Making the Most of Bio-Controls: Mode of Action and Compatibility -Biological Fungicides- Greg Rogers Field Development Manager Certis USA

Making the Most of Bio-Controls: Mode of Action and Compatibility -Biological Fungicides- Greg Rogers Field Development Manager Certis USA

Biological Fungicides Bacillus Fungicides SAR/ISR Inducers

Bacillus Fungicides B. subtilis Serenade(s) - Strain QST 713 Bayer B. subtilis var amyloliquefaciens Taegro ECO Strain FZB24 - Syngenta B. amyloliquefaciens Double Nickel Strain D747 - Certis Serifel Strain MBI 600 - BASF Stargus Strain F747 Marrone Bio Innovations

B.s and B.a Fungicides All are native soil-borne organisms Happiest in the soil! B.s and B.a closely related species All products are different strains of bacillus Compatibility claims vary Double Nickel No Antibiotics Coppers are ok - CAVEAT No PAA/H2O2 s

Foliar or fruit application: Antimicrobial metabolites (lipopeptides) kill pathogenic fungi and bacteria by destroying their cell walls and cell membranes Lipopeptides produced during fermentation ( in the bag ) Additional lipopeptides produced as spores germinate upon application Epiphytic colonization (Competition) Systemic acquired resistance (SAR) Soil application: Forms biofilm on root surfaces Barrier to infection by pathogens Lipopeptides may kill pathogens on application Induced systemic resistance (ISR) to pathogens Plant growth promotion (PGPR)

Host colonization by Bacillus amyloliquefaciens Colonization of tomato root hairs by Bacillus amyloliquefaciens (soil application). Colonization of kiwifruit pollen by Ba D- 747 after spray application. Untreated 1DAT 3DAT 6DAT Colonization of pollen Untreated Treated Solid arrows indicate fully colonized root hairs. Dashed arrows indicate non-colonized root hairs. Source: Nihorimbere et al. FEMS Microbiol. Ecol. 79 (2012): 176-191. Source: G. Balestra et al. 2014. L Informatore Agrario 22: 50-53.

Tomato 1100 1050 Bacterial spot (Xanthomonas perforans) ab ab 1069 1058 Balm, FL IND-2016-236 Means with the same letter are NSD @ P= 0.05 1000 AUDPC 950 900 cd cd 934 930 d 906 d 927 850 800 Untreated control Cuprofix 2 lb + Penncozeb 1 lb (1-6) Taegro 4 oz (1-6) Actigard 0.5 oz (1-6) Serenade Opti 14 oz (1-6) Variety: Tycoon Treatments: 6 weekly applications from Sep 13 Oct 18, 100 gal/ac AUDPC based on severity ratings on Oct 3, 28, and Nov 3, trial inoculated on Aug 31 Cooperator: G. Vallad, Univ FL Double Nickel 55 0.75 lb (1-6)

Tomato 140 135 130 125 120 115 110 105 Marketable fruit yield (lbs/10 plants) abc 121.3 a-d a-d 114.6 112.7 ab 123.5 123.1 Balm, FL IND-2016-236 Means with the same letter are NSD @ P= 0.05 ab a 130.5 100 Untreated control Cuprofix 2 lb + Penncozeb 1 lb (1-6) Taegro 4 oz (1-6) Actigard 0.5 oz (1-6) Serenade Opti 14 oz (1-6) Double Nickel 55 0.75 lb (1-6) Variety: Tycoon Treatments: 6 weekly applications from Sep 13 Oct 18, 100 gal/ac AUDPC based on severity ratings on Oct 3, 28, and Nov 3, trial inoculated on Aug 31 Cooperator: G. Vallad, Univ FL

Tomato Bacterial speck (Pseudomonas syringae) 25 a 23.8 20 Geneva, NY IND-2016-248 Means with the same letter are NSD @ P= 0.05 % Severity 15 10 5 bcd 13.5 def 9.3 ef 8.5 f 6.8 0 Untreated Control Regalia 4 qt/ac + Actinovate AG 12 oz/ac Actinovate AG 12 oz/ac Regalia EC 4 qt/ac Variety: BHN 589 Treatments: 4 weekly applications from Jun 27 Jul 19, 40 gal/ac @ 40 psi Disease was assessed on Jul 11, percent effected leaf area Cooperator: C. Smart/H. Lang, Cornell Univ Double Nickel LC 32 fl oz/ac

Rhizoctonia in Potatoes 450 400 b a-f Total Yield- CWT per acre a-d a 350 g 300 250 200 Untreated Control Quadris 0.6 oz. per 1000 ft. Priaxor 0.48 oz per 1000 ft. Double Nickel LC 2.2 oz per 1000 ft. Kirk, Schafer, Rosenzweig & Steere: Michigan State University, East Lansing, MI. Trial: MSU: Horticultural Experimental Station, Clarksville, 2014. RCB design, 4 reps, one application spray in furrow at planting, very high infection rate of Rhizoctonia. Means with same letters NSD at 0.05. CER-2014-068

LC 320 310 300 290 280 270 260 250 292 Rhizoctonia Control in Potatoes Mean Yield cwt/acre 271 315 NSD 0.05 304 240 AGTIV Quadris 2.08 SC Double Nickel 55 0.41 kg/acre Serenade Soil Bruxelles, Manitoba. At plant over seed furrow application. NSD at 0.05 level. IND-2016-185

SAR/ISR Inducers LifeGard Certis USA Bacillus Mycoides isolate J Regalia Marrone Bio Innovations Extract of Reynoutria sachalinensis, (Giant Knotweed) ActiGard (Not NOP or OMRI) - Syngenta Vacciplant Helena Laminarin an oligosaccharin

Priming of Plant Defenses Defense priming: An induced state in which plants respond to very low levels of a stimulus in a more rapid and robust manner than unprimed plants. Priming for enhanced defense is a component of SAR. Defense priming is associated with elevated levels of PR-proteins. Adapted from Conrath et al. 2015. Ann. Rev. Phytopathol. 53: 97 119. primed not primed Defense activity stimulus pathogen attack Time Source: www.slideshare.net/dhanyaagri/primeome-a-molecular-approach-towards-defensepriming

Plant Defense against Pathogens Spore hypha haustoria Oxidative burst ROS = Danger signal Protection of tomato leaves against Alternaria alternata by foliar spray of salicylic acid (SA) four days after inoculation with Alternaria spores. SA H 2 O M. Esmailzedah et al. 2008. J. Biol. Sci. 8(6): 1039 1044. Lee et al. 2015. The Plant Cell 27: 3425 3438.

Induced Defense against Pathogens *Microbe-associated molecular patterns MAMP* Oxidative burst Pathogen receptors Inactive oligomer Active monomer Plant cell wall & membrane DANGER! PR proteins NPR1 = Non-expresser of Pathogenesis- Related proteins Pathogenesis-related (PR) proteins: Coded by host plant genes expressed only in pathological or related situations. Some are antimicrobial, others function to signal infection to nearby cells. Family Type member Properties 1 PR-1 anti-oomycete 2 β-1,3-glucanase endogluconase 3 Chitinase endochitinase 4 Chitinase endochitinase 5 Thaumatin-like fungal membrane disruption 6 Proteinase-inhibitor inhibits pathogen proteinases 7 Endoproteinase inhibits pathogen proteinases 8 Chitinase endochitinase 9 Peroxidase liginification 10 Ribonuclease-like digests RNA 11 Chitinase endochitinase 12 Defensin plasma membrane disruption 13 Thionin plasma membrane disruption 14 Lipid Transfer Protein plasma membrane disruption

BmJ Mode of Action ASM Oxidative burst BmJ Pathogen Inactive oligomer Active monomer PR proteins No direct antagonistic effects on plant pathogens. LifeGard spores triggers the SAR process by directly activating NPR1 protein in 3 hours Mode of Action similar to acibenzolar-s-methyl (ASM): Actigard, Blockade, Bion (Syngenta) - BUT No phytotoxity observed in 20 years of trials. LifeGard response persists 18 21 days (vs. ~14 days for ASM). MAKES THE PLANT RESISTANT! Systemic sugar beet apoplastic PR protein activity 6 days after treatment. References: Bargabus et al. 2002. Physiol. Mol. Plant Pathol. 61: 289-298 Bargabus-Larson & Jacobsen. 2007. J. Sugar Beet Res. 44: 17-33.

Regalia Mode of Action Activates Plants Natural Defenses Induced Systemic Resistance (ISR) Inhibits Pathogen Growth Strengthens Cell Walls Promotes Plant Growth Independent of disease control, increases leaf chlorophyll content

Regalia Mode of Action Summary Oxidative Burst Pathway Phenylpropanoid Pathway Pathogen killing Signaling L-phenylalanine H 2 O 2 ROS PAL Chitinase, β-1,3- glucanase. O 2 - Cinnamate NADPH oxidase complex P-coumarate 4-Coumaroyl-CoA CHS Degradation of fungal cell wall O 2 Papilla Caffeic acid Chalcone CHI/ CHS Lignin, CHI Wall-bound phenolics, Ferulate Flavones Wall strengthening and fungitoxicity Soluble phenolics etc. Sinapate Anthocyanins Isoflavones Pathogen Flavonoid phytoalexins Isoflavanones

How to use ISR/SAR Compounds APPLY EARLY!! 1) Stand Alone (Rarely!) 2) In a program replacing other products (Most Common) 3) In addition to a program (Special Cases)

Potatoes

Potato Early Blight 0.40 RAUDPC a Hancock, WI CER-2016-139 0.35 ab ab b LG replaced 9 fungicide apps Standard check b 0.30 Untreated check LifeGard 1.6 oz/a (1-10) 10 weekly applications 13 Jul - 7 Sep. Bravo WS 1.5 pt/a (1-10) Bravo 1.5 pt (1-8) Priaxor 4.5 fl oz (3,6) Endura 3.5 oz (5,7) Dithane 2 oz + Super Tin 2.5 oz (9,10) Tractor-mounted spray boom delivering 35 GPA at 40 psi through 4 nozzles/row. LifeGard applied at concentration of 10 7 cfu/ml (4.5 oz/100 gal). Cooperator: A. Gevens & S. Jordan, Univ. Wisconsin LifeGard 1.6 oz alt. Bravo WS 1.5 pt/a (5 apps ea.)

Potato Late Blight 700 600 500 AUDPC a b Freeville, NY CER-2016-140 Means sharing the same letter are not significantly different @ P=0.05 400 300 LG replaced 3 Bravo apps 200 100 0 Untreated check LifeGard 4.5 oz/100 gal (1-6) c LifeGard (1,3,5) Bravo WS (2,4,6) c Bravo WS 1.5 pt/a (1-6) c Revus Top 7 fl oz (3,5) Ridomil Gold 2.5 pt (4) Variety: Katahdin Artificially inoculated with P. infestans (US-24) at 1 day after 2 nd application. Cooperator: H. Mayton & W.E. Fry, Cornell Univ.

Potato White mold 9.0 8.0 7.0 White mold RAUDPC 8.4 6.8 Aberdeen, ID CER-2017-064 Means were not significantly different @ P = 0.05 6.0 5.0 4.0 3.0 2.0 1.0 3.5 3 0.0 Untreated Check LifeGard 1.3 oz/a (AE) alt Luna TQ 11.2 fl oz/a (C) alt Bravo WS 1.5 pt/a (G) Double Nickel LC 1 qt/a (ACEG) Variety: Russet Burbank Treatments applied at 7d intervals starting July 18 through Aug 29. Tank mix size of 2 liters, applied at 25 gal/ac @ 30 psi Cooperator: P. Wharton, Univ. Idaho LifeGard 1.3 oz/a (ABCD)

Potato 4% 3% % PVY Incidence in tubers (winter grow-out) 3.10% 2.62% Antigo, WI CER-2016-141 2% 1% 0% 1.44% 1.10% 1.29% 0.76% 0.37% 0.00% 1 2 3 4 5 6 7 8 UTC Lifegard Lifegard Lifegard - - Actigard L Actigard H - Aphoil 2% Aphoil 4% Aphoil 2% Aphoil 4% Aphoil 2% Aphoil 2% (bi-wkly) (bi-wkly) (bi-wkly) (wkly) (wkly) (3 app) (3 app) LifeGard applied at 1.78 oz./acre in 28 GPA. No statistically significant differences between treatments (P > 0.05). Cooperator: R. Groves, Univ. Wisconsin

Cucurbits

Downy mildew in cucumbers David Langston, University of Georgia, Tifton, Georgia 70 60 a a CER-2013-055 DM Severity (%) 50 40 30 20 10 b b 0 Untreated check LifeGard only (6 app) LifeGard (3) alt Fungicides (3) Fungicide program (6) Treatment Spray sequence (weekly intervals) A B C D E F LifeGard only LifeGard LifeGard LifeGard LifeGard LifeGard LifeGard LifeGard alt Fungicides LifeGard Zampro + Manzate LifeGard Ranman + Manzate LifeGard Zampro + Manzate Fungicide program Zampro + Manzate Ranman + Manzate Zampro + Manzate Ranman + Manzate Zampro + Manzate Ranman + Manzate LifeGard applied at 4.5 oz. per 100 gal., 40 GPA.

DM Severity (%) 100 80 60 40 20 Downy mildew in summer squash Ken Pernezny, UFL, Fort Pierce, Florida a a b IND-2006-073 Total of 9 applications at weekly intervals. b 0 Untreated check LifeGard only LifeGard alt Bravo WS Previcur alt Bravo WS LifeGard applied at 4.5 oz. per 100 gal., 32-64 GPA, dependent on plant size.

Fruiting Vegetables

Bacterial leaf spot in tomato Mills River, NC CER-2012-034 Appl Standard program LifeGard program Actigard program 1 Endura, Kocide, Actigard BmJ Actigard 2 Kocide BmJ Actigard 3 Endura, Kocide, Actigard BmJ Actigard 4 Kocide BmJ, Manzate, Kocide Actigard, Manzate, Kocide 5 Endura, Kocide, Actigard, Bravo BmJ, Manzate, Kocide, Bravo Actigard, Manzate, Kocide, Bravo 6 Kocide, Bravo BmJ, Manzate, Kocide, Bravo Actigard, Manzate, Kocide, Bravo 7 Endura, Kocide, Actigard, Bravo BmJ, Manzate, Kocide, Bravo Actigard, Manzate, Kocide, Bravo 8 Kocide, Bravo BmJ, Manzate, Kocide, Bravo Actigard, Manzate, Kocide, Bravo 9 Endura, Bravo BmJ, Manzate, Kocide, Bravo Actigard, Manzate, Kocide, Bravo 10 Ranman BmJ, Manzate, Kocide, Bravo Actigard, Manzate, Kocide, Bravo 11 Endura, Bravo BmJ, Manzate, Kocide, Bravo Actigard, Manzate, Kocide, Bravo Untreated check Grower standard LifeGard program Actigard program

Bacterial leaf spot in tomato Mills River, NC CER-2012-034 a c Application rates: LifeGard WG: 3.5 oz/100 gal (8 10 6 cfu/ml) Kocide 3000 (Cu(OH) 2, Kocide L.L.C.): 1.75 lb/ac Manzate DF (Zn/Mn/EBDC, UPI): 1.5 lb/ac Bravo Weather Stik (chlorothalonil, Syngenta): 1.5 pt/ac Endura (boscxalid, BASF): 3.5 oz/ac Ranman (cyazofamid, FMC Corp.): 2.75 oz/ac Actigard (ASM, Syngenta): 0.5 oz/ac

Tomato 200 180 160 140 Bacterial spot (Xanthomonas perforans) a 180.6 Immokalee, FL CER-2016-087 Means with the same letter are NSD @ P= 0.05 AUDPC 120 100 80 60 40 20 0 b-e 53.5 Untreated Control Cueva 2 qt/ac + Double Nickel LC 1 qt/ac (A-M) 39.0 35.9 Actigard 0.5 oz/ac (A-M) Kocide 2 lb/ac + Manzate 2 lb/ac (F-M) Kocide 1.75 lb/ac + Manzate 1.5 lb/ac + SS (A-M) Variety: Charger Treatments: 13 applications made at 90 gal/ac from Sep 23 to Dec 13 AUDPC values calculated from 2 rating dates; Dec 9, and Dec 15 Disease severity was considered light despite two inoculations (Oct 26 and Nov 4) Cooperator: Pam Roberts, Univ FL cde de e 27.1 LifeGard 4.5 oz/100 gal (A-M) Kocide 2 lb/ac + Manzate 2 lb/ac (F-M)

Tomato Early & Late Blight 100 80 60 40 20 0 % Defoliation a Untreated check b LIFEGARD only Efficacy (% reduction relative to UTC) indicated for each treatment. 76% 90% c LIFEGARD program 86% c Standard program 50 40 30 20 10 0 Mkt fruit (ton/a) Yield increase (ton/a) over UTC indicated for each treatment. c Untreated check 25 b LIFEGARD only 35 a LIFEGARD program 31 ab Standard program Untreated check LIFEGARD Standard program K. Ivors, NC State University, Mills River, North Carolina

Thank you! Greg Rogers Elkon, MD 443-967-4010