🦠 Biopesticide Formulations

This lesson builds core elective concepts in BSc Agriculture with practical applications and exam-oriented clarity.

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Biopesticide Formulations

Classification of Biopesticides

Biopesticides are categorized into three major groups by the USEPA and Indian regulatory system:

1. Microbial biopesticides — contain a microorganism (bacterium, fungus, virus, protozoan) as the active ingredient
2. Biochemical biopesticides — naturally occurring compounds that control pests by non-toxic mechanisms (plant extracts, pheromones, insect growth regulators)
3. Macro-organisms (biocontrol agents) — predatory insects, parasitoids (Trichogramma spp., Chrysoperla)

This lesson focuses primarily on microbial biopesticides — the most commercially significant category.

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Bacillus thuringiensis (Bt)

Bt is the world's most widely used microbial biopesticide, accounting for over 95% of all biopesticide sales globally.

Strains and Target Pests

Bt Subspecies	Key Cry Proteins	Target Order	Example Pests
Bt var. kurstaki (Btk)	Cry1Aa, Cry1Ab, Cry1Ac	Lepidoptera	Spodoptera, Helicoverpa, DBM
Bt var. israelensis (Bti)	Cry4Aa, Cry11Aa, Cyt1Aa	Diptera	Mosquito larvae, blackfly
Bt var. tenebrionis (Btt)	Cry3Aa	Coleoptera	Colorado potato beetle
Bt var. aizawai	Cry1Ca, Cry1Da	Lepidoptera	Diamond back moth, resistant strains

Mode of Action

1. Insect ingests Bt spores/crystals → alkaline gut pH (pH 9–10) solubilizes crystal
2. Protoxin (130–140 kDa) cleaved by gut proteases → active toxin (60–70 kDa)
3. Active toxin binds to cadherin receptors on midgut epithelium
4. Oligomerization → pore formation → cell lysis → gut paralysis → death within 24–72 hours
5. Specificity due to receptor binding — receptors absent in vertebrates, non-target invertebrates

Bt Formulation Types

Wettable Powder (WP):

• Composition: fermentation-dried biomass (spores + crystals) + inert carrier (kaolin) + wetting agent (sodium lignosulfonate) + dispersant
• Particle size: <50 μm for uniform suspension
• Potency expressed as IU/mg (International Units) or LC50 against test insect
• Advantages: easy storage, longer shelf life than liquid

Suspension Concentrate (SC):

• Aqueous suspension of Bt biomass
• Additives: surfactants (Tween 80), anti-freeze agents (propylene glycol), thickeners (xanthan gum)
• Advantages: no dust hazard; easy to measure and mix
• Shelf life: 18–24 months at room temperature

Emulsifiable Concentrate (EC):

• Oil-based; used for trunk injection in forest pest management
• Emulsifiers allow mixing with water at application

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NPV (Nuclear Polyhedrosis Virus)

NPV (Nucleopolyhedrovirus) is a host-specific insect virus that causes lethal infection by replicating in the insect's nucleus. It is one of the safest biopesticides known.

Key NPV Products in India

Virus	Target Insect	Crop
Spodoptera litura NPV (SLNPV)	S. litura (tobacco caterpillar)	Groundnut, soybean, vegetables
Helicoverpa armigera NPV (HaNPV)	H. armigera (American bollworm)	Cotton, chickpea, tomato
Achaea janata NPV	A. janata (castor semilooper)	Castor

Mode of Action

1. Larva ingests occlusion bodies (OBs) with food
2. Alkaline midgut pH dissolves polyhedrin protein → releases virions
3. Virions infect midgut epithelial cells → spread systemically
4. Replicates in nucleus → fills cell with new OBs
5. Larva becomes flaccid, skin ruptures → releases billions of OBs onto foliage
6. Death in 4–7 days; slower than chemical insecticides

Production (In Vivo Method — NBAII Protocol)

NPV can only be produced in living insect larvae (no in vitro culture possible for most NPVs):

1. Rear Spodoptera larvae on semi-artificial diet (castor/groundnut leaves or agar-based)
2. Virus inoculation — Smear virus suspension (10^6 OB/ml) on leaf surface; larvae feed
3. Collect dead/dying larvae (liquefied stage, day 5–7)
4. Homogenize in distilled water, filter through muslin
5. Centrifuge to separate OBs (15,000 rpm, 20 min)
6. Count OBs under microscope using hemocytometer
7. Quality standard: ≥1.5 × 10^9 OBs/ml for SC formulation

Formulation

• SC formulation: OB suspension + glycerol (anti-desiccant) + UV protectants
• UV protectants: Optical brighteners (e.g., Tinopal UNPA-GX, Leucopur) — absorb UV, re-emit in visible range; protect OBs from UV degradation; also enhance per os infectivity
• WP formulation: Spray-dried OB suspension on kaolin + starch
• Storage: 4°C; shelf life 6 months (liquid), 12 months (WP at room temperature)

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Entomopathogenic Fungi

Entomopathogenic fungi kill insects by cuticle penetration — they do not need to be ingested, making them effective against sucking pests that cannot be reached by Bt or NPV.

Beauveria bassiana

• Target pests: Whitefly, thrips, aphids, diamondback moth larvae, BPH in rice, termites
• Infection mechanism: Conidial attachment to cuticle → germination → appressorium formation → mechanical + enzymatic (protease, lipase, chitinase) penetration → hyphal growth fills body cavity → Beauvericin toxin production → death in 5–10 days
• White halo of conidia on dead insect is diagnostic

Production (Solid substrate fermentation):

1. Sterilize rice grains or sorghum grains (autoclaved)
2. Inoculate with Beauveria bassiana starter culture
3. Incubate at 25°C, 85% RH for 14 days in polypropylene bags
4. Dry at 30–35°C (do not exceed 40°C — kills spores)
5. Grind to fine powder; pass through 100-mesh sieve
6. Quality: ≥2 × 10^8 conidia/g; germination rate ≥80%

WP formulation: Dried conidia + talc + kaolin + wetting agent (Tween 80) + dispersant

Metarhizium anisopliae

• Target pests: Rhinoceros beetle (Oryctes rhinoceros), white grubs, termites, locusts
• Production: Similar to Beauveria — solid substrate on rice/sorghum
• Quality standard: ≥2 × 10^8 conidia/g; germination ≥75%
• Green muscardine on dead insects is diagnostic

Isaria fumosorosea (syn. Paecilomyces fumosoroseus)

• Emerging biopesticide against whitefly, thrips, spider mites
• USEPA first approval 2003

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Trichoderma Formulations

Trichoderma spp. are the most widely used fungal biocontrol agents against soil-borne plant pathogens in India.

Species and Target Pathogens

Trichoderma species	Target pathogen	Crop
T. viride	Fusarium oxysporum, Rhizoctonia solani	All crops
T. harzianum	Sclerotium rolfsii, Pythium spp.	Groundnut, vegetable crops
T. asperellum	Fusarium wilt	Tomato, banana
T. virens	Pythium spp.	Cotton, soybean

Mode of Action

1. Mycoparasitism — Coils around pathogen hyphae; secretes cell-wall degrading enzymes (glucanase, chitinase)
2. Antibiosis — Produces volatile and non-volatile antibiotics (viridian, gliotoxin, trichoviridin)
3. Competition — Outcompetes pathogens for space and nutrients in rhizosphere
4. ISR (Induced Systemic Resistance) — Primes plant defense pathways

Formulation Types

Talc-based (most common in India):

• Carrier: purified talc (pH 7.0; sterilized)
• Minimum quality: 10^8 CFU/g
• Shelf life: 6 months at room temperature
• Application: seed treatment (4–6g/kg seed) + soil application (2.5 kg/ha in FYM)

Liquid formulation:

• Fermentation broth; minimum 10^8 CFU/ml; shelf life 12 months

Wettable Powder (WP):

• Spray-dried conidia on silica carrier; suitable for foliar and drench application

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Pseudomonas fluorescens Formulations

P. fluorescens is a Gram-negative PGPR with dual role: plant growth promotion + biocontrol.

• Biocontrol mechanism: Production of hydrogen cyanide (HCN), 2,4-diacetylphloroglucinol (2,4-DAPG), pyoluteorin — inhibit soil-borne pathogens; ISR induction
• PGPR activity: Phosphate solubilization, siderophore production, IAA synthesis
• Formulation: Talc-based (10^8 CFU/g) or liquid; similar to Trichoderma
• Target diseases: Damping-off, root rot, bacterial wilt suppression

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Biopesticides Registered in India (Major Products)

Biopesticide	Active Ingredient	Target Pest	Crop	Dose
Biobit/Delfin	Bt kurstaki	Lepidopterous pests	Cotton, vegetables	1–2 kg/ha
Achook	Azadirachtin 0.15%	Sucking pests, Lepidoptera	All crops	2–5 ml/L
Helicovex	HaNPV (1.5×10^9 OB/ml)	Helicoverpa armigera	Cotton, chickpea	250 ml/ha
Spodonim	SLNPV	Spodoptera litura	Groundnut, soybean	250 ml/ha
Boveril	Beauveria bassiana	Whitefly, thrips	Vegetables, cotton	1 kg/ha
Metariz	Metarhizium anisopliae	White grubs, rhinoceros beetle	Sugarcane, coconut	2.5 kg/ha
Ecostin	Trichoderma viride	Fusarium, Rhizoctonia	All crops	4g/kg seed

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Overview

Microbial biopesticide formulations span a spectrum from bacterial spore-crystal products (Bt) to obligate viral pathogens (NPV) and fungal entomopathogens. Each has distinct production requirements, formulation challenges, and target specificity. The key challenges are maintaining viability, protecting from UV degradation, and ensuring shelf stability — challenges that modern formulation science increasingly addresses through encapsulation and protective additives.

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Summary Cheat Sheet

Topic	Key takeaway
Main focus	Classification, production, and formulation of microbial biopesticides including Bt, NPV, entomopathogenic fungi, and Trichoderma.
Section context	Revise this lesson with the rest of Biopesticide Technology for stronger conceptual continuity.