Bacterial insecticides beyond Bacillus thuringiensis

Abstract

Bacterial insecticides have emerged as a biotechnological strategy for controlling insect pests in crops, primarily due to the ineffectiveness of synthetic pesticides, which have led to resistance in insect populations due to their overuse. For many years, Bacillus thuringiensis has been recognized as the most promising microorganism for this purpose, while other entomopathogenic bacteria, which have demonstrated efficacy against various insect orders, have been largely overlooked. These entomopathogenic bacteria employ diverse mechanisms to control insect pests, including damage via contact or ingestion, disruption of intestinal cell integrity, interference with the insect's central nervous system, and alterations to reproductive processes. Among the insecticidal compounds produced by these bacteria are Cry homologous toxins, non-homologous toxins, biosurfactant-type compounds, macrocyclic lactones, lipopeptides, chitinases, and other metabolites that remain underexplored. This paper provides a comprehensive overview of the current state of lesser-known entomopathogenic bacteria utilized in the development of these biopesticides. It discusses the advantages and disadvantages of these biotechnological products, the mechanisms of action of entomopathogenic bacteria, genetic engineering strategies aimed at enhancing these biopesticides, and the application of these bacteria in commercial production. Additionally, the paper reviews patents related to this field and examines the role of insecticidal bacteria as inducers of systemic resistance (ISR) in plants, highlighting their potential for the development of commercial bioproducts.

Introduction

The intensification of agriculture driven by population growth is becoming increasingly apparent, with projections indicating that agricultural production must increase by 70% by 2050 to meet rising food demands (Sunjka et al. 2022). However, this intensification is accompanied by significant economic losses worldwide, primarily due to plant pathogens that have developed resistance to traditional pesticides, which can result in losses of up to 42% in major crops (Glare et al. 2012; Aneja et al. 2016; Samada et al. 2020).

Currently, several alternatives exist for managing plant pathogens, including the development of resistant crop varieties and the use of synthetic agrochemicals. However, the latter approach has notable limitations. Environmental problems stemming from excessive chemical use in agroecosystems have become evident, leading to issues such as the emergence of pest resistance, the elimination of beneficial microorganisms and non-target organisms, the rapid proliferation of secondary pests, and the accumulation of pesticide residues in food, which poses potential health risks, including cancer and fetal damage (Gupta and Dikshit 2010; Singh et al. 2014; Mishra et al. 2015; Hakim et al. 2020; Kumar et al. 2021; Perez et al. 2011; Hernandez et al. 2022).

In light of these challenges, biopesticides have been developed as biotechnological products that offer an ecological and sustainable alternative to chemical pesticides in agroecosystems (Glare et al. 2012; Prabha et al. 2017; Hakim et al. 2020). Research indicates that biopesticides can outperform synthetic pesticides when applied at optimal times and concentrations (Shah et al. 2013). Furthermore, they effectively reduce product losses without compromising quality and are considered environmentally friendly due to their biodegradable nature (Leng et al. 2011; Kumar et al. 2021).

The United States Environmental Protection Agency (EPA) categorizes biopesticides into three groups based on their active ingredients: plant-incorporated protective agents (PIPs), which are produced through transgenic methods that induce the synthesis of natural insecticidal compounds in plant tissues; biochemical biopesticides, which are derived from natural compounds produced by insects and plants; and microbial pesticides, which are based on bacteria, fungi, viruses, or microalgae (Gupta et al. 2010; Seiber et al. 2014; Nathan et al. 2014; Mishra et al. 2015; Aneja et al. 2016; Kachhawa 2017; Lengai et al. 2018; Hakim et al. 2020) (Fig. 1).

Fig. 1

Types of biopesticide according to the United States Environmental Protection Agency (EPA)

Bacterial insecticides are natural formulations derived from live bacteria or their metabolites, specifically designed to control insect pests through highly specific mechanisms while exhibiting low toxicity to non-target organisms, including beneficial insects (Thakore 2006; Mazid et al. 2011; Nathan et al. 2014; Kumar and Singh 2015; Hakim et al. 2020).

Historically, most commercial products have been developed from the bacterium Bacillus thuringiensis (Bt). In fact, products based on the genus Bacillus represent the most significant class of commercially available crop protection products (Jacobsen et al. 2004; Samada et al. 2020). However, other notable bacterial genera have not received adequate recognition. This review will focus on the exploration of alternative entomopathogenic bacteria as a foundation for formulating biopesticides. It will discuss insecticidal molecules and their modes of action, highlight some commercial products, outline genetic engineering strategies employed in their production, and present patents awarded or in progress related to these products and their role as inducers of systemic resistance (ISR) in plants as part of the state of the art.

Bacterial insecticides: advantages and disadvantages

Bacterial insecticides were first developed in the 1950s and, due to their ease of mass production, became the first microbial pesticides to be commercialized. For over 40 years, these products have been in use and currently account for 90% of biopesticides marketed globally (Nathan et al. 2014; Osman et al. 2015; Tabashnik and Carrière 2017; Villareal et al. 2017; Kumar et al. 2021). While these insecticides offer advantages such as high effectiveness and specificity, they also face challenges, including low stability, high production costs, and inconsistent results in field trials (Cooping and Menn 2000; Gullino et al. 2005; Hynes and Boyetchko 2006; Gupta et al. 2010; Xu et al. 2011; Chandler et al. 2011; Leng et al. 2011; Glare et al. 2012; Gasic and Tanovic 2013; Bhattacharjee and Dey 2014; Siever et al. 2014; Ritika and Utpal 2014; Mishra et al. 2015; Mnif et al. 2015; Kumar and Singh 2015; Tijjani et al. 2016; Aneja et al. 2016; Shiberu et al. 2016; Kachhawa 2017; Lengai et al. 2018; Samada and Tambunan 2020; Hakim et al. 2020; Kumar et al. 2021) (Fig. 2).

Fig. 2

Advantages and disadvantages of using bacterial insecticides

Entomopathogenic bacteria as insecticides

By the 1980s, approximately 100 bacteria had been identified as entomopathogenic, yet only four Bacillus thuringiensis, Bacillus popilliae, Bacillus lentimorbus, and Bacillus sphaericus had been extensively studied as agents for insect control (Miller et al. 1983). Among these, Bacillus thuringiensis (Bt) has received the most attention, leading to the development of commercial products. Bt is a Gram-positive, spore-forming bacterium that is non-pathogenic to humans and typically found in soil and the guts of various insects, particularly Lepidoptera. Globally, there are around 175 registered biopesticides with approximately 700 active compounds available for use (Hakim et al. 2020). In the United States, over 150 commercial products are available, while the European Union market features only 60 analogous products (Kumar 2021).

Bacteria used as pesticides can be either Gram-positive or Gram-negative. Gram-positive bacteria can be further classified into endospore-forming and non-endospore-forming types, with endospore-forming bacteria being the most widely studied for commercial applications (Kumar et al. 2021) (Fig. 3).

Fig. 3

Classification of entomopathogenic bacteria used as biopesticides

Currently, a wide diversity of bacteria is recognized for their potential use in the production of bioinsecticides. Most of these bacteria belong to the families Bacillaceae, Pseudomonadaceae, Enterobacteriaceae, Lactobacillaceae, Micrococcaceae, and Streptococcaceae (Ruiu 2015; Kachhawa 2017; Azizoglu et al. 2020). Notable species within the Bacillaceae family include Bacillus subtilis, Bacillus pumilus, Bacillus amyloliquefaciens, Bacillus megaterium, and Bacillus licheniformis, as well as Paenibacillus popilliae and Brevibacillus laterosporus (Mnif et al. 2015; Villareal et al. 2017). Additionally, species from the Pseudomonadaceae family, such as Pseudomonas entomophila and Pseudomonas aeruginosa, along with those from the Yersiniaceae family like Yersinia entomophaga, are also noteworthy (Raaijmakers and Mazzola 2012; Prabha et al. 2017; Villareal et al. 2017; Kumar et al. 2021). Furthermore, insecticidal bacteria of the genera Xenorhabdus and Photorhabdus form a symbiotic relationship with entomopathogenic nematodes from the genera Steinernema and Heterorhabditis. During infection, the nematode acts as a vector, transporting the bacteria to the larva of the target insect, where the bacteria are disseminated into the hemolymph, leading to septicemia (Chattopadhyay et al. 2004) (Table 1).

Table 1 Entomopathogenic bacteria used for the production of bacterial insecticides

Mechanisms of action of entomopathogenic bacteria

Entomopathogenic bacteria employ various mechanisms to infect, colonize, and ultimately eliminate their insect hosts. The primary strategies include inducing contact damage, producing toxins that disrupt the insect's digestive system, generating molecules that target the insect's nervous system, and interfering with reproduction and development. These mechanisms can operate independently or in synergy, enhancing their overall effectiveness (Jurat-Fuentes and Jackson 2012).

Production of contact damage

Chitinases are enzymes that degrade chitin, a structural polysaccharide found in the exoskeletons of insects and crustaceans. Chitin plays a crucial role in preventing water loss and forming a protective barrier against pathogens and environmental stressors (Veliz et al. 2017). The degradation of chitin can weaken the exoskeleton, rendering the insect unable to support its own weight and increasing its susceptibility to microbial infections. Additionally, the presence of these enzymes can disrupt the molting process, leading to improper formation of the new exoskeleton, which may result in deformities, inability to shed the old exoskeleton, or death during molting (Winssy et al. 2024). Bacteria that produce such enzymes include Serratia spp. and certain actinomycete species such as Streptomyces spp. (Kwak et al. 2015).

Production of gut-level damage

Many entomopathogenic bacteria produce lethal toxins, which can be proteins, peptides, or secondary metabolites that interfere with vital physiological processes in insects. Approximately 90% of bacterial insecticides are derived from endotoxin-producing bacteria that enter the insect when larvae consume plant leaves (Mishra et al. 2015; Kachhawa 2017). Once ingested, these bacteria are lysed by the alkaline environment of the gut, releasing toxins as inclusions or inactive protoxins. These protoxins are then solubilized and activated by proteases, such as trypsin (Drobniewski et al. 1989; Ruiu et al. 2018). During activation, peptides from the N-terminal and C-terminal regions are cleaved by proteases, generating the active toxin, which binds to receptors on the membranes of intestinal epithelial cells. This binding triggers a conformational change in the toxin, forming crystalline inclusions that create pores or ion channels within the receptors, leading to cell lysis and disruption of transport processes. Consequently, the insect becomes paralyzed, ceases feeding, and ultimately dies from starvation within 48 h (Jisha et al. 2013) (Fig. 4).

Fig. 4

Toxin production and damage to the insect gut

Some variants of insecticidal toxins exhibit species-specific activity (Nathan et al. 2014; Aneja et al. 2016; Kachhawa 2017; Jurat et al. 2012). Variations in the gene sequences of these toxins can lead to different affinities for gut receptors, resulting in distinct insecticidal activities among various bacteria (Pigott et al. 2007). The molecules studied from the aforementioned bacteria display significant chemical diversity, with some not yet fully characterized. This challenges the notion that only proteins homologous to the Cry proteins of Bacillus thuringiensis (Bt) possess insecticidal properties. While most mechanisms of action for these entomopathogenic bacteria involve ingestion or contact, the specifics remain unclear for other species, particularly regarding their biological targets, which include insects from the orders Lepidoptera and Coleoptera. Utilizing sporulated bacteria in product development enhances viability and stability over time (Villareal et al. 2017).

Examples of diseases caused by Cry-like proteins include Cbm 17.1 and Cbm 17.2, produced by Paraclostridium biofermentans, which are highly effective against flies and mosquitoes (Qureshi et al. 2014). Additionally, Paenibacillus popilliae is responsible for milky spore disease in beetles, characterized by an opaque white coloration due to the accumulation of sporulating bacteria in the hemolymph. This disease can be fatal if the infective dose of spores is sufficiently high during the early stages of larval development (Grady et al. 2016).

Another spore-producing bacterium, Lysinibacillus sphaericus, is a facultatively aerobic, terminally spore-forming, and motile bacterium found in soil and aquatic environments. It utilizes amino acids and organic acids as carbon sources, as it lacks genes for sugar-degrading enzymes. This bacterium can accumulate hydrocarbons and immobilize heavy metals due to its paracrystalline S-layer. Strains of Lysinibacillus sphaericus have shown high larvicidal efficacy, producing a toxin similar to that of Bt, which typically damages the epithelial microvilli in the insect midgut. This leads to the cessation of feeding and ultimately the insect's death (Kumar et al. 2021).

Involvement of the insect nervous system and behavior

Certain bacteria can impact the nervous systems of insects as direct pathogens, behavior-modifying symbionts, or as causative agents of specific neurological diseases (Table 1). For instance, Streptomyces rubrisoli and Saccharopolyspora spinosa produce macrocyclic lactones that affect insect nervous systems, leading to paralysis and eventual death. Examples of these molecules include the avermectins—such as emamectin, avermectin, ivermectin, and abamectin and milbemycins, including milbemectin. Both classes of lactones have been utilized as antiparasitics in animals and for insect control in various crops (Pérez-Cogollo et al. 2018).

These compounds reach nerve endings and trigger the release of gamma-aminobutyric acid (GABA), causing GABA-activated Cl⁻ ion channels to open. This hyperpolarizes the membrane potential of nerve cells, blocking signal transmission to the insect's peripheral nervous system and inhibiting neurotransmitter release, ultimately resulting in paralysis (Martin et al. 2002; Khan and Khan 2023). Unlike other avermectins, spinosins activate nicotinic receptors on the postsynaptic membrane through an unknown mechanism, leading to nervous system excitation, prostration, and paralysis. Some avermectins may also affect other physiological systems in target organisms. For example, ivermectin can impair muscle function and excretion in parasites, facilitating the clearance of infestations (Gonzalez et al. 2009; Lumaret et al. 2012). Marques et al. (2020) evaluated a spinosad-based formulation called Tracer® on the stingless bee foragers, Plebeia lucii Moure (Apidae: Meliponini), and found that bee locomotion, both walking and flying, was reduced with increasing spinosad concentrations.

Interference with insect reproduction and development

Wolbachia pipientis is a Gram-negative bacterium belonging to the order Rickettsiales that affects the reproductive systems of its hosts. This endosymbiotic bacterium predominantly resides in the gonads of various insects, with some estimates suggesting that approximately 16% of arthropods harbor Wolbachia spp. It can also infect spiders and mites (Kaur et al. 2021). Wolbachia is known to induce several reproductive processes, including feminization, parthenogenesis, male killing, and cytoplasmic incompatibility (Shropshire et al. 2020).

Feminization leads to genetic males developing phenotypically as females, though the molecular mechanisms underlying this process remain unknown. In cases of parthenogenesis, uninfected species typically exhibit arrhenotokous parthenogenesis due to their haplodiploid sex determination system. However, virgin mothers infected with Wolbachia produce all-female offspring from their unfertilized eggs, switching from arrhenotokous to telitokous parthenogenesis. Male killing results in a female-biased sex ratio in several arthropods by selectively eliminating males (Fukui et al. 2015). Additionally, this bacterium has potential applications in controlling nematodes and parasites, such as Plasmodium spp., the causative agents of malaria (Bourtzis et al. 2014).

Burkholderia cepacia is an aerobic, motile, non-fermenting, Gram-negative bacterium known for its significant metabolic versatility. It is important for colonizing soil, water, and plants and serves as a symbiont in legume root nodules (Rojas-Rojas et al. 2019). This bacterium has successfully colonized the guts of insects, leading to reduced egg deposition. In contrast, Spiroplasma spp., belonging to the group Mollicutes, are helicoid-shaped bacteria that lack a cell wall and are obligate symbionts in certain insects. They feed on trehalose, the predominant sugar in insect hemolymph, causing symptoms such as tremors, paralysis, and behavioral changes. In Drosophila melanogaster, infection with Spiroplasma melliferum has been associated with increased mortality of male progeny during embryonic or larval stages and a female-biased sex ratio in subsequent generations, although the underlying mechanism remains unclear (Anbutsu and Fukatsu 2011).

Genetic engineering strategies for the improvement of genes encoding insecticidal molecules

Advancements in molecular biology, genetic engineering, and protein engineering have facilitated the development of bacterial insecticides (Azizoglu et al. 2020). Initial genetic modifications of biopesticides employed recombinant DNA technology and protein engineering in Bacillus thuringiensis, particularly in the subspecies Kurstaki, to create strains with novel insecticidal proteins effective against codling moths in maize (All et al. 1994; Federici et al. 2005). As of 2012, there were no commercially available biopesticides based on genetically modified microorganisms due to regulatory constraints (Glare 2012). However, genes encoding insecticidal toxins have now been isolated, characterized, manipulated, and expressed in various organisms to create new combinations of toxins with broader activity (Azizoglu et al. 2020). Recombinant DNA technologies have been employed to enhance insecticidal efficacy by increasing protein synthesis and facilitating the creation of new endotoxin combinations (Karabörklü et al. 2018). Furthermore, genetic engineering can enhance the resilience of entomopathogens to adverse environmental conditions, potentially increasing their effectiveness in the field (Azizoglu et al. 2020).

These approaches have primarily targeted entomopathogenic bacteria from the genera Bacillus, Lysinibacillus, and Pseudomonas (Azizoglu et al. 2020). A notable study reported the transformation of the cry1Ab gene from Bacillus thuringiensis strain LM-466 into B. subtilis and B. licheniformis strains, evaluating their insecticidal activity against Tuta absoluta larvae, with an LC50 comparable to that of the original B. thuringiensis strain (Theoduloz et al. 2003). Other cry and cyt genes from B. thuringiensis have been transferred to Lysinibacillus sphaericus strains, resulting in tenfold increased toxicity against Culex spp. (Federici et al. 2003).

Research has also focused on Serratia entomophila, where a plasmid carrying the sepABC genes, responsible for inducing feeding cessation and mortality in Costelytra zealandica larvae, was inserted into Escherichia coli. The recombinant strains displayed the desired insecticidal traits (Hurst et al. 2004). An alternative approach involves the transconjugation of endotoxin-encoding genes in plasmids or cloning Bacillus thuringiensis genes for expression in alternative hosts such as Pseudomonas fluorescens, a common plant-associated bacterium, or other endophytic microorganisms (Federici et al. 2005).

Liu et al. (2010) identified the toxin production gene (tccC) in Pseudomonas taiwanensis, which was overexpressed in E. coli, resulting in increased mortality in Drosophila melanogaster. In another study, the chiABC gene from Serratia marcescens was inserted and overexpressed in E. coli, yielding recombinant chitinases that exhibited up to 80% insecticidal activity against Malacosoma neustria and Helicoverpa armigera (Danışmazoğlu et al. 2015). Finally, Zhang et al. (2020) conducted genome reduction of Bacillus amyloliquefaciens LL3 through metabolic engineering to enhance surfactin production, a lipopeptide biosurfactant with potential insecticidal properties. The original strain’s sfrA operon, crucial for surfactin production, was optimized by removing unnecessary genomic regions, promoting rapid growth and high operon expression (Zhang et al. 2020).

Another strategy for plant protection against insects involves transferring genes encoding insecticidal proteins to crops. This approach minimizes the need for continuous insecticide application, as it facilitates the constant production of Bt proteins (Federici et al. 2005). However, studies indicate that only genes derived from Bacillus thuringiensis have been effectively transferred to various plant species such as cotton, tomato, and eggplant, yielding economic benefits for growers (Leng et al. 2011; Nathan et al. 2014; Siever et al. 2014; Mishra et al. 2015). In contrast, research involving other entomopathogenic bacterial species remains in its early stages.

While recombinant DNA technology offers the potential to modify insecticide modes of action by altering host specificity or enhancing efficacy, regulatory restrictions on the use of genetically modified organisms (GMOs) have made research in this area less appealing for commercial product development. Concerns about the potential for gene flow to wild species, development of insect resistance, and negative impacts on beneficial organisms further complicate the landscape (Azizoglu et al. 2020).

Bacterial insecticides as commercial products

The European Union currently leads the global market for commercial biological control products, offering incentives for the registration of low-risk biological control agents (Glare 2012). In contrast, bacterial insecticides in the United States are regulated by the Division of Biopesticides and Pollution Prevention within the Environmental Protection Agency (EPA) (Leahy et al. 2014). Despite existing regulations, there is a pressing need for enhancements to current legislation (Kumar et al. 2021) to streamline the registration process for new products, especially in developing countries. The lengthy and costly nature of current registration procedures poses significant challenges (Kumar et al. 2016a, b).

While biopesticides represent only 5% of plant protection products worldwide, their growth has accelerated in recent decades. Hakim et al. (2020) report that there are currently 175 biopesticides registered globally, encompassing 700 active substances. The biopesticide market was valued at approximately $3 billion in 2013 and is projected to reach $4.5 billion by 2023 (Lahlali et al. 2022; Hernández et al. 2022). Notably, products derived from Bacillus, Burkholderia, Pseudomonas chlororaphis, and Serratia entomophila are among the most effective against various orders of insects (Table 2).

Table 2 Commercial products made from different entomopathogenic bacteria

The development of a commercial product is a complex, multi-step process that begins with the selection of promising microorganisms. These are initially evaluated through in vitro tests against various pathogens using techniques such as disc diffusion, agar well diffusion, and the poisoned food technique before undergoing larger-scale assessments (Jahangiriana et al. 2013). It has become evident that many microorganisms deemed promising at the in vitro level often fail to perform effectively in greenhouse formulations, rendering them unfeasible as commercial products (Hynes and Boyetchko 2006).

The active component may consist of metabolites produced by a microorganism. In such cases, various analytical techniques, including thin-layer chromatography (TLC), high-performance liquid chromatography (HPLC), and gas chromatography coupled with mass spectrometry (GC–MS), are employed for characterization (Lengai et al. 2018). Following this, the fermentation stage is critical, requiring careful consideration of the type of fermentation (solid or submerged), culture medium composition, oxygen transfer, incubation temperature, collection timing, and additional treatments (Montazeri and Greaves 2002).

Next, the formulation of the active compound begins. At this stage, it is essential to optimize the combination of active ingredients, carrier materials, emulsifiers, surfactants, and other components to enhance stability and efficacy while minimizing degradation from environmental factors during field application (Lengai et al. 2018; Hynes and Boyetchko 2006). The initial step involves selecting an appropriate carrier that facilitates the controlled release of the active ingredient. Inert materials, such as petroleum distillates, starch, talc, clay, and water, serve as stabilizers to prolong the shelf life of the product. Additionally, emulsifying agents or surfactants, such as soap, may be incorporated to enhance effectiveness (Lengai et al. 2018). Adjuvants that conserve moisture and protect the active ingredient from UV radiation or desiccation must also be selected based on microbial physiology studies (Burges 1998; Boyetchko et al. 2002; Hynes and Boyetchko 2006).

Another important consideration during formulation is enhancing the controlled release of the active compound by testing various product presentations. For example, slow-release granules or spray formulations for foliar applications could improve coverage with the active agent (Glare 2012). Despite progress in these areas, unexpected challenges in the fermentation and formulation processes, as well as inadequate market management, have led to unfinished products or the withdrawal of products from the market (Hynes and Boyetchko 2006).

While advancements have been made in developing commercial products based on various entomopathogenic bacteria, Bacillus thuringiensis (Bt)-based products maintain a competitive edge. This advantage stems from years of research on Bt, which has elucidated its biology, mechanisms of action, and spectrum of efficacy. Such foundational knowledge has informed improvements in product manufacturing, including the selection of suitable carriers for controlled release, effective application methods, strategies for extending product shelf life, and safety assurances. These factors contribute to lower production costs and increased profitability for manufacturers. However, despite these advances, limitations remain, including rapid deactivation upon light exposure, short duration of activity, low lethality, and high sensitivity to environmental conditions (Ayilara et al. 2023). Continued research is essential to overcome these challenges and establish competitive alternatives for managing insect pests.

Patents related to bacterial insecticides

To date, patents related to the production of bacterial insecticides based on entomopathogenic bacteria other than Bacillus thuringiensis (Bt) are limited. This scarcity may be attributed to the insufficient large-scale production studies of these alternative bacterial inputs and the challenges associated with optimizing their formulations. The existing patents primarily focus on the production of insecticidal metabolites derived from certain species of phytopathogenic bacteria or improvements in the synthesis of these compounds through genetic engineering processes. The countries most active in this field include the United States and the European Union (Table 3).

Table 3 Patents related to bacterial insecticides based on other entomopathogenic bacteria

Entomopathogenic bacteria that can induce systemic resistance (ISR) in plants

Induction of systemic resistance (ISR) is a physiological state that enhances a plant's defense capacity, elicited by plant growth-promoting rhizobacteria (PGPR). These bacteria can induce local resistance and subsequently transfer it to other parts of the plant, achieving global induction (Ilham et al. 2019). In response to pathogen or insect attacks, as well as abiotic stresses, plants can rapidly activate various cellular defense mechanisms, including increased activity of chitinases, β-1,3-glucanases, and peroxidases, along with the accumulation of phytoalexins and the formation of protective layers composed of lignin and hydroxyproline-rich glycoproteins (Archana et al. 2011; Jatoi et al. 2019). Among the genera recognized as inducers of systemic resistance are some insecticidal bacteria previously discussed in this review, underscoring their potential relevance as bioproducts.

Wei et al. (1996) conducted greenhouse studies that demonstrated the ability of the PGPR Bacillus pumilus INR7 to promote plant growth and induce systemic resistance against cucumber diseases. When applied as a seed treatment, this strain significantly increased protection against anthracnose caused by Colletotrichum orbiculare. Furthermore, a combined inoculation of Bacillus pumilus INR7, Curtobacterium flaccumfaciens MEl, and Bacillus subtilis GB0 effectively controlled cucumber angular leaf spot caused by Pseudomonas syringae pv. lachrymans (Raupach et al. 2000).

Li et al. (2020) evaluated rhizospheric isolates from tobacco and found that B. pumilus strain S2-3–3 significantly reduced disease severity in tobacco by inducing systemic resistance while also promoting growth through the production of indoleacetic acid (IAA). In bell pepper plants, disease severity decreased when roots were drenched with strain S2-3–2, leading to enhanced plant weight and chlorophyll content compared to untreated controls. Ilham et al. (2019) highlighted selected strains of Bacillus amyloliquefaciens (I3) and Trichoderma harzianum in inducing systemic resistance in Arabidopsis thaliana, revealing that these microorganisms can trigger defense pathways associated with salicylic acid and jasmonic acid, unlike plants treated with chemical elicitors.

Abdelkhalek et al. (2020) reported the antiviral activity of Bacillus licheniformis strain POT1 against alfalfa mosaic virus (AMV) in potato plants. Dual foliar applications of crop filtrate 24 h before and after inoculation with AMV proved most effective, resulting in an 86.79% reduction in viral accumulation and improvements in various growth parameters. Transcriptional analysis indicated that thirteen genes related to phenylpropanoid, chlorogenic acid, and flavonoid biosynthetic pathways were induced after treatments, with anthocyanin, a type of flavonoid, playing a crucial role in plant defense against viral infection.

Bharathi et al. (2004) found that Bacillus subtilis provided protection against rot and progressive death of chili bell pepper (Capsicum annuum) fruits caused by Colletotrichum capsici, and PGPR significantly enhanced seed germination and seedling vigor. Saravanakumar et al. (2007) evaluated bioformulations of PGPR (Pseudomonas sp. and Bacillus subtilis) for their efficacy against vesicular blight disease (Exobasidium vexans) in tea (Camellia sinensis) under field conditions, finding that foliar applications consistently reduced the incidence of blight disease.

Wang et al. (2015) identified and characterized a protein elicitor secreted by Brevibacillus laterosporus strain A60, named PeBL1. When expressed in Escherichia coli, this recombinant protein induced a hypersensitive response (HR) and systemic resistance in Nicotiana benthamiana, triggering a cascade of plant defense responses, including reactive oxygen species (ROS) production and phenolic compound deposition. In a related study, Jatoi et al. (2019) reported that the protein inducer PeBL2 (17.2 kDa) encoded by the PeBL2 gene could similarly induce HR in tobacco, generating ROS and systemic resistance against Botrytis cinerea.

Shabanamol et al. (2017) explored the biocontrol mechanisms of Lysinibacillus sphaericus, a diazotrophic endophyte of rice, against the rice sheath blight pathogen Rhizoctonia solani. This endophyte induced systemic resistance, achieving 100% growth inhibition of the fungal pathogen through the production of volatile organic compounds and siderophores, as well as hydrogen cyanide (HCN) and ammonia. Under greenhouse conditions, foliar and soil applications of L. sphaericus significantly reduced disease incidence by accumulating defense enzymes, including peroxidases and polyphenol oxidases, and enhancing phenolic compound levels. Kumar et al. (2016a, b) found that inoculation of tobacco plants with Paenibacillus lentimorbus B-30488 increased the accumulation of defense-related enzymes in response to cucumber mosaic virus infection.

Kim et al. (2008) reported that root colonization by Pseudomonas chlororaphis O6 in cucumber induced ISR against Corynespora cassiicola. Molecular studies identified the cucumber galactinol synthetase gene (CsGolS1), with transcription levels increasing upon treatment with strain O6, correlating with heightened resistance to Botrytis cinerea and Erwinia carotovora. Khalimi et al. (2011) evaluated formulations of P. aeruginosa to enhance growth and induce ISR in soybean plants against soybean stunting virus under greenhouse conditions. These formulations significantly increased plant growth, reducing disease incidence from 15 to 80%, while untreated plants exhibited up to 90% infection rates.

Finally, Ezziyyani et al. (2017) demonstrated that inoculating bell pepper stems with Phytophthora capsici and treating the roots with antagonists Burkholderia cepacia and Trichoderma harzianum together induced a defensive response. Stem infection resulted in a hypersensitive reaction, but necrosis was slowed in plants treated with both antagonists, evidenced by increased production of proteins with β-1,3-glucanase activity, a component of the hypersensitive defense system.

Conclusion

The production of commercial bacterial bioinsecticides is a complex process that requires extensive research and multiple stages of development. While a microorganism may demonstrate effectiveness in laboratory settings (in vitro), it may not perform as expected in field conditions. Challenges in scaling up production and formulating these microorganisms need to be addressed to ensure viable market management. Despite these hurdles, the production of bacterial bioinsecticides has significantly increased worldwide, particularly in developed countries. Current market offerings predominantly include mixtures of bacteria, such as various species within the genus Bacillus (e.g., B. chitinosporus, B. mycoides, B. pumilus, and B. subtilis). These bacteria produce toxins analogous to Cry proteins, which can cause intestinal damage and starvation in target insects. Additionally, certain actinomycetes, like Streptomyces species, cause contact damage, while bacteria such as Streptomyces rubrisoli and Saccharopolyspora spinosa produce macrocyclic lactones that affect the insect nervous system. Other notable microorganisms, including Wolbachia pipientis, Burkholderia cepacia, and Spiroplasma melliferum, can disrupt the reproductive systems of their hosts. With the advancement of genetic engineering, there has been manipulation of genes encoding insecticidal toxins across various organisms to create novel combinations that enhance efficacy, broaden the spectrum of action against additional insect species, and increase resistance to environmental factors. Moreover, efforts are underway to transfer genes encoding insecticidal proteins into plants, which could reduce the need for repeated insecticide applications in crops. In addition to their insecticidal properties, some of these bacteria can also enhance systemic resistance (ISR) in plants, thereby increasing interest in developing commercial bioproducts. However, legislative changes are necessary to streamline the product registration process, which is currently lengthy and costly in many countries. Such reforms are essential for bacterial bioinsecticides to compete with traditional chemical insecticides on price, achieve mass production, and contribute positively to soil health and environmental sustainability. Furthermore, additional research is required to provide scientific evidence that addresses regulatory concerns about genetically modified organisms, particularly regarding potential gene flow to wild species, insect resistance development, and ecological impacts. This approach will help mitigate resistance and apprehension among regulatory bodies, facilitating the responsible release of beneficial microbial products.