Bacillus Species in Agriculture: Functional Traits, Biocontrol Performance, and Regulatory Safety Assessment

Abstract

Bacillus species are among the most widely used microbial agents in agricultural biocontrol, reflecting their ecological resilience, functional diversity, and long history of practical application. The antagonistic activity of Bacillus spp. against plant pathogens and their plant growth–promoting effects are well established. However, these biological functions are frequently considered in isolation from safety evaluations and regulatory decision-making, resulting in a fragmented evidence base. This review addresses this gap by providing an integrated synthesis of agriculturally relevant Bacillus taxa, explicitly linking biocontrol performance with strain-level safety considerations and regulatory assessment. This review focuses on the principal groups currently applied in agriculture, including the Bacillus subtilis lineage, notably B. amyloliquefaciens, B. velezensis, B. pumilus, and B. licheniformis, as well as B. thuringiensis and Cytobacillus firmus. Key mechanisms underlying biocontrol efficacy are examined alongside evidence from greenhouse and field applications. These mechanisms include the production of secondary metabolites and volatile compounds, biofilm formation, rhizosphere colonisation, and the induction of plant defence responses. Attention is given to environmental and operational factors that influence the consistency of performance. A central contribution of this review is the integration of functional evidence with safety-relevant considerations, such as realistic metabolite exposure, antimicrobial resistance potential, and ecological effects. Regulatory approaches in the European Union, the United States, and selected Organisation for Economic Co-operation and Development countries are compared to illustrate how such evidence informs risk assessment and supports the sustainable use of Bacillus-based biocontrol agents in modern agriculture.

1. Introduction

Microbial technologies have become essential tools in modern agriculture, as rising climate and economic pressures challenge global food production systems [1,2,3]. The increasing frequency of droughts and heat waves, together with the deterioration of soil quality, is placing growing pressure on agricultural production systems. At the same time, the demand to minimise the use of synthetic pesticides and fertilisers [4,5,6] has driven increasing interest in biological alternatives across Europe, North and South America, Asia, and parts of Africa.

Bacillus species exhibit a combination of features, including ecological ubiquity and physiological resilience, that distinguishes them from other microbial groups [7,8,9]. The ability of Bacillus species to form long-lasting endospores enables them to withstand industrial processing, long-distance transportation and highly fluctuating field conditions. As a result, Bacillus-based inoculants are generally more robust and reliable under practical field use than non-spore-forming microbial products [7,10,11]. These properties have been the driving force behind the international commercial development of products based on strains of B. subtilis, B. amyloliquefaciens, B. velezensis, B. pumilus, B. licheniformis, B. nakamurai and C. firmus.

Bacillus species possess agricultural value as they perform various well-established biological functions. They suppress pests through the production of diverse secondary metabolites, including lipopeptides (surfactins, iturins and fengycins) [12,13,14], polyketides (difficidin and bacillaene) [12], and ribosomally synthesised peptides such as bacteriocins, including subtilin, subtilosin A and plantazolicin [15,16]. In addition, Bacillus species produce multiple volatile organic compounds, including 2,3-butanediol, acetoin and dimethyl disulfide, which further contribute to their biocontrol activity [17,18]. These biological compounds protect crops from nematode pathogens throughout different agricultural systems [19,20]. Bacillus species form biofilms and compete for space and resources on plant roots, while also modulating plant hormone signalling and enhancing nutrient availability. These activities promote plant growth and contribute to improved crop performance under stressful environmental conditions [12,21,22]. Specific bacterial variants trigger whole-plant resistance, which alters plant defence systems without an actual pathogen attack. Together, these characteristics have made Bacillus species key components of biological control systems for greenhouse crops in the European Union (EU). They are also widely applied in large-scale maize, rice and soybean production systems across the Americas and Asia [23,24]. Taken together, these functional traits underpin the agronomic relevance of Bacillus-based products. Their widespread and increasing use, however, also necessitates robust safety evaluation and appropriate regulatory oversight, which are addressed in the following section.

The rapid increase in Bacillus-based products on the market underscores the need for a comprehensive and transparent safety evaluation. Current taxonomic alterations of the B. subtilis operational group, and the development of genome-based systematics, have enhanced the species demarcation [25,26,27]. However, significant strain-level diversity remains, particularly regarding the biosynthetic gene clusters, metabolite repertoires, and AMR genes [28,29,30]. It is therefore necessary to separate agricultural strains from the B. cereus operational group since the latter includes strains that produce emetic and enterotoxins [25,31,32]. Regulators have responded to this by enhancing the standards for identity verification, metabolite characterisation, toxicological testing, and environmental risk assessment [33,34]. These are the principles upon which the regulations in the EU, the United States (US and some regions of the Organization for Economic Co-operation and Development (OECD) are based. However, the specific data requirements for strain identity, genomic characterisation, metabolite assessment, toxicological testing, and environmental fate differ across jurisdictions.

This review synthesises current scientific evidence on agriculturally relevant Bacillus species. It focuses on the functional traits that support their use in crop protection, including pathogen suppression, plant growth promotion and rhizosphere colonisation. These biological functions are examined in conjunction with contemporary approaches to safety assessment and regulatory oversight. The analysis integrates evidence on agronomic performance with data on potential risks. It addresses the production of bioactive secondary metabolites, the presence and mobility of antimicrobial resistance determinants, and interactions with non-target organisms and soil microbial communities. Attention is given to how such factors are interpreted within existing regulatory frameworks across major jurisdictions. By linking strain-level biological characteristics with regulatory practice, this review aims to support informed strain selection and product development. It further seeks to facilitate risk-based decision-making for the sustainable and climate-resilient use of Bacillus-based biocontrol agents.

2. Taxonomy, Genomic Features and Ecological Niches of Bacillus spp.

Agriculturally essential members of the Bacillaceae are dominated by two major evolutionary lineages, which possess unique ecological environments and biological characteristics. B. subtilis operational group comprises B. subtilis, B. atrophaeus, B. vallismortis, B. mojavensis and other species that are widely investigated and used in crop protection [35,36]. The members of this group inhabit soils, the phyllosphere, and the rhizosphere, where they establish themselves effectively and generate antimicrobial secondary metabolites [13,37,38]. The B. cereus operational group includes B. thuringiensis. This species of insect-associated life cycles strongly influences its ecological behaviour, while insecticidal toxins are maintained through plasmid retention [39]. The environmental and taxonomic distinctions between Bacillus spp. are internationally acknowledged and reflected in distinct, dominant biocontrol applications for each group.

In addition to these well-established lineages, the B. licheniformis operational group represents a distinct evolutionary branch within the broader B. subtilis operational group. This group, comprising B. licheniformis and B. paralicheniformis, has been consistently resolved as a separate lineage in phylogenomic studies [40,41]. Analyses based on average nucleotide identity and core-genome comparisons separate it from the B. subtilis and B. amyloliquefaciens operational groups, even though these taxa overlap phenotypically [42,43]. Genomes of B. licheniformis operational group members are typically enriched in genes encoding extracellular hydrolytic enzymes, reflecting adaptation to saprophytic lifestyles in nutrient-rich soils, composts, and decaying plant material [43,44]. Secondary metabolite biosynthetic gene clusters are present but generally less diverse than those found in the B. amyloliquefaciens group, indicating a mode of ecological success driven primarily by rapid resource exploitation, competitive exclusion, and high thermotolerance rather than extensive antibiotic production [45,46]. These environmental and genomic characteristics underpin the use of B. licheniformis group strains in enzyme-based agricultural formulations and selected biocontrol applications, while also shaping regulatory assessments that emphasise strain-level qualification.

The B. pumilus operational group constitutes a phylogenetically distinct lineage that includes B. pumilus and closely related taxa such as B. safensis. Comparative genomic studies indicate that members of this group typically possess relatively compact and streamlined genomes, enriched in genes associated with stress tolerance, DNA repair and protection against oxidative damage [47,48]. This genomic configuration aligns with their frequent recovery from environmentally exposed niches, including arid soils, high-UV habitats and plant surfaces [49,50]. In contrast to the rhizosphere-specialised B. subtilis and B. amyloliquefaciens groups, B. pumilus group members typically behave as opportunistic colonisers, with ecological success driven by pronounced persistence and efficient sporulation rather than long-term competitive dominance in the rhizosphere [51,52]. Consistent with this environmental strategy, reported biocontrol effects are most often linked to the production of antimicrobial peptides and the induction of plant defence responses, rather than to extensive or diverse secondary metabolite repertoires [53,54]. Collectively, these genomic and ecological characteristics help to explain both the episodic use of B. pumilus group strains in agricultural applications and the relatively cautious, strain-specific regulatory assessments applied to this lineage.

The B. amyloliquefaciens operational group, comprising B. amyloliquefaciens, B. velezensis, and B. siamensis, has become a model for studying how plant-associated Bacillus spp. evolve [36,55]. Phylogenomic analyses reveal that most previous strains classified as separate subspecies are closely related to B. velezensis [56,57]. This has resulted in worldwide taxonomic revisions based on nucleotide identity thresholds and core-genome phylogenies [36,55]. The same classification changes have been applied to B. firmus, which has been reclassified to the Cytobacillus firmus group based on international phylogenomic evidence of its distinction from the core Bacillus clade [25]. These changes are now reflected in major reference databases worldwide.

Genomic traits play a central role in explaining the global agricultural relevance of Bacillus spp., as they encode the metabolic, ecological and adaptive capacities that underpin their performance in field environments [36,55]. B. amyloliquefaciens OG species contain some of the highest concentrations of biosynthetic gene clusters reported for Gram-positive bacteria. These systems, like nonribosomal peptide synthetase (NRPS) and polyketide synthase (PKS), produce surfactins, fengycins, iturins, difficidin, macrolactins, and bacilibactin [45,58]. Within this group, B. velezensis consistently stands out as one of the most biosynthetically active species [59,60]. B. subtilis shares a similar genomic organisation, although it usually carries fewer accessory biosynthetic clusters [61,62]. In contrast, B. licheniformis and B. pumilus operational groups have genomes that are more streamlined and adapted to stress tolerance. These differences are more evident in their stress tolerance than in broad antimicrobial biosynthesis [63,64]. For example, BGC composition patterns unique to each species appear consistently across isolates from various parts of the world [7,65].

Members of the B. subtilis group are commonly found in the rhizosphere across diverse agricultural systems worldwide. They form long-lasting spores, germinate in response to plant exudates, and produce biofilms that persist on root surfaces [66,67]. Studies in tropical, temperate, and semi-arid agroecosystems often identify B. amyloliquefaciens and B. velezensis as among the dominant rhizosphere colonisers in crops such as rice, maize, wheat, soybean, and various horticultural species [68,69,70]. They are successful in competition because they can adapt to different environments and interact with other soil microorganisms through metabolites [71,72]. Species such as B. pumilus and B. licheniformis exhibit extensive ecological distributions, encompassing marine habitats, arid soils, and constructed environments globally [73,74]. B. mojavensis was first found in North American desert soils, but it is now known to be a seed-borne endophyte in cereals and forage crops on several continents [75,76]. B. nakamurai is less extensively studied than other Bacillus spp. that live in the rhizosphere, but it fits the general ecological profile of these species [75,76,77] (

Table 1. Comparative Overview of Agriculturally Relevant Bacillus Species Complexes: Functions, Mechanisms of Action, Key Traits, and Regulatory Status.

Representative Species	Agricultural Functions	Mode of Action	Applications	Key Traits	Regulatory Status
B. subtilis operational group
B. subtilis	Biocontrol; plant growth promotion	Llipopeptide production, nutrient competition, and induced systemic resistance	Seed treatment, soil and foliar applications, biofungicides	Strong rhizosphere coloniser; stable endospore formation; well-characterised secondary metabolite biosynthesis	Multiple strains authorised 1
B. atrophaeus	Biocontrol; soil health support	Antimicrobial metabolite production; competitive exclusion	Seed coatings, soil amendments	High environmental persistence; genetic stability; historically used as a non-pathogenic reference strain	Long history of safe use; evaluated at strain level 1
B. vallismortis	Plant growth promotion; biocontrol	Lipopeptide production; rhizosphere colonisation	Biofertilisers, biocontrol formulations	Close phylogenetic relation to B. subtilis; efficient root colonisation;	Limited number of authorised 4 strains
B. mojavensis	Biocontrol (Fungi)	Production of antifungal lipopeptides; nutrient competition	Soil and seed treatments	Endophytic potential; strong antifungal activity; ecological adaptation to arid soils	Case-by-case evaluation 3
B. pumilus operational group
B. pumilus	Biocontrol; stress tolerance enhancement	Antimicrobial peptides; induction of plant defence responses	Seed treatment, foliar sprays	High tolerance to UV and oxidative stress; robust spore resistance	Some strains are authorised 3; others are subject to additional scrutiny
B. safensis	Plant growth promotion; stress mitigation	Plant hormone modulation; niche competition	Biofertilisers, inoculants	Extreme-environment adaptability; low virulence profile; limited metabolite diversity	Limited use 4; strain-specific assessment
B. cereus operational group
B. thuringiensis	Insect pest control	Cry and Vip toxin production	Bioinsecticides, transgenic crops	Highly specific insecticidal proteins; plasmid-encoded toxin genes	Widely authorised 1 control applications
B. cereus	Limited biocontrol potential	Antimicrobial metabolites; competition	Rarely used due to safety concerns	Potential human toxin production; close relation to pathogenic strains	Generally excluded 1; strict strain-level assessment required
B. mycoides	Soil colonisation; minor biocontrol	Competitive exclusion; rhizosphere effects	Experimental soil applications	Distinctive filamentous colony morphology; strong soil persistence	Not generally authorised
B. licheniformis operational group
B. licheniformis	Biocontrol; enzyme production; plant growth promotion	Antimicrobial metabolites; enzyme-mediated nutrient mobilisation	Biofertilisers, biopesticides	High extracellular enzyme secretion; heat-tolerant spores	Limited number of authorised 4 strains
B. paralicheniformis	Biocontrol; soil health	Like B. licheniformis; antimicrobial activity	Soil amendments	Genetically distinct but phenotypically like B. licheniformis	Limited number of authorised 4 strains
B. amyloliquefaciens operational group
B. amyloliquefaciens	Biocontrol; plant growth promotion	Lipopeptide and polyketide production; ISR induction	Seed treatment, soil and foliar use	Rich secondary metabolite gene clusters; strong biofilm formation	Included on EFSA QPS list (with qualifications); PPP authorisation strain-specific
B. velezensis	Strong biocontrol activity; yield enhancement	Broad-spectrum secondary metabolites; biofilm formation	Commercial biopesticides	High genomic investment in antimicrobial biosynthesis; superior rhizosphere competence	Multiple strains authorized 1 for agricultural use
B. siamensis	Disease suppression; growth promotion	Antifungal metabolites; competition	Biofertilisers	Emerging taxon; metabolite profiles like B. velezensis	Under evaluation 2 in selected jurisdictions
B. nakamurai	Biocontrol (limited data)	Antimicrobial metabolites	Experimental applications	Recently described species; limited toxicological data	Limited regulatory status 4
C. firmus operational group (formerly B. firmus)
C. firmus	Nematode control; soil health	Nematicidal metabolites; rhizosphere competition	Soil treatments, nematicides	Alkaliphilic physiology; strong nematicidal activity; reclassified genus	Authorised 1 on a strain-specific basis

Regulatory status descriptors are indicative and reflect the general regulatory situation of strain-based plant protection products across major jurisdictions. Authorised ¹—refers to strains that have obtained market authorisation as active substances or microbial plant protection agents in at least one major regulatory framework (e.g., the European Union or the United States). Under evaluation ²—indicates strains for which regulatory dossiers are currently under assessment or have been submitted in selected jurisdictions. Case-by-case evaluation ³—refers to taxa for which regulatory acceptance depends strictly on individual strain-level identity, genomic characterisation and safety assessment, without any generic approval at species or operational-group level. Limited regulatory status ⁴—indicates that only a small number of strains have been assessed or authorised, or that use remains restricted to specific applications or regions.

).

In contrast, B. mojavensis and B. nakamurai are associated with stress-tolerant and endophytic life strategies that support persistence within plant tissues, particularly under environmentally challenging conditions [75,78]. Although reclassified outside the core Bacillus clade, C. firmus is considered here because it continues to be regulated and applied alongside Bacillus spp. in agricultural practice. Its agronomic relevance is linked to a specialised ecological niche defined by interactions with plant-parasitic nematodes, which underpins its use across a range of cropping systems [30,79]. B. thuringiensis represents a distinct insect-associated lineage whose agricultural importance derives from the production of insecticidal toxins, a feature that has driven its widespread adoption in biological pest control [80]. Together, these taxonomic, genomic and ecological distinctions provide the foundation for understanding how different Bacillus lineages express functional traits relevant to agricultural performance, which are examined in the following section. Specifically, the contrasting genome content, biosynthetic gene cluster repertoires and ecological niches of the major operational groups explain observed differences in rhizosphere colonisation capacity, metabolite production profiles and the consistency of disease suppression under field conditions.

3. Functional Traits Relevant for Agricultural Performance

3.1. Antagonistic Traits and Pathogen Suppression

Bacillus species contribute to crop protection mainly through the production of antimicrobial metabolites. These metabolites suppress plant pathogens and influence the structure of rhizosphere microbial communities. Lipopeptides such as surfactins, iturins, and fengycins act on microbial membranes and cellular integrity. Their activity results in inhibition of fungal growth and modulation of developmental processes, including biofilm formation [81,82]. This multifunctionality is particularly pronounced in B. velezensis and B. amyloliquefaciens, which frequently synthesise multiple lipopeptide families simultaneously [13,83,84]. Additionally, secondary metabolites, including difficidin, bacillaene, and macrolactins, interfere with intracellular processes in bacteria and fungi, thereby extending antagonistic activity beyond membrane disruption [85,86]. In addition to lipopeptides, Bacillus species produce a range of secondary metabolites, including difficidin, bacillaene, and macrolactins. Bacteriocins further contribute to competitive fitness. Their activity is mainly directed towards closely related Gram-positive taxa. This selectivity supports niche stability while limiting broader impacts on beneficial microorganisms [87,88].

Volatile organic compounds (VOCs) provide an additional, indirect mechanism of action. Compounds such as 2,3-butanediol diffuse through soil pore spaces and can affect both microbial interactions and plant physiological responses [89,90]. Experimental evidence indicates that VOCs can reduce pathogen activity and contribute to induced systemic resistance. They have also been associated with enhanced root development under controlled conditions. Antimicrobial metabolites and VOCs act together as important drivers of microbiome structure. Their combined activity is associated with community configurations that correlate with reduced disease pressure [91,92,93].

Biofilm formation integrates these chemical traits into a coordinated ecological strategy. Biofilms enhance tolerance to abiotic stress and promote persistence on plant surfaces. They also facilitate the local accumulation of antimicrobial compounds [94,95]. In B. subtilis, surfactin-regulated developmental pathways control the production of extracellular polysaccharides and amyloid fibres. These components are essential for biofilm architecture and stability [96,97,98]. Closely related taxa, including B. velezensis, form robust and persistent biofilms. This trait is considered a key factor supporting more consistent field performance under variable environmental conditions [95,99,100].

3.2. Plant Growth Promotion and Nutrient Mobilisation

Beyond antagonistic activity, Bacillus spp. promote plant growth through several mechanisms related to nutrient acquisition and hormonal regulation. Many strains are reported to produce indole-3-acetic acid (IAA), which stimulates root branching and enhances nutrient uptake. Other strains synthesise cytokinins that influence shoot development and delay senescence. Through modulation of phytohormone balance, Bacillus spp. contribute to improved root system architecture and increased overall plant vitality [101,102].

Phosphate solubilisation is a particularly important trait in low-input and phosphorus-limited agricultural systems. Bacillus species release organic acids, phosphatases, and chelating compounds that convert insoluble mineral phosphates into plant-available forms [103,104,105,106]. Although these bacteria do not fix atmospheric nitrogen, they can indirectly support plant nitrogen nutrition. This occurs through effects on root exudation patterns, stimulation of microbial mineralisation processes, and promotion of interactions with diazotrophic partners. The combined effect of these nutrient-related mechanisms enhances nutrient availability and uptake under abiotic nutrient constraints, resulting in improved plant growth and productivity in nutrient-limited soils [103,107].

3.3. Abiotic Stress Resistance and Ecophysiological Effects

Bacillus spp. is frequently associated with enhanced plant tolerance to abiotic stress. Plants colonised by Bacillus spp. often show physiological responses linked to improved membrane stability and increased antioxidant enzyme activity. Changes in water balance regulation have also been reported. These responses contribute to greater resilience under drought, salinity, temperature fluctuations, and oxidative stress [108,109].

Certain taxa contribute additional specialised functions through distinct ecological strategies. C. firmus produces enzymes that degrade the eggs and cuticles of plant-parasitic nematodes, interrupting their development and reducing root damage [19,20,110,111]. In contrast, B. thuringiensis employs Cry and Vip proteins to target insect pests, representing a highly specialised form of biological control [23,24,80,109]. These diverse mechanisms illustrate the broad functional versatility of Bacillus and related Bacillaceae taxa in agricultural systems.

Agriculturally relevant Bacillus species display a coordinated set of chemical, structural and ecological traits that support plant health and productivity. Their effectiveness is best explained by the interaction of multiple complementary mechanisms rather than by any single defining function. This integrated mode of action provides a useful framework for understanding the role of Bacillus-based applications in sustainable farming systems.

These functional traits include the production of antimicrobial secondary metabolites, biofilm-mediated persistence in the rhizosphere and the modulation of plant physiological processes. The agronomic significance of these traits ultimately depends on whether they result in consistent disease suppression and measurable yield benefits under greenhouse and field conditions. The following section, therefore, examines how these coordinated characteristics translate into effective biocontrol performance in experimental and commercial settings (Figure 1).

4. Biocontrol Performance of Bacillus spp. in Agricultural Systems

4.1. Consistent Patterns of Disease Suppression Across Crops and Regions

Across diverse agricultural systems, Bacillus species have been reported to reduce plant disease incidence. This effect is observed across different climatic zones and soil types. Bacillus spp. strains establish persistent root-associated populations, which—as evidence from temperate, tropical, and semi-arid systems confirms—occur with sufficient consistency. This consistency is generally attributed to conserved mechanisms of antagonism, competitive exclusion, and rhizosphere competence [21,112,113].

One frequently reported observation is the suppression of major fungal pathogens, including Botrytis spp., Fusarium spp., Rhizoctonia spp., Alternaria spp., and Sclerotinia spp. Such effects have been reported in horticultural, cereal, and vegetable production systems across different regions. While the magnitude of disease reduction varies between studies, strains, and cropping systems, the direction of the effect is generally positive [114,115,116,117,118].

Across field trials, disease suppression is most often linked to the combined action of membrane-active lipopeptides, intracellularly acting polyketides, volatile organic compounds, and sustained rhizosphere colonisation, rather than from any single mechanism operating in isolation [12,119], and persistent root-associated populations [120,121]. Membrane-active lipopeptides, such as surfactins, iturins, and fengycins, remain central to this activity [28,122,123]. Their effects are boosted by polypeptides such as difficidin and bacillaene, which interfere with intracellular processes and extend inhibition to pathogens that are less vulnerable to membrane disruption alone.

4.2. Fungal and Bacterial Pathogens: Shared and Divergent Control Patterns

Although a shared functional toolkit underlies the suppression of both fungal and bacterial pathogens, comparative analyses point to recurring differences in dominant mechanisms. In fungal pathosystems, membrane-active lipopeptides are most consistently identified as key drivers of inhibition. Their effects are often reinforced by biofilm-mediated accumulation at the root surface, which increases local compound concentrations and persistence [122,123].

In contrast, suppression of bacterial phytopathogens such as Xanthomonas spp., Pseudomonas spp., Clavibacter spp., Erwinia spp., and Ralstonia spp. more consistently depends on polyketides, bacteriocins, siderophores, and interference with quorum sensing. These mechanisms are repeatedly reported across rice, vegetable, and perennial crop systems, indicating that antibacterial activity relies more heavily on intracellular disruption and nutrient competition than on membrane destabilisation alone [114,115,116,117,118]. Volatile compounds further extend antagonistic effects beyond zones of direct contact. They suppress pathogen growth and interfere with microbial communication networks. This pattern has been reported in both controlled experiments and field-based studies [124,125].

4.3. Nematode Suppression as a Multi-Mechanism Interaction

Control of plant-parasitic nematodes, particularly Meloidogyne spp., illustrates how Bacillus-mediated biocontrol often results from layered mechanisms rather than single-target effects. Evidence from banana, soybean, and greenhouse production systems indicates that suppression commonly involves multiple processes. These include hydrolytic enzymes that damage egg masses and cuticles, lipopeptides that disrupt protective lipid layers, and changes in root exudate composition that interfere with host-finding behaviour [111,126].

Importantly, plant-mediated responses make a substantial contribution to these outcomes. Induced systemic resistance has been shown to strengthen root tissues and increase defensive enzyme activity [127,128]. In parallel, biofilm formation along the root axis creates additional physical and biochemical barriers to nematode penetration. This multi-layered interaction helps to explain why nematode suppression is increasingly pursued through microbial consortia rather than single-strain interventions.

4.4. Greenhouse Versus Field Performance

A common pattern across studies is the divergence between greenhouse and field performance. Under controlled conditions, Bacillus spp. routinely achieve high levels of disease suppression, reflecting reduced ecological constraints, stable colonisation, and favourable conditions for metabolite production [53,56,129].

Field performance, by contrast, is more variable and repeatedly constrained by temperature fluctuations, moisture extremes, nutrient heterogeneity, and competition with indigenous microbiota. In semi-arid regions, moisture limitation impedes biofilm formation, whereas in nutrient-rich temperate soils, resident microbial communities often reduce colonisation efficiency [130,131,132]. Nevertheless, studies consistently report improved field outcomes when agronomic practices, compatible formulations, and organic amendments are used to help mitigate these constraints.

For example, Mendis et al. [133] demonstrated that B. firmus I-1582 and B. amyloliquefaciens QST713 achieved stable and high root colonisation under controlled conditions, whereas substantially lower and more heterogeneous colonisation levels were observed under non-sterile field conditions, reflecting strong environmental and microbiome-mediated constraints. Similarly, large-scale multi-location field trials in soybean reported by Vasconcelos et al. [134] showed that yield and stress-mitigation benefits associated with Bacillus-based inoculants varied markedly between sites and seasons, despite consistently positive effects observed in controlled or semi-controlled experiments [133,134].

4.5. Environmental Drivers That Recurrently Limit Efficacy

Across production systems, soil texture, pH, moisture availability, and organic matter content are repeatedly identified as key environmental determinants of biocontrol efficacy. Sandy soils are often associated with reduced biofilm stability and limited metabolite retention. In contrast, dense clay soil can restrict oxygen availability and slow microbial metabolic activity. Loam soils most frequently support effective biocontrol performance by providing a balance of aeration, moisture retention, and nutrient availability [135,136].

Moisture availability is a particularly influential factor across production systems. It affects bacterial motility, root colonisation dynamics, and the regulation of secondary metabolism. Organic matter generally enhances microbial activity and persistence. At the same time, higher organic inputs increase microbial competition, which underscores the importance of functional integration into existing microbial networks rather than reliance on competitive dominance. These recurring constraints explain why effective Bacillus-based biocontrol most reliably arises from alignment between microbial traits, soil conditions, plant physiology, and rhizosphere community structure [53,136,137]. While these studies demonstrate substantial biocontrol potential, the deployment of Bacillus-based products must also be evaluated in the context of safety, exposure and regulatory requirements, which are addressed in the following section.

5. Metabolites of Potential Concern and Safety Assessment

The safety assessment of Bacillus-based biocontrol agents has evolved from basic pathogenicity screening to integrated evaluation of metabolic potential, genetic determinants, and ecological behaviour under field-relevant exposure conditions. This shift is reflected in modern regulatory practice in the European Union (Regulations 1107/2009 and 2019/1381) and in other jurisdictions, where microbial agents are increasingly assessed with respect to metabolite hazard profiles, exposure pathways, and potential contributions to antimicrobial resistance [138,139,140].

Agriculturally relevant Bacillus spp. can produce diverse bioactive metabolites. In a safety context, however, the key determinants of concern are (i) whether a metabolite class is linked to well-established mammalian toxicity, and (ii) whether field application leads to realistic exposure at biologically relevant doses, considering environmental fate and persistence. Species within the B. subtilis operational group (e.g., B. subtilis, B. amyloliquefaciens, B. velezensis, B. pumilus, B. licheniformis, B. nakamurai) generally show favourable profiles because they lack the emetic and enterotoxigenic determinants associated with the B. cereus operational group, and because their metabolites typically exhibit constrained exposure routes and rapid degradation in environmental matrices [141,142,143] (Figure 2).

5.1. Toxicologically Relevant Metabolites

A central, repeatedly confirmed toxicological distinction among Bacillus taxa used in agriculture is the separation between the B. cereus operational group and the B. subtilis complex [144,145]. Cereulide, together with enterotoxins such as HBL (Haemolysin BL), NHE (Non-Haemolytic Enterotoxin) and CytK (Cytotoxin-K), is restricted to B. cereus OG and toxigenic lineages, and these toxins have well-defined adverse effects in mammals [144,145]. Reported LOAEL (Lowest Observed Adverse Effect Level) values for cereulide are in the µg/kg range and rodent LD₅₀ values fall below 10 µg/kg, supporting the conclusion that even low exposures can be relevant in a hazard context [146,147]. Consequently, regulatory systems generally exclude B. cereus group organisms from plant protection uses unless strain-level genomic evidence demonstrates the absence of toxin biosynthetic capacity [145,148].

In contrast, species within the B. subtilis operational group lack these canonical mammalian toxin determinants, shifting the safety question from “presence of known high-hazard toxins” to “margin of safety for bioactive metabolites under field exposure.” In this context, amphiphilic lipopeptides (e.g., surfactins, iturins, fengycins) are often treated as the primary “metabolites of potential concern” because membrane activity is a plausible hazard mechanism at sufficiently high concentrations [37,149]. Importantly, multiple lines of evidence indicate that field-relevant exposure is far below effect thresholds: haemolytic activity is typically observed only at ≥50–100 µg/mL, whereas environmental residues after field application are reported orders of magnitude lower (often ng/g soil), and exposure is further constrained by adsorption and rapid degradation [150,151,152]. Toxicological studies with purified lipopeptides report high NOAEL values (including oral NOAELs > 100 mg/kg bw/day in rodents for selected compounds) and limited evidence for dermal irritation or systemic uptake, consistent with a wide margin of safety relative to plausible dietary or operator exposure [150,153]. Polyketides such as difficidin and bacillaene can be biochemically potent antimicrobials, but available toxicological datasets similarly indicate low hazard at high administered doses and limited persistence under environmentally relevant conditions, which again constrains realistic exposure [88,152]. Collectively, these patterns support risk conclusions driven less by intrinsic hazard and more by exposure limitation and environmental fate.

Regulators increasingly recognise that laboratory metabolite profiles may overestimate or misrepresent metabolite expression in situ. Accordingly, metabolite characterisation under plant–soil microcosm conditions is increasingly emphasised, and for widely used strains (e.g., QST 713; PTA-4838) in situ analyses support the conclusion that expressed metabolites align with ecological function while remaining distinct from profiles associated with mammalian toxicity under realistic exposure [154,155,156].

5.2. Risks Associated with Antimicrobial Resistance Genes

Antimicrobial resistance is now a core component of microbial agent evaluation, reflecting One Health integration into modern regulation [157,158]. Bacillus spp. often displays intrinsic resistance to specific antibiotic classes (e.g., β-lactams, polymyxins), which is typically chromosomally encoded, evolutionarily stable, and broadly distributed in soil microbiomes, and is therefore not considered a transferable hazard in regulatory risk assessment [157,159].

Regulatory scrutiny focuses primarily on acquired and potentially mobile antimicrobial resistance determinants. Current dossier practice relies on whole genome sequencing to identify plasmid- or transposon-associated AMR genes. Phenotypic susceptibility testing is then used to confirm functional expression of detected determinants [160,161,162]. For widely used strains (e.g., B. amyloliquefaciens QST 713; B. velezensis FZB42; C. firmus I 1582), reported resistance profiles are generally consistent with intrinsic characteristics rather than transferable determinants, supporting a low AMR concern under current regulatory criteria [159,160,163].

Although Bacillus can exhibit natural competence, multiple ecological barriers limit horizontal gene transfer in soil (DNA degradation, niche separation, low cell density, and fitness costs), and available field-oriented evidence has not supported the view that approved Bacillus biopesticide strains act as reservoirs of transferable AMR [164,165]. On this basis, regulatory authorities generally characterise AMR risk as minimal when strains lack mobile resistance determinants.

5.3. Ecological Safety

Ecological safety assessments examine potential effects on non-target organisms, soil microbial communities, and key biogeochemical processes. Bacillus strains commonly used in agriculture show low toxicity to non-target arthropods, vertebrates, and aquatic organisms. These effects are observed at exposure levels that exceed typical agricultural application rates. Such findings are consistent with limited environmental exposure and rapid attenuation following application [166,167,168].

At the soil community level, inoculation with Bacillus spp. most often results in transient and selective modulation rather than sustained disturbance. High-throughput sequencing studies across multiple crops indicate that introduced strains frequently decline within weeks after application. These studies also show that effects tend to be targeted towards pathogen-associated taxa, with no consistent suppression of beneficial microbial groups [169,170,171]. Concerns that Bacillus spp. metabolites might inhibit functionally critical groups (e.g., mycorrhizae, nitrogen fixers) that have thus far received limited experimental support; where effects are observed, they are generally context-dependent and appear unlikely to drive long-term ecological perturbation given rapid metabolite degradation and constrained persistence.

5.4. Toxicological Data, NOAEL, and ADI Considerations

Across regulatory systems, toxicological datasets generally support the conclusion that Bacillus strains used in agriculture represent low hazard across exposure routes, consistent with limited ability to proliferate in mammalian tissues and rapid elimination following ingestion [172,173]. Rodent studies conducted under OECD Test Guidelines commonly report high NOAEL values for spore preparations, and dermal and inhalation studies designed to simulate operator exposure typically indicate low toxicity and limited sensitisation potential [88,173].

Because canonical mammalian toxin determinants are absent from B. subtilis complex, and because exposure to bioactive metabolites is low under realistic residue scenarios, establishment of an ADI is often considered unnecessary. Where quantitative thresholds are needed, purified metabolite studies are used to define conservative margins of safety. For surfactin, NOAEL values > 100 mg/kg bw/day provide substantial safety margins relative to conservative dietary exposure estimates, which are typically orders of magnitude lower [88,174]. Comparable safety margins have been reported for iturins and fengycins, and the extensive toxicological evaluation of Cry proteins provides additional context supporting low mammalian hazard for microbial metabolites used in agriculture [175,176].

Synthesised across genomic, toxicological, and ecological evidence, B. subtilis complex generally fulfill criteria associated with low-risk microbial agents when strain-level identity and hazard determinants are addressed. Their metabolites, while central to biological function, are characterised by exposure levels and persistence profiles that are consistently below thresholds associated with toxicological concern. Beyond hazard and risk considerations, effective use of Bacillus-based agents also depends on industrial production, formulation stability and application practices, which are discussed in the next section.

In addition to their use in plant protection, several Bacillus strains belonging to the B. subtilis operational group have been evaluated and authorised for use as probiotic or functional food and feed additives for humans and animals. Repeated-dose and subchronic oral toxicity studies conducted for such applications consistently report an absence of adverse effects and support high NOAEL values, providing an independent and complementary line of evidence for the low mammalian hazard of these taxa. This extensive history of safe oral exposure further supports the risk-based conclusion that agriculturally used Bacillus strains, when appropriately identified and assessed at the strain level, do not pose relevant toxicological concern [145,177].

6. Industrial Production, Formulation, and Application Challenges

The commercial development of Bacillus-based biocontrol agents requires translation of strain-level biological potential into products that remain stable during production, storage, transport, and application. Unlike synthetic pesticides, microbial products are sensitive to process conditions across the entire pipeline, from bioreactor cultivation to performance in the soil environment. In an industrial context, this sensitivity imposes constraints on reproducibility, formulation design, and scale-up decisions [53,56,129].

6.1. Upstream Production and Scale-Up Constraints

Industrial production is generally robust because Bacillus spp. tolerate fluctuating oxygen levels and can grow on diverse substrates. However, commercial manufacturing requires not only high biomass yields but also the production of physiologically uniform spores with predictable germination behaviour and field persistence. Spore quality depends on tight control of oxygen transfer, pH, nutrient availability, and the timing of sporulation induction [178,179,180]. Scale-up also introduces mechanical stresses that can shift physiology and product characteristics. Foaming and shear stress remain recurrent challenges because industrial aeration and mixing regimes can increase foam formation and influence sporulation dynamics. Antifoam agents mitigate foam but can alter surface properties and downstream formulation behaviour [181,182,183,184].

A further recurring industrial challenge concerns trait stability across repeated fermentation cycles. Even when genetic stability is maintained, expression of key functional traits can vary with process parameters. Such variation may result in reduced functional output following scale-up. This issue is particularly evident under production regimes that promote rapid or aggressive sporulation, where metabolite yields may decline relative to laboratory-scale cultivation. To address these risks, manufacturers typically rely on master seed banking, batch-to-batch quality control, and routine metabolite profiling to preserve functional consistency [53,56,137].

6.2. Formulation Stability and Shelf-Life Limitations

Formulation plays a decisive role in determining whether industrially produced spores remain viable and functionally effective during storage and distribution. Although spores are intrinsically resilient, viability and performance are strongly affected by moisture, temperature fluctuations, carrier choice, and the physicochemical compatibility of excipients. Stability losses are especially problematic in humid and warm environments where water activity increases during storage and transport [132,137,185]. Dry formulations generally improve shelf stability by reducing water activity. However, they require appropriate carriers to maintain dispersibility and to protect spores from humidity and thermal stress. Liquid formulations are often preferred for drip irrigation and spray applications. At the same time, they introduce additional challenges, including sedimentation control, compatibility with adjuvants, and the risk of unintended germination during storage [186,187].

6.3. Application-Stage Losses and Agronomic Compatibility

At application, performance depends on whether viable propagules reach the appropriate root or foliar microhabitat under conditions that permit germination and establishment. Soil moisture is repeatedly identified as a decisive factor: drought conditions constrain motility and colonisation, whereas excessive irrigation can redistribute propagules away from the rhizosphere. Salinity can further reduce establishment and survival [53,132,182].

Compatibility with standard agronomic inputs is a persistent applied constraint. Tank mixes with copper-based compounds, certain fungicides, and high-salt fertilizers can reduce viability or disrupt establishment, even when spores remain nominally “stable” in storage. These interactions help explain why high efficacy under greenhouse conditions does not always translate to consistent field outcomes unless application timing, formulation, and local management practices are aligned [53,129,183].

6.4. Regulatory Constraints on Process Flexibility

Regulatory systems increasingly require that industrially produced strains remain equivalent to the strains assessed during authorisation, including genetic identity, the absence of harmful determinants, and consistency of key biological properties. This creates a practical constraint: process innovation must be incremental, because substantial shifts in production conditions that alter trait expression or measurable profiles can trigger additional justification requirements [182,188].

In practice, this means that producers must design manufacturing pipelines around strain biology rather than treating the organism as a fully “engineerable” input. This is one reason why repeated inoculation strategies, compatible formulation platforms, and integrated pest management positioning have become central to commercial success [53,132]. Industrial production and deployment of Bacillus-based products involve a continuous trade-off between biological responsiveness and engineering control. Products perform most reliably when upstream production, formulation stability, and field application conditions are designed to support physiological requirements for germination, persistence, and competitive establishment. When performance fails, it often reflects mismatches introduced along the production–application continuum rather than intrinsic limitations of Bacillus biology [53,135,182]. These practical constraints further interact with regulatory requirements that govern strain identity, manufacturing consistency and product authorisation, as outlined in the following section.

7. Regulatory Landscape for Bacillus spp. in the EU, US and Globally

Regulation of microbial biocontrol agents has evolved rapidly over the past decade. This evolution reflects a broader shift in agricultural policy towards biological alternatives to synthetic pesticides. Bacillus-based products occupy a prominent position within this transition. They are widely recognised as low-risk agents. At the same time, they challenge regulatory frameworks that were originally developed for static chemical substances. Authorities are therefore required to reconcile chemical risk assessment paradigms with the dynamic biology of living microorganisms [189,190,191]. As a result, regulatory approaches to Bacillus spp. differ substantially across jurisdictions. These differences influence both the pace of market access and the nature of the scientific evidence required.

7.1. European Union: Metabolite- and Genome-Centred Regulation

In the European Union, Regulation (EC) No. 1107/2009 forms the legal backbone of microbial pesticide authorisation. It is complemented by transparency and data governance requirements introduced under Regulation (EU) 2019/1381. In practice, these instruments are operationalised through the European Food Safety Authority‘s (EFSA) detailed guidance on microorganisms, which has progressively aligned microbial dossiers with the structure and depth traditionally required for chemical active substances [192].

Applicants are expected to provide unambiguous strain identity supported by multilocus or whole-genome sequencing, comprehensive genomic annotation, and a detailed assessment of metabolite production under conditions representative of agricultural use. Emphasis is placed on the identification of secondary metabolites, their potential toxicological relevance, and their behaviour in soil and plant-associated environments [193]. EFSA has repeatedly clarified that microorganisms cannot be treated as “black boxes”; instead, regulators seek evidence linking in vitro observations to realistic field exposure scenarios [194,195,196].

Case histories illustrate how this regulatory philosophy is applied in practice. Assessments of C. firmus highlight increased scrutiny of antimicrobial resistance determinants. In these cases, EFSA required evidence that detected resistance markers were intrinsic and not associated with mobile genetic elements [33,197]. Reviews of B. amyloliquefaciens strains, later taxonomically resolved as B. velezensis, underscore the regulatory importance of phylogenomic clarity. Closely related taxa may differ in metabolic capacity and ecological behaviour, with direct implications for risk assessment and strain characterisation [56,195,198,199]. Long-standing assessments of B. thuringiensis have further shaped regulatory expectations. This research established benchmarks for environmental persistence and non-target exposure to biologically active proteins. Such precedents continue to inform the assessment of non-insecticidal Bacillus species.

7.2. United States: Risk-Proportionate, Biology-Focused Assessment

In contrast, the US Environmental Protection Agency (EPA) applies a regulatory model centred on infectivity, pathogenicity, and ecological behaviour rather than exhaustive metabolite characterisation [200]. For most Bacillus spp., including B. subtilis, B. pumilus, and B. velezensis, dossiers focus on acute toxicity, pathogenicity and clearance studies, alongside a limited set of ecotoxicological tests demonstrating the absence of adverse effects on non-target organisms [201,202,203].

Detailed metabolomic analyses are typically requested only when a strain is known to produce metabolites of recognised human-health relevance or when genetic modification is involved. This approach relies heavily on historical safety data, decades of environmental exposure, and consistent evidence that these organisms do not proliferate in mammalian tissues or exhibit systemic infectivity [204]. Therefore, Bacillus-based products often reach the United States market more rapidly than the European Union market. This difference reflects contrasting regulatory philosophies and procedural approaches rather than fundamentally different conclusions regarding product safety.

7.3. OECD and Global Convergence Efforts

OECD guidance has sought to bridge these contrasting regulatory cultures by promoting weight-of-evidence approaches that integrate ecological behaviour, functional identity, and exposure potential [191,205]. Many countries in the Asia–Pacific region, including Japan, the Republic of Korea, and increasingly China, draw on OECD frameworks while adapting requirements to national agricultural priorities. Implementation across the region remains heterogeneous. For example, Brazil’s Ministry of Agriculture (MAPA) refers to OECD principles but applies them with a degree of flexibility. This approach reflects the widespread use of microbial inoculants in large-scale soybean and horticultural production systems. As OECD guidance is advisory rather than legally binding, national authorities retain discretion in balancing data requirements against perceived risk [206,207,208] (Figure 3).

7.4. Persistent Divergences: Metabolites and Antimicrobial Resistance

Despite ongoing convergence efforts, substantial differences remain, particularly regarding metabolite assessment and antimicrobial resistance. In the European Union, applicants are required to evaluate metabolite expression under realistic agricultural conditions. This often involves soil incubation studies, advanced metabolomics, and quantitative exposure assessments [209,210]. For lipopeptide-producing species such as B. velezensis, EFSA routinely requests data on the presence, concentrations, and toxicological relevance of surfactin, iturin, and fengycin homologues. These assessments typically cover both soil compartments and plant surfaces [211,212].

In the US and Canada, similar data are requested only when a specific hazard is anticipated. AMR assessment follows a comparable pattern. While all major jurisdictions screen for resistance determinants, only the EU mandates a systematic assessment of mobility potential, interpreting AMR through a One Health lens even when direct human exposure is unlikely [213,214]. The EPA, by contrast, focuses on whether resistance traits influence pathogenicity or infectivity [215,216].

7.5. Implications for Global Commercialisation

The practical consequence of these regulatory asymmetries is that a Bacillus strain approved in the US, Brazil, or Japan may still require several additional years of data generation before gaining EU authorisation. This delay reflects differences in hazard conceptualisation rather than evidence of increased risk. To navigate this landscape, multinational developers increasingly prepare parallel dossiers: one aligned with the EU’s emphasis on genomics, metabolomics, and AMR, and another tailored to the EPA’s biologically oriented risk model [217,218].

At the same time, experience from Asia suggests that emerging regulatory systems often look to the EU’s stringent framework when drafting new microbial pesticide rules, particularly regarding transparency and genomic characterisation. In this way, regulation not only evaluates Bacillus spp. biocontrol technologies but actively shapes research priorities and data generation strategies.

Several commercially available plant protection products are already based on strains belonging to the B. subtilis operational group, including formulations derived from B. subtilis strain QST 713 (e.g., Serenade^®), and products based on B. amyloliquefaciens or B. velezensis strains (e.g., Double Nickel^® and related formulations). These products illustrate the practical translation of the functional traits discussed above into registered and widely used biological control agents (

Table 2. Comparison of regulatory approaches for Bacillus-based biocontrol agents across major jurisdictions. Adapted from [175,217,218].

Aspect	European Union	United States	OECD Countries
Primary regulatory focus	Comprehensive strain identity, genomics and metabolite characterisation	Infectivity, pathogenicity and ecological behaviour	Weight-of-evidence integrating identity, function and exposure
Role of genomics	Mandatory strain-level genomic characterisation	Used mainly to support identity and safety	Recommended as supporting evidence
Metabolite assessment	Systematic evaluation under realistic agricultural conditions, including exposure and fate	Requested mainly when a specific hazard or genetic modification is suspected	Generally addressed in a flexible, case-by-case manner
AMR assessment	Screening and evaluation of mobility potential under a One Health perspective	Focus on relevance to pathogenicity or infectivity	Addressed within broader risk characterisation frameworks
Regulatory philosophy	Hazard and exposure driven, aligned with chemical-style data structures	Risk-proportionate and biology-focused	Advisory guidance with national discretion
Legal status of guidance	Binding regulatory framework	Binding national regulatory framework	Non-binding guidance

).

Across regulatory systems, a consistent conclusion is that strains within the B. subtilis operational group are low-risk organisms with no history of human pathogenicity, no requirement for consumer exposure thresholds such as acceptable daily intake (ADIs), and no evidence of ecological disruption when used according to label. The divergence lies not in risk conclusions, but in the evidentiary pathways required to reach them. In the European Union, regulatory practice places strong emphasis on comprehensive genomic, metabolic, and environmental characterisation of microbial agents, whereas in the United States, regulatory practice tends to emphasise tiered data, case-specific data requirements that reflect anticipated exposure scenarios and use conditions. Reconciling these regulatory cultures is a key challenge for the global expansion of microbial biocontrol, requiring continued dialogue between science and policy to ensure protection goals are met without unnecessarily fragmenting the field [145,219,220]. Against this regulatory background, important scientific and methodological gaps remain that limit both performance predictability and long-term risk assessment, as discussed in the next section.

8. Knowledge Gaps and Future Perspectives

Despite substantial progress in the development and deployment of Bacillus-based biocontrol agents, important uncertainties remain regarding the predictability of their performance across heterogeneous agricultural systems [113,221]. These limitations do not reflect a lack of demonstrated efficacy, which has been reported across multiple crops and regions. Rather, they arise from the inherent biological and ecological complexity of Bacillus species. Functional expression is strongly influenced by soil properties, plant–microbe interactions, microbial competition, and climatic variability [151,222]. As microbial products are subject to increasing regulatory scrutiny and wider field adoption, these unresolved factors become central not only to performance reliability but also to long-term risk assessment [113].

A persistent challenge concerns the relationship between taxonomy, genomics, and functional behaviour. Although phylogenomic revisions of the B. subtilis and B. amyloliquefaciens OG have substantially improved taxonomic resolution, discrepancies between historical nomenclature and genome-based classification remain widespread [154,222]. As a result, strains continue to be reported under outdated or inconsistent species names, complicating cross-study synthesis and introducing ambiguity into regulatory dossiers. More fundamentally, extensive strain-level variation in biosynthetic gene clusters often shaped by horizontal gene transfer limit’s ability to infer metabolite production or ecological function directly from species identity or genome annotation. While genomics provides insight into biosynthetic potential, it remains an imperfect predictor of functional expression in complex soil environments [102,223].

These limitations are particularly apparent when laboratory characterisation is contrasted with field behaviour. The production of surfactins, iturins, fengycins, polyketides, and volatile compounds by Bacillus species is well documented under controlled conditions. In agricultural soils, however, metabolite expression is substantially more variable. Nutrient availability, moisture, temperature, plant-derived substrates, and microbial competition jointly shape in situ metabolite profiles. As a result, field outcomes often diverge from laboratory expectations [113,223,224].

Consequently, metabolite profiles generated under laboratory conditions frequently fail to reflect realistic field exposure scenarios [223,225]. Although regulatory authorities increasingly acknowledge this discrepancy, the limited availability of integrated field-based metabolomics combined with microbiome analyses continues to constrain robust ecological and toxicological assessment [113,223].

Knowledge gaps are also pronounced with respect to ecological interactions and longer-term effects. Short-term studies generally indicate low acute toxicity and limited disruption of non-target organisms. However, data addressing repeated applications, cumulative exposure, and persistence across multiple growing seasons remain limited [226,227]. Indigenous microbial communities may either resist colonisation or accommodate introduced strains, resulting in highly variable persistence patterns. It therefore remains unclear whether Bacillus spp. inoculants function primarily as transient biological inputs or contribute to more subtle, long-term restructuring of microbial communities through sustained metabolite production. This uncertainty is likely to be amplified under climate-driven changes in soil conditions.

Antimicrobial resistance is a related area of uncertainty. Although commercial Bacillus strains typically harbour only intrinsic, non-transferable resistance determinants and comply with current regulatory requirements, soils constitute dynamic genetic environments. The possibility that repeated high-density inoculation could influence selection pressures within indigenous microbial communities over extended timescales is not fully addressed by current assessment frameworks, which rely predominantly on genomic screening and short-term susceptibility testing [160,228,229,230].

Interpretation is further complicated by methodological fragmentation across studies. Comparability is limited by variation in inoculum density, soil type, irrigation regime, experimental design, and analytical endpoints. Inconsistent definitions of colonisation and persistence, along with the absence of standardised methods for in situ metabolite quantification, further hinder synthesis. While field trials are necessarily adapted to local agronomic conditions, this contextual specificity reduces reproducibility and contributes to the persistent gap between laboratory and field performance [133,134,231,232].

Addressing these challenges will require a shift from predominantly empirical screening towards more predictive, mechanistic approaches. Field-based multi-omics strategies integrating genomics, transcriptomics, metabolomics, and microbiome profiling are needed to link biosynthetic potential with realised ecological function [233,234]. In parallel, improved markers of viability and metabolic activity are required to assess post-application performance across contrasting soils and climatic regimes [235,236,237]. Advances in computational tools, including AI-assisted analysis of NRPS and PKS gene clusters combined with environmental datasets, offer promising avenues for forecasting metabolite production and strain behaviour in specific agronomic contexts [46,233,234].

Further progress is expected from the rational design of microbial consortia and continued advances in formulation technology. An increasing body of evidence suggests that multi-strain systems may provide greater ecological stability and functional resilience than single-strain products, particularly under variable field conditions. Adaptive formulations, controlled-release carriers, and microencapsulation strategies, therefore, remain important tools for translating biological potential into consistent agricultural performance. These developments support a gradual shift towards Bacillus-based biocontrol approaches that are more reliable, context-sensitive, and scalable within sustainable farming systems.

9. Conclusions

The evidence reviewed in this article indicates that agriculturally relevant Bacillus species constitute a well-established and scientifically substantiated group of microbial agents whose performance derives from the interaction of multiple biological mechanisms rather than from any single defining trait. Their ability to suppress pathogens, promote plant growth, and improve tolerance to abiotic stress reflects conserved genomic features, metabolite profiles that are diverse yet largely predictable, and ecological strategies that support effective rhizosphere colonisation and persistence.

In parallel, advances in phylogenomics, metabolite characterisation, and environmental exposure assessment have enabled more precise and proportionate approaches to safety evaluation, allowing low-risk taxa within the B. subtilis complex to be clearly distinguished from toxigenic members of the B. cereus operational group. These developments have contributed, across regulatory systems, to a shift away from generic pathogenicity screening towards strain-level risk assessment frameworks that better reflect the biology of living microbial products.

Remaining limitations in production, formulation stability, and field performance are most often associated with the alignment between microbial traits, environmental conditions, and application practices rather than with intrinsic biological constraints. The available evidence supports the conclusion that Bacillus-based products, when appropriately selected, formulated, and regulated, serve as a robust and adaptable component of sustainable and climate-responsive agricultural systems, while continued integration of genomic, ecological, and regulatory knowledge will be essential to ensure their long-term reliability and acceptance.