Increasing Application of Multifunctional Bacillus for Biocontrol of Pests and Diseases and Plant Growth Promotion: Lessons from Brazil

Abstract

The microbial genus Bacillus inhabits a diverse range of environments and is widespread across all global biomes, with a significant presence in soil habitats. In agriculture, Bacillus strains play multifaceted roles, serving as biocontrol agents against pests and diseases, and promoting plant growth by facilitating nutrient availability and enhancing stress tolerance. Through mechanisms such as phosphate solubilization, ACC-deaminase activity, and synthesis of phytohormones and siderophores, Bacillus spp. contribute to soil health and crop productivity, in a new approach of regenerative agriculture. The ability of Bacillus spp. to solubilize phosphate makes essential nutrients more accessible to plants, while ACC-deaminase activity helps plants withstand environmental stresses. Additionally, the synthesis of phytohormones can stimulate plant growth and development, and siderophores may facilitate the uptake of nutrients such as iron by plants. As the agricultural industry embraces Bacillus-based formulations for pest management and crop enhancement, future research holds promising prospects for optimizing their applications and harnessing their full potential in agroecosystems. Continued exploration of Bacillus spp. diversity and their interactions with plants and soil microbiota will further advance sustainable agricultural practices. This review contributes to understanding how Bacillus strains can revolutionize agriculture by enhancing soil health, increasing crop productivity, and providing effective biological solutions against pests and diseases. The successful application of Bacillus-based technologies in millions of hectares in Brazilian agriculture demonstrates the synergy between the need for more sustainable agricultural practices and the use of bio-inputs.

1. Introduction

The genus Bacillus consists of Gram-positive, rod-shaped, motile bacteria that are aerobic or facultatively anaerobic [1]. Under abiotic stress conditions, such as high temperature, radiation, dehydration, and exposure to certain chemicals, spore formation occurs [2]. Spore formation, or bacterial sporulation refers to the production of metabolically inactive resistance structures until they are subjected to minimal conditions for vegetative structure development [3].

In the mid-1870s, while working at the University of Breslau, Ferdinand Cohn isolated a small, motile, aerobic bacterium from hay infusions, naming it Bacillus subtilis (meaning “subtle rod”). From this isolate, Cohn described the life cycle of Bacillus, detailing spore formation, its heat resistance, and the germination process. However, the genus and the species did not gain scientific importance until 1876, when Robert Koch described a phenotypically similar organism as Bacillus anthracis, highlighting the importance of the genus [4].

Upon contact with a mammal, B. anthracis, the causative agent of anthrax, produces toxins due to the presence of genes located on plasmids, resulting in a severe disease [5]. Scientifically, this species is also important because it was the first experimental model for the development of central postulates of infectious diseases and the first to outline the role of macrophages in cellular immunity [6,7].

The genus Bacillus is ubiquitous, meaning that it can be found in a wide variety of habitats, including soil [8], where it may represent up to 95% of the population of Gram-positive bacteria [9]. The diversity is so great that compared to the Enterobacteriaceae family—which has dozens of genera—it is already possible to find an equivalent number of species [10]. A phylogenetic tree based on the 16S rRNA sequences highlights the outstanding diversity of Bacillus spp. Comparative genomic studies and complementary data have revealed the presence of genetically distinct groups that have evolved from a common ancestor. These analyses, which were conducted with hundreds of Bacillus genomes, aimed to clarify the evolutionary relationships and classification of species. As a result, a significant set of species has been reclassified into several new genera: Alkalicoccus, Alkalihalobacillus, Alteribacter, Caldalkalibacillus, Caldibacillus, Calidifontibacillus, Cytobacillus, Domibacillus, Ectobacillus, Evansella, Ferdinandcohnia, Gottfriedia, Heyndrickxia, Lederbergia, Litchfieldia, Margalitia, Mesobacillus, Metabacillus, Neobacillus, Niallia, Peribacillus, Priestia, Robertmurraya, Rossellomorea, Salibacterium, Salisediminibacterium, Schinkia, Siminovitchia, Solibacillus, Sutcliffiella, and Weizmannia [11,12,13,14,15,16]. However, they will all be treated here as Bacillus, and the new reclassification is included in parentheses.

The diversity of the genus encompasses species related to diseases, such as Bacillus cereus, which is notably associated with food poisoning [5], B. anthracis, and others. However, it also harbors biotechnologically promising species, such as Bacillus safensis, Bacillus endophyticus, and B. subtilis which have applications in the pharmaceutical industry [17], Bacillus amyloliquefaciens in the feed industry [18], Bacillus licheniformis for environmental remediation [19], and various species with significant roles in agriculture [20].

Globally, in the agricultural market, there is a tradition of using Bacillus for biological control of pests and diseases, owing to its ability to produce antimicrobial, nematicidal, and insecticidal compounds [21]. Brazil stands out as an important market for biocontrol, and Bacillus composes the large majority of the formulations—about 80% of the market, in 2023. The composition is as follows: B. subtilis (32.8% of the total), B. licheniformis (26.2%), B. amyloliquefaciens (8.2%), Bacillus paralicheniformis (6.5%), Bacillus thuringiensis (4.9%), and Bacillus velezensis (1.6%) [21,22].

In addition to their ability to synthesize biocidal molecules to protect against pests and diseases or trigger plant defense responses, there are Bacillus species with other plant growth-promoting properties responsible for increasing the tolerance of host plants to abiotic stress conditions, producing phytohormones, and making nutrients available to plants [21,22]. Regarding Bacillus spp. with prominent applicability in agriculture, a group known as plant growth-promoting bacteria (PGPB) encompasses species that play a significant role in plant development, especially in economically important crops [23].

The development of a plant’s aboveground portion is highly dependent on the underground root system. In the rhizosphere, the zone of plant-microorganism communication, there is continuous interaction between roots and microbial communities established in the soil, as well as populations introduced for crop management [24]. Soil represents the primary reservoir for the potential bacterial community of the rhizosphere [25], and through root exudates, plants attract rhizospheric microorganisms to colonize the root surface and/or internal tissues, while microorganisms—via the production of phytohormones, the release of nutrients, or induction of responses to various stresses—can promote plant growth [21].

Many PGPBs, by producing phytohormones, volatile organic compounds, and secondary metabolites, play a crucial role in the architecture and growth of roots and/or root hairs [26]. With increased surface area for water and nutrient absorption by plants, there are advantages in growth and resource utilization efficiency under multiple soil constraints [27], which characterize a significant indicator of agricultural sustainability [28]. Bacillus species can enhance the productivity and quality of various agricultural crops by providing nutrients in forms assimilable by plants, activating physiological and molecular processes in plants, or through the production of metabolites and compounds beneficial to plant development [23], in addition to controlling pests and diseases [29].

The action of plant growth-promoting microorganisms can be involved in biocontrol or directly promoting plant growth (Figure 1). Examples of beneficial properties to plant growth include phosphate solubilization, 1-aminocyclopropane-1-carboxylic acid (ACC) deaminase activity, and synthesis of phytohormones and siderophores. As an indirect property of plant development, biocontrol encompasses the production of antimicrobial metabolites and hydrolytic enzymes, quorum quenching, competition for nutrients and space, production of siderophores, and promotion of induced systemic resistance (ISR).

Traditionally, the primary application of Bacillus in agriculture has been as a biological control agent. However, in recent years, its use as a PGPB has been growing. Interestingly, the number of reports showing multifunctionality at the strain level is increasing. Therefore, the same strain of Bacillus can be used for biocontrol and to promote plant growth. Bio-inputs based on this multifunctionality can represent a very interesting approach towards a new regenerative agriculture. This review presents the main microbial processes of Bacillus that may improve plant growth (Figure 1), the uses of Bacillus in agriculture, and prospects for the future.

2. Applications of Bacillus spp. in Agriculture as Biocontrol

Biological control represents a sustainable strategy for combating plant pests and diseases. This process, which uses antagonists to pests and diseases, is reported as one of the most promising alternatives to chemical control [30,31]. Microorganisms-based products containing live cells or microbial metabolites have been used as biopesticides worldwide because they are generally safer for non-target organisms, including humans, have reduced persistence in the environment, and are potentially acceptable for use in organic agriculture [32].

Reports of disease control promoted by Bacillus are not new, as the insecticidal properties of B. thuringiensis (Bt) have long been recognized [33]. Although some reports indicate that the Egyptians were aware of the insecticidal properties of what was likely Bt, the microorganism was isolated by Ernst Berliner only in 1911, in Japan, from a diseased larva of the flour moth. In 1954, Thomas Angus demonstrated that the crystalline protein inclusions produced by B. thuringiensis during sporulation were responsible for the insecticidal action. In Brazil, in 1994, there were also reports of products based on B. subtilis, responsible for controlling coffee (Coffea spp.) rust [34].

B. thuringiensis insecticidal products were first marketed in France in the late 1930s and are still considered safe because specific formulations only harm a narrow range of insect species. Furthermore, the incorporation of Bt genes related to the insecticidal crystal protein (ICP) into various agronomically important crops represented a significant advancement in the management of various agricultural pests [33]. However, any possibility of gene incorporation must be investigated for potential toxicity. While acknowledging the agronomic benefits of GMOs (Genetically Modified Organisms), it is imperative to conduct analyses of risk assessments due to potential impacts. Studies such as the one conducted by Seralini [35] have raised questions about the long-term toxicity of these organisms. Therefore, comprehensive evaluations are necessary to ensure the safety and sustainability of GMO adoption in agriculture.

An advantage of the genus is that it not only inhibits the development of the pathogen but also restores, at least in part, the microbial community altered by the presence of the disease-causing microorganism in plants [36]. For example, the strain FZB42 of B. amyloliquefaciens subsp. plantarum, when associated with lettuce (Lactuca sativa L.), did not affect the root microbiome and contributed to the restoration of the original structure of the community that was previously altered by the pathogen Rhizoctonia solani [36]. Therefore, Bacillus may highly contribute to soil health.

Bacillus species are renowned for their ability to produce a diverse array of components of significant biotechnological value [37]. The use of bio-inputs based on Bacillus spp. for pests and diseases control in plants has proven to be effective in various agronomically important crops due to a range of specific mechanisms associated with each species. These mechanisms include the production of antibiotic metabolites, hydrolytic enzymes, quorum quenching, competition for space and nutrients, production of siderophores, and ISR. Several examples are shown in

Table 1. Mechanism of actions of Bacillus spp. as a biocontrol agent in crop protection.

Species of Bacillus	Crop	Mechanism	Pathogen	Study
Bacillus amyloliquefaciens JDF3; Bacillus subtilis RSS-1	Glycine max	Inhibition of ribosomal activity	Fungi Phytophthora sojae	[38]
Bacillus (Priestia)megaterium Sneb207	Glycine max	Induction of systemic resistance	Nematode Heterodera glycines	[39]
Bacillus sp. P12	Phaseolus vulgaris	Synthesis of lipopeptide isoforms: kurstakin, surfactin, iturin, polymyxin, and fengycin	Fungi Macrophomina phaseolina	[40]
Bacillus halotolerans QTH8	Triticum aestivum	Synthesis of lipopeptides: iturin, surfactin, and fengycin	Fungi Fusarium pseudograminearum	[41]
Bacillus velezensis BM21	Zea mays	Cytoplasmic necrosis and disintegration of pathogen organelles	Fungi Fusarium verticillioides; Thanatephorus cucumeris; Typhula incarnata; Fusarium oxysporum; Pythium graminicola; Rhizoctonia solani	[42]
Bacillus sp. BMH	Oryza sativa	Induction of systemic resistance	Fungi Pyricularia oryzae	[43]
Bacillus velezensis FJAT-46737	Solanum lycopersicum	Secretion of lipopeptides: iturins, fengycins, and surfactins	Fungi Ralstonia solanacearum	[44]
Bacillus subtilis K4-4; Bacillus subtilis GH3-8	Citrus sinensis	Production of HCN, siderophores, bacillomycin, iturin, fengycin	Fungi Fusarium solani	[45]
Bacillus pumilus	Fragaria × ananassa Duchesne	Systemic induction of resistance	Fungi Rhizoctonia solani; Fusarium solani; Pythium sp.	[46]
Bacillus velezensis MS20	Zea mays	Synthesis of surfactin	Fungi Rhizoctonia solani	[47]
Bacillus (Weizmannia) coagulans; Bacillus globisporus; Bacillus pumilus; Bacillus subtilis; Bacillus (Niallia) circulans; Bacillus cereus; Bacillus (Weizmannia) coagulans; Bacillus cereus	Gossypium barbadense	Systemic resistance induction and/or antibiosis	Fungi Rhizoctonia solani; Macrophomina faseolina; Sclerotium rolfsii; Pythium sp.; Fusarium oxysporum; Fusarium solani; Fusarium moniliforme	[48]
Bacillus velezensis ZW-10	Oryza sativa	Synthesis of peroxidase, protease, and cellulase	Fungi Magnaporthe oryzae	[49]

. An interesting observation from this table is that the strains and even the species are distinct, indicating that bioprospecting for specific conditions is a path that has led to success.

2.1. Antimicrobial Metabolites

Bacteriocins are ribosomal-synthesized proteins or peptides with bactericidal action against species that may be closely related to the producing bacteria and exhibit variable biochemical properties, inhibitory spectra, and mechanisms of action [50]. Most often, these molecules are targeted against competitive microorganisms; thus, providing a selective advantage to their producers [51].

Bacillus spp. is a relatively abundant source of antimicrobials, as many species of this genus synthesize antimicrobial peptides [52,53]. One example is of antibiotics of the iturin group reported in two strains of B. subtilis, PRBS-1 and AP-3, which inhibited the growth of five important pathogenic fungi of soybean (Glycine max (L.) Merr.) seeds [54]. Another example is the endophytic Bacillus strain DMW1, which has the ability to control Phytophthora sojae and Ralstonia solanacearum due to the presence of 12 biosynthetic gene clusters of secondary metabolites [55].

2.2. Hydrolytic Enzymes

The accumulation of hydrolytic enzymes, such as chitinases and β-1,3-glucanase, which degrade the cell walls of pathogenic fungi formed by chitin and β-1,3-glucan, represents a common defense mechanism of plants [56]. Microorganisms can also produce chitinases as an important biocontrol attribute in the rhizosphere [57]. In addition to the production of hydrolytic enzymes by the biocontrol agents themselves, PGPB can induce the production of chitinases in the host plant, accentuating the plant’s defense response [58].

Strains of Bacillus that produce chitinases have been used as post-harvest biocontrol agents; for example, peaches (Prunus persica (L.) Batsch) treated with B. cereus AR156 showed a lower disease incidence and smaller lesion diameter compared to the control when in contact with Rhizopus stolonifer. In this study, it was found that the treatment with B. cereus AR156 notably improved chitinase and β-1,3-glucanase activities [59]. Another Bacillus species, Bacillus pumilus, including subspecies B. pumilus HR10, B. pumilus SS-10.7, B. pumilus MCB-7, B. pumilus INR7, B. pumilus SE52, SE34, SE49, B. pumilus RST25, B. pumilus JK-SX001, and B. pumilus KUDC1732 are capable of suppressing phytopathogens such as Arthrobotrys conoides, Fusarium solani, Fusarium oxysporum, Sclerotinia sclerotiorum, R. solani, and Fagopyrum esculentum [60].

2.3. Quorum Quenching

Through quorum sensing systems, bacteria change their behavior when a threshold concentration of signaling molecules is exceeded [58]; that is, it is a communication pathway between bacterial cells. Pathogenic bacteria use quorum sensing to assess the size of their population and regulate the timing of entry into the apoplast or plant cell [61,62]. However, besides cross-communication by producing the same signaling molecules, bacteria can degrade each other’s signals, also known as quorum quenching.

Some strains belonging to the genus Bacillus, such as those studied by Caicedo and colleagues [63]—Bacillus sp. SJ13 and Bacillus sp. SJ15—can degrade the diffusible quorum signaling factor, cis-11-methyl-2-dodecenoic acid [58]. This signal is involved in regulating virulence in Xanthomonas spp. and Xylella fastidiosa [64]. Bacillus toyonensis AA1EC1, capable of degrading quorum signaling molecules such as N-acyl-homoserine lactones (AHLs), significantly attenuated the virulence of relevant phytopathogens, reducing symptoms of soft rot in potatoes (Solanum tuberosum L.) and carrots (Daucus carota L.) [65].

2.4. Competition for Nutrients and Space

Among the main resources necessary for microbial survival are nutrients and space, which vary between environments, so that microorganisms will compete for restricted components [66]. For example, the available carbon source may be the limiting factor for the development of a population [67]. As they grow and produce more biomass, microbial groups expand in space and compete with others to colonize areas where nutrients are more abundant [66].

The action of B. subtilis strains B006 and B010 in inhibiting F. oxysporum and F. solani—pathogens causing soybean root rot was studied by Guo and colleagues [68]; the control efficiency was higher than 63%, in addition to increasing soybean yield. The authors report that the rapid spread of bacterial colonies may be an important mechanism of biocontrol for these strains, due to their greater efficiency in nutrient competition. Mates and colleagues [69] evidenced that B. velezensis GF267 is a multisite antagonist and, among its properties of inhibiting pathogenic microorganisms, the strain stands out in competition with Xanthomonas perforans, for example.

2.5. Production of Siderophores

For most microorganisms, iron (Fe) is related to essential cellular processes. Some microorganisms have highly efficient Fe-acquisition systems to capture Fe from the environment under restriction conditions. In many cases, this involves the secretion and internalization of extracellular ferric chelators called siderophores [70]. Siderophores produced by a microorganism can bind to Fe with high specificity and affinity, making it unavailable to other microorganisms and thus limiting their growth [71].

This strategy may be involved in biological control against plant pathogens [72]. For example, the B. subtilis strain MF497446 was considered effective in siderophore production. When inoculated alone or in combination with Pseudomonas koreensis MG209738, it induced resistance to late wilt disease caused by Cephalosporium maydis in maize (Zea mays L.), resulting in increased plant growth and grain yield [73]. A siderophore-producing B. subtilis (CWTS 5) with multiple plant growth-promoting properties effectively inhibited R. solanacearum through secondary metabolites identified via LC-MS analysis, reducing disease severity and enhancing plant growth in Solanum lycopersicum L., with genomic insights supporting its use as a biocontrol agent and plant growth promoter [74].

2.6. Induction of Systemic Resistance

If defense mechanisms are triggered by a stimulus prior to pathogen infection, the disease may be less severe due to the ISR, which is a state of increased defensive capacity developed by a plant when adequately stimulated. This stimulus can be of microbial origin, and resistance is expressed throughout the plant, not just at the site of contact with the inducer [75].

Species of the genus Bacillus sensitize plant immune systems to increase protection without directly activating costly defenses [76]. B. subtilis IAGS174 is potentially a control agent for Fusarium wilt, and the methyl ester of phthalic acid produced by the bacterium is the determinant of ISR, which can effectively trigger defense responses in tomatoes (Solanum lycopersicum L.) [77]. Yadav and colleagues [78] identified that B. subtilis NBRI-W9 simultaneously activates systemic acquired resistance (SAR) and ISR against Fusarium chlamydosporum NBRI-FOL7, thereby increasing wilt resistance in tomato plants.

3. Importance of Bacillus spp. in Agriculture as Plant Inoculants or Biofertilizers

Unlike biocontrol, which encompasses a well-defined set of properties attributed to Bacillus spp., characteristics related to plant growth promotion have begun to be studied recently. Reports show that the association of strains of this genus with different crops can contribute to nutrient supply, production of phytohormones, or impart tolerance to various abiotic stresses. Some Bacillus strains effective in pathogen biocontrol can also benefit plants through growth-promoting properties, as demonstrated by B. amyloliquefaciens WS-10 in studies associated with tobacco (Nicotiana tabacum L.), with proven nutrient solubilization ability [79,80].

The benefits obtained from inoculation with Bacillus strains will depend on their compatibility with the crop and/or other associated microorganisms. For example, in soybean inoculated with Bradyrhizobium spp., the synthesis of phytohormones and molecules of biocontrol by B. subtilis strains PRBS-1 and AP-3 contributed to controlling seed-pathogenic fungi, improving root growth and nitrogen-fixation parameters, and increasing grain yield [54,81]. However, the strains of Bacillus showed antibiosis to Bradyrhizobium, requiring the use of either formulated metabolites or mutants of Bradyrhizobium tolerant to Bacillus [81].

The use of Bacillus strains, either alone or in association with Trichoderma, has shown prominent results in biomass accumulation in soybean, rice (Oryza sativa L.), cowpea (Vigna unguiculata (L.) Walp), and maize crops, demonstrating their potential as a growth promoter, particularly efficient in the latter two crops, in terms of increased shoot and root dry mass [82]. Data from Nain et al. [83] support the results of Chagas et al. [82] regarding cowpea cultivation, adding the observed benefits in seed germination, leaf area, shoot and root length, number of pods, and grain yield per plant when cowpea seeds were inoculated with Bacillus sp. strain RM-2.

The benefits that Bacillus species can provide in the development of various agronomically important crops are diverse, through phosphate solubilization [84], ACC-deaminase production [85], phytohormones [86], and siderophores [87], which will be discussed below.

Table 2. Role of Bacillus spp. in augmenting the growth of economically important agricultural crops.

Species of Bacillus	Crop	Mechanism	Effect	Study
Bacillus amyloliquefaciens SQR9	Zea mays	Increase in soluble sugar content; efficiency of peroxidase/catalase activity and glutathione content; reduction in Na+ levels in the plant	Tolerance to saline stress	[88]
Bacillus (Priestia) aryabhattai SRB02	Glycine max	Production of abscisic acid, indole acetic acid, cytokinin, and different gibberellic acids	Tolerance to thermal, oxidative, and nitrosative stress	[89]
Bacillus subtilis BEB-lSbs	Lycopersicon esculentum	Not identified	Increase in fruit productivity and quality	[90]
Bacillus pumilus	Lycopersicon. esculentum cv Jinpeng 10	Adaptation of leaf gas exchange rates, stomatal density, and endogenous levels of ABA	Efficiency of water use under water deficiency	[91]
Bacillus sp. wp-6	Triticum aestivum	Alteration of alpha-linolenic acid metabolism, amino acids, and flavonoid synthesis	Increase in fresh weight of shoot and root	[92]
Bacillus velezensis JB0319	Lactuca sativa	Superoxide dismutase and lactoperoxidase activity; decrease in malondialdehyde accumulation and increase in osmotic regulator substance accumulation of proline	Increase in lettuce shoot biomass, root length, and alteration of rhizosphere bacterial community	[93]
Bacillus amyloliquefaciens RWL-1	Oryza sativa	Production of abscisic acid, glutamic acid, and proline	Increase in productivity and saline stress tolerance	[94]
Bacillus (Priestia) megaterium EGE-B-1.4.a; Bacillus (Peribacillus) simplex EGE-B-1.2.k; Bacillus subtilis EGE-B.24.4i; Bacillus subtilis EGE-B.26.1; Bacillus (Priestia) megaterium EGE-B.10.3.F; Bacillus subtilis EGE-B.3.P.5	Lycopersicon lycopersicum cv. Target F1; Capsicum annuum var. cv. Kekova F1; Solanum melongena cv. Faselis F1	P solubilization, IAA production; improvement in radicle and hypocotyl development; increase in plant growth, enhancing root and stem growth	Promotion of seed germination and vegetative development	[95]
Bacillus (Priestia) aryabhattai LAD	Zea mays	P solubilization	Increase in shoot length, total root length, and main root thickness	[96]
Bacillus cereus YL6	Glycine max; Triticum aestivum; Brassica rapa (Chinensis Group)	Solubilization of inorganic and organic P; production of indole-3-acetic acid (IAA) and gibberellin (GA)	Increase in soybean and wheat biomass in pot experiments; increased growth and yield of Chinese cabbage	[97]
Bacillus proteolyticus Cyn1; Bacillus safensis Cyn2	Phaseolus vulgaris	Production of NH3, ACC deaminase, biofilm; P solubilization; secretion of catalase enzyme and siderophores;	Tolerance to abiotic stresses	[98]
Bacillus sp. LrM2	Avena sativa	Production of ACC deaminase and antioxidant enzymes	Tolerance to saline stress, shoot growth, and root system development	[99]

presents more examples of these contributions.

3.1. Phosphate Solubilization

The process of phosphate solubilization involves making the nutrient phosphorus (P) available through the association of phosphate-solubilizing microorganisms in substrates containing poorly soluble sources of phosphate. In general, in tropical soils, although P may be present in the soil, it is practically unavailable for plant absorption due to its complexation affinity with metal ions [100]. Introducing phosphate-solubilizing microorganisms into the system facilitates the transformation of insoluble phosphates in the soil through various mechanisms, including the secretion of organic acids, enzyme production, and siderophore excretion—this process chelates metal ions and forms complexes, making phosphates available for plant uptake. However, although important, this latter pathway is not the primary mechanism responsible for phosphate solubilization [101].

The conversion of PO₄³⁻ (non-absorbable) to HPO₄²⁻ and H₂PO₄⁻ (absorbable) primarily occurs through the release of metabolites, such as organic acids that lower the pH of the medium and release soluble phosphate [102,103,104]. This solubilization occurs through the respiratory pathway of direct oxidation, operating on the external surface of the bacterial cell membrane [105]. The organic acids produced release mineral P because of the exchange of the phosphate anion for the organic acid anion, and/or they can complex/chelate cations such as Fe, Al, and Ca in the rhizosphere [106]. The organic acids commonly released by phosphate-solubilizing microorganisms are gluconic acid [107,108], oxalic acid, citric acid [109], and lactic acid [110]. Most phosphate-solubilizing microorganisms belong to the genus Bacillus [111]. In the study conducted by Saeid et al. [112], the solubilizing exudates produced by Bacillus consisted of five organic acids: gluconic, lactic, acetic, succinic, and propionic.

Another pathway for P availability is through mineralization, which occurs by the production of phosphatases that cause dephosphorylation of organic P compounds in the soil, breaking the phosphoester or phosphoanhydride bonds. Many soil microorganisms can perform this function, including Bacillus [113,114]. Unlike phosphatases produced by plants, microbial-origin phosphatases have a higher affinity for P from organic compounds [115].

3.2. ACC-Deaminase Activity

The molecule ACC is the biochemical precursor of ethylene—a plant hormone responsible for fruit ripening, seed germination, cell expansion and differentiation, flowering, leaf and flower senescence, and fruit abscission [116].

ACC present in the roots or rhizosphere can be metabolized by bacteria producing the enzyme ACC-deaminase. This enzyme alters the pathway of ethylene formation, thereby reducing the level of this hormone in the roots. By lowering ethylene levels, ACC-deaminase prevents the inhibition of root growth and promotes overall plant growth. Additionally, by reducing ethylene production, the enzyme decreases the plant’s susceptibility to various stresses, such as drought, salinity, and pathogens attacks [117,118,119,120]. Bacteria producing the enzyme are attracted to the ACC content produced by plants and released in exudates, establishing plant-bacteria interactions in the rhizosphere [121].

The contribution to stress tolerance by bacteria producing ACC-deaminase has been observed in various abiotic stress conditions, such as flooding [122], drought [123], salinity [124], flower senescence [125], metal pollution [126], and pathogens attacks [127]. For example, there are reports of the ability of B. licheniformis K11 to reduce ethylene concentration in pepper plants (Capsicum annuum L.), cleaving ACC under water stress and, therefore, promoting plant growth [128].

3.3. Production of Phytohormones

Many PGPBs synthesize plant growth-regulating hormones [129]. Indole-3-acetic acid (auxin or IAA) is an example of a phytohormone that controls a wide range of functions in plant development and acts as a key component in root architecture formation, through differentiation of root vascular tissue, regulation of lateral root initiation, polar positioning of root hairs, and root gravitropism [130]. However, when phytohormones are present in high concentrations, the effects on plants can be deleterious [131], as indicated by the study of Silva et al. [132], where a few days after the application of the herbicide 2,4-D (an auxin mimetic) at the V4 and V6 stages, visual symptoms of toxicity were observed in the aerial part of soybean plants in treatments with doses equal to or greater than 20 g/ha.

Nevertheless, generally, the synthesis of IAA by PGPB does not harm the development of the associated crop. This fact can be explained due to the intimate relationship formed between the plant and the PGPB. Many PGPBs capable of synthesizing IAA are dependent on tryptophan—the precursor amino acid of IAA—which can be produced by the plant in association [133]. However, when PGPBs with a high capacity to synthesize IAA are applied in inoculation at high cellular concentrations, a decrease in plant growth can be observed, indicating the need for further investigation [134].

The production of IAA by Bacillus strains can contribute to the development of different crops. For example, the rooting efficiency of kiwifruit (Actinidia deliciosa Planch.) stem cuttings was favored in the presence of Bacillus sp. strain RC03 and Bacillus (Peribacillus) simplex strain RC19, due to the production of IAA, maximizing the yield of rooted clonal cuttings in nurseries and allowing the reduction in the use of synthetic/chemical rooting products [135]. Another example was reported in the cultivation of strawberries (Fragaria × ananassa Duch.) by Chebotar et al. [136], where B. velezensis BS89 contributed significantly to the production of IAA, reflecting on the development and yield of the plants. For soybean, the synthesis of IAA and abscisic acid (ABA) by B. subtillis strains PRBS-1 and AP-3 improved root growth parameters [54].

3.4. Production of Siderophores

Despite their contribution to biological control, the production of siderophores can also contribute to plant nutrition, as some organisms can form biogenic chelators to complex the predominant oxidized form of iron (Fe³⁺) present in the aerobic environment and alter the bioavailability of Fe [137]. By this process, the nutrient that was previously unavailable can be reduced to Fe²⁺ and assimilated by both plants and some microorganisms [138].

The synthesis of siderophores such as pyoverdine, hydroxamates, and ferrioxamines by microorganisms in the rhizosphere region can contribute to up to a threefold increase in the efficiency of iron transport during root growth and plant meristem development [139].

In addition to iron availability for plants, siderophore production may also be related to the solubilization of iron phosphate, due to the affinity and formation of Fe chelators, releasing phosphate [140]. For example, Bacillus sp. WR12, capable of producing siderophores, significantly increased root length and dry mass, leaf chlorophyll content, and Fe content in wheat (Triticum aestivum L.) seedlings [141].

4. Bacillus spp. in Brazil: A Successful Case in Agriculture

Soybeans have played, and still play, a fundamental role in the development and advancement of the use of bio-inputs in Brazil. The biological nitrogen fixation with Bradyrhizobium spp. and the biological control of soybean caterpillars with B. thuringiensis stand out as the first successful cases [142,143].

The relationship between the soybean crop and the use of bio-inputs in Brazil is evident not only historically but also in practical terms. The demand for sustainable agricultural management has driven the search for solutions based on beneficial microorganisms, such as those of the genus Bacillus. These bio-inputs not only have contributed to pest and disease control but have also promoted plant growth. The application of Bacillus-based technologies in Brazilian agriculture exemplifies the synergy between the need for more sustainable agricultural practices and the rich history of soybean culture as a driver of innovation in the national agricultural sector [144].

Bacillus-based inoculants are particularly interesting due to their ability to form spores that can persist in fields for long periods under different climatic conditions, and they can also be produced and stored for longer periods than non-spore-forming bacteria, allowing longer marketing periods [145,146]. Despite Bacillus being the most abundant genus in the rhizosphere [147], for agricultural crops, the concentration of colony-forming units (CFUs) needed to benefit yields is obtained through the annual supply of bio-inputs. Some examples of products marketed in Brazil based on Bacillus are listed in

Table 3. Main bio-inputs based on Bacillus spp. commercialized in Brazil in 2023.

Species of Bacillus	Commercial Name	Mechanism	Crop	Marketed by
Bacillus subtilis CNPMS B2084 (=BRM034840) Bacillus (Priestia) megaterium CNPMS B119 (=BRM033112)	Biomaphos	Phosphate solubilizer	Zea mays; Glycine max	Bioma
Bacillus subtilis BRM 2084 Bacillus (Priestia) megaterium BRM 119	Solubphos	Phosphate solubilizer	Zea mays; Glycine max	Simbiose
Bacillus subtilis CNPMS B2084 (=BRMO34840) Bacillus (Priestia) megaterium CNPMS B119 (=BRMO33112)	Omsugo P	Phosphate solubilizer	Glycine max	Corteva
Bacillus subtilis CNPMS B2084 (-BRMO34840) Bacillus (Priestia) megaterium CNPMS B119 (=BRMO33112)	Omsugo Eco	Phosphate solubilizer	Saccharum officinarum	Corteva
Bacillus licheniformis CCTB07	Bioprince	Growth promoter	Zea mays	Biotrop
Bacillus pumilus CCTB05 Bacillus subtilis CCTB04 Bacillus amyloliquefaciens CCTB09	Biotrio	Growth promoter	Zea mays Glycine max Lactuca sativa	Biotrop
Bacillus subtilis BV09	Biobaci	Microbiological nematicide	Any crop with the following targets: Meloidogyne incognita, Meloidogyne javanica, Meloidogyne exígua, Meloidogyne paranaenses, Pratylenchus zeae, and Fusarium oxysporum	Vittia
Bacillus amyloliquefaciens UMAF6614	Veraneio	Microbiological nematicide	Any crop with the following targets: Meloidogyne incognita, Meloidogyne javanica, and Pratylenchus zeae	Koppert
Bacillus amyloliquefaciens SIMBI BS 10 CCT 7600	Nemacontrol	Microbiological nematicide	Any crop with the following targets: Heterodera glycines, Meloidogyne exigua, Meloidogyne incógnita, Pratylenchus brachyurus, and Sclerotinia sclerotiorum	Simbiose
Bacillus pumilus CNPSo 3203	Caravan	Microbiological fungicide	Any crop with the following targets: Septoria glycines, Corynespora cassiicola, and Cercospora kikuchii	Koppert
Bacillus amyloliquefaciens FZB45	Phosbac	Phosphate solubilizer	Zea mays; Glycine max	Andermatt
Bacillus (Priestia) aryabhattai CMAA 1363	Auras	Growth promoter (tolerance to drought)	Zea mays; Glycine max	NOOA Ciência e Tecnologia Agrícola

. It is worth emphasizing that the Brazilian market of bio-inputs based on Bacillus for biocontrol represents over 80% of all doses commercialized [21,22], with applications estimated in about 40 million hectares. Additionally, the new market of Bacillus bio-inputs for plant growth promotion has reached over 6 million hectares in only five years. Overall, the Brazilian market of bio-inputs expects an increase of 110% in the next five years.

The intimate relationship between bacteria and plants provides mutual benefits for both organisms. A single plant species can benefit from various growth promotion mechanisms, which can be expressed by different bacterial genera or species. For example, the association of different Bacillus species in a single formulation has shown synergy, as in the case of the composition containing B. pumilus CCTB05, B. subtilis CCTB04, and B. amyloliquefaciens CCTB09, registered as a plant growth-promoter inoculant [148].

Bacillus spp. were evaluated for their ability to enhance sugarcane (Saccharum spp.) growth by improving P availability. Selected strains, including B. licheniformis MGB2281, showed promising results in phosphatase activity and plant growth, highlighting its potential as an effective inoculant for sustainable agriculture [149]. Also, Castelo Souza et al. [150] highlighted the potential of Bacillus (Priestia) aryabhattai to enhance crop resilience in semi-arid tropical regions of Brazil. The strains promoted leaf gas exchange efficiency and plant growth, and helped to mitigate salt and water stress in maize [150].

Bacillus strains isolated from cotton (Gossypium hirsutum L.) roots in Brazilian fields showed potential in promoting plant growth by improving physical, phytochemical, and macronutrient parameters in cultivars FM 985 and TMG 47; the strains enhanced plant height, biomass, root volume, and various biochemical markers [151]. In addition, the use of the B. amyloliquefaciens strain FZB45 (Phosbac product) was found to significantly increase shoot dry mass, root dry mass, and grain yield in both maize and soybean; the increases showed a positive correlation with P uptake by the crops [152].

The most successful example of Bacillus spp. used as PGPB in Brazil is relatively recent. The formulation containing B. subtilis strain BRM 2084 and B. (Priestia) megaterium strain BRM 119, has a proven capacity for phosphate solubilization, synthesizing phytohormones, and other mechanisms that facilitate nutrient absorption [153]. The product has been shown to be beneficial for the growth and nutritional and physiological status of crops such as the common bean (Phaseolus vulgaris), maize, soybeans, and sugarcane. In addition, these strains enhance maize productivity and P uptake via seed treatment. Oliveira-Paiva and colleagues [154] evaluated their efficacy in diverse Brazilian soils, emphasizing their roles in P solubilization, indole-3-acetic acid production, biofilm formation, and enzymatic activities, while showing compatibility with other beneficial microorganisms. These results are corroborated by recent studies by Souza et al. [155], De Sousa et al. [156], Oliveira-Paiva et al. [157], and as exemplified in Figure 2, in the study by Oliveira et al. [158]. The first commercial bio-input carrying these two strains was released for the maize crop in 2019, and it has already been registered for use in maize, soybean, common bean, and sugarcane.

Due to substantial yield increases and other significant benefits driven by Bacillus species, important cost reductions can be experienced in various cropping systems. For example, bio-input with B. cereus promoted the growth of coconut (Cocos nucifera (L.) Arecaceae) seedlings, enhanced seedling quality, and reduced nursery time, reducing costs by 10%, and highlighting its economic efficiency [159]. The study by Oliveira et al. [158] found that the productivity gains from inoculation exceeded costs in most locations evaluated. Maize showed increased productivity in all sites, while soybeans had gains in 175 out of 181 locations, with average increases of 8.6% for maize and 6.3% for soybeans (Figure 2).

Another notable success is the use of B. (Priestia) aryabhattai (strain CMAA1363). When applied as a seed treatment to maize, this microorganism significantly enhanced plant growth and productivity, with increases ranging from 5.9% to 43.7%, proving to be an effective inoculant to the crop [160]. Noteworthy, the strain was released and announced as a Bacillus that increases plants’ tolerance to drought.

In relation to the comparative performance of Bacillus-based biopesticides with other biologicals and chemical products, in studies performed outside Brazil, the application of B. velezensis strain BUZ-14 to grafted grapevine (Vitis vinifera L.) had a remarkable protective effect against vascular necrosis caused by Neofusicoccum parvum and Diplodia seriata, outperforming traditional products based on Thichoderma harzianum and Trichoderma atroviride [161]. Compared to commercial fungicides, B. velezensis BUZ-14 emerged as a highly effective biocontrol agent. Similarly, B. amyloliquefaciens Bac 28.3 exhibited comparable efficacy to commercial fungicides in controlling Botrytis cinerea in tomatoes, highlighting it as a promising alternative [162].

5. Future Perspectives and Biosafety Measures for the Use of Bacillus in Agriculture

The market value of Bacillus-based bio-inputs was estimated to be at least USD 18 billion in 2020, with expectations for continued growth in the coming years, particularly in the agricultural sector [163]. The use of Bacillus spp. in agriculture is expanding, not only for biological control of pests and diseases but also for promoting plant growth due to a variety of mechanisms, with an emphasis on plant nutrition and tolerance to drought. As research advances, new strains of Bacillus are expected to be discovered and developed, further enhancing their applications in various crops and environmental conditions. Biotechnology can also play a crucial role in optimizing existing strains, increasing their effectiveness and adaptability, and new biotechnological tools such as genomic editing may be promising to obtain elite strains.

The potential of Bacillus species is vast and continues to be explored, both in the prospecting of new species and strains and in the evaluation of their compatibility with crops grown on a large scale. This field of research offers promising prospects for the discovery of new applications and for deepening the understanding of interactions between bio-inputs based on Bacillus and crops of interest. However, it is essential to pay attention to the dynamics of these organisms in real environmental conditions and how species will adapt and perform their expected functions. This includes understanding how beneficial members interact with each other to establish a stable community and enhance their collective performance.

When using Bacillus species in agriculture, it is important to implement minimum biosafety measures to ensure safety and efficacy. First, rigorous strain identification and characterization should be conducted to confirm the absence of pathogenic or harmful traits. Second, environmental risk assessments must be performed to evaluate potential impacts on non-target organisms and ecosystems. Third, compliance with regulatory guidelines and obtaining necessary approvals from relevant authorities are essential. Additionally, proper handling, storage, and application procedures should be established to prevent contamination and ensure consistent results. Continuous monitoring and post-application assessments are recommended to track the long-term effects and efficacy of Bacillus-based products. Implementing these biosafety measures helps ensure that Bacillus applications contribute positively to sustainable agriculture without compromising environmental or human health.

It is also important to highlight that questions about the cost comparison of biological and chemical treatments are often raised. In Brazil, the costs of bio-inputs toward plant nutrition are always lower than chemical fertilizers, which are mostly imported. In general, costs are also lower for biopesticides. However, even if there were no differences in costs of applying bio-inputs based on Bacillus in the replacement of pesticides or chemical fertilizers, the environmental benefits should also be valued and certainly are always highly favorable to the biologicals.

As new discoveries emerge in this field, regulatory frameworks play a crucial role in promoting the use of these less harmful options for chemical fertilizers and pesticides. Studies and reviews conducted in various regions, such as the work by Nihorimbere and colleagues [164] focusing on the African context, underscore the necessity for favorable regulatory frameworks to facilitate the adoption of these technologies, particularly in underdeveloped and developing regions worldwide. These frameworks not only ensure safe and effective deployment of biocontrol agents and sustainable agricultural practices, but also foster innovation and economic development in agriculture.

6. Conclusions

Bacillus spp. can greatly contribute to agricultural production by promoting environmentally sustainable practices. The use of Bacillus in biocontrol to reduce the incidence of pests and diseases has been well-known for a long time, with many commercial bio-inputs available at the market. The benefits related to plant growth promotion by Bacillus are more recent and not yet widely spread. Considering the biotechnological potential to produce phytohormones, improve nutrient availability, and act as a biological control offered by the genus, it is pertinent to prospect new strains and develop new bio-inputs, expanding their use to several crops. The use of Bacillus-based formulations as multifunctional bio-inputs, for both biological control and plant growth promotion represents an innovative approach that may have great success at the market.