Live Microbial Bioinsecticides

IR-4 Western Region Biopesticide Training Workshop

Fort Collins, Colorado April 25 - 26, 2017

Michael B. Dimock Certis USA

Entomopathogenic fungi

Baculoviruses

Bacteria Paenibacillus popilliae (Milky Spore) - Japanese beetle

Microsporidia Nosema locustae (NoloBait, Semaspore) - Grasshoppers

Live Microbial Insecticides

Bioinsecticides covered in this presentation: Activity depends on infection & replication in host Registered & commercially available in USA Used for plant protection

Mycoinsecticides: Entomopathogenic Fungi as Biopesticides

Beauveria bassiana

www.potatobeetle.org

Isaria fumosorosea (formerly Paecilomyces fumosoroseus)

P. Avery, Univ. of Florida

Metarhizium anisopliae www.ffpri.affrc.go.jp

Mode of Action: Fungal Infection

Figure 6.9 from Vega, F.E. and H.K. Kaya (2012). Insect Pathology, 2nd ed.

Mycoinsecticides

Conidium

Appressorium

Growth of Isaria on surface of whitefly egg.

Lacey et al. 1999. Biocontr Sci Technol 9: 9-18..

Fungus penetrates & kills insect, but may not form visible mycelium on outside of cadaver.

Whitefly nymph killed by Isaria

Little or no fungus evident on dead nymphs

Visual Symptoms of Fungal Infection

Point of hyphal penetration

Whitefly nymph Aphid

Dark spots visible at points of infection.

“Silent mortality” as cadavers darken & desiccate.

Mycosis and secondary spread under favorable conditions (high RH).

Mycoinsecticides

Leaf on moist filter paper in Petri dish ~24 hr

Fungus Strain Products Formulations Registrant

EPA-Registered Products Mycoinsecticides

*Now recognized as M. brunneum.

Metarhizium anisopliae F52* Met52 Granular (soil incorp.)

Oil dispersion (EC) Novozymes

Isaria fumosorosea

Apopka 97 PFR-97, Preferal Water-dispersible granule Certis USA

FE 9901 NoFly Wettable powder Futureco

Beauveria bassiana

GHA BotaniGard, Mycotrol

Wettable powder LAM International

Oil dispersion (ES)

ATCC 74040 Naturalis-L Flowable liquid Troy BioSciences

ANT-03 Bioceres Wettable powder Anatis

Bioprotection Water-dispersible briquette

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.

Target Arthropods Mycoinsecticides

Food crops: • Vegetables • Cucurbits • Grapes • Berries • Tree fruits & nuts • Herbs & spices • Seed crops • Mushrooms*

Target Crops/Use Sites Mycoinsecticides

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.

*Some products

Residue & Labor Management Mycoinsecticides

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)

Compatibility, Storage & Handling Mycoinsecticides

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.

Compatibility, Stability, Storage Mycoinsecticides

May be affected by formulation (liquid/oil vs. dry)

Consult labels, tech bulletins, & registrants!

USDA-ARS

Spores (conidia)

Beauveria bassiana F. Ihara/NARO (Japan)

Spores (conidia)

Metarhizium anisopliae

Hyphal growth from germinated blastospores

Blastospores

P. Avery, Univ. of Florida

Isaria fumosorosea

May be affected by spore type: • Beauveria & Metarhizium: Conidia produced on solid media • Isaria: Blastopores produced in submerged (liquid) fermentation

Effects on Beneficials

Active Ingr. Product Label statements on bees

B. bassiana

BotaniGard, Mycotrol Potentially pathogenic/may harm beneficial insects & honeybees. Bioceres

Naturalis

I. fumosorosea PFR-97, Preferal Do not apply when bees actively foraging

NoFly None (greenhouse use only)

M. anisopliae Met52 None

Mycoinsecticides

Effects on predators, parasitoids: I. fumosorosea - Extensively tested, broadly compatible

Biobest Side Effects Manual (www.biobestgroup.com) B. bassiana - Generally compatible, some adverse effects M. anisopliae?

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)

Resistance Management Mycoinsecticides

Control Bb GHA

Nymph of Bemisia tabaci biotype Q infected with Beauveria bassiana (right) compared to untreated control nymph (left).

Zhu & Kim, 2011. Biocontrol Science & Technology 21(12): 1471 - 1483.

No known cases of resistance to commercial mycoinsectides. • But: Insects do have immune defenses against pathogens.

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

Considerations in Using Mycoinsecticides

Baculoviruses: Highly Specific Microbial Insecticides

Granulovirus (GV)

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

Nucleopolyhedrovirus (NPV)

Healthy beet armyworm

~3 days post-infection

NPV

~7 - 10 days post-infection

~10 - 14 days post-infection

Type Products Registrant Target insects Crops/Use sites

GV

Cyd-X, Cyd-X HP Certis USA

Codling moth Pome fruits, stone fruits, walnuts

Carpovirusine Arysta

ViroSoft BioTEPP

Madex HP Andermatt Codling moth & OFM

NutGuard, FruitGuard AgriVir Indianmeal moth Dried fruit, nuts, stored grain, seeds

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.

NPV

Gemstar Certis USA Corn earworm, tomato fruitworm, bollworm, tobacco budworm

Vegetables, berries, cucurbits, corn, cotton, potatoes, soybeans, flowers, many others

Heligen AgBiTech

Helicovex Andermatt

Spod-X Certis USA Beet armyworm

Spexit Andermatt

Loopex Andermatt Cabbage looper Vegetables

TM-Biocontrol US Forest Service

Douglas fir tussock moth Forest & shade trees

Gypcheck Gypsy moth

Residue & Labor Management Viral Insecticides

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

Compatibility, Stability, Storage Viral Insecticides

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!

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

Resistance Management Viral Insecticides

IPM Considerations Viral Insecticides

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.

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.

Considerations in Using Viral Insecticides

Codling Moth Granulovirus in Apples

Infected as late instar or with low dose

Failed entry (“sting”)

Infected as early instar

Viral Insecticides

Charmillot & Pasquier 2007

Tree banding to monitor adult emergence

Results after first use

Results over multiple generations

Randomized complete block with 4 replicates. Applied in 0.1” overhead sprinkler irrigation

from 20 Aug - 10 Sep.

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

Accepted limit

Alternating program of NPV and insecticide resulted in better control at significantly lower cost compared to a insecticide-only program.

8 appl Entrust Total 16 oz

$332/A

4 appl Gemstar Total 16 fl oz

4 appl Entrust Total 4 oz

$153/A

Univ. Minnesota

Untreated control

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

Gemstar 4 oz/A Every 5 days

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.

Strategy for NPV vs. Bollworm & Budworm in Cotton

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).

USDA-ARS photos

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.

More Considerations in Using Viral Insecticides

Highly virulent compared to other microbials • Lethal dose for 1st instar larva: 1 or 2 OB’s. • Objective: “An OB in every bite.”

Most of the kill from a single virus spray results from larval feeding in the first few hours after application.

Other Considerations

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.

Live Microbial Insecticides

Common to all live microbial insecticides:

Useful References

Lacey, L. & H. Kaya (2007) Field Manual of Techniques in

Invertebrate Pathology, 2nd ed. Springer, Dordrecht, NL.

ISBN: 978-4020-5932-2

Lacey, L., ed. (2012) Manual of Techniques in Invertebrate Pathology,

2nd ed. Academic Press, NY.

ISBN: 978-4020-5932-2 Lacey, L., ed. (2017) Microbial Control of Insect & Mite Pests.

Academic Press, NY. ISBN: 978-4020-5932-2

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

Academic Press, NY. ISBN: 978-0-12-384984-7