Recent Advances in Biopesticide Research and Development with a Focus on Microbials

3.1 Mechanism of action and efficacy of biopesticides

How do they work? Biopesticides work through various mechanisms, such as 1) biochemical disruption,-where they interfere with the biochemical processes of pests, affecting their growth, development, or reproduction; 2) microbial activity-infecting or parasitizing pests; 3) predation and competition-acting as natural predators or competitors of pests to limit their population growth; and 4) repellency-deterring pests from attacking crops (Essiedu et al. 2022). They can play a crucial role in IPM strategies and contribute to sustainable agricultural practices by reducing the environmental impact and minimizing the risk of pesticide resistance (Essiedu et al. 2022), thereby complying with the 3 pillars of climate-smart agriculture and the synergies with the SDGs (FAO 2024) (Figure 1).

5.1 Biopesticide categories

Biopesticide is a generic term generally applied to a substance derived from nature, such as a botanical or semiochemical, that may be formulated and applied like a conventional chemical pesticide and that is normally used for short-term pest control (USEPA 2020). Naturally derived or laboratory-made chemicals with structures and functions similar to naturally occurring ones are known as biochemical pesticides. They differ from traditional pesticides in their origin (source) and how they control or kill pests (O’Brien and Jones 2009). Microbial pesticides are natural pest control agents harnessing the power of microscopic living organisms (Clemson 2007). These “living bullets” can be either spores or active organisms, often specifically chosen for their pathogenic nature towards targeted pests. Common examples include biofungicides (Trichoderma, Pseudomonas, Bacillus, Colletotrichum), bioherbicides (Phytophthora, Cylindrobasidium, Colletotrichum acutatum), and bioinsecticides (Bt) (MacGregor 2006; Gupta 2010). Microbial biopesticides can originate from naturally occurring or genetically modified bacteria, fungi, algae, viruses, or protozoans. They subdue pests through a diverse range of strategies (toxic metabolites, disease-causing, competitive exclusion, and other modes of action), depending on the specific microbe (Clemson 2007).

Microbial biopesticides offer diverse delivery methods to effectively reach crop targets. These methods include live organisms, dead organisms, and spores (O’Brien and Jones 2009). Globally, microbials account for 41% of all BCAs used worldwide, macrobiotics comprise 33% of the global market, and other natural products represent the remaining 26% (Guillon 2003). Based on their origin or active ingredients, they can be classified as bacterial-based biopesticides, fungal biopesticides, viral biopesticides, nematodes, and protozoan-based biopesticides.

5.2 Bacterial based biopesticides

Among all microbial pesticides, bacterial biopesticides are the most widely used. These versatile microbes attack pests in multiple ways, primarily targeting insects. While mostly used against various orders of insect pests in agriculture, bacterial biopesticides can also curb the growth of plant-harming bacteria and fungi (O’Brien and Jones 2009; Jouzani and Valijanian 2017). Bacterial pathogens need direct contact with their target to be effective, often requiring ingestion. They secrete endotoxins, protein-based toxins specific to the targeted pest that wreak havoc on their digestive system (Schallmey and Singh 2004). Bacillus thuringiensis (Bt) dominates the biopesticide market, accounting for a whopping 90% share in the USA alone (Chattopadhyay and Bhatnagar 2004). Bacillus thuringiensis is the most common species used commercially to control a diverse range of insect pest species, including lepidopterans, hemipterans, and coleopterans in agriculture, forestry, and even medicine since its discovery in 1901 (Mazid 2011). It is a spore-forming bacterium with several strains that produce diverse insecticidal crystal proteins, often targeting different pests ( Table 1). The crystal proteins are toxic and, once ingested, damage the gut tissues, leading to paralysis of the gut. The infected insects stop feeding and eventually die from gut impairment and starvation. Bt var. kurstaki specifically targets caterpillars, while the genes encoding Cry proteins have been transferred into crops like cotton, hence reducing reliance on chemical pesticides (Mazid 2011). Commercial Bt-based products are available in various forms, such as powders containing a combination of dried spore and crystal toxins or formulations in liquid suspensions (Boyetchko et al. 2020). Bacillus thuringiensis’s high target specificity and environmental safety make it an ideal complement to traditional chemical pesticides for insect pest control (Roy and Moktan 2007; Kumar 2012). Figure 2 illustrates the effects of Bacillus thuringiensis on insect larvae.

Figure 2. Effects of Bacillus thuringiensis (Bt gene and Cry protein) on insect larvae (Singh et al. 2019).

Recombinant DNA technology has been used to develop several new Bt-based products. MVP^TM and M-Trackr^TM, for example, were created using Mycogen Corporation’s CellCap^® encapsulation method. This procedure entails removing a gene from Bt that encodes the delta-endotoxin protein, incorporating it onto a plasmid, and inserting it into a Pseudomonas fluorescens strain (Pitterna et al. 2009; Legwaila et al. 2015). Before being killed by heat and chemical treatment, the recombinant cells were cultured in aerobic culture and induced to express delta-endotoxin. The dead bacterial cells in the aqueous formulation acted as microcapsules, protecting the fragile Bt toxin from destruction in the environment.

Pathogenic bacteria have also been considered for biological weed control. However, one of the challenges to using phytopathogenic bacteria for biological weed control is the necessity of free water for dispersal and wounds or natural holes for bacterial penetration into the weed plant (Zidack et al. 1992; Johnson et al. 1996). Previously, researchers have studied Xanthomonas campestns pv. Poae for the control of annual bluegrass (Pea annua L.) and discovered that cutting or mowing turfgrass allows bacteria to enter the plant (Imaizumi et al. 1997). Furthermore, bacteria administered at a rate of log CFU/mL at high water levels (400 mL/m²) reduced disease severity in annual bluegrass by more than 90%. Silwet L-77 (0.2%), an organosilicon surfactant, increased bacterial penetration and entrance into plant stomata and hydathodes (Johnson et al.1996; Boyetchko et al. 2020). A low surface tension of 30 dynes/cm or less is necessary to convey liquid into a leaf’s stomata. Silwet decreases the surface tension of water to 20 dynes/cm. Compared to plants sprayed with bacteria without the surfactant, the application of Pseudomonas syringae pv. Tagetes with this surfactant facilitated the penetration and entry of bacteria into stomata and hydathodes, significantly increasing disease severity and incidence (Johnson et al. 1996).

Bacterial biopesticides are environmentally safe and precise alternatives to traditional pest control methods. They offer several advantages, including safety for humans, wildlife, and beneficial insects. These biopesticides can be used in conjunction with other pest control strategies, including some chemical methods. Additionally, they leave no harmful residues, don’t harm pollinators and other valuable organisms, and can be applied close to harvest time. The additional benefit is that they can self-propagate, extending their effectiveness beyond the initial application and into subsequent growing seasons (Meenatchi and Aditi 2021). Bacterial biopesticides, while effective, come with a few disadvantages. For instance, they only target specific species or groups of insects, leaving other pests to survive and continue causing damage. This means that additional biopesticides are needed for the other pests. Hence, they should be applied alongside other IPM strategies. Finally, the effectiveness of microbial insecticides can be influenced by factors like ultraviolet radiation and heat, so it’s important to apply them strictly according to the label (Meenatchi and Aditi 2021).

5.3 Fungal biopesticides

Mycopesticides include naturally occurring fungi and fungi cell components. These fungi attach to their targets through sticky spores. These spores then germinate and form microscopic tubes that pierce the insect’s skin, releasing potent hydrolytic enzyme mixtures along with toxins (Langner and Göhre, 2016). This internal invasion disrupts the insect’s physiology, leading to its eventual death. The fungi then grow outward from the dead insect, producing new spores to continue the cycle, hence perpetuating natural pest control (Pineda et al. 2007; Gabarty et al. 2014). The role of hydrolytic enzymes, especially chitinases, in the killing process, and the possible use of chitin synthesis inhibitors are prime research areas. Popular commercially available mycoinsecticides are often derived from species like Beauveria bassiana, Metarhizium anisopliae, Isaria fumosorosea, Hirsutella thompsonii, Phytophthora palmivora, Alternaria cassia, and Lecanicillium spp. ( Table 1). Notably, Beauveria bassiana and Metarhizium anisopliae are two common ascomycetes known for their broad effectiveness against pests like aphids, beetles, grasshoppers, and caterpillars. These fungi are typically applied as conidia (spores) or mycelium, which then sporulate after application, ensuring sustained pest control (Lacey et al. 2015). Cylindrobasilium laeve for the control of Acacia mearnsii (black wattle) and Colletotrichum acutatum for the control of Hakea sericea (silky hakea) are some of the available mycoherbicides for integrated alien and invasive species management.

Fungal biopesticides hold immense promise as environmentally safe options against a diverse range of insect and mite pests. They possess several desirable traits for biocontrol: they selectively target pests, leaving beneficial insects like bees and predators unharmed. Additionally, they generally pose no threat to the growth or development of other beneficial organisms like earthworms and springtails. This makes them ideal candidates for IPM and sustainable agricultural practices, promoting biodiversity and protecting the environment (Goettel et al. 2008; Kim and Goettel 2011; Koike et al. 2011). Several fungi have been successfully mass-produced and formulated for commercial application as mycoinsecticides, paving the way for their widespread use in pest control (Chandler et al. 2008).

The remarkable potential of entomopathogenic fungi is actively being harnessed for use in IPM programs. Mass production techniques and ecological approaches are constantly refined to optimize their effectiveness and ensure seamless integration into sustainable agricultural practices (Chandler et al. 2011; Lacey et al. 2015). Furthermore, promising results have been achieved by combining fungal biopesticides with traditional insecticides, often leading to significantly boosted pest mortality rates. For example, studies have shown that combining B. bassiana with low-dose insecticides enhances potato beetle control, while its synergy with neem oil proves effective against tobacco thrips eggs and nymphs (Chandler et al. 2011; Lacey et al. 2015; Sarwar 2015).

Fungi-based biopesticides boast several advantages over other biocontrol methods. Their broader host range allows them to tackle a diverse range of pests across fields, greenhouses, storage facilities, and soil, unlike bacteria and viruses with limited targets (Amruta et al. 2019). Commercially important fungi like Beauveria, Metarhizium, Lecanicillium, and Isaria are surprisingly easy to mass produce, requiring significantly less substrate compared to alternative methods. These fungi are highly productive, delivering powerful, targeted pest control while reducing the impact on the environment. They pose no risk to beneficial organisms, readily biodegrade, and integrate seamlessly into IPM programs. Additionally, their persistence in the environment offers long-lasting protection against pest outbreaks, making them a potentially cost-effective solution for sustainable agriculture (Naveenkumar et al. 2017; Rajaput et al. 2019; Sufyan et al. 2020; Upamanya and Bhattacharyya 2020).

Fungi-based biopesticides, however, face some environmental hurdles. Optimal spore germination and penetration into insect cuticles often require humidity levels exceeding 80%, depending on the specific fungal species. Temperature fluctuations and exposure to UV radiation can significantly impact their survival. Germinating conidia, essential for active pest control, are often delicate and susceptible to environmental damage. Conversely, the production of durable resting spores for long-term persistence can be expensive compared to other microbial methods like bacteria-based biopesticides (Sabaratnam 2002; Kolombet et al. 2008; Kordali et al. 2008; Li et al. 2014). Figure 3 illustrates the mode of action of fungi-based biopesticides, highlighting the steps by which these biopesticides attack target insect pests (Figure 3).

Figure 3. Mode of action of fungi-based biopesticides.

5.4 Viral biopesticides

Baculoviruses (BVs) are tiny, naturally occurring viruses that have emerged as powerful allies in the fight against insect pests. They belong to a group known as entomopathogenic viruses, specifically targeting and infecting insects and other arthropods. Unlike harmful conventional chemical pesticides, baculoviruses pose no threat to humans, wildlife, or beneficial insects, making them an environmentally safe option for pest control (Cory 2000; Jackson and Crawford 2005; Ramle et al. 2005; Moscardi et al. 2011), and are considered as the most commercial viral biopesticides (Granados 1986; Moore and King 1987). Baculoviruses involve circular supercoiled double-stranded DNA genomes, which range from 80 to 180 kbp (Rohrmann 2013). Within the Baculoviridae family, scientists have identified three main subgroups: nuclear polyhedrosis viruses (NPVs), cytoplasmic polyhedrosis viruses (CPVs), and granulosis viruses (GVs). These subgroups differ in the structure and number of their protective protein coats, called occlusion bodies (Moscardi et al. 2011). These bodies allow the virus to survive outside the host, waiting for its next target. NPVs and CPVs form polyhedral bodies with numerous virus particles, while GVs have smaller, granular bodies containing just one particle. Notably, each subgroup employs a unique method to uncoat and establish infection within their host: NPVs in the nucleus, CPVs in the cytoplasm, and GVs within the nuclear pore complex (Cory 2000; Moscardi et al. 2011).

Baculoviruses offer highly specific targeting, primarily focused on lepidopteran larvae and hymenopterans (butterfly and moth) pests that damage crops like cotton, rice, and vegetables. One example is Heliothis zea nucleopolyhedrosis virus (HzNPV), the first commercially successful broad-spectrum viral insecticide. Interestingly, HzNPV demonstrates versatility, effectively controlling pests across various crops like soybeans, sorghum, maize, tomatoes, and beans. While other entomopathogenic viruses exist, such as tetraviruses and cypoviruses, their use in crop protection remains limited compared to the widespread adoption of baculoviruses (Sarwar 2015). A comprehensive summary of viral biopesticides and their target pests is presented by (Usta 2013; Singh et al. 2019; Meenatchi and Aditi 2021).

Figure 4 shows how viral replication and infection take place and attack their target host. After infecting a target cell, viral replication in the nucleus or cytoplasm unfolds in three distinct phases: early (0-6 hours), second (6-24 hours), and very late (24-72 hours). During the late phase, the virus forms protective protein coats called occlusion bodies (Obs) or virions. These tiny packages containing multiple virus particles can lead to natural outbreaks (enzootics) that drastically reduce pest populations. However, sunlight poses a challenge: occlusion bodies can rapidly lose potency when exposed to ultraviolet radiation (UV) between 280-320 nm (Killick 1990). Interestingly, studies show that plastic greenhouses can help mitigate this issue by filtering out over 90% of harmful UV-B rays, boosting infection rates in larvae (Lasa et al. 2007).

Figure 4. Mode of action of viral biopesticides.

The virions enter the midgut cell nucleus, at which point the virus replicates within the nuclei of susceptible tissue cells, and tissue susceptibility varies greatly between viruses, with some NPVs being capable of infecting almost all tissue types and most GVs being tissue-specific replications. In the terminal stage of infection, the insect liquefies and thus releases polyhedral, which can infect other insects upon ingestion. A single caterpillar at its death may contain over 109 occlusion bodies from an initial dose of 1000. Under optimal conditions, target pests may be killed in 3-7 days, but when the condition is not suitable, death may be caused in 3-4 weeks (Kalamkoff 2004).

The virions enter the midgut cell nucleus, at which point the virus replicates within the nuclei of susceptible tissue cells, and tissue susceptibility varies greatly between viruses, with some NPVs being capable of infecting almost all tissue types and most GVs being tissue-specific replications. In the terminal stage of infection, the insect liquefies and thus releases polyhedral, which can infect other insects upon ingestion. A single caterpillar at its death may contain over 109 occlusion bodies from an initial dose of 1000. Under optimal conditions, target pests may be killed in 3-7 days, but when the condition is not suitable, death may be caused in 3-4 weeks (Kalamkoff 2004).

Several studies have been conducted to explore ways to improve the effectiveness of viral biopesticides. Certain formulations, like stilbene, can enhance susceptibility to NPV infection by disrupting the insect’s digestive barrier (Okuno et al. 2003) or inducing cell death in the midgut (Dougherty et al. 2006). Additionally, researchers are investigating genetically engineered viruses. For example, vAcTaITX-1 and vAcDTX9.2, derived from specific spiders, show promise as commercial biopesticides against lepidopteran pests (Hughes et al. 1997). While viral biopesticides offer numerous advantages over conventional chemical pesticides, including specificity and environmental safety, they face certain challenges for large-scale adoption. Producing them efficiently, particularly recombinant viruses, can be costly and labor-intensive, requiring specialized equipment and lengthy procedures (Gupta 2010; Lacey et al. 2015). Despite these hurdles, organizations like IPM centers and state agricultural departments are actively supporting the development and small-scale production of these promising biopesticides.

What is the main advantage of viral biopesticides? Viral biopesticides offer a safer alternative, protecting human health, wildlife, and even beneficial insects. These targeted pathogens would not trigger resistance in pests, making them a lasting solution. Moreover, they seamlessly work with other pest control methods for a comprehensive defense (Singh et al. 2019; Meenatchi and Aditi 2021). While viral biopesticides offer enticing, environmentally safe alternatives to chemical pesticides, they come with limitations. Unlike the broad spectrum of some conventional chemical insecticides, their use often remains targeted to specific pest species. Additionally, successful pest control can be a slow process, as infections may take a significant amount of time to become lethal and can be easily deactivated by high temperatures and ultraviolet radiation. Understanding these vulnerabilities is crucial for choosing the right pest control strategy (Singh et al. 2019; Meenatchi and Aditi 2021) for the targeted pest and bioclimatic zone.

5.5 Nematodes as biopesticides

Entomopathogenic nematodes (EPNs) are classed as microbial pesticides, even though they are multicellular. EPNs are tiny soil-dwelling organisms that live in the water films around soil particles, fit nicely into IPM programs because they are considered nontoxic to humans, relatively specific to their target pests, and can be applied with standard pesticide equipment (Shapiro-Ilan et al. 2006). These beneficial parasites help maintain the balance in the ecosystem by targeting specific pests without harming other organisms (Kachhawa 2017). Two main families of EPNs, Steinernematidae, and Heterorhabditidae, have been enlisted in the fight against pests (Bhat and Chaubey 2020). Species like S. carpocapsae, S. thermophilum, H. bacteriophora, and H. indica are commercially available and actively deployed in pest control programs in India (Meenatchi and Aditi 2021). Commercial biopesticides based on EPNs contain non-feeding, third-stage infective juveniles (IJs) as the active ingredients. Heterorhabditis and Steinernema are mutualistically associated with bacteria of the genera Photorhabdus and Xenorhabdus, respectively (Ferreira 2014).

So, how do these tiny organisms take down their targets? EPNs, in their infective juvenile stage, barely millimeters long, use their keen senses to locate insects through carbon dioxide emissions, vibrations, and other chemical cues. They then wriggle their way into the insect’s body through natural openings like the mouth, anus, or breathing holes. Once inside, the nematodes release their symbiotic bacterial companions, setting off a natural pest control cascade. These bacteria produce toxins that quickly kill the insect within a week. The EPNs then feast on the insect’s liquefied remains, multiplying within the host to create new generations of tiny warriors. Finally, once their feast is over, the next generation of infective juveniles emerges, ready to seek out their next victim and continue the cycle (Figure 5). Under ideal conditions, the impact of EPNs becomes visible within 5-7 days. Look for browning or tanning of insects infected by Steinernematidae, while those taken down by Heterorhabditidae turn a distinctive red (Gupta 2010; Koul 2011; Ruiu 2018).

Figure 5. Mode of action of Entomopathogenic nematodes.

The juvenile stage releases cells of their symbiotic bacteria from their intestines into the hemocoel. The bacteria multiply in the insect hemolymph, and the infected host usually dies within 24 to 48 hours. After the death of the host, nematodes continue to feed on the host tissue, mature, and reproduce (Bedding 1982). The progeny nematodes develop through four juvenile stages to the adult. Depending on the available resources, one or more generations may occur within the host cadaver, and a large number of infective juveniles are eventually released into the environment to infect other hosts and continue their life cycle (Bedding 1982). Nematodes enter the body cavity of insects and release their symbiotic bacteria into the host's intestine.

Advantages of EPNs: EPNs offer a double win: safety and effectiveness. These organisms pose no threats to human health, plants, or animals, requiring no protective gear or waiting periods. Moreover, they leave no harmful residues, making them a sustainable choice for healthy crops and clean environments. Their target list reads like a bug buffet: cranberry girdlers, root weevils, webworms, and even wood borers tremble at their approach. However, these tiny heroes have their preferences. Moist soil around roots, protection from harsh UV, and short stints with other pest control methods are necessary for them to thrive, ensuring their targeted pest control doesn’t disrupt the natural ecosystem (Ruiu 2018; Singh et al. 2019; Meenatchi and Aditi 2021).

The disadvantages of EPNs: EPNs are highly susceptible to adverse weather conditions. Harsh UV rays and sizzling temperatures can quickly deactivate these tiny heroes, making their effectiveness dependent on fickle environmental conditions. Compared to their chemical counterparts, EPNs come with a hefty price tag. This, along with their short shelf life, makes them a less financially enticing option for some applications. EPNs can’t reach aerial pests, limiting their range of action. Additionally, relying solely on EPNs can disrupt the natural predator-prey balance, requiring repeated applications to maintain pest control. Furthermore, EPNs thrive in moisture. Pre- and post-irrigation, along with application during cooler hours, are essential for their success (Meenatchi and Aditi 2021).

5.6 Protozoan biopesticides

Protozoans, also known as microsporidians, are intracellular parasites that can only survive by living inside other cells. They are found almost everywhere and can attack certain insects, like lepidopteran and orthopteran, making them useful in IPMs. Examples include Nosema sp. and Vairimorpha sp. However, despite their pest-specific nature and slow-acting properties, the use of protozoa as biopesticides is not as effective as other organisms, such as bacteria, viruses, and fungi. They can cause chronic and debilitating effects on their targets, but their success rate is lower compared to other biopesticides (Meenatchi and Aditi 2021). Protozoans have a specific mode of action. Microsporidia infects the European corn borer, Ostrinia nubilalis, by being eaten by insects. The spores germinate in the midgut region, and the sporoplasm is then injected into the midgut cells. The spores then spread to different tissues and organs, multiply, and cause tissue breakdown and septicaemia (Senthil-Nathan 2015).

7.1 Biopesticide formulation requirements and challenges

The effectiveness of a biopesticide hinges on the biological activity of its active microorganism or metabolite. In its raw state, this active ingredient requires formulation to enable its practical handling, storage, and application (Harald 2019). A crucial distinction between conventional (chemical) pesticide formulation and biopesticide formulation lies in the biopesticide’s living nature. Biopesticides are sensitive to storage conditions and environmental factors, necessitating careful formulation to preserve their biological viability (Harald 2019). Formulating biopesticides faces several challenges, including ensuring market viability, ease of production and application, product stability during storage and transport, and long-term viability and efficacy of the biopesticide (Lumsden et al. 1995). Contributing factors to their limited commercial success include production difficulties, sensitivity to environmental stressors, and lack of appropriate formulation (Powell and Jutsum 1993). Another challenge is the slime layer produced by gram-positive bacteria like Bacillus spp. during endospore formation. This slime can clog sprayer filters and nozzles, hindering application. While removing the slime through methods like centrifugation is possible, it may also remove valuable components contributing to the biopesticide’s effect (Boyette et al. 1996).

The goal of formulation is to improve product stability, bioactivity, delivery, and integration into pest management systems. Successful formulations also prioritize user convenience, compatibility with equipment and practices, and effectiveness at practical rates. Environmental factors like temperature, moisture, and UV exposure significantly influence efficacy, particularly for foliar-applied biopesticides (Alan 1988). Soil properties, moisture, temperature regimes, and microbial competition impact soil-applied biopesticides. All these factors need careful consideration during formulation development.

7.2 Types of biopesticide formulations

7.2.1 Formulation of bacteria biopesticide

Several recent studies highlight the growing use of bacteria-based biofungicide formulations as a safe and effective complement to conventional chemical pesticides (Cook 1993; Lumsden et al. 1995; Elad 1995). Bacillus-based products are already boosting crop yields in various countries, from China to the United States (Zhang et al. 1996). These wettable powders, compatible with existing fungicides, target diseases caused by Rhizoctonia and Fusarium in a range of crops like cotton, legumes, vegetables, and ornamentals.

Mass production of these biofungicide bacteria formulations typically involves deep-tank liquid fermentation. However, in some cases, methods like semisolid or solid-state fermentation might be more suitable. Key factors influencing both bacterial growth and their beneficial metabolite production include the medium’s nutrient composition and optimal growth conditions (Paau 1988). Cost-effectiveness and readily available components are prioritized. The final formulation, usually involving additional ingredients after fermentation, comes in various forms like solids, slurries, powders, or granules. Long-term viability during transport and storage (at least 4 months) is crucial (Paau 1988). Formulation significantly impacts the biofungicide’s performance by enhancing bacterial survival after application. This involves creating a protective environment that maximizes the bacteria’s potential for successful colonization and disease control. Choosing the right formulation can help ensure consistent results in the field.

The products are designed as wettable powders and can be used with various seed treatment fungicides ( Table 1). Agrobactenum tumefaciens is available commercially in Australia, the United States, and New Zealand and is formulated as a concentrated liquid or moist peat-based product or supplied as a non-formulated agar culture (Kerr 1980; Elad 1995). After being suspended in water, the bacteria can be applied to the seeds, cuttings, roots, or root wounds of susceptible orchards and ornamental plants as a dip, spray, or drench. Agrobacterium tumefaciens, available as a liquid, peat-based product, or agar culture, combats diseases in orchards and ornamental plants through dips, sprays, and drenches. Streptomyces griseoviridis (Mycostop), formulated as a wettable powder, tackles damping-off and root rot in vegetables and ornamentals caused by Fusarium, Phomopsis, and Pythium. It can be applied dry to seeds or as a liquid drench and is compatible with chemical pesticides (Muir 2023). Three Burkholderia assian strains (Blue Circle, Deny, and Intercept) come in liquid and fight a wider range of enemies. These bacterial warriors target fungi like Fusarium, Phytophthora, and Pythium alongside nematodes, including Globodera rostochensis, Heterodera glycines, and Hoplolaimus columbus (Kumari et al. 2022).

Most commercial bioinsecticide formulations today harness the power of Bacillus thuringiensis (Bt), a friendly, spore-forming bacterium. This gram-positive warrior wields a powerful weapon: delta-endotoxin proteins that wreak havoc on the mid-guts of susceptible insect larvae (Cannon 1993) ( Table 1). Bt proteins are highly specific, only affecting a few species in the Lepidoptera (butterflies and moths), Coleoptera (beetles), and Diptera (flies) families. Bt biopesticides come in an array of formulations, from concentrated liquids to handy dusts, catering to different application needs.

7.2.2 Formulation of fungal biopesticides

Fungicide formulations primarily include filamentous fungi (e.g. Gliocladium virens and Trichoderma harzzanum), yeast-like fungi (e.g. Pseudozymajlocculosa (Bélanger 1997) and Tllletiopsis pallescens (Urquhart and Punja 1997). These biofungicides are used to control root-infecting pathogens (e.g. Pythlum, Rhizoctonia), and foliar fungal pathogens (e.g. powdery mildew (Bélanger 1997; Urquhart and Punja 1997; Punja 1997) and Botrytzs (Elad 1995). Environmental conditions, such as temperature and moisture, can impact the growth and survival of fungal biofungicides. Various formulations have been developed for the application of spore inocula, including granules, pellets, dust, and wettable powders. These formulations can be applied directly or suspended in water or oil, as is the case with Cylindobasidium leave (Muir 2023).

Granular formulations not only protect against desiccation but also offer a food base for the fungus. Meanwhile, powders can be easily sprayed and can provide coverage for large areas. An example of this is the large-scale aerial application of sawdust covered in Colletotrichum acutatum spores for the control of Hakea sericea (Jacobson and Azevedo 2023). It is also possible to treat seeds with liquids or dust for the application of these biopesticides. Moreover, spore formulations in inverted emulsions have been tested for yeasts, such as Tilletiopsis (Urquhart and Punja 1997).

The use of alginate prill has been successfully developed to create a granular formulation of Gliocladium virens (Soil Gard), which helps control root-infecting fungi in potting media (Lumsden et al. 1995). Similarly, Trichoderma powder or dust formulations with pyrophyllite clay (Pyrax) have also been proven to be effective (Jayaraj and Radhakrishnan 2006). To produce the necessary biomass, appropriate nutrient substrates are used in large-scale deep tank fermenters. The resulting product can either be used wet or dried before formulation (Lumsden et al. 1995). Most of the factors that affect product development for fungi are like those for bacteria.

Entomopathogenic fungi are a fascinating group of soil-dwelling microorganisms that play a crucial role in biological control. These fungi are capable of infecting and killing insects through cuticle penetration. They don’t need to be ingested by the insect hosts; instead, they directly invade through the insect’s outer covering. This unique mode of action allows them to control a wide range of insect pests, including sucking insects. However, they cannot effectively combat viruses and bacteria similarly (Shishupala 2022). Entomopathogenic fungi are a type of fungi that can target and control pests without harming beneficial organisms such as pollinators. They are an environmentally safe alternative to traditional chemical pesticides for managing agricultural pests. Some of the most well-researched fungal species with bioinsecticidal properties include Akanthomyces muscarius, Beauveria bassiana, Cordyceps fumosorosea, Purpureocillium lilacinum, Verticillium, and the Metarhizium anisopliae species complex (Shishupala 2022). A recent study by Luo et al. (2022) isolated a new entomopathogenic fungus from an adult cadaver of D. citri. The fungus was identified as Cordyceps fumosorosea based on its morphology and ITS sequence analysis. The researchers named this specific isolated C. fumosorosea SCAU-CFDC01. The study assessed the pathogenicity of the strain against D. citri nymphs and adults in both laboratory and greenhouse settings.

Various fungi can be used to control different types of insects. Verticillium lecanil can control aphids, Beauveria bassiana can control whiteflies, locusts, and beetles, Metarhizium anisopliae and M. flavoviride can control mosquitos and locusts respectively, while Lagenidium gigantem can control mosquito larvae (Lacey and Goettel 1995; Bateman 1997; FAO 2017; Savita 2019). These fungi can be applied to insects in different ways, such as wettable powders, emulsions, specks of dust, baits, or traps, or even added to the soil (Auld 1992; Feng et al. 1994; Goettel et al. 1995; Inglis et al. 1996) or used in indoor residual spraying. It is important to formulate these fungi to protect against environmental conditions such as moisture, temperature, UV damage, and desiccation. For example, B. bassiana conidia on foliage can be damaged by UV-B radiation, which is part of the sunlight spectrum (Caudwell and Gatehouse 1996a; Daoust and Pereirn 1986). In field conditions, these entomopathogens can be applied at ultralow volumes (ULV) to increase their effectiveness and protect against UV damage (McGUIRE and Shasha 1992; Moore et al. 1993; Inglis et al. 1996). To improve survival and shelf-life, sunlight blockers (such as clay) or UV-B absorbing compounds (such as Tinopal) can be added to inoculum formulations or starch encapsulation (Pereira and Roberts 1991; McGuire and Shasha 1992; Caudwell and Gatehouse 1996b).

7.2.3 Formulation of mycoherbicides

Environmental factors, such as temperature and moisture, can greatly impact the effectiveness of Mycoherbicides. The moisture needed for the disease to develop is often determined by the amount of dew that occurs. “DeVine” is the first registered Mycoherbicide, which is a liquid formulation consisting of chlamydospores of Phytophthora palmivora (Burnett et al. 1974; TeBeest and Templeton 1985). This product has a limited shelf life of only 6 weeks when refrigerated due to its instability. Another Mycoherbicide called “Collego” (Colletotrichum gloeosporioides f.sp. aeschynomene) is formulated as dried spores and is available in a wettable powder (Boyette 1994). The commercialized formulation of Colletotrichum acutatum is formulated as dried spores that are mixed with water when ready to apply, and Stumpout is Cylindrobasidium laeve formulated as dried spores in an iron carrier that is mixed with standard cooking oil when ready to apply (Muir 2023).

There are several ways to improve the effectiveness of mycoherbicides, which are used to control weeds (Boyette et al. 1996). One way is to use adjuvants and other additives that can help spores grow, make the pathogen more stable, change the environment, or make it possible to use bioherbicides on more plants (Boyette 1994). For example, a fungus called Colletotrichum truncatum can kill a weed called hemp sesbania, but it needs a lot of moisture to work (Boyette et al. 1993). Adding unrefined corn oil to the biocontrol agent can help it work better, so less moisture is needed. This makes it easier to use the bioherbicide and reduces the amount of spray needed (Boyette 1994).

Surfactants are ingredients used in formulations to help wet plants by reducing surface tension and improving the dispersal of fungal spores in spray droplets. Some surfactants used include Tween 20 with Fusarlum lateritum, nonoxynol with Alternaria macrospora and A. assia, and sorbitol with Colletotrichum coccodes (Boyette 1994). However, evaluating the inhibitory or stimulatory effects on spore-germination and infection is important before selecting appropriate surfactants and ensuring that the surfactants do not contain pesticide derivatives. Water-in-oil invert emulsions have been used with foliar fungal biopesticides to provide a favorable environment for germination and infection (Connick et al. 1990; Daigle et al. 1990; Auld and Morin 1993; Boyette et al. 1996). Using inverted emulsions (Boyette et al. 1993) has been found significantly to improve the efficacy of C. truncatum and Alternaria assia. However, inverted emulsions are very viscous and may demonstrate phytotoxicity in some target plants (Walker and Boyette 1985; Amsellem et al. 1990; Goettel et al. 1995). Connick et al. (1990) developed an inverted emulsion with improved water-retention properties that was less viscous. Additionally, vegetable oils can enhance the efficacy of mycoherbicides, such as Colletotrichum orbiculare, for the control of spiny cocklebur (Auld 1992) and Collototrichum acutatum for the control of Hakea sericea and H. gibbose. No phytotoxicity and improvements in the spread of the inverted emulsion were observed.

Solid-based formulations of mycoherbicides have been developed for weeds that are infected at or below the soil surface, which is more suitable for preemergence mycoherbicides (Daigle and Connick 1990; Boyette et al. 1996). These formulations can provide a food base for the pathogen, act as a buffer in extreme environmental conditions, and retain inoculum that may not be easily washed away. One such formulation, known as “PESTA,” uses a wheat-gluten matrix consisting of liquid inoculum, semolina wheat flour, and kaolin. This formulation includes fungal agents like C. truncatum, A. crassa, and Fusarium lateritium and can be applied as preemergent and soil-incorporated treatments (Boyette et al. 1984). The shelf life of the product can be improved by manipulating the water activity and sucrose content. Other solid substrates used to formulate mycoherbicides include bran, wheat kernels, cornmeal/sand, vermiculite (Auld and Morin 1993; Boyette et al. 1996), and sawdust (Muir 2023). For example, cornmeal/cornmeal sand was used to formulate mycelium, micro- and macroconidia, and chlamydospores of Fusarium solani f.sp. cucurbitae for the control of Texas gourd. This pre-emergent granular formulation provided 96% control of the weed (Boyette et al. 1984).

7.2.4 Formulation of viruses

Baculoviruses have been studied as a potential solution for controlling insect pests that belong to the Lepidoptera, Hymenoptera, and Coleoptera families (Gory and Bishop 1995). These viruses have several advantages, such as high specificity, not harming beneficial insects, and persisting in the environment, which makes long-term control of insect pests possible without damaging the environment. Nuclear polyhedrosis viruses (NPVs) and granulosis viruses (GVs) are examples of baculoviruses. However, there are some limitations to using these biopesticides, such as their slow speed of biological activity, low stability under UV light, and difficulties in production (Powell and Jutsum 1993).

The stability of the baculoviruses is often a function of viability, but it is not a significant problem for small-scale field trials as the viruses can be collected from macerated larvae, mixed with water, and stored for short periods through refrigeration. However, these systems are not suitable for large-scale production and application (Gory and Bishop 1995). The formulation of these viruses is an important aspect of product development, but researchers have not given it as much attention as bacteria and fungi.

Baculoviruses are usually available in the form of concentrated wettable powders. The biocontrol product called “Elcar” for corn earworm (Helicoverpa zea) NPV can be spray-dried or air-dried after being diluted with an inert carrier. The gypsy moth (Lymantria dispar L.) NPV is freeze-dried either with carbohydrates or by acetone precipitation (Gory and Bishop 1995). The virus can be inactivated by UV radiation, especially wavelengths of 290-320 nm. UV protectants like reflections or absorbers can be added to formulations to stabilize baculoviruses. Several dyes, such as lissamine green, acridine yellow, alkali blue, and mercurochrome, have been used as UV protectants, especially to absorb UV-A irradiation (Shapiro 1995). Optical brighteners (fluorescent brighteners), which are commonly used in soaps, detergents, and fabric softeners, also absorb UV light. They have been shown to significantly reduce the photodegradation of NPVs and enhance viral activity (Shapiro 1992; Dougherty et al. 1995; Shapiro 1995). Care must be taken to ensure that these optical brighteners do not adversely affect the environment due to residues and residuality.

There are two types of biopesticide formulations: liquid and dry, which differ based on their physical state ( Figure 7). Dry formulations (dust powders, granules, seed dressers, wettable powders, and wettable) dispersible powder. They are produced using various technologies, including spray drying, freeze drying, or air drying, with or without a fluidized bed (Li 2002). They typically require binders, dispersants, and wetting agents for optimal efficacy and application. Liquid formulations can be water-based, oil-based, polymer-based, or combinations. Water-based formulations, like suspension concentrates, suspo-emulsions, capsule suspensions, and ultra-low volume liquids, require additional inert ingredients for stability and performance (Boyetchko et al. 1999). These include stabilizers, stickers, surfactants, coloring agents, antifreeze compounds, and additional nutrients. Care must be taken to ensure that these inert ingredients do not adversely affect the environment due to residues and residuality and do not contain pesticide derivatives.

Figure 7. General workflow of the procedures involved in the development of microbial biocontrol formulation.

Straight line arrows indicate process (steps), and broken lines indicate categories or classifications.

8.1 Seed treatments and soil applications

Seed treatments and soil applications involve various methods for applying biopesticides to protect seeds and enhance crop establishment. Seed coatings and treatments apply biopesticides onto the seed surface, providing early protection and improving crop establishment (Khan et al. 2020). Incorporating biopesticides into soil amendments can improve soil health, control soil-borne pests, and enhance plant growth, offering sustainable pest management and environmental benefits. Other methods include soil drenching, which involves applying biopesticides as a liquid solution directly to the soil around the plant base. This technique targets pests in the soil, such as nematodes and soil-borne pathogens. Biopesticide-based soil fumigation utilizes the controlled release of compounds to control pests and diseases in the soil, providing an alternative to chemical fumigation but with reduced environmental impacts. Rhizosphere application focuses on applying biopesticides directly to the root zone of plants. This technique targets pests that interact with plant roots, such as soil-dwelling insects and nematodes. These seed treatment and soil application methods offer effective pest management while minimizing environmental impacts and fate.

8.2 Factors affecting the efficacy of biopesticides

Various challenges and limitations, including environmental conditions such as temperature, humidity, and sunlight, can influence by the efficacy of biopesticides during pest control. Some biopesticides may require specific temperatures or moisture levels for optimal performance. Additionally, biopesticides often exhibit narrow target specificity that is effective against specific pests but not others. This limitation reduces their broad-spectrum efficacy, and it may be necessary to use multiple biopesticides to target different pests (Gurr et al. 2017). Furthermore, the formulation and storage conditions of biopesticides can affect their stability and shelf life. Factors such as pH, temperature, and exposure to light can degrade the effectiveness of biopesticides over time (Riga et al. 2017).

It’s important to note that these challenges and limitations can vary depending on the specific biopesticide and pest control scenario. By considering and addressing these factors, it is possible to optimize the efficacy of biopesticides in pest control practices. The efficacy of biopesticides can be influenced by the application methods used. Factors such as spray volume, droplet size, and coverage play a crucial role in the distribution and effectiveness of the biopesticide on the target pests (Chandler et al. 2011). Ensuring that the application method is properly calibrated is important to achieve adequate coverage and contact with the pests (International Symposium on Food Safety and Control).

Biopesticides may have a shorter persistence compared to chemical pesticides, which may require more frequent applications. Their residual activity on plants or the environment can be limited, necessitating careful timing and repeated treatments. Proper timing of application is essential to target pests during vulnerable stages of their life cycle thus, it is critical to fully understand the pest’s lifecycle before implementing a biopesticide programme. In addition to the challenges mentioned earlier, several other factors can limit the efficiency of biopesticides. These include regulatory approval, which can be a complex and time-consuming process. Limited availability and accessibility of biopesticides can pose challenges for farmers (Biondi et al. 2021). And their compatibility with integrated pest management can be a complex issue. Lack of standardization and quality control protocols may affect the consistent performance of biopesticides. Furthermore, biopesticides can be more expensive compared to chemical pesticides, affecting their cost-effectiveness. The efficacy of biopesticides can vary depending on factors such as pest population density, pest development stage, and resistance mechanisms. Pests can develop resistance to specific biopesticides over time, which can reduce their effectiveness. Therefore, it is important to monitor pest populations and implement IPM strategies that incorporate the use of different biopesticides and other pest control measures to mitigate the development of resistance.