The Significance of Bacillus spp. in Disease Suppression and Growth Promotion of Field and Vegetable Crops

Abstract

Bacillus spp. produce a variety of compounds involved in the biocontrol of plant pathogens and promotion of plant growth, which makes them potential candidates for most agricultural and biotechnological applications. Bacilli exhibit antagonistic activity by excreting extracellular metabolites such as antibiotics, cell wall hydrolases, and siderophores. Additionally, Bacillus spp. improve plant response to pathogen attack by triggering induced systemic resistance (ISR). Besides being the most promising biocontrol agents, Bacillus spp. promote plant growth via nitrogen fixation, phosphate solubilization, and phytohormone production. Antagonistic and plant growth-promoting strains of Bacillus spp. might be useful in formulating new preparations. Numerous studies of a wide range of plant species revealed a steady increase in the number of Bacillus spp. identified as potential biocontrol agents and plant growth promoters. Among different mechanisms of action, it remains unclear which individual or combined traits could be used as predictors in the selection of the best strains for crop productivity improvement. Due to numerous factors that influence the successful application of Bacillus spp., it is necessary to understand how different strains function in biological control and plant growth promotion, and distinctly define the factors that contribute to their more efficient use in the field.

1. Introduction

Plant diseases, caused by various microorganisms, including fungi, bacteria, viruses, nematodes and protozoa, affect agricultural production and result in major yield losses [1]. Approximately 20–40% of losses in crop yield are caused by pathogenic infections [2]. Different strategies have been used to reduce the occurrence of plant diseases including pesticides, less susceptible cultivars, crop rotation, and other control measures, but their efficacy is usually insufficient due to the survival and resistance of soil-borne pathogens [3]. Moreover, the excessive use of synthetic pesticides has adverse effects on the environment and living organisms, and also disturbs ecosystem functioning and decreases agricultural sustainability [4].

Nowadays, research is directed to environmentally friendly alternatives for controlling plant pathogens and improving crop production, which are recommended within an integrated crop management system (ICMS) [5]. As an important component of an ICMS, biological control is defined as the use of beneficial organisms to reduce the negative effects of plant pathogens and promote positive responses by the plant [6]. The most common approach to biological control is the selection of antagonistic microorganisms, studies on their mechanisms of action and development of a biocontrol preparation [7].

Bacillus species are among the most investigated biocontrol agents i.e., biopesticides which contribute to suppression of plant pathogens by antagonism and/or competition [8]. Inhibition of pathogen growth by Bacillus spp. entails the involvement of mechanisms such as competition for nutrients and space, production of antibiotics, hydrolytic enzymes, siderophores, and/or inducing systemic resistance [9]. Bacillus spp. also can act as biofertilizers or biostimulators, either by facilitating the uptake of certain nutrients from the environment (nitrogen fixation, phosphate solubilization), or by providing the plant with a compound (biosynthesis of plant hormones) [10].

Hence, Bacillus spp. represent an alternative to plant growth enhancement agrochemicals, i.e., synthetic pesticides and fertilizers. The beneficial effects of Bacillus spp. on plant growth and yield have been demonstrated in several agricultural crops including wheat, maize, soybean, sunflower, common bean, tomato, pepper, potato, cucumber, and many others [11]. Application of Bacillus spp. to increase the productivity of field and vegetable crops is limited by variability among the results obtained in the laboratory, greenhouse and field [12]. In fact, when reintroduced by plant/soil inoculation, only 1 to 2% of rhizobacteria exert a beneficial effect on plant growth [13]. Due to numerous factors that affect the effectiveness of Bacillus spp., it is necessary to understand how different strains deploy biocontrol and growth-promoting actions in plants, and clearly determine the traits and manner for selecting the best acting strains [12].

This review summarizes the different mechanisms utilized by Bacillus spp. in plant disease control and plant growth promotion, and focuses on the new approaches to the improvement of beneficial plant–Bacillus interactions and Bacillus spp. efficiency in the field.

2. Genus Bacillus

The genus Bacillus was established in 1872 by Cohn and encompasses over 200 described species and subspecies belonging to the Firmicutes phylum. Based on the morphological characteristics, bacteria of this genus are described as rod-shaped, Gram-positive, aerobic or facultatively anaerobic, and catalase-positive [14]. Due to their broad physiological ability and capability to form endospores, Bacillus spp. are resistant to adverse environmental conditions and omnipresent in a wide range of habitats, including soil. Bacillus spp. represent the predominant soil and rhizosphere bacteria, where they comprise up to 95% of the Gram-positive bacterial populations [15]. Furthermore, they are among the most widespread endophytic bacteria [16].

Bacillus is a large and diverse group of non-pathogenic and pathogenic bacteria. Most species of Bacillus, as well as their products, are considered safe for intended use in the environment [17]. These bacteria are preferred for commercialization for their ability to secrete several bioactive metabolites, produce extremely tolerant endospores, and grow rapidly in different media [18]. Consequently, they maintain viability and can be easily formulated and stored [19]. Populations of Bacillus spp. can successfully persist in the soil and plant rhizosphere without any perdurable effects on other bacterial populations [20]. Commercial Bacillus-based preparations are developed and distributed worldwide and contain beneficial strains of Bacillus subtilis, Bacillus amyloliquefaciens, Bacillus pumilus, Bacillus licheniformis, Bacillus megaterium, Bacillus velezensis, Bacillus cereus, Bacillus thuringiensis, etc., [21].

3. Mechanisms of Biological Control

3.1. Antibiotics

The antagonistic activities of Bacillus spp. are frequently related to the production of secondary metabolites with antibiotic properties. These compounds mainly involve peptides with low molecular weight that are generated ribosomally (bacteriocins) or non-ribosomally (lipopeptides, peptides, polyketides).

Bacteriocins are ribosomally synthesized peptides produced by numerous bacteria which might be useful against pathogenic and antibiotic-resistant bacteria [22]. Bacteriocins act against target cells by interfering with the synthesis of the cell wall or by forming pores in the cell membrane [23]. The antimicrobial mechanisms of bacteriocins are usually directed against the species which are the same or closely related to the producers, with a narrow spectrum of action. Nevertheless, due to the production of bacteriocins, Bacillus spp. exhibit a broad-spectrum of antibacterial activity [24]. Some reports identify bacteriocins and bacteriocin-like substances (BLSs) (amylolysin, amysin, subtilin, subtilosin A, subtilosin B, thuricin) isolated from various Bacillus spp., including B. amyloliquefaciens, B. subtilis, B. thuringiensis, B. cereus, and B. coagulans [25]. Isolation and characterization of bacteriocins and BLSs may have an important use in the biological control of the pathogenic bacteria. However, Bacillus spp., which produce non-ribosomally synthesized lipopeptides and peptides, exhibit much stronger antimicrobial activity [26].

Cyclic lipopeptides (LPs), well known for their antagonistic action against a wide range of plant pathogens, are the most thoroughly studied antibiotic compounds in Bacillus spp. [27]. These peptides are synthesized by large non-ribosomal peptide synthetases (NRPSs) [28]. The primary mechanisms of LPs’ actions usually involve an interaction with the cell membrane of the target pathogens, causing changes in its structure and permeability through disruption, solubilization or formation of ion-conducting pores [26]. It has also been demonstrated that LPs interact with intracellular structures, such as DNA [29]. Recent investigations have shown that LPs influence colonization and persistence of Bacillus in the rhizosphere, and stimulate plant defense mechanisms [30].

The most important cyclic LPs from Bacillus spp. are represented by surfactin, iturin and fengycin families [27]. These antibiotics consist of amino acids, amino- or hydroxyl-fatty acids with different lengths of hydrocarbon chains. The surfactin family (surfactin, lichenysin, pumilacidin, halobacilin, bamilocyn) are heptapeptides, identified in B. subtilis, B. amyloliquefaciens, B. licheniformis, B. pumilus and Bacillus coagulans. Surfactins act as both antifungal and antibacterial agents. The iturin family (iturin, mycosubtilin, bacillomycin, bacillopeptins, mixirins, mojavensin, subtulene) consists of heptapeptides produced by B. subtilis, B. amyloliquefaciens, Bacillus circulans, B. pumilus, and Bacillus vallismortis. Iturins display inhibitory effects on a wide range of fungi, but are less active against bacteria. The fengycin family (fengycin, plipastatin, maltacin) are decapeptides of which production was detected in B. subtilis and B. amyloliquefaciens. Fengycins are useful in protecting plants against fungal pathogens [26,27,28].

Other non-ribosomally synthesized LPs include kurstakins, bacitracins, polymyxins, gramicidins, and tyrocidines. The kurstakins are cyclic or linear heptapeptides that are specific to the B. cereus and B. thuringiensis, with the ability to destabilize biological membranes of both bacteria and fungi [31]. Bacitracins are cyclic decapeptides produced by B. licheniformis, B. subtilis, and B. sonorensis, of which activity is primarily directed against Gram-positive bacteria [32]. Polymyxins are cyclic decapeptides produced by Paenibacillus polymyxa (Bacillus polymyxa), which inhibit Gram-negative bacterial cells. Gramicidins and tyrocidines are cyclic decapeptides produced by Bacillus brevis, active against a broad range of Gram-negative and Gram-positive bacteria [33].

Several Bacillus spp. are known to produce other non-ribosomally synthesized antibiotics, such as peptides (bacilysin, rhizocticin, amicoumacin, mycobacillin, diketopiperazines) and polyketides (bacillaene, dihydrobacillaene, difficidin, macrolactin), with diverse antifungal and antibacterial activities [34].

The most commonly used biocontrol agents, B. subtilis and B. amyloliquefaciens, dedicate 4–5% and 8.5% of total genetic capacity to synthesis of secondary metabolites, with the potential to produce more than two dozen structurally diverse antimicrobial compounds [27,35]. Nowadays, gene clusters encoding for bacteriocins, as well as peptides and polyketides, can be readily identified in genomic sequences by genome mining. In total, 583 putative bacteriocin gene clusters were identified from 328 strains of 57 Bacillales species, while 1231 putative non-ribosomal antimicrobial gene clusters were detected and sub-grouped into 23 types of peptide and five types of polyketide compounds distributed over 49 species of Bacillales [36].

Numerous studies revealed a broad antimicrobial effect by Bacillus spp. due to production of antibiotics (

Table 1. Biocontrol mechanisms exhibited by Bacillus species.

Bacillus Species	Mechanism (s)	Target Pathogen (s)	Plant Disease	Reference
Bacillus amyloliquefaciens	Bacteriocins, surfactin, fengycin	Various pathogenic bacteria, Fusarium oxysporum, Fusarium avenaceum, Mucor sp.	Several diseases of field and vegetable crops	[24]
Bacillus amyloliquefaciens	Iturin	Verticillium dahliae	Wilt of cotton	[30]
Bacillus amyloliquefaciens/Bacillus subtilis	Iturin, surfactin/surfactin, fengycin	Fusarium graminearum	Head blight of wheat	[39]
Bacillus subtilis	Surfactin, Lytic enzymes	Fusarium spp.	Clove rot of garlic	[40]
Bacillus subtilis	Iturin, fengycin	Pythium ultimum	Damping-off of bean	[41]
Bacillus sp., Bacillus amyloliquefaciens	Surfactin, iturin, fengycin, siderophore	Sclerotinia sclerotiorum	White mold of common bean	[42]
Bacillus pumilus, Bacillus amyloliquefaciens	Lipopeptides	Pseudomonas syringae pv. aptata	Leaf spot disease of sugar beet	[43]
Bacillus subtilis	Iturin, surfactin, plipastatin, bacillomycin, difficidin	Gaeumannomyces graminis var. tritici	Take-all of wheat	[44]
Bacillus licheniformis	Surfactin	Magnaporthe grisea	Blast disease of rice	[45]
Bacillus subtilis	Chitinase	Rhizoctonia solani	Stem canker and black scurf of potato	[47]
Bacillus amyloliquefaciens	Protease	Fusarium oxysporum f. sp. lycopersici	Wilt disease of tomato	[48]
Bacillus amyloliquefaciens	Siderophores, cellulase, lipase, protease, chitinase	Clavibacter michiganensis	Bacterial canker of tomato	[49]
Bacillus sp.	Protease, glucanase, chitinase, siderophores	Fusarium verticillioides	Stalk and ear rot of maize	[50]
Bacillus velezensis	Protease, Chitinase, Cellulase, Glucanase	Botrytis cinerea	Gray mold disease of pepper	[51]
Bacillus subtilis	Siderophores	Fusarium oxysporum f.sp. capsici	Wilt of pepper	[52]
Bacillus subtilis	Siderophores, lytic enzymes	Bipolaris sorokiniana	Spot blotch of wheat	[53]

). Bacillus spp. mostly produce LPs from one family, while a few strains were identified as co-producers of different LPs [37]. Furthermore, antimicrobial activity of Bacillus spp. relies on the proportion and diversity in the production of antibiotics [38]. Fusarium clove rot of garlic, as well as head blight of wheat, were successfully suppressed by B. subtilis and/or B. amyloliquefaciens, due to LPs production [39,40]. In another study, B. amyloliquefaciens was defined as a producer of bacteriocins, surfactin, and fengycin, and was proven as a very potent biocontrol agent against numerous Gram-positive and Gram-negative bacteria, as well as Fusarium oxysporum, Fusarium avenaceum, and Mucor sp. [24]. Ongena et al. [41] found that iturin and fengycin produced by B. subtilis, which contributed to antifungal activity against Pythium ultimum. Han et al. [30] showed that iturin-producing B. amyloliquefaciens was effective in the biocontrol of Verticillium dahliae. Similarly, lipopeptides from Bacillus sp. and B. amyloliquefaciens such as surfactin, iturin, and fengycin, were responsible for antifungal activity against Sclerotinia sclerotiorum [42]. When tested for its biocontrol potential, B. amyloliquefaciens and B. pumilus LPs producing strains were very effective in the reduction in Pseudomonas syringae pv. aptata infection of sugar beet [43]. Additionally, Yang et al. [44] established that B. subtilis was able to suppress Gaeumannomyces graminis var. tritici infection of wheat through the production of LPs, namely iturin, surfactin, plipastatin, bacillomycin, and difficidin. Antifungal lipopeptide produced by B. licheniformis, determined as surfactin, was very successful against Magnaporthe grisea, a causative agent of rice blast [45]. Antifungal activity against Rhizoctonia solani, Pythium aphanidermatum, and Sclerotium rolfsii was attributed to B. pumilus because of the production of lipopeptide pumilacidin from the surfactin family [46].

3.2. Lytic Enzymes

Antimicrobial activity of Bacillus spp. could also be due to the production of hydrolytic enzymes such as chitinases, chitosanases, glucanases, cellulases, lipases, and proteases, which efficiently hydrolyze the major components of the fungal and bacterial cell walls.

Chitinases (EC 3.2.1.14) are glycoside hydrolases (GHs) which degrade the β-1,4-glycosidic bonds in chitin, the second most abundant naturally available polysaccharide after cellulose, and the main component of the fungal cell wall [54]. Bacteria primarily produce chitinases in order to degrade chitin for its utilization as an energy source, whereas some bacterial chitinases are prospective biological control agents against a variety of plant diseases caused by phytopathogenic fungi [47,55]. Chitosanases (E.C. 3.2.1.132) are GHs which catalyze the hydrolytic degradation of the β-1,4-glycosidic bonds in the chitin derivative-chitosan [56]. Chitosanases are important for the extensive carbon and nitrogen recycle [57]. Since chitosan is also found in fungal cell walls, chitosanase-producing Bacillus spp. may be used as biocontrol agents to prevent plant infection caused by pathogens [58]. Glucanases are GHs which hydrolyze glycosidic bonds present in α-glucans and β-glucans. α-1,3-glucan is not indispensable cell wall component, but plays an important role in some fungi during cell separation and vegetative growth [59], while β-1,3-glucan is the second major component of the fungal cell wall after chitin [60]. The primary role of cell wall glucans in fungi is structural, but they may also be degraded and used as nutritional sources. Bacillus spp. are a rich source of α-1,3-glucanase (EC 3.2.1.84) and β-1,3-glucanase (EC 3.2.1.39). The enzymes have previously been isolated from Bacillus brevis, B. licheniformis, B. subtilis, B. circulans, and Bacillus halodurans [61]. Besides chitin and glucan, the skeleton of fungal cell walls contains cellulose, lipids and proteins. Bacterial cellulases, lipases and proteases may, therefore, play a significant role in the cell wall lysis that occurs during pathogen–Bacillus interactions [48].

Successful cell wall degradation depends on the activity of more than one enzyme. Chitinase activity is preceded by, or coincides with, the hydrolytic activity of other enzymes, especially glucanases. Mixtures of hydrolytic enzymes with complementary modes of action may be required for maximum efficacy, while correct combinations of enzymes may increase antifungal activity [62].

Recently, several reports have documented the production of lytic enzymes from Bacillus spp. biocontrol agents (Table 1). Chitinase-producing B. subtilis was effective against Rhizoctonia solani [47]. Crude and purified protease of B. amyloliquefaciens showed efficacy in biocontrol of Fusarium oxysporum [48]. The potential of B. amyloliquefaciens for biocontrol of Clavibacter michiganensis ssp. michiganensis was attributed to the production of lytic enzymes (cellulase, lipase, protease, chitinase) [49]. Hydrolytic enzymes (protease, glucanase, chitinase) produced by Bacillus sp. were responsible for a strong inhibitory activity against Fusarium verticillioides causing stalk and ear rot of maize [50]. The strength of hydrolase activity (protease, chitinase, cellulase, glucanase) was the key factor of B. velezensis in control of pepper gray mold caused by Botrytis cinerea [51]. Generally, it has been found that strains of Bacillus spp. which have the ability to produce cell wall hydrolases are more effective in the suppression of plant pathogens [63]. In search of efficient biocontrol agents, isolation and characterization of enzyme-producing Bacillus spp. should be done in order to achieve maximum survival of bacteria under detrimental environmental conditions and intrusion of pathogens [40,64].

3.3. Siderophores

Siderophores are metal-chelating, non-ribosomal peptides with low molecular weight produced by some microorganisms and plants, especially under iron starvation conditions [65]. Iron (Fe) is an essential element for different biological processes such as oxygen metabolism, DNA and RNA syntheses, electron transfer, and enzymatic processes. The primary role of siderophores is to chelate Fe, allowing its solubilization and extraction from minerals and organic compounds. The significance of siderophores in biological control is based on competition for Fe in order to reduce its availability for pathogens [9]. Furthermore, microbial siderophores can be reduced to donate Fe to the transport system of a plant or chelate Fe from soils, and then, do a ligand exchange with phytosiderophores, thus, providing plants with this essential element so as to enhance their growth [66]. In addition to Fe, siderophores also have the ability to bind a variety of metals in the environment, thereby acting as bioremediation agents [67].

Siderophores are grouped into three main families, depending on the functional group, including hydroxamates, catecholates, and carboxylates [9]. Most of the bacterial siderophores are catecholates, such as bacillibactin produced by several Bacillus spp. including B. subtilis, B. amyloliquefaciens, B. cereus, B. thuringiensis, etc., [68]. Besides bacillibactin, Bacillus spp. produce a wide variety of siderophores such as pyoverdine, pyochelin, schizokinen, petrobactin, etc., [69]. Bacillus spp. were better producers of siderophores than other bacterial isolates from the maize rhizosphere [70]. Siderophores produced by Bacillus spp. have been involved in suppression of several plant diseases (Table 1). For instance, siderophore-producing B. subtilis reduced the incidence of Fusarium wilt, and enhanced the growth and yield of pepper [52]. Several studies indicated synergistic antimicrobial effects of siderophores along with lipopeptides and/or lytic enzymes [42,49,50]. Similarly, B. subtilis is a promising biological control agent against Bipolaris sorokiniana due to production of siderophores, chitinase, and cellulase [53].

4. Systemically Induced Disease Resistance

Plants adapt to constant pathogen exposure through defense mechanisms. Resistance to pathogens, developed after proper stimulation, represents an improvement in the defense capacity of the plant. Infected plants increased their levels of signaling molecules which coordinate the activation genes for appropriate syntheses, followed by preventive structural and histological changes, preventative chemical substances (phenols and other products of secondary metabolism), and in other ways [71,72].

Plant defense mechanisms, such as induced systemic resistance (ISR), can be initiated by external agents before infection or triggered by a localized infection, resulting in systemic acquired resistance (SAR) [73]. Both biotic and abiotic factors have been used for inducing ISR in plants against different plant pathogens. ISR is promoted by non-pathogenic rhizobacteria, and is mostly dependent on the jasmonate (JA) and/or ethylene (ET) signaling pathways [74], while SAR is mediated via a salicylic acid (SA)-dependent process. SAR also activates specific sets of defense-related genes associated with the production of pathogenesis-related proteins (PR), while ISR is not accompanied by the activation of these genes [75]. The defense mediated by ISR is significantly weaker than that obtained by SAR. However, ISR and SAR together provide better protection, indicating that they can act additively in inducing resistance to pathogens [72].

Rhizobacteria promote ISR in plants through the production of various metabolites such as antibiotics, siderophores, volatile organic compounds (VOCs), and others [76]. Bacillus spp. are among the most studied rhizobacteria that trigger ISR in plants (

Table 2. Induced systemic resistance elicited by Bacillus species.

Bacillus Species	Target Pathogen (s)	Plant Disease	Reference
Bacillus amyloliquefaciens	Tomato spotted wilt virus, Potato virus Y	Wilt disease of tomato	[78]
Bacillus cereus	Botrytis cinerea	Gray mold disease of field and vegetable crops	[79]
Bacillus subtilis	Rhizoctonia solani	Sheath blight of rice	[80]
Bacillus amyloliquefaciens	Xanthomonas axonopodis pv. vesicatoria	Leaf spot disease of pepper	[81]
Bacillus megaterium	Mycosphaerella graminicola	Septoria tritici blotch of wheat	[82]
Bacillus subtilis, Bacillus amyloliquefaciens	Fusarium moniliforme	Ear, stalk, and root rots of maize	[83]
Bacillus subtilis	Alternaria solani, Phytophthora infestans	Early and late blight of tomato	[84]
Bacillus spp.	Pyricularia oryzae	Blast disease of rice	[85]
Bacillus sp.	Colletotrichum capsica	Anthracnose of chili	[86]
Bacillus sp.	Rhizoctonia solani, Fusarium oxysporum	Root rot and wilt of soybean	[87]
Bacillus subtilis	Fusarium oxysporum f. sp. cucumerinum	Root rot of cucumber	[88]
Bacillus sp.	Plasmopara halstedii	Downy mildew of sunflower	[89]

), while being capable of inducing resistance against several pathogens in the same plant [77]. B. amyloliquefaciens induced salicylic acid-dependent resistance in tomato plants, reduced the incidence of Tomato spotted wilt virus, and delayed systemic accumulation of Potato virus Y [78]. Application of B. cereus significantly reduced disease incidence caused by Botrytis cinerea through activation of ISR [79]. Chandler et al. [80] showed that B. subtilis triggered ISR in rice against Rhizoctonia solani via jasmonic acid (JA) and ethylene (ET), as well as abscisic acid (ABA) and auxin signaling. The same authors reported an indispensable role of B. subtilis LPs, namely fengycin and surfactin, in the induced defense state. ISR promoting B. amyloliquefaciens produced VOCs and significantly reduced spot disease caused by Xanthomonas axonopodis pv. vesicatoria in pepper [81]. The ability of B. megaterium to reduce Septoria tritici blotch severity, caused by Mycosphaerella graminicola, was the result of a combination of different mechanisms, including ISR [82]. Bacillus endophytes of maize may protect host plants by producing antifungal lipopeptides that inhibit Fusarium moniliforme as well as by inducing the systemic acquired resistance [83].

Bacillus spp. can elicit ISR by inducing the synthesis of antioxidant defense enzymes. Host enzymes induced by B. subtilis include peroxidase (POX), polyphenol oxidase (PPO), phenylalanine ammonia-lyase (PAL), and superoxide dismutase (SOD). Increased synthesis of antioxidant defense enzymes results in ISR against early and late blight in tomato seedlings [84]. Similarly, Rais et al. [85] showed that Bacillus spp. enhanced the SOD, POX, PPO, and PAL activities in infected rice, thus, alleviating Pyricularia oryzae-induced oxidative damage and suppressing blast disease incidence. The antagonistic Bacillus sp. suppressed anthracnose disease of chili by the activation of defense-related enzymes and the accumulation of phenolic compounds [86]. Similarly, Bacillus sp. enhanced growth promotion and protection against Rhizoctonia solani and Fusarium oxysporum by the eliciting of defense-related enzymes (PAL, POX, PPO) in soybean [87], while B. subtilis was capable of impairing disease incidence, promoting seedling growth and increasing activities of antioxidant enzymes (POD, PPO, PAL) in cucumber plants [88]. The induction of resistance to Plasmopara halstedii by Bacillus sp. strain was accompanied by the accumulation of defense-related enzymes (PAL, POX, PPO) in sunflower [89].

5. Mechanisms of Plant Growth Promotion

5.1. Nutrient Availability

Bacillus spp. produce numerous metabolites which can increase nutrient availability to plants, and thus, directly promote plant growth and yield. Most of the plant essential nutrients are supplied through mineral fertilization, a practice which causes major economic losses, as well as posing significant problems to the environment. The use of biofertilizers which contain N₂-fixing and/or P-solubilizing Bacillus spp. is a reasonable approach to reducing the negative impacts of synthetic fertilizers without compromising food safety [5,17]. N₂-fixing and P-solubilizing Bacillus spp. are directly related to nutrient uptake and the subsequent growth promotion in different plants (

Table 3. Plant growth-promoting mechanisms exhibited by Bacillus species.

Bacillus Species	Mechanism (s)	Treated Plant (s)	Effect	Reference
Bacillus pumilus	N2-fixation	Maize	Increase the total N content and dry biomass	[93]
Bacillus sp.	N2-fixation	Maize	Increased seed germination and root volume	[96]
B. megaterium, B. subtilis, B. simplex	P-solubilization	Eggplant, pepper, tomato	Promoted seed germination and vegetative growth	[98]
Bacillus subtilis	P-solubilization	Cucumber	Increased plant growth, total accumulation of P and P uptake	[99]
Bacillus subtilis	IAA, GA	Tomato	Enhanced shoot and root growth, seedling vigor and leaf area, higher levels of hormones	[84]
Bacillus methylotrophicus	GAs	Lettuce	Increased shoot length, shoot fresh weight, leaf width, proteins, amino acids, macro and micro minerals, carotenoids and chlorophyll a	[100]
Bacillus subtilis	CKs	Lettuce	Increased plant shoot and root weight, higher CKs levels	[101]
Bacillus megaterium	CKs	Common bean	Promoted growth of seedlings	[102]
Bacillus spp.	IAA, CKs, GAs, ABA	Soybean	Better growth and higher proline contents	[103]
Bacillus subtilis	IAA, ACC deaminase	Tomato	Increased shoot and root biomass and chlorophyll (a and b) contents	[104]
Bacillus aryabhattai	ABA, IAA, CKs, GAs	Soybean	Longer roots and shoots, higher hormone levels, better stress tolerance	[105]
Bacillus amyloliquefaciens	ABA	Rice	Increased growth and stress tolerance	[106]

).

Nitrogen (N) is essential for plant growth, albeit largely unavailable in its atmospheric form (more than 80%) [90]. Biological nitrogen fixation (BNF) is carried out by several groups of microorganisms that are able to absorb elemental nitrogen from the atmosphere and form compounds, which serve as plant nutrients [91]. The microorganisms produce the enzyme nitrogenase in order to catalyze the conversion of molecular dinitrogen (N₂) to ammonia (NH₃), which is subsequently taken by plant roots and assimilated in amino acids. BNF provides Earth’s ecosystems with about 200 million tons N per year [92]. The nitrogen-fixing microorganisms are either free-living or symbiotic. Several PGPR, including Bacillus spp. can decrease chemical fertilizer-N use and increase plant growth and yield through asymbiotic nitrogen fixation. BNF by rhizobacteria has been reported to contribute up to 12–70% of total N uptake in agricultural crops. The study of Kuan et al. [93] provided evidence that B. pumilus can fix atmospheric N₂ and significantly increase the total N content and dry biomass of maize. Ding et al. [94] suggested that the nifH gene could be detected in both the Bacillus and Paenibacillus genera. Similarly, the study of Xie [95] reported nitrogenase activities of several Bacillus spp. including B. megaterium, B. cereus, B. pumilus, B. circulans, B. licheniformis, B. subtilis, B. brevis, and B. firmus. Szilagyi-Zecchin et al. [96] reported that endophytic Bacillus spp. were positive for the nitrogen fixation ability evaluated through the acetylene reduction assay and amplification of nifH gene. Increased relative abundance of Bacillus spp. in rice plants under the conditions of low nitrogen suggest the potential contribution of their BNF [97].

In addition to nitrogen, the plant growth directly depends on phosphorus (P). However, a high amount of P (more than 80%) is fixed in soil and is unavailable for plant uptake due to adsorption, precipitation or conversion [107]. Microorganisms that dissolve organic and inorganic phosphates belong to the group designated as Phosphate Solubilization Microorganisms (PSM) [108]. These microorganisms solubilize insoluble inorganic P and mineralize insoluble organic P [109]. Mechanisms of inorganic phosphate solubilization by microorganisms involve the production of organic and inorganic acids, siderophores, protons, hydroxyl ions, and CO₂, which chelate cations or reduce pH in order to release P [110]. Mineralization of organic phosphate occurs due to the synthesis of extracellular enzymes such as phosphatases, phytases, and phospholipases [111].

Plant/soil inoculation with PSM is a promising strategy for the enhancement of plant absorption of P, while Bacillus spp. are among the most powerful PSM. Saeid et al. [112] showed that solubilizing exudates produced by Bacillus (B. subtilis, B. megaterium, B. cereus) are composed of gluconic, lactic, acetic, and succinic acids, confirming strong correlation between the total concentrations of organic acids and the amounts of released phosphorus. Isolates of B. megaterium, B. subtilis, and B. simplex, exhibited P-solubilizing ability by producing acetic, propionic, isobutyric, isocaproic, caproic, and heptanoic acids, and had positive effects on the seed germination and vegetative growth parameters of eggplant, pepper, and tomato [98]. Tao et al. [113] suggested that P-solubilization and P-mineralization could coexist in the same Bacillus strain. Similarly, inoculation with B. subtilis increased plant growth, and total accumulation of P and P uptake by cucumber plants [99].

5.2. Phytohormone Production

Bacillus spp. may directly increase plant yield through mechanisms that impart the production of phytohormones or plant growth regulators (PGRs), such as auxins, cytokinins, gibberellins, ethylene, and abscisic acid. Plant hormones are organic substances that influence the physiology and development of plants at very low concentrations. Plant hormone biosynthesis by Bacillus spp. has been directly related to subsequent growth promotion in different plants (Table 3).

Auxins are a group of plant hormones that stimulate plant growth, mainly through the regulation of cell division, cell elongation, and tissue differentiation. The main naturally occurring auxin is indole-3-acetic acid (IAA) [114]. Different bacteria, including Bacillus spp., have the ability to produce IAA and use this phytohormone to interact with plants as part of their colonization strategy, including phytostimulation and circumvention of basal plant defense mechanisms [80]. Production of IAA is widespread among soil bacteria, and approximately 80% of rhizobacteria have been estimated to produce IAA [115]. The in vitro application of IAA-producing Bacillus strains on plant roots resulted in increases in root length as well as the number of lateral roots [116]. B. subtilis was reported to enhance shoot and root growth, seedling vigor and leaf area of tomato, while higher levels of gibberellins and IAA were detected in treated plants [84]. Recent studies demonstrated that Bacillus spp. play a major role in controlling endogenous IAA levels in plant roots by regulating the auxin-responsive genes, thereby causing changes in root architecture [117].

Gibberellins (GAs) are a group of plant hormones that affect many developmental processes in higher plants, including seed germination, stem elongation, flowering, and fruiting. Gutierrez-Manero et al. [118] documented the production of gibberellins by B. pumilus and B. licheniformis. The beneficial effect of Bacillus methylotrophicus on plants due to the secretion of an array of gibberellins was confirmed by increasing the percentage of seed germination of lettuce, muskmelon, soybean, and vegetable mustard [100]. The same authors established that GA-producing bacterial strain increased shoot length, shoot fresh weight, leaf width, proteins, amino acids, macro and micro minerals, carotenoids and chlorophyll in lettuce.

Cytokinins (CKs) are a group of plant hormones that play a key role in promoting cell division, or cytokinesis, in plant roots and shoots. They are important regulators of other physiological and developmental plant processes such as seed germination, apical dominance, nutrient mobilization, and leaf senescence. Plants and microorganisms produce about 30 compounds from the group of CKs. It has been found that 90% of phosphate-dissolving rhizobacteria have the ability to produce CKs in vitro [119]. Arkhipova et al. [101] reported the ability of B. subtilis to produce CKs, while inoculation of lettuce plants increased the cytokinin content of both shoots and roots, as well as plant shoot and root weight. Ortíz-Castro et al. [102] reported that B. megaterium promoted the growth of Arabidopsis thaliana and Phaseolus vulgaris seedlings through CKs production. Naz et al. [103] also perceived that cytokinin-producing species, such as Bacillus and others, stimulated the growth of soybean plants.

Ethylene is a gaseous plant hormone that mainly regulates maturation and senescence processes, as well as response to biotic and abiotic stresses. In addition to plants, ethylene production was established in bacteria and fungi, but little has been reported on how ethylene-producing microorganisms affect plant growth. Several PGPR, including Bacillus spp., synthesize the enzyme 1-aminocyclopropane-1-carboxylate (ACC) deaminase that modulates ethylene levels in plants which might otherwise become growth inhibitory [120]. The enzyme ACC deaminase (3.5.99.7) cleaves ACC (direct precursor of ethylene biosynthesis in plants) into ammonia and α-ketobutyrate. Bacteria characterized by ACC deaminase activity can help maintain plant growth and development under stress conditions (drought, salt, flooding and anoxia, the presence of pathogens or contaminants) [121]. The interaction of plants with ACC deaminase-producing bacteria may be expected to promote plant growth during plant processes associated with local increase in ethylene concentration, like flower wilting or symbiosis establishment [122]. Although ACC deaminase activity has been described in many Bacillus strains [104,123], ACC deaminase genes (structural gene acdS and the regulatory gene acdR) could not be identified in 271 completely sequenced strains belonging to the Bacilli class, including many soil and plant-associated Bacillus and Paenibacillus species [124].

Abscisic acid (ABA) is a plant hormone with an important role in many plant physiological processes, including seed germination and stress tolerance. Park et al. [105] showed that Bacillus aryabhattai produced significant amounts of ABA, IAA, CKs, and GAs in culture, while inoculated soybean plants had high levels of phytohormones, longer roots and shoots, and better tolerance to heat, oxidative, and nitrosative stress. The bacterial endophyte B. amyloliquefaciens has been found to produce ABA and increase plant growth and resistance to salinity stress [106].

6. Efficient Use and New Approaches

6.1. Isolation and Identification

Prior to characterization and selection in laboratory and in greenhouse/field conditions, the search for effective strains requires isolation and identification of preferred Bacillus species from different sources. Bacillus spp. are the predominant soil, rhizosphere and endophytic bacteria [15,16]. Considering a very small proportion of beneficial microorganisms in the rhizosphere, their isolation, multiplication, and inoculation into the plant/soil trigger microbiological processes and intensify overall microbial activity [70]. Thus, only a few Bacillus spp. of about 200 within the genus exhibit multiple plant growth-promoting traits and might be useful in formulating inoculants [21]. Identification of isolated Bacillus spp. is of great importance because their beneficial traits are characteristic of certain species. Accordingly, it is necessary to use methods that can quickly and reliably test a large number of Bacillus spp. as the potential plant growth promoters and biological control agents.

Determination of morphological, physiological and biochemical traits is a long and often unreliable process. The most accurate method for examining the diversity of Bacillus spp. is their identification and characterization at the molecular level. The NCBI (National Center for Biotechnology Information) and RDP (Ribosomal Database Project) databases contain 2611 individual 16S rDNA sequences originating from 175 different species of Bacillus, of which only 1586 have been identified to the species level [125]. In addition to standard molecular methods such as 16S rRNA analysis, RFLP (Restriction Fragment Length Polymorphism), RAPD (Random Amplified Polymorphic DNA), rep-PCR (Repetitive element sequence-based Polymerase Chain Reaction), MLSA (Multilocus Sequence Analysis), etc., different PCR methods with species-specific primers are increasingly used for reliable differentiation of Bacillus spp. [126].

6.2. Characterization and Selection

The characterization of bacteria includes determination of numerous traits in laboratory, while selection of potential biofertilizers and biopesticides involves testing their effectiveness on the plant–soil system in greenhouse and/or field. The tests require a lot of time, which makes it impossible to examine a large number of strains. Given that no individual or combined traits can reliably predict the effectiveness in biocontrol and plant growth promotion, Akinrinlola et al. [90] suggested greenhouse pot tests as the first criterion for bacterial strain selection, instead of screening bacteria for multiple traits. According to reports, the effectiveness of Bacillus spp. frequently varies depending on specific plant and soil conditions, which can constrain their ability to colonize the rhizosphere and express beneficial traits [12].

The efficiency of inoculation is usually higher when bacteria are isolated from the rhizosphere of plant species, and/or soil that will be inoculated, suggesting that the growth-promoting ability of the strains is highly related to certain plant species and soil types [12]. Furthermore, the efficiency of Bacillus spp. from the rhizosphere is higher compared to those from the bulk soil, while both the rhizosphere and endophytic bacteria possess various beneficial traits regarding the number and the production amount of these characteristics [127]. Knowledge of biocontrol and plant growth promotion mechanisms of Bacillus spp. is very important for their intended use, for instance, the use of LPs and hydrolase-producing strains in the suppression of pathogen infection, or P-solubilizing and N₂-fixing strains in P and N-deficient soils. Their efficiency as individual and combined plant/soil inoculants in different environments needs to be established through continuous selection of effective isolates in greenhouse and field trials.

6.3. Plant–Bacillus Interactions

Successful application of Bacillus spp. in the field also depends on plant–Bacillus interactions and it can be limited by poor colonization of the rhizosphere [128]. Bacillus spp. require 24 h to form a biofilm, which contributes to root colonization of Bacillus spp. and extends their beneficial effects in the soil [129]. Transcriptomic analysis of the B. amyloliquefaciens genome revealed numerous genes included in rhizosphere habituation and plant-beneficial traits, such as plant polysaccharide utilization, cell motility and chemotaxis, secondary antibiotics synthesis, and plant growth promotion-relevant clusters [130]. Gao et al. [128] demonstrated that both chemotaxis and swarming motility are important in tomato root colonization by B. subtilis, while the part of swarming is greater than that of chemotaxis.

However, root colonization is more effectual in indigenous strains of Bacillus than in laboratory or commercial strains. Emerging strategies such as microbiome engineering and breeding of microbe-optimized crops can directly or indirectly detect, modulate and enhance the traits and ways for better performance of Bacillus strains [3,18]. The genes involved in root colonization and plant–Bacillus interactions, are induced by the presence of root and seed exudates [129,130,131]. New research in plant–bacteria interactions uncovers plant capability to shape their rhizosphere and endorhiza microbiome [127]. Recent studies of the rhizobiome and the utilization of next-generation sequencing (NGS) techniques, combined with proteomics, metagenomics, metabolomics, etc., will assist to elaborate on these interactions, including how this relationship affects plant health and growth [132].

6.4. Bacillus-Based Preparations

In recent years, the distribution of commercial Bacillus-based preparations has significantly increased worldwide (

Table 4. Examples of commercial Bacillus-based preparations.

Bacillus Species	Preparation	Plant (s)	Company
Bacillus subtilis	Serenade®	Vegetables, fruits	AgraQuest Inc., USA
Bacillus subtilis	Companion®	Legumes, vegetables, maize, and others	Growth Products Ltd., USA
Bacillus subtilis	Kodiak®	Legumes, cotton, and others	Gustafson Inc., USA
Bacillus subtilis	Cease®	Several crops	BioWorks Inc., USA
Bacillus subtilis	Subtilex®	Vegetables, legumes, cotton, and others	Becker Underwood, Inc., USA
Bacillus subtilis	Pro-Mix®	Soybean, ornamentals, and others	Premier Horticulture Inc., Canada
Bacillus subtilis	FZB24®	Several crops	ABiTEP GmbH, Germany
Bacillus subtilis	Bio Safe®	Legumes, vegetables, cotton	Lab. Biocontrole Farroupilha, Brazil
Bacillus subtilis	Ecoshot®	Vegetables, legumes, fruits, and others	Kumiai Chemical Industry, Japan
Bacillus subtilis	Biosubtilin®	Cereals, vegetables, legumes, oilseeds, cotton, and others	Biotech International Ltd., India
Bacillus amyloliquefaciens	BioYield®	Legumes, vegetables, tobacco	Gustafson Inc., USA
Bacillus amyloliquefaciens	Rhizocell GC®	Cereals, sugar beet	Lallemand Plant Care, France
Bacillus amyloliquefaciens	RhizoVital®42, RhizoVital®42TB	Vegetables, cereals, ornamentals	ABiTEP GmbH, Germany
Bacillus pumilus	Ballad® Plus	Cereals, oilseeds, sugar beet, sweet corn	AgraQuest Inc., USA
Bacillus pumilus	Yield Shield®	Legumes, cereals, vegetables, sugar beet, cotton	Bayer CropScience, USA
Bacillus pumilus	Sonata®	Vegetables, fruits	AgraQuest Inc., USA
Bacilluslicheniformis	EcoGuard®	Several crops	Novozymes A/S Denmark, Novozymes Biologicals, USA
Bacillus velezensis	Botrybel®	Vegetables, fruits	Agricaldes, Spain
Bacillus megaterium	Symbion-P®	Cereals, legumes, oilseeds, vegetables	T. Stanes & Co. Ltd., India
Bacillus sp.	Sublic®	Several crops	ELEP Biotechnologies, Italy
Bacillus spp.	Bacillus SPP®	Several crops	Bio Insumos Nativa, Chile

). In addition to their beneficial influence on plants, effective strains of Bacillus spp. should be able to persist in the environment and be stable and viable for extended storage and purposeful use in the field. Resistance and stability are among the major limitations of Bacillus-based preparations. These bacteria are suitable for commercialization due to their ability to secrete various metabolites, produce endospores, and grow rapidly in different media [17,18,19,20]. Endospores of Bacillus spp. can not only endure adverse environmental conditions but survive all processing phases during production. In order to enhance sporulation and synthesis of preferable metabolites, production of Bacillus-based preparations should be optimized at each stage, which implies selection of appropriate strains or consortium of strains, as well as cultivation and formulation process [133].

Selection of appropriate Bacillus strains must be performed so as to avoid competition, especially if a preparation contains more than one species. For instance, interspecies competition between biofilms of the soil-residing bacteria B. subtilis and related Bacillus species could negatively affect their formulation and efficient use [134]. Nutrient sources such as carbon, nitrogen, inorganic salts and additional substances, as well as environmental factors such as temperature, pH value and O₂ supply, influence growth in addition to the production of spores and metabolites in Bacillus species [135]. Bacillus spp. are suitable for preparation as either solid or liquid formulations, with the addition of different carriers, stabilizers, protectants and other supplements [133]. Further research should find the best possible production technology for each bacterial strain or bacterial combination, while taking into account the cost-effectiveness of Bacillus-based products.

7. Conclusions

Bacillus spp. represent an environmentally friendly strategy for crop production improvement through different mechanisms of biological control, biofertilization and biostimulation. Although possibilities to use Bacillus spp. for disease incidence reduction and crop production improvement are well known, their application is not a widespread practice, mostly because of inconsistent efficiency under different conditions. The ability of Bacillus spp. to exhibit beneficial traits depends on the interaction of bacteria with plant and/or pathogen, and the environment. Given the great economic and ecological importance of Bacillus spp., it is necessary to increase the number of practically important species and find advanced methods for their rapid and comprehensive research and efficient application.