Development of Serenade as a biopesticide against plant bacterial diseases COST 873 Thierry Merckling, Denise Manker, Manuele Ricci, Sarah reiter October 26-27, 2009 Cetara
Agenda Introducing AgraQuest Serenade : Mode of action Field Experience in bacterial diseases control Situation in Europe Future developments
Headquarters - Davis, California 3 October 27, 2009 CONFIDENTIAL
Discovery & Development Highest hurdles for development and in-licensing of products. Efficacy, safety & consistency. 4 October 27, 2009 CONFIDENTIAL
R&D Organization Experienced & diverse team of 40 scientists and professionals: Entomology Plant Pathology Fermentation Microbiology Analytical Chemistry Formulation Informatics Regulatory Product Development 5 October 27, 2009 CONFIDENTIAL
2009 AgraQuest, Inc. SERENADE, SONATA, RHAPSODY, & BALLAD are registered trademarks of AgraQuest, Inc. These trademarks are registered in the U.S.Patent and Trademark Office as well as in the intellectual property offices of numerous other countries worldwide. The SERENADE and RHAPSODY products are protected by U.S. Patent Nos. 6060051, 6103228, 6291426, 6417163, and 6638910. patents in numerous other countries. In addition, these products are protected by The SONATA and BALLAD products are covered by U.S. Patent Nos. 6245551, 6586231, and 6635245 and by patents in numerous other countries. BARITONE is a trademark of AgraQuest, Inc. The BARITONE product is manufactured and distributed subject to EPA Reg. No. 62637-5-69592. BARITONE Bio-Insecticide is currently registered for use in the following states: Arizona, California, Colorado, Florida, Georgia, Idaho, Maryland, Michigan, Nevada, North Carolina, Oregon, South Carolina, Virginia and Washington Products comprising the Muscodor fungus are protected by U.S. Patent No. 6,911,338 and are the subject of numerous pending patent applications worldwide. AgraQuest owns the following product registrations: SERENADE MAX - EPA Reg. No. 69592-11; SERENADE ASO - EPA Reg. No. 69592-12; SONATA - EPA Reg. No. 69592-13. These products are also registered in numerous other countries worldwide. 6
What is SERENADE?
SERENADE Fungicide and Bactericide SERENADE is a highly effective broad spectrum fungicide and bactericide bundling several modes of action for a wide spectrum of control with little potential for resistance Based on the proprietary active ingredient, Bacillus subtilis QST713; a naturally occurring strain Each bag or jug contains chemical compounds produced by QST713 during fermentation, as well as spores QST713 is unique in its production of both antifungal and antibacterial compounds During production, we control fermentation parameters in order to optimize output of these compounds 8 October 27, 2009 CONFIDENTIAL
Disease Control 1. Broad anti-fungal activity 4 Modes of Action Lipopeptides kill fungal cells They are highly stable, resistant to elevated temp and ph extremes Synergistic activity of 3 different classes 2. Anti-bacterial activity 3+ major chemical classes Activity on gram positive/negative pathogens Stable through spray drying Plant Activation 3. Plant Defense mechanisms Elicits plant responses - SAR (Systemic Acquired Resistance) or ISR (Induced Systemic Resistance) 4. Growth Promoting 9 Enhances yield Improves quality
1. Anti-fungal activity Only QST 713 produces this unique group of three lipopeptide classes Each peak is a unique compound which contributes to anti-fungal activity RT:0.01-30.01 100 Relative Abundance 90 80 70 60 50 7.47 Iturins 8.88 Surfactins 23.80 10.91 23.52 24.58 NL: 1.07E8 Base Peak F: + c Full ms 40 30 20 10 0 1.66 Agrastatins/ Plipastatins 25.71 11.22 9.30 19.02 20.10 17.79 6.27 20.19 11.92 5.50 16.75 26.90 15.41 3.73 4.93 12.62 28.93 2 4 6 8 10 12 14 16 18 20 22 24 26 28 30 Time (min) 10 CONFIDENTIAL
2. Anti-bacterial Activity QST 713 produces at least 3 classes of anti-bacterial compounds The classes have different modes of anti-bacterial action Compounds are not used in human or animal health Block bacterial cell protein production and cell wall formation Efficacy against broad range of bacterial pathogens Clavibacter, Pseudomonas, Xanthomonas, Erwinia Lipopeptides are not involved in anti-bacterial control Macrolactins Difficidins Bacillicine 11
3. Plant Activating SERENADE triggers plants internal defenses and physiological responses Activated when SERENADE contacts the plant The effect is systemic responses are triggered throughout the plant even when a small area is treated SERENADE induces resistance to disease Proven with bioassay and RNA analysis Activated pathway is distinct from salicylic acid (SAR, systemic acquired resistance) and jasmonic acid (ISR, induced systemic resistance) pathways 12
Plant Health Induced Resistance Measurement of PR gene expression Clear induction of PR-1 by SERENADE on untreated leaves Water Elicitine SERENADE Day after treatment 0 4 Elicitine (positive control) is a known inducer of pathogenesis related proteins such as PR-1 White indicates RNA expression Induction of PR-1 gene expression on tobacco leaves treated by water, Elicitin and SERENADE. RT-PCR were carried out on mrna extracted after 0 and 4 days of incubation from untreated upper leaves (SAR project) of tobacco plants. 13
Tomato Xanthomonas perforans **Error bars represent 95% confidence intervals 14
4. Plant Growth Promotion SERENADE applied at planting stimulates growth Tomato seeds treated with SERENADE in furrow when planted in sterile soil at different field rates (oz/acre) Treatment was to soil (not as seed treatment) Plants are compared at same days after planting This is not an anti-fungal (soil was disease-free) nor a fertilizer effect 0 oz. 4 oz. 8 oz. 16 oz. SERENADE applied to tomatoes in furrow at seeding, pathogen free (70215) 15 CONFIDENTIAL
Spectrum of Activity: Major Global Crops / Diseases CROP Grape Vine, table Tomato, pepper, eggplant Cucumber, Melon, squash, lettuce Strawberry, Soft fruits, Pome fruit Stone fruit Beans, Onions, garlic Potato, asparagus, carots, Ornamentals Roses, Chrysanthemums Citrus Banana Mango DISEASE Botrytis, sour rot, powdery mildew Botrytis, early blight (alternaria) Powdery mildew (leveillula) bacterial leaf spot (xanthomonas) Powdery mildew, Botrytis, downy mildew Leaf drop (sclerotinia) Botrytis, anthracnose Apple scab (venturia), fire blight (erwinia),powdery mildew Monilia, Bacterial spot (xanthomonas) White mold (sclerotinia), Neck rot (Botrytis) Leaf spots (erwinia, xanthomonas, pseudomonas) Bacterial wilt (ralstonia) Canker Black sigatoka Anthracnose, Powdery mildew
Bacterial Control Spectrum Crop Disease Common Name Pome Erwinia amylovora Fire blight Tomato / Pepper Blueberry Peaches Citrus Xanthomonas campestris pv vesicatoria Pseudomonas syringae Pseudomonas syringae pv syringae Xanthomonas arboricola pv pruni Xanthomonas axonopodis pv citri Bacterial leaf spot Bacterial speck Bacterial blight Bacterial leaf spot Citrus canker Note: Approved uses vary depending on region and regulatory authority 17
Results of field trials
Provides Fire Blight control on Apples, and helps reduce resistance G.W. Sundin, Mich State Univ., East Lansing, MI 2005. 20
Controls Fire Blight on Apples AgriAssistance, Wayne Co., NY 2006. Erwinia amylovora. (60303) 21
Controls Fire Blight on Pears S. Lindow, Univ. of California Extension, CA 2004. Erwinia amylovora. (40190) 22
Serenade against fire blight TIMING AT FLOWERING: apply Serenade at 10-20% flowering in order to prevent the frequently asymptomatic multiplication of the pathogen on the flower; bees can contribute to the spread of B. subtilis spores among flowers. Application rate: either 1 single application at 2-3 kg/ha or 2-3 successive applications at 1 kg/ha during flowering (also in tank mixture with fungicides). UNDER CONDITIONS FAVOURABLE TO INFECTION (hail, storms, days at high risk of infection due to temperature and humidity conditions, etc.) and at APPEARANCE OF SECONDARY BLOSSOMS (pear) Application rate: 2-3 kg/ha, also in tank mixture with copper-based products, both at a reduced rate (Serenade at 1.5 kg/ha).
Bacterial Spot Control on Tomatoes SERENADE MAX spray program NCSU recommended spray program Dr. Kelly Ivors, 2006 NCSU Mountain Hort. Crops REC Untreated Check 24
Pseudomonas Syringae (Bacterial Speck) on Tomatoes a ab b b b D. Hicks, Hulst Research Farm, Hughson, CA. 2005. Pseudomonas syringae. (50110) 25
Controls Bacterial Leaf Spot on Tomatoes a 1 SERENADE 1lb + Kocide 2lb (A, C, E) + Biotune (B, D, F) Quadris 6.2oz 2 Tanos 8oz + Manzate 2lb (A, C) + Kocide 2000 1.5lb Manzate 2lbs + Kocide 1.5lb Tanos 8oz + Bravo 2pt + Kocide 1.5lb (E) Bravo 2pts + Kocide 1.5lb (F) (A, C, E) 3 Endura 3oz (B, D, F) Bravo 2pt (B, D) c bc bc D. Egel, SW Purdue Agricultural Center -2006. Application timing: A = 6/15, B = 6/29, C = 7/06, D = 7/18, E = 7/28, F = 8/03. Xanthomonas campestris (60318) 26
Provides excellent control of Blight in High Bush Blueberry when applied 3 times at 2 or 3kg/ha a b b ab ICMS Inc, Canada 2008. Three applications timed to bud swell, bud break and visible buds. (80152) 27
Provides control of Citrus Canker on Grapefruit a bc ab c Charles Mellinger, Glades Crop Care, Palm Beach Co., FL- 2006. Nine applications, 14 day intervals, starting March 28. Rating Aug 16. Same letters NSD, 0.1, Student- Newman-Keuls. AgraQuest generated data. Aug. 16 evaluation date. (60402) 28
Controls Canker on Peaches c b b Prof. Bazzi, Univ. of Bologna 200 Xanthomonas arboricola pv. Pruni 7. Serenade applied one day before artificial inoculation and one day after inoculation. Severity rated two weeks after second application. (70640) 29
Controls Bacterial Canker in Cherries Gary Grove, Washington State Univ., Prosser, WA 2006. Pseudomonas syringae. (50118) 30 SERENADE 2lb acre at Fall 90% leaf drop, Spring popcorn and Spring late dormant. SERENADE Program: 1) SERENADE 2lb + NutriPhite 2qt + New Cop 50 at 2.5lb at Fall 90% leaf crop 2) SERENADE 2lb + NutriPhite 2qt at Spring Popcorn 3) SERENADE 2lb + NutriPhite 2qt at Spring late dormant Cuprofix Disperss 20DF 2.5lb at Spring late dormant.
SERENADE Registrations: Europe Registration granted SWEDEN Registration submitted FINLAND NORWAY No Registration ESTONIA DENMARK LITHUANIA LATVIA REPULIC OF IRELAND UNITED KINGDOM NETHERLANDS GERMANY POLAND BELARUS BELGIUM CZECH REPUBLIC UKRAINE SLOVAKIA FRANCE SWITZERLAND ITALY AUSTRIA SLOVENIA HUNGARY ROMANIA BULGARIA PORTUGAL SPAIN GREECE TURKEY 31
Serenade uses for bacterial diseases control in Europe Tomato Pseudomonas syringae, Xanthomonas Italy, France (*) Pome fruit Erwinia amylovora Italy, France (*), Germany, Switzerland, Stone fruits Xanthomonas arboricola Italy Tree nuts Xanthomonas France Tropical fruits Bacterial diseases France
SERENADE: recognized by industry as a resistance management tool Novel, low resistance risk mode of action designated by FRAC (2009 update) Contact agent Major target is cell membrane (not a protein). No simple mutation that can make cell resistant MOA TARGET SITE AND CODE GROUP NAME CHEMICAL GROUP COMMON NAME COMMENTS FRAC CODE F: lipids and membrane synthesis F6: microbial disrupters of pathogen cell membranes B. subtilis Bacillus subtilis and the fungicidal lipopeptides they produce Bacillus subtilis strain QST 713 No resistance reported. Assumed to be low risk based on mode of action of nonspecific membrane disruption 44 33 CONFIDENTIAL
Conclusion Serenade largely used in programs and Integrated Crop management program against bacterial diseases R&D effots to characterize the mode of action Large base of trials and data which support the use of Serenade Strong commitment to continue to develop biopesticides and new solutions for bacterial disease management
Thank you 35