1.1 Microbial Pesticides

(microbial pesticide/microbial pest control agent, MPCA)

Pesticides whose active ingredients are live microorganisms, such as naturally occurring or genetically modified bacteria, fungi, and viruses.

1.2 Active Ingredients of Microbial Pesticides

(microorganism active substance)

Microorganisms and their associated metabolites that contribute to the control of harmful organisms may be either live or inactivated.

1.3 Microbial Pesticide Technical Grade Material

(Technical grade of MPCA)

Microbial raw materials used in the formulation of microbial pesticides represent a highly purified form of these products. Aside from substances employed during production to support microbial growth or reproduction, as well as those used for standard purification or preparation, they typically contain no additional additives or excipients. In specific cases, to facilitate storage and transportation, small amounts of preservatives, stabilizers, or diluents may be added.

1.4 Microbial Pesticide Formulations

(microbial pest control product, MPCP)

Pesticide formulations that use live microbial agents as the active ingredient are registered or labeled for the control of harmful organisms.

1.5 Host/Host Organism

(host)

An animal (including humans), plant, or microorganism that provides a habitat or nutrients for other organisms (parasites).

1.6 Proliferation/Reproduction

(multiplication)

The ability of microorganisms to replicate or increase in number during the infection process.

1.7 Colonization

(colonization)

The ability of microorganisms to proliferate or persist in the environment—whether on the surface of a host organism (e.g., the skin) or on the surfaces of internal organs (such as the gut or lungs), or within the host’s external milieu (e.g., the skin) or internal milieu (tissues and organs like the gastrointestinal tract)—is referred to as colonization. When microorganisms colonize a particular organ or site, they may remain there for an extended period; their population size can remain stable, increase, or decline gradually. Colonizing microorganisms may be harmless, or they may be functional or pathogenic.

1.8 Persistence

(survival)

Active microorganisms remain viable on the surfaces or within the bodies of plants and animals, or in the soil, for a specified period after application.

1.9 Infection

(infection)

The process by which a pathogenic microorganism is introduced into or gains entry to a susceptible host, regardless of whether it elicits a pathological effect or disease. The microorganism must enter the host’s body—typically at the cellular level—and be capable of replicating to generate new infectious units. Simply ingesting a pathogen does not constitute infection.

1.10 Intrusion

(invasion)

The process by which microorganisms enter the host organism, such as by penetrating the epidermal layer or intestinal epithelial cells. “Primary invasiveness” is a characteristic of pathogenic microorganisms.

1.11 Pathogenicity

(pathogenicity)

The ability of microorganisms to cause disease in their hosts and/or to inflict harm on them. Many pathogenic bacteria exert pathogenicity through the combined effects of virulence and invasiveness, or of virulence and colonizing capacity. However, in some cases, diseases caused by invasive pathogens result from aberrant responses of the host’s defense system.

1.12 Infectiousness

(infectivity)

The ability of microorganisms to breach or evade the host’s natural barriers, enter the host, and remain viable or replicate. The nature of an infection may vary in severity, site, and quantity, but it does not depend on whether it elicits pathological effects or clinical manifestations.

1.13 Ecological Niche

(ecological niche)

The unique ecological niche of a particular species is understood in terms of the actual physical space it occupies within a community or ecosystem and the functions it performs.

1.14 Symbiotic Phenomenon

(symbiosis)

An interaction between two organisms in which one organism is closely associated with the other, benefiting both.

2 Classification Terms

2.1 Bacterial Pesticides

(bacterial pesticide)

A single-celled prokaryotic microbial pesticide with insecticidal and antibacterial bioactivity. It is extremely small in size, exhibits a simple morphology, reproduces asexually by binary fission, and is the most widely distributed and abundant group in nature.

2.1.1 Spores (Endospores)

(endospore)

Bacterial spores are dormant structures that appear as round, oval, or rod-shaped. Each spore gives rise to a single bacterium within the cell and exhibits exceptional heat resistance and strong refractivity, making it easy to count under a microscope. Spore-forming bacteria are the most abundant in microbial pesticide formulations, including Bacillus thuringiensis and Bacillus subtilis, among others.

2.1.2 Cyst

(cyst)  

Under adverse environmental conditions, certain bacteria form a round, dormant structure by thickening the entire cell wall and losing water; this structure is resistant to desiccation but not to heat.

2.1.3 Vegetative Body

(vegetative)

A single living organism can typically form a colony on an appropriate culture medium.

2.2 Fungal Pesticides

(fungal pesticide)

Eukaryotic microbial pesticides with bioactive properties such as insecticidal and disease‑preventive effects. They typically maintain their populations through asexual and sexual reproductive spores, with common reproductive structures including conidia, blastospores, chlamydospores, and mycelial vegetative hyphae.

2.2.1 Spores

(spore)

Fungal reproductive structures come in various forms.

2.2.1.1 Conidia

(conidium)

Asexual reproductive cells, which are colorless or pigmented, are produced by conidiogenous cells on conidiophores—either terminal or lateral, arranged in chains or clusters, and varying in shape and size, being either single‑celled or multicellular. This is the most common spore morphology found in fungal biopesticides. Common examples include Beauveria bassiana and Metarhizium anisopliae.

2.2.1.2 Arthrospores

(blastospore)

They are produced by budding: the cell membrane and cytoplasm protrude outward from a specific region of the cell wall, with one nucleus remaining in the mother cell and the other entering the protrusion, thereby forming the bud. These spores exhibit diverse morphologies and are characteristic of asexual reproduction in filamentous fungi; yeasts, Cryptococcus, and Candida can also produce such spores.

2.2.1.3 Chlamydospores

(chlamydospore)

Asexual dormant fungal spores, characterized by thick walls and long lifespans, are produced through the enlargement (protoplasmic condensation) and division of the mycelium, enabling them to withstand adverse environmental conditions.

2.2.2 Mycelial Vegetative Body / Nutritional Hyphae

(vegetative hypha)

A single tubular filament is the structural unit of most fungi.

2.2.3 Microspores

(microsporidium)

An ovoid, infectious parasite that develops and divides within host cells, capable of repeated asexual multiplication; it belongs to the lower fungi and is commonly referred to as a microsporidian, formerly known as a microsporidium.

2.3 Viral Pesticides

(viral pesticide)  

A non-cellular form of a viral particle composed of nucleic acid and a protein capsid, which uses insects as hosts, exhibits pathogenicity toward its host, and functions as a microbial pesticide with both direct and indirect biocontrol activities against harmful insects.

2.3.1 Baculoviridae

(Baculoviridae)

2.3.1.1 Nucleopolyhedrovirus

(nucleo polyhedrovirus, NPV)  

It contains one or more (monocapsid或多capsid) polyhedral virus particles—such as icosahedral, tetrahedral, pentahedral, and hexahedral forms—with diameters ranging from 0.5 to 15 μm. These particles are found within the host cell nucleus, and their nucleic acids comprise segmented genomes. This category represents the most widely used and most heavily registered class of viral microbial pesticide products; common examples include the cotton bollworm nucleopolyhedrovirus.

2.3.1.2 Granulovirus

(granulovirus, GV)

It contains a round or oval granular body that harbors nucleopolyhedrovirus particles—small in size and typically encased in a membrane enclosing a single‑stranded nucleocapsid. The inclusion bodies are located either in the nucleus or in the cytoplasm, and the viral genome is double‑stranded DNA. In the pesticide sector, its applications and registered formulations rank second only to those of nucleopolyhedroviruses; common examples include the diamondback moth granulovirus.

2.3.2 Reoviridae

(Rroviridae)

2.3.2.1 Polyhedrosis Virus

(cytoplasmic polyhedrovirus, CPV)  

It contains multiple virus particles, typically spherical polyhedra with hexagonal or quadrangular faces (diameter 0.5–10 μm). Inclusion bodies proliferate in the host cell cytoplasm, and the nucleic acid is segmented double-stranded RNA. Common examples include the pine caterpillar nuclear polyhedrosis virus, among others.

2.3.3 Parvoviridae

(Parvoviridae)

2.3.3.1 Concentrated Nuclear Virus

(densovirus, DV)

A small, icosahedral (diameter 18–30 nm) non-enveloped single-stranded DNA virus particle that replicates in the host cell nucleus and, during the early stages of infection, induces the formation of densely stained chromatin domains within the nucleus. Common examples include the nucleopolyhedrovirus of the American cockroach.

3.1 Toxin Protein Content

(toxin protein content)

The content of the insecticidal toxin protein, which is generated by alkaline hydrolysis of the parasporal crystals in Bacillus thuringiensis (Bt) products, is typically determined by SDS–polyacrylamide gel electrophoresis (SDS-PAGE), with results expressed as a percentage (%).

3.2 Bacterial Content

(microbial content)

The content of the active bacterial or fungal ingredient, in the form of viable microbial cells (including mycelia or spores), per unit mass or volume of a microbial pesticide product, is typically determined by plate count assay and expressed in CFU/g or CFU/mL.

3.2.1 Colony-Forming Units

(Colony forming units, CFU)

A colony, consisting of one or more cells—such as conidia or hyphae—grown on a culture medium and visible to the naked eye, is used to estimate the content of the active ingredient in live fungal or bacterial biopesticides.

3.3 Spore Content

(spore content)

The content of the fungal active ingredient in the form of viable spores per unit mass or volume of a microbial pesticide product is typically determined using the hemocytometer method. Spore count = measured number of spores × viability rate, with units expressed as spores/g or mL.

3.3.1 Viable Spore Rate

(percentage of alive spores)

Under specified culture conditions, the percentage (%) of spores that germinate—defined as having a germ tube length exceeding half the spore length—relative to the total number of spores is referred to as the spore germination rate.

3.4 Inclusion Bodies

(occlusion body, OB / polyhedral inclusion body, PIB)

After infecting a host, the virus replicates extensively within the host cells, forming aggregates of non-crystalline, inactive protein particles with high refractive indices, enclosed by a protective membrane. These structures, known as inclusion bodies, consist of viral particles, a protein matrix that immobilizes the virions, and a protective outer envelope. In an alkaline environment, the inclusion bodies dissolve, releasing the viral particles, which then reinfect the host. Inclusion bodies serve as the primary vehicle for horizontal transmission of microbial viruses and can be visualized under an optical microscope as distinct particulate structures.

3.4.1 Number of Inclusions

(OB content/PIB content)

The number of occlusion‑body virus particles contained per unit mass or volume of a microbial pesticide product. For polyhedra viruses, the hemocytometer method is commonly used; for granulovirus, either the bacterial counting plate method or real-time fluorescent quantitative PCR may be employed. The unit is expressed as (OB or PIB)/g or mL. Typically, OB is used for granuloviruses, while PIB or IB is used for polyhedra viruses; however, internationally, OB is now the predominant designation.

3.5 Virulence/Biological Potency (Ratio)

(virulence/biopotency (ratio))

The lethal or inhibitory efficacy of a microorganism against its target pest is assessed by the severity of disease symptoms observed in the host or recipient following inoculation with a specified dose of the microorganism, and its potency or bioactive properties are determined through bioassays. The half‑lethal dose (LD ₅₀ ), half-lethal time (LT ₅₀ ), expressed as the mortality percentage or inhibition rate (%), among others. Active units also include International Toxicity Units (ITU) and International Units (IU). Bioassay potency is determined by comparing the biological responses elicited by a test sample and a reference standard of a microbial pesticide, thereby calculating the ratio that reflects the bioassay potency or biological activity of the microbial pesticide.

3.5.1 International Toxicity Units/International Units

(international toxic unit, ITU/international unit, IU)

In bioassays, the smallest unit that can reliably indicate a given level of toxicity is referred to as a “toxicity unit” or simply a “unit.” The standard unit established by international pesticide‑related organizations (or through international consensus) is the International Toxicity Unit (ITU) or the International Unit (IU). These two units are essentially equivalent; it is recommended that biological potency be expressed consistently using the ITU, which more accurately reflects the product’s toxicity and helps avoid unnecessary confusion. The key to this approach lies in the preparation of reference standards and the validation of their potency. The toxicity or biological potency of microbial pesticides can be determined by comparative bioassays against reference samples with well‑defined International Toxicity Units or International Units, with results reported as the number of International Toxicity Units (or International Units) per unit weight or volume.

4.1 Microbial Contaminants

(microbial contaminants)

In microbial pesticide products, any microorganisms or substances other than the active microbial ingredient (or its metabolites) are considered contaminants or impurities. Microbial mutants or substitutes are likewise regarded as contaminants or impurities.

4.1.1 Amount of Contaminating Microorganisms

(number of microbial contaminants)

In microbial pesticide products, the total number of microorganisms other than the active ingredient (also referred to as the total colony count of miscellaneous microorganisms).

4.1.2 Rate of Contaminating Microorganisms

(percentage of microbial contaminants)

In microbial pesticide products, the percentage of non‑active microorganisms (such as bacteria and fungi) relative to the total microbial population.

4.2 Relevant Secondary Compounds/Metabolites

(relevant secondary compound/metabolite)

Any secondary compound or metabolite that, from a toxicological perspective, poses a hazard to human or animal health and/or to the environment. Some toxins may also be regarded as related secondary compounds or metabolites.

4.2.1 Secondary Compounds

(secondary compound)

A compound produced by microorganisms that is not essential for their growth, expansion, or reproduction.

4.3 Chemical Impurities

(chemical impurities)

Any chemical substance in a microbial pesticide product other than the active ingredient and substances essential to the manufacturing process.

4.4 Moisture/Drying Loss

(water/loss in weight)

Methods for expressing the moisture content of microbial pesticides generally fall into two categories: (1) Moisture: the ratio of water mass to the total mass of the sample; and (2) Loss on drying: the ratio of the mass loss after heating under specified conditions to the original mass of the sample, typically accounting for moisture and a small fraction of low‑volatility substances. This method applies only to solid pesticides. Although the two approaches differ in their analytical procedures, their underlying concepts are similar; in microbial pesticide standards, the latter is preferred due to its relative simplicity.

5.1 Plate Count Method/Colony-Forming Unit Method

(plate colony counting method/colony forming unit (CFU) method)

A method for determining the number of viable colony-forming units per unit mass or volume of a microbial pesticide product, by serially diluting fungal and bacterial samples to appropriate concentrations, uniformly spreading or pouring them onto culture agar plates, incubating at an optimal temperature, and then indirectly quantifying the visible colony count either by visual inspection or using an electronic counter; the unit is expressed as CFU/g or CFU/mL.

5.1.1 Method for Determining the Amount and Rate of Contaminant Fungi

(number or percentage of microbial contaminants determination)

In microbial pesticide products, the determination of contaminant microbial counts or contamination rates typically employs the plate count method. Contaminant microbial counts are expressed in CFU/g or CFU/mL, while contamination rates are reported as a percentage (%).

5.2 Hemocytometer Method/Direct Counting Method

(blood counting chamber method/direct counting method)

Under an optical microscope, the number of individual microorganisms—such as fungi or polyhedral viruses—can be directly counted using a hemocytometer. Typically, a suspension is prepared by serial dilution to an appropriate concentration, and after counting, the spore or inclusion body content per unit mass or volume of the product is calculated, expressed in units of spores/g or mL, or OB (PIB)/g or mL.

5.2.1 Bacterial Counting Chamber Method

(battery counting chamber method)

Under an optical microscope, the number of individual microorganisms—such as small particulate viruses—can be directly observed and counted using a bacterial counting chamber. Typically, a suspension is prepared by diluting the sample to an appropriate factor, and after counting, the number of inclusion bodies per unit mass or volume of the product is calculated; the units are usually expressed as OB/g or mL.

5.3 Real-Time Fluorescent Quantitative PCR Method

(real-time fluorescence quantitative PCR method)

By designing primers targeting specific conserved regions of known‑concentration standards—plasmids or genomic DNA containing the target gene—and performing quantitative real‑time PCR, a standard curve is generated to quantify the copy number of the nucleic acid–specific conserved fragment. The copy number of the conserved fragment obtained from an unknown sample via quantitative real‑time PCR is then calculated, allowing estimation of the number of inclusion bodies (expressed as OB/g or mL) or viral particles (expressed as viral particles/g or mL) per unit mass or volume of the microbial pesticide product. This method is primarily used for the detection of granulovirus and nucleopolyhedrovirus‑based pesticides.

5.4 Method for Determining Toxin Protein Content

(toxin protein content determination)

In Bacillus thuringiensis (Bt), the parasporal crystals are hydrolyzed under alkaline conditions to yield insecticidal toxin proteins. Based on differences in protein molecular weight, these proteins are separated from contaminant proteins in the sample by SDS‑PAGE gel electrophoresis; the toxin protein content in the Bt microbial pesticide product is then quantified using electrophoretic image scanning, expressed as a percentage (%).

China does not use this data to indicate the Bt‑product content. In the United States, Bt product labels list toxin protein content as one of the component‑content metrics and also specify bioassay potency data.

5.5 Methods for Determining Virulence or Biological Potency (Ratio)

(virulence/biopotency (ratio) determination)

A method that assesses the biological potency or bioactivity of microbial pesticides by comparing the biological responses of test samples and reference standards using bioassay techniques. The unit is expressed as International Toxicity Units (ITU) per milligram or International Units (IU) per milligram. This method is primarily used for determining the active‑ingredient content in products such as Bacillus thuringiensis preparations—including the Israeli subspecies—and Bacillus sphaericus, as well as for supplementary assays of the “activity” of certain viral active ingredients and for specific identification tests.

Virulence or biological potency = (reference standard ITU/mg or IU/mg) × reference standard LC ₅₀ /Specimen LC ₅₀ , with units expressed as ITU/mg or IU/mg; biological potency ratio = reference standard LC ₅₀ /Specimen LC ₅₀ ×100%, with the unit expressed as a percentage (%).

In summary, the hemocytometer method (including the bacterial counting chamber method) and the plate‑count method are currently the primary approaches for determining the active ingredient content in microbial pesticides. Real-time fluorescent quantitative PCR, while promising, remains less widely adopted by industry due to its stringent technical requirements and relatively high equipment and assay costs; it is presently applicable only to granuloviruses and nucleopolyhedroviruses, though it holds considerable potential for future development. The toxin‑protein assay is suitable exclusively for Bacillus thuringiensis‑based products. The virulence/bioassay method is primarily used for Bacillus thuringiensis products—including the Israeli subspecies—as well as for Bacillus sphaericus and similar formulations; despite certain limitations in product range, it accounts for the largest share of assays. Meanwhile, in viral products, bioassay‑based potency determination serves mainly as a supplementary “in vivo” verification or identification test.