Biopesticides: Production, Formulation and Application Systems

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International Journal of Current Microbiology and Applied Sciences ISSN: 2319-7706 Volume 9 Number 10 (2020) Journal homepage: http://www.ijcmas.com

Review Article https://doi.org/10.20546/ijcmas.2020.910.453

Varsha Bharti* and Shahida Ibrahim

Division of plant pathology, Sher-e- Kashmir University of Agricultural Science and Technology of Jammu Chatha, India

*Corresponding author

ABSTRACT

Increasing public concern about the potential damage of chemical inputs in agricultural production systems has challenged industry to develop new and effective pest management

and control strategies against insect pests, diseases and weeds. These new strategies must

K e yw or ds be less harmful to the environment than the current, chemical-based ones, and they must also safeguard the health of agricultural workers and consumers. Bio-pesticides are the Biopesticides , formulated form of active ingredients based on microorganisms such as bacteria, viruses, Microb ial fungi, nematodes or naturally occurring substances, including plant extracts and component , semiochemicals. Not all the natural products are biopesticides; some are chemical Semiochemicals pesticides if they act on nervous system of the pest. The formulation process leads to a

Article Info final product by mixing the microbal component with different carriers and adjuvants for better protection from environmental conditions, greater survival of the biological agents,

as well as improved bioactivity and storage stability. Biopesticide formulation can be Accepted: 25 September 2020 divided into liquid and dry formulation. Application of products must be easy, economical, Available Online: effective, and timely to the appropriate site of action. The application of biopesticides ﬁts 10 October 2020 the modern strategy of integrated pest management (IPM) which combines all suitable control techniques harmoniously with one another and integrates them with other crop production practices, to suppress pest populations below economic injury levels, while maintaining the integrity of the ecosystem.

Introduction mainly for environmental concerns. Degraded soils and groundwater pollution has resulted Agriculture has had to face the destructive in nutritionally imbalanced and unproductive activities of numerous pests like fungi, weeds lands. Violative pesticide residues also and insects from time immemorial, leading to sometimes raise food safety concerns among radical decrease in yields. With the advent of domestic consumers and pose trade chemical pesticides, this crisis was resolved to impediments for export crops. Therefore, an a great extent. But the over dependence on eco-friendly alternative is the need of the hour chemical pesticides and eventual uninhibited (Gupta and Dikshit, 2010). use of them has necessitated for alternatives

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The United States Environmental Protection can control many different kinds of pests, Agency (EPA) defines biopesticides as, although each separate active ingredient is “certain types of pesticides derived from such relatively specific for its target pest. For natural materials as animals, plants, bacteria, example, there are fungi that control certain and certain minerals”. They pose less threat to weeds, and other fungi that kill specific the environment and to human health. The insects. The most widely known microbial most commonly used biopesticides are living pesticides are varieties of the bacterium organisms, which are pathogenic for the pest Bacillus thuringiensis, or Bt, which can of interest. These include biofungicides control certain insects in cabbage, potato, and (Trichoderma), bioherbicides (Phytopthora) other crops. Bt produces a protein that is and bioinsecticides (Bacillus thuringiensis). harmful to specific insect pest. Certain other There are few plant products also which can microbial pesticides act by out-competing now be used as a major biopesticide source. pest organisms. Microbial pesticides need to Plant-incorporated protectants include be continuously monitored to ensure that they substances that are produced naturally on do not become capable of harming non-target genetic modification of plants. Such examples organisms, including humans (Mazid, 2011). are incorporation of Bt gene, protease inhibitor, lectines, chitinase etc into the plant Plant- Incorporated-Protectants (PIPs) genome so that the transgenic plant synthesizes its own substance that destroys PIPs are pesticidal substances that plants the targeted pest. The potential benefits to produce from genetic material that has been agriculture and public health programmes added to the plant. For example, scientists can through the use of biopesticides are take the gene for the Btpesticidal protein, and considerable (Kandpal, 2014).The interest in introduce the gene into the plants own genetic biopesticides is based on the advantages material. Then the plant, instead of the Bt associated with such products which includes bacterium manufactures the substance that Inherently less harmful and less destroys the pest. Both the protein and its environmental load. Designed to affect only genetic material are regulated by EPA; the one specific pest or, in some cases, a few plant itself is not regulated (Mazid, 2011). target organisms. Often effective in very small quantities and often decompose quickly, Biochemical pesticides thereby resulting in lower exposures and largely avoiding the pollution problems. These are naturally occurring substances such When used as a component of Integrated Pest as plant extracts, fatty acids or pheromones Management (IPM) programs, biopesticides that control pests by non-toxic mechanisms. can contribute greatly (kandpal, 2014). Conventional pesticides, by contrast, are synthetic materials that usually kill or Types of biopesticides inactivate the pest. Biochemical pesticides include substances that interfere with growth Biopesticides fall into three major categories: or mating, such as plant growth regulators, or substances that repel or attract pests, such as Microbial pesticides pheromones. Because it is sometimes difficult to determine whether a natural pesticide Microbial pesticides contain a microorganism controls the pest by a non-toxic mode of (bacterium, fungus, virus, protozoan or alga) action, EPA has established a committee to as the active ingredient. Microbial pesticides determine whether a pesticide meets the

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criteria for a biochemical pesticide (Mazid, thuringiensis active against agriculturally 2011). important insects. Similarly there are several hundred isolates of B. sphaericus, 25 percent Biopesticides in India of which are larvicidal to mosquito larvae. (Moazami, 2000). The most important single Biopesticides represent only 2. 89% (as on need for the production of microbial 2005) of the overall pesticide market in India insecticides is a supply of reproducible, and is expected to increase drastically in reliable, authentic cultures of the coming years. In India, so far only 12 types of microorganism. The principle bacteria used in biopesticides have been registered under the the control of insects (B.t. and B.s.) are Insecticide Act, 1968 (Table 1). Neem based relatively easy to maintain pesticides, Bacillus thuringensis, NPV and Trichoderma are the major biopesticides Liquids produced and used in India. Whereas more than 190 synthetics are registered for use as Coconut milk (waste product), crude sugar, chemical pesticides. Most of the biopesticides e.g., jaggery, whey (waste product), molasses, find use in public health, except a few that are corn steep liquid, inorganic nitrogen and used in agriculture (Table 2). Besides, i) (NH4)2SO4 (Moazami, 2000). transgenic plants and ii) beneficial organisms called bio-agents: are used for pest Material of plant origin management in India (Kandpal, 2014). Legumes and other seeds, chick peas, peanuts, Production lima beans, cowpeas, soya beans, bambara beans, kidney beans, cotton seed meal, peanut Mass production technology cake, soy peptone, cotton seed, hydrolysate, There are two major step for inoculum horse beans, lentils; cereals, corn, guinea corn production millets, wet mash from breweries, wheat flour, wheat bran carbohydrate, dextrin, Solid state fermentation maltose, sucrose, glucose Plant extracts, Liquid state fermentation potato tubers, sweet potato roots, minced citrus peels, ground seed of dates, carrots; Solid state fermentation: when organism Tubers, cassava, yams, sweet potatoes; Yeast grown on the surface of the medium. powder, fodder yeast. (Moazami, 2000) Example: nutrient agar, potato dextrose agar. Materials of animal (non mammalian) Liquid state fermentation: when organism origin: Fishmeal grown the beneath the surface of the medium. Materials of mammalian origin: Blood, Example: Nutrient broth, potato dextrose chicken slaughterhouse residue broth (Askew and Laing, 1993). Minerals: Minerals are essential in the Production of Bacillus thuringiensis(B.t) nutrition of organisms. Five metallic ions are and Bacillus sphaericus (B.s) considered to be particularly important in the growth and sporulation of bacilli: Mg++, Culture Maintenance and Preservation Mn++, Fe++, Zn++, and Ca++. These are all normally present in the carbon and nitrogen There are over a thousand strains of B. 3933

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sources used in fermentations and there may are not formed. Rather, ammonia accumulates be no need to include these ions in the in the broth, probably due to deamination of fermentation media (Moazami, 2000). amino acids. The final pH may range from 8.0 to 9.0 depending upon the protein content of Fermentation the medium. It is possible to control the pH by the addition of acid, and this may enhance The fermentation of the different isolates of toxin production by some strains but not by B.t., regardless of subspecies, have some others. general characteristics in common. They all use sugar (usually glucose, molasses, or The "log-phase" of any bacterial fermentation starch), producing acid during the is that period during which the organism is fermentation. In general, they have similar vigorously growing and rapidly dividing. This requirements for proteins or protein first phase lasts 16-18 hours. Sporulation is hydrolysates, can use NH4+ salts, and complete 20-24 hours after inoculation, respond similarly to minerals. However, the although the cells have not yet lysed. Lysis is individual isolates are unique entities, and a complete by 35-40 hours (Moazami, 2000). particular medium that may support good growth or toxin production by one isolate may Use of new genetic-engineering technology be less satisfactory for another. Different isolates of B.t. may produce toxins with Biological control is the most important different spectra of activities. alternative to chemical pesticides in protecting crops from pests, pathogens, and After sterilization, the pH of the fermentor is weeds. Major breakthroughs in molecular pH 6.8-7.2. Immediately after inoculation, the biology and biotechnology since the early pH falls steadily as the glucose is utilized, 1980s indicate that quick improvement in the reaching a pH of about 5.8-6.0 after 10- 12 competitive ability of biological control hours. At this point, the pH starts to rise at the methods is possible and the biopesticide scan same rate that it fell, reaching pH 7.5 after 25 play a major role in crop protection in the hours. future. It has become possible to improve some of the critical properties that earlier The rise in pH slows gradually, reaching a pH hampered the usefulness of many biocontrol of 8.0 after about 30 hours. The pH may agents. Valuable genes from completely continue to rise, reaching pH 8.8 after 50-60 unrelated organisms can now be utilized for hours. With some cultures, the initial drop in biological control purposes. pH may only reach pH 6.4-6.6. In such fermentations, there may be little or no Biological control using recombinant DNA increase in pH as the fermentation continues, (genetic engineering) technology can be reaching a pH of 8.0 at about 30 hours. The achieved in several different ways: control pH may continue rising, reaching a pH of 8.8 agents may be improved; crop plants can be after 50-60 hours. engineered to carry better resistance genes; or organisms associated with the plant may be The pH in B.s. fermentations in contrast to modified to provide protection. All these B.t. fermentations moves continuously approaches have successfully been used in upward throughout the growth and several different ways experimentally. sporulation of the bacteria. Since the bacteria do not use sugars as a source of carbon, acids Product development has been very active in the area of incorporating resistance genes 3934

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mainly from Bt-directly into plants. Successes the root systems of crops, has been include potato, tomato, tobacco, and cotton. engineered to express Bacillus thuringiensis General root colorizing bacteria of plants (Bt) toxins, and thus provide continuous have also been engineered to produce protection against such pests as corn insecticidal toxins, which protect against pests rootworm. The genes for all the major such as the corn rootworm. proteins that account for the insecticidal properties of Bt have been cloned and Another bacterium living in the vascular sequenced. Now we have nucleotide tissues of corn has also been modified to give sequences for more than 20 Bt genes that protection against the corn borer. None of encode proteins active against leopidopterans, these modified plants or associated organisms eight genes encoding proteins active against is available commercially yet. Similar dipterans, and two genes encoding proteins approaches are used for the biological control active against coleopterans (Moazami, 2000). of plant pathogens and weeds, but research has been most active in the area of insect To increase the environmental stability and control (Moazami, 2000). effectiveness of the various Bt toxins in the field, genes encoding proteins active against Engineering biological control agents beetles and caterpillars have also been cloned into the rhizobacterium Pseudomonas The genetic improvement of biological agents fluorescens. After fermentation, the bacteria is a relatively new concept. For this, a great are killed and the cell walls hardened deal must be known about the biology, chemically. The endotoxins are thereby ecology, and behaviour of the organism. This microencapsulated, resulting in insecticides is a very crucial step (Moazami, 2000) with greatly enhanced residual activity. Large-scale field trials with this product have Engineering crop plants been performed, and the product obtained full registration in 1991 (Moazami, 2000). The first published reports of successful engineering of crop plants to produce Through genetic-engineering techniques, the insecticidal or antifeedant proteins appeared Autographa californica multinucleocapsid in 1987. The crop plants were tobacco and nucleopolyhedrosis virus (AcMNPV) has tomato, producing the delta endotoxin of been engineered to kill insects more quickly Bacillus thuriengensist o make them resistant by expressing either enzymes or toxins soon against caterpillars. To date, transgenic crop after host invasion. Of particular interest is plants have been produced of at least 27 the possibility of making viruses produces different species, including potato, cabbage, insect neurohormones, which can cause rapid sugarbeet, rice, soybeans, corn, rapeseed, physiological disruptions in minutely defined sunflower, walnut and poplar (Moazami, target hosts. This strategy is in its early stages 2000). of development, but there is little doubt that within the very near future we will have Instead of being inserted directly into the crop viruses with extended or specifically designed plant genome, the protective insecticidal host ranges, capable of killing insects within genes can be engineered into associated 24 to 48 hours. These genetically engineered organisms. Two bacteria have been viruses should have an advantage for use successfully tested for this purpose. against hosts that are not easily controlled by Pseudomonas fluorescens, which colonizes Bt.(Moazami,2000).

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so that it is easily delivered to the target Very little is known about the genetics of in the most appropriate manner and entomopathogenic fungi. The first form. transformation system for an To protect the agent from harmful entomopathogenic fungus was developed environmental factors at the target site, using Metarhiziumanisopliae protoplasts thereby increasing persistence. mixed with a fungicide-resistant plasmid. A To enhance activity of the organism at the benomyl-resistant strain of M.anisoplieaehas target site by increasing its activity, thus been obtained. Fungal enzymes involved reproduction, contact and interaction in the penetration of the insect cuticle have with the target pest or disease organism now been identified. Knowledge of these (Seaman, 1990; Mollet, 2001). genes and gene products will eventually lead to the possibility of genetic alteration of Regarding their physical state, biopesticide fungal pathogens that possess those formulations can be divided two formulations. genes.Transformation systems for some fungi exist already and may soon be applied to the Liquid formulations entomopathogenic species(Moazami,2000). Dry formulations.

Formulation Liquid formulations can be water-based, oil- based, polymer-based, or combinations. Formulation refers to the preparation of a Water-based formulations (suspension product from an active ingredient by the concentrate, suspo-emulsions, capsule addition of certain active (functional) and suspension, etc.) require adding of inert non-active(inert) substances (Grewal, 2005). ingredients, such as stabilizers, stickers, surfactants, coloring agents, antifreeze Principles of formulation compounds, and additional nutrients.

Formulated organisms are suspended in a Dry formulations can be produced using suitable carrier which is supplement by different technologies, such as spray drying, additives to maximize survival in store, freeze drying, or air drying either with or optimize application to the target and protect without the use of fluidized bed. They are the organisms after application. In contrast to produced by adding binder, dispersant, chemical active ingredients, they are wetting agents, etc. (Tadros, 2005; Brar, particulate and live or proteinous in nature, 2006; Knowles, 2008). making them relatively sensitive to storage conditions and the environment (Bergis and Biopesticides are usually formulated as: dry jones,1998). There are a number of formulations for direct application – dusts amendments used in preparation of (DP), seed dressing formulations – powders formulation and some important ones are for seed dressing (DS), granules (GR), micro listed below (Table 3). granules (MG), dry formulations for dilution in water – water dispersible granules (WG), Four basic function of formulation and wettable powders (WP); liquid formulations for dilution in water – To stabilize the organism during distribution emulsions, suspension concentrates (SC), oil and storage. dispersions (OD), suspo-emulsions (SE), cap- To aid handling and application of the product sule suspensions (CS); ultra low volume

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formulations (Knowles, 2005, 2006). range 100-1000 microns for granules and 100-600 microns for micro granules) are Dusts made from mineral materials (kaolin, attapulgite, silica, starch, polymers, dry Dusts (DP) are formulated by sorption of an fertilizers and ground plant residues) (Tadros, active ingredient on finely ground, solid 2005). Concentration of active ingredient mineral powder (talc, clay, etc.) with particle (organisms) in granules ranges from 5-20%. size ranging from 50-100 µm. Dusts can be The active ingredient either coats the outside applied directly to the target, either of the granules or is absorbed into them. mechanically or manually. Inert ingredients Granule products are very simply for this formulation are anticaking agents, manufactured, their active ingredient is ultra violet protectants and adhesive materials processed by mixing a powder blend with a to enhance adsorption. Concentration of small amount of water to form a paste which active ingredient (organism) in dust is usually is then extruded and dried if necessary. 10%. Another way of production is applying a liquid active ingredient to coarse absorptive Although they have positive effects under material. After that granules can be coated certain circumstances, they also pose serious with resins or polymers to control the rate of inhalation hazard for users. This is an old effectiveness of active ingredient after formulation type that had been used for many application. Granular biopesticides are mostly years before granules were developed and used to apply products to soil in order to they became restricted on the account of their control weeds, nematodes, and insects living adverse health impact on users. Other dusts in soil, or for plant uptake by root. Once are manufactured very simply and they are applied, granules release their active still used today in many parts of the world ingredient slowly. Some granules require soil (Knowles, 2001). moisture to release their active ingredient (Knowles, 2005; Lyn, 2010). Powders for seed treatment (DS) Wettable powders (WP) Powder for seed treatment are formulated by mixing an active ingredient, powder carrier Wettable powders (WP) are dry, finely and accompanying inert to facilitate product ground formulations to be applied after adherence to seed coats. This type of suspension in water. Wettable powders are formulation is applied to seeds by tumbling produced by blending an active ingredient seeds with the product designed to adhere to with surfactant, wetting and dispersing agents them. Powders for seed treatment are a very and inert fillers, followed by grinding to a old type of formulation, a traditional product required particle size (about 5 microns). form for coating seeds, and they also contain These products can raise serious health and a red pigment as a safety marker for dressed safety issues for manufacturers because of seed (Woods, 2003). their dustiness, which can cause inhalation and skin and eye irritation problems if strict Granules (GR) safety precautions are not taken. For these reasons and because of their dustiness during Granules (GR) are similar to dust application, wettable powders are gradually formulations, except that granular particles suppressed by suspension concentrates or are larger and heavier. Coarse particles (size water dispersible granules, which have been

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the most widely used pesticide formulations instability the proper choice of emulsifiers for (Knowles, 2005). Regarding solid stabilization is extremely important. In the biopesticide formulations, much attention has case of invert emulsions, losses due to been focused on WPs because of their long evaporation and spray drift are minimal storage stability, good miscibility with water because oil is the external phase of the and convenient application using formulation (Brar, 2006). However, lower conventional spraying equipment (Brar, shelf stability and occasional phyto- toxicity 2006). Water dispersible granules (WG) have may affect the overall performance of been developed to overcome problems of emulsions. Studies are currently being dustiness of powder formulations. conducted to screen a variety of oils and emulsifying agents in order to improve initial Water dispersible granules (WG) invert emulsion formulations for biopesticides (Verner, 2007). Water dispersible granules are designed to be suspended in water, i.e. granules break up to Suspension concentrate (SC) form uniform suspension similar to that formed by a wettable powder. Compared to Suspension concentrate (SC) is a mixture of a powder products these WGs are relatively finely ground, solid active ingredient dust-free, and with good storage stability. dispersed in a liquid phase, usually water. The Water dispersible granules can be formulated solid particles are not dis- solved in liquid using various processing techniques, such as phase, so that the mixture needs to be agitated extrusion granulation, fluid bed granulation, before application to keep particles evenly spray drying, etc. distributed. The composition of suspension concentrate is complex and it contains The products contain wetting agent and wetting/dispersing agents, thickening agents, dispersing agent similar to those used in antifoaming agents, etc. to ensure a required wettable powders, but the dispersing agent is stability. They are produced by a wet grinding usually at higher concentration. Water process and have particle size distribution dispersible granules are usually more ranging from 1-10 µm. During the grinding expensive than older types of formulations process, inert ingredients adsorbed onto (dusts, wettable powders) but their safety and particle surfaces prevent re-aggregation of greater convenience regarding application small particles. make them still desirable for many users (Knowles, 2008). These small particles often exhibit improved bioefficacy in use because greater particle Emulsions surface area offers easier access of the active ingredient to plant tissues. Because they are Emulsions consist of liquid droplets dispersed water-based, they offer many advantages, in another immiscible liquid (dispersed phase such as of pouring and measuring, safety to droplet size ranges from 0.1 to 10 µm). the operator and the environment, and Emulsion can be oil in water (EW), which is a economy. Therefore they are becoming a very normal emulsion, or water in oil (EO), an popular type of formulation (Woods, 2003; invert emulsion. Both products are designed Knowles, 2005). to be mixed with water before use. To avoid

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Table.1 Biocontrol agents

S. No. Name of the Biopesticide

1. Bacillus thuringiensis var. israelensis

2. Bacillus thuringiensis var. kurstaki

3. Bacillus thuringiensis var. galleriae

4. Bacillus sphaericus

5. Trichoderma viride

6. Trichoderma harzianum

7. Pseudomonas fluoresens

8. Beauveriabassiana

9. NPV of Helicoverpa armigera

10. NPV of Spodopteralitura

11. Neem based pesticides

12. Cymbopogan

Table.2 Different bio pesticides used against various plant pathogens

Trade name Biocontrol Class Target disease References organism Rhapsody/ Bacillus subtilis Fungicide , Rhizoctonia, Marrone ,2002 Serenade soil foliage Fusarium, Quarles, 2005 Alternaria, Aspergillus Blightban Pseudomonas Fungicide Fire blight, Quarles,2011 A506 fluorescens Bactericide frost damage, bunch top Green light Neem oil Fungicide, Powdery mildew, Cao et al,2010 rust, anthracnose, leaf spot Madex HP Codling moth Insecticide Fruit moth Arthurs et al, granulosis Virus 2004. Soil Gard Trichoderma virens Fungicide Pythium, Rose et al, 2004. Rhozoctonia and root rot

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Table.3 Different type of amendments and example materials for formulation

Amendment type Examples Liquid carriers Vegetable oil Mineral carriers Kaolinite clay, diatomaceous earth. Organic carriers Grain flours Stabilizers Lactose ,sodium benzoate Nutrients Molasses ,peptone Binders Gum Arabic, carboxymethy cellulose Desiccants Silica gel, anhydrous salts Thickeners Xanthan gum Surfactants Tween 80 Dispersants Microcrystalline cellulose UV protectants sunscreens Oxybenzone Light blockers Lignin (PC 1307) Stickers Pregelatinized corn flour

Table.4 Different Sprayer technology used for application of control agents against pests

Sprayer / application technology Control agent Pest References Electrostatic knapsack mistblower; Bt Diamondback Perez et al. (1995) hydraulic. moth Spinning discs Nematode Diamondback Mason et al. (1998,, moth 1999) Grooved and smooth spinning Nematodewith Leaf miner Piggott (2000), discs polyacrylamide Piggott et al. (2000) Refrigerated aerial l spraying Predator pupae Trichogramma Bouse and Morrison (1985) Dispersing pheromones in Insectsex Fruit moth Atterholt et al. parafﬁn-wax capsules pheromones (1994)

Oil dispersions (OD) same way as suspension concentrate. Inert ingredients for this type of formulation should Oil dispersions (OD) are dispersions of solid be carefully selected to prevent instability active ingredients in non-aqueous liquid problems (Verner, 2007). intended for dilution before use. The non- aqueous liquid is most often an oil, the best Suspo-emulsions (SE) choice is some kind of plant oil. In that way retention, spreading and penetration can be Suspo-emulsions (SE) can be considered as a improved. Oil dispersion provides several mixture of suspension concentrate and important characteristics, such as an ability to emulsion. The product is very demanding to deliver water sensitive active ingredients and formulate because it is necessary to develop a an ability to use an adjuvant fluid instead of homogenious emulsion component water which can increase and broaden pest simultaneously with a particle suspension control. This formulation is produced in the component which will remain stable in the

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final formulation of the product. Careful active ingredient which is extremely soluble selection of appropriate dispersing and in crop-compatible liquid (ultra-low volume emulsifying agents is necessary to overcome liquid). UL products are not intended for the problem of heteroflocculation between dilution with water before use and often solid particles and oil droplets. In addition, contain surface active agents and drift control extensive storage stability testing of this additives. Ultra-low volume liquids are easy formulation is necessary (Knowles, 2008). In to transport and use. UL liquid biopesticides spite of the complexity of this formulation, can be formulated in a similar way using a the use and importance of suspo-emulsions suspended biocontrol agent as an active has been remarkable and will continue to ingredient (Woods, 2003). increase. Application system Capsule suspension (CS) Delivery of products must be easy, Capsule suspension (CS) is a stable economical, effective, timely to the suspension of micro-encapsulated active appropriate site of action, and compatible ingredient in an aqueous continuous phase, with current agronomic practices and intended for dilution with water before use. equipment. Formulated microbes can be Bio-agent as its active ingredient is delivered to seed, seed pieces, tubers cuttings encapsulated in capsules (coating) made of seedlings, transplants mature plants, or soil, gelatin, starch, cellulose and other polymers. these application methods are In that way the bio-agent is protected from extreme environmental conditions (UV Seed treatment radiation, rain, temperature, etc.), and its residual stability is enhanced due to slow For optimal protection of germinating seeds (controlled) release. The most frequently and seedlings against disease, the applied method of encapsulation uses the biofungicides need to be delivered in a principle of interfacial polymerization. manner that allows the organism(s) to Encapsulation in microcapsules has been colonize the spermospere and the developing extensively used to give smaller size and high rhizosphere at a density that is high enough to efficiency to fungal biopesticide formulations suppress the pathogen (Cook and Baker, (Winder, 2005; Brar, 2006). Microcapsule 1983). Biocontrol agents can be percoated or suspensions need to be stabilized with encapsulated onto the seed, mixed with the surfactants and thickeners in the same way as seed at the planting, applied in-furrow, or suspension concentrate and similar additives incorporated into the soil-mix or seed bed are used. Despite clear benefits of this (Thomashow and Weller,1990). controlled release formulation, its commercial development is rather slow. The slow Precoating of seed usually involves progress is partly due to the complexity of formulations of dry powders or oil-and formulation and partly to its high production polymer-based liquids with dormant microbes cost (El-Sayed, 2005; Chen, 2013). that are capable of surviving a period of desiccation (Pauu, 1988). Additives, such as Ultra low volume liquids (UL) xanthum gm and gum Arabic are sometimes used to increase adhesion of the microbial Ultra-low volume liquids (UL) are product to the seed. A specialized seed- formulations with very high concentration of coating process, termed seed encapsulation,

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involves enveloping the seed, microbe and the biocontrol agent. This creates a possibly other components such as pesticides “suppressive soil,” making subsequent or micronutrients, in a gelatinous or polymer colonization by other less beneficial gel-matrix, thereby prolonging survival of organism’s difficult (Lumsden et al., 1995). microbial agents on seed. An example of a Dust, powder and granular formulations can seed encapsulation product is GEL-COAT, be broadcast and incorporated into soil,where which is an alginate hydrogel preparation as wettable powder, water-dispersible patented as a delivery system for granular, and liquid formulations can be entomopathogenic nematodes. The seed delivered in furrow (Lewis,1991). Soil encapsulation method of delivery has the application may also be a useful method for distinct advantage of user safety and reduced controlling overwintering pathogen environmental hazard, since the active propagules in soil. For example, the product ingredients are effectively sealed until they CONTANS, a water dispersible granular are released during seed germination. Factors formulation of the hyperparasite to consider in selecting a formulation for Coniothyrium minitans can be incorporated coating seeds include inoculum density into soil to reduce the number of achievable on the seed, stability of the Sclerotiniasclerotiorum (Boyetchko et al., coating, both for microbe viability and coat 1996). integrity, and the feasibility and cost of production (McIntyre and Press, 1991). In greenhouse crops, a simple yet effective Formulations consisting of fine dusts or method of delivering biocontrol agents to soil powders, wettable powders, or liquids can be or growth medium is by direct injection into applied to seed with or without sticker an irrigation system, such as overhead boom materials at the time of planting. Delivery at or spaghetti systems. This type of delivery is the time of planting usually ensures a high advantageous in that it allows precise control number of viable microbes and may allow of the concentration and total volume of growers to apply the product directly into the microbial suspension being applied, and plant box. Drawbacks to this delivery method requires minimal labour to treat large include possible variability in efficacy numbers of plants. The one drawback to this resulting from a reliance on the grower’s type of delivery system is that it requires ability to apply the seed treatment correctly specialized injection equipment (Boyetchko et and the extra task for growers (Boyetchko et al., 1996). al, 1996). Treatment of plants Soil treatment Biocontrol products can also be applied to If seed treatment is not a practical option, e.g., plant roots, wounds and foliage by drenching, if direct inoculation onto seed is harmful to dipping or spraying. Formulated bacteria can the microbe due to dessication, or presence of be applied directly to roots as a dip or drench inhibiting compounds (Gindrat, 1979), (Funk, 1997).Spores of the biofungicide biocontrol agents can be applied to soil. Soil Phelbia gigantea in an aqueous suspension treatment is most effective when the agents can be brushed onto freshly cut stumps of are applied as a post-fumigation treatment or pine to prevent entry of Heterobasidion at time of planting. In sterile soil or growth annosum (Rishbeth, 1975), thereby protecting mixes, colonization by pathogens may be exposed wounds. Alternatively, spores can be reduced by establishing a high population of incorporated into chain saw oil so that they

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are delivered at the same time the tree is electrostatic rotary atomizer has been used to harvested. Formulations of bacteria or fungi apply Verticilliumlecanii, an used as foliar sprays vary according to the entomopathogenic fungus, to successfully crop to be treated, the pest to be controlled, control the aphid Aphis gossypii. In and the anticipated delivery system. The two addition,ultralow-volume equipment, such as formulations most commonly used for foliar spinning disk sprayers, are now commonly sprays are liquids and slurries, with the used for application of baculoviruses in slurries usually reconstituted from either dry forests (Cory and Bishop,1995). or moist carrier-based formulations. Emulsifiers, stickers, spreaders, and other In conclusion adjuvants and additives aid in application, dispersal and adhesion of the microbes on Organic food indicates huge scope for growth plant surfaces, and protect the microbes from of Bio-pesticides sector in India. At the adverse environmental conditions, such as same time increasing population can be desiccation, unfavourable pH, and UV fed by organic farming dependence is a radiation (Harvey, 1991 and Shieh, 1995). A big question and unless organic farming broad range of spray application equipment yield can be brought equal to that of and techniques is available for applying conventional farming involving the use chemical pesticides (Table 4) including high of agrochemicals etc, the organic volume (1000 L/ ha), medium volume (350 farming may not be feasible at the L/ha), low to very low volume (3-150L/ha), moment. and ultra low volumes (0.5-3.1 L/ha), Rich traditional knowledge base available controlled droplet application, and with the highly diverse indigenous electrostatic spraying (Auld, 1992). If control communities in India may provide agents are to be applied using the same valuable clues for developing newer and techniques, formulations must have the effective bio pesticide. necessary physical properties. A new more active strain of B.t was produced which has increased the performance Steinke and Akesson found that surface and acceptance of commercial products tension and viscocity of the suspension to be and broadened its use against other sprayed are important factors in reducing insect pests. droplet size and maintaining the necessary Commercial biopesticides should be dispersion and control of droplets. Density of economical to produce, have persistent the suspension was not an important factor. storage stability, be easy to handle, mix Successful application of biocontrol agents and apply, and provide effective control using different spray techniques has been of target pests. achieved. For example, Bt-based products have been applied to numerous crops using References conventional ground or aerial spraying methods. Highly concentrated ultra-low Arthurs, S.P. and Lacey, L.A. 2004. Field volume liquid formulations of Bt-based evaluation of commercial formulations products have also been used to control insect of the codling moth granulosis virus: pests on such crops as cotton and banana persistence of activity and success of (Sheih, 1995). And to control spruce bud seasonal applications against natural worm over large areas of coniferous infestations of codling moth in Pacific forests(Bryant,1994). A low –volume Northwest apple orchards. Biological

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How to cite this article:

Varsha Bharti and Shahida Ibrahim. 2020. Biopesticides: Production, Formulation and Application Systems. Int.J.Curr.Microbiol.App.Sci. 9(10): 3931-3946. doi: https://doi.org/10.20546/ijcmas.2020.910.453

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