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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