The Potential of Beneficial Microbes for Sustainable Alternative Approaches to Control Phytopathogenic Diseases

Abstract

Sustainable agricultural practices are essential for eradicating global hunger, especially in light of the growing world population. Utilizing natural antagonists, such as fungi and bacteria, to combat plant diseases, rather than relying solely on synthetic chemical pesticides, which pose significant risks to the environment and human health, is known as biocontrol. Microbial biological control agents (MBCAs) have proven effective against phytopathogens and are increasingly embraced in agricultural practices. MBCAs possess several beneficial traits, including antagonistic potential, rhizosphere competence, and the ability to produce lytic enzymes, antibiotics, and toxins. These biocontrol mechanisms directly target soil-borne pathogens or indirectly stimulate a plant-mediated resistance response. The effectiveness of MBCAs in managing plant diseases depends on various mechanisms, such as hyperparasitism, antibiosis, competition for nutrients or space, disruption of quorum-sensing signals, production of siderophores, generation of cell wall-degrading enzymes, and the induction and priming of plant resistance. Formulating effective biopesticides requires optimal conditions, including selecting effective strains, considering biosafety, appropriate storage methods, and ensuring a prolonged shelf life. Therefore, formulation is crucial in developing pesticide products, particularly concerning efficacy and production costs. However, several challenges must be addressed to ensure the successful application of biological control, including the shelf life of biopesticides, slower efficacy in pest management, inadequate awareness and understanding of biocontrol methods, regulatory registration for commercialization, and suitable agricultural applications. This review clarifies the principles of plant disease biocontrol, highlighting the mechanisms of action and functionality of MBCAs in biocontrol activities, the formulation of biopesticides derived from microorganisms, and the challenges and barriers associated with the development, registration, commercialization, and application of biopesticides.

1. Introduction

Phytopathogens pose a significant threat to global crop productivity. Thus, various agricultural diseases necessitate effective management strategies to ensure healthy food for the world’s growing population [1]. Pests and diseases lead to 20–40% annual losses in global crop production, including food crops. These losses often contribute to malnutrition and hunger, affecting food security and health worldwide. Natural resources influence preventive measures to reduce food losses and can lead to environmental degradation [2]. With rising global concerns regarding the risks tied to pesticide residues in food and the environment, the use of synthetic pesticides for managing plant diseases has gradually decreased, even though the chemical approach remains more prevalent than other control methods. The use of synthetic pesticides is discouraged because of the increasing prevalence of pesticide-resistant strains, as well as consumer and retailer demand for agricultural products with very low or zero chemical residues, alongside stringent international regulations concerning permissible chemical residue levels, registration, and the ecotoxicological impacts of synthetic pesticides [3]. Emerging trends in plant disease management and crop protection focus on reducing reliance on conventional pesticides while mandating the implementation of integrated pest management concepts outlined in national regulations. Hence, there is growing interest in safe, effective, long-term alternatives and sustainable strategies to traditional pesticides [4].

Alternative approaches, such as biological controls that use microorganisms to suppress the phytopathogens, have the potential to significantly reduce the pollution and harmful effects of synthetic chemicals on the environment [5]. Beneficial microbes such as bacteria and fungi [biocontrol agents] seem to be the most promising methods for maintaining plant health, environmental biosafety, and the quality of crop production. The last few decades have seen abundant research devoted to assessing the efficacy of numerous carefully chosen MBCAs against a wide range of phytopathogenic fungi. Therefore, MBCA-based bioproducts have been developed and commercialized [3,6]. In terms of biopesticides, promising developments have occurred, particularly with the successful application of some antagonistic MBCAs, specifically Bacillus spp., Burkholderia spp., Pseudomonas spp., and Trichoderma sp. against phytopathogens that cause soil-borne and foliar diseases such as Fusarium spp., Pythium spp., Rhizoctonia solani, Agrobacterium radiobacter var radiobacter, Phytophthora spp., and Erwinia spp. [5].

MBCAs can protect plants against disease invasions through numerous mechanisms. They can use one or more of these mechanisms to suppress plant diseases and interact with pathogens directly or indirectly [7]. MBCAs protect plants directly from pathogens by releasing antimicrobial materials, which affect the pathogens virulence and its competition for nutrients and space. Several MBCAs produce and secrete metabolites such as bacteriocins, lipopeptides, biosurfactants, cell-wall degrading enzymes, antibiotics, or volatile compounds that exhibit antimicrobial activity by decreasing the metabolic activity or growth of phytopathogens [4,7]. Additionally, MBCAs can interfere with the quorum sensing (QS) system of phytopathogens by degrading enzymes or inhibiting the production of signal molecules to initiate infections. For example, the production of QS inhibitors like chitinases, pectinases, and lactonases degrades QS signal molecules, suppressing pathogen infection and reducing disease symptoms [8].

Furthermore, MBCAs can suppress pathogen infection through competitive exclusion of phytopathogens by inhibiting their growth without killing them. Highly competitive MBCAs can efficiently survive and colonize the infection site with a nutrient absorption system that is more effective than that of pathogens, such as siderophores [9]. MBCAs can interact directly with phytopathogens through antibiosis or hyperparasitism. Hyperparasites attack and indirectly destroy resting spores within the structures of fungal pathogens and the bacterial cells of pathogens [7]. Additionally, MBCAs can indirectly protect plants by inducing defense mechanisms or promoting vegetative growth. Understanding the mechanisms behind an MBCA’s protective effect will aid in optimizing biocontrol and creating ideal conditions for the interaction between the MBCA, the pathogen, and the host. It will also assist in designing suitable formulations and application techniques to enhance plant health and promote sustainable agriculture [4]. However, the development and commercialization of new biopesticides face significant challenges, such as low market availability, inconsistent efficacy in preserving plants in the field, the complexity of the registration processes, and the reluctance of growers to accept biopesticides [3,5].

The aim of this review is to supply information on plant disease biocontrol, with emphasis on the mechanisms of action and function of MBCAs in biocontrol activity, formulations of biopesticides based on microorganisms, and challenges associated with the development, registration, commercialization, and application of biopesticides.

2. Diversity of Microbial Biological Control Agents (MBCAs)

Different MBCAs were used for plant pathogen control according to their biocontrol potential (

Table 1. The potential of Bacillus and Pseudomonas species as biocontrol agents against various phytopathogen attacks.

MBCAs	Target Pathogen	Host Plant	References
Bacillus licheniformisBL06	Phytophthora capsici	Pepper	[13]
Bacillus thuringiensis	Sclerotinia sclerotiorum	Sclerotiniose/Brassica campestris L.	[14]
Bacillus subtilis26DCryChS	Stagonosporanodorum Berk.	Wheat	[15]
Bacillus thuringiensisGBAC46	Aphelenchoidesbesseyi	Rice	[16]
Bacillus SubtilisATCC6633	Fusarium graminearum and Fusarium verticillioides	Wheat and Maize	[17]
Bacillus atrophaeusGBSC56	Meloidogyne incognita	Tomato	[18]
Bacillus atrophaeusTS1	Fusarium graminearum	Wheat	[19]
Bacillus velezensisDMW1	Phytophthora sojaeand Ralstonia solanacearum	Tomato and Soybean	[20]
Bacillus amyloliquefaciensEZ1509	Sclerotinia sclerotiorum	Rape Seed and Tabaco	[21]
Bacillus velezensisBvel1	Botrytis cinerea	Pepper and Grape	[22]
Pseudomonas fluorescensZX	Botrytis cinerea	Grapes	[23]
Pseudomonas putidaBP25	Magnaporthe oryzae	Rice	[24]
Pseudomonas fluorescens	Mucor piriformis	Apple	[25]
Pseudomonas simiae PICF7	Verticillium dahliae	Olive plants	[26]
Pseudomonas fluorescensEPS817	Phytophthora cactorum	Strawberry plants	[27]
Pseudomonas parafulva JBCS1880	Xanthomonas axonopodispv. glycines	Soybean	[28]
Pseudomonas rhodesiaeGC-7	Meloidogyne graminicola	Rice	[29]
Pseudomonas fluorescens CHA0	Meloidogyne javanica	Tomato and Cucumber	[30]

and

Table 2. Biocontrol capability of Trichoderma against some phytopathogens.

Species	Target Pathogen	Disease	Crop	Reference
T. harzianum, T. asperellum, T. longibrachiatum	Meloidogyne incognita	Root-knot nematode	Tomato	[31]
T. viride	Macrophominaphaseolina	Charcoal rot	Strawberry	[32]
T. harzianum, T. asperellum, T. longibrachiatum, T. viride.	Fusarium oxysporum f. sp. capsici	Fusarium wilt	Pepper	[33]
T. longibrachiatum, T. harzianum	Fusarium oxysporum f. sp. ciceris	Fusarium wilt	Chickpea	[34]
T. citrinoviride	M. incognita	Root-knot	Tomato	[35]
T. longibrachiatum	Pythium ultimum	Damping-off, root rot	Melon	[36]
T. asperellum	Sclerotium cepivorum	White rot	Onion	[37]
T. asperellum	F. oxysporum	Fusarium wilt	Stevia	[38]
T. asperellum	Sphaerothecafuliginea	Powdery Mildew	Cucumber	[39]
T. afroharzanium	Plasmopara viticola, Uncinula necator	Downy mildew, Powdery mildew	Grapes	[40]

). There are many species of bacterial MBCAs, including many genera with different species, such as Bacillus subtilis, B. thuringiensis [Bt], Agrobacterium radiobacter strain 84, Pseudomonas fluorescens, and Serratia marcescens. On the other hand, fungal MBCAs contain Beauveria bassiana, Fusarium verticillioides, Gliocladium spp., Trichoderma spp., Verticillium lecanii, and Streptomyces [10]. Nematophgus fungi also include: Arthrobotrys robustus, Aspergillus niger, Nematophthora gynophila, Verticillium chlamydosporium, and some species of Hirsutella, Acrostalagmus, and Harposporium [11]. Other mycoviruses and bacteriophages are also reported as effective as MBCAs against specific plant pathogens [12].

3. Advantages of Using Microbial Biological Control Agents (MBCAs)

Microbial biological control agents (MBCAs) are microorganisms with a strong ability to reduce various plant pathogens [7]. Many species of MBCAs effectively manage plant pathogens, and their products can be formulated for plant disease control [1]. Given the harmful effects of chemical pesticides—such as toxicity for workers, air pollution, carcinogenicity, ozone depletion, and contamination of plants, animals, and groundwater—MBCAs are viewed as an alternative to these chemicals, offering various advantages (Figure 1), including enhanced safety for humans, animals, and environmental health [41]. MBCAs are more target-specific and result in little to no residual effects. As biopesticides for plant diseases, MBCAs and their products are non-pathogenic to non-target organisms. They can also be part of integrated control program strategies for managing plant disease and other components that help sustain and develop their populations in the environment with minimal disruption and interference [7].

4. Mode of Action

Microbial biocontrol agents utilize various mechanisms against harmful plant pathogens, including mycoparasitism, antibiotics, bacteriocins, siderophores, lytic enzymes, and additional indirect methods (e.g., competition and quorum quenching) (Figure 2). The primary mode of action for biocontrol involves not only mycoparasitism and antibiosis but also the induction of plant resistance to stress and the enhancement of plant growth [42,43]. Moreover, a significant aspect of MBCAs is their capacity to induce systemic resistance in plants; in certain instances, biocontrol is achieved through the release of antibiotics, such as T. virens against Rhizoctonia solani in cotton damping off, while mycoparasitism occurs using T. harzianum against Pythium spp. Various genetic and mutational analyses confirm their role in inducing plant resistance [44,45]. Therefore, exploring alternative action mechanisms could help ensure the safety of biological control methods for plant diseases.

4.1. Hyperparasitism

Indeed, the interaction process between two organisms—host and parasite—involves obtaining nutrients from one organism by another organism and is called parasitism. Simultaneously, this interaction is termed hyperparasitism when the host is also a parasite, such as a plant pathogen [7]. This type of interaction is reported among fungi and is a common phenomenon in nature. In some biotrophic fungi, the hyperparasite primarily depends on the status of the host fungus (plant pathogen) and acquires nutrients from host cells via haustoria without killing the host plant [3]. This strategy is crucial in plant disease management. However, it faces challenges in mass production as a commercial biocontrol agent because it relies on the substrate of living host fungus mycelium [1,5].

On the other hand, necrotrophic hyperparasites obtain nutrients from dead hyphal cells or host spores after killing them, along with organic matter. This capability enables these organisms to function as commercial MBCAs with large-scale production [3]. The primary process of parasitic mechanisms starts with the excretion of cell-wall-degrading enzymes and/or secondary metabolites very close to the host cell that degrade the cell wall, followed by the disorganization of the cytoplasm. The most commonly found enzymes in degradation are chitinases, proteases, and β-1,3-glucanases, or cellulases, in hyperparasites of oomycetes [7]. Researchers have previously investigated the phenomenon of hyperparasitism. Jeffries mentioned that about 30 hyperparasitic species belonging to 16 different genera of fungi are registered against Rhizoctonia solani [46]. Particular interest has been related to the use of hyperparasites. Hyperparasitism has been reported in approximately 30 fungal species against rust disease pathogens such as Cladosporium uridinicola and Alternaria alternata against Puccinia violae and P. striiformis f. sp. tritici, respectively. Meanwhile, the germ tubes of A. alternata penetrate the urediospores of the fungus P. striiformis f. sp. tritici, causing them to collapse completely [47].

Trichoderma is one of the most fortunate mycoparasites. This fungus has structures that help in attachment and infection, and then it kills its host. Cell-wall-degrading enzymes (CWDEs) are often used with antimicrobial secondary metabolites [7]. Recognition of the fungal host causes changes in the transcriptional and gene expression of the “molecular weapons” that play a role in the attachment and lysis of hosts, such as certain CWDEs. During the Trichoderma-mediated mycoparasitism process, the mycoparasitism-related gene families, like prb1 and ech42, are upregulated. The activation of CWDEs releases oligosaccharides and oligopeptides by the host, which are recognized by Trichoderma receptors and thereby act as inducers [48]. The attack by Trichoderma leads to further increased degradation and permeability of host cell walls in the attacked part and death. The synergistic transcription of several genes implicated in cell wall degradation during the interaction between Trichoderma atroviride, Phytophthora capsici, and Botrytis cinerea has been reported [49].

In the case of Trichoderma parasitism on nematodes, the gelatinous matrix (GM) plays a significant role in the parasitism processes and nematode-fungus interactions. Studies have shown that a gelatinous matrix benefits fungal attachment, enhances the parasitic abilities of fungal isolates, and that fungus uses it as a source of nutrients. Fungal conidiospores can attach to nematode eggs, J2s, and egg masses in contact with the gm; otherwise, gm-free eggs and J2s are nearly unattached by the fungal conidiospores [50,51]. Trichoderma species and isolates vary in their attachment and parasitism abilities, reflecting the specificity of parasitism processes. Conidia attachment and parasitism processes were observed by detecting the green fluorescent protein-expressing Trichoderma spp. using scanning electron microscopy (SEM) in vitro. Scanning showed fungal and parasitic behavior, including tight attachment of spores and hyphae, coiling of hyphae around J2s, and formation of appressoria-like structures [50].

Recently, Pasteuria penetrans has been suggested as a promising biocontrol agent against several economically critical plant-parasitic nematodes, particularly root-knot nematodes (Meloidogyne spp.) [52]. During the movement of the host nematode juveniles through the soil, endospores of the Pasteuria, which are a persistent survival stage, attach to the cuticle of the host nematode. Subsequently, the bacterium’s endospores infiltrate the juvenile nematode’s body after the juvenile has penetrated the roots and commenced its feeding activity. The bacterium grows vegetatively inside the nematode’s body, suppressing and inhibiting the female nematode’s egg production. Bacteria mature and sporulate, filling the female’s body with spores. Eventually, the female body ruptures, releasing thousands of endospores into the soil, which can then infect other nematode juveniles. [53]. Nematophagous fungi are considered biocontrol agents against plant-parasitic nematodes. This fungus penetrates the worm cuticle or eggshell after a solid adhesion to the host surface. Arthrobotrys oligospora is a nematode-trapping fungus that enters the nematode by forming complex three-dimensional networks [54]. The trapping process commences with the adhesion, penetration, and immobilization of the trapped nematodes. [55]. Controlling plant-parasitic nematodes using this fungus has yielded intriguing results against root-knot nematodes, as documented by numerous researchers [56,57].

4.2. Antibiosis via Antimicrobial Metabolites

Microorganisms also employ alternative mechanisms of antibiosis, including the secretion of antimicrobial metabolites. Antimicrobial metabolites are low-molecular-weight, organic, secondary-metabolite compounds synthesized by various organisms. These compounds inhibit the growth or metabolic activities of other microorganisms and are released in minimal quantities into the environment. Different substances are produced during the antibiosis process, such as antibiotics, bacteriocins, and organic acids [58].

4.2.1. Antibiotics

Various microorganisms synthesize antibiotics. Approximately 8700, 4900, and 2900 distinct antibiotics are produced by actinomycetes, fungi, and bacteria [59]. It is supposed that the plurality of antibiotics produced in the environment is still unknown [60]. Some microbes are the primary source of secreting and producing single or multiple compounds with antibiotic properties, which are considered an effective means against pathogens of plant disease. Researchers worldwide are concerned about antibiotic production as the primary mode of action of many biocontrol agents [61]. These scientific achievements led to the detection of many antibiotic-producing microorganisms, matching biocontrol strategies for plant diseases [61]. Bacteria synthesize antibiotics ribosomally or non-ribosomally in the bacterial cells and secrete them in their adjacent niche [62]. Production of metabolites with broad-spectrum antimicrobial activity was confirmed with different bacteria belonging to Agrobacterium, Pseudomonas, Bacillus, Serratia, Pantoea, Streptomyces, Stenotrophomonas, and other genera. Different derivatives are extracted and evaluated under laboratory and field conditions, and they present antibacterial, antifungal, antihelminthic, and antiviral effects. For example, many derivatives such as subtilin, sublancin, bacilysin, subtilosin, iturin, Tas A, fengycin, chlorotetain, bacillaene, and surfactin have been released from Bacillus alone. In addition, from Pseudomonas, diverse derivatives like aeruginosin, butyrolactones, ecomycins, pyocyanine, zwittermycin A, pyoluteorins, 2,4-diacetylphloroglucinol (DAPG), cepaciamide A, kanosamine, rhamnolipids, viscosinamide, and other derivatives were produced [63,64]. Streptomyces produce dioctatin, streptomycin, kasugamycin, actinomycin D, and azalomycin B [65,66]. Antibiotic agrocin 84 is produced by Agrobacterium radiobacter K84, and it is used successfully against A. tumefaciens, the causal organism of crown gall disease, and is produced commercially in some countries [67]. Phenazine and DAPG were secreted by Pseudomonas putida WCS358r (genetically engineered strains), suppressing wheat plant diseases [68].

The concentration of antibiotics in the rhizosphere is lower than that observed in artificial culture media, indicating a potential degradation in natural environments or ineffective bactericidal substances. This phenomenon may reduce growth and/or facilitate communication among various microorganisms [60]. Yet, these antibiotics play a critical role in microorganisms’ development in the soil, and their production is essential for the biological control of plant diseases [69]. These genera could release unknown secondary metabolites with expected antimicrobial activity, based on the genomic information. Furthermore, Pseudomonas has been widely shown to have a high ability to produce various antibiotic compounds. Previously, experiments reported the bioactivity of six classes of Pseudomonas antibiotic compounds against soil-borne pathogens such as pyrrolnitrin, phloroglucinols, pyoluteorin, phenazines, and cyclic lipopeptides [70]. In addition, some antagonistic fungi can release antimicrobial substances. Trichoderma and Clonostachys [formerly Gliocladium] produce viridin, 6-PAP, gliotoxin, and various antimicrobial compounds [71]. These released antibiotics present antagonistic interactions with plant disease-causing microbes, leading to fungosis and sporogenic inhibition, damaging of macromolecules, disturbing cellular membrane permeability, lyse fungal hyphae, and impeding electron transport [72].

Another biological activity of antibiotics is the activation of plant systemic resistance, especially under in vivo conditions. These results were confirmed since they showed a decreased effect of these metabolites at low concentrations towards plant pathogens in the rhizosphere compared to their high antagonistic activity under laboratory conditions [73]. Trichoderma produces different metabolites, such as antibiotics, phytotoxins, and mycotoxins, which help antagonism by hyperparasitism, antibiosis, or competition. It releases antibiotics, such as viridin, gliotoxin, glucanases, chitinase, and chitobiase enzymes [74].

The production of several secondary metabolites with different biological activities by Trichoderma species has been reported (

Table 3. Biological activity of bioactive compounds/secondary metabolites produced by Trichoderma.

Secondary Metabolites	Trichoderma spp.	Biological Activity					References
		Antifungal	Antibacterial	Anti-Nematode	Cytotoxic	Mycotoxin
Atrichodermone A, B, C	T. atroviride				+		[76]
bisabolan-1,10,11-triol Dechlorotrichodenone-C	T. asperellum		+				[77]
Cerebroside A, D	T. saturnisporum Trichoderma spp.		+				[78]
cycloneodiol oxide Epicycloneodiol oxide	T. harzianum T. koningiopsis		+				[79]
Harzianopyridone	T. harzianum	+					[80]
Koninginin C	T. koningii	+					[81]
6-pentyl-2H-pyran-2-one	T. atroviride T. harzianum T. koningii T. viride	+		+			[82]
Mycotoxin T2	T. lignorum	+				+	[83]
Koninginin E, B, A	T. harzianum T. koningii	+					[84]
Nafuredin	T. citrinoviride Trichoderma sp.		+				[79]
Trichodermin	T. polysporum T. sporulosum T. virens, T. reesei	+				+	[83]

). The antimicrobial inhibitory effect of microorganisms’ secondary metabolites on fungal hyphal growth or spore germination can be measured by rearing these microorganisms in culture media and then using the dual plate technique by testing their released metabolites in the medium as supernatants or the purified concentrated metabolite. Using the dual cultures technique to study the inhibitory effect in a liquid or solidified medium presents some benefits. For instance, it is a fast technique, highly reproducible, an efficient resource, and easy to measure germinated spore percentages and colony sizes. Obtained inhibition zones showed biocontrol efficacy. In vitro experiments and assays are used to screen new antagonists to detect the antagonistic effect by evaluating the antimicrobial metabolites under laboratory conditions in artificial environments. This helps estimate the mechanism compared to other mechanisms, which cannot be assessed using an in vitro assay technique. In vitro, assays may prove the significance of antibiosis in biocontrol by accepting different modes of action. The dual cultures technique’s main downside is that secondary metabolites are produced and released based on medium composition and nutrient concentration [7]. Studies show that in standard nutrient media, nutrients are around 100 times richer than in the rhizosphere. In addition, bulk soils are less nutrient-rich, containing bacteria at a concentration 10–1000 times less than the rhizosphere [75].

4.2.2. Bacteriocins

Bacteriocins are ribosomally synthesized antimicrobial peptides that usually target related microorganisms [85,86]. Researchers have recently paid attention to bacteriocins as new antimicrobial compounds and considered them immune-modulating agents. The excessive use of antibiotics over a long period has allowed infectious organisms to adapt to antibiotics and reduce their effectiveness. Bacteriocins represent a potential agent for antibiotic-resistant bacteria, referring to their broad- or narrow-spectrum activity [87]. Gram-positive and gram-negative bacteria are able to produce bacteriocins, and several of these bacteriocins are effective against many different pathogenic bacteria [88,89]. In addition, bacteriocins can be used as food preservatives, such as nisin, which regulatory agencies have legally approved as a food preservative. Despite a lack of studies on bacteriocins as biocontrol methods for plant diseases, there is growing regard for their potential application towards antibiotic-resistant bacteria. Bacteriocins and their strains effectively inhibit pathogenic bacteria in various food matrices and vegetables [90]. Bacteriocins can be as efficient in agricultural applications, which provides a healthy microbiome for many cultivated crops [91]. Bacillus subtilis 14B showed Bac 14B bacteriocin, which participates in biological control properties towards Agrobacterium spp, the causal organism of crown gall [92].

In gram-positive bacteria, the mode of action of bacteriocins is to inhibit bacteria by targeting the bacterial cell envelope. Class I bacteriocins inhibit lipid II on the bacterial cell membrane, which leads to the inhibition of peptidoglycan synthesis in the bacteria. Other bacteriocins form pores to inhibit or kill the target bacterium. Class II bacteriocins, like lactococcin A, are linked with the pore-forming receptor mannose phosphotransferase system (Man-PTS). Nisin and other members of class I bacteriocins inhibit peptidoglycan synthesis and form pores. Other class I peptides, such as thiopeptides and bottromycins, control gram-positive bacteria by attacking the translation process [93]. In gram-positive bacteria, bacteriocins inhibit their target bacteria by interacting with protein, DNA, and RNA metabolism. For instance, MccJ25 inhibits RNA polymerase, microcin B17 (MccB17) inhibits DNA gyrase, and MccC7-C51 inhibits aspartyl-tRNA synthetase. However, exemptions were noticed with MccE492, whose mechanism is via pore formation [93].

4.3. Competition for Nutrients or Space

Microorganisms possess additional strategies to protect plants without relying on their natural defenses. The limitation and shortage of nutrients or spaces in rhizosphere-associated surfaces result in competition and limit the resident soil microbes adapted to these special conditions [94]. The competition process requires highly effective biocontrol agents from native microflora. Moreover, genetic and environmental factors may influence the colonization process between plant roots and specific biocontrol agents [95]. For example, secreted organic metabolites from roots [root exudates] are considered the lively origin of organic carbon to soil microorganisms [96]. Accordingly, soil-encircled plant roots become a prosperous place for microbial diversity, multiplicity, and ecological interactions [97]. In these conditions, microorganisms adapted to survive by competing with each other for nutrients, space, and sometimes elements. Since plant surfaces and the rhizosphere have limited space, competition for niches was suggested as a microorganism’s strategy for protecting plants [98]. The likelihood of this mechanism increases because the root exudates are not stable throughout plant development and the root [99]. Competition for niches and nutrients involves the mode of action of disease and the biological control of fruits by yeast [100]. There are important things for effective biocontrol agents in colonization, such as the following: Firstly, they can thrive and multiply in the presence of large numbers of microorganisms and phytopathogens. Secondly, they have the ability to build an effective compound with high inhibitory effects towards numerous neighboring organisms. Thirdly, the ability to produce a biofilm, which forms a physical barrier, prevents communication between competitors and colonized surfaces. For example, Rhodotorula mucilaginosa decreases apple fruit colonization by Penicillium expansum and Botrytis cinerea via quick competition for available nutrients [101]. This mechanism is reported in the biological control of soil-borne diseases such as Verticillium dahlia in eggplant [102]. Biofilm formation plays a considerable role in this mechanism by obstructing the plant surface from pathogen infestation [103]. In addition, competition between organisms for organic carbon is considered a limiting factor in the soil [104].

Competition for nutrients was reported as essential in the biological control of soil-borne diseases, such as Fusarium wilt and Pythium damping-off. Previous results showed that the tested microorganisms produced no metabolites that suppressed P. aphanidermatum. At the same time, the growth suppression was correlated with glucose concentration in the medium and was associated with cucumber protection against P. aphanidermatum. In another case, in fruit postharvest diseases, carbon can be the most limiting factor on fruit surfaces [105]. Trichoderma, as a biocontrol agent, can compete with plant pathogens for carbon, nitrogen, and iron as nutritional sources and acts against soil-borne pathogens of plants. The competence of Trichoderma strains in the rhizosphere leads them to colonize plant root surfaces and compete with other microorganisms for the nutrients secreted from the roots [106].

4.4. Elimination of Quorum-Sensing Signals

Quorum-sensing (QS) is an interaction mechanism between species that distinguishes bacteria and fungi. Quorum-quenching (QQ) (interruption of QS signaling) is an interaction mechanism of interspecies, and even acts cross-kingdom [107]. Studies have shown that it can be used as a mechanism of antagonism in bacteria or for protection against pathogens such as eukaryotes. QS is a cell-to-cell signaling mechanism observed in density-dependent microbe populations [108]. It is one of the most well-studied cell-to-cell communication mechanisms in microorganisms [109]. Communication and coordination of social behavior are essential in community life. This means that QS is a pathway for sensing the existence of similar bacteria, while QQ refers to any disturbance of intercellular communication. The QS signal is broken in different ways: (1) stopping production of signaling molecules; (2) interventions with the binding of signal receptors in a bacterial cell or competition with signal molecules known as receptor analogs; (3) prohibiting target genes that should have been exciting by the QS signal, and (4) enzymatic devastation or inactivation of signaling molecules [110].

During reproduction, once bacterial cells have reached an optimum density, a specific set of genes turns over to alter cell metabolism, and bacteria begin to synthesize and produce chemical molecules known as autoinducers [111]. The molecules bind to cell receptors in the surroundings and then generate a signaling pathway that leads to population-wide changes in gene expression and eventually the unified growth of cells with high virulence. Activating the quorum sensing at a particular point can arrest the phytopathogen virulence and prohibit plant growth inhibition [107]. Some plant growth-promoting rhizobacteria produce lactonase enzymes that can be used as quenching tools. Thus, treating some seedlings with this bacterium helps activate the inhibitory pathway to ward off plant pathogens. Previous studies on plant pathogenic species showed that the inactivation of QS in Erwinia carotovora affects virulence [112].

4.5. Siderophores

Siderophores are high-affinity compounds for ferric ions with low molecular mass, produced by fungi and bacteria [113]. These compounds effectively chelate with the Fe³⁺ ion in the soil and transport it to the bacterial cell, which can then attach to the bacterial membrane receptor for use in metabolism and growth [114]. Different types of siderophores are based on the oxygen ligands for Fe [III] coordination: catecholate, hydroxamate, carboxylate, and mixed types [115]. The hydroxamate type comprises the most common group of siderophores found in nature, synthesized by both bacteria and fungi. Certain bacteria produce the catecholate type exclusively; some bacteria, such as Staphylococcus and Rhizobium, along with specific fungi, generate the carboxylate type [116]. Consequently, the number of siderophores that exist can bind to a significant portion of the low Fe³⁺ ion concentration in the soil, preventing surrounding plant pathogens from acquiring sufficient iron for their growth and reproduction. However, the host plant is less affected by iron deficiency in the soil than plant pathogens, partly due to its ability to uptake ferric-siderophore complexes and release their reductive iron for plant growth [117].

4.6. Production of Cell-Wall-Degrading Enzymes

The main constituents of fungal cell walls include about 20% chitin, around 50–60% glucans, and proteins represent about 20–30%. Disruption or degradation of the main components of the cell wall leads to fungal suppression. Thus, microbial strains that are able to secrete lytic enzymes are potent and efficient candidates for controlling phytopathogens and developing biopesticides [118]. Microorganisms release various extracellular hydrolytic enzymes, which are crucial in suppressing phytopathogens. Microbial chitinase and β-1,3-glucanase hydrolyze and destroy both chitin and β-1,3-glucan, respectively, which are two principal components of several fungal cell walls, suppressing phytopathogens [119]. Microbial strains, such as Rhizobium, B. subtilis, B. thuringiensis, B. cereus, and Serratia marcescens, are capable of releasing hydrolytic extracellular enzymes that act on hydrolyzing different polymeric compounds such as proteins, chitin, glucan, DNA, cellulose, and hemicellulose of phytopathogens. These enzymes are efficient in the suppression of phytopathogens like Fusarium oxysporum, Rhizoctonia solani, Sclerotium rolfesii, and others by hyphal curling and swelling in the hyphae, as well as affecting the cell wall structural integrity of the target pathogen [120]. Among the hydrolytic enzymes, glucanases, chitinases, cellulases, and proteases play a significant role in the suppression of phytopathogens due to their capacity to lyse and degrade, disrupting the structural integrity of fungal cell walls. Therefore, these enzymes are used to biocontrol fungal diseases, such as F. oxysporum and S. rolfsii [121]. Trichoderma asperellum has antagonistic activities against plant diseases caused by Fusarium species, which decrease the fungal growth of phytopathogens by 65–74%, as well as suppress spore germination by 30–75% through releasing hydrolytic enzymes such as β-1,3-glucanase and chitinase and destroying the cell walls of phytopathogens [122]. Enzymes known as chitinases can hydrolyze chitin polymers found in the insect cuticles and fungal cell walls by lysing the β-[1,4]-glycosidic bond and releasing chitooligosaccharides (COS) and N-acetyl-d-glucosamine [GlcNAc] [123]. Chitinase enzymes produced by Bacillus licheniformis NM120-17 and B. thuringiensis NM101-19 exhibited the potential to lyse the cell wall of different soybean fungal pathogens such as F. oxysporum, Aspergillus sp., Pythium sp., Verticillium sp., Rhizopus sp., and Rhizoctonia sp. [124]. Various rhizospheric microorganisms release chitinase enzymes with antifungal activity, including bacteria such as Serratia, Xanthomonas, Pseudomonas, Chromobacterium, Aeromonas, Klebsiella, and Streptomyces, and fungi like Penicillium, Trichoderma, and Agaricus [125]. The antifungal mechanism of β-1,3-glucanases and β-1,6-glucanases depends on their ability to lyse glucosidic bonds of both β-1,3-glucans and β-1,6-glucans, respectively, resulting in the degradation of the principal structural constituent of the fungal cell wall [β-glucans] into glucose monomers [123]. β-glucanase enzymes secreted by B. subtilis CW14 suppressed the mycelial growth and spore germination of Aspergillus ochraceus and decreased fungal infection in soybean plants by 96% [126]. The exterior layer of the fungal cell wall consists of glycoproteins, which are considered a suitable target for protease enzyme activity that hydrolyzes this structural protein into small peptides, resulting in cell lysis, as well as cellular leakage [123]. Therefore, protease enzymes play a crucial role in the suppression of phytopathogenic fungi, where B. amyloliquefaciens exhibited protease-mediated antagonism against Fusarium semitectum, F. oxysporum, Macrophomina phaseolina, and Alternaria alternate [127]. However, the antagonist can produce more than one enzyme, such as B. velezensis, that suppresses mycelial growth and spore germination of Colletotrichum gloeosporioides [Anthracnose] in walnut plants through the combined effect of β-1,3-glucanase, chitinase, and protease enzymes [128]. Similarly, T. asperellum ZJSX5003 released β-glucanases, chitinase, and protease against F. graminearum, the causative agent of stalk rot of maize [127]. Cellulase enzymes play a vital role in the biocontrol of oomycetes by the degradation of cell walls, which primarily consist of cellulose, into simple monomers like hexose and pentose, resulting in cell deformation and lysis and cellular contents leakage [123,129]. For instance, cellulase enzymes secreted by B. velezensis 6–5 showed more than 90% inhibition against the causal agent of potato blight disease (Phytophthora infestans) [129].

4.7. Induced Plant Resistance and Priming

Plants use various chemical and physical defense mechanisms to fend themselves against phytopathogens. Enhancing plant resistance is one of the most effective agronomic techniques to avoid biotic losses in crop yield. Inducible resistance mechanisms supplement constitutive mechanisms like cuticles [7]. When beneficial microbes colonize plant roots or necrotizing pathogens attack plants, a unique physiological condition called “priming” is induced. The diverse cellular defense strategies triggered by the invasion of pathogens and insects or in response to abiotic stress are stimulated faster and stronger in primed plants [130]. Antagonistic microorganisms can provide systemic resistance in plants against various plant diseases by inducing resistance and bio-priming. Applying non-pathogenic microorganisms (priming) to plants might reduce the effects of biotic and abiotic stresses and, in some instances, even harm from insects and nematodes [3]. Several beneficial microorganisms can induce plant resistance or enhance the immunity response of plants without direct interaction with pathogens through natural products and chemical compounds such as microbial metabolites, synthetic chemicals, and microbe-associated molecular patterns (MAMPs) [3,7,131]. Induced resistance can occur locally or systemically throughout the plant by chemical signals [131]. Several secondary products [metabolites] contribute to signal transduction, compounds, and catalytic activities, such as salicylic acid, nitric oxide, and acetylsalicylic acid, which have characteristics that induce immunity and enhance resistance in host plants [132]. After pathogen infection, these substances generate systemic acquired resistance (SAR) in the host plant and can be released by numerous non-pathogenic microorganisms, including rhizobacteria [131]. Likewise, these compounds commonly exist in plant tissues, but their distribution varies significantly among species and genotypes of the same species [132]. It is remarkable that some of these induced substances reduce plant diseases and enhance plant vigor simultaneously, due to the increased phytohormone production [131]. MBCAs can develop plant systemic resistance, which leads to the accumulation of structural barriers and elicits various biochemical and molecular defense responses in the plants. This activity requires pathway signalization of phytoalexins, phytohormones, and defense enzymes like phenolic compounds, phenylalanine ammonia-lyase, pathogenesis-related (PR) proteins, and chitinase [5].

The molecules of defense signaling in plants include salicylic acid (SA), which contributes to SAR and defense against phytopathogens, jasmonic acid (JA), and ethylene, which are both thought to be involved in defense against necrotrophic phytopathogens and advantageous in plant-microbe interactions [5,133]. Disease suppression by resistance induction in the host plants is an alternative mechanism of MBCAs that occurs as a result of the secretion of elicitors such as volatiles, antibiotics, and proteins by MBCAs that activate gene expression of the SA pathway or the JA/ethylene pathway [134]. One of these processes, SAR, occurs through SA and usually expresses PR-proteins involving various enzymes. A second scenario, called “induced systemic resistance” (ISR), is induced by JA and/or ethylene, which are formed after applications of certain non-pathogenic rhizobacteria [135]. Generally, plant systemic resistance includes ISR, which is induced by non-pathogenic microorganisms, and SAR, which is caused by pathogenic microorganisms [5]. ISR is a prominent mechanism by which rhizospheric bacteria and fungi prime the entire plant body to enhance resistance and defense against a diversity of diseases, as well as insect herbivores [5,136]. Plants employ long-distance systemic signaling to shield distal tissue following ISR activation by triggering a strong, rapid immune responses to phytopathogen invasion [137]. Therefore, MBCAs can suppress plant diseases and pests by activating the plant’s immune system. The ability of many MBCAs to produce ISR has been observed in the past. In this context, soil treatment using the cell-free culture filtrate of T. longibrachiatum AD-1 led to the upregulation of defense-related genes PR1, PR2, PR3, and PR4 in Phaseolus vulgaris. It protected it against root rot disease caused by Fusarium spp [138]. Beneficial microorganisms, like species belonging to the genera of both Bacillus and Pseudomonas, can assist plants in developing broad-spectrum resistance against diseases via defense response stimulation [5]. Several microorganisms, such as P. fluorescens, B. amyloliquefaciens, B. cereus, B. atrophaeus, and Trichoderma spp., are effective MBCAs against bacterial, fungal, and viral invasion through ISR activation in plants [137,139]. The application of B. amyloliquefaciens Ba13 effectively decreased the incidence of tomato yellow leaf curl virus disease by activating ISR in plants [137]. The beneficial microorganisms can induce early plant ISR processes, including enhanced activities of defense-related compounds, like peroxidase, polyphenol oxidase, and phenylalanine ammonia-lyase, accumulating reactive oxygen species (ROS), chitinase, and β-1,3-glucanase [137]. ISR is activated in the host plants by cyclic lipopeptide antibiotics and siderophores. In addition, microbial volatiles are related to systemic resistance induction in the plant through an ethylene-dependent pathway [134]. On the other hand, SAR is an efficient SA-dependent mechanism that offers long-term protection against many biotrophic pathogens and pests by increasing SA levels and triggering the expression of pathogenesis-related (PR) genes [5]. These PR proteins involve different enzymes that can lyse invading cells directly, induce localized cell death, or strengthen the boundaries of cell walls to resist infections [134].

5. Formulation and Development of Biopesticides

Fermentation and formulation of biopesticides are interconnected processes that influence the product’s efficacy and overall performance. Effective biopesticide production necessitates several optimal conditions, including the selection of robust and effective strains, biosafety considerations, proper storage, rigorous quality control, extended shelf life, and advanced application technology [140]. Various biopesticide formulations are available, which consist of active ingredients [microorganisms, spores, secondary metabolites], adjuvants, carriers, and inert materials that serve as substrates for the growth of microorganisms while delivering the active components to their intended locations. These elements should protect the final product from adverse environmental conditions, enhance the activity and dispersion of the biopesticides, and maintain the stability of the pesticides during manufacture, processing, and storage [140,141]. The formulation process is crucial in the development of pesticide products, particularly concerning effectiveness and production costs. Industry secrets often protect formulation processes and technologies, resulting in limited information being available about them. Most MBCAs protect plants against fungal diseases based on the viability of cells and the subsequent biological processes underlying their mechanisms. Therefore, the primary objective of a microbe-based biopesticide formulation should be to stabilize these microbial cells, keeping them viable in reasonable quantities over extended periods from storage through usage. The formulation process significantly impacts microbial viability, as cells endure various stresses during this phase [3]. Typically, the primary objectives of the formulation process include: (i) maintaining MBCA stability during distribution, storage, and shelf life, (ii) aiding in product handling and application, (iii) ensuring the viability and effectiveness of microorganisms at the application site, and (iv) protecting the microorganisms against detrimental environmental influences [141]. Presently, numerous alternative formulation methods exist, allowing for the assessment and selection of a method that minimally impacts the viability of each microbe. Pesticides can be produced using a variety of approaches, including dry formulations (powders for seed dressing, dusts, granules, micro granules, wettable powders, and dispersible granules), liquid formulations for dilution in water [oil dispersions, emulsions, capsule suspensions, suspension concentrates], and ultralow volume formulations. However, commercially available biopesticides exist worldwide in liquid, wettable powder, and granular formulations [142]. A formulated product mainly contains or may include the following components: (1) viable microorganisms or their secondary metabolites as active ingredients, (2) inert materials serving as carriers to support and deliver the MBCA to the target site, and (3) adjuvants, such as stabilizers, surfactants, protectants, adherents, and additional nutrients, which preserve the activity and functionality of the MBCA (active ingredient) against ultraviolet radiation, desiccation, and high temperatures, as well as enhance product distribution and dispersal within the intended environment [141]. The growth medium is often combined with protective materials to mitigate the negative effects of harsh conditions on viable microbial cells. Stability during storage can be improved through pre-formulation treatments, including the adoption of suitable growth conditions. Furthermore, chemical additives or appropriate packaging are vital in maintaining formula stability and microbial cell viability [3,143]. Many MBCA formulations have been developed, including liquid and solid formulas. Generally, liquid formulations are simpler and less expensive to produce than solid formulations, although solid formulations can enhance MBCA stability [141]. Typically, dry formulas in powder or granule form are preferred over liquid formulas due to their longer shelf lives and easier transportation and storage. Most granular or powder formulas can also be transformed into liquid or water-based suspensions suitable for root-dip, spray, or drench applications [144]. The final product of biopesticides may take four possible forms: liquid, slurry, powder, or granular. In the case of solid formulas, inert carriers are classified into two categories: organic (such as cellulose, lignin, polysaccharides, humic acids, starch, and skim milk) and inorganic (including clay, vermiculite, silica, and zeolite). Dehydration of microbial biomass is necessary in formulating solid products, which can be achieved through vacuum or freeze drying [3,145]. Liquid formulations consist of microbial cell suspensions enhanced with ingredients that improve their stability, adhesion, surfactant, and dispersal properties. Liquid formulations have the advantage of being easier to handle than solid ones [3]. The formulation process also serves two additional significant purposes: it facilitates the handling and application of the biopesticide and boosts the persistence of the MBCA at the target site post-application. Consequently, various adjuvant materials are commercially available for these functions, enhancing solubility in water, promoting uniform dispersion of MBCA cells on plant surfaces, and preserving microbial viability against abiotic stresses following application [140].

6. Challenges of Biopesticides

Given the rising worries about the excessive application of chemical pesticides, the increased importance of plant pathogens as a result of food demand increase, climate change, the emergence of novel invasive strains, and pesticide resistance by pathogenic strains has led to expanded biological control of plant diseases in the sustainable agriculture systems [146]. Although biological control provides a potent and effective alternative to chemical techniques in suppressing phytopathogens, several challenges remain to be faced and overcome. Biocontrol entails the addition of non-native living microorganisms, which may have serious environmental consequences. For instance, the non-native introduced species may become invasive and harm the environment [1,5]. Furthermore, using MBCAs has not always been effective, most likely due to shifting environmental circumstances. For instance, Trichoderma sp. only displayed predatory behavior in environments with restricted nutritional availability. Trichoderma species do not attack the phytopathogen Rhizoctonia solani in compost, which provides the agent with nutrients [5]. Various other difficulties are linked to the application of MBCAs, such as the shelf life of biopesticides, which are highly biodegradable. Additionally, only a tiny percentage of pests in the field are controlled by pesticide-based methods. Therefore, they might not be effective against all pests [147]. Biopesticides are facing numerous challenges, such as formulation improvement, slower pest management, acceptance, registration for commercialization, and proper usage in agriculture [140]. However, they have many advantages, including easy handling, rapid biodegradability, effectiveness against target pests, lower toxicity, and reduction of resistance development in the pest [140,148]. These difficulties are covered briefly below.

6.1. MBCAs: From the Laboratory to the Field

The evaluation and selection of promising strains of MBCAs are achieved in specialized laboratories for the development of biopesticides that are potent and effective against phytopathogens in agriculture. These strains are typically chosen based on their capacity to combat infections, availability, host range, ease of biomass production, simplicity of formulation, and application in the field [1]. The strains of MBCAs with the lowest LC50 and LT50 are evaluated in controlled field trials after being tested in a greenhouse. Accredited laboratories and private sector partners carry out formulation and field-effectiveness experiments and conduct the required mammalian and ecological toxicity tests and quality assurance for the eventual commercialization of MBCA products [1,149]. Field bioassays are necessary to verify the effectiveness of selected products [5]. Formulation methods, storage environment, and shipment impact biopesticide stability. Therefore, optimizing the production of MBCA strains, improving the formulation approaches, extending the MBCA product’s shelf life, and realizing widespread usage via affordable manufacturing companies are essential to achieving significant success in the biopesticides strategy [150]. Government authorities regulate the large-scale application of biopesticides in the field by granting permission and authorization. The regulatory portfolio of MBCA registration is typically an adjusted version of chemical pesticides with a risk evaluation that involves mode of action, toxicological and ecotoxicological assessments, and host range [1,5]. These requirements represent a challenge for regulators due to the difficulty of determining the appropriate biopesticides that ensure safety, effectiveness, and consistency standards [1].

6.2. Limited Number of Registered MBCA Products

Although the efficacy of biopesticides is well-documented, MBCAs now represent less than 5% of the commercial value of the crop protection sector [1,131]. The limited amount of authorized MBCA products strongly correlates with the low level of technology transfer, suggesting that the agricultural system, particularly in developing nations, has not yet realized its full economic potential [5]. Identifying, characterizing, and licensing potential microorganisms requires more time and academic-industry cooperation [151]. Additionally, several moral and legal concerns exist with exploiting natural resources to control plant diseases (such as MBCAs), which may impact an area’s biodiversity [152]. In this context, new MBCA populations and species are prohibited from entering some nations. However, the commercial use of biopesticides in protected conditions like greenhouses is much easier because more isolated, regulated, and controlled environments are available and have fewer adverse ecological effects [153]. For the registration of a single strain (MBCA) for utilization at a commercial level, companies would need to carry out extensive efficacy trials with statistical significance for each disease/crop in each region. As a result of this limitation, there is a dearth of MBCA products in Europe and Asia. Furthermore, European farmers have authorized the use of only a limited number of these products for the biocontrol of plant diseases [146].

6.3. Lack of Awareness and Knowledge of Biopesticides

As opposed to chemical pesticides, which are more dependable and predictable, growers may make little financial gain through biopesticide application [154]. Occasionally, farmers are unfamiliar with practical technological advancements and are not encouraged to utilize the biocontrol technique. Positive campaigns, such as free conferences, training sessions, and community discussions, could raise awareness of using MBCAs in some farming communities [153]. Microorganism-based biocontrol offers significant advantages regarding the decrease in chemical pesticide usage in the agriculture sector. Biocontrol management directly affects farmers’ costs and revenue and indirectly affects their financial benefits due to its effects on agricultural biodiversity and environmental sustainability [150]. However, a lack of knowledge and awareness has prevented growers from using commercial biopesticides to their full potential. To renovate the farming community’s confidence in the application of biopesticides, it is necessary to reinforce the concept of biological management [1].

6.4. Legislative Procedure and Commercialization

Although biocontrol agents are becoming increasingly common in managing crop diseases, they still account for only 1% of agricultural control approaches; in contrast, synthetic pesticides represent about 15% of plant disease control. Biopesticide commercialization is a multi-step process that faces several challenges. Like synthetic pesticides, MBCAs must pass risk assessments before being granted a license for commercialization [67,155]. Based on a risk evaluation, the European Regulation (EC) No 1107/2009 establishes guidelines for the commercialization of plant protection products. In Europe, a list of approved microorganisms for use in biocontrol is provided under Regulation (EC) No 540/2011. Consequently, obtaining commercial authorizations for biopesticides (like synthetic pesticides) requires labor-intensive procedures involving several tests, such as efficiency, environmental, and toxicological assessments. Furthermore, toxicological investigations are often either nonexistent or too costly and take significantly longer for local firms in many cases [1,41]. However, the current trend toward reducing chemical pesticide use and simplifying the approval procedure for low-risk compounds may boost the manufacturing and commercialization of MBCAs globally [5]. In addition, other challenges are faced by biopesticides, which can be summarized as follows.

(1) The application of MBCAs presents significant challenges in delivering them to the appropriate location at the right time, in sufficiently dense concentration to be effective, and then ensuring they remain in place. (2). Farmers doubt the effectiveness of MBCAs. (3) MBCAs are only effective against specific diseases. (4) They require intensive labor. (5) Their effect on plant diseases is slow. (6) Currently, they are not available in larger quantities. (7) Environmental conditions influence the efficacy of MBCAs. (8) They are less effective than synthetic fungicides. (9) Only a limited number of MBCAs are usable and available in specific areas. (10) The biocontrol approach is preventive rather than curative. (11) It requires skilled and qualified individuals to multiply and supply MBCAs without contamination. (12) MBCAs have an extremely short shelf life (for example, 3 months for P. fluorescens). (13) The necessary population of MBCAs must be maintained at the proper level for practical application and checked regularly at specified intervals. (14) A biocontrol agent can sometimes become a pathogen [134].

7. Conclusions

In recent years, there have been notable advancements in understanding Biological Control Agents (BCA), which focus on developing commercial products to manage bacterial and fungal diseases. The utilization of microorganisms for biological control represents a compelling alternative approach for the suppression of plant diseases. Furthermore, it aligns with the objectives of sustainable agricultural systems. As individuals become more health-conscious, biological surfaces emerge as an effective, safe, and environmentally friendly option for mitigating plant diseases. A comprehensive understanding of disease epidemiology, cropping systems, ecology, population dynamics, and the biology of Microbial Biological Control Agents (MBCAs), along with the interrelations among these elements, is essential for the success of the biocontrol process. Understanding the modes of action or mechanisms underlying antagonist-pathogen interactions will be critical, as this knowledge could provide a robust foundation for identifying and developing more potent biocontrol agents. Since MBCAs are non-detrimental to plants, their formulations must promote the growth and activity of the microbes they contain. Furthermore, various developments, including the optimization of formulation and distribution strategies, scaling of manufacturing processes, ensuring adherence to legal regulations, and improving cost-effectiveness, are essential prior to their effective utilization. A prominent challenge associated with microbial-based biopesticides is their restricted efficacy against a broad spectrum of pathogenic organisms, resulting in chemical plant protection compounds being the sole alternative in particular situations. Moreover, the necessity to create biopesticides with prolonged shelf lives constitutes another substantial challenge.