Horticulturae 10 00805

horticulturae
Review
A Review on Biocontrol Agents as Sustainable Approach for
Crop Disease Management: Applications, Production,
and Future Perspectives
Anshika Tyagi 1,† , Tensangmu Lama Tamang 1,† , Hamdy Kashtoh 1,† , Rakeeb Ahmad Mir 2 ,
Zahoor Ahmad Mir 3 , Subaya Manzoor 4 , Nazia Manzar 5 , Gousia Gani 6 , Shailesh Kumar Vishwakarma 5 ,
Mohammed A. Almalki 7, * and Sajad Ali 1, *
1 Department of Biotechnology, Yeungnam University, Gyeongsan 38541, Republic of Korea;

[email protected] (H.K.)
2 Department of Biotechnology, School of Life Sciences, Central University of Kashmir, Ganderbal 191201, India
3 Department of Plant Science and Agriculture, University of Manitoba, Winnipeg, MB R2M0TB1, Canada;
[email protected]
4 Division of Plant Pathology, Faculty of Agriculture SKUAST-K, Wadura, Sopore 193201, India
5 Plant Pathology Lab, ICAR-National Bureau of Agriculturally Important Microorganisms,
Maunath Bhanjan 275103, India
6 Division of Basic Science and Humanities, Faculty of Agriculture-SKUAST-K, Wadura, Sopore 193201, India
7 Department of Biological Sciences, College of Science, King Faisal University,
Al-Ahsa, Al Hofuf 31982, Saudi Arabia
* Correspondence: [email protected] (M.A.A.); [email protected] (S.A.)
† These authors contributed equally to this work.
Abstract: Horticultural crops are vulnerable to diverse microbial infections, which have a detrimental
impact on their growth, fruit quality, and productivity. Currently, chemical pesticides are widely em-
Citation: Tyagi, A.; Lama Tamang, T.; ployed to manage diseases in horticultural crops, but they have negative effects on the environment,
Kashtoh, H.; Mir, R.A.; Mir, Z.A.; human health, soil physiochemical properties, and biodiversity. Additionally, the use of pesticides
Manzoor, S.; Manzar, N.; Gani, G.; has facilitated the development and spread of resistant pathovars, which have emerged as a serious
Vishwakarma, S.K.; Almalki, M.A.; concern in contemporary agriculture. Nonetheless, the adverse consequences of chemical pesticides
et al. A Review on Biocontrol Agents on the environment and public health have worried scientists greatly in recent years, which has
as Sustainable Approach for Crop led to a switch to the use of biocontrol agents such as bacteria, fungi, and insects to control plant
Disease Management: Applications, pathogens. Biocontrol agents (BCAs) form an integral part of organic farming, which is regarded as
Production, and Future Perspectives.
the future of sustainable agriculture. Hence, harnessing the potential of BCAs is an important viable
Horticulturae 2024, 10, 805. https://
strategy to control microbial disease in horticultural crops in a way that is also ecofriendly and can
doi.org/10.3390/horticulturae10080805
improve the soil health. Here, we discuss the role of the biological control of microbial diseases in
Academic Editors: Livio Torta, crops. We also discuss different microbial-based BCAs such as fungal, bacterial, and viral and their
Graciela Dolores Ávila-Quezada and role in disease management. Next, we discuss the factors that affect the performance of the BCAs
Irasema Vargas-Arispuro under field conditions. This review also highlights the genetic engineering of BCAs to enhance their
Received: 5 June 2024 biocontrol efficiency and other growth traits. Finally, we highlight the challenges and opportunities
Revised: 4 July 2024 of biocontrol-based disease management in horticulture crops and future research directions to boost
Accepted: 26 July 2024 their efficacy and applications.
Published: 30 July 2024
Keywords: horticulture; biocontrol agents; diseases; pesticides; production; metabolic engineering
Copyright: © 2024 by the authors.

Licensee MDPI, Basel, Switzerland.
1. Introduction
This article is an open access article
distributed under the terms and
Horticultural crops are an important source of income with high nutritional, medicinal,
conditions of the Creative Commons
and industrial significance [1]. Farmers’ adaptation to horticultural farming has signif-
Attribution (CC BY) license (https:// icantly increased in recent times because of the huge demand, substantial income, and
creativecommons.org/licenses/by/ technological advancement. Agriculture and economic diversification can be accelerated
4.0/). via horticulture farming [1]. However, the production of horticulture crops is affected by
Horticulturae 2024, 10, 805. https://doi.org/10.3390/horticulturae10080805 https://www.mdpi.com/journal/horticulturae

Horticulturae 2024, 10, 805 2 of 31
both biotic and abiotic stresses [2]. Among them, phytopathogens pose serious threats
to horticultural crops across the globe. For instance, fungal pathogens such as Alternaria
spp., Phytophthora infestans, Septoria lycopersici, Fusarium oxysporum f. sp. lycopersici, Botry-
tis cinerea, and Verticillium dahliae have been found to significantly affect the quality and
yield production of horticulturally important crops [3–7]. In addition, a large number
of seed-born fungal pathogens viz., Macrophomina phaseolina, Colletotrichum, Fusarium,
Ascochyta pinodes, and Sclerotinia sclerotiorum have been reported to impair crop growth
and yield [6–10]. On the other hand, many bacterial diseases like canker, soft rot, leaf
spot/spot, wilt, blight, speck, and brown spot affect the fruit quality and crop productiv-
ity [11,12]. Moreover, different types of viruses cause severe diseases in horticultural crops
by hampering their physiological processes and hindering growth, leading to higher yield
losses [13,14].
Currently, the majority of the farmers use pesticides as a standard method of managing
disease in horticultural crops, which has huge implications on human health and the
environment. The excessive application of pesticides has polluted water bodies, leading to
a growing apprehension regarding the spread of toxic chemicals to humans [15]. Despite the
use of antifungals, on a global scale, growers lose about 10–23% of crops due to infestations
of fungal pathogens [16]. The situation is further worsened by an additional 20% crop loss
during the post-harvest process [16]. On the other hand, for the management of bacterial
disease, antibiotics are used, which have also become a major concern due the emergence
of antibiotic resistant pathovars. For instance, strAB streptomycin-resistance genes have
been found in Pseudomonas syringae, Erwinia amylovora, and Xanthomonas campestris [17].
Previous study has shown that bacterial plant pathogens have developed resistance against
a wide range of antibiotics such as streptomycin and tetracycline [17]. Therefore, there is a
need to find a viable alternative to address this problem and manage bacterial pathogens
in horticulture. Although transgenic technology has also been adapted for improving
disease resistance in horticulture crops, apart from several benefits such as developing
resistant plant varieties, transgenic plants raise serious concerns regarding health risks and
environmental concerns [18].
BCAs can play a vital role in disease management and increase food production in
a more sustainable way [19]. However, researchers should also thoroughly investigate
the range of BCAs to reduce the use of synthetic chemicals that are damaging the balance
in ecosystems as well as address pathogen resistance to BCAs. The use of pesticides
has expanded during the last decade due to their contribution to improving agricultural
productivity by controlling the prevalence and persistence of plant pathogens [20]. Previous
studies support the potential benefits of pesticides to control plant pathogens [21,22].
Eventually, there is a strong anticipation that scientific endeavors will strongly need to
mitigate the use of hazardous pesticides to contain the peril they pose to human well-being
and disturbance in the ecosystem. All of the above-mentioned strategies and their potential
constraints have forced scientists to rely on natural methods of controlling plant pathogens.
One such approach is the use of BCAs, which have a huge number of benefits and the
least constraints. In addition to the use of disease-resistant cultivars, the intervention of
BCAs has played a pivotal role in integrated pest management strategies to reduce the use
of chemical-based pesticides. Specific BCAs systematically penetrate the target pathogen
without affecting the crop plant [23]. Additionally, the public perception of using BCAs
as natural and environmentally friendly makes them more convenient for agricultural
use. The BCAs confer disease management by enhancing the immunity of crop plants,
secreting antimicrobial compounds that inhibit pathogen prevalence and growth [23]. The
importance of BCAs and the principle of synergically assessing their efficiency, efficacy,
durability, precision, and environmental safety further widen the scope for their use in
our agricultural systems. The current review article is a compendium to address the
contribution of BCAs in controlling plant pathogens and also highlights the potential
benefits of BCAs in comparison to chemical pesticides.
2. Biocontrol as a Viable Approach for Controlling Disease in Horticulture Crops

BCAs have emerged as a viable tool not only for controlling pathogens and pests, but
also in improving plant growth and soil health, which make them an important tool in
maintaining the one-health principle [24]. However, several enhancements such as scal-
ing up production, optimizing formulation techniques and delivery methods, increasing
cost-effectiveness, and meeting regulatory requirements are necessary before they can
be successfully implemented in the field [25]. The global scientific community is at pace
with exploring BCAs in terms of understanding the mechanisms behind their strategies
to control pathogens [26]. BCAs include bacteria, viruses, fungi, insects, mites, nema-
todes, yeasts, and protozoa, which control pathogens through a wide range of biological
mechanisms [27]. Upon interaction with the target pathogen, BCAs modulate their genetic
machinery to produce arsenals of compounds to circumvent the pathogen dominance and
disease prevalence [28,29]. Bacterial species such as Bacillus thuringiensis, Bacillus subtilis,
Bacillus amyloliquefaciens, Bacillus licheniformis, Bacillus velezensis, Bacillus pumilus, and Bacil-
lus mojavensis are used as BCAs against plant fungal pathogens [30–32]. The biocontrol
mechanism mediated by these bacterial species includes the production of antibiotics,
siderophore production, and the production of antimicrobial metabolites including lytic
enzymes, and most importantly, inducing systemic resistance mechanisms against specific
pathogens [33]. In addition, BCAs also trigger the production of growth phytohormones
such as cytokinins (CK), indole-3-acetic acid (IAA), and gibberellins (GA) as well as the de-
fense hormones salicylic acid (SA), abscisic acid (ABA), and jasmonic acid (JA) to improve
growth and adaptive responses during stress conditions [34,35]. Moreover, some BCAs
also produce metabolic intermediates such as ACC deaminase, the enzyme that degrades
1-aminocyclopropane-1-carboxylic acid (ACC) to regulate the ethylene concentration under
biotic stress conditions [35]. Consequently, these reports suggest that BCAs operate multi-
ple mechanisms to control pathogens in addition to improving the growth and metabolism
in crop plants [36]. Furthermore, we show how BCAs use a multifaceted mode of action for
controlling plant pathogens or pests in Figure 1.
Many antimicrobial compounds (AMCs) largely belonging to secondary metabolites
are known to be secreted by microbes such as bacteria, fungi, and actinomycetes [37]. The
classes of antimicrobial agents produced by bacteria include non-ribosomal lipopeptides
and peptides (NRPs), ribosomal peptides (RPs), and polyketides (PKs) [38]. A large class of
NRPs belongs to cyclic lipopeptides (LPs), which exert their action against a wide range
of fungal and bacterial pathogens. This class of antibacterial agents mediates their action
by attaching to a membrane to cause perforation, resulting in the leakage of ions, which
is followed by depolarization of the membrane, where, once entering the cells, it inhibits
the replication, transcription, and translation of bacterial pathogens [39]. Similarly, several
types of LPs exhibit antifungal properties and exert their action by disrupting cell walls
by synthesizing chitin and (1–3)-β-D-glucan synthases, which in turn disrupts osmotic
regulation and the morphological architecture of fungal pathogens [40]. In addition, the
Bacillus species contains three families of LPs like iturin, fengycin, and surfactin, which
possess both antifungal as well as antibacterial properties [29,41]. Hussain et al. [42] recently
reported that the LPs iturin A and bacillomycin F produced by the Bacillus siamensis Sh420
strain inhibited the growth of Fusarium graminearum. Similarly, previous studies have
shown that iturin A and bacillomycin F from the B. siamensis JFL15 strain showed strong
antifungal activity against different plant fungal pathogens such as Colletotrichum nymphaeae,
Rhizoctonia solani, and Magnaporthe grisea [43]. The β-1,3-glucanase and chitinase secreted
by B. siamensis QN2MO-1 contribute to the antifungal activities against Fusarium wilt in
tomato plants, and also promotes growth and improves fruit quality [44]. LPs have a great
potential as biocontrol elements synthesized by bacterial species to enhance agricultural
productivity. For instance, the bacteriocins belonging to RP have been reported to exhibit
broad-spectrum antimicrobial activities against closely related bacterial pathogens by
disrupting the cell wall and damaging cytoplasmic membranes [38,45]. Several Bacillus
species produce some important bacteriocins like amisin, amylolysin, ericin, entianin,
thuricin, subtilin, subtilosin A, and subtilosin B, which are used as potential BCAs against
a wide range of bacterial pathogens [38,46]. Bacteriocins like leuconocin S and leucocin
A-UAL are produced by Gram-positive and Gram-negative bacterial species, even though
their lysis mechanisms completely differ. The most widely accepted mechanism includes
the absorption of bacteriocins by cell surfaces, followed by the inhibition of cell wall
Horticulturae 2024, 10, x FOR PEER REVIEW 4 of 32
synthesis, enhancing the permeabilization of the cell membrane and the inhibition of
RNases and DNases [45,47].
Figure 1.1. Schematic

Figure diagram depicting
Schematic diagram depicting the
the defense
defense mechanism
mechanism operated
operated by
by BCAs
BCAs against
against plant
plant
pathogens. The production of bioactive molecules by BCAs mediates a diverse range of
pathogens. The production of bioactive molecules by BCAs mediates a diverse range of defense defense
responses against pathogens. They produce antibiotics, siderophores, antimicrobial metabolites,
responses against pathogens. They produce antibiotics, siderophores, antimicrobial metabolites, and
and induce systemic resistance mechanisms against specific pathogens. This defense response
induce systemic resistance mechanisms against specific pathogens. This defense response consequently
consequently improves plant growth and regulates the defense mechanisms against a wide range
improves plant growth and regulates the defense mechanisms against a wide range of pathogens.
of pathogens.
Polyketides and other peptides are another class of antimicrobial agents produced
Many antimicrobial compounds (AMCs) largely belonging to secondary metabolites
by BCAs that possess antifungal and antibacterial activities [46,48]. For instance, oxyd-
are known to be secreted by microbes such as bacteria, fungi, and actinomycetes [37]. The
ifficidin and difficidin, produced by Bacillus methylotrophicus DR-08, exert antibacterial
classes of antimicrobial agents produced by bacteria include non‐ribosomal lipopeptides
properties against R. solanacearum, the causative agent of bacterial wilt in tomato plants [49].
and peptides (NRPs), ribosomal peptides (RPs), and polyketides (PKs) [38]. A large class
Additionally, macrolactin and bacillomycin D, isolated from B. amyloliquefaciens NJN-6,
of NRPs belongs to cyclic lipopeptides (LPs), which exert their action against a wide
were found to possess antifungal properties against F. oxysporum and R. solanacearum in
range of fungal and bacterial pathogens. This class of antibacterial agents mediates their
banana [50]. The polyketide viz. bacillaene, difficidin, and macrolactin, produced by B. amy-
action by attaching to a membrane to cause perforation, resulting in the leakage of ions,
loliquefaciens DSBA-11, inhibited the growth of the Ralstonia pseudosolanacearum pathogen in
which is followed by depolarization of the membrane, where, once entering the cells, it
tomato plants [51]. Chen et al. [52] reported that the bacterial strain B. velezensis SDTB038
inhibits the replication, transcription, and translation of bacterial pathogens [39]. Simi‐
possessed antifungal properties against Fusarium crown and root rot in tomatoes by syn-
larly, several
thesizing types of
metabolites LPs
like exhibit antifungal
bacillaene, properties
bactin, bacilysin, and exert
difficidin, theirmacrolactin
fengycin, action by dis‐H,
and surfactin. A wide range of fungal species have previously been reported which
rupting cell walls by synthesizing chitin and (1–3)‐β‐D‐glucan synthases, in turn
to be used as
disrupts osmotic regulation and the morphological architecture of fungal pathogens
microbial formulations to contain the pathogenesis of pathogenic fungi Botrytis, Fusarium, [40].
In addition,
Sclerotinia, and the Bacillus[53].
Pythium species
The contains
biocontrolthree families
of these of species
fungal LPs likerelies
iturin,
onfengycin,
mechanisms and
surfactin, which possess both antifungal as well as antibacterial properties
such as mycoparasitism, the synthesis of antimicrobial metabolites, and competition for [29,41].
Hussain et
nutrients al.space
and [42] recently
[54,55]. reported that the LPs iturin A and bacillomycin F produced by
the Bacillus siamensis Sh420 strain biocontrol
The Trichoderma species-based inhibited the growth of
mechanism Fusarium
relies graminearum.
on antibiosis, Simi‐
mycopara-
larly, previous
sitism, studies
competition have shown
for resources and that iturin
space, and A and bacillomycin
inducing F fromThe
defense systems. themechanism
B. siamensis
JFL15
of strainrelies
antibiosis showed strong
on the antifungal
production activityand
of primary against different
secondary plant fungal
metabolites pathogens
that are critical
such as Colletotrichum nymphaeae, Rhizoctonia solani, and Magnaporthe grisea [43]. The
β‐1,3‐glucanase and chitinase secreted by B. siamensis QN2MO‐1 contribute to the anti‐
fungal activities against Fusarium wilt in tomato plants, and also promotes growth and
improves fruit quality [44]. LPs have a great potential as biocontrol elements synthesized
by bacterial species to enhance agricultural productivity. For instance, the bacteriocins
belonging to RP have been reported to exhibit broad‐spectrum antimicrobial activities
in controlling the growth and division of pathogens. Reports suggest that Trichoderma spp.,
releases around 390 non-volatile metabolites, some of which include caffeic acid, cathequin,
ferulic acid, gliotoxin, gliovirin, heptelidic acid, nematolin, sepedonin, 3,4,15-scirpenetriol,
and viridian [56]. For instance, Trichoderma spp. helps in nutrient and water uptake, en-
hances the metabolism of plants, and induces defense signaling pathways by establishing a
positive interaction with crop plants [57]. Strains of the fungal species Trichoderma harzianum
have been characterized and identified as potential candidates to control F. oxysporum f. sp.
lactucae (Fol) in baby lettuce (Lactuca sativa L. var acephala) [58].
3. Factors Affecting the BCAs Activities and Strategies to Overcome Limitations of

BCA Effectiveness
Despite huge efforts, the impact of biocontrol has remained reasonably insignificant in
managing to control diseases in crops in comparison to its synthetic counterparts [23]. Most
importantly, BCAs face greater challenges when transforming their roles from experimental
conditions to crop fields exposed to various environmental factors [59]. For instance, the
type of plant species and genotype have a significant role in the effectiveness of BCAs [60].
Umer et al. [61] reported that the production of antimicrobials from BCAs as well as their
colonization and induction of systematic resistance is dependent on the type of plant
and varies from species to species. Consequently, for a targeted application of BCAs,
scientists must uncover the complexities of host plants, BCAs, and pathogens to avoid
less effectiveness and failure [62]. The pathogen also greatly influences the biocontrol
potential of specific types of BCAs. Reports suggest that each pathogen interacts with the
host plant differently in terms of pathogenesis and prevalence due to the ecological fitness
and genetic variability in specific pathogens [63,64]. Additionally, a large number of pests
have developed resistance to BCA antimicrobials. For example, the resistance of several
pests such as Diabrotica virgifera, Busseola fusca, Helicoverpa zea, Spodoptera frugiperda, and
Pectinophora gossypiella has been reported toward Bt toxins [59]. The nature of BCAs and
the class of biocontrol agents they release is another factor that is critical for efficacy as
potential BCAs. Consequently, to understand the nature of BCAs, it is critical to investigate
the distribution pattern of BCAs in the rhizosphere [63,64]. Furthermore, the mode of
action of BCAs, pathogen selection, and the ability to resist harsh climatic conditions play
defining factors in the success of BCAs in disease management [65].
Environmental conditions such as biotic and abiotic factors play a pivotal role in the
ability of BCAs to kill specific pathogens [59]. It is very important to select a specific type of
BCA that has higher stability and efficacy in a wide range of environmental conditions like
heat stress, drought stress, salinity stress, type of soil, and competition with other biotic
species [66]. Understanding the factors that influence the BCAs and their efficacy is critical
before their application in crop fields. Currently, the efficacy, viability, and susceptibility
of the BCAs to stressful environmental conditions is one of the major problems that has
limited its expansion [23]. Considering this limitation, the efficacy of biocontrol BCAs can
be increased by preparing small liquid formulations. For instance, to control the devastating
impact of Sclerotium rolfsii, Sutthisa et al. [67] prepared 5% trehalose-Trichoderma asperellum
MSU007 as a liquid formulation. Another strategy to enhance the efficacy of BCAs is
to combine them with fungicides in order to weaken and stress the pathogens to render
them more susceptible to subsequent attacks by BCAs [68]. For instance, the combination
of Fusarium sp. isolates with a benzimidazole fungicide improved the effectiveness of
Fusarium sp. in controlling the Fusarium wilt of carnation or cyclamen [69]. Consequently,
by employing a combination of BCAs and synthetic chemicals, the duration of effectiveness
will be increased, and therefore, the use of fungicides or antibacterial chemicals will be
reduced, leading to the sustainable agricultural production of crops. The combinatorial
approach of using nanoparticles and BCAs is another versatile method for enhancing the
efficacy of BCAs. For example, Vehapi et al. [70] used a formulation based on nanoparticles
(BpNPs), polyvinyl alcohol (PVA), and sodium alginate (SA) in combination with B. pumilus
as plant protection against Colletotrichum gloeosporioides infestation to inhibit postharvest
fungal decay disease in crop plants. The huge number of inclusive studies demonstrates
the potential of BCAs to enhance agricultural productivity at a global scale [71]. However,
the affectivity of BCAs is determined by the number of crops per farm, the size of the farms,
and the cultivation strategies [72,73]. Our existing agricultural practices must be redesigned
to favor biocontrol applications. Apart from the use of BCAs, monitoring measures are also
important to make decisions regarding the time of application, preventive measures, and
the use of pathogen-resistant varieties are crucial to decrease the use of synthetic chemicals
for pest control. Additional considerations include the choice of cropping system and
specific environmental conditions that help avoid pest pressure and disease progression.
4. Fungal Species as BCAs: Mechanisms and Applications

Fungal species are one of the important groups of BCAs that are commonly used to con-
trol pests and pathogens mainly due to their target specificity, quick generation time, and
relatively high reproduction rate [74]. Many genera of fungi have been utilized extensively
as efficient BCAs against fungal phytopathogens such as Alternaria [75] Penicillium [76],
Aspergillus [77], Fusarium [78], Rhizoctonia [79], Colletotrichum [80], and other devastating
pests such as insects and nematodes [81]. Trichoderma spp. is the most well-known fun-
gal antagonist being studied and employed as a microbial fungicide. Their integration
promotes sustainable agricultural practices and aids in the efficient management of plant
diseases [82].
Numerous fungi, particularly Trichoderma, have been found to have broad-spectrum
antagonistic activity against a variety of phytopathogens [75–80]. Trichoderma is a common
genus of fungus organisms that live in soil and function as mycoparasites, saprotrophs,
and plant symbionts [82]. Filamentous fungi of this genus have been studied in detail and
used as BCAs against agricultural plant diseases [83]. In order to provide effective plant
disease management, a great deal of research has been conducted in the last 10 years on the
direct and indirect control potential of BCAs against phytopathogens. In 1794, Trichoderma
was initially isolated from soil and decaying organic materials [84]. Nowadays, Trichoderma
is the source of more than 60% of effective biofungicides used worldwide [85]. Several
antibacterial secondary metabolites including viridiofungin, trichokonin, and lysozyme
have been identified from T. harzianum. These compounds have shown an antibacterial
activity against bacterial plant pathogens such as Clavibacter michiganensis and Erwinia
amylovora [86]. R. solanacearum, a causing agent of bacterial wilt disease in tomato, chilli,
and other crops, is responsible for enormous agricultural losses globally [87]. Many
approaches have been studied for the management of R. solanacearum. The crude extract
of T. harzianum could inhibit the growth of R. solanacearum when in vitro and in planta
experiments were conducted on tomato plants. Disruption of bacterial cells was confirmed
by scanning electron microscopy [88]. Applications of the T. asperellum isolates T4 and T8 in
the field delayed the onset of wilt, reduced the incidence of R. solanacearum-caused illness,
and improved tomato plant growth and yield [89].
The fungal strain BCP, also called Acremonium strictum, has the ability to hinder B.
cinerea growth by severely lysing the host hyphae [90]. Using light microscopy, the lysis of B.
cinerea mycelia was verified [90]. Fusarium wilt was found to be effectively controlled by the
Fo47 strain of F. oxysporum, which was recognized as nonpathogenic. It was also discovered
that Fo47 could raise the bioactivity of chitinase, β-1,3-glucanase, and β-1,4-glucosidase in
tomato plants, indicating that Fo47 could cause resistance in tomatoes [91]. Upon applying
a mixture of T. harzianum and rice straw (RST) and oil palm (EFB) compost to okra plants,
the plants exhibited resilience against Choanephora wet rot disease. Up to 85.04% less
illness severity was observed when T. harzianum was suspended in water [92]. Jaihan
et al. [93] used the extracts of entomopathogenic fungus Ophiocordyceps sobolifera against
Colletotrichum spp. in chili plants and found that it significantly inhibited the mycelial
growth and conidial germination of all the tested Colletotrichum spp. under in vitro con-
ditions. A previous study showed that the coexpression of β-1,3- and β-1,6-glucanase
genes in T. virens significantly inhibited pathogens such as R. solani, Rhizopus oryzae, and
Pythium ultimum (Oomycota, Chromista) [94]. In a field study, commercial formulations

of T. virens (Soilgard) and T. harzianum (Rootshield) were used to prevent Fusarium wilt in
tomato plants, where it was found that these formulations prevented 62 and 68% of the
disease, respectively [95]. Fusarium wilt of potatoes (F. oxysporum) was better controlled
by T. harzianum in a greenhouse setting compared to a field setting, but a higher yield
was obtained in the field [96]. Previous study has reported that gliotoxin, an important
secondary metabolite of Trichoderma virens T23, suppressed Sclerotium rolfsii, which causes
destructive soilborne disease in many plants [97]. Isolates of T. harzianum, T. viride, and T.
spirale showed different inhibitory effects against the mycelial growth of R. solani and F. oxys-
porum f. sp. phaseoli of bean in laboratory, greenhouse, and field conditions. T. harzianum
formulation-treated plots had a yield comparable to the healthy control. Boureghda and
Bouznad [98] reported that three strains of T. atroviride and T. harzianum (Th. 16) were
the most effective isolates in protecting chickpea seedlings against Fusarium wilt among
several isolates of T. atroviride, T. harzianum, and T. longibrachiatum. They also observed that
an increase in the vegetative growth of the tested plants was correlated with a decrease
in the severity of the disease. Tsai et al. [99] reported that among five Trichoderma strains
isolated from Anoectochilus rhizospheres, a conidial formulation of a strain of T. asperellum
mixed with carboxymethyl cellulose (CoCMC) had an excellent ability in controlling the
stem rot disease (F. oxysporum) of Taiwan Anoectochilus and could completely protect the
tested plants against this disease for 9 weeks. Schubert et al. [100] examined the potential of
Trichoderma spp. as a wound treatment for controlling wood decay fungi in urban trees and
reported that T. atroviride (T-15603.1) could be successfully applied as a biological wound
treatment against wood decay fungi. Another previous study reported that Penicillium
citrinum and Aspergillus terreus showed biocontrol activity against the Sclerotium rolfsii
pathogen by inducing jasmonic acid and salicylic accumulation in sunflower plants [101].
Among the fungal BCAs, Trichoderma is the most widely used BCA across the globe.
However, there is need to explore novel fungal BCAs with growth promoting traits as well
as effective biocontrol activity that will boost agricultural productivity.
Mechanism of Action
Fungal BCAs can employ various mechanisms such as parasitism, competition for
nutrients and space, prevention of pathogen colonization in specific host tissues, antibiosis,
and the induction of plant resistance against diseases to target pathogens, thereby inhibiting
their growth, sporulation, and spread within infected plants [45,55]. Multiple approaches
have been developed to investigate the activity mechanisms of selected BCAs [102]. In-
terestingly, some BCAs initially thought to hinder pathogen development through my-
coparasitism or antibiotic production were later found to induce systemic resistance in
plants against pathogens [82]. Besides protecting plants from diseases, certain fungal
BCAs have the ability to increase plant growth, leading to increased biomass and yield.
Hyperparasitism is a fungal propensity characterized by direct antagonistic interactions,
whereby other microorganisms eliminate a pathogen [59]. Sometimes, a fungal species
can be parasitic on different species of fungi; this tendency is called mycoparasitism [103].
When cucumbers in greenhouse conditions were treated with conidial suspensions of the
hyperparasite Ampelomyces quisqualis, they were found to be effective against Sphaerotheca
fuliginea, a causative agent of powdery mildew of cucumber. A. quisqualis parasitized S.
fuliginea extensively in studies with commercial cucumber crops [104]. Previous studies
have reported that Trichoderma spp. are useful in the biocontrol of R. solani, the main fungus
that causes damping-off and root rot in many crops [105,106]. Due to their hyperparasitic
nature, Trichoderma spp. are among the most studied fungi as BCAs because they effectively
protect crops against a wide range of plant pathogens. On the other hand, antagonistic
relationships, or antibiosis, are biological interactions between two or more organisms that
are harmful to one or more of them. Antagonistic fungi, for instance, produce antimicro-
bial compounds to inhibit the growth of pathogenic fungi in their vicinity [107]. Fungi
produce one or more antimicrobial compounds and other secondary metabolites with
antibiotic activity [108]. The most studied fungal species for antibiosis is Trichoderma [109],
and Gliocladium spp. [110] and T. virens (syn. Gliocladium virens) secrete gliotoxin and
gliovirin [111,112]. A previous study reported that the inoculation of strawberry plants
with Trichoderma spp. controlled B. cinerea infection as well as improved growth [113].
Competition occurs when two organisms vie for nutrients or space. Certain species
of fungi and yeasts can impede phytopathogens through competitive mechanisms, dimin-
ishing nutrient availability. Fungus can inhibit the pathogens through the suppression of
the spore germination rates and decrease the germ tube development of pathogens [114].
Endophytic fungi can reside inside the plant tissues; this can be either cellular interspace
or intracellular space [115]. Competition for essential nutrients often leads to pathogen
starvation, a common cause of microorganism mortality, consequently facilitating the
biological control of fungal phytopathogens [116]. Trichoderma spp. produce various sec-
ondary metabolites including nonribosomal peptides, polyketides, peptaibols, pyrones,
siderophores, and volatile and non-volatile terpenes, which possess pharmaceutical and
biotechnological significance [117]. The symbiotic association of Trichoderma with plant root
systems enhances mineral and water uptake while conferring resistance against pathogenic
organisms. Under iron-deficient conditions, many fungi secrete siderophores, iron-binding
ligands facilitating the mobilization of environmental iron [118]. These siderophores en-
hance the rhizosphere competence of T. asperellum strain T34, rendering it effective as a
BCA against F. oxysporum f. sp. lycopersici (Fol) on tomato plants [100]. For instance, the
efficient biocontrol of Pythium and F. oxysporum mediated by Trichoderma was found to be
associated with the bioavailability of iron [119].
Trichoderma spp. has gained significant attention in the field of agriculture due to
its potential as a BCA against various phytopathogens. Its effectiveness lies not only in
its direct antagonistic action against pathogens, but also in its ability to trigger various
regulatory mechanisms in plants, leading to enhanced disease resistance [82]. The T. asperel-
loides PSU-P1 strain was able to inhibit the growth of Stagonosporopsis cucurbitacearum by
expressing PR genes, chitinase, and the glucanase enzyme. S. cucurbitacearum is responsible
for disease in muskmelon. Scanning electron microscopy (SEM) analysis confirmed that
T. asperelloides PSU-P1 mediated the destruction of S. cucurbitacearum hyphae [120]. The
biocontrol of Sorghum anthracnose, caused by Colletotrichum graminicola, with T. asperellum
and T. harzianum was demonstrated in one study. In this study, seeds bio-primed using
the T3 (T. asperellum) isolate resulted in the highest increased activities of the antioxidant
enzymes, with superoxide dismutase showing a 36.63% increase, peroxidase exhibiting a
43.59% increase, and polyphenol oxidase showing a 40.96% increase at 48 h post pathogen
inoculation. Following the 15th day of pathogen inoculation, lignification was increased in
the sorghum roots in most treatments, indicating a reinforcement of the defense mecha-
nism. The field trials with T3 were found to be most effective [80]. Many other species of
Trichoderma have been reported for their prominent plant growth promotion and biocontrol
properties. The noticeable species of Trichoderma are T. atroviride [121], T. harzianum [122], T.
koningii [123], T. hebeiense [124], T. longibrachiatum [125], T. polysporum [126], T. reesei [127], T.
virens [128], T. viride [129]. T. koningii, and B. megaterium, either alone or in combination have
been tested for controlling root-knot nematodes and F. oxysporum in potato plants [130].
Induced resistance (IR) stands out as a crucial mechanism of biocontrol in plants and is
effective against both soilborne and foliar pathogens, as highlighted by Hossainet al. [131].
This resistance mechanism hampers pathogen growth and spread by triggering the secretion
of defense-related enzymes like chitinases, proteases, and peroxidases [132]. The fungi
Penicillium and Trichoderma have been shown to induced systemic resistance (ISR against
pathogens. Previous study has reported that the inoculation of plant growth-promoting
fungi (PGPF) in tobacco plants not only increased their growth, but also induced immunity
against potato virus Y (PVY) infection. In comparison to non-inoculated plants, the treated
plants exhibited better growth, reduced virus concentrations, and activated genes associated
with defense, indicating the efficacy of PGPF in boosting plant protection [133].
Salicylic acid (SA), produced by the T. harzianum T39 strain, has been shown to induce
resistance against B. cinerea in bean plants [134]. Applying a BCA directly to a diseased plant
part exhibited ISR, while using dead cells of the BCA showcased local induced resistance
(IR). For instance, the use of dead cells of T39 can hinder powdery mildew infection in
cucumber and B. cinerea infection in tobacco, pepper, and beans. Additionally, inoculating
the cucumber seedlings’ leaves, and roots with T. harzianum led to increased peroxidase
and chitinase activity [135]. Root endophytic fungal isolates KB2S2-15 (ectomycorrhiza)
and KA2S1-42 (Pleosporales) showed the complete suppression of P. infestans sporangia
germination, with a slightly lower inhibition with KB1S1-4. Pre-treating leaflets with a
5% extract from these endophytes resulted in the total suppression of P. infestans mycelial
development and late blight symptoms. This suggests that these biocontrol candidates
could be used to control late blight disease [136]. Different fungal BCAs for disease
management in sustainable agriculture are shown in Table 1.
Table 1. List of fungal BCAs for disease management in plants.
Fungal Species Mode of Action Disease/Host Reference

Ampelomyces quisqualis Hyperparasitism Effective against powdery mildew in grapes [137]
Induction of systemic
resistance in pepper Effective against blight and fruit rot of peppers
Trichoderma harzianum [138–140]
plants along with (Phytophthora capsica)
capsidol accumulation
Aureobasidium pullulans Antagonism Effective against gray mold of strawberry [141–144]
Manage blue mold (Penicillium italicum) on citrus
Pichia guilliermondii Antagonism [145–147]
fruits
Cladosporium cladosporioides
Antagonism Manage white rust disease in chrysanthemum [148]
Cladosporium pseudocladosporioides
Candida famata Antagonism Manage Penicillium digitatum in orange fruits [145,149]
T. harzianum Antagonism Fusarium crown and root rot (FCRR) of tomato [150]
T. atroviride T95 Mycoparasitism Botryosphaeria berengeriana f. sp. piricola [151]
T. harzianum (Th.J.89-2), T. viride
(TV.J.92-1 and ITCC-1433), Antagonism Dry root rot chilli (R. solani) [152]
T. auxeoviride
T. koningii Mycoparasitism Sclerotinia sclerotiorum [153]
Trichoderma longibrachiatum Antagonism Sclerotium cepivorum [154]
Fusarium root and stem rot disease
Trichoderma asperellum Antagonism [155,156]
Apple replant disease
T. viride Mycoparasitism F. oxysporum f. sp. adzuki and Pythium arrhenomanes [157]
T. viride Antagonism Phytophthora infestans [158]
Alternaria alternata, Alternaria solani, Colletotrichum
T. viride Antagonism spp., Fusarium solani, Fusarium equiseti, and F. [159]
oxysporum
T. virens Mycoparasitism Pythium ultimum [160]
T. virens Mycoparasitism Phytophthora erythroseptica [161]
Cell wall-degrading
enzymes, synthesis of Potential to control Penicillium expansum, B. cinerea,
Aureobasidium pullulan [162,163]
antifungal compounds, Aspergillus niger, and Rhizopus stolonifer in grapes
and mycoparasitism
C. gloeosporioides and R. vinctus Panama wilt of banana [164]
Table 1. Cont.
Fungal Species Mode of Action Disease/Host Reference

Seed rot and damping-off of several seed- and
Mycofungicide soilborne plant pathogens like P. infestans (late blight
Chaetomium globosum [165–167]
compound production of potato), Pythium ultimum (Pythium damping-off of
sugar beet), Alternaria raphani, and A. brassicola
Apple rot, apple collar rot, and crown rot
Paecilomyces Antagonism [168,169]
(Phytophthora cactorum)
Aspergillius versicolor lm6–50 Antagonism Powdery scab in potato (Spongospora subterranea) [170]
In the past two decades, there has been significant progress in utilizing fungi as
biological control agents (BCAs), with several commercially available BCA products already
on the market. The future expansion of fungal BCAs hinges on the successful development
of resting spores and robust mycelia. However, BCAs alone may not suffice to control
all types of plant diseases across diverse conditions. While the mechanisms of action
for some BCAs are becoming clearer, further research and development are necessary to
better understand their behavior. The genetic transformation of fungi holds promise for
enhancing BCA performance under varying environmental conditions, however, potential
risks related to their application into the environment require thorough investigation to
establish acceptable implementation guidelines.
5. Bacteria as BCAs for Plant Disease Management

Beneficial bacteria such as Bacillus, Bradyrhizobium, Paenibacillus, Pseudomonas, Acineto-
bacter, Azotobacter, Azospirillum, Rhizobium, and Streptomyces are known to promote not only
plant growth, but also act as portent BCAs [171,172] that use different strategies to combat
pathogen attack. For example, they can secrete diverse antimicrobial compounds such as
antibiotics, lipopeptides, biosurfactants, microbial volatile compounds, bacteriocins, and
cell-wall degrading enzymes against different bacterial and fungal pathogens [29,172–174].
On the one hand, they can trigger the host immune system by acting as elicitors or priming
agents. Additionally, BCAs may potentially disrupt the pathogens’ quorum sensing (QS)
system by preventing or enzymatically breaking down the formation of signal molecules
that initiate infections [174,175]. When it comes to nutrition acquisition, highly competitive
bacterial BCA can outcompete the pathogen by using low-molecular-weight siderophores
that have an affinity for ferrous iron to colonize and persist in the infected site [175]. The
first use of bacteria as a BCA for plant protection was Bacillus thuringiensis [176]. Similarly,
after the discovery of penicillin as an antibiotic to control bacterial pathogens, Staphylococcus
was the first to revolutionize the scientific community [177]. The primary bacteria utilized
as BCAs in horticulture crops are strains of Bacillus, Pseudomonas, and Serratia [173]. The
significant contribution of microbial pathogens in controlling major diseases in horticul-
ture crops is widely recognized. The bacteria release secondary metabolites to inhibit the
growth of fungal pathogens and serve as potential biopesticides. Bacillus species are the
most commonly used biopesticides and they also enhance plant defense by activating the
induced systemic response (ISR) (Figure 2). Ongena et al. [178] reported that bean and
tomato plants with a high concentration of Bacillus in their rhizosphere exhibited enhanced
disease resistance against B. cinerea.
Biological control agents show great promise in managing diseases and reducing the
spread of pathogens at the site of infection. BCAs are integrated into the soil to improve
the diversity of beneficial microorganisms in the vicinity of the rhizosphere. It restricts
pathogen growth through various processes including parasitism and the production
of lytic enzymes. Some bacteria emit volatile organic compounds such as acetoin, 2,3-
butadial, and 3-hydroxy-2butanone that stimulate defense enzymes and restrict pathogen
growth [179]. Similarly, the VOCs produced by P. fluorescens WR-1 have restricted the
growth of R. solanacearum in tomato plants [180]. Applying beneficial microorganisms
(BCAs) to root exudate enhances plant defense by activating the induced systemic resistance
(ISR) pathway and promoting the expression of defense-related genes. The root colonization
by BCAs is an important feature in the delivery of secondary metabolites and cell-wall
degrading enzymes. Additionally, the application of Pseudomonas strain PCL1751 to tomato
plants improves their resistance to the root rot pathogen F. oxysporum [181]. In another
study conducted by Schuhegger et al. [182], it was found that Serratia liquefaciens and
Pseudomonas putida have the ability to stimulate induced systemic resistance (ISR) in tomato
plants, providing defense against Alternaria alternata. Induced systemic resistance is a
crucial aspect of disease resistance as it facilitates the activation of defense hormones
and ultimately results in the production of resistance genes. In Table 2, we summarize
the list of potential bacterial BCAs that have been used against different pathogens in
sustainable agriculture.
Table 2. Role of bacterial BCAs to control pathogens in different crop systems.
Bacterial Pathogen Dose/CFU

S. No. Disease Crop Mode of Action Reference
Strain against (g−1 /mL−1 )
Bacillus Colletotrichum Damage cell
Anthracnose 1 × 108
1. altitudinis gloeospori- Tea permeability and [183]
disease cfu/mL.
GS-16 oides integrity
Bacillus Bean, potato, Inhibition mycelium
2. B. cinerea Grey mold 106 cfu g−1 [184]
thuringiensis and rapeseed growth
Bacillus
Colletotrichum Anthracnose Apple and Inhibition mycelium
3. subtilis 108 cfu/mL [185]
spp. diseases hot pepper growth
GYUN-2311
Inhibits fungal and
Bacillus Magnaporthe 2 × 107 oomycete hyphae
4. Rice blight Rice [186]
velezensis S4 oryzae cfu·mL−1 growth and alters
appressoria
Fusarium Banana Interacts directly with
Pseudomonas
5. oxysporum f. Fusarium Banana ~107 host cells via flagella, [187]
aeruginosa 91
sp. cubense wilt pili and lipoproteins
Induction of defense
related enzymes,
Pseudomonas Xanthomonas
Bacterial production of
6. aeruginosa oryzae pv. Rice 109 cfu mL−1 [188]
leaf blight 4-hydroxy-2-
BRp3 oryzae
alkylquinolines (HAQs)
along with siderophores
Pseudomonas
Botrytis 2.5 × 108 cfu
7. aeruginosa Gray mold Tomato Type III secretion system [189]
cinerea mL−1
CQ-40
Pseudomonas
Pythium Damping- Lettuce and
8. strain 7.47 × 108 cfu Inhibit mycelial growth [190]
ultimum off Cucumber
IALR1619
Downregulation of
Bacillus Ralstonia Bacterial 1 × 108 genes associated with
9. Tomato [191]
velezensis solanacearum wilt cfu/mL spore germination and
growth
Inhibit Rhizoctonia
Bacillus
Rhizoctonia Foliar 3.7 ± 0.2 × cerealis mycelial growth
10. subtilis Wheat [192]
cerealis blight 1011 cfu/g and induce mycelial
XZ18-3
swelling and rupture.
Horticulturae 2024,
Horticulturae 2024, 10,
10, 805
x FOR PEER REVIEW 12 of 32
12 of 31
2. Schematic
Figure 2.
Figure Schematicdiagram
diagramshowing
showing thethe
rolerole
of bacterial biocontrol
of bacterial agents
biocontrol in plant
agents in disease resistance.
plant disease re‐
The induction of defense hormones triggers the production of SA and jasmonic
sistance. The induction of defense hormones triggers the production of SA and jasmonic acidacid (JA), which in(JA),
turn
confersin
which resistance to fungal
turn confers infections.
resistance The role
to fungal of secondary
infections. metabolites
The role secreted
of secondary by beneficial
metabolites microbes
secreted by
beneficial
leads to themicrobes
activationleads to thesystemic
of induced activation of induced
resistance systemic
(ISR). They resistance
can also produce(ISR). They
diverse can also
antimicrobial
produce
compounds diverse antimicrobial
such as compounds biosurfactants,
antibiotics, lipopeptides, such as antibiotics, lipopeptides,
microbial biosurfactants,
volatile compounds, micro‐
bacteriocins,
bial volatile compounds, bacteriocins, and cell‐wall degrading enzymes
and cell-wall degrading enzymes to inhibit pathogen growth and disease progression. to inhibit pathogen growth
and disease progression.
6. Viruses as Biocontrol Agents
Biological
Viruses arecontrol
importantagents BCAsshow thatgreat promisea in
act against managing
wide range ofdiseases
microbial and reducingand
pathogens the
spread of pathogens at the site of infection. BCAs are integrated into
pests [193]. Viruses often carry a negative reputation due to their association with diseases the soil to improve
the
and diversity
pandemics. of However,
beneficial itmicroorganisms
is important to note in the
thatvicinity of the of
the majority rhizosphere.
viral speciesItarerestricts
harm-
pathogen
less or even growth
helpful,through
playingvarious processes
crucial roles including[194].
in ecosystems parasitism
As the andmostthe production
abundant of
entities
lytic enzymes.
on Earth, virusesSome bacteriato have
are believed emit avolatile organic
significant compounds
ecological impact insuch as acetoin,
maintaining the
2,3‐butadial, and 3‐hydroxy‐2butanone
balance of organism populations. All threethat stimulate
domains defense
of life are enzymes
susceptible andinfection;
to viral restrict
pathogen
however, many endocellular bacteria such as phytoplasmas are conspicuously immunere‐
growth [179]. Similarly, the VOCs produced by P. fluorescens WR‐1 have to
stricted the growth
viral infection. Sinceofviruses
R. solanacearum
are frequentlyin tomato plants
regarded as [180]. Applying
the ultimate beneficial
parasites and micro‐
do not
organisms
usually cause (BCAs) to rootharm
detectable exudate enhances
to their plantare
hosts, they defense by activating
appealing candidates theforinduced sys‐
BCAs [195].
temic
A resistance
century (ISR) pathway
ago, Mallmann and promoting
and Hemstreet the expression
[196] initially proposed of thedefense‐related genes.
use of bacteriophages
The root against
as BCAs colonization by BCAs
bacteria. is an researchers
Currently, important feature in the delivery
are interested of secondary
in investigating phagesme‐as
tabolites
BCAs forand cell‐wall
bacterial degrading
disease control, enzymes.
primarily Additionally,
because of the fast application of Pseudomonas
development of antibi-
otic resistance
strain PCL1751in tobacterial
tomato plantspathogensimprovesthat has
theirresulted
resistancein serious
to the rootagricultural
rot pathogenloss [197].
F. ox‐
Interestingly,
ysporum [181].phages are viruses
In another study that only infect
conducted bacteria; they
by Schuhegger et do
al. not directly
[182], it washarmfoundplants
that
or mammals.
Serratia On the
liquefaciens other
and hand, theputida
Pseudomonas utilization
have ofthethe mycovirus
ability Cryphonectria
to stimulate inducedhypovirus
systemic
1resistance
(CHV1), (ISR)
which inwas found
tomato to induce
plants, providinghypovirulence in theAlternaria
defense against ascomycetous fungus
alternata. Cry-
Induced
phonectriaresistance
systemic parasitica,iswas the first
a crucial instance
aspect of a virus
of disease being aassuccessful
resistance BCA
it facilitates theinactivation
fungi [193].
of
Since then, CHV1 has been used to impede the spread of chestnut
defense hormones and ultimately results in the production of resistance genes. In Table 2, blight in forests and
orchards
we [198]. the
summarize Thelist
first
ofinstances
potential of virus biocontrol
bacterial BCAs thatinhave insects
been were
usedtheagainst
applications of
different
baculoviruses to manage populations
pathogens in sustainable agriculture. of lepidopteran insects [199]. Viruses can also be
employed to manage viral disease in plants via cross protection. Here, attenuated plant
virus strains are used as a BCA in order to safeguard crops from pathogenic strains of
the same or similar viral species. The basis for this crop protection technique is the cross
protection/interference phenomena, which was initially studied in tobacco plants infected
spread of chestnut blight in forests and orchards [198]. The first instances of virus bio‐
control in insects were the applications of baculoviruses to manage populations of lepi‐
dopteran insects [199]. Viruses can also be employed to manage viral disease in plants via
cross protection. Here, attenuated plant virus strains are used as a BCA in order to safe‐
Horticulturae 2024, 10, 805 guard crops from pathogenic strains of the same or similar viral species. The basis for this
13 of 31
crop protection technique is the cross protection/interference phenomena, which was in‐
itially studied in tobacco plants infected with a yellow strain of the tobacco mosaic virus
with
(TMV).a yellow
Figurestrain of theviral
3, shows tobacco mosaic
BCAs virus
employ (TMV).strategies
different Figure 3, shows viral BCAs
to safeguard employ
plants from
different
pathogenstrategies
attacks. to safeguard plants from pathogen attacks.
Figure 3. Schematic
Schematic diagram
diagram showing
showing the
the role of viruses as BCAs in controlling plant pathogens
and pests.
and pests. (A)
(A) Depicts
Depicts plants
plantswith
withviral
viralbiocontrol
biocontrolagents
agentsthat
thatprotect
protectthem
them from
from pathogens
pathogens byby us‐
using
ing diverse
diverse strategies
strategies suchsuch as activating
as activating immuneimmune signaling
signaling pathways,
pathways, infecting
infecting pathogens
pathogens to their
to alter alter
their virulence, triggering pathogen cell lysis, and cross infection by activating memory resistance.
virulence, triggering pathogen cell lysis, and cross infection by activating memory resistance. (B) De-
(B) Depicts plants without viral BCAs being challenged by pathogens, which leads to disease de‐
picts plants without viral BCAs being challenged by pathogens, which leads to disease development
velopment and ultimately host death.
and ultimately host death.
6.1. Biocontrol
6.1. Biocontrol Potential
Potential ofof Mycoviruses against Fungal
Mycoviruses against Fungal Plant
Plant Pathogens
Pathogens
Mycoviruses are obligate parasites found in the majority of economically significant
plant‐pathogenic fungi.Latent
plant-pathogenic fungi. Latentinfections
infections areare often
often linked
linked to mycovirus–fungus
to mycovirus–fungus interac‐
interactions;
tions; however,
however, mycovirus
mycovirus infectioninfection can potentially
can potentially be advantageous
be advantageous or deleterious
or deleterious to the
to the fungal
fungalThey
host. host. They
have have aofvariety
a variety genome of types,
genome buttypes, butcommon
the most the mostones
common ones are dou‐
are double-stranded
ble‐stranded
RNA (dsRNA)RNA (dsRNA)
or positive- or positive‐ and
and negative-sense negative‐sense
single-stranded single‐stranded
RNA (ssRNA+/ RNA
−) genomes.
(ssRNA+/−) genomes.
Single-stranded Single‐stranded
DNA (ssDNA) genomesDNA (ssDNA)
are far genomes
less common and are
are far less common
conspicuously and
absent
are conspicuously
from dsDNA viruses absent fromThese
[200,201]. dsDNA haveviruses
a mono- [200,201]. These have genome,
or multi-segmented a mono‐nakedor mul‐or
ti‐segmented [202].
encapsidated genome, naked or encapsidated
Mycovirus-infected hypovirulent [202]. Mycovirus‐infected
Cryphonectria hypovirulent
parasitica was effectively
used in the past
Cryphonectria to control
parasitica was chestnut
effectively blight,
used which led totoan
in the past increased
control investigation
chestnut into
blight, which
mycoviruses and their
led to an increased potential for
investigation into the biocontrol of
mycoviruses pathogenic
and fungus
their potential forinthe
plants across
biocontrol
different pathosystems
of pathogenic fungus in[203].plantsBased
across on different
artificial transection
pathosystems methods, it wason
[203]. Based found that
artificial
mycoviruses have broad
transection methods, hostfound
it was rangesthatandmycoviruses
induce hypovirulence.
have broadFor instance,
host ranges species that
and induce
are phylogenetically close to C. parasitica such as C. radicalis, C. havanensis, C. cubensis, and
Endothia gyrosa were artificially infected using the synthetic transcript of CHV1-EP713,
which showed that CHV1 was not only able to multiply, but also induced hypovirulence
as well as other phenotypic changes in all of these fungal strains [204,205]. Previous
reports have shown that CHV1 can also reduce the virulence and growth of important pant
pathogens such as Valsa ceratosperma and Phomopsis G-type, highlighting their potential
as BCAs [206]. The infection of C. parasitica, Glomerella cingulate, and V. ceratosperma with
mycovirus Rosellinia necatrix partitivirus 1 (RnPV1) also inhibited growth and reduced their
virulence [207]. On the other hand, Xiao et al. [208] used purified virions of mycovirus
Sclerotinia sclerotiorum partitivirus 1 (SsPV1) to infect B. cinerea, which also showed growth
alteration, stable persistent infection, and the induction of reduced virulence. Lee et al. [209]
used protoplast fusion to transmit mycovirus FgV1-DK21 to F. graminearum, F. asiaticum,

F. oxysporum f. sp. lycopercisi, and C. parasitica. All of these hosts allowed FgV1-DK21 to
proliferate, even the distantly related C. parasitica, where it resulted in growth changes
and decreased pathogenicity, which were different from those seen in CHV1 infections.
These studies further support the notion that mycoviruses are potential BCAs and could be
applied to the control of a wide range of plant fungal pathogens.
6.2. Phage-Based Biobactericides

Plant pathogenic bacteria also pose a serious threat to crop productivity. Currently,
many bacterial pathogens have developed antibiotic resistance, which is a major concern
to the environment and human health. For example, Xanthomonas campestris, Erwinia
amylovora, and Pseudomonas syringae have been found to harbor antibiotic resistance genes
(strAB) that confer resistance against streptomycin [210]. To overcome this problem, har-
nessing phage-based BCAs is a viable strategy for controlling bacterial pathogens in sustain-
able agriculture. Bacteriophages, often known as phages, are viruses that cause bacterial
hosts to degrade by selectively infecting and multiplying within them as antimicrobial
agents. In order to control bacterial disease and increase crop yields, phage therapy is a
viable, effective strategy against resistant bacterial plant pathogens [211]. The U.S. Envi-
ronmental Protection Agency (EPA) has approved four distinct phages from Omnilytics
as active ingredients for bactericides since 2005. The first cocktail, known as AgriPhage,
guards tomato and pepper plants in both greenhouses and open fields from bacterial spot
and speck by including bacteriophages against X. campestris pv. vesicatoria and P. syringae.
Similarly, Omnilytics has created concoctions to combat fire blight on pear and apple trees
as well as bacterial canker disease on citrus and tomatoes [195]. Unfortunately, the literature
lacks specific information regarding the phage composition and anticipated reductions in
symptom development of the current cocktails; however, preliminary formulations of the
X. campestris pv. vesicatoria phage cocktail were found to reduce tomato plant bacterial spot
disease severity by an average of 17% [212]. Five greenhouse trials found that a cocktail
against X. citri subsp. citri comprising of at least some of the Omnilytics phages might lower
citrus canker disease severity by an average of 59% [213]. A&P Inphatec, a subsidiary of Ot-
suka Pharmaceutical, developed XylPhi-PD, a phage product that the EPA licensed in 2021
to guard grapevines in California against Pierce’s disease, which is brought on by Xylella
fastidiosa [194]. In a four-site field study conducted in California, the business revealed that
the constant injection of XylPhi-PD in the xylem reduced disease incidence by 57%. The
Hungarian government has given Enviroinvest permission to distribute the phage cocktail
Erwiphage Plus domestically as an anti-E. amylovora bactericide. This medicine is modified
every year to prevent the development of resistance, and it can only be used under very
specific circumstances during the flowering season. Each year, emergency authorization is
granted for as few as 120 days, spanning from mid-March to mid-July [214]. An engineered
cocktail of four lytic phages has been reported as an effective BCA of X. fastidiosa and its
associated infections including PD, olive-quick decline syndrome, and oleander, coffee, and
almond leaf scorch [215,216]. Ahmad et al. [217] showed that the filamentous phage XacF1
caused loss of virulence, reduced EPS production, restricted motility, and delayed growth
rate in Xanthomonas axonopodis pv. citri, the causative agent of citrus canker disease, which
further highlights its biocontrol potential against bacterial pathogens. On the other hand,
phages such as vRsoP-WF2, vRsoP-WR2, and vRsoP-WM2 were also identified as effective
BCAs against R. solanacearum [218]. Two particular phage species, E. amylovora Siphoviridae
phage (PhiEaH1) [214], and PhiEaH2 [219], were isolated locally and effectively decreased
the incidence of fire blight cases in field studies.
6.3. Cross-Protection
Cross-protection is a strategy that pre-immunizes plants against mild virus strains
in order to fight viral infections [195]. Plants are shielded against secondary infection by
other viral strains by cross protection. McKinney was the first to demonstrate the cross-
protection effect in tobacco plants chronically infected with a tobacco mosaic virus (TMV)
light green isolate, which decreased the onset of yellow symptoms caused by a TMV yellow
mosaic isolate [220]. There has been some success using it to manage viral illnesses such as
cross-protecting tomatoes against mild strains of the tobacco mosaic virus, papaya against
minor strains of the papaya ring spot virus, and citrus against moderate strains of the
citrus tristeza virus [221]. On the other hand, yellow symptoms were not suppressed by the
TMV moderate dark green isolate. Many viruses such as the potato virus X, potato leafroll
virus, and citrus tristeza virus (CTV) have been shown to exhibit cross protection [222].
“Plant vaccines” are another term for attenuated isolates. Sap from appropriate leaves can
be made to proliferate an attenuated strain in order to provide a real plant vaccination.
Next, approximately ten day old seedlings are mechanically given this sap along with
carborundum in their cotyledons [223]. An attenuated virus cannot infect another viral
species that is closely related to it once it has infected a whole plant. As a result, attenuated
viruses are crucial agents of biological control. Salaman [224] initially looked into the
cross-protective effects of plant viruses in 1933 and discovered that a moderate isolate of
potato virus X (PVX) may prevent a severe PVX isolate from becoming infected. Holmes
subsequently created a mild isolate of TMV by heating a virulent strain and observed that
in plants infected with the mild isolate, the symptoms brought on by infection with the
virulent isolate were reduced [225]. For field studies involving over 2000 citrus rootstocks,
a mild isolate of CTV was created in 1951 [226]. The findings suggested that citrus trees
protected against more severe CTV isolates were infected with milder isolates. Posnette
and Todd [227] employed a similar study approach when they tested a mild isolate of
cacao virus 1A in an African area where swollen shoot disease was common. In that
study, only 35 out of 416 trees infected with the mild isolate exhibited severe symptoms
of infection compared to 273 out of 387 uninfected trees that displayed severe symptoms.
This outcome was interpreted as proof that inoculating cacao trees with a moderate viral
isolate may successfully protect them in the real world. As a result, attenuated viruses have
been the subject of both fundamental and applied research for many years. In addition,
cross-protection against the pepino mosaic virus has been recently created and widely
used in Europe [228]. These strains of the virus also received separate authorization from
the European Food Safety Agency in 2015. Since then, cross-protection has been used in
open-field settings to guard against significant viral diseases caused by the papaya ringspot
virus in papaya [229] and the citrus tristeza virus (CTV) in citrus species [229]. In South
Africa, Peru, and the U.S., cross protection has been proven to be an effective management
strategy for the citrus stem pitting disease caused by CTV [230].
7. Biocontrol Agents’ Production and Formulations

7.1. Production of BCAs
A critical factor that must be considered when selecting a BCA for commercial devel-
opment is the availability of a cost-effective manufacturing and stabilization technology
that yields an optimally effective form of the antagonist [23]. More studies on the practical
aspects of mass production and formulation need to be undertaken to make new biocontrol
products stable, effective, safer, and more cost effective [231]. One essential stage in creating
effective BCAs for market use is the production of BCAs. The basic objective of industrial
scale-up processes, which involve either liquid or solid phase fermentation, is to produce a
high amount of biomass at the lowest feasible cost [231]. This is typical procedure to create
bacteria and yeasts by liquid fermentation in a continuously stirred tank in order to attain
a high aeration. However, a lot of molds are mostly made in a solid state. Regardless of
the mass production method, the main objective is to have the highest yield at the lowest
cost [232]. The practical efficacy of a BCA greatly depends on the quality of the inoculant,
itself a function of the production and formulation processes [233]. The two main processes
in production optimization are determining the composition of the medium and enhancing
the growing conditions. To manage the BCA production operation and attain an excessive
number of cells, create secondary metabolites, or both, it is vital to comprehend their mode
of action. Erlenmeyer flasks are typically used in a low-volume laboratory setting for the
first phase, which involves evaluating a huge number of substances and concentrations.
After that, 2 to 5 L laboratory bioreactors are used to properly calibrate the growth settings.
Ultimately, 100–300 L pilot plant bioreactors will be used for the initial large-scale man-
ufacturing, and it will not be hard to scale up the operation to commercial requirements
if the pilot plant results are satisfactory. In specifically made plastic bags (VALMIC® )
encompassing turba:vermiculite (1:1 w/w), the generation of Penicillium frequentans strain
Pf909, a BCA in the management of brown rot in stone fruit, was developed [234]. By
applying potato extract, V-8 juice, molasses, and wheat fiber, the BCA Trichoderma spp. was
grown in both liquid form [235] and on solid media containing various cereals such as
sorghum and millet [236]. As an example, the formation of the conidia of the antagonist
Ampelomyces quisqualis has been shown in a variety of liquid media including a sugar-based
medium enhanced with shrimp shell powder [237] and potato dextrose broth adjusted
with 2.5% glycerol [238]. In order to guarantee a high, strong, and productive microbial
population, careful research is required to enhance the growth conditions (temperature,
pH, agitation, aeration, initial inoculum, and process duration) after the optimal growth
medium for each microorganism has been determined [239]. A valuable product can be
recovered after fermentation by focusing on acquiring cells (or spores), the supernatant
containing the released metabolites, or even both, depending on the BCA. One instance of
BCA that is recommended is the recovery and formulation of B. amyloliquefaciens [240], as
both forms are involved in its mode of action to manage diseases. This includes both the
vegetative and endospore cells as well as the produced metabolites. Microorganisms are
typically present in low concentrations and are combined with other molecules from which
they must be removed through a number of steps. These processes include centrifugation,
flocculation, and filtering (pressure, rotary vacuum drum, and flocculation) [241].
7.2. BCA Formulation

When developing a formulation, a number of factors need to be considered including
the cost of production and acquisition and compatibility with agricultural machinery [242].
As the link between fermentation and field application, the formulation is sometimes seen
as the main obstacle to the commercialization of BCAs [243,244]. Both liquid and solid BCA
formulations come in a wide range of varieties. Solid formulations can increase the BCA
stability, although liquid formulations are typically simple and less expensive to produce.
A formulated product is primarily made up of, or may contain: (1) the active ingredient,
which is synthesized microorganism cells and secondary metabolites, (2) carriers, which
are often inert materials that carry and deliver the active ingredient in the target site, and
(3) adjuvants, which are substances that protect the active ingredient from high tempera-
tures, desiccation, ultraviolet radiation, and other environmental stresses while also facili-
tating the product’s spread and dispersal in the intended environment. The microbe must
be compatible with all adjuvants employed in the formulation process and not negatively
affected by any of them [245].
7.2.1. Liquid Formulation

In order to maximize the product’s viability and improve the BCA’s adhesion, sur-
factant, and dispersion capabilities, liquid formulations combine whole cultures or cell
suspensions with additives like stabilizers, colorants, and extra nutrients [246]. Compared
to formulations based on solids, these formulations are less expensive and simpler to
process. Particles can be suspended in a variety of liquids such as oil or water, dispersants,
surfactants, suspender components, or a carrier liquid in some liquid compositions. When
the processed culture is combined with emulsifiers, surfactants, and/or mineral or veg-
etable oils that facilitate their subsequent dispersion in water, the mixture is referred to
as oil-based. The oils that are utilized must not be harmful to humans, plants, microor-
ganisms, and animals. Since oils provide a defensive effect that lengthens the shelf life
of microorganisms, it is generally thought that oil-based formulations are appropriate for
foliar applications in dry ambient circumstances [247]. The development of the filamentous
fungus T. harzianum, an efficient opponent to prevent Botrytis rot in apples, is an illustration
of this system and was conducted by emulsion [248]. The yeast Hanseniaspora guilliermondii
isolate YBB3 was developed as a liquid formulation based on glycerol to manage Aspergillus
rot in grapes. This formulation proved to be more effective than solid formulations that
mixed the biomass produced with talc/kaolin powder alone or modified with yeast extract,
sucrose, and sodium alginate [249].
7.2.2. Solid Formulation

Although dried products have short viability rates due to both the thermal and dehy-
dration stresses that occur during the drying operation, they are nevertheless a practical
solution because of their low production costs and ease of storage and transportation [250].
As a result, every isolate requires a technique that is experimentally tailored to it. The
following methods of dehydration are efficient: fluidized bed drying, spray drying, and
freeze-drying. Vacuum desiccation, often known as freeze-drying or lyophilization, is a
technique for dehydrating labile goods. In short, the sampled liquid needs to be chilled
until the solutes crystallize, then the non-crystallizing solutes create an amorphous matrix,
and the freezable solution water finally turns into ice. Next, the water is removed from
the amorphous matrix by sublimating the ice in vacuum-controlled conditions. Ultimately,
the product’s moisture content is disported [251]. This method is frequently employed for
the conservation of microorganisms, particularly those kept in microbiology collections.
According to Prakash et al. [252], it keeps microorganisms viable for over twenty years and
does not require specific low-temperature settings for continued management. By eliminat-
ing the moisture from the liquid droplets, a product can be transformed from a liquid to a
solid state in the form of powder through spray-drying, also known as atomization. The
scattered droplets (10–200 m), which are mostly created by pressure nozzles, pneumatic
type atomizers, or rotary wheel/disc atomizers, are combined with low-humidity hot
air (150–170 ◦ C) inside the chamber. Subsequently, rapid mass transfer and related heat
reactions cause the moisture to swiftly escape as vapor from the suspended droplets. Until
the required particle properties are attained, the droplets continue to dry inside the drying
chamber. Ultimately, external apparatus like cyclones and/or bag-filter houses are used to
separate the dried particles from the drying air and collect them afterward [253]. The only
BCAs that can be created with this method are those that can withstand high temperatures
such as those that can produce thermoresistant endospores. B. amyloliquefaciens strain
CPA-8, created to suppress Monilinia spp. in nectarines and peaches, has shown some
promising effects [253]. Powders, granules, and spheres are dried, granulated, and coated
using fluidized bed technology [254]. Its foundation is the strong interactions that occur
between particles. Via a perforated plate, heated air that has been filtered and perhaps de-
humidified (between 35 and 45 ◦ C) enters the product, counteracting gravity and fluidizing
the material that has been previously sucked or placed into the granulator. However, in
order to use this product, it must first be extruded and then chopped into short rods or
“pellets”. Filters retain small particles that are transported by the air flow to the cylindrical
expansion chamber, allowing them to return to the conical product chamber. As the process
proceeds, this cycle is mostly controlled by changes in the product’s temperature and the
fluidization air flow [254]. Because this technology uses relatively mild temperatures, takes
little time, and is inexpensive, it is especially well-suited for BCAs that are sensitive to
heat. Numerous BCAs including Epicoccum nigrum and Penicillium oxalicum have been
successfully conserved using this technique [255].
The process of encapsulation involves the microorganism being placed inside a specific
matrix or capsule that may be biodegradable if it is made of polyacrylamides, guar gum,
gum arabic, sodium alginate, chitosan, or other biocomposites [256]. Vemmer et al. [227,257]
demonstrated that these matrices are effective carriers because they enclose living cells
and shield them from harsh environmental elements like UV rays and rain. Additionally,
González et al. [258] reported that these microcapsules are easily manipulable, can be kept
long-term in room temperature storage, and are excellent for BCAs that require a delayed
release such those placed in soil. The alginate encapsulation of Gliocladium virens (Soil
Gard) [259] and the encapsulations of B. thuringiensis in shell-core hydrocapsules, which
have a liquid center surrounded by a polymer membrane [260], are two examples of this
type of formulation.
8. Role of Genetic Engineering for Improving BCAs Traits

The use of BCAs for disease and pest management has significantly increased in
recent times because of the negative impact of chemical pesticides on soil fertility, the
environment, and human health. However, there are many challenges while using BCAs
such as their environmental stability, efficacy, pest or disease suppression ability, host
range, target selection, reducing the ecological risk, and improving the mass production or
storage [261,262]. Recent developments in genomics and biotechnology have facilitated
improvements in the above traits in BCAs and also aids in the identification of novel
BCAs, their characterization, and the genetic by-products [261–263]. The availability of
microbial genome data has opened new dimensions for the discovery of a large array
of BCAs [263]. The integrated approaches of omics technologies will provide a strong
foundation for in-depth insights regarding the interactions between BCAs and their hosts.
Through the application of omics approaches such as the genomics, transcriptomics, and
proteomics of BCAs, pathogens, and crop plants, a specific set of interactome could reveal
the key mechanisms and factors influencing the efficacy and applicability of an organism
as a BCA [264,265]. The efficacy of BCAs can be increased using genetic engineering. For
instance, in Pseudomonas fluorescens F113, the loss of the sadB, wspR, and kinB genes results
in increased motility and improved root colonization, demonstrating good biocontrol ac-
tivity against Phytophthora cactorum in strawberry plants and Fusarium oxysporum f. sp.
Radicis-lycopersici in tomato plants [266]. Conversely, significant mycosubtilin production
resulting from the overexpression of the mycosubtilin gene utilizing the Staphylococcus
aureus constitutive promoter in Bacillus subtilis strain ATCC 6633 enhances Pythium aphani-
derm suppression [267]. Introduction of the chitinase gene in Bacillus thuringiensis 3023 from
Serratia marcescens showed broad biocontrol activity against various pests [268]. Geneti-
cally modified Pseudomonas fluorescens strain BL915 with high antipathogenic substances
(APS) like pyrrolnitrin showed strong biocontrol activity against soilborne and seedling
pathogens [269]. Similarly, the overexpression of Cry genes in B. thuringiensis showed
strong insecticide activity against susceptible and Bt-resistant insects in tobacco plants [270].
Genetically-modified Pseudomonas spp., through the introduction a translational enhancer
from Bacillus confers strong biocontrol activity. Genome shuffling has been used in Bacillus
subtilis, Streptomyces melanosporofaciens, and Streptomyces bikiniensis for the improvement in
biocontrol traits against Fusarium oxysporum f. sp. melonis, Phytophthora infestans, Fusarium
oxysporum f. sp. Cucumerinum [271], Streptomyces melanosporofaciens [272], and Streptomyces
bikiniensis [273]. Recently, the Sarocladium oryzae mutant strain was found to efficiently
control Rhizoctonia solani and Sclerotinia sclerotiorum pathogens [274]. Transformation of the
Serratia marcescens chitinase gene into Pseudomonas fluorescens improved the biocontrol effi-
ciency against R. solani on bean seedlings [275]. Similarly, the introduction of pyrrolnitrin
coding genes from Pseudomonas protegens Pf-5 to P. synxantha 2–79 showed strong biocontrol
activity against wheat pathogens. It has been demonstrated that T. virens cotton strains
containing two copies of the ech42 gene exhibited increased antagonistic activity against R.
solani [276]. Sun et al. [277] genetically modified the Clonostachys rosea strain, which showed
strong biocontrol activity against Sclerotinia rot of soybean. To increase its effectiveness
against soilborne infections, a genetically engineered strain of Pseudomonas putida was
developed with phz or phl biosynthesis gene loci, which produced constitutively phenazine-
1-carboxylate (PCA) or 2,4-diacetylphloroglucinol (DAPG) [278]. Another study found that
once the trichodermin gene (tri5-trichodiene synthase) was cloned and overexpressed in T.
brevicompactum, it produced higher trichodermin, which exhibited strong biocontrol activity
against Aspergillus fumigatus and Fusarium spp. Ma et al., 2003. A previous study showed
that T. virens ∆tvk1 mutants showed improved biocontrol efficacy against R. solani along
with the overproduction of lytic enzymes and the increased expression of genes linked to
mycoparasitism [279]. These studies further support the notion that genetic engineering
is the key to developing long-term effective BCAs. Although genetic engineering has
been a major driver for BCA development, it has some possible health risks and lateral
gene flow to non-target animals, which might pose threats to the environment. In this
regard, genome editing is an alternative for improving the traits of BCAs more precisely
and effectively without endangering the environment. The use of CRISPR and Cas genes
has expedited the creation of versatile and economical genomic engineering toolkits that
rely on the programmable targeting of CRISPR–Cas technologies [280]. Genome editing
using CRISPR/Cas has been used for improving diverse biocontrol traits in Trichoderma
species [281]. In future, CRISPR/Cas based gene editing for the improvement of biocon-
trol traits is the most promising tool for developing future climate resilient, efficient, and
ecofriendly BCAs in sustainable agriculture.
An understanding of the biosynthetic pathway of antimicrobial metabolites in BCAs is
important for improving their biocontrol efficiency. For example, the antibiosis action of BCAs
is often achieved by the synthesis of low-molecular-weight chemicals that either directly or
indirectly impede the development of pathogens. These encompass a wide range of chemical
groups including polyketides [282], terpenes [283], and peptides [284,285]. Thus, metabolic
engineering is needed to increase the secondary metabolite production of BCAs, which may
potentially result in the synthesis of new antimicrobial compounds. Metabolic engineering
holds great promise for the development of future BCAs, offering innovative strategies to
enhance their efficacy, specificity, and environmental sustainability. Modern techniques
Horticulturae 2024, 10, x FOR PEER REVIEW 21 such
of 32
as plasmid engineering, RNAi interference, and CRISPR-based genome editing utilized to
modify the T. hazrium strain for strain enhancement seems promising (Figure 4).
Figure 4.4. Schematic

Figure Schematic diagram
diagram highlighting
highlighting the
the role
role of
of different
different molecular
molecular methods
methods for
forimproving
improving
the biocontrol traits in BCAs for disease and pest management. (a) gene editing, (b) CRISPR/Cas9,
the biocontrol traits in BCAs for disease and pest management. (a) gene editing, (b) CRISPR/Cas9,
(c) RNAI, (d) DNA methylation and (e) Metabolic engineering.
(c) RNAI, (d) DNA methylation and (e) Metabolic engineering.
9. Conclusions
In sustainable agriculture, BCAs are currently being examined as alternatives to
synthetic pesticides to manage plant pathogens and pests, mainly due to their safety and
ecofriendly nature. These include naturally occurring microorganisms such as fungus,
bacteria, and viruses, which are commercially produced as biopesticides and have a va‐
9. Conclusions
In sustainable agriculture, BCAs are currently being examined as alternatives to
synthetic pesticides to manage plant pathogens and pests, mainly due to their safety and
ecofriendly nature. These include naturally occurring microorganisms such as fungus,
bacteria, and viruses, which are commercially produced as biopesticides and have a variety
of agricultural applications in biological and integrated pest management programs. BCAs
use diverse mechanisms such as parasitism, antibiosis, or competition and induce the plant
defense system to manage diseases or pests. They also play a vital role in improving plant
growth and soil fertility. However, the identification and development of a novel class of
biocontrol agents possessing increased potency, high productivity in fermenters, extended
shelf life, room temperature storage capacity, and excellent compatibility with other control
strategies are required for the sustainable management of plant diseases. On the other
hand, BCAs must be evaluated for any negative impacts on crops, consumers, and the
environment before they can be approved and commercially produced. To further clear
the regulatory road and guarantee biosafety, it is also necessary to comprehend how BCAs
affect the natural microbiome, which includes the soil and plant microbiomes. Before they
may be effectively used in the field, a number of modifications must be made including
improving the formulation and delivery strategies, scaling up manufacturing, adhering to
regulatory standards, and enhancing cost-effectiveness. Further investigations on certain
underdeveloped areas such as the creation of next generation BCAs and the application of
biotechnology in conjunction with “omics” approaches to enhance biocontrol efficacy are
also necessary for the future development of successful biological control strategies. In this
regard, genetic engineering using CRISPR genome editing have the potential to decrease
undesirable traits in BCAs and introduce new, desirable traits, which can act against
a broad spectrum of pathogens and pests in different crop systems. In climate change
scenarios, biological control agents may lose their efficacy if there is a higher fluctuation in
temperature, humidity, frequency of rain, and other weather conditions. Therefore, it is
important to develop climate resilient biocontrol agents in sustainable agriculture.
Author Contributions: Conceptualization, M.A.A. and S.A.; Methodology, A.T., T.L.T., H.K., R.A.M.,
Z.A.M., S.M., N.M., G.G., S.K.V., M.A.A. and S.A.; Software, A.T., T.L.T., H.K., R.A.M., Z.A.M., S.M.,
N.M., G.G., S.K.V., M.A.A. and S.A.; Validation, A.T., T.L.T., H.K., R.A.M., Z.A.M., S.M., N.M., G.G.,
S.K.V., M.A.A. and S.A.; Formal analysis, A.T., T.L.T., H.K., R.A.M., Z.A.M., S.M., N.M., G.G., S.K.V.,
M.A.A. and S.A.; Investigation, M.A.A. and S.A.; Resources, A.T., T.L.T., H.K., R.A.M., Z.A.M., S.M.,
N.M., G.G., S.K.V., M.A.A. and S.A.; Data curation, A.T., T.L.T., H.K., R.A.M., Z.A.M., S.M., N.M.,
G.G., S.K.V., M.A.A. and S.A.; Writing—original draft preparation, A.T., T.L.T., H.K., R.A.M., Z.A.M.,
S.M., N.M., G.G., S.K.V., M.A.A. and S.A.; Writing—review and editing, A.T., T.L.T., H.K.; R.A.M.,
Z.A.M., S.M., N.M.; G.G., S.K.V., M.A.A. and S.A.; Visualization, A.T., T.L.T., H.K., R.A.M., Z.A.M.,
S.M., N.M., G.G., S.K.V., M.A.A. and S.A.; Supervision, M.A.A. and S.A.; Project administration,
M.A.A. and S.A.; Funding acquisition, M.A.A. and S.A. All authors have read and agreed to the
published version of the manuscript.
Funding: This research received no external funding.
Data Availability Statement: Not applicable.
Conflicts of Interest: The authors declare no conflicts of interest.
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1 Department of Biotechnology, Yeungnam University, Gyeongsan 38541, Republic of Korea;

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Horticulturae 2024, 10, 805. https://doi.org/10.3390/horticulturae10080805 https://www.mdpi.com/journal/horticulturae

2. Biocontrol as a Viable Approach for Controlling Disease in Horticulture Crops

Figure 1.1. Schematic

3. Factors Affecting the BCAs Activities and Strategies to Overcome Limitations of

4. Fungal Species as BCAs: Mechanisms and Applications

Pythium ultimum (Oomycota, Chromista) [94]. In a field study, commercial formulations

Table 1. List of fungal BCAs for disease management in plants.

Fungal Species Mode of Action Disease/Host Reference

Fungal Species Mode of Action Disease/Host Reference

5. Bacteria as BCAs for Plant Disease Management

Table 2. Role of bacterial BCAs to control pathogens in different crop systems.

Bacterial Pathogen Dose/CFU

used protoplast fusion to transmit mycovirus FgV1-DK21 to F. graminearum, F. asiaticum,

6.2. Phage-Based Biobactericides

7. Biocontrol Agents’ Production and Formulations

7.2. BCA Formulation

7.2.1. Liquid Formulation

7.2.2. Solid Formulation

8. Role of Genetic Engineering for Improving BCAs Traits

Figure 4.4. Schematic

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