Nature’s Antimicrobial Arsenal: Non-Ribosomal Peptides from PGPB for Plant Pathogen Biocontrol

Abstract

Non-ribosomal peptides (NRPs) are a diverse group of bioactive compounds synthesized by microorganisms, and their antimicrobial properties make them ideal candidates for use as biocontrol agents against pathogens. Non-ribosomal peptides produced by Plant-Growth-Promoting Bacteria (PGPB) have gained interest for the biocontrol of plants’ bacterial and fungal pathogens. In this review, the structure and mode of action of NRPs, including their characterization and the characterization of NRP-producing microorganisms, are discussed. The use of NRPs in soilless agriculture and their potential as part of a sustainable plant disease control strategy are also highlighted. In addition, the review debates the commercial aspects of PGPB’s formulations and their potential as a biocontrol agent. Overall, this review emphasizes the importance of NRPs derived from PGPB in the biocontrol of plant pathogens and their potential to be used as an environmentally friendly and sustainable plant disease control strategy.

1. Introduction

Food is an essential requirement for human survival, and its production has been a matter of concern since prehistoric times. In the modern era, ensuring food security through sustainable strategies has become a major focus of the global research community [1]. Agriculture, as the main source of food, has undergone significant improvements in the last few decades, mainly emphasizing crop production and quality [2]. To meet the expanding demands of a growing population, advanced agricultural tools and improved crop varieties have also been introduced [3]. According to the Food and Agriculture Organization (FAO, 2021) of the United Nations Economic and Social Council, pests cause significant losses in global crop production, with estimates ranging from 20 to 40 per cent annually. Plant diseases alone cost the global economy around USD 220 billion each year, while invasive insects result in losses of around USD 70 billion. These losses have a significant impact on food security, economic stability, and the livelihoods of farmers worldwide [4].

When environmental factors impact a plant’s physiological processes, it can become vulnerable to infection, leading to changes in its structure, growth, and function, among other parameters [5]. Plant diseases are classified as infectious or non-infectious, depending on the nature of the disease-causing agents. Microorganisms such as bacteria, fungi, protozoa, and viruses can cause infectious diseases in plants. Bacteria, in particular, are ubiquitous and can be pathogenic to plants, animals, and humans. Many bacteria have genetic information contained in DNA molecules other than those of the bacterial chromosome, called plasmids, and although plasmid DNA does not carry the essential genetic information for the life of the bacterium, it does carry genes that give it new phenotypic properties and that, in some cases, are useful for their adaptation to growth in certain environments; for example, the b-lactamase enzymes that confer resistance to penicillins are encoded in plasmids [6]. It allows bacteria to acquire new traits rapidly, and it is also responsible for inducing antibiotic resistance among pathogenic bacteria [7].

In terms of evolution, fungi are much older than plants. The coexistence duration of plants and fungi can be compared to the evolutionary age of higher plants [8].

More than 90% of vascular plant species form some type of mycorrhizal association with fungi [9]. Mycorrhizal fungi form mutualistic associations with plant roots, providing nutrients and water to the plant in exchange for carbohydrates produced by the plant through photosynthesis [10].

This association is thought to have played a crucial role in the evolution of terrestrial plants, allowing them to colonize nutrient-poor soils and adapt to a terrestrial environment [11]. However, some fungi are also responsible for disturbing the balance between mutually beneficial relationships of plants and fungi by infecting the plant and acting as biotrophs, hemibiotrophs, or necrotrophs [12]. It has been observed that the majority of plant diseases are caused by soil-borne bacteria and fungi [13].

For the effective control of plant diseases caused by bacterial or fungal pathogens, the use of chemical agents has been popular. But, they do pose serious risks like resistance among pathogens, affecting non-target organisms, and entering the food chain to affect the ecosystem [14,15,16]. Primarily, the uncontrolled use of chemicals can permanently degrade soil quality. Furthermore, there are high economic costs associated with this approach, and several pesticide/insecticide-exposure-associated health problems can develop after they enter the food chain [15].

Therefore, it is imperative to devise efficacious and sustainable strategies to eliminate or curtail the use of synthetic and chemical disease control agents. In this regard, experts are focusing on microbial biocontrol as it offers environmentally friendly solutions [17,18]. Bacterial antagonists not only mitigate the deleterious effects of pathogens but also produce several organic compounds that are effective against a range of bacterial or fungal pathogens [19]. Of particular interest are microbial agents such as plant-growth-promoting bacteria (PGPB), which possess biological mechanisms to combat pathogenic growth [20]. These bacteria establish a symbiotic association with the plant, exerting beneficial effects by enhancing nutrient uptake, producing and releasing phytohormones, bolstering plant tolerance to environmental stressors, and suppressing plant diseases by adversely affecting phytopathogens [21]. Thus, these organisms are recognized as pivotal agents helpful in sustainable agriculture.

PGPBs colonize the rhizosphere and promote plant growth through various mechanisms and suppress plant diseases [22]. They are known to produce a variety of compounds that can be useful for the biocontrol of plant diseases [23,24] as well as combating the pollution load [25]. They can produce compounds with antimicrobial activity that inhibit the growth of pathogenic microorganisms. For example, many strains of Pseudomonas are reported to produce pyoluteorin, which has a broad-spectrum antimicrobial activity against a range of plant pathogens including fungi and bacteria. Pyoluteorin has been studied extensively as a potential biocontrol agent for managing plant diseases [26]. It has shown promise in field trials against a range of plant pathogens, including Fusarium [27], Rhizoctonia [28], and Phytophthora [29]. Pyoluteorin is considered a safe and environmentally friendly alternative to chemical pesticides for managing plant diseases, and further research is underway to explore its potential applications in agriculture. There are several other compounds such as hydrogen cyanide (HCN), and volatile organic compounds (VOCs) are secreted that can also deter potential pathogens [30].

In addition to several antimicrobial substances, non-ribosomal peptides (NRPs) have gained a lot of attention in the past decade. NRPs are a large group of structurally diverse natural products (Figure 1) that are synthesized by enzymes called non-ribosomal peptide synthetases (NRPSs) [31]. They are produced by certain bacteria, e.g., Bacilllus subtilis and Pseudomonas fluorescens are significantly useful for the biocontrol of plant diseases [32,33].

The objective of this review article is to explore the contribution of NRPs in the biocontrol of bacterial and fungal plant pathogens. Our study encompasses a comprehensive analysis of NRP synthesis, its mode of action, and characterization. In addition, we highlight the pivotal role that NRPs play in establishing sustainable plant disease control strategies and implementing biocontrol using PGPB in soilless agriculture. Furthermore, we deal with valuable insights into the commercial aspects of PGPB’s formulations, thereby providing a more comprehensive understanding of the subject matter.

2. Potential Non-Ribosomal Peptides for Biocontrol

Unlike ribosomal peptides, NRPs possess complex structures and exhibit a variety of biological activities. The structure of NRPs is highly diverse and complex. NRPs possess intricate and often non-linear architectures [34]. They are incorporated with a short stretch of amino acid building blocks including both proteinogenic and non-proteinogenic residues, which contribute to their structural variability [35]. NRPs can contain unique non-peptide bonds, such as ester, thioester, and amide linkages, formed through the actions of NRPSs. Many NRPs adopt cyclic structures, adding stability and rigidity to their conformation [36]. NRPs undergo modifications, such as oxidation, glycosylation, acylation, and phosphorylation, that further enhance their structural diversity [37]. Tailoring reactions mediated by specific enzymes introduce additional modifications and functional groups. The combination of these factors results in the remarkable diversity of NRPs, allowing them to exhibit a range of biological activities and making them attractive candidates for drug discovery and therapeutic applications [38]. Among the various NRPs, iturin, surfactin, and fengycin are some notable examples (Figure 2).

• Iturin

Iturin is a cyclic lipopeptide compound that belongs to the family of secondary metabolites known as iturins. It is a kind of cyclic lipopeptide (CLP) produced by Bacillus subtilis and other closely related bacteria [32]. Many strains of B. subtilis are known to produce iturin and it has a broad-spectrum inhibitory effect on many plants’ pathogenic fungi and bacteria [39]. Many different members of the iturin family have common structures, but they also have their characteristic structures. The most important representatives of the iturin family are iturin A, C, D, and E, and the mycobacterium subtilisin and bacteriomycin variants (D, F, L) [40]. In nature, iturin A consists of eight variants, each named Iturin A1-A8 [41]. These isomers are often produced as mixtures with molecular weights ranging from 1029 to 1084 Da [42]. The production of iturin by Bacillus subtilis is considered a significant trait that contributes to its biocontrol and plant-growth-promoting properties [43]. Iturin has garnered considerable attention due to its diverse biological activities and potential applications in the agriculture and pharmaceutical industries [39].

The structure of iturin consists of a cyclic peptide ring linked to a fatty acid chain [44]. The peptide ring is composed of seven amino acids, with the amino acid sequence varying among different iturin variants. The fatty acid chain, typically consisting of 14 to 17 carbon atoms, is responsible for the amphiphilic nature of iturin, enabling it to interact with both hydrophobic and hydrophilic environments [45]. The specific structure of iturin contributes to its various bioactive properties, including antifungal, antibacterial, and surfactant activities [46].

The genes encoding iturin are typically found within the genome of bacteria belonging to the Bacillus genus. Specifically, the genes responsible for the biosynthesis of iturin are part of the non-ribosomal peptide synthetase (NRPS) gene cluster. NRPS genes are involved in the production of various peptide-based compounds, including iturin. Iturins do vary slightly between different strains of Bacillus and are biosynthesized by a cluster of genes including ituA, ituB, ituC, and ituD [47]. These genes encode the enzymes necessary for the assembly of the iturin peptide and the subsequent modifications that occur during its biosynthesis [48]. The arrangement and organization of the iturin-encoding gene clusters vary within the strains and species [49].

The mode of action of iturin involves its interaction with cellular membranes, leading to disruption and subsequent cell death [50]. Iturin’s amphiphilic nature allows it to insert into the lipid bilayer of cell membranes. The hydrophobic fatty acid chain of iturin embeds into the hydrophobic region of the lipid bilayer, while the cyclic peptide ring remains in the aqueous environment [51]. Once inserted, iturin causes changes in the membrane’s physical properties, including fluidity and permeability. Iturin molecules aggregate and form channels or pores within the membrane, resulting in the leakage of ions, metabolites, and other essential components from the cell. This disruption of the membrane’s integrity leads to cell lysis and ultimately the death of the target organism [52]. The membrane-disrupting activity of iturin is primarily attributed to its ability to interact with ergosterol, a sterol found in the cell membranes of fungi, or with other membrane components in bacteria. Iturin molecules preferentially bind to ergosterol or certain lipids present in bacterial membranes, disrupting their normal function and compromising cell viability [53]. Iturin A has been reported to severely damage the plasma membranes by forming a large pore in the cell of Fusarium graminearum at MIC = 5 µg/mL and to successfully inhibit the hyphal growth [54]. Iturin A has also been reported to stimulate oxidative stress, leading to mitochondria damage and the eventual destruction of the pathogen cell [55].

Iturin’s potential applications extend beyond agriculture and encompass areas such as pharmaceuticals and cosmetics. Its surfactant properties make it a valuable ingredient in various formulations, including detergents and emulsifiers. The ability of iturin to promote plant growth and induce systemic resistance (IRS) in plants has led to its utilization as a biocontrol agent in integrated pest management strategies. In inference, iturin is a cyclic lipopeptide produced mostly by Bacillus subtilis, with diverse biological activities and potential applications. Its structure, detection methods, and characterization techniques enable scientists to explore its properties and understand its mode of action. By further investigating iturin’s potential and optimizing its production, it may hold promise for addressing challenges in agriculture, healthcare, and other industries.

b.

Surfactin

Surfactin, a well-studied biosurfactant, is produced by various Bacillus species such as B. amyloliquefaciens, B. subtilis, and B. pumilus [56]. It possesses unique physicochemical properties due to its amphiphilic structure, enabling foaming, emulsifying, hydrophobic surface modification, and chelation abilities [57]. Its structure comprises a peptide loop of seven amino acids and a variable-length β-hydroxy fatty acid chain. The hydrophilic characteristic and negative charge of the peptide loop, consisting of glutamic acid and aspartic acid residues, contrast with the major hydrophobic domain formed by the valine residue facing the fatty acid chain [58]. Surfactin exhibits hydrophobic interactions within micelles below the critical micellar concentrations (CMCs), allowing the fatty acid tail to extend freely into the solution [59].

Notably, surfactin acts as a broad-spectrum antibiotic with detergent-like activity, increasing the permeability of cell membranes in bacteria irrespective of their Gram stain classification [60]. Its minimum inhibitory concentration (MIC) ranges from 12 to 50 μg/mL [61]. Additionally, surfactin has gained attention for its antifungal properties, particularly against phytopathogenic fungi such as Fusarium, Lasiodiplodia, Colletotrichum, Botryosphaeria, Aspergillus, and Penicillium [62].

c.

Fengycin

Fengycin is a lipopeptide compound produced by certain strains of Bacillus subtilis, a common soil bacterium. It belongs to the cyclic lipopeptide family and is known for its biocontrol activity against plant pathogens [63,64]. Fengycin has a complex structure consisting of a cyclic heptapeptide ring linked to a β-hydroxy fatty acid chain. This unique structure contributes to its biological activities [65].

The major source of fengycin is Bacillus subtilis, a Gram-positive bacterium widely distributed in the environment, including the soil, water, and plant rhizosphere. Several strains of Bacillus subtilis, as well as genus Bacillus, e.g., B. subtilis F29-3, and B. amyloliquefaciens MEP₂18, are known to produce fengycin [66,67]. These strains can colonize plant roots and establish a beneficial relationship with plants, promoting growth and protecting them from pathogenic infections [68,69,70].

Fengycin demonstrates strong biocontrol activity against a variety of plant pathogens, particularly fungal pathogens [71]. It acts by inhibiting the growth and development of pathogenic fungi, thereby protecting plants from diseases. Fengycin disrupts the integrity of fungal cell membranes, leading to their lysis and subsequent death [72]. It is effective against several fungal genera, including Fusarium, Alternaria, and Botrytis, which are notorious for causing devastating crop diseases [73].

The biocontrol activity of fengycin is of great interest in the field of agriculture as it provides an eco-friendly and sustainable approach to disease management [74,75]. By making optimal utilization of fengycin-producing strains of Bacillus, the dependency on chemical fungicides can be reduced, which often have harmful effects on the environment and can contribute to the development of pesticide resistance in pathogens [15]. Its selectivity toward fungal pathogens and its minimal impact on beneficial organisms make it a valuable tool for integrated pest management strategies [76].

Apart from these three, several other NRPs are discussed in the application section of the manuscript.

3. Synthesis, Mode of Action, and Characterization

• Synthesis of NRPs

Non-ribosomal peptides are small peptides that are synthesized by non-ribosomal peptide synthetases (NRPSs). Unlike other peptides, NRPs are not dependent on messenger RNA translation by ribosome. The synthesis of NRPs is mediated by NRPS biosynthetic gene clusters (BGCs), which possess specific domains generally referred to as modules. These modules contribute to specific amino acids and modify the growing peptide, resulting in the generation of short peptide structures with diverse chemical modifications, including cyclic, branched, and non-proteinogenic amino acids [77]. Additionally, oxidation, reduction, and dehydration reactions can also occur during NRP synthesis.

The NRPSs are exclusive to one type of peptide compound, and the modules within NRPS BGCs usually have an initiation module, elongation module, and termination module (Figure 3). The initiation module involves formylation or N-methylation, adenylation, thiolation, and peptide carrier proteins with attached 4’-phospho-pantetheine. The elongation modules include condensation to form the amide bond, cyclization into thiazoline or oxazolines, N-methylation, adenylation, thiolation, and epimerization into D-amino acids. Lastly, the termination module involves termination by a thioesterase or reduction to a terminal aldehyde or alcohol [78,79]. The final peptide product is frequently subject to modifications, such as glycosylation, acylation, halogenation, or hydroxylation, which are often carried out by enzymes associated with the synthetase complex [80]. These enzymes are typically encoded within the same operons or gene clusters [81]. Non-modular NRPS enzymes are more prevalent in bacterial systems, where they are often arranged in clusters [77]. These nonmodular NRPS enzymes consist of one or two A-PCP-C modules or may lack complete A-PCP-C modules and instead have a single A-domain or an A-PCP unit followed by a variety of C-terminal domains. However, such multiple repeating modules are commonly referred to as A-PCP-C domains. These multi-modular NRPSs are commonly found in fungal genomes, where these modules that are involved in peptide assembly are connected within a single enzyme [82].

In bacterial systems, biosynthesis can be performed by mono- or bi-modular NRPSs. These proteins interact with other NRPS proteins to activate amino acids and transfer the activated substrate to either a C domain in the same NRPS or a different NRPS [83]. This type of biosynthesis is known as nonlinear biosynthesis [84]. The distribution of mono/bi modular NRPS subfamilies across taxonomic groups suggests that these structures may have originated in ancient times, potentially predating the divergence of eubacteria and fungi. NRPSs can also be fused to a polyketide synthase (PKS) unit, creating a single polypeptide that combines both types of synthetases [85].

Several PGPB and also some fungi have been reported to produce a wide range of NRPs such as surfactin, iturin, fengycin, and polymyxin that have been associated with diverse biocontrol activity against a number of plant pathogens [86]. The U.S. Food and Drug Administration (FDA) and the U.S. Environmental Protection Agency have authorized their use as commercial applications (EPA) [82].

b.

Mode of action

NRPs have been found to exhibit diverse mechanisms of action against bacterial pathogens, making them valuable therapeutic agents for the study of bacterial diseases among plants. The most common method of action that NRP follows is action on the cell wall and its synthesis machinery. It is one of the mechanisms of action of NRPs against bacteria that inhibits cell wall synthesis. NRPs such as vancomycin, daptomycin, and bacitracin are known to inhibit the biosynthesis of bacterial cell walls by targeting key enzymes involved in peptidoglycan synthesis [87,88]. These NRPs bind to specific targets in the cell wall synthesis pathway, leading to the inhibition of bacterial growth and cell division.

Vancomycin is a glycopeptide NRP produced by a soil bacterium Amycolatopsis orientalis; it binds to the D-alanyl-D-alanine terminus of the peptidoglycan precursor, thereby inhibiting its incorporation into the growing cell wall. The binding of vancomycin to the peptidoglycan precursor prevents the formation of cross-links between peptidoglycan chains, leading to the inhibition of cell wall synthesis and bacterial growth [89]. Similarly, daptomycin is a lipopeptide antibiotic that binds to the bacterial membrane and causes depolarization of the membrane potential, leading to the disruption of membrane function and cell death [90]. Bacitracin is a cyclic peptide antibiotic that inhibits the dephosphorylation of the peptidoglycan precursor, leading to the accumulation of the precursor and the inhibition of cell wall synthesis [91]. Bacitracin binds to the undecaprenyl-pyrophosphate carrier molecule, which is responsible for the transport of the peptidoglycan precursor across the cell membrane [92]. The binding of bacitracin to the carrier molecule prevents the transport of the precursor, leading to the inhibition of cell wall synthesis and bacterial growth. Fengycin is also one of the potent antimicrobial substances. C16-Fengycin has been reported to be involved in both cell wall destruction and the accumulation of reactive oxygen species (ROS), which may activate the High-Osmolarity Glycerol Mitogen-Activated Protein Kinase (HOG-MAPK) pathway [93]. Fengycin BS155 affects the mitochondrial membrane potential (MMP), elevates the ROS bursts, and simultaneously negatively affects the ROS-scavenging enzymes [72].

Another mechanism by which NRPs act on the pathogen is the disruption of membrane function. NRPs such as polymyxins and gramicidin are known to disrupt the structure and function of bacterial cell membranes. Polymyxins are a group of CLP antibiotics that interact with the lipid components of the bacterial cell membrane, leading to the formation of ion channels and pore structures that disrupt the membrane’s integrity and lead to cell death [94]. Polymyxins have been shown to bind to the lipid A component of the bacterial cell membrane, leading to the disruption of the membrane structure and the release of intracellular contents [95]. Daptomycin has also been shown to bind to the bacterial cell membrane and form channels that allow the influx of ions, leading to the disruption of the membrane potential and the loss of membrane integrity [96].

Gramicidin is a CLP compound that forms channels in the bacterial cell membrane, leading to the disruption of membrane function and cell death. Gramicidin has been shown to bind to specific lipid components of the bacterial cell membrane, leading to the formation of ion channels and the influx of ions, which disrupts the membrane potential and leads to the loss of membrane integrity [97]. For example, gramicidin A (gA) forms a channel in lipid membranes, which allows H⁺ ions conduction through the lipid bilayer of pathogens [98]. Surfactin also exhibits a range of antimicrobial mechanisms. It can cause the disintegration of cell membranes or osmotic pressure imbalance, leading to the leakage and lysis of lipid membranes [58].

NRPs are also reported to interfere with nucleic acid integrity. Bacillomycin D, which is a potent antifungal compound and effectively combats Aspergillus ochraceus (a common fungal contaminant in food samples), affects the hyphal distortion and sporular disruption in A. ochraceus, leading to decreased fungal growth. A study has reported that bacillomycin D binds to the DNA of A. ochraceus and affects its integrity, which can ultimately affect its growth. The study also found that bacillomycin D caused breaks in A. ochraceus DNA and inhibited its repair mechanisms, leading to cell death [99]. Fengycin BS155 is reported to induce chromatin condensation, resulting in an upregulation of DNA-repair-related protein expression and cleavage of poly (ADP-ribose) polymerase (PARP) [72].

NRPs have also been found to modulate bacterial virulence by affecting key virulence factors such as quorum sensing and biofilm formation [100]. NRPs such as agrimophol and fengycin have been shown to inhibit quorum sensing in Gram-positive bacteria, leading to the inhibition of virulence factor production and the attenuation of bacterial virulence [101].

NRPs exhibit diverse mechanisms of action against bacterial pathogens. NRPs can inhibit cell wall synthesis, disrupt membrane function, interfere with nucleic acid integrity, affect protein synthesis, and modulate bacterial virulence. The unique mechanisms of action of NRPs make them potential agents for the biocontrol of bacterial and fungal pathogens of plants.

c.

Characterization

The modular structure of NRPs allows for the incorporation of various amino acids and other functional groups, thereby producing a range of chemical structures and biological activities. The characterization of NRPs is important for understanding their biological function, as well as for the discovery of new natural compounds with potential antagonistic activity and promising scope in the biocontrol of plant pathogens.

Bioassays are an important and primary approach for the identification of NRPs. It involves the testing of NRPs for biological activity, such as antimicrobial (for biocontrol) or anticancer activity (many NRPs have reported anti-cancer activities). The two most common bioassays that can be used are disk diffusion assays and broth dilution assays. However, further mass culturing for harvesting possible NRPs is carried out.

The second step in the characterization of NRPs is the isolation and purification of metabolites from the selected potential microbes. NRPs can be obtained from mass culture broths of microorganisms by various extraction techniques, such as solvent extraction [102,102], solid-phase extraction [103], and liquid–liquid extraction. After extraction, the crude extract is subjected to a series of purification steps, such as column chromatography, high-performance liquid chromatography (HPLC), and preparative thin-layer chromatography (TLC), to obtain pure NRPs [104]. The purity of the NRP is determined by analytical HPLC or TLC.

• Mass spectrometry (MS) is a powerful technique for the analysis of NRPs [105]. MS allows for the determination of the mass-to-charge ratio (m/z) of the NRP and its fragments [106]. This information helps identify the exact chemical structure of the NRP, as well as to determine its molecular weight and the presence of various functional groups [107]. Several types of MS techniques can be used for the analysis of NRPs, including matrix-assisted laser desorption/ionization (MALDI-TOF) MS [108], electrospray ionization (ESI) MS [109], and tandem mass spectrometry (MS/MS) [110].

MALDI MS is a soft ionization technique that is particularly useful for the analysis of large biomolecules, such as NRPs. In MALDI MS, the sample is mixed with a matrix material and deposited on a target plate. The sample is then irradiated with a laser, which causes the matrix to vaporize and ionize the sample molecules. The resulting ions are then detected and analyzed [111]. Electron spray ionization (ESI) MS is another soft ionization technique that is commonly used for the analysis of NRPs. In ESI MS, the sample is ionized in a solution containing a volatile buffer and injected into a mass spectrometer. The sample is then subjected to an electric field, which causes the sample ions to be generated and detected [112]. MS/MS can be performed using various fragmentation methods, such as collision-induced dissociation (CID) [113] and electron transfer dissociation (ETD) [109].

• The use of nuclear magnetic resonance (NMR) spectroscopy has also been reported in NMR characterization; however, it can be tedious, error-prone, and require substantial quantities of purified material [114]. This can be especially problematic since NRPs are often produced by microorganisms that are difficult to cultivate, making it challenging to obtain enough material for NMR-based sequencing [115]. As a solution, a new nanomolar scale approach to NRP sequencing is needed. A recent study has claimed to identify three compounds, keanumycins A-C, from Pseudomonas sp. QS1027 using NMR that is found to have significant potential against phytopathogen Botrytis cinerea [116].
• X-ray crystallography has also been employed often for the analysis of NRPs that have complex three-dimensional structures [117,118] or are difficult to analyze using other techniques [119]. However, the technique requires the NRP to be crystallized, which can be challenging for some NRPs [35].
• Genome mining is a relatively new approach to the identification of NRPs. It involves the analysis of the genome sequence of microorganisms to identify BGCs for NRPSs and other biosynthetic enzymes [120]. It can also be used to predict the chemical structures of NRPs based on the presence of specific biosynthetic genes. There are several computational tools available for genome mining, such as antiSMASH and NRPSpredictor [121]. These tools can be used to predict the chemical structures of NRPs, as well as to identify potential new NRPs.

To sum up, the characterization of NRPs from bacteria or fungi is a complex and multidisciplinary process that requires a combination of analytical techniques (Figure 4). Isolation and purification, bioassays, mass spectrometry, nuclear magnetic resonance spectroscopy, X-ray crystallography, and genome mining are all important tools for the characterization of NRPs.

4. Application of NRPs in Sustainable Agriculture

The NRPs produced by PGPB can offer the biocontrol of plant pathogens through a variety of mechanisms. One of the primary mechanisms is directly inhibiting the growth and development of plant pathogens. NRPs can do this by interfering with key cellular processes such as DNA replication, protein synthesis, and cell wall synthesis in the target pathogen [122]. In addition to their direct effects on plant pathogens, NRPs can also indirectly promote plant health and defense against pathogens. For example, some NRPs can exhibit IRS in plants, e.g., surfactin, bacillomycin, and fengycin provided IRS among Arabidopsis plantlets against P. syringae pv. tomato (Pst DC3000) and Botrytis cinerea [123], Priming with such PGPR could certainly be more effective in mitigating the pathogen attacks [124]. NRPs produced by PGPBs make them promising candidates for the biocontrol of plant pathogens, with the potential to reduce the reliance on traditional chemical pesticides and antimicrobial agents and promote more sustainable agricultural practices.

Some of the NRPs, like surfactin and iturin, are potential plant disease biocontrol agents as they exhibit antimicrobial properties, and some other NRPs such as daptomycin and polymyxin are used as antibiotics to handle animal and human diseases that are caused by pathogenic bacteria. [24]

The potential benefit of using NRPs is that they may have a lower risk of causing resistance compared to traditional pesticides. Several plant pathogens have developed resistance to chemical pesticides over time, making them less effective for disease control [125]. NRPs, however, have a more complex structure and mode of action compared to traditional pesticides, which may make it more difficult for pathogens to develop resistance [126]. Apart from this, NRPs may be more environmentally friendly compared to traditional pesticides [127]. Many traditional pesticides certainly have negative impacts on non-target organisms and the environment. NRPs, on the other hand, are typically produced by naturally occurring microorganisms and may have a lower toxicity to non-target organisms [122].

In this section, we discuss the NRPs for controlling bacterial and fungal plant pathogens.

4.1. Impact on Bacterial Pathogens

NRPs are produced by a variety of bacteria and fungi, and several NRP molecules have strong antimicrobial properties against bacterial and fungal pathogens of plants. NRPs can be highly specific in their activity, targeting specific bacterial species or strains, making them a promising alternative to broad-spectrum antibiotics that are susceptible to gaining antibiotic resistance [126]. Studies have demonstrated the effectiveness of NRPs in controlling a range of bacterial pathogens of the plant, viz., several strains of Pectobacterium, Pseudomonas, Ralstonia, and Xanthomonas [24]. For example, bacteria of the genus Burkholderia inhabit diverse ecological niches and play important roles in the environment through the secretion of various secondary metabolites including modular NRPs and polyketides (PKs). These metabolites represent a class of broadly biomedical and bioregulatory important natural products, such as antibiotics, siderophores, anticancer agents, and biopesticides, which can protect ecological niches from competitors and are considered a new source that can be used to promote plant growth [128].

Bacillus genera are one of the leading genera that have been widely studied for the production of potential NRPs. The major bioactive molecules the Bacillus genera have been reported to synthesize are NRPs/lipopeptides, polyketide compounds, bacteriocins, and siderophores [32,129]. Bacillus lipopeptides have a highly complex biosynthetic mechanism activated via NRPSs. It is a large enzyme complex with a modular structure with each module responsible for the incorporation of a specific amino acid. In Bacillus subtilis strains, approximately 4–5% of the total genome is dedicated to the synthesis of secondary metabolites having the potential to generate over 20 structurally distinct antimicrobial compounds [32]. Many species of the Pseudomonas genus are also potent biocontrol agents that can produce cyclic lipopeptides (CLPs) along with quinolones, siderophores, phenazines, phloroglucinol, hydrogen cyanide, and rhamnolipids. There are varieties and differences in secondary metabolite production even within the same Pseudomonas strains [130].

A list of such NRPs is provided that demonstrates the biocontrol of bacterial pathogens of the plant in

Table 1. NRPs compounds against the bacterial plant pathogen biocontrol.

S. No.	Source Organism	Compound	Assessment Method	Plant-Pathogen	Disease Caused	Reference
1	B. porteri HB1.4B Brevibacillus RS1.1 Brevibacillus DP1.3A Brevibacillus HB2.2	Uncharacterized NRPs and NRP-PK hybrids.	Antagonist activity was identified through in vitro antibacterial activity and gene clusters were identified after whole-genome sequencing, analysis, and annotations	Clavibacter michiganensis	Bacterial wilt and canker of tomato	[131]
2	B. porteri HB1.1 Brevibacillus DP1.3A			Xanthomonas campestris	black rot in cruciferous	[131]
3	B. subtilis TY-1	surfactin, bacillibactin, and fengycin	Identified using AntiSMASH	Ralstonia solanacearum	Bacterial wilt of tobacco	[132]
4	Pseudomonas strain CHA0	orfamide A	Direct contact inhibition, indirectly using supernatant assays Whole-genome sequencing and AntiSMASH	R. solanacearum	bacterial wilt in a range of host plants, e.g., potato, tobacco, tomato	[133]
5	Sphingomonas, Pseudoxanthomonas and Stenotrophomonas	Not Specified	NPRS BGC enrichment was confirmed using metagenomic studies. Pathogen inhibition was further confirmed in pot experiments	R. solanacearum	bacterial wilt in a range of host plants, e.g., potato, tobacco, tomato	[134]
6	Gordonia sp. TD-4	Not Specified	NPRS BGC enrichment was confirmed using metagenomic studies	Not Specified	Not Specified	[135]
7	Pseudomonas strain CHA0	orfamide A	Direct contact inhibition, indirectly using supernatant assays. Whole-genome sequencing and AntiSMASH	R. solanacearum	bacterial wilt in a range of host plants, e.g., potato, tobacco, tomato	[133]
8	B. subtilis EH11 Paenibacillus sp. EDO6 B. endophyticus FH5	Surfactin, fengycin, bacillibactin, petrobactin, lichenysin, and bacillaene.	In vitro inhibition assay of bacterial pathogens and NRP bio-clusters were confirmed by genome-based analysis	Erwinia carotovora	soft rot disease of potato, carrot, and cabbage	[136]
9	B. subtilis EH11 Paenibacillus sp. EDO6 B. endophyticus FH5			P. syringae	bacterial blight and bacterial speck among several plants	[136]
10	Brevibacillus laterosporus MG64	Bogorols K	MIC = 4 μg/mL LC-MS/MSantiSMASH	X. campestris pv. campestris NCCB92058	Black rot disease in crucifers	[24]
		Bogorols L	MIC = 2 μg/mL LC-MS/MSantiSMASH	X. campestris pv. campestris NCCB92058
		Brevibacillin	MIC = 2 μg/mL LC-MS/MSantiSMASH	X. campestris pv. campestris NCCB92058
11		Brevibacillin	MIC = 2 μg/mL LC-MS/MSantiSMASH	X. translucens pv. graminis LMG587	leaf streak in wheat and cereal crop.	[24]
		Bogorol L	MIC = 8 μg/mL LC-MS/MSantiSMASH	P. syringae pv. tomato DC3000
12	P. mediterranea Strain S58	Fengycin, Crochelin A, Entolysin, Orfamide B, Siderophore, Syringomycin	Antagonistic test and Disease control assay confirmed the biocontrol potential. NRPS BGCs were identified, encoding for NRPs	P. syringae pv. tabaci	Wild fire, Angular leaf spot in Tobacco	[137]
13	Lysobacter enzymogenes LE16	surfactin	antiSMASH	P. syringae pv. tabaci	tobacco wildfire disease	[138]
14	B.safensis F4	Surfactin	MIC = 1.56 mg mL−1 (crude biosurfactant)Identified using HPLC	P. savastanoi	olive knot disease	[139]
15		Surfactin	MIC = 3.125 mg mL−1 (crude biosurfactant)Identified using HPLC	Agrobacterium tumefaciens	Crown gall	[139]
16	B. amyloliquefaciens SQR9	Fengycin	In vitro inhibition assay	P. syringae pv. tomato DC3000 (Studied on Arabidopsis)	Model organism for studying plant–bacterial interactions. This strain can infect Arabidopsis too	[123]

.

4.2. Impact on Fungal Pathogens

Phytopathogenic fungi are amongst the most common infectious agents in plants and one of the principal plant infections that cause significant monetary and production losses. Plant pathogenic fungi have been the most diverse and deadly class of economic and ecological dangers throughout the history of agriculture [140]. Many secondary metabolites produced by microorganisms have been reported to act as antifungal agents against phytopathogenic fungi [141,142,143].

However, some strains of Bacillus and Pseudomonas produce short peptide compounds that are synthesized non-ribosomally such as bacilysin, mycobacillin, bacillomycin, mycosubtilin, iturins, fengycins, and surfactins that have antimicrobial properties [32,34]. These NRP compounds are antagonistic to numerous additional plant pathogenic fungi, including Fusarium. graminearum, F. oxysporum, F. solani, and Rhizoctonia solani [54,144].

NRPs like surfactin produced by Bacillus have exhibited potent antifungal action against Trichoderma reesei, T. atroviride, F. moniliforme, F. oxysporum, and F. solani [145,146]. Similarly, mycosubtilin produced by B. subtilis has shown a significant inhibition potential of Pythium infection in tomato seedlings [147]. Numerous research studies have documented the impact of NRPs on the biocontrol of fungal pathogens. However, this article aims to emphasize the latest findings regarding the use of NRPs as a biocontrol measure against pathogenic fungi. To facilitate understanding, a table is included below to provide additional information (

Table 2. NRP compounds and their action against fungal plant pathogen biocontrol.

S. No.	Source Organism	NRP Compound	Assessment Method	Pathogen	Disease Caused	Reference
1	B. velezensis 2211	Bacillomycin D (bmyA), fengycin (fenB)	Direct Inhibition study Gene expression of NRPS BGCs	Colletotrichum fructicola	The causal agent of anthracnose and soft rot in avocado fruits	[148]
2	B. subtilis Strain UD1022	-	Direct and indirect antifungal assay NRP mutant strains (srfAC ¯, ppsB ¯, ppsB ¯, srfAC ¯, and sfp ¯) were used to validate the antagonism of the strain UD1022	Clarireedia jacksonii	Dollar spot of the grass	[149]
3	B. subtilis Strain UD1022	Surfactin	Direct antagonism by plate inhibition assay spo0A_ (surfactin-encoding gene) mutated strain	Ascochyta medicaginicola StC 306-5	Black stem of Alfalfa and Medicago truncatula.	[150]
4	Burkholderia vietnamiensis strain WPB	Occidiofungin	Dual plate inhibition assay In silico identification of NRPS BGCs	Gaeumannomyces graminis var. tritici	Take all diseases, roots of grass and cereal plants	[151]
5	B. velezensis S141	surfactin, bacilysin, and bacillomycin D	Dual culture assays Transcriptomic analysis of genes encoding surfactin, bacilysin, and bacillomycin D	Cercospora canescens	leaf spot disease of amaranth	[152]
6	Pseudomonas spp. SK2, and SK3	Obafuorin and cupriachelin	Plate-based inhibition assay In vitro tube assay with wheat plant Whole-genome sequencing and antiSMASH Extracted ion chromatograms (EIC) and detected by LC/MS	F. oxysporum	yellowing, stunting, and death of seedlings and yellowing and stunting of older plants	[153]
7	Pseudomonas spp. SK2, and SK3	Obafuorin and cupriachelin	Plate-based inhibition assay In vitro tube assay with wheat plant Whole-genome sequencing and antiSMASH Extracted ion chromatograms (EIC) and detected by LC/MS	Verticillium dahliae	verticillium wilt causes the leaves to curl and discolor.	[153]
8	B. subtilis PTS-394	Surfactin, Iturin, and Fengcyin	Plate-based inhibition assay Biocontrol study on the plant was studied on pot and field too. MALDI-TOF-MS	F. solani	pepper root rot	[154]
9	Alternaria sp. CC-3	NRPS BGCs were identified	Direct antagonist study was conducted (52.5% inhibition) Primer-based amplification	Ophiognomonia leptostyla	walnut anthracnose or walnut black spot	[155]
10	B. subtilis strain Y17B	surfactin	61.3% inhibition on plate assay. Primer-based amplification of BGCs and UPLC Q TOF mass spectrometry analysis	Alternaria alternata	Leaf spots, rots, blights and affects other plant parts in over 380 host plants	[156]
11	Bacillus. velezensis KS04AU.	Surfactin	confrontation assay of Petri plate, BGCs for surfactin were identified by antiSMASH	F. oxysporum f. sp. radicis-lycopersici ZUM2407 (Forl ZUM2407)	Fusarium wilt disease in Tomatoes causes heavy loss	[157]
12	Paenibacillus sp. strain (UY79)	fusaricidin B, tridecaptin,	Direct inhibition assay Whole-genome sequencing, AntiSMASH	F. verticillioides A71.	seedling blight, or stalk or ear rot in maize	[158]
13	Dickeya solani MK10	Solanimycin	Direct inhibition assay antiSMASH UHPLC/Q-TOF MS	V. dahliae	verticillium wilt in many host plants	[159]
14	B. subtilis (B. velezensis QST713)	Ericin + NRPS	Biocontrol assay antiSMASH	T. aggressivum	green mold disease	[160]
15	Bacillus subtilis (B. velezensis RC 218)	Iturin, Fengycin	Bioassay, LC-MS, whole-genome sequencing, antiSMASH3.0	F. graminearum	Fusarium head blight	[161]

). It highlights the compounds with their source, method of characterization, and antagonism to the pathogen with appropriate citations. This table includes several recent investigations worth highlighting.

4.3. Biocontrol in Soilless Agriculture

Soilless agriculture has gained significant attention over the years due to its numerous advantages over traditional soil-based agriculture, including the better control over nutrient uptake and plant growth, and reduced disease incidence. However, soilless agriculture is also associated with a higher risk of plant disease, especially when compared to conventional soil-based agriculture. In response, the use of PGPBs has emerged as a viable solution to promote plant growth and control disease incidence in soilless agriculture. NRPs have been identified as effective biocontrol agents, with the potential to reduce plant disease incidence in soilless agriculture. NRPs have been shown to play an important role in plant disease control. In soilless agriculture, plants are grown in a sterile environment, which makes them more susceptible to disease. Additionally, the use of hydroponic systems can lead to the build-up of pathogens, as the recirculating nutrient solution can harbor pathogens that can infect plants.

In this perspective, the use of NRPs producing PGPB can provide an effective means of controlling plant diseases in soilless agriculture. By inhibiting the growth of plant pathogens, NRPs can reduce the incidence of disease, thus promoting plant growth and increasing yield. Additionally, NRPs may also enhance plant defenses against pathogens, further reducing the risk of disease. Recent research studies have shown successful implementations of PGPB in hydroponics, as demonstrated in

Table 3. NRP compounds and their potential application for biocontrol of plant pathogens in soilless agriculture (hydroponics).

S. No.	NRP Compound	Source of NRP (Only Bacteria)	Pathogen	Plant Name	Reference
1	Purified surfactin	B. velezensis GA1	Athelia rolfsii	Peanut	[162]
2	Iturin, bacillomycin D, surfactin, and fengycin	B. amyloliquefaciens DHA55	F. oxysporum f. sp. niveum (fon)	Watermelon	[163]
3	Surfactin, Iturin A	Bacillus spp.	P. syringae pv. maculicola MAFF 302783	Cabbage	[164]
4	Lokisin	P. koreensis 2.74 (CBS 125413)	Pythium ultimum	Tomato	[165]
5	Surfactin	B. velezensis	Botrytis cinerea	Tobacco	[166]
6	Surfactin A, surfactin B, Surfactin C	B. velezenis 1B-23 or Bacillus sp. 1D-12	Clavibacter michiganensis subsp. michiganensis	Tomato	[167]
7	Surfactin	B. velezensis 32a	Agrobacterium tumefaciens C58	Tomato	[168]
8	Phenylacetic acid (PAA) and methylphenyl acetate (MPA)	B. mycoides BM02	F. oxysporum f. sp. lycopersici (Fol)	Tomato	[169]

.

The PGPB’s application can also enhance plant growth by promoting optimal root growth and increasing biomass, chlorophyll content, and leaf surface area. Additionally, it has been found to confer tolerance to both abiotic and biotic stress factors, as well as improve nutrient uptake [86].

The colonization of PGPB is more effective in soil systems compared to hydroponic systems [87,88]. While soilless agriculture has its benefits, such as the ability to control the nutrient supply and minimize the presence of pests and diseases, there are certain limitations. For instance, the substrates used in soilless systems to support roots are not as versatile as soil. Moreover, soil also hosts a variety of beneficial organisms, such as nematodes and earthworms, which aid in the cycling of nutrients and promote advanced colonization, leading to better plant growth. In soilless systems, nutrient availability mainly relies on external sources like commercial nutrients. However, nutrients alone cannot provide optimal growth for plants, which is why PGPB plays a crucial role in such a system [89]. The NRPs directly help in the biocontrol of antibacterial and antifungal properties (Table 4) and also act as a biostimulator by ensuring the presence of compounds such as antioxidants and enzymes, aiding in plant growth [170]. Siderophores and plant hormones are also significantly helpful in plant growth and optimal health [171]. PGPB metabolites also indirectly contribute to plant growth [172].

Numerous studies have been conducted on PGPB in soil systems, and the results have been promising, which highlights their potential benefits in soilless farming as well [173]. Hence, its application in such systems is an exciting opportunity for promoting plant growth and deserves further investigation.

Table 4. Application of various plant-growth-promoting bacteria on different plants in soilless culture.

S. No	Type of PGPB	Media	Crops	References
1.	Bacillus sp, Halobacillus sp., B.gibsonii, Staphylococcus succinus, Zhihengliuella halotolerans, Oceanobacillus oncorhynchi, Exiguobacterium aurantiacum, B.atrophaeus, Zhihengliuella sp., Halomonas sp., Virgibacillus picturae, Oceanobacillus sp., and Thalassobacillus sp	Hydroponic	Triticum aestivum	[174]
2.	Bacillus amyloliquefaciens, B. brevis, B. circulans, B. coagulans, B. firmus, B. halodenitrificans, B. laterosporus, B. licheniformis, B. megaterium, B. mycoides, B. pasteurii, B. subtilis, and Paenibacillus polymyxa	Hydroponic floating system	Lactuca sativa L. var. Crispa	[175]
3.	Enterobacter hormoechei	Hydroponics	Cucumis sativus	[176]
4.	Acinetobacter calcoaceticus	Hydroponics	Lactuca sativa	[177]
5.	BFD160 Enterobacter asburiae, TFD26 P. koreensis, and BFS112 P. linii	Soilless culture comprising rock wool blocks placed in plastic pots containing perlite and peat (1:1)	Cucumis melo L	[178]
6.	Pseudarthrobactr chlorophenolicus BF2P4-5	Cocopeat media	S. lycopersicum	[179]
7.	Flavobacterium crocinum HYN0056T	Soilless culture comprising cocopeat (51.5%), peat moss (10%), Vermiculite (13%), perlite (15%), zeolite (10%), humic acid (0.1%), fertilizer (0.4%)	Arabidopsis thaliana	[180]
8.	Pantoea dispersa, Pantoea ananatis, Burkholderia arboris, Burkholderia pyrrocinia, and Burkholderia pyrrocinia	Cocopeat substrate	Solanum melongena	[181]
9.	B. megaterium TV-91C, Pantoea agglomerans RK-92, and B. subtilis TV17C	Peat	Brassica oleracea var. capitata ‘Yalova1’	[182]
10.	B. amyloliquefaciens, B. subtilis, B. pumilus, and B. sphaericus	Peat moss	Cucurbita pepo and S. lycopersicum	[183]
11.	Rhizobacterium, B. subtilis	Perlite	Lactuca sativa ‘Partavousi’	[70]
12.	Bacillus sp.	Hydroponics	Zea Mays	[184]
13.	P. pseudoalcaligenes and Bacillus subtilis	Hydroponics	Glycine max L.	[185]
14.	Sinorhizobium meliloti and P. fluorescence	Soilless media containing sand and sterile perlite (v/v, 2:1)	Medicago sativa L.	[186]

Table 4. Application of various plant-growth-promoting bacteria on different plants in soilless culture.

S. No	Type of PGPB	Media	Crops	References
1.	Bacillus sp, Halobacillus sp., B.gibsonii, Staphylococcus succinus, Zhihengliuella halotolerans, Oceanobacillus oncorhynchi, Exiguobacterium aurantiacum, B.atrophaeus, Zhihengliuella sp., Halomonas sp., Virgibacillus picturae, Oceanobacillus sp., and Thalassobacillus sp	Hydroponic	Triticum aestivum	[174]
2.	Bacillus amyloliquefaciens, B. brevis, B. circulans, B. coagulans, B. firmus, B. halodenitrificans, B. laterosporus, B. licheniformis, B. megaterium, B. mycoides, B. pasteurii, B. subtilis, and Paenibacillus polymyxa	Hydroponic floating system	Lactuca sativa L. var. Crispa	[175]
3.	Enterobacter hormoechei	Hydroponics	Cucumis sativus	[176]
4.	Acinetobacter calcoaceticus	Hydroponics	Lactuca sativa	[177]
5.	BFD160 Enterobacter asburiae, TFD26 P. koreensis, and BFS112 P. linii	Soilless culture comprising rock wool blocks placed in plastic pots containing perlite and peat (1:1)	Cucumis melo L	[178]
6.	Pseudarthrobactr chlorophenolicus BF2P4-5	Cocopeat media	S. lycopersicum	[179]
7.	Flavobacterium crocinum HYN0056T	Soilless culture comprising cocopeat (51.5%), peat moss (10%), Vermiculite (13%), perlite (15%), zeolite (10%), humic acid (0.1%), fertilizer (0.4%)	Arabidopsis thaliana	[180]
8.	Pantoea dispersa, Pantoea ananatis, Burkholderia arboris, Burkholderia pyrrocinia, and Burkholderia pyrrocinia	Cocopeat substrate	Solanum melongena	[181]
9.	B. megaterium TV-91C, Pantoea agglomerans RK-92, and B. subtilis TV17C	Peat	Brassica oleracea var. capitata ‘Yalova1’	[182]
10.	B. amyloliquefaciens, B. subtilis, B. pumilus, and B. sphaericus	Peat moss	Cucurbita pepo and S. lycopersicum	[183]
11.	Rhizobacterium, B. subtilis	Perlite	Lactuca sativa ‘Partavousi’	[70]
12.	Bacillus sp.	Hydroponics	Zea Mays	[184]
13.	P. pseudoalcaligenes and Bacillus subtilis	Hydroponics	Glycine max L.	[185]
14.	Sinorhizobium meliloti and P. fluorescence	Soilless media containing sand and sterile perlite (v/v, 2:1)	Medicago sativa L.	[186]

4.4. Future Prospective

NRP-producing bacteria have been isolated from soil, the rhizosphere, and plant tissues. They are known to possess several biological activities, including antimicrobial, antifungal, and antiviral properties. A major and diverse potential application of NRP-producing bacteria in sustainable agriculture is as biocontrol agents for plant pathogens. B. subtilis and Pseudomonas have been popularly studied bacteria for biocontrol, as they produce effective NRPs against a range of plant pathogens. The production of NRPs by bacteria can be influenced by various factors, including bacterial strain, growth conditions, and the presence of other microorganisms in the environment. They can produce NRPs that can inhibit the growth of plant pathogenic fungi and bacteria. This is effective in controlling plant diseases and reducing the use of chemical pesticides. NRPs are also specific to certain plant pathogens and can inhibit their growth and spread.

In addition, NRP-producing PGPB is also conferred with plant-growth-promoting traits that enable nutrient solubilization, phytohormones, and the development of root structure architecture. They are also capable of degrading xenobiotic compounds such as persistent organic pollutants (PCBs, PAHs, and pesticides), heavy metal remediation, and improving plant resistance to biotic and abiotic stresses. The PGPBs enable IRS among the plants and are also significantly crucial for plant health and enhanced crop productivity. The PGPB play a significant role in soil health and the ecosystem, important for sustainable agriculture as the production of NRPs and subsequent pathogen control can influence the composition and diversity of soil microbial communities, which have positive impacts on overall soil health and nutrient cycling.

However, there are challenges associated with the use of NRP-producing bacteria in sustainable agriculture. One challenge is cost-effectiveness, as microbial preparation for biocontrol can be expensive and time-consuming. Further requirements of genetically engineered microorganisms for enhanced biocontrol can also negatively affect cost-effectiveness. They also raise concerns about potential risks to the environment and human health without appropriate risk assessment studies and approvals.

The development of effective and efficient methods for the large-scale production and application of NRPs/NRP-producing PGPBs to plants and the production of NRPs by bacteria can be influenced by various environmental factors.

NRPs produced by PGPB have potential applications in sustainable agriculture for biocontrol agents, plant-growth-promoting agents, and to improve soil health. However, the challenges associated with their production and delivery must be addressed to ensure their effectiveness and safety in agricultural practices.

4.5. Commercial Aspects of PGPB Formulations for Biocontrol Strategies

PGPB formulations are faster becoming popular in agriculture as a means of improving plant growth, health, and yield. These formulations contain live PGPBs that can colonize the roots of plants and promote their growth by improving nutrient uptake, reducing stress, and suppressing pathogens. However, as with any commercial product, various aspects need to be considered in the development and marketing of PGPB formulations. There are many examples of PGPB formulations that have been successfully developed and commercialized (

Table 5. Commercial PGPB formulations and their bacterial ingredients.

Brand Name	PGPB Used	Manufacturer	Country of Origin
Taegro 2	B. subtilis var. amyloliquefaciens Strain FZB24	Novozymes	Denmark
Rhizolizer	B. amyloliquefaciens along with a fungus Trichoderma harzianum	Locus Agricultural Solutions	United States
LALGUARD M52 OD	B. subtilis	Lallemand Plant Care	Canada
RHIZOVITAL® 42	B. amyloliquefaciens	Andermatt	Canada
GeumanoControl	Not Disclosed	Bio-lide	Poland
CEASE	B. subtilis strain QST 713	BioWorks Inc.	United States
Nutri-Life Platform	Consortium of Bacillus, Pseudomonas, and Trichoderma	Nutri-Tech Solutions	Australia
Serenade® SOIL	Bacillus subtilis QST 713	Bayer	Canada
Subtilex®	B. subtilis strain MBI 600	Bioglobal	Turkey
Rizovital 42	Bacillus amyloliquefaciens	Organic Crop Protectants (OCP)	Australia

). Some of them are listed below:

An important aspect of the commercialization of PGPB formulations is the selection of the bacterial strains to be incorporated into the product. Several strains of PGPBs have shown excellent results in terms of plant growth and yield; still, it is crucial to assess and recognize the most effective ones for the crops and soil application where the product will be used. It requires extensive research and testing for the potential of PGPBs to control plant pathogens along with other plant-growth-promoting traits such as nitrogen fixation, nutrient acquisition, and phytohormones production. Field trials are far more crucial than evaluating the effectiveness under actual environmental conditions and determining the optimal application rates and methods.

Another important aspect of such formulations is the preparation itself. For the PGPB to be effective and formulation to be successful, it must be devised in a way that allows PGPBs to survive storage, transportation, and application in varying environmental conditions. This can be achieved through various means, such as encapsulation or blending in compatible matrices, or by incorporating the PGPB into a carrier such as vermiculite or peat moss. The formulation must also be compatible with the intended application method, whether that be through seed treatment, soil application, or foliar spray.

The production and distribution of PGPB formulations also involve an understanding of various commercial aspects, viz., quality control, regulation/policy framework, and marketing. Quality control is essential to ensure that the PGPB are viable and effective throughout the product’s shelf life, and this requires rigorous testing and monitoring of production processes.

Regulation is another important criterion, as PGPB formulations may be subject to different regulations in different countries or regions. This includes compliance with regulations governing the use of genetically modified organisms (GMOs) or the use of microorganisms in agriculture.

Marketing is a decisive aspect of PGPB formulations, as successful marketing can help to increase the adoption and sales of the product. This entails a strong sense of the target market, including the crops and regions where the product will be most effective, as well as the benefits and limitations of the product. Effective marketing can include advertising, trade shows, and other promotional activities, as well as partnerships with distributors or retailers to increase product availability and visibility.

To infer, the commercial aspects of PGPB’s formulations are complex and require careful consideration of many factors, including its selection, formulation, production, regulation, and marketing. However, with careful planning and execution, PGPB formulations have the potential to revolutionize agriculture by improving plant growth and health in a sustainable and environmentally friendly manner.

5. Conclusions

It is evident that NRPs are promising candidates for biocontrol agents against bacterial and fungal pathogens. The structural diversity among NRPs and their mode of action make them significantly effective against several plant pathogens, including those that have developed resistance to conventional chemical treatments and antimicrobial agents. The growing concern of sustainable agriculture has led to many front advances in research on NRPs and its prospecting. Subsequently, the methods of characterization of NRPs have been rapidly advancing, making it easier to identify and optimize their bioactivity. Moreover, the application of PGPBs in soilless agriculture has shown great potential in enhancing plant growth and productivity. Hence, prospecting them for biocontrol in soilless agriculture is correspondingly promising.

The forthcoming NRPs in biocontrol are highly promising, with increasing interest from the commercial sector for their use in agriculture. However, more research is still needed to fully understand the mechanisms of action of NRPs and to optimize their formulations, production, and delivery. Overall, NRPs represent a promising avenue for the sustainable and effective biocontrol of plant pathogens, and further research into their potential is substantially realistic in sustainable agriculture.