Live Microbial Bioinsecticides

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1 Live Microbial Bioinsecticides Michael B. Dimock Certis USA IR-4 Western Region Biopesticide Training Workshop Fort Collins, Colorado April 25-26, 2017

2 Live Microbial Insecticides Bioinsecticides covered in this presentation: Activity depends on infection & replication in host Registered & commercially available in USA Used for plant protection Entomopathogenic fungi Baculoviruses Bacteria Paenibacillus popilliae (Milky Spore) - Japanese beetle Microsporidia Nosema locustae (NoloBait, Semaspore) - Grasshoppers

3 Mycoinsecticides: Entomopathogenic Fungi as Biopesticides Beauveria bassiana P. Avery, Univ. of Florida Isaria fumosorosea (formerly Paecilomyces fumosoroseus) Metarhizium anisopliae

4 Mycoinsecticides Mode of Action: Fungal Infection Appressorium Conidium Growth of Isaria on surface of whitefly egg. Lacey et al Biocontr Sci Technol 9: Figure 6.9 from Vega, F.E. and H.K. Kaya (2012). Insect Pathology, 2 nd ed.

Mycoinsecticides Visual Symptoms of Fungal

of hyphal penetration Little or no fungus

kills insect, but may not form visible mycelium

Dark spots visible at points of infection.

Mycosis and secondary spread under favorable

5 Mycoinsecticides Visual Symptoms of Fungal Infection Whitefly nymph killed by Isaria Point of hyphal penetration Little or no fungus evident on dead nymphs Fungus penetrates & kills insect, but may not form visible mycelium on outside of cadaver. Dark spots visible at points of infection. Silent mortality as cadavers darken & desiccate. Mycosis and secondary spread under favorable conditions (high RH). Whitefly nymph Aphid Leaf on moist filter paper in Petri dish ~24 hr

6 Mycoinsecticides EPA-Registered Products Fungus Strain Products Formulations Registrant Beauveria bassiana Isaria fumosorosea GHA Apopka 97 BotaniGard, Mycotrol PFR-97, Preferal Wettable powder Oil dispersion (ES) Water-dispersible granule LAM International ATCC Naturalis-L Flowable liquid Troy BioSciences ANT-03 Bioceres Wettable powder Water-dispersible briquette Anatis Bioprotection Certis USA FE 9901 NoFly Wettable powder Futureco Metarhizium anisopliae F52* Met52 Granular (soil incorp.) Oil dispersion (EC) Novozymes *Now recognized as M. brunneum.

7 Mycoinsecticides Target Arthropods On all or most labels: Aphids Mealybugs Plant bugs Psyllids Spider mites Thrips Whiteflies On some labels: Ants Beetles Caterpillars Chinch bugs Leafminers Orthoptera Root weevils Ticks White grubs Etc.

8 Mycoinsecticides Target Crops/Use Sites Food crops: Vegetables Cucurbits Grapes Berries Tree fruits & nuts Herbs & spices Seed crops Mushrooms* *Some products Non-food crops: Cut flowers Foliage plants Bedding plants Shrubs Turf (lawns)* Other ornamentals Forestry* NoFly: Indoor (greenhouse) use only. Naturalis: Outdoor use only. Others labeled for both indoor/outdoor use.

9 Mycoinsecticides Residue & Labor Management All are exempt from residue tolerance (no PHI or MRLs) 4 hour REI (12 hours for NoFly) Signal word: CAUTION PPE requirements: Applicator & other handlers Mixer/loader/applicator Long sleeve shirt Dust/mist filtering respirator Long pants Protective eyewear Shoes & socks Waterproof or chemical resistant gloves (some)

10 Mycoinsecticides Compatibility, Storage & Handling Compatibility: effect on spore viability in tank mix Most insecticides, oils, and adjuvants OK Fungicides generally not OK for mixing, OK for separate application Copper hydroxide OK to mix with some Wait 2-7 days before/after application of fungicides Storage conditions: Cool or refrigeration preferred Apply within [4-24] hours of mixing with water.

Mycoinsecticides Compatibility, Stability, Storage

Metarhizium: Conidia produced on solid media

fermentation Spores (conidia) Spores (conidia)

blastospores USDA-ARS Beauveria bassiana F.

11 Mycoinsecticides Compatibility, Stability, Storage May be affected by spore type: Beauveria & Metarhizium: Conidia produced on solid media Isaria: Blastopores produced in submerged (liquid) fermentation Spores (conidia) Spores (conidia) Blastospores Hyphal growth from germinated blastospores USDA-ARS Beauveria bassiana F. Ihara/NARO (Japan) Metarhizium anisopliae P. Avery, Univ. of Florida Isaria fumosorosea May be affected by formulation (liquid/oil vs. dry) Consult labels, tech bulletins, & registrants!

12 Mycoinsecticides Effects on Beneficials Active Ingr. Product Label statements on bees B. bassiana BotaniGard, Mycotrol Bioceres Naturalis Potentially pathogenic/may harm beneficial insects & honeybees. I. fumosorosea PFR-97, Preferal Do not apply when bees actively foraging NoFly M. anisopliae Met52 None Effects on predators, parasitoids: None (greenhouse use only) I. fumosorosea - Extensively tested, broadly compatible Biobest Side Effects Manual ( B. bassiana - Generally compatible, some adverse effects M. anisopliae?

Distinct, nonchemical mode of action Mix/rotate with other insecticides to manage

13 Mycoinsecticides Resistance Management No known cases of resistance to commercial mycoinsectides. But: Insects do have immune defenses against pathogens. Distinct, nonchemical mode of action Mix/rotate with other insecticides to manage resistant pests. (e.g. Bemisia Q biotype, Colorado potato beetle) Low risk of cross resistance Synergism with some chemistries (Pyrethrins, IGRs) Nymph of Bemisia tabaci biotype Q infected with Beauveria bassiana (right) compared to untreated control nymph (left). Zhu & Kim, Biocontrol Science & Technology 21(12): Control Bb GHA

Considerations in Using Mycoinsecticides Contact insecticides (good coverage important). 3-7 days required to kill most target pests. Sensitive to environmental conditions.

14 Considerations in Using Mycoinsecticides Contact insecticides (good coverage important). 3-7 days required to kill most target pests. Sensitive to environmental conditions. High temperature (>85 F) Low relative humidity (<80%) Direct sunlight Mycosis ( fuzzballs ) Not necessary for product efficacy. Secondary spread under optimal conditions. May not work well in crops with heavy fungicide use. Effect of spore type Conidia - hydrophobic, may benefit from oil/surfactant Blastospores - hydrophilic, less need for adjuvant

15 Granulovirus (GV) Baculoviruses: Highly Specific Microbial Insecticides Nucleopolyhedrovirus (NPV) Infect and kill only Lepidoptera larvae. Infective by ingestion only. Virus embedded in protein occlusion bodies (OB) OBs dissolve in alkaline midgut, releasing virus Replicates in midgut cells, spreads throughout host NPV Healthy beet armyworm ~3 days post-infection ~7-10 days post-infection ~10-14 days post-infection

Viral Insecticides in the USA All produced in vivo in mass-reared host larvae. Most are aqueous SC formulations of insect homogenate containing OB s.

16 Viral Insecticides in the USA All produced in vivo in mass-reared host larvae. Most are aqueous SC formulations of insect homogenate containing OB s. Type Products Registrant Target insects Crops/Use sites GV NPV Cyd-X, Cyd-X HP Carpovirusine ViroSoft Certis USA Arysta BioTEPP Codling moth Madex HP Andermatt Codling moth & OFM Pome fruits, stone fruits, walnuts NutGuard, FruitGuard AgriVir Indianmeal moth Dried fruit, nuts, stored grain, seeds Gemstar Heligen Helicovex Spod-X Spexit Certis USA AgBiTech Andermatt Certis USA Andermatt Corn earworm, tomato fruitworm, bollworm, tobacco budworm Beet armyworm Loopex Andermatt Cabbage looper Vegetables TM-Biocontrol Gypcheck US Forest Service Douglas fir tussock moth Gypsy moth Vegetables, berries, cucurbits, corn, cotton, potatoes, soybeans, flowers, many others Forest & shade trees

17 Viral Insecticides Residue & Labor Management Exempt from residue tolerance (no PHI or MRLs) 4 hour REI Signal word: CAUTION PPE requirements: Applicator & other handlers Mixer/loader/applicator Long sleeve shirt Dust/mist filtering respirator Long pants Protective eyewear (some) Waterproof gloves (chemical resistant for some) Shoes & socks

18 Viral Insecticides Compatibility, Stability, Storage Compatible with most insecticides, fungicides, adjuvants. Avoid highly alkaline conditions (buffer if ph 9). Avoid silicone spreaders OB s follow wetting front (non-uniform deposition) Oil-based adjuvants, spreader/stickers, pinolenes are OK Mixing with copper fungicides may reduce potency Most require refrigeration for storage 1 year. Some OK for 3-6 months at room temperature. Consult labels, tech bulletins, & registrants!

19 Viral Insecticides Resistance Management Codling moth resistance to CpGV in Europe since 2005 Organic orchards relying solely on frequent virus applications. Solutions: New CpGV isolates, in vivo production in resistant larvae. No confirmed field resistance to CpGV in North America Mostly used in programs with conventional chemical insecticides. Spinosad formulation (Entrust) for use in organic programs. Several different CpGV isolates already in commercial use. No known cross-resistance with other insecticides Unique mode of action: Resistant larvae still susceptible to viral infection Application to transgenic Bt crops

harm to pollinators Potential for horizontal & vertical transmission Viral epizootics common in nature

20 Viral Insecticides IPM Considerations No adverse effects on nontarget organisms Infect and kill only certain Lepidoptera larvae No direct effects on predators, parasitoids No secondary pest outbreaks No harm to pollinators Potential for horizontal & vertical transmission Viral epizootics common in nature Carryover to next generation or season Areawide management programs with mating disruption, crop-free periods, etc.

21 Considerations in Using Viral Insecticides Effective against Lepidoptera larvae only. Most effective against small larvae (L1 - L2). Slower than many insecticides: ~3 d to stop feeding, 7 d to kill. Larger larvae: higher dose, cause more damage before death. Must be ingested by larvae Uniform spray coverage important. Spreader-sticker may help (oil-based, pinolene, molasses). Avoid silicone spreaders (OB s follow wetting front). Limited persistence ~ 2 day half-life in direct sun - UV screen or spray late in day. Repeat applications: New growth, sustained oviposition/egg hatch. Moderately rainfast when allowed to dry.

22 Viral Insecticides Codling Moth Granulovirus in Apples Results after first use Failed entry ( sting ) Infected as early instar Infected as late instar or with low dose Results over multiple generations Charmillot & Pasquier 2007 Tree banding to monitor adult emergence

Helicoverpa zea in Organic Sweet Corn A. Schreiber/Eltopia, WA Univ.

1 overhead sprinkler irrigation from 20 Aug - 10 Sep.

Accepted limit Untreated control Gemstar 4 oz/a Every 5 days Entrust 2 oz/a Every 5 days Entrust 2

23 Helicoverpa zea in Organic Sweet Corn A. Schreiber/Eltopia, WA Univ. Minnesota Randomized complete block with 4 replicates. Applied in 0.1 overhead sprinkler irrigation from 20 Aug - 10 Sep. 8 appl Entrust Total 16 oz $332/A 4 appl Gemstar Total 16 fl oz 4 appl Entrust Total 4 oz $153/A Accepted limit Untreated control Gemstar 4 oz/a Every 5 days Entrust 2 oz/a Every 5 days Entrust 2 oz/a Every 3 days Gemstar 4 fl oz alt. Entrust 1 oz/a Every 3 days Alternating program of NPV and insecticide resulted in better control at significantly lower cost compared to a insecticide-only program.

formation: Use NPV to reduce larvae numbers and damage in

Tank mix with chemical ovicides or insecticides if desired.

24 Strategy for NPV vs. Bollworm & Budworm in Cotton USDA-ARS photos Before square formation: Use NPV to reduce larvae numbers and damage in vegetative crop stages. Tank mix with chemical ovicides or insecticides if desired. Early season use may result in reduced infestation later due to NPV epizootic. After square formation: Use chemical insecticides as main tactic to protect fruiting structures. Tank mix with NPV to kill insecticide-resistant larvae. Use NPV for pupa busting (reduce carryover to next crop).

More Considerations in Using Viral Insecticides Highly

instar larva: 1 or 2 OB s. Objective: An OB in every bite.

from sunlight. High temperature of concern only during storage.

25 More Considerations in Using Viral Insecticides Highly virulent compared to other microbials Lethal dose for 1 st instar larva: 1 or 2 OB s. Objective: An OB in every bite. Once ingested, the virus persists inside the host, protected from sunlight. High temperature of concern only during storage. Once applied, high temperatures promote more rapid uptake & replication of virus in the host. Most of the kill from a single virus spray results from larval feeding in the first few hours after application.

Live Microbial Insecticides Other Considerations Common to all live microbial

Repeated use may lead to population reduction over time (epizootic) Highly

26 Live Microbial Insecticides Other Considerations Common to all live microbial insecticides: Microbial control via inundative (vs. inoculative) release. Repeated use may lead to population reduction over time (epizootic) Highly infectious (risk of cross-contamination in trials). Physical separation of plots (e.g. buffer rows) Order of application & evaluation: Untreated check Standard check Low dose High dose Sanitation of spray equipment, tools, etc. (e.g. bleach) Shallow or no rate response compared to chemical insecticides. Application frequency often more important than rate/acre. Symptoms can be early indicators of efficacy.

Useful References Vega, F. & H. Kaya (2012) Insect Pathology, 2 nd ed.

Kaya (2007) Field Manual of Techniques in Invertebrate Pathology, 2 nd ed.

(2012) Manual of Techniques in Invertebrate Pathology, 2 nd ed.

27 Useful References Vega, F. & H. Kaya (2012) Insect Pathology, 2 nd ed. Academic Press, NY. ISBN: Lacey, L. & H. Kaya (2007) Field Manual of Techniques in Invertebrate Pathology, 2 nd ed. Springer, Dordrecht, NL. ISBN: Lacey, L., ed. (2012) Manual of Techniques in Invertebrate Pathology, 2 nd ed. Academic Press, NY. ISBN: Lacey, L., ed. (2017) Microbial Control of Insect & Mite Pests. Academic Press, NY. ISBN:

BioCeres WP AzaGuard. IR-4 Bio-Pesticide Workshop, Presented by: Manuel Campos

BioCeres WP AzaGuard. IR-4 Bio-Pesticide Workshop, Presented by: Manuel Campos BioCeres WP AzaGuard IR-4 Bio-Pesticide Workshop, 2016 Presented by: Manuel Campos BioCeres WP Active Ingredient: Beauveria bassiana strain-ant-03-20.0% w/w Shelf Life: 6 months Environmental Hazards: