ACTINOVATE Features of a New Biofungicide Undergoing EU Registration 12

ACTINOVATE Features of a new biofungicide undergoing EU Registration 12

Carolina Fernández1, Ángela Sarro1, José Manuel Lara1 & Matthew Kowalski2

6th Annual Biocontrol Industry Meeting (ABIM) Lucerne, Switzerland October 24‐26, 2011 CONTENTS

PART 1: BIOFUNGICIDE ACTINOVATE

Active ingredient Biological properties and risk assessment Characteristics of SP formulation

PART 2: SUITABILITY FOR IPM

Side effects on non‐target organisms Efficacy against fungal and bacterial diseases Compatibility with PPPs PART I – BIOFUNGICIDE ACTINOVATE HISTORY: Streptomyces lydicus WYEC 108 (ACTINOVATE®)

Discovery: 1991 Isolation from rhizosphere from linseed plant roots (Crawford et al., 1993) Screening of new actinomycetes to control damping‐off (Phytium ultimum) Selection of WYEC 108: Stable prolific sporulator (15‐35 ºC / pH 4.0‐8.0)

Patenting & Licensing 1992: IRF (University of Idaho): plant growth / yield enhancer 1995: US patent ofC WYE 108 to control plant pathogens 1998: worldwide licensing to Natural Industries (US)

Registration 2004: USA (EPA 73314‐1) 2007: Canada (PMRA PRD2007‐10) 2005‐2011: Turkey, Vietnam, Ecuador, Trinidad (in progress: Australia, Taiwan, Thailand, Costa Rica) 2010: EU (RMS: The Netherlands) 2011: Completeness Check Decision 2011/253/EU of 26 April 2011 PART I – BIOFUNGICIDE ACTINOVATE ACTIVE INGREDIENT

Taxonomy Streptomyces lydicus strain WYEC 108 Family: Noctuoidea; Orden: Actinomycetales; Class: Actinobacteria; Phylla: Actinobacteria Kingdom: Bacteria

Origin Rhizosphere linseed plant (South Downs, UK)

Distribution Widely distributed in most agricultural soils worldwide

Deposited ATCC 55445

Target Root damping‐off and foliar fungal pathogens

Growing Te Typical mesophylic specie (15‐35ºC; optimum 30ºC)

Growing pH 4.0 –8.0 PART I – BIOFUNGICIDE ACTINOVATE MODE OF ACTION

Competition For the space and nutrients.

Mycoparasitism The bacteria breaks down the cell walls of pathogenic fungus mycelium by excreting lytic enzymes (chitinases, glucanases, peroxidases)

Antibiosis Production and release of antimicrobial secondary metabolites

Fusarium

WYEC 108

Fusarium PART I – BIOFUNGICIDE ACTINOVATE ADDITIONAL BENEFITS

High siderophore production: Improve mineral & nutrient uptake: a) Promotes plant growth even in the absence of pathogen b) Plant more vigorous /able to survive stressful conditions

Bactericide: Firelight (Erwinia amylovora), Citrus Canker (Xanthamonas axonopodis), Walnut Blight (Xanthomonas arboricola), Rice Leaf Blight (Xanthomonas oryzae), Bacterial spot (Xanthamonas perforans)

Xanthomonas Erwinia PART I – BIOFUNGICIDE ACTINOVATE

TOXICOLOGY: Acute Mammalian Toxicity

Guideline Study Toxicity Results Category 152‐30 Acute Oral IV 5 male + 5 female rats (Sprague‐Dawley)

OPPTS 870.1100 Toxicity No rats died. LD50 > 5.000 mg/kg 152‐32 Acute Pulmonary N/A S. lydicus was not toxic, infective, or pathogenic (rats). OPPTS 885.3150 Toxicity /Pathogen. Clearance: liver‐lymph nodes 7d, kidneys 14d, lungs 28d 152‐33 Acute Injection N/A S. lydicus was not toxic, infective, or pathogenic (rats). OPPTS 885.3200 Toxicity / Pathogenicity Clearance: blood‐kidney‐lymph nodes 14d, lungs 21d, liver‐spleen 28d 152‐34 Acute Dermal Irritation IV 3 rabbits dosed (500 mg) OPPTS 870.2500 Study Very slight erythema in 1 rabbitt with clearance by 24h 152‐35 Acute Eye Irritation IV 3 rabbits dosed (0.1mL) OPPTS 870.2400 Study Conjunctival irritation was reduced by 24 h OECD 471, 1997 Reverse Mutation Assay No Salmonella typhimurium (Ames test) mutagen No genotoxic in vitro; Irrelevant to mammals PART I – BIOFUNGICIDE ACTINOVATE FORMULATION CHARACTERISTICS

Active Ingredient Streptomyces lydicus strain WYEC 108 Concentration 1x107 cfu/g (0.0371% TGAI) Physical State Solid powder (SP) Colour White pale pH (CIPAC MT 75) 6.65 (1% p/v) Stability (Tª) 1 year (35ºC)

4ºC RT

1,00E+09 1,00E+08 1,00E+07 1,00E+06 (CFU/mL)

1,00E+05 1,00E+04 1,00E+03 1,00E+02 VIABILITY 1,00E+01 1,00E+00

0 50 100 150 200 250 300 350 DAYS PART I – BIOFUNGICIDE ACTINOVATE TARGET PATHOGENS

SOIL DISEASE FOLIAR DISEASE

Fusarium Laveillula Pythium Spahaeroteca Rhizoctonia Erisiphe Phytophthora Oidium Sclerotinia Venturia Armillaria Botrytis Alternaria

Fusarium Fusarium + Phytoph. Phytoph. + Rhizoctonia Rhizoctonia + Pythium Pythium + ACTINOVATE ACTINOVATE ACTINOVATE ACTINOVATE PART II – SUITABILITY FOR IPM EFFECT ON NON‐TARGET ORGANISMS

Guideline Group Target Test Remarks

OPPTS Invertebrate: Dietary effect No risk 885.4380 Honey bees NOEC > 17980 µg/mL (≈ x20 Apis mellifera commercial dose) OPPTS Vertebrate: Avian Oral Pathog./ No risk Terrestrian 885.4050 Nothern Bobwhite Toxicity Test (1.25x108 cfu/kg body for 5d) Organisms Colinus virginatus OPPTS Non‐Target Plants ‐ Efficacy trials Absence of adversed effects 885.4300 ‐ Databases ‐ Background levels OPPTS Invertebrate: Chronic Toxicity NOEC (survival) = 3.1x103cfu/mL 850.1300 y Acuatic Daphnia magna (21‐d static renewal NOEC (reprod.) = 1.6x103cfu/mL 885.4240 Organisms test) OPPTS Vertebrate: Acute Toxicity Test No risk 6 850.1075 Rainbow Trout (96‐h static test) LC50 > 1x10 cfu/mL Oncorhynchus myskiss NOEC > 1x106 cfu/mL

‐ Potential Ecological Risk Minimal PART II – SUITABILITY FOR IPM DIRECTION FOR USE

Crop Soil Foliar

Vegetables 300 ‐ 500 g/Ha 250 ‐ 700 g / Ha

200 – 500 g / 300 ‐ 1200 mL Seed coating (per 50 g seeds)

3 kg/Ha (initially) Turf 1 kg/Ha (every 6‐8 wks) Cut flowers, 100‐200 g/500 L bulbs and pot 500 g/250 L (woody plants) 250 ‐ 700 g / Ha plants 200‐300 g/50 kg bulbs PART II – SUITABILITY FOR IPM EFFICACY TRIAL: Phytium and Rhizoctonia

Petunia and Geranium (Reddy, 2007, Alabama, US)

Petunia ‐ Pythium Geranium ‐ Rhizoctonia

5 5 4,5 4,5 4 4 3,5 3,5 Untreated Control Untreated Cont 5 3 5

‐ 3 ‐ 0 0

Pathogen Control Pathogen Cont 2,5 2,5 ACTINOVATE ACTINOVATE Scale 2 Scale 2 Chemical Standard Chemical Stand 1,5 1,5 1 1 0,5 0,5 0 0 Vigor Root‐rot Severity Vigor Root‐rot Severity

‐ Preventive teatment 7 days before transplant and inoculation with pathogen ‐ Damping‐off was evaluated 45 days after transplanting.

‐Root‐rot severity was reduced by ACTINOVATE ≈ same level than Chemical Standard ‐Vigor was increased by ACTINOVATE (PGPR) PART II – SUITABILITY FOR IPM EFFICACY TRIAL: Rhizoctonia solani

Potato crop (Chapingo, México)

Disease Incidence (%) Disease Severity (%)

100 50

90 45

80 40 35 70 65% 61% Pathogen Control 72% Contr 60 30 70% 76% ACTIN 67% 80% 75% ACTINOVATE (1 kg/Ha) 80% 79% 50 25 76% 82% 77% 79% ACTINOVATE (1.5 kg/Ha) 81% ACTIN 82% 20 81% ACTIN 40 ACTINOVATE (2 kg/Ha) 85% 30 15 20 10

10 5

0 0

36 DAT 71 DAT 110 DAT 36 DAT 71 DAT 110 DAT ‐ ACTINOVATE preventively applied to potato seeds

‐Dose‐response effect high dose = best results PART II – SUITABILITY FOR IPM EFFICACY TRIAL: Rhizoctonia solani

Turf vr. Raleigh (Schlesselman & Bruno, 2006, Wharton, Texas, US)

Disease Incidence (%)

45 40 35 30 25 20 15 10

5 0

CONTROL Chemical 1ACTINOVATEChemical 2Chemical 3

‐ Two spray applications

‐ ACTINOVATE efficacy was similar than efficacy showed by chemical standards PART II – SUITABILITY FOR IPM

EFFICACY TRIAL: Fusarium oxysporium fsp cyclaminis (FOC) Cyclamen (*Elmer & McGovern, 2004, Gainesville, Florida US)

120 Trial I 100

0 AUDPC Dry Weight %VD

FOC T (1DAT) + ACT (7DAT) + FOC (14 DAT) F (1DAT) + ACT (7DAT) + FOC (14 DAT)

AUDPC: Area Under the Disease Progressive Curve ; %VD: % Vascular Discoloration T: Thiophanate methyl; F: Fludioxonil;

‐ Applying ACTINOVATE after a preventive fungicide showed promise supression of FOC

*Published: Crop Protection PART II – SUITABILITY FOR IPM

EFFICACY TRIAL: Fusarium oxysporium fsp cyclaminis (FOC) Cyclamen (*Elmer & McGovern, 2004, Gainesville, Florida US)

120 Trial II 100 Trial II

AUDPC DryWeight %VD FOC FOC (1 DAT) + AZ‐ACT (7DAT) + ACT (14 DAT) FOC (1 DAT) + AZ (7DAT) + AZ (14 DAT) FOC (1 DAT) + F ‐ ACT (7DAT) + ACT (14 DAT) FOC (1 DAT) + F (7DAT) + F (14 DAT) AUDPC: Area Under the Disease Progressive Curve ; %VD: % Vascular Discoloration F: Fludioxonil; Az: Azoxystrobin

‐ACTINOVATE rotated with a chemical fungicide showed a better suppresion of FOC than the chemical alone

*Published: Crop Protection PART II – SUITABILITY FOR IPM COMPATIBILITY WITH PPPs

COMPATIBLE with ACTINOVATE NON‐COMPATIBLE with ACTINOVATE

100 90 100 80 90 (%) (%)

70 80 60 70 60 50 50 40 40 30 30 CO MPATIBILITY COMPATIBILITY 20 20 10 10 0 0

40% 80%

60% 2,5% 25% Methyl 50% 24% Sulphur Citrus Copper W ettable Copper Tiofanate phosphite 15% potassium Bupirimate oxychloride Cymoxanile extract Triadimenol oxychloride Thiram Procimidone Miclobutanil Ciproconazol Kasugamycin Dimethomorph

Fungicide Fungicide‐Bactericide Fungicide Fungicide‐Bactericide

‐Ten of them affected less than 80% the viability of ACTINOVATE spores. CLOSING REMARKS

ACTINOVATE (Streptomyces lydicus strain WYEC 108)

• Efficient broad range biofungicide (bactericide)

• Plant Growth Promoter even in the absence of pathogen

• IPM tool (low risk substance compatible with other PPPs) Thank you www.futurecobioscience.com