 2004

Principal Components Analysis of Principal Components Analysis of 11HH--NMR SpectraNMR Spectra

12

14

A_14

A_15

6

8

10

G_1

H_1 H_2

I_4

0

2

4

PC2

A_1

A_2

A_3A_4A_5

A_6A_8

A_9A_10A_11A_12A_13 A_18A_19

A_20

A_21

A_22A_23A_24

A_25

A 26

A_27 A_28A_29A_30

A_31A_32 A_33A_34A_36

A 37A_38A_39A 40

A_41

A 42

A_43B_1

B_2C_1

D_1

F_1

I_1I_2

I_3

J_1

K_2

L_1L_2

-6

-4

-2_A_26 _A_35A_37A_40 A_42

E_2

E_3 K_1

-12

-10

-8 A_16A_17

E_1

-10 0 10 20PC1

HPLC Ultahydrogel Profile with PSA AssayAcadian SSEP 4318 (A1-04)

0.25

0.3

139.4

S

Very large 1,000kDa 500kDa 100kDa 50kDa 10kDa 1kDa

0.15

0.2so

rban

ce @

480

-490

nm

137.4

138.4

Inde

x O

utpu

t mV

PSA AssayRI Output

0.05

0.1

PSA

Assa

y, A

b

135.4

136.4

Refra

ctiv

e

015 17 20 22 25 27 30 32 35 37 40 42

Elution time, min

134.4

HPLC Ultahydrogel Profile with PSA AssayLSE Pressurized Cook

0.7

0.8

0.9

1

480-

490n

m

138.6

140.6

t mVPSA Assay

RI Output

Very large 1,000kDa 500kDa 100kDa 50kDa 10kDa 1kDa

0.3

0.4

0.5

0.6

A As

say,

Abs

orba

nce

@ 4

134.6

136.6

Refra

ctiv

e In

dex

Out

putp

0

0.1

0.2

15 18 20 23 25 28 30 33 35 38 40 43

Elution time, min

PSA

130.6

132.6

Points to Consider

� Different seaweed products are made in� Different seaweed products are made in different ways.Diff d h diff bi i i i� Different products have different bioactivities

� Need to identify these actives in various ymarine plant products

� Need to connect activities to specific plant� Need to connect activities to specific plant responsesGenetic/molecular responses� Genetic/molecular responses

Hypersensitive Vitamins

A ti id t

ypresponse

Vitamins

Hormones(stimulants /Anti-oxidants (stimulants /

retardants

PhenolicsOrganic

AcidsAmino acids

PolyaminesPhytoalexins Polyamines

Betaines SalicilatesJasmonatesGABA Jasmonates

Brassins

Stimuli

Stress ResistanceStress ResistanceS id di t i i b tS id di t i i b t

s/g

Superoxide dismutase in creeping bentgrassSuperoxide dismutase in creeping bentgrass

15

20

00 u

nits

5

10

ty (x

100

DM)

0

5

D ac

tivit

Low N High N

SOD

Treated Control

Virginia Polytechnical University, 1997Virginia Polytechnical University, 1997

Ascorbate peroxidase activity in Ascorbate peroxidase activity in tall fescuetall fescuetall fescuetall fescue

3500y

250030003500

se a

ctiv

ityw

t.)

100015002000

pero

xida

sol

e/h/

g fr.

0500

E+ E-scor

bate

(µ

mo

E+ E-As

Seaweed No Seaweed

Virginia Polytechnical UniversityVirginia Polytechnical University, 1997Virginia Polytechnical University, 1997

P/S capacity in creeping bentgrassP/S capacity in creeping bentgrassI h t th ti ffi i

0 390)

Improves photosynthetic efficiency

0.2

0.25

0.3ty

(Fvm

6

0.1

0.15

0.2

c ca

paci

t

0

0.05

0.1

synt

hetic

0Low N High N

Phot

o

Treated Control

Virginia Polytechnical University, 1997Virginia Polytechnical University, 1997

Resistance to Disease StressResistance to Disease Stress

University of Avignon, 1998

Resistance to Disease StressResistance to Disease Stress

University of Avignon, 1998

Seaweed extract:At least 2 applications‘Primer’ & ‘Activator’

Soil Applied

Increased

Greenerfoliage

Primer & Activatorroot & shootgrowth

(Fe & Laminarin + other microbe ‘food’)

P d l t

Feeds microbes

Produce plantgrowth stimulatingcompoundsRE

Alarm reactioninduced SAR

Direct induced SAR

(fluorescent Pseudomonads)

SiderophoreFe

ß-1,3-glucanase

induced SAR

Greater resistance Siderophore

production

Laminarin(Antagonism)

es sta ceto soilbornefungal diseases

ß-1,3-glucanases

(Fungal hyphae)Laminarin

(Antagonism)ß-1,3-glucans

Less disease (Antagonism)

Root Application Dec, 2002

Less diseasesymptomsOn plants (Also compete for space, nutrients, water & may produce antibiotics)

Acadian Seaplants R&DAcadian Seaplants R&DFocusFocus

I d th & litI d th & lit•• Increased crop growth & quality:Increased crop growth & quality:¾¾ From plant establishment to harvestFrom plant establishment to harvest

•• Enhanced crop resistance to abiotic and biotic Enhanced crop resistance to abiotic and biotic stresses:stresses:¾¾ D ht li it h t hilli f t tD ht li it h t hilli f t t l i tl i t¾¾ Drought, salinity, heat, chilling, frost, waterDrought, salinity, heat, chilling, frost, water--logging, etclogging, etc¾¾ Disease and InsectsDisease and Insects

•• Provide commercially relevant technical Provide commercially relevant technical knowledge: knowledge: ¾¾ Market support for Market support for AscophyllumAscophyllum productsproducts..

Acadian Seaplants R&DAcadian Seaplants R&Dpp

•• Bioassay testing for bioactivityBioassay testing for bioactivity•• Processing developmentProcessing development•• Formulation of active fractionsFormulation of active fractions•• ShelfShelf--life studieslife studies•• Greenhouse efficacy testingGreenhouse efficacy testing•• Local replicated field trialsLocal replicated field trials•• Target market replicated field trialsTarget market replicated field trials•• Commercialization and release of informationCommercialization and release of information

MCSB: Product Testing

0.4a

Maize Chilling Stress Bioassay

ce (F

v/Fm

)0.3

a

abbbb

bhy

ll Flu

ores

cenc

0.2c

Chl

orop

h

0 0

0.1

0.0

Control (distilled water)SSEP Control (Batch 1186)ASL

ControlExtract 1

ASL Liquid Seaweed Concentrate 29%, pH 8 (May 17, 2005; Batch 5118)ASL Liquid Seaweed Concentrate 29%, pH 8 (May 20, 2005; Batch 5131)Beijing Leili (Spain, October 2004 (rec'd))Beijing Leili (USA, March 2005 (rec'd))Algifert (Norway, March 2005 (rec'd))

Additional extract 1Additional extract 2Additional extract 3

Extract 2Extract 3Extract 4Extract 5Extract 6

Figure 20. Bioactivity (Maize Chilling Stress Bioassay (MCSB)) of controls (applied at 100 mg/l or equivalent. Different letters indicate significantly different means between treatments (α = 5%). Error bars (fractions) represent standard error (SE). (n = 30)

Extract concentration

Figure 2. Effect of batch 5054 on lettuce hypocotyls elongation in the dark after 96 hours.

Extract concentration

Figure 1. WinRhizo root scanner and associated computer software analyze plant roots for important characteristics of root physiology such as root surface area, root diameter, root volume, p y gy , , ,and the number of root tips. .

ArabidopsisSSEP 5221

p-4.5oC for 48 hoursArabidopsis canSurvive -2.6oC

Control

Total Chlorophyll Content after freezing

0.9000

1.0000

p y g

0.7000

0.8000

h W

t)

0.5000

0.6000

hyll

(mg

per g

Fre

s

0.3000

0.4000

Tota

l Chl

orop

h

0.1000

0.2000

0.0000Control 1186 5221

Treatments

Enhanced Salinity Stress ResistanceEnhanced Salinity Stress ResistanceEnhanced Salinity Stress ResistanceEnhanced Salinity Stress Resistance

Control SSEP

5221150 mM NaCl

5221

Enhanced Salinity Stress ResistanceEnhanced Salinity Stress ResistanceSSEP1186

Step 1A LMW HMW MethanolStep 1B

Pellet

Step 2A F2A1 F2A2 F2A3 F2A4 F2A5

Alginatesremoved

Alginates retained

HexaneStep 2B

removed ChloroformStep 3B

Ethyl acetateStep 4B Water

150mM NaCl 150mM NaCl + Methanol extract

0 h 24 h 48 hControl INA LA 5221 INA LA 5221

PR-5

PR-1

PR-3

PR-1

Actin

18S

• INA = control treatment known to affect PR gene expression• SSEP 5221 had no effect on PR1, PR3 or PR5 gene, g

expression.

Disease (Pseudomonas syringae) development in A bid i l tArabidopsis plants treated with SSEP 5221

Cntrl 1g/L 0.5 g/L 0.1 g/LCntrl 1g/L 0.5 g/L 0.1 g/L

Comparison of diseaseComparison of disease symptoms in leaves on plants treated with various concentrations of SSEP 5221

Cntrl 1g/L 0.5 g/L 0.1 g/LCntrl 1g/L 0.5 g/L 0.1 g/L

Ascophyllum nodosum

ASL Extracts

Fractions & compoundsASL Extracts

MolecularBiology

Bioassays

Bioactivity

BiochemicalMode of Action Discovery &y

Mode of Action

Field Trials ProgramField Trials Programh fi ld i l i l j i i lh fi ld i l i l j i i lResearch field trials, coop trials, joint trialsResearch field trials, coop trials, joint trials

Traditionally Responsive Crops� Grapes� Solanaceaes: Tomato, Peppers, Potato,

Tobacco, Eggplant, others � Apples, Strawberries, Cherries pp , ,� Stone fruits, Citrus

Other Responsive Crops� Rice (India, US, Thailand, China)� Watermelon (USSE, bioassays)� Avocados (California)� Cotton (US, International)� Olives (Spain, Greece, Australia)� Tropical crops (bananas)� Almonds� Strawberry through drip

Developing Crops� Legumes (alfalfa, beans)� Field crops (cereals, soya)p ( , y )� Grass seed production� Blueberries� Blueberries

Novel Application Methods:Novel Application Methods:Rates and Timings

� Low rate applications (10-50% of our current recommendations)� Especially Asia and S. America

� High rate applications (50% to 100% more)� Developed countries: integrated programs

� Broadcast vs Tree-Row volume (TRV)� Newer delivery systems (impregnation)� Co-applications (fertigation)� Co applications (fertigation)

Generalized Recommendations Generalized Recommendations –– Application Timings and Application Timings and Targets:Targets:gg

Applications to foliage

Seed Transplant Two applications4 weeks apart

When needed mixed with fungicides,Insecticides, nutrients and other inputs Post-harvest

(Soak)( )

(Treat/ Soak)

Applications to Roots

Growers juggling questionGrowers juggling questionGrowers juggling questionGrowers juggling question

•• B ll 1 S iB ll 1 S i•• Ball 1: ScienceBall 1: Science¾¾How many times should I apply? How many times should I apply? ¾¾LittlLittl OftOft¾¾LittleLittle--OftenOften

•• Ball 2: EconomicsBall 2: Economics¾¾ H ti I ff d t l ?H ti I ff d t l ?¾¾ How many times can I afford to apply? How many times can I afford to apply? ¾¾ Consider ROIConsider ROI

•• Ball 3: PracticalBall 3: Practical•• Ball 3: PracticalBall 3: Practical¾¾ How many times will I actually apply?How many times will I actually apply?¾¾ Tank mixes, routines, weather, labourTank mixes, routines, weather, labour, , ,, , ,

ControlControlPlot: 3 x 10’Plot: 3 x 10’Crop: Processing tomatoesCrop: Processing tomatoes

AcadianAcadianAcadianAcadianPlot: 3 x 10’Plot: 3 x 10’Crop: Processing tomatoesCrop: Processing tomatoes

ControlControlPlot: 10’ rowPlot: 10’ rowCrop: Green PeppersCrop: Green PeppersRep: 3Rep: 3

TreatedTreatedPlot: 10’ rowPlot: 10’ rowCrop: Green PeppersCrop: Green PeppersRep: 2Rep: 2Rep: 3Rep: 3 Rep: 2Rep: 2

Chart 10: Acadian on Strawberries - Ventura County, Winter 2007 - Cumulative Marketable Production Net Return by Pick Day

$22,000.00

$24,000.00

$14,000.00

$16,000.00

$18,000.00

$20,000.00

er A

cre

$4,000.00

$6,000.00

$8,000.00

$10,000.00

$12,000.00

Dol

lars

pe

Break even point approximately $18,000

$0.00

$2,000.00

$ ,000 00

1/11

/200

7

1/15

/200

7

1/19

/200

7

1/24

/07

1/27

/07

2/1/

07

2/6/

2007

2/9/

2007

2/14

/200

7

2/17

/200

7

2/21

/200

7

2/24

/200

7

2/26

/200

7

3/1/

2007

3/5/

2007

3/8/

2007

3/10

/200

7

3/13

/200

7

3/16

/200

7

3/19

/200

7

3/22

/200

7

3/24

/200

7

3/27

/200

7

3/30

/200

7

4/2/

2007

4/5/

2007

4/7/

2007

4/11

/200

7

4/13

/200

7

4/18

/200

7

4/21

/200

7

4/25

/200

7

4/30

/200

7

5/4/

2007

5/9/

2007

1 2 3 4 5 6 7 8 9 10 11 12 12 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35

Pick DayGrower Standard Acadian

Effect Of Acadian, Soil-Applied Post Veraison On Rachis Length In CRIMSON and RED GLOBE Grapes, Fresno, CA. 2002

ACADIAN

30

CONVENTIONAL

25

15

20

5

10

R hi T t l L th (CM) CRIMSON R hi T t l L th (CM) RED GLOBERachis Total Length (CM) CRIMSON Rachis Total Length (CM) RED GLOBE

Tobacco

A: TreatedB: Control

BEANS UNDER WATER STRESS

TREATEDCONTROL

Benefits of Benefits of AscophyllumAscophyllump yp yTake Home MessagesTake Home Messages

•• Benefits through foliar applications:Benefits through foliar applications:•• Improved plant nutrition, growth and Improved plant nutrition, growth and

d l td l tdevelopmentdevelopment

•• Improved fruit quality and quantityImproved fruit quality and quantity

•• Improved efficiency of crop inputsImproved efficiency of crop inputs

•• Enhanced disease insect and nematodeEnhanced disease insect and nematodeEnhanced disease, insect and nematode Enhanced disease, insect and nematode repressionrepression

Benefits of Benefits of AscophyllumAscophyllum

•• Benefits through soil applicationsBenefits through soil applications ::

p yp yTake Home MessagesTake Home Messages

Benefits through soil applicationsBenefits through soil applications ::¾¾Stimulate plant beneficial microbes and Stimulate plant beneficial microbes and

increase soil suppressiveness to diseasesincrease soil suppressiveness to diseasesincrease soil suppressiveness to diseasesincrease soil suppressiveness to diseases

¾¾ Induce a direct SAR effect via the rootsInduce a direct SAR effect via the roots

L d t b tt t bli h tL d t b tt t bli h t•• Leads to better crop establishment, Leads to better crop establishment, improved crop health and ultimately improved crop health and ultimately increased productivity plus profitability (to increased productivity plus profitability (to growers)growers)

Benefits of Benefits of AscophyllumAscophyllump yp yTake Home MessagesTake Home Messages

•• Ascophyllum nodosumAscophyllum nodosum extract is a extract is a general general biostimulantbiostimulant¾¾(vitamin(vitamin--like effects)like effects)¾¾Substantial research shows SAR benefitsSubstantial research shows SAR benefits

•• Can be used many times during the seasonCan be used many times during the season¾¾Foliar and/or soil applicationsFoliar and/or soil applications

•• Stimulates growth and development at time of Stimulates growth and development at time of applicationapplication

•• Can use often without toxicity problemsCan use often without toxicity problemsy py p

Benefits of Benefits of AscophyllumAscophyllume e ts oe e ts o scop y uscop y uTake Home MessagesTake Home Messages

•• Seaweed products can be made in different ways Seaweed products can be made in different ways (e.g. species, processing and extraction)(e.g. species, processing and extraction)¾¾May result in different activitiesMay result in different activities¾¾May result in different activities.May result in different activities.

•• Identifying active ingredients in Identifying active ingredients in AscophyllumAscophyllum extractsextracts¾¾FootFoot printing molecular profiles of our productsprinting molecular profiles of our products¾¾FootFoot--printing molecular profiles of our productsprinting molecular profiles of our products

•• Connecting activities with specific plant responsesConnecting activities with specific plant responses¾¾ In controlled bioassaysIn controlled bioassays¾¾ In controlled bioassaysIn controlled bioassays¾¾ In the fieldIn the field

•• Delivering bestDelivering best--use application information touse application information toDelivering bestDelivering best--use application information to use application information to growers and endgrowers and end--users on ongoing basisusers on ongoing basis

Stimulate proteins and growth promotinggrowth promoting

compounds