16 Bio-pesticides 2

Dr. Gajender Singh and Dr. Sunil Mittal

1.Introduction

2.Microbial Biopesticides

2.1.Bacterial Biopesticides

2.2.Viral Biopesticides

2.3.Fungal Biopesticides

2.4.Nematode Biopesticides

2.5.Protozoan Biopesticides

3.Plant Incorporated Protectants

4.Biochemical Biopesticdes

4.1. Pheromones

4.2.Natural Plant products

1.Introduction

Bio-pesticides as defined in the previous chapter are the pesticides of biological origin. The demand for bio-pesticides in place of synthetic pesticides is increasing very rapidly due to their potential benefits for the environment and mankind. The advantages, disadvantages, challenges, history, market scenario (National and intenational), regulations, availability of bio-pesticides have been discussed in the previous module (No. 18) titled “Bio-pesticidse”. The present module is focused on the various types of bio-pesticides and their mechanism/mode of action. Although, the bio-pesticides can be classified in many ways depending on their mode of action, physic-chemical properties, their physical nature, active ingredient, mode of use, based on target pest etc. but the most commonly used and convenient classification is based on their source. A general classification based on the source of the bio-pesticides is shown in figure 1.

Fig 1: Classification of bio-pesticides

2. Microbial pesticides

The major group of broad spectrum bio-pesticides consists of microbial pesticides. These pesticides do not act on non-target species. The active ingredient present in the microbial pesticides may be a virus, fungi, protozoan, or bacteria. They are known to suppress the pest by producing a toxic compound that is inhibitory to the pest growth.

There are approximately 200 microbial bio-pesticides available in more than thirty countries with their affiliation to Organization for Economic Co-operation and Development (OECD). Bacterial, fungal, viral, predator and other bio-pesticides claim 74%, 10%, 5%, 8% and 3%, of the bio-pesticide market, respectively. Microbial bio-pesticides are delivered to the crop plants either in live, dead or sporulated form. Table 1 gives an account of the number of microbial bio-pesticides registered in different countries.

It has been observed that the registrations for microbial bio-pesticides are increasing throughout the world. With the advancement of the technology, more and more microbes and their byproducts are being explored for their use as bio-pesticides. Microbial bio-pesticides are further sub-divided into five types depending on the type of microorganism:

1. Bacterial bio-pesticides
2. Viral bio-pesticides
3. Fungal bio-pesticides
4. Nematode bio-pesticides
5. Protozoan bio-pesticides

2.1. Bacterial bio-pesticides

The general form of microbial bio-pesticides is represented by bacterial bio-pesticides. The most commonly and widely used microbial bio-pesticide is Bacillus thuringiensis (Bt). These bacteria produce cry proteins. These cry proteins produce toxins and kill the insects by binding to their mid gut receptor. Bt bio-pesticide is prepared from the strains of dendrolimus, kurstaki and galeriae.

These bacterial bio-pesticidescan be divided into four categories:

• Crystalliferous spore formers that include Bacillus thuringiensis
• Obligate pathogens that include Bacillus papiliae
• Potential pathogens like Serratia marcesens
• Facultative pathogens like Pseudomonas aeruginosa

The main reason for the wide acceptance of spore formers for commercialization is their bio efficacy and safety. Their primary targets are insects. However, they can be used for bio control of unwanted viruses, bacteria, fungi etc. As an insecticide, their specific targets are moths, butterflies, beetles, mosquitoes and flies. In case of insects, they are effective only if ingested but in case of pathogenic species of bacteria and virus, these colonize in the plant and wipes out the pest. Table 2 briefly describes the different bacterial bio-pesticides, target pests and their mode of action. Further the mode of action of Bt gene against insects is represented diagrammatically in figure. 2.

Viral bio-pesticides

Viruses have a unique property of host specificity. Due to his property, they can infect closely related species. For example, bacteriophage (a virus) can be used as a pesticide if it is attacking disease causing bacteria. Omnilytics (USA Company) has developed phage products for the treatment of bacterial diseases in plants such as bacterial spot of peppers and tomatoes. More than seven hundred insect infecting viruses have been isolated from various orders of insects like lepidoptera, coleoptera, diptera, hymenoptera and orthoptera. Some of the viruses that have been commercialized for virus control bio-pesticides are Granulosis virus, Nuclear polyhedrosis virus, poxviruses, DNA containing baculoviruses and RNA containing retroviruses. These viruses are used globally for pest control (field crop and vegetable pests). They have high efficiency against plant-chewing insects, gypsy moths, sawflies, and pine caterpillars also. Commercially used virus for the designing of phage pesticides is Baculovirus. Nuclei of the host insect cells are the place where baculoviruses develop.

When the host insect feeds on baculoviruses, the infectious viral particles are liberated inside the pest and get activated due to suitable survival conditions. Once the baculoviruses enter the larval gut, the protein overcoat of the virus undergoes disintegration within a short time. Thereafter, the viral DNA infects the cells of digestive system. After few days of infection, the digestive system of larvae becomes weak and the larvae cannot digest the food. As a result, larvae die. These baculoviruses pose no threat to mammals, fish, plants, birds and non-target insects. Sudden outbreak of baculoviruses within host population can control the disease completely. Baculoviruses have a major advantage that they can provide a suitable alternate to chemical pesticides and antibiotics. Among all the microbial bio-pesticides, baculoviruses claim 6% of the market share alone.

Mechanism of viral pathogenesis

• Virus gets replicated inside the nucleus or cytoplasm of target cells and expresses itself there.
• Expression occurs in three phases i.e., early phase (0-6 hr after infection), late phase (6-24 hr after infection) and very late phase (up to 72 hr after infection).
• Virus infected nuclei results in the production of several polyhedral and number of granules within a single cell.
• As a result, enzootics are created that are responsible for the depletion of pest population.
• A significant impact is created on the economic threshold of pest due to decline in pest population.

Viral bio-pesticide example: Cydiapomonella Granulosis virus against codling moth that causes damage to fruits like apples and pears.

Fungal Bio-pesticides

Many horticultural and field crops become prone to diseases caused by fungal pathogens. This leads to the loss in the crop quantity. In order to get rid of fungal diseases, farmers make the intensive use of fungicides without knowing their further effects. Fungicides result in the deposition of toxic compounds that cause harm to living beings and environment. Moreover, the fungal pathogens develop resistance to a particular fungicide after few generations.

Pathogenic fungi can be used to control certain type of pests. Pathogenic fungi are amphibious in nature i.e., they can survive both in aquatic and terrestrial habitats. Pathogenic fungi are known as entomopathogenic fungi when they are linked with insects. Entomopathogenic fungi are good microbial bio-pesticides since they have multiple mechanisms for pathogenesis. Their target point is the gut epithelium or integument of the pest insect. Within the integument, the fungi create their conidia. Some fungi such as streptomycetes act against insects by the production of some toxins like novobiocin, actinomycin A and cycloheximide. Example of entomopathogenic fungi include Beauveria bassiana that infects green vegetable bug and green and brown mirids. Now the question arises that how fungi attack insects? The answer is simple that fungi have the ability to penetrate the cuticle of insects.

The mode of action of fungal bio-pesticides is variable and depends primarily upon target pest. Fungal bio-pesticides have one major advantage over bacterial and viral pesticides that their efficacy doesn’t depend upon their ingestion. They require optimum conditions for their proliferation such as cool temperature and humid soil.

Examples of fungal bio-pesticides:

Trichoderma harzianum used against plant pathogens is a fungal bio-pesticide that has antagonistic action against other fungi such as Fusarium, Rhizoctonia, Pythium and some soil-borne pathogens.

Muscodor albus fungal bio-pesticides as a replacement of methyl bromide for post-harvest treatments.

Aspergillus flavus strain AF36 fungicide for cotton. Other strains of Aspergillus produce aflatoxin, a liver carcinogen. However, aflatoxin is not produced by AF36 strain of Aspergillus flavus.

Mechanism of action of Trichoderma

• Infects the major tissue of fungal pathogen.
• Enzymes are secreted by the Trichoderma that dissolves the cell wall of fungal pathogen.
• Trichoderma consumes the cell contents of target fungus and its spore multiplication takes place.
• Mycotrol ES and Naturalis L are registered bio-pesticides from Beauveria bassiana, against mealybugs, aphids, leafhoppers, thrips and weevils.

2.4. Nematode bio-pesticides

Entomopathogenic nematodes (EPN) represents other group of microbes that can be used for pest control including white grubs, gnats, weevils and some species of Sesiidae family. These organisms are responsible for suppression of stem borers and soil borne pests.

• Steinernema and Heterorhabditis genus contain most commonly used nematode as infective juveniles (IJs) that attack the host. IJs have various routes for entry inside the host such as cuticle, spiracles, anus and mouth.
• Bacterial symbionts are released inside the haemocoel of host.
• Hosts get killed within 24-48 hr after the release of symbionts.

Large biomass of EPN can be obtained under invitro and invivo conditions using either liquid fermentation or solid media.

2.5. Protozoan bio-pesticides

Protozoan pathogens have not been declared successful as bio-pesticides. Microsporidia belonging to the genera Nosema and Vairimorphahave some capacity to attack the orthopteran and lepidopteran insects. Hoppers have been reported to be more sensitive to microsporidia as compared to other insects.

Examples of protozoan bio-pesticides

Cruiser

Heteromask

Nema-BIT

Grubstake

3. Plant Incorporated Protectants (PIPs)

PIPs are pesticidal substances produced by the plants due to their genetic modification with a specific gene able to produce the respective substance. Since, these crops/plants possess foreign gene from different species, these are also called GM crops. The most widely used genetic material is isolation of gene from bacteria Bacillus thuringiensis (Bt) and its introduction it into genetic material of crop/plant. The Bt modified plant starts producing toxic cry proteins which act as pesticide when the plant is fed by the pest. The common examples are the Bt cotton, Btbrinjal, Bt tomato, Bt corn etc. Initially, the Bt plants were having only limited acceptance, but in the last 15 years, the acceptance of genetically modified (GM) crops has increased dramatically.

Environmental Protection Agency (EPA) is the nodal agency which ensures that PIP products should be carefully tested on feed crops and human food to meet human safety standards. EPA only regulates protein and its genetic material but not the plant itself. EPA evaluates for short term (allergenicity, eye and skin irritation, and toxicity) and long term (reproductive and nervous system disorders, birth defects, and cancer) health impacts on humans, environment, non-target species, resistance etc. The table 3 gives an account of list of PIPs active ingredients registered during 2007-2017 as per EPA.

4. Biochemical bio-pesticides

The chemicals derived from natural products which act against the target pest in a non-toxic way are termed as biochemical bio-pesticides. The substances that interfere with the mating or growth process, or attract or repel the pests are also included in the category of biochemical bio-pesticides. The biochemical bio-pesticides can be mainly divided into 2 types depending on their mode of action.

A. Pheromones

B.Natural Plant Products (Essential Oils, Phenolic compounds etc.)

1 Pheromones

Pheromones are volatile chemicals emitted by living organisms used to send messages to individuals – usually of the opposite sex – of the same species. Pheromones released by one organism travel through the air or water medium and reach the second organism of same species, where they are detected by the receiver. In case of Insects, the pheromones are recognized by the antennae on the head of receiver insect. Pheromones can range from small hydrophobic molecules to water-soluble peptides. These are classified in two groups:

i). Primer effect Pheromones: These pheromones are regulated through the gustatory (test) sensilla and induce the chain of the physiological effects. They have very limited value in insect pest control.

ii). Release effect Pheromones: These pheromones are regulated through the olfactory (smell) sensilla. The sex, aggregation and alarm pheromones comes under this category and widely used in the insect pest management.

a). Sex Pheromones: Sex pheromones are chemicals released by female insects to attract males for mating. These chemicals have been used most extensively in combination with lepidopteran pests. These pheromones are used in place of light traps to selectively attract insects or disruption in pest mating.

Pheromone traps: Pheromone traps are basically the chemicals which attract the insect inside the mechanical structures called traps. Different types of insects including flies, pantry

moths, pantry beetles and cockroaches are captured by these traps. Different types of traps are used for different type of insects. On the basis of shape, size and composition they are classified as Funnel Traps, Grain Probe Traps, Pitfall Traps, Sticky Traps, Box Traps, Wing Traps, Delta Traps, Diamond Traps, and Discreet Trap. Some Process used in the Insect management through Pheromone traps are as follow.

Mating disruption

Mating disruption involves the use of sex pheromones to prevent male insects finding females and mating. In this method false or synthetic pheromone that is identical to the natural version is released from the dispensers placed throughout the crop to be protected. The male get disoriented or confused to detect the plume of calling female. In Germany and Switzerland more than 20 percent of the grape growers use this technique and produce wine without using insecticides. In the United States, mating disruption has proven effective in codling moth, navel orange worm, pink bollworm, Oriental fruit moth, European grape moth, and grapevine moth.

b). Aggregation pheromones: These are produced by insects such as wood-invading beetles to indicate to others the presence of a good food source. The use of aggregation pheromones reported in the management of the boll weevil (Anthonomus grandis (Boheman), pea and bean weevil (Sitona lineatus (L.)) and stored product weevils (Sitophilus zeamais (L).

c). Alarm pheromones: These are produced by insects that are under attack from a predator and this leads to a movement by the insects away from the source of production. They are highly volatile and having low molecular weight, hence used very limited in insect management.

4.2. Plants and their Products (Essential Oils, Phenolic compounds etc.)

Plants have been employed as pesticides since ancient times. Prehistorically, plant parts like leaves, flowers, stem, roots etc. were used as insect repellents or pesticides in agricultural fields and storage. The use of leaves of Azadirachta indica, flower buds of Chrysanthimum species, parts of Eucalyptus species, Ocimum plants are common examples of plants used historically for their pesticidal properties. With the advancement of science, it has been discovered that several higher plants have the ability to synthesis and produce numerous secondary metabolites. These metabolites can be extracted and has potential to be used as pesticides. These common metabolite chemicals from plants can be classified as essential/volatile oils and phenolics and are described below:

i). Essential Oils

Essential Oils (EOs) are secondary plant metabolites present in their parts like leaves, root, wood, bark, stem, flowers, and fruits, etc. The presence of different types of complex organic compounds which could be classified under monoterpines, sesquiterpene, phenols, simple alcohols, ketones, coumarins, etc. are responsible for their fragrance or aroma. These are volatile in nature. These have been reported as insect repellent and potential inhibitor of the growth of various types of fungus, microbs as well as the larvae of insects and moths and weeds. EOs as well as their pure components (eg. Eucalyptol, menthol, eugenol, ocimine, myrcene) possess pesticidal properties, hence these are regarded as Botanical Pesticides. The EOs as botanical pesticides have following advantages:

• Eco Friendly: It has low toxicity against non-target organisms, including humans.
• High effectiveness against: a wide range of pests and diseases of agricultural and medical importance.
• Multiple mechanisms of action; due to the large number of active ingredients in each blend, the development of resistance is less likely.
• Low health risk during application due to low toxicity of residues.

Mode of action of EOs

The EOs and their pure components act by different mechanisms in different type of pests. The inhibitory mechanism of EOs for insect control is not completely understood but these probably act as a neurotoxic agent for the insects. These act on the nervous system by disturbing the ion transport and release of acetylcholine esterase. These also interrupt the functioning of octopamine (neurotransmitter) which results in total breakdown of nervous system in insects.

Antimicrobial activity can be either microstatic (inhibition of the growth) or microbicidal (killing of microbes). The efficacy and mechanism of EOs depend on their composition and the type of microbe. Overall, the mechanism involves a series of biochemical reactions inside the cell, breakdown of membrane integrity and increased permeability, disruption of membrane transport and other metabolic regulatory functions, depletion of the ATP etc.

The EOs are also allelopathic in nature and hence explored for their herbicidal properties. The EOs inhibit seed germination and plant growth in weeds. The germination inhibition may be the consequence of blockage in the water uptake, disruption of the activity of metabolic enzymes that are involved in the glycolysis and oxidative pentose-phosphate pathways. The volatile constituents alter the electron flow into cytochrome pathway which leads to the decreased ATP production and alteration of energy-demanding cell processes.

The EOs are also used as a preservatives of grains, fruits and vegetables during their storages. Various EOs are reports as main active ingredients in packaging materials used during packaging and storage. List of some EOs is given in the table

ii). Phenolic compounds and other biochemicals

Apart from the EOs, the plant products like pyrethrins, nicotine, picrotoximn, propolis, azadirachtin and fusapyrone etc. are also used for the insect pest management. Pyrethrins are the most common natural compounds known for their potent insecticidal property. These are organic compounds derived from bud of the Chrysanthemum cinerariifolium flower. Propolis is another natural resinous substance obtained from leaf buds and bark of poplar and conifer trees. Propolis contains protein, amino acids, vitamins, minerals and flavonoids. It has antibiotic activity, antibacterial and antifungal activity. Propolis has been found to inhibit the postharvest pathogens B. cinerea and P. expansum. Nicotine and picrotoximnals are used as insecticides historically. Both act as a neurotoxins, nerve axons and synapses. Another extensively used plant product extracted from the seed kernel of neem is azadirachtin. It contributed much for the development of several commercial bio-pesticide formulations. More than 100 commercial products are developed with azadirachtin including Margosan-O, Bio-neem, Azatin, Neemies, Safer’s ENI, Wellgro, RD-Repelin, Neemguard, Neemark, Neemazal, Nimbin, Nimbicidine etc., which are used successfully in many parts of the world. Apart from these, some other natural compounds like juvenile hormones, β– asarone, ryanodine have etc. are also used commercially for insect control.