Advancing Phoma macrostoma selective bioherbicide to commercialization. Stuart Falk & Karen Bailey August 20, 2012

Transcription

1 Advancing Phoma macrostoma selective bioherbicide to commercialization Stuart Falk & Karen Bailey August 20, 2012

2 Discovery Photos from Ag Canada White tip disease* Wanuskewin isolate of Phoma macrostoma *proposed 2

3 Phoma macrostoma Discovery From small chlorotic and necrotic lesions on Canada thistle growing in fields, pastures, and roadsides Disease surveys Five provinces in Canada (AB, SK, ON, NB, NS) 10 biocontrol isolates identified by CBS as P. macrostoma var. macrostoma Granular formulation applied to soil Selective herbicide for control of broadleaf weeds with pre- and postemergent activity (no effect on crabgrass) Photos from Ag Canada 3

4 Historical Agriculture and Agri-Food Canada published technology brief Requested proposals for development partner Oct 1, 2001 Scotts Canada selected as partner March 19, 2002 COLLABORATIVE RESEARCH AND DEVELOPMENT AGREEMENT Natural Broadleaf Weed Control BETWEEN: HER MAJESTY THE QUEEN IN RIGHT OF CANADA AND: SCOTTS CANADA LTD SOLE LICENSE AGREEMENT FOR NATURAL BROADLEAF WEED CONTROL: BIOHERBICIDE PHOMA MACROSTOMA BETWEEN: HER MAJESTY THE QUEEN IN RIGHT OF CANADA Canada AND SCOTTS CANADA LTD. The Company July 7,

5 Requirements for commercialization Intellectual property protection or trade secret Research to support registration (for biopesticides) Product label and direction for use Characterization of microbial pest control agent Manufacturing method Disclosure of ingredients Analytical method Mammalian toxicology Environmental toxicology Efficacy (CA DPR & PMRA) Commercially viable manufacturing method Economically viable business case Simple, sustainable and significant product 5

6 Patents Fungal isolates and biological control compositions for the control of weeds Bailey & Derby, AAFC, Saskatoon Issued Patents New Zealand No July 13, 2006 Australian No June 12, 2008 Europe* No June 2, 2010 *Belgium, Austria, the Netherlands, United Kingdom, Germany, Italy, Sweden, Spain, Ireland, and France Mexico No September 22, 2010 Japan No October 1, 2010 Canada No. 2,448,890 Dec. 7, 2010 U.S. No. 8,211,830 July 3,

7 CIP Patents On July 25, 2006, a continuation-in-part (CIP) was filed to protect the genetic identification of the genes responsible for weed control in isolates of Phoma macrostoma Issued CIP s US No. 7,772,155 August 10, 2010 South Africa No August 29, 2009 Mexico No October 1, 2010 Pending CIP s Canada (Application # ) Europe (Application # ) China (Application # ) Hong Kong (Application # ) AFLP Fingerprint* Bioherbicidal isolates are readily differentiated from nonbioherbicidal isolates *Pitt et al,

8 Host pathogen interaction In nature, the geographic distribution of P. macrostoma is widespread In nature, it is a weak or wound pathogen at low frequency (less than 3%) mostly on woody hosts Bioherbicidal isolates of P. macrostoma have only been found from Canada thistle (= White tip disease) When applied by inundative release, the high numbers of pathogenic propagules lead to weed death Two modes of action Direct invasion of roots with close proximity to host vascular tract** Uptake of the active metabolite from product and live fungus Macrocidin ** Bailey et al, 2011 Phoma antibody (blue): Dandelion root (green) Phoma stained (red): Dandelion vascular tract (green) 8

9 Discovery of the macrocidins Natural bleaching herbicides Joint project between AgCanada and Dow AgroSciences Macrocidins are first reported cyclic tetramic acids Isolated by LC from liquid culture and identified by X-ray diffraction A major compound (A) and a minor (B) were shown to be herbicidal Physical properties consistent with phloem mobility H O H N H O O O OH OH HN Tetramic acid Graupner, P. R., et al. (2003). The macrocidins: Novel cyclic tetramic acids with herbicidal activity produced by Phoma macrostoma. Journal of Natural Products 66(12): O O Macrocidin A Mol. Wt. 357 C 20 H 23 NO 5 H Macrocidin B Mol. Wt. 373 C 20 H 23 NO 6 macrocidin B = 13-hydroxymacrocidin A 9

10 Detection of macrocidins hplc HPLC method developed to identify macrocidins (pre A, A, Z, post Z) Scotts modified AgCanada method to use a readily available standard Method does not use purified macrocidin as standard Results reported as relative hplc/100 mg (i.e., hplc x 10 = MU/g) Rate calculations (MU/m 2 ) allow quantification of field rates Macrocidins occur at concentrations of ~ 2-5% in barley culture 10

11 Mode of Action Phoma macrostoma produced macrocidins MOA unknown Not HPPD inhibition (Graupner et al, 2006) Causes bleaching Requires repeat application HPPD = p-hydroxyphenylpyruvate dioxygenase Dandelion Mesotrione (based on Callistemon citrinis) HPPD inhibitor prevents carotenoid formation Chlorophyll and cell membranes destroyed Causes bleaching Requires repeat application Macrocidin A Mesotrione Dandelion 11

12 Environmental Fate DNA specific marker for bioherbicidal trait in Phoma macrostoma On roots and in soil profile (1-8 cm) No horizontal movement (30-60 cm) Phoma DNA is gone from the soil after 12 months Field testing one year after application confirms that sensitive plants are safely grown Source: Zhou et al 2003 Photo from Ag Canada 12

13 Efficacy for PMRA/DPR submission Turf Broadleaf Weed Spectrum and Turf Safety Dandelion control from 30 field trials Years of research 129 Efficacy trials 69 Phytotoxicity trials Weeds Dandelion Clover Plantain Chickweed Canada Thistle Black Medic Buckhorn Spotted Spurge CA Bur clover Cudweed Ragweed Chamomile Wild Mustard English Daisy Turf type Kentucky bluegrass Perennial ryegrass Annual ryegrass Red fescue Tall fescue Bentgrass Smooth brome grass Meadow brome grass Timothy grass Turfgrass mixtures Bermudagrass Other proposed uses for consumer garden (non -food) and professional hort % Weed control days days Regression 56 days Regression 84 days Spot treatment 2-3 apps/yr MU/g 400 Pre-emergent single app/yr MU/g 400 Post-emergent 2-3 apps/yr MU/g 400 Macrocidin units/square metre MU/m MU/m MU/m oz/8 in diam lb/m 3.4 lb/m

14 Efficacy: Pre-emergent dandelion % Reduction (Abbott's) Dandelion MU/m /14/ /14/ /14/2007 Treated Untreated Photos from Scotts Miracle-Gro 14

15 Efficacy: Post-emergent dandelion % Reduction in cover Dandelion MU/m WBG 0 14-Jun 12-Jul 9-Aug 6-Sep Two applications June/July Treated Photos from Scotts Miracle-Gro Untreated 15

16 Mammalian Toxicology Summary Study Acute oral infectivity and toxicity Acute pulmonary infectivity and toxicity Intraperitoneal (IP) infectivity Acute dermal toxicity Acute dermal irritation Reporting of hypersensitivity incidence Primary Eye Irritation Acute oral toxicity Rat EP Conclusions Non-pathogenic and non-toxic Non-pathogenic and non-toxic Non-pathogenic and non-toxic EPA Toxicity Category IV for acute dermal effects. EPA Toxicity Category IV for dermal irritation. No incidences of hypersensitivity. EPA Toxicity Category III for eye irritation effects. EPA Toxicity Category IV for acute oral effects. 16

17 EcoToxicology Summary Study Avian Oral: Toxicity/ Pathogenicity Wild Mammal Toxicity/Pathogenicity Freshwater Fish Terrestrial arthropods Non-target Insect and Honey bee Testing Aquatic arthropods (Freshwater aquatic invertebrate testing) Terrestrial Plants Aquatic Plants (Tier 1) No toxicity or pathogenicity. A waiver has been submitted. Status No signs of infection. The observed 32-day NOEC is 2.04 x 10 5 times greater than the worst case EEC. For honey bee, ladybird beetle, parasitic hymenopteran and green lacewing larvae there was no evidence of pathogenicity, sublethal effects or mortality. The LC50 value was greater than the highest concentration tested, 1,000,000 CFU Phoma macrostoma /ml. On Daphnia the 21-day LOEC is 1.27 x 10 4 times greater than the worst case EEC. No effect on monocots. However, most plants in Brassicaceae, Asteraceae, and Leguminosae had reduced germination, exhibited chlorosis, and had high seedling mortality. On Lemna the 7-day NOEC is equivalent to 5.11 x 10 4 times greater than the worst case EEC. 17

18 Regulatory Status Canada: PMRA granted conditional registrations on June 13, 2011 United States: EPA granted conditional registrations on January 13, 2012 California: DPR proposed decision to register on July 6,

19 Early product manufacturing Dry (Solid State) Fermentation Typical fungal colonization after 4 days incubation at 23 C Bag inoculation system: inoculum suspension, peristaltic pump, injection hose Photos from Ag Canada Harvest: trays are ready to place in drying racks 19

20 Formulating a prototype end product Making dough, extruding, spheronize, dry, sieve Photos from Ag Canada 20

21 Solid fermentation: Rationale AgCanada based their process on mushroom spawn production solid state fermentation This is the best method for the short term most research data from AgCanada uses an existing viable commercial process Scotts partner is major mushroom spawn producer Looking to expand into biological pesticides A willing commercial partner prepared to invest capital Has state of the art process which allows much faster batch turnover DVT reactor allows single vessel sterilization, cooling (vacuum), and mixing prior to bagging 21

22 Commercial production is closely related to laboratory production Solid State Fermentation Inoculum Prep Inoculation of Grain ( Cook ) Incubation Granulation 7 14 days 2 hours days 2 hours Scotts owned/operated *Must be co located with fermentation steps because of high Mushroom spawn producer owned/operated water content (50 70%) and refrigeration requirements Variables: Grain Type, Preconditioning, Moisture content, Temperature, Rest period Inoculum Concentration & Volume Mixing duration and velocity Additives (Flow Aids, nutrients, ph adjustment, or other supplements) Mushroom spawn bag size, location of breath strips Bag Fill Weight / Bag Volume Incubation Temperature, Air exchange, Humidity Granulation Process Drying temperature and duration Moisture content 22

23 Pilot Test Capabilities 23

24 LBI large scale bulk inoculation process 24

25 Liquid Fermentation: The future? Liquid fermentation with filamentous fungi offers unique challenges feasibility studies completed Need viable fungus along with macrocidins (biopesticide) many commercial biopesticide products only spores or biomass need an improved detection method for macrocidin in liquid May be more cost effective and higher yielding? needs verification 10L Liquid fermenter 25

26 Requirements for commercialization Intellectual property protection or trade secret Research to support registration (for biopesticides) Product label and direction for use Characterization of microbial pest control agent Manufacturing method Disclosure of ingredients Analytical method Mammalian toxicology Environmental toxicology Efficacy (CA DPR & PMRA) Commercially viable manufacturing method Economically viable business case Simple, sustainable and significant product 26