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1 ACADIAN SEAPLANTS LIMITED ADVANCES IN THE USE OF ASCOPHYLLUM NODOSUM SEAPLANT EXTRACTS FOR CROP PRODUCTION LINKING LABORATORY AND FIELD RESEARCH Jeffrey Norrie, Ph.D., P.Ag. CPH Acadian Seaplants Ltd., Dartmouth, Nova Scotia, Canada

2 Major Processing Facilities Prince Edward Island New Brunswick Pennfield, NB Nova Scotia Maine, USA Yarmouth, NS Cornwallis, NS Charlesville, NS Head Office Dartmouth, NS

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5 Hana--nori Hana

6 Alimentation animale

7 Ascophyllum nodosum Norwegian Kelp or Knotted wrack

8 Bay of Fundy

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11 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 Marketing Get best-use information into the hands of growers and market partners

12 R&D Challenges Different countries, different crops, different varieties, different growing conditions (soil, water, atmosphere, crop programs) Research field trials, coop trials, joint trials Draw on expertise from greater scientific community to examine seaweed products to understand best use strategies

13 Ascophyllum nodosum Biostimulant-Nutrient activity Stimulate proteins and growth promoting compounds Enzymes production Mediate cellular processes Defensive proteins Structural proteins Anti-oxidants Growth response (cell division and Cause-effect differentiation) bioassays Foliar nutritional component Cell filling Fruit filling Protection against biotic and abiotic stress Different fractions Different active ingredients Different growth stages Different crops Interactions with other crop inputs Better yields Bud retention and fruit set Longer shelf life Higher fruit number Better fruit quality

14 Challenge Sensitivity How do we show differences between treatments? Remove experimental error Cause and effect Provide scientific explanations Data Data Data

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17 ASL R&D Center for Innovation Cornwallis Business Park, Clementsport, Nova Scotia

18 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.

19 What is Ascophyllum Seaweed AC Complex mix of: Extract? Plant growth regulators Auxins, Gibberellins, Cytokinins, i Betaines, Amino acids Organic acids Carbohydrates Vitamins Macro- and micro-nutrients

20 Plant Growth Hormones CYTOKININS Trans-zeatin dihydro-zeatin dihydro-zeatin riboside trans-zeatin riboside Isopentyladenosine isopentyladenoside AUXINS Indole acetic acid ABSCISSIC ACID GIBBERELLINS GA 3 GA 4

21 Other Bioactives of Interest Oligosaccharides Polyphenolics

22 Betaine Levels (ppm) Sample GB ABAB AVAB A. nodosum M SSEP

23 Acadian Seaplants R&D What kinds of projects are we looking at? Active ingredient identification: HPLC technologies Carbohydrate profiling Plant Hormones Gas Chromatography/Mass spectrometry Specific molecules NMR technologies Comparative profiles of molecular constituents In vivo testing

24 NMR Spectra of ASL Extracts Localities and Seasons Ingomar NS Sep 2004 Barrington NS Jun 2004 Bear Point NS Mar 2004 Glenwood NS Jan 2004

25 Principal Components Analysis of 1 H-NMR Spectra 14 A_15 12 A_14 10 I_4 8 G_1 6 H_1 H_2 4 K_2 PC B_2 F_1 L_1 B_1 C_1 L_2 I_3 A_2 A_19 A_43 A_9 A_21 A_4 A_10 A_5 A_11A_12 A_13 A_23 A_18 A_25 A_41 A_31 A_27 A_22 A_6 A_29 A_28 A_38 A_39 A_32 A_30A_20 A_24 A_33 A_34 A_36 I_1I_2 J_1 A_26 A_8 A_40 A_37 A_42 A_35 D_1-4 E_3 K_1 E_ A_16 A_ E_ PC1

26 HPLC Ultahydrogel Profile with PSA Assay Acadian SSEP 4318 (A1-04) 0.3 Very large 1,000kDa 500kDa 100kDa 50kDa 10kDa 1kDa nm PSA Assay RI Output Index Output mv PSA Assay, Ab Refractive Elution time, min HPLC Ultahydrogel Profile with PSA Assay LSE Pressurized Cook Very large 1,000kDa 500kDa 100kDa 50kDa 10kDa 1kDa PSA Assay, nm PSA Assay RI Output t mv Refractive Index Output Elution time, min 130.6

27 Points to Consider Different seaweed products are made in different ways. Different products have different bioactivities i i i Need to identify these actives in various marine plant products Need to connect activities to specific plant responses Genetic/molecular responses

28 Hypersensitive response Vitamins Hormones Anti-oxidants id t (stimulants / retardants Phenolics Organic Acids Amino acids Phytoalexins Polyamines Betaines GABA Salicilates Jasmonates Brassins

29 Stimuli

30 Stress Resistance Superoxide dismutase in creeping bentgrass SOD activit ty (x units s/g DM) Low N High N Treated Control Virginia Polytechnical University, 1997

31 Ascorbate peroxidase activity in tall fescue se activity wt.) peroxidas ole/h/g fr. scorbate (µmo As E+ E- Seaweed No Seaweed Virginia Polytechnical University, 1997

32 P/S capacity in creeping bentgrass Improves photosynthetic th ti efficiency i Photo synthetic capacit ty (Fvm6 90) Low N High N Treated Control Virginia Polytechnical University, 1997

33 Resistance to Disease Stress University of Avignon, 1998

34 Resistance to Disease Stress University of Avignon, 1998

35 Soil Applied Increased root & shoot growth Greener foliage Seaweed extract: At least 2 applications Primer & Activator (Fe & Laminarin + other microbe food ) Direct induced SAR Greater resistance sta to soilborne fungal diseases Alarm reaction induced SAR ß-1,3-glucanases Fe RE Feeds microbes (fluorescent Pseudomonads) Siderophore production (Antagonism) (Fungal hyphae) Produce plantl growth stimulating compounds ß-1,3-glucanase Laminarin Less disease symptoms On plants Root Application Dec, 2002 ß-1,3-glucans (Antagonism) (Also compete for space, nutrients, water & may produce antibiotics)

36 Focus Acadian Seaplants R&D Increased crop growth & quality: From plant establishment to harvest Enhanced crop resistance to abiotic and biotic stresses: Drought, salinity, it heat, chilling, frost, water-logging, etc Disease and Insects Provide commercially relevant technical knowledge: Market support for Ascophyllum products.

37 Acadian Seaplants R&D Bioassay testing for bioactivity Processing development Formulation of active fractions Shelf-life life studies Greenhouse efficacy testing Local replicated field trials Target market replicated field trials Commercialization and release of information

38 MCSB: Product Testing Maize Chilling Stress Bioassay 0.4 a Chloroph hyll Fluorescenc ce (Fv/Fm) c b b b ab b Control Control (distilled water) SSEP ASL Extract Control 1 (Batch 1186) ASL Extract Liquid 2 Seaweed Concentrate 29%, ph 8 (May 17, 2005; Batch 5118) ASL Extract Liquid 3 Seaweed Concentrate 29%, ph 8 (May 20, 2005; Batch 5131) Beijing Additional Extract Leili 4 extract (Spain, 1October 2004 (rec'd)) Beijing Additional Extract Leili 5 extract (USA, March (rec'd)) Algifert Additional Extract (Norway, 6 extract March (rec'd)) 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)

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

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41 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,, and the number of root tips..

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44 Arabidopsis -4.5 o C for 48 hours Arabidopsis can Survive -2.6 o C SSEP 5221 Control

45 Total Chlorophyll Content after freezing h Wt) Total Chloroph hyll (mg per g Fres Control Treatments

46 Enhanced Salinity Stress Resistance Control SSEP 150 mm NaCl 5221

47 Enhanced Salinity Stress Resistance SSEP 1186 Step 1A LMW HMW Step 1B Methanol Pellet Step 2A F2A1 F2A2 F2A3 F2A4 F2A5 Step 2B Hexane Alginates removed Alginates retained Step 3B Chloroform Step 4B Ethyl acetate Water

48 150mM NaCl 150mM NaCl + Methanol extract

49 0 h 24 h 48 h Control INA LA 5221 INA LA 5221 PR-5 PR-3 PR-1 18S Actin INA = control treatment known to affect PR gene expression SSEP 5221 had no effect on PR1, PR3 or PR5 gene expression.

50 Cntrl 1g/L 0.5 g/l 0.1 g/l Disease (Pseudomonas syringae) development in Arabidopsis i plants treated with SSEP 5221 Comparison of disease symptoms in leaves on plants treated with various concentrations of SSEP 5221 Cntrl 1g/L 0.5 g/l 0.1 g/l

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52 Ascophyllum nodosum ASL Extracts Fractions & compounds Molecular Biology Bioassays Bioactivity Biochemical Mode of Action Discovery & y Mode of Action

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54 Field Trials Program Research field trials, i l coop trials, i l joint i trials i l

55 Traditionally Responsive Crops Grapes Solanaceaes: Tomato, Peppers, Potato, Tobacco, Eggplant, others Apples, Strawberries, Cherries Stone fruits, Citrus

56 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

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

58 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)

59 Generalized Recommendations Application Timings and Targets: Applications to foliage Seed Transplant Two applications 4 weeks apart When needed mixed with fungicides, Insecticides, nutrients and other inputs Post-harvest (Soak) (Treat / Soak) Applications to Roots

60 Growers juggling question Ball 1: Science How many times should I apply? Little Little-Often Ball 2: Economics How many times can I afford to apply? Consider ROI Ball 3: Practical How many times will I actually apply? Tank mixes, routines, weather, labour

61 Plot: 3 x 10 Control Crop: Processing tomatoes Acadian Plot: 3 x 10 Crop: Processing tomatoes

62 Control Plot: 10 row Crop: Green Peppers Rep: 3 Rep: 2 Treated Plot: 10 row Crop: Green Peppers

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68 $24, $22, Chart 10: Acadian on Strawberries - Ventura County, Winter Cumulative Marketable Production Net Return by Pick Day Dollars pe er Acre $20, $18, $16, $14, $12, $10, $8, $6, $4, $2, Break even point approximately $18,000 $0.00 1/11/2007 1/15/2007 1/19/2007 1/24/07 1/27/07 2/1/07 2/6/2007 2/9/2007 2/14/2007 2/17/2007 2/21/2007 2/24/2007 2/26/2007 3/1/2007 3/5/2007 3/8/2007 3/10/2007 3/13/2007 3/16/2007 3/19/2007 3/22/2007 3/24/2007 3/27/2007 3/30/2007 4/2/2007 4/5/2007 4/7/2007 4/11/2007 4/13/2007 4/18/2007 4/21/2007 4/25/2007 4/30/2007 5/4/2007 5/9/ Grower Standard Pick Day Acadian

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70 Effect Of Acadian, Soil-Applied Post Veraison On Rachis Length In CRIMSON and RED GLOBE Grapes, Fresno, CA ACADIAN CONVENTIONAL Rachis Total llength (CM)CRIMSONCRIMSON Rachis Total llength (CM) RED GLOBE

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72 Tobacco B: Control A: Treated

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74 BEANS UNDER WATER STRESS CONTROL TREATED

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79 Benefits of Ascophyllum Take Home Messages Benefits through foliar applications: Improved plant nutrition, growth and development Improved fruit quality and quantity Improved efficiency of crop inputs Enhanced disease, insect and nematode repression

80 Benefits of Ascophyllum Take Home Messages Benefits through soil applications : Stimulate plant beneficial microbes and increase soil suppressiveness to diseases Induce a direct SAR effect via the roots Leads to better crop establishment, t improved crop health and ultimately increased productivity plus profitability (to growers)

81 Benefits of Ascophyllum Take Home Messages Ascophyllum nodosum extract is a general biostimulant (vitamin-like effects) Substantial research shows SAR benefits Can be used many times during the season Foliar and/or soil applications Stimulates growth and development at time of application Can use often without toxicity yp problems

82 Benefits e of Ascophyllum Take Home Messages Seaweed products can be made in different ways (e.g. species, processing and extraction) May result in different activities. Identifying active ingredients in Ascophyllum extracts Foot-printing molecular profiles of our products Connecting activities with specific plant responses In controlled bioassays In the field Delivering best-use application information to growers and end-users on ongoing basis

83 Stimulate proteins and growth promoting compounds