# Bio-pesticides and Bio-fertilizers

Ajeet Kumar

► To cite this version:

Ajeet Kumar. Bio-pesticides and Bio-fertilizers. Master. Bio-pesticides and Bio-fertilizers, DR. RAJENDRA PRASAD CENTRAL AGRICULTURAL UNIVERSITY PUSA, SAMASTIPUR-848125, BIHAR, INDIA, India. 2025, pp.1-14. hal-05124501

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Submitted on 6 Aug 2025

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Distributed under a Creative Commons Attribution 4.0 International License
Dr. Rajendra Prasad Central Agricultural University
Pusa (Samastipur) - 848125, Bihar

*A lecture notes*

*On*

LSN-8-BP-Ajeet Kumar/ASS-307/Bio-Pesticides-specific matter

<figure><img src="image_2.png" /></figure>

COURSE TITLE: Bio-pesticides and Bio-fertilizers

*(Elective course)*

COURSE NO.: ASS-307

CREDIT HOUR: 3 (2+1)

YEAR/SEMESTER: 3rd YEAR / 6th SEMESTER

Dr. AJEET KUMAR

Assistant Professor-cum-Scientist (Soil Science)

Department of Microbiology

Collage of Basic Sciences and Humanities (CBS&H),

Dr. Rajendra Prasad Central Agricultural University, Pusa (Samastipur)

June 2025
# LSN-8-BP-Ajeet Kumar/ASS-307/Bio-Pesticides-specific matter

Dr. AJEET KUMAR<fnref n="1" />

A fascinating and economically important area of applied microbial ecology is the biological control of pests and disease-causing agents using microorganisms. Biological control using microorganisms to initiate disease in pest populations has the potential of reducing agricultural reliance on chemical pesticides.

The negative interactions (amensalism, predation and parasitism) among microbial populations and between microbes and higher organisms have always formed a natural basis for the biological control of pests and pathogens, and biological control methods take advantage of those relationships.

Biological methods to control populations of disease causing organisms and pests are based on modification of host and or vector population, modification of reservoirs of pathogens, and the direct use of microbial pathogens and predators. Biotechnology may improve approaches for controlling pest and disease causing populations.

## Biological Control

Biological control is the reduction of inoculum density or disease producing activities of a pathogen or parasite in its active or dormant state by one or more organisms. This is accomplished naturally or through manipulation of the environment/host/antagonist or by mass introduction of one or more antagonists.

## Principles of Biocontrol

**Biocontrol** is based on antagonism. Which is further based on competition (for nutrients and space), parasitism (by production of volatile non-volatile antibiotics) and hyperparasitism. Competition In the soil, the dormant propagules of fungi are subjected to intense microbial competition when nutrients become available, e.g. Ammonium sulphate solution improves the biocontrol of Heterobasidium annosum by *Trichoderma viridae*.

**Hyperparasitism** It is the attack of a secondary parasite on primary parasite (parasitism of one microbe by another) observed among airborne pathogens. Rusts are often parasitised by

<footnote n="1">¹ Assistant Professor-cum-Scientist (Soil Science-Soil Microbiology, Soil fertility, Soil Chemistry), Department of Microbiology, College of Basic Sciences & Humanities (CBS&H), RPCAU, Pusa-848125, Bihar. E-mail: ajeet.sri@rpcau.ac.in</footnote>
Enderluca carices and powdery mildews by Ampelomyces quisqualis. Cladosporium species parasitise both rust and powdery mildews.

**Antibiosis** It depends on competitive saprophytic ability of a fungus. Sterile soil does not produce antibiotic substances. Several phylloplane fungi such as Aureobasidium, Alternaria, Botrytis and Helminthosporium have been shown to produce antibiotics.

**Microbial Insecticides:**

Pests and diseases can be effectively controlled without chemical pesticides by good crop management practices.

**Integrated Pest Management (IPM):**

It is a law that emphasises the need to restrict insect population rather than to eradicate them so that the pest population is kept at a non-injurious level. IPM is a pest management system that in the context of the associated environment and the population dynamics of the pest species utilises all suitable techniques and methods in a compatible manner as possible to maintain the pest population at levels below those causing economic injury.

Population of pathogenic or predatory microorganisms that are antagonistic towards a particular pest population provide a natural means of controlling pest population and preparations of such antagonistic microbial populations are called microbial pesticides or microbial insecticides.

An effective microbial pesticide possesses the following characteristics:

1. The microbial pathogen must be virulent and cause disease in the pest population when properly applied at the recommended concentration.

2. Pathogen should not be susceptible to environmental conditions. After application, it should survive long until the infection is established within the pest population.

3. Resistant stages of the pathogen like spores or cysts are best for use.

4. The pathogen should be rather specific for the pest population and must not cause disease in the non-pest population. It should rapidly establish disease in the pest population so as to minimise destruction caused by the pest.

5. The microbial pesticide should be harmless to humans and other valued plant and animal population.

The various types of microbial insecticides include

(a) Bacterial pesticides
(b) Viral pesticides and

(c) Fungal pesticides.

(a) Bacterial Pesticides

Several bacterial pathogens that have been used as insecticides include

a) Endospore forming: *Bacillus* and *Clostridium* species

b) Non-endospore forming: species of *Pseudomonas*, *Enterobacter*, *Proteus*, *Serratia*, *Xenorhabdus*.

Of the potential bacterial pesticides, *Bacillus thuringiensis* has been most extensively studied.
*Bacillus thuringiensis* has been successfully tested against more than 140 insect species
(Lepidoptera, Hymenoptera, Diptera and Coleoptera).

At present, there are 12 groups of B. thuringiensis. All strains produce protein crystal inclusion bodies which act as endotoxin. They are toxic factors and are called parasporal bodies. These crystals dissolve under alkaline condition. They are not soluble in water under neutral or acidic condition. The midgut contents of the caterpillar larvae (pests) are alkaline. On ingestion, the crystal dissolves in the midgut fluid and gets digested particularly by the proteolytic enzyme present in the midgut fluid. This digested protein crystal attacks the cementing substances which are present in the gut wall, thus loosening the epithelial gut wall which helps continuous diffusion of liquid from the gut into the blood making the blood of the insect highly alkaline and leading to gut paralysis. The parasporal bodies are highly toxic for caterpillars with an LD50 value of < 0.9 mg/ g of larvae. The process of crystal synthesis and spore formation proceed simultaneously. The toxin production by a culture can be enhanced by controlling many factors.
*Bacillus thuringiensis* toxin genes have been introduced via recombinant DNA technology into the genome of plants or plant associated microorganisms.

Method of Production Submerged fermentation

Preparation of inoculum

The inoculum is prepared using media containing beet molasses, corn steep solids and calcium carbonate.

Production medium

Production medium enhances the production of toxins within the cell which happens
during sporulation. The fermentable carbohydrate and nitrogen available for growth are
exhausted at the same time after the commencement of sporulation. Submerged fermentations
have been mainly used to produce flowable formulations of spore crystal complex to be used as
sprays.

Semisolid fermentation

Medium consists of dextrose, yeast autolysate and a potassium source. This medium is used for propagating the culture in flasks. The production medium to produce spores and toxins contain wheat bran and solid substrate along with other nutrients. This is an aerobic fermentation with an incubation period of 36-48 hours. This fermentation gives a wettable powder or dust form which is got by just drying and grinding the bran cake. This product is more stable than flowable one but it cannot be used in the wet form since bran begins to swell when wetted.

Bacillus thuringiensis in the Field

Insecticides based on *B. thuringiensis* are used for short-term control. They do not persist from year to year. During crop spraying, it is essential to make sufficient coverage on a leaf surface. But the *B. thuringiensis* spray must be protected from inactivation by sunlight since it is sensitive to ultra-violet light. In order to avoid this, *emulsifying agents*, stickers or binders and UV protectants can be used in the formulation. Some commercial names of *B.thuringiensis* are **DIPEL, DOOM**.

*Bacillus popilliae* and *B. lentimorbus* are potent insecticides causing Milky disease in Japanese beetle. Following the ingestion of viable spores by larvae, germination takes place in the gut and the vegetative cells penetrate the gut wall and enter the body cavity where they multiply. During this time, they remove all the nutrients from the blood and lead to the death of the larva.

(b) Viruses As Insect Pests

Insect viruses are encased in protein coats which are insoluble in water. These protein
crystals serve to protect the virus particles and due to the presence of these protein crystals, the
infective viruses remain active even under normal storage condition for many years. Specificity
of the virus-host relationship makes viruses an ideal candidate for use in the control of specific
pest population. There are two major groups of insect viruses:

1. **Polyhedrosis virus:** where many virus particles are embedded in each protein crystal. When they occur in the host cell nucleus, they are called nuclear polyhedrosis virus (**NPV**), and when they occur in the host cytoplasm they are called cytoplasmic polyhedrosis virus (**CPV**).
2. **Granulosis virus:** where only one virus is contained in each protein crystal. They either develop in the nucleus or cytoplasm of the host cells (NGV or CGV). NPV and CGV come under the group of **Baculoviruses** and they are the most extensively studied insect viruses.

Infection is caused by ingestion of contaminated food containing NPV / GV followed by cell invasion beginning in the midgut. The GV are sprayed over the leaves and they enter the larvae feeding on the foliage. The viruses multiply fast in the larvae and make them lethargic. Affected larvae become sluggish and stop feeding and die. For example, NPV controls the pest Helicoverpa armigera. The affected larvae hang upside down from the leaves and twigs in a characteristic way and a brownish fluid oozes from them. This is a highly infective fluid and is readily disseminated amongst the healthy insect population. The spread takes place by wind, rain, etc. TAIVIRIDAE is a commercial NPV pesticide used to control cotton pest bollworm and budworm.

**Production:**

The specificity and obligate parasitism of insect viruses makes their mass production possible only on live insects, e.g. production of bollworm-budworm NPV (Baculovirus helothis). Bollworm larvae are raised on a semi-synthetic diet containing a water-based mixture of casein, sucrose, wheat germ, growth factors, etc. Agar is used to solidify the diet. Chemicals like formalin, sorbic acid, aureomycin are used to inhibit bacteria, yeasts and fungi. The diet is dispersed hot from a large tank to plastic trays and sealed. These are inoculated by caterpillars from an insectary and incubated under controlled conditions to produce a mass of larvae. A known volume of virus is sprayed on the diet which replicates on the caterpillars to the extent of 5000-10000 times in 5-7 days. The infected caterpillars are suctioned and then treated with water, filtered, centrifuged, precipitated and spray dried. After quality control testing, the preparation is packaged and sold.

**(c) Fungal Pesticides**

Insect mycoses are caused by Phycomycetes, Ascomycetes, Basidiomycetes and Deuteromycetes, e.g. *Beauvaria*, *Metarrhizium*, *Entomophthora*, *Hirsutella*.

**Biological Control Agents**

*Trichoderma* is a cosmopolitan biological control agent which is a saprophyte.

* It has rapid growth rate.

* It sporulates abundantly.
* It produces antibiotics (**gliotoxin** and **viridian**).

* It is a well-known antagonist.

Commercial product **BINABTSEEPIC** can be easily applied in soil in the form of granules for control of soil-borne diseases.

Fungi used as insecticides include the following:

1. **Beauveria bassiana**: This common soil fungus has a broad host range that includes many beetles and fire ants. It infects both larvae and adults of many species. One or more companies currently are developing preparations of this fungus for EPA registration. Understanding the interactions between *Beauveria bassiana* and other soil microorganisms may be the key to successful use of this fungus.

2. **Nomuraea rileyi**: In soybeans, naturally occurring epidemic infections of *Nomuraea rileyi* cause dramatic reductions in populations of foliage-feeding caterpillars.

3. **Vericillium lecanii**: This fungus (once sold under the trade name Vertelec®) has been used in greenhouses to control aphids and whiteflies.

4. **Lagenidium giganteum**: This aquatic fungus is highly infectious to larvae of several mosquito genera. It cycles effectively in the aquatic environment (spores produced in infected larvae persist and infect larvae of subsequent generations), even when mosquito density is low.

5. **Hirsutella thompsonii**: Although preparations of this pathogen were once registered by the US EPA and marketed under the trade name Mycar®, it is no longer available commercially. *Hirsutella thompsonii* is a pathogen of the citrus rust mite.

## Some examples of fungal agents

*Trichoderma harzianum* is successfully used as a bio-control agent for diseases caused by *Rhizoctonia solanii* on beans and tomato. T. harzianum is the most active antagonistic agent in the case of *Fusarium* species.

## Control of Airborne Diseases

*Botrytis cinerea* was controlled when treated with Trichoderma species on the senescent (aged) floral parts. Their colonisation prevented the saprophytic establishment of *B. cinerea* on the vine (hyperparasitism). Their establishment on the floral parts limit the development of
disease on the branches. Spore suspension of *Trichoderma* sp. decrease the number of rust pustules caused by *Puccinia*.

## Seed-borne diseases

*T. harzianum* and *T. koningii* when applied to pea seeds resulted in reduction in the incidence of pre-emergence of damping off of the pea.

## Fruit diseases

Botrytis rot of strawberry was controlled with *Trichoderma*. *Anthracnose* by *Colletotrichum* was also effectively controlled by *Trichoderma* sp.

## Mechanism of Control

Antibiotics may play a crucial role in microbial antagonism. Lytic enzymes like chitinase and β-1-3, glucanase also help in controlling the diseases.

## Mode of Application

Mycelial preparation of *T. harzianum* is more effective in comparison to spore preparation of biological control caused by *Sclerotinia* spp. Wheat bran/ peat is colonised by *Trichoderma* sp. A liquid medium is used to inoculate the bran/ corn meal along with other nutrient ingredients like glucose, sodium chloride, calcium carbonate and soybean meal.

Semi-solid fermentation is preferable for most of the fungi. Since the dry conidia bran mixture is relatively stable. Embedding propagules of the bio-control agents in a matrix formed by sodium alginate (binder) and clay appear to be an effective method of delivery of *T. viridae*. It can also be applied as a seed coating with various adhesives/ gels. Some commercial products available are ANTAGON, BIOCURE-F, BIODERMA, TRICHOCAN, etc. Those commercial formulations can be introduced into organic manures, composts, dry cow dung, farmyard manure, neem cake etc.
Fill in the Blank Questions based on the above contents:

1. Biological control involves reducing inoculum density or disease-producing activities of a pathogen by one or more ________.

2. Biocontrol is based on antagonism, including competition, parasitism, and ________.

3. ________ is the law that emphasizes restricting insect populations rather than eradicating them.

4. Microbial insecticides can be categorized into (a) bacterial pesticides, (b) viral pesticides, and (c) ________ pesticides.

5. An effective microbial pesticide should be specific to the pest population, harmless to humans and other valued organisms, and possess resistant stages such as ________ or cysts.

6. Bacillus thuringiensis produces protein crystal inclusion bodies known as ________ bodies, which act as endotoxins.

7. In insect viruses, Polyhedrosis viruses embed many virus particles in each protein crystal, while Granulosis viruses contain ________ virus in each crystal.

8. In the context of Integrated Pest Management (IPM), what does IPM stand for? ________ Pest Management.

9. Trichoderma harzianum is a biological control agent used for diseases caused by ________ solanii on beans and tomatoes.

10. The mode of application for mycelial preparation of *Trichoderma harzianum* is more effective than spore preparation in controlling diseases caused by ________ spp.

Answer:

1. Organisms

2. Hyperparasitism

3. Integrated pest management (ipm)

4. Fungal

5. Spores

6. Parasporal

7. One

8. Integrated

9. Rhizoctonia

10. Sclerotinia
**Multiple Choice Questions:**

1. Biological control is based on antagonism, which includes:

a) Symbiosis

b) Competition, parasitism, and hyperparasitism

c) Mutualism

d) Commensalism

2. What is the primary characteristic of an effective microbial pesticide?

a) High cost

b) Broad-spectrum toxicity

c) Harmful to non-pest populations

d) Specificity to the pest population and harmlessness to humans and valued organisms

3. Which bacterial pathogen has been most extensively studied as an insecticide?

a) *Bacillus cereus*

b) *Clostridium botulinum*

c) *Bacillus thuringiensis*

d) *Pseudomonas aeruginosa*

4. Insect viruses are categorized into:

a) Polyhedrosis and Granulosis viruses

b) Retroviruses and Coronaviruses

c) Adenoviruses and Herpesviruses

d) Lytic and Lysogenic viruses

5. What is the law that emphasizes restricting insect populations rather than eradicating them?

a) Eradication Law

b) Pest Control Law

c) Integrated Pest Management (IPM)

d) Insect Limitation Law

6. Which fungal pesticide is used as a bio-control agent for diseases caused by Rhizoctonia solanii on beans and tomatoes?

a) Beauvaria

b) Metarrhizium

c) Entomophthora

d) Trichoderma harzianum

7. How do Polyhedrosis viruses differ from Granulosis viruses?

a) Polyhedrosis viruses contain one virus in each crystal, while Granulosis viruses embed many virus particles.

b) Granulosis viruses contain one virus in each crystal, while Polyhedrosis viruses embed many virus particles.

c) Both Polyhedrosis and Granulosis viruses contain many virus particles in each crystal.

d) Both Polyhedrosis and Granulosis viruses contain one virus in each crystal.

8. What is the primary mode of application for mycelial preparation of Trichoderma harzianum?
a) Submerged fermentation

b) Spray application

c) Seed coating

d) Semisolid fermentation

9. Which commercial product is an example of a mycelial preparation of Trichoderma harzianum?

a) DIPEL

b) DOOM

c) ANTAGON

d) TAIVIRIDAE

10. What is the primary advantage of using fungal pesticides in biological control?

a) Broad-spectrum toxicity

b) Rapid growth rate

c) Harmfulness to humans

d) Specificity and obligate parasitism

**Answers:**

1) Competition, parasitism, and hyperparasitism

2) Specificity to the pest population and harmlessness to humans and valued organisms

3) c) Bacillus thuringiensis

4) Polyhedrosis and Granulosis viruses

5) Integrated Pest Management (IPM)

6) Trichoderma harzianum

7) b) Granulosis viruses contain one virus in each crystal, while Polyhedrosis viruses embed many virus particles.

8) Semisolid fermentation

9) c) ANTAGON

10) d) Specificity and obligate parasitism
True or False type Question:

<table><thead><tr><td>Sl. No.</td><td>True or False statement</td><td>Answer</td></tr></thead><tbody><tr><td>1)</td><td>Biological control involves the use of chemicals to eliminate pests</td><td>False.</td></tr><tr><td>2)</td><td>The negative interactions among microbial populations are not relevant to biological control</td><td>False</td></tr><tr><td>3)</td><td>Bacillus thuringiensis is a bacterial pathogen extensively studied as an insecticide, primarily due to its resistance to environmental conditions</td><td>False</td></tr><tr><td>4)</td><td>Insect viruses, specifically Polyhedrosis and Granulosis viruses, are encased in protein coats that are soluble in water</td><td>False.</td></tr><tr><td>5)</td><td>Trichoderma harzianum is a fungal biological control agent known for its slow growth rate and inability to produce antibiotics</td><td>False</td></tr><tr><td>6)</td><td>Integrated Pest Management (IPM) aims to eradicate insect populations completely</td><td>False</td></tr><tr><td>7)</td><td>The specificity and obligate parasitism of insect viruses make their mass production feasible only on live insects</td><td>True</td></tr><tr><td>8)</td><td>Antibiotics play a crucial role in microbial antagonism, aiding in the control of diseases</td><td>True</td></tr><tr><td>9)</td><td>Microbial control in agriculture is significant for its potential harm to humans and other valued plant and animal populations</td><td>False</td></tr><tr><td>10)</td><td>Biological control relies on negative interactions, such as amensalism, predation, and parasitism, for managing pests and diseases.</td><td>True</td></tr><tr><td>11)</td><td>Bacillus thuringiensis produces parasporal bodies that act as exotoxins, causing gut paralysis in pests.</td><td>False (endotoxins)</td></tr><tr><td>12)</td><td>Integrated Pest Management (IPM) focuses on eradicating insect populations to ensure agricultural productivity.</td><td>False (restricting insect populations)</td></tr><tr><td>13)</td><td>Trichoderma harzianum is a bacterial biological control agent</td><td>False (fungus)</td></tr></tbody></table>
<table><tr><td></td><td>effective against diseases caused by Rhizoctonia</td><td></td></tr><tr><td>14)</td><td>Insect viruses, specifically Polyhedrosis and Granulosis viruses, are encapsulated in protein crystals that protect them and remain active for many years.</td><td>True</td></tr><tr><td>15)</td><td>The specificity of viruses makes them ideal candidates for the control of specific pest populations.</td><td>True</td></tr><tr><td>16)</td><td>Microbial insecticides should not be specific for the pest population, allowing a broader range of control.</td><td>False (specific for the pest population)</td></tr><tr><td>17)</td><td>Azolla pinnata is a bacterium commonly found in ponds, ditches, and rice fields, used as a biofertilizer for rice.</td><td>False (water fern)</td></tr><tr><td>18)</td><td>The role of Trichoderma harzianum as a bio-control agent includes rapid growth, abundant sporulation, and production of antibiotics like gliotoxin and viridian.</td><td>True</td></tr><tr><td>19)</td><td>The microbial pesticide <i>Bacillus thuringiensis</i> remains active from year to year and persists in the environment.</td><td>False</td></tr></table>

**Short Type Questions:**

1. Define Biological Control.

2. What are the three negative interactions that form the basis for biological control?

3. Explain the principles of Biocontrol.

4. Name the three categories of microbial insecticides.

5. What are the characteristics of an effective microbial pesticide?

6. Describe the role of parasporal bodies in *Bacillus thuringiensis*.

7. Differentiate between Polyhedrosis and Granulosis viruses.

8. What does IPM stand for, and what is its main emphasis?

9. Which bacterial pathogen has been extensively studied as an insecticide, and what is its primary toxic factor?

10. How does *Trichoderma harzianum* act as a bio-control agent?

**Long Type Question:**
1. Explain the role of microbial agents in biological control, detailing the categories of microbial insecticides, their characteristics, and the principles underlying their use. Provide examples and discuss the significance of microbial control in agriculture.

Answer:

Biological control is a method of managing pests and diseases using microorganisms to initiate disease in pest populations, thereby reducing the reliance on chemical pesticides. The negative interactions among microbial populations and between microbes and higher organisms serve as the basis for biological control. There are three main categories of microbial insecticides: bacterial pesticides, viral pesticides, and fungal pesticides.

An effective microbial pesticide should be specific to the pest population, survive environmental conditions, possess resistant stages like spores, and be harmless to humans and valued organisms. *Bacillus thuringiensis*, a bacterium, has been extensively studied. Its parasporal bodies act as endotoxins, causing gut paralysis in pests.

Insect viruses, categorized into Polyhedrosis and Granulosis viruses, are encased in protein crystals that protect them. They cause infections when ingested by pests, leading to lethargy and death. Integrated Pest Management (IPM) emphasizes restricting insect populations rather than eradication.

*Trichoderma harzianum*, a fungal biological control agent, is effective against diseases caused by *Rhizoctonia solanii*. It competes, produces antibiotics, and acts as an antagonist, providing a sustainable approach to disease management.

Microbial control in agriculture is significant as it reduces the need for chemical pesticides, minimizes environmental impact, and helps maintain pest populations below economically damaging levels. This approach aligns with sustainable and environmentally friendly agricultural practices.