Review

microorganisms
Review
Biological Control of Plant Pathogens: A Global Perspective
Rachid Lahlali 1, * , Said Ezrari 1,2 , Nabil Radouane 1,2 , Jihane Kenfaoui 1,2 , Qassim Esmaeel 3 ,
Hajar El Hamss 1 , Zineb Belabess 4 and Essaid Ait Barka 3, *
1 Phytopathology Unit, Department of Plant Protection, Ecole Nationale d’Agriculture de Meknès, Km10,
Rte Haj Kaddour, BP S/40, Menkes 50001, Morocco; said.ezrari@usmba.ac.ma (S.E.);
nabil.radouane@usmba.ac.ma (N.R.); jihane.kenfaoui@usmba.ac.ma (J.K.); hajar.elhamss@gmail.com (H.E.H.)
2 Laboratory of Functional Ecology and Environmental Engineering, Sidi Mohamed Ben Abdellah University,
P.O. Box 2202, Route d’Imouzzer, Fez 30000, Morocco
3 Unité de Recherche Résistance Induite et Bio-Protection des Plantes-EA 4707-USC INRAE1488,
Université de Reims Champagne-Ardenne, 51100 Reims, France; qassim.esmaeel@univ-reims.fr
4 Plant Protection Laboratory, Regional Center of Agricultural Research of Oujda, National Institute of
Agricultural Research, Avenue Mohamed VI, BP428 Oujda, Oujda 60000, Morocco; zineb.belabess@inra.ma
* Correspondence: rlahlali@enameknes.ac.ma (R.L.); ea.barka@univ-reims.fr (E.A.B.);
Tel.: +212-55-30-02-39 (R.L.); +33-3-2691-3441 (E.A.B.)
Abstract: The increase in the world population has generated an important need for both quality and
quantity agricultural products, which has led to a significant surge in the use of chemical pesticides
to fight crop diseases. Consumers, however, have become very concerned in recent years over the
side effects of chemical fungicides on human health and the environment. As a result, research into
alternative solutions to protect crops has been imposed and attracted wide attention from researchers
worldwide. Among these alternatives, biological controls through beneficial microorganisms have
gained considerable importance, whilst several biological control agents (BCAs) have been screened,

among them Bacillus, Pantoea, Streptomyces, Trichoderma, Clonostachys, Pseudomonas, Burkholderia, and
Citation: Lahlali, R.; Ezrari, S.; certain yeasts. At present, biopesticide products have been developed and marketed either to fight
Radouane, N.; Kenfaoui, J.; Esmaeel, leaf diseases, root diseases, or fruit storage diseases. However, no positive correlation has been
Q.; El Hamss, H.; Belabess, Z.; Barka, observed between the number of screened BCAs and available marketed products. Therefore, this
E.A. Biological Control of Plant review emphasizes the development of biofungicides products from screening to marketing and
Pathogens: A Global Perspective. the problems that hinder their development. Finally, particular attention was given to the gaps
Microorganisms 2022, 10, 596. observed in this sector and factors that hamper its development, particularly in terms of efficacy and
https://doi.org/10.3390/ legislation procedures.
microorganisms10030596
Academic Editor: James F. White Keywords: biological control; biological control agents; crop diseases; antagonist
Received: 21 February 2022

Accepted: 7 March 2022
Published: 9 March 2022
1. Introduction
Publisher’s Note: MDPI stays neutral Modern agriculture is continuously developing and evolving. After the generalization
with regard to jurisdictional claims in
of the use of chemical fertilizers and pesticide products, which allowed a considerable
published maps and institutional affil-
increase in yield in the twentieth century, the rise of biotechnologies and new cultivation
iations.
techniques is underway [1]. The next challenge to be met is to feed around 9 billion people
by 2050 [2,3]. In this context, one of the major concerns is to expand food production
capacities, including those derived from plants, while preserving the environment [4].
Copyright: © 2022 by the authors.
Nowadays, countries are striving to expand their food production to meet their needs [5].
Licensee MDPI, Basel, Switzerland. The increase in the production of a given crop is often linked to the improvement of
This article is an open access article cultivation techniques, particularly the use of more productive cultivars with resistance to
distributed under the terms and main diseases [6].
conditions of the Creative Commons Crop protection, which is still largely achieved by applying chemical products, is also
Attribution (CC BY) license (https:// in a transitional phase [7]. Therefore, the gradual integration of new practices, taking into
creativecommons.org/licenses/by/ account the agricultural production system, requires not only the environmental dimension
4.0/). but also the socio-economic dimension [8]. Nevertheless, these cultures are often subjected
Microorganisms 2022, 10, 596. https://doi.org/10.3390/microorganisms10030596 https://www.mdpi.com/journal/microorganisms

Microorganisms 2022, 10, 596 2 of 33
to parasitic attacks that farmers are still forced to control below the threshold of harmfulness
to survive and be efficient [9]. In addition, ongoing growth in productivity and international
trade boosts the incidence of certain diseases, thus requiring the application of more
pesticides. Subsequently, these pesticides increase environmental pollution and build up
chemical residues in the treated ecosystem [10]. Other alternatives such as genetic pathways
offer interesting control methods from a practical point of view, but also strengthen the
risks of the emergence of resistant genes in the pathogen [11]. Other alternatives such as
biological controls using microorganisms are a possible way to minimize the pollution and
nuisances associated with the use of synthetic chemicals and greatly reduce their negative
impact on the environment [12,13].
The concept of biocontrol has caused an important technological, economic, and
political debate aiming to develop sustainable agriculture at a lower ecological cost [14,15].
Accordingly, different countries have implemented a protective plan that can reduce around
50% of used pesticides [16]. These measures unequivocally illustrate a major awareness of
the accumulation of toxic residues in the environment and the various links within the food
chain. They also indicate the lack of alternatives to reduce the reliance of the agricultural
sector on pesticides. In this context, it appears crucial to deepen our knowledge about
biocontrols to improve their use and efficiency [15]. For all of these reasons, research is
progressing well towards a perspective of biological control, based on the application of
microbial inoculum, that could be added to the other aforementioned ways to develop a
strong strategy to fight plant diseases.
Promising achievements in terms of biological control have emerged, especially after
the successful use of certain antagonistic biocontrol agents (BCAs), in particular Pseu-
domonas spp., Bacillus spp., Burkholderia spp., and Trichoderma sp. against pathogens causing
foliar and soilborne diseases like Agrobacterium radiobacter var radiobacter, Erwinia spp.,
Fusarium spp., Rhizoctonia solani, Phytophthora spp. and Pythium spp. diseases [13,17].
Additional BCAs have showed an antagonistic effect against a wide spectrum of diseases,
namely bacterial species such as Burkholderia spp., Paenibacillus spp., Pantoea spp., Serratia
spp., Streptomyces spp., and fungal species such as Aspergillus spp., Beauveria spp., Fusarium
spp., Penicillium spp., and Phoma spp. Furthermore, in addition to halting the pathogen
proliferation, many of these BCAs can also directly promote plant growth [9,12,13,17,18].
However, in most cases, the efficacy of BCAs has always been found to be lower than that
of synthetic fungicides. This might be due to the complexity of the rhizosphere and the
need to apply a high amount of BCAs to cover the entire rhizosphere [19]. This can only
happen if the BCA is applied continuously and consecutively. In addition, the way BCAs
are formulated and applied can also affect their efficacies [20].
Even though the research for new microorganisms as potential biopesticides has
increased, biopesticides have been produced in limited numbers. However, small and
medium companies are starting to adapt to this new emerging market. The North American
region has the largest amount of the biopesticide market, reaching USD 539 million in 2015
and predicted to attain USD 1.67 billion by 2022 [21]. Research on biological control using
microorganisms is experiencing and gaining remarkable momentum, although applications
in the field are still limited. The factors limiting the use of BCAs in the field include the
inconsistent efficiency in protecting plants under field conditions, decreased availability in
the market, and the wide unacceptance of BCAs by farmers.
In this paper, we discuss the progress made on the prospecting of biocontrol agents,
their development, and their mechanisms of action, with a special emphasis on legislative
procedures and factors affecting their application and marketing development.
2. Rhizosphere as a Potential Reservoir of Biopesticides

The rhizosphere is the soil area that surrounds living roots and is influenced by plant
root exudates. It is one of the most important microbial hotspots in the soil, with much
greater process rates and more intensive interactions [22,23]. The rhizosphere contains a
complex microbial community called the rhizosphere microbiome. This microbial consor-
tium lives around plant roots and is formed up of many microorganisms, some of which
are beneficial such as bacteria, algae, and fungi [24]. Because it is a source of utilizable
carbon, the rhizosphere provides a supportive environment for crucial and intensive inter-
actions between plants, soil, microorganisms, and soil microfauna [25]. Plants can recruit
a unique beneficial rhizosphere microorganisms, which can help them to attenuate the
disease activity and make them more resistant to environmental stressors [26].
Understanding the role of rhizosphere microorganisms in pest and disease control
seems to be a rising research field, as evidenced by the high increase in studies conducted
between 2000 and 2019. When the term “rhizosphere” was added to a Google Scholar
search with the keywords “microorganisms”, “control”, “pest”, and “diseases”, the num-
ber of records retrieved increased from roughly 5000 (2000–2005) to 8500 and >20,000
(2006–2010 and 2011–2019, respectively). In the absence of this term, records dropped from
roughly 17,000 to 15,000 in the most recent period [27]. In the present review, we have ex-
tracted bibliometric data to perform a deep analysis and establish a network of worldwide
distribution-related articles on biocontrol agents. The bibliometric data were extracted
from the SCOPUS database (https://www.scopus.com/, accessed on 22 December 2021)
using the specific keywords ‘Rhizobacteria’ OR ‘Endophytes’ OR ‘Biocontrol agents’, from
which 1150 documents were obtained. The bibliometric analysis was performed using
different bibliometric indices, including the most popular used keywords, countries, and
the top journals, and was constructed using the VOSviewer processing software (v1.6.9.,
Leiden University, Leiden, The Netherlands) (Figure 1A). The network analysis showed
the worldwide distribution of related articles to biocontrol agents, which revealed the
relationship between keywords found and allows the obtainment of a comprehensive
perspective of the current research of this area (Figure 1B).
Figure 1. Bibliometric analysis of 1150 articles published on biological control according to the Scopus
database using specific keywords such as “Rhizobacteria” OR “Endophytes” OR ”Biocontrol” (A)
and the network analysis of their worldwide distribution (B); the larger the circle, the more intense
the scientific activity.
Since the use of pesticides to control pests and diseases has been linked to environmen-
tal, ecological, and human health risks, it has become necessary to seek out eco-friendly
biological agents known as biopesticides [28,29] to ensure biological control. The latter,
often known as “biocontrol”, is a method of reducing or eliminating the impact or damage
produced by a specific pest or weed by releasing a biocontrol agent, such as a predator,
herbivore, or pathogen [30]. To handle plant diseases, biological control is considered a
promising and reliable alternative to the use of synthetic fungicides [27,31]. Several billions
of dollars are being spent on biocontrol research, and the number of biocontrol drugs that
are available for various plant crops is rapidly expanding [32]. Biopesticides are microbial
or products generated from microbes, plants, and other biological organisms aiming to
control plant pests [29,33].
Biopesticides have long-term potential in improving sustainable agriculture [29]. There
are currently many (more than 440 species) control agents available for various pests [34].
BCAs are used to control plant diseases in crops, and they involve a variety of mecha-
nisms [35]. Van Lenteren et al. [34] presented the first list of BCAs registered worldwide,
which includes bacteria, fungi, mycoviruses, and bacteriophages [34]. Certain antagonistic
microorganisms have been identified from the rhizosphere of numerous agricultural plants
to inhibit some plant diseases and hence reduce the need for agrochemical pesticides.
In the last two decades, researchers have been studying the use of these specialized
antagonistic microorganisms in the biological control of soil-borne diseases. Biopesticides
are available inside the rhizosphere to combat pests and microbial diseases due to the close
connection of root-colonizing probiotic microorganisms with plant host cells [36]. Several
bacteria, fungi, protozoa, and nematodes have recently demonstrated antagonistic activity
that could be employed in the biocontrol of root and foliar diseases in a variety of crops,
and also insect pests [37,38]. Antibiotics, bacteriocins, siderophores, hydrolytic enzymes,
and other secondary metabolites produced by these beneficial rhizosphere microorganisms
inhibit pathogenic bacteria and fungi [28,32].
Direct antagonism of potential pests through the production of biopesticides is one of
the features of plant-probiotic microorganisms that contribute to plant health. Antagonistic
fungi such as Trichoderma spp., as well as bacteria from the genera Pseudomonas, Bacillus,
and Streptomyces, account for the majority of rhizosphere microorganisms commonly used
in biocontrols [27].
2.1. Beneficial Bacteria

Most plants grown in the field are colonized by various rhizosphere bacteria [39].
Some plant-associated bacteria are classified as beneficial microorganisms, based on their
effects on plant performance. Among these free-living bacteria, plant-growth-promoting
rhizobacteria (PGPR) thrive freely in the rhizosphere soil [25,33] and produce a variety of
antifungal metabolites and plant growth-promoting features [25]. Based on the ability or
behavior of the crops and biocontrol agents, PGPRs also act as biopesticides [33]. PGPRs
are bacterial species including Alcaligenes, Azospirillum, Arthrobacter, Acinetobacter, Bradyrhi-
zobium, Bacillus, Burkholderia, Enterobacter, Erwinia, Flavobacterium, Pseudomonas, Rhizobium,
Azorhizobium, Bradyrhizobium, Allorhizobium, Sinorhizobium, Frankia, Mesorhizob, Azoarcus, Ex-
iguobacterium, Methylobacterium, Paenibacillus, and Pantoea [29,40–48]. In reaction to diverse
chemicals found in root exudates, beneficial bacterial communities in the soil are chemoat-
tracted towards plant roots. These beneficial associations boost plant health and crop
productivity by increasing nutrient availability, releasing plant growth-stimulating hor-
mones, decreasing pathogen/pest-caused diseases, or improving the environmental stress
resistance [39]. Antibiotics, endotoxins, bacteriocins, siderophores, hydrolytic enzymes,
hydrogen cyanide (HCN), phenazine-1-carboxylic acid (PCA), 2,4-diacetylphloroglucinol
(DAPG), and other secondary metabolites produced by certain rhizosphere bacteria kill
pathogens, thus preventing disease development [39,43,44,47,49–54]. Some PGPRs utilized
in the control of plant pests and diseases are discussed below.
Bacillus species have a long history in biocontrol and crop growth-promoting appli-
cations [55]. Bacillus thuringiensis (Bt) is the most commercially successful biopesticide on
the market [29,56]. Bt produces endotoxins [51,52], which are toxic and can be exploited as
biopesticides as well as a source of genes for the development of insect-resistant transgenic
plants [52]. During their stationary phase of growth, Bt strains produce crystalline proteins
(δ-endotoxins) that are toxic to lepidopterous, coleopterous, and dipterous insects as well
as mites, nematodes, protozoa, and flukes [51]. Insect bioassays were performed on the
spore crystal mixtures of 12 Bt isolates, and it was found that the isolate F8.IIPR has the
maximum toxicity against Spilosoma obliqua Walker (100%), Olepa ricini Fabricius (92%),
and Helicoverpa armigera Hubner (100%) larvae [57]. Bacillus subtilis (BCB-19) and Bacillus
megaterium (SB-9) induced considerable larval death and growth inhibition in both H.
armigera and Spodoptera litura [58]. Bacillus amyloliquefaciens LMR2, Bacillus halotolerans (SF3
and SF4), and Bacillus mojarvensis SF16 were recently isolated from soil of fire blight host
plants in different Moroccan regions for their higher efficacy in reducing apple fire blight
disease [48]. Monilinia fructigena and Monilinia laxa, which cause brown rot disease of fruits,
are controlled by B. amyloliquefaciens SF14 and B. amyloliquefaciens SP10, respectively. In a
semi-commercial large-scale study, the efficacy of these strains was found to be comparable
to that of two commercial BCAs, but slightly lower than that of a commercial synthetic
fungicides [47,59]. The considerable biological control activity of a Bacillus velezensis strain
named ZW10, as well as its ability to boost host defenses, make it a potential biopesticide
for rice blast biocontrol caused by the fungus Magnaporthe oryzae [60]. The commercially
available B. amyloliquefaciens FZB42 product has considerably decreased lettuce bottom
rot caused by R. solani. This reduction was due to the secretion of surfactin and other
FZB42-non-ribosomally synthesized secondary metabolites in the lettuce rhizosphere. Sub-
sequently, plant regulation genes are triggered and expressed to protect plants against the
pathogen R. solani [53].
In a dual culture bioassay, endophytic Bacillus strains obtained from cotton roots have
demonstrated an inhibitory efficacy against Verticillium dahliae strain VD-080. Scanning
electron microscopy examination underlined mycelial disintegration, curling, and shrink-
age of V. dahliae hyphae after treatment with methanolic extracts of isolated endophytes.
Furthermore, when compared to control treatments, cotton plants treated with two Bacillus
strains (HNH7 and HNH9) showed a considerable reduction in verticillium wilt severity.
Moreover, the expression of some defense-related genes was significantly higher in plants
treated with Bacillus strains and inoculated with VD-080 [54].
Pseudomonas chlororaphis isolates are used as biopesticides in agriculture as they protect
plants from various microbial diseases, insects, and nematodes. These isolates directly
suppress microbial pathogens, insects, and nematodes by producing a variety of metabo-
lites [36]. P. chlororaphis PcO6, isolated from the roots of dryland, enhanced plant health and
was used as a biofertilizer and BCA in agriculture. Plant growth is stimulated by an array of
metabolites generated by this bacterium through direct pathogen antagonism and induction
of systemic resistance in the plant. The mechanisms by which specific bacterial metabolites
create protection against pathogenic microorganisms, insects, and nematodes have been
identified in studies on PcO6. The role of a global regulatory system, the Gac/Rsm regulon,
in conferring protection against plant pathogens has been highlighted [61]. The Gac/Rsm
system network in PcO6 communicates the capacities required to adapt to various stressors
and enhance survival while also maintaining plant health by priming stress responses.
When the bacterium reaches a critical cell density, the Gac/Rsm network is activated,
causing the microbe to switch its nutrition consumption and create protectants instead of
adding cell mass. Other systems influencing gene expression are linked to the Gac/Rsm
system. Antimicrobials are produced when the Gac/Rsm network in PcO6 is activated,
which may help in sustaining plant root colonization and protecting the plant against
microbial diseases. PcO6 impacts microbiomes and plant health in the rhizosphere by
secreting antimicrobials and volatiles. The bacterium forms a protective biofilm on the
root surface, which acts as a physical shield and a water-holding gel [61]. Antifungal
compounds such as bacteriocin, HCN, and siderophore were produced by Pseudomonas
aeruginosa isolated from the banana field rhizosphere. Phytopathogens such as Aspergillus
niger, Aspergillus flavus, Fusarium oxysporum, and Alternaria alternata were suppressed by
the isolate’s bacteriocinogenic, siderophoregenic, and HCN rich broth. Bacteriocin has a
toxic effect on bacteria, whereas siderophore and HCN inhibit fungal phytopathogens. It is
worth mentioning that none of the helpful rhizobia were inhibited by these compounds. In
comparison to copper-based systemic chemical fungicide, the P. aeruginosa isolate showed
higher antifungal activity and a lower minimum inhibitory concentration [43]. The ability
of Pseudomonas sp. LBUM 223 to control common scab potato caused by Streptomyces scabies
was proven via PCA synthesis. This PCA synthesis is critical for LBUM 223’s capacity
to control common scabs of potato, limit pathogen growth, and inhibit the expression of
important pathogenicity genes [50]. Similarly, Lanteigne et al. [49] have identified DAPG
and HCN as the chemicals responsible for Pseudomonas sp. LBUM300’s capacity to inhibit
the growth of Clavibacter michiganensis subsp. michiganensis in vitro and regulate the devel-
opment of bacterial canker in tomato under soil conditions. In both H. armigera and S. litura,
Pseudomonas spp. (SB-21) was reported to induce considerable larval death and growth
inhibition [58].
2.2. Fungi and Yeasts

Several fungal species have been identified as insect pest entomopathogens, as well
as plant endophytic fungi [62]. Trichoderma is a ubiquitous fungus genus found as soil
inhabitants, plant symbionts, saprotrophs, and mycoparasites [63]. This genus contains
filamentous fungi that have been extensively investigated and utilized in agriculture as
a biocontrol agent against phytopathogens due to its capacity to compete and parasitize
them (mycoparasitism), among other mechanisms of action [62–64], as well as mitigating
unfavorable growth conditions [63]. Trichoderma is by far the most studied fungal biocontrol
agent, with some species having previously been commercialized as biopesticides or biofer-
tilizers [63,64]. The use of Trichoderma as a BCA for insect pests has been discussed in recent
years, both directly and indirectly [62]. Trichoderma has been shown to directly suppress
insect pests by parasitism and the generation of secondary insecticidal metabolites [62,65],
antifeedant chemicals, and repelling metabolites. Indirectly, by activating systemic plant
defense systems, attracting natural enemies, and parasitizing insect-symbiotic microorgan-
isms. As a result, Trichoderma use in agriculture is effective not only against plant pathogens
but also against insect pests, making it a promising future alternative for the development
of sustainable agriculture [62]. Trichoderma aggressivum isolates were collected from nu-
merous Agaricus bisporus growing substrates from farms in Castilla-La Mancha (Spain).
This fungus had high antagonistic activity against a variety of phytopathogens, greater
than 80%. In a detached leaves assay, the most effective isolate, T. aggressivum f. europaeum
TAET1, has completely suppressed the mycelial growth of fungal pathogens Botrytis cinerea,
Sclerotinia sclerotiorum, and Mycosphaerella melonis and inhibited S. sclerotiorum sclerotia
germination. This excellent compatibility with chemical fungicides may allow this isolate
to be used in combination with various pest management strategies [66]. Trichoderma as-
perellum TaspHu1, an isolate obtained from Juglans mandshurica Maxim. rhizosphere soils in
China improved tomato seedling resistance to A. alternata leaf spot disease [67]. Trichoderma
spp. from olive rhizosphere soils in northern Algeria had great biocontrol potential against
V. dahliae, the causal agent of wilting on olive trees. These isolates showed an effective
potential in reducing the in vitro mycelial growth of this pathogenic fungus. By using the
confrontation assay method, Trichoderma isolate T12 inhibited V. dahliae isolates at a rate
of 69% [68]. Trichoderma spp. has been proven to be effective in suppressing Sclerospora
graminicola, the causal agent of pearl millet downy mildew disease. Their prominent role
indirectly suppressing the pathogen in the rhizosphere and establishing systemic resistance
has been well demonstrated [69].
Furthermore, yeasts are often used in commercial formulations because of their biocon-
trol capacity. They are already available for postharvest use on a variety of crops, including
pome and citrus fruits. Because of their great ability for quick colonization and inhibition
of pathogens, as well as their potential for competition for nutrients, physical interaction,
and the generation of lytic enzymes, these microorganisms have been shown to have a
significant BCA activity [70].
Millan et al. [71] investigated the in vivo and in vitro effects of 69 yeast strains isolated
from Spanish vineyards against Alternaria alternata, Penicillium expansum, and B. cinerea,
as well as soil-borne diseases V. dahliae and Fusarium oxysporum, to find a potential yeast
with high biocontrol capabilities against postharvest diseases and wilt diseases. As a result,
Wickerhamomyces anomalus Wa-32 reduced the severity of disease caused by V. dahliae up
to 40%, and that due to F. oxysporum up to 50%. Furthermore, the postharvest assays
revealed a high biocontrol performance against P. expansum and B. cinerea (up to 86 and
97% reduction in disease severity, respectively). Furthermore, W. anomalus Wa-32 and two
Metschnikowia pulcherrima strains showed significant action against B. cinerea (Mp-22 and
Mp-30). However, according to the same study, Candida lusitaniae Cl-28, Candida oleophila
Co-13, Debaryomyces hansenii Dh-67, and Hypopichia pseudoburtonii Hp-54 were the most
efficient against P. expansum, according to the study. This was also observed with grapes
and apples [71].
3. BCAs Modes of Action

BCAs are applied in the disease management of plant pathogens, where they act
via a variety of different modes of action to control plant pathogens. Understanding the
mechanisms behind the protective effects of BCAs will facilitate the optimization of control
and will allow the use of more efficient strains in the correct environment [72,73]. These
mechanisms might be used alone or in combination by the BCA to control plant disease
directly and indirectly (Figure 2).
Figure 2. Possible modes of action of biological control agents.
3.1. Direct Mode of Action

In the direct way of disease control, BCAs act through a direct antagonistic effect on the
pathogen, encompassing (i) antibiosis, (ii) parasitism, and (iii) reducing pathogen virulence,
and (iv) infection pressure by competition [9,74]. BCAs act through antibiosis that relies
on BCAs secretion of allelochemicals which could either be diffusible metabolites such as
lipopeptides, bacteriocins, antibiotics, biosurfactants, and cell-wall-degrading enzymes
or microbial volatile compounds, which interfere with the metabolism of phytopathogen
and thereby inhibit the pathogen development [9,75,76]. There are two types of parasitism:
necrotrophic parasitism and biotrophic parasitism. In the first type, the BCA kills the host
cells before or just after the invasion of the target pathogen and uses its released nutri-
ents [77]. Whilst in the second type of parasitism, the development of the BCA is favored by
a living rather than a dead host structure [78]. Depending on the BCA, we can distinguish
the parasitism of viruses on bacteria (bacteriophages), bacteria on fungi (mycophagy),
and fungi on fungi (mycoparasitism) [9,75,76]. BCAs may also reduce pathogen virulence
by secreting enzymes that interfere with pathogenicity factors, including pectinases and
chitinases, which reduce pathogen virulence [76]. Finally, BCAs can reduce pathogen
infection pressure through the competition for nutrients and space where both BCAs and
pathogens compete with each other. This will help BCAs to establish themselves in the
environment through the physical occupation of the site, biofilm formation resulting in
reduced colonization of roots by the pathogen, and secretion of the essential micronutrients
chelating such as siderophores as well as the characteristics of BCAs, which have more
efficient uptake system for micronutrients in the case of the pests [72,79].
3.1.1. Involvement of Lytic Enzymes

Plants are probably primed for SAR by some Trichoderma spp., but the full pathway
remains inactivated until a subsequent pathogen/parasite attack [80]. That is the case
for example of Trichoderma asperellum (T203) which was used to prime cucumber against
angular leaf spot caused by Pseudomonas syringae pv. Lachrymans. Disease symptoms de-
creased by up to 80%, equating to a two-orders-of-magnitude drop in bacterial cell densities
in leaves of T. asperellum-treated plants. Bacterial cell multiplication in these plants was
inhibited. The accumulation of mRNA of two defense genes, the phenylpropanoid pathway
gene expressing phenylalanine ammonia-lyase (PAL) and the lipoxygenase pathway gene
encoding hydroxyperoxide lyase (HPL), was linked to BCAs protection. This was further
corroborated by the accumulation of secondary phenolic compounds, which demonstrated
a six-fold increase in the capacity of inhibition of bacterial growth in vitro [81].
Trichoderma asperellum, which was isolated from banana wilt-affected plantations,
has anti-pathogenic properties based on a variety of mechanisms. One of them includes
antibiosis, which is used by a variety of pathogenic fungi, belonging to Fusarium genus.
T. asperellum has been proven to decrease phytopathogen growth by 65–74% and impede
spore germination by 30–75%. Furthermore, T. asperellum releases mycolytic enzymes
(chitinase and β-1,3, glucanase), which may be able to destroy phytopathogen cell walls.
In pathogen-induced cultures, the accumulation of respective transcripts and enzymatic
activity of both chitinase and β-1,3, glucanase were significantly higher [82]. In the stimula-
tion of the plant immune system by biocontrol fungi against RKNs, ET- and SA-responsive,
genes were upregulated by glucanase and chitinase activity, whilst downregulated by genes
encoding antioxidant enzymes [80].
3.1.2. Antimicrobial Molecules

Antimicrobial molecules are secondary metabolites that belong to a diverse category
of organic, low-molecular-weight compounds produced by microorganisms and are toxic
to the growth of other microorganisms or metabolic processes [83]. Antibiotics and related
natural compounds can be produced by nearly all living organisms as they are secondary
metabolites. They are produced by both prokaryotic and eukaryotic (plant and animal
kingdom) species, within unicellular bacteria and eukaryotic fungus. Generally, filamentous
actinomyces are the most common and varied producers of antibiotics. Furthermore,
amongst prokaryotic and unicellular bacteria, Bacillus and Pseudomonas species are the most
common producers [84].
3.1.3. Biofilm Formation

Understanding how to use BCAs effectively is critically important in sustainable agri-
culture. The exact mechanisms, chemical or physical, involved when crops are attacked by
infectious pathogens are therefore the first steps to determine the factors influencing the
efficacity of biocontrol against plant diseases. Physical mechanisms of BCAs, for example,
include techniques triggered at the molecular level to physically protect the plant because
of antagonistic interactions with host plants. Understanding these mechanisms of action
of BCAs would help develop biofungicides products with superior biocontrol properties.
BCAs from either the same species or different species can physically interact with surfaces
to form complex multicellular assemblies or aggregates of microorganisms also called,
biofilms. These biofilms provide various advantages including increased resistance to
certain biotic stresses [85]. The microbial cells adhere to each other’s surface through
a complex matrix medium comprising a variety of extracellular polymeric substances
(EPS) including exopolysaccharides, proteins, and DNA, known as signaling molecules.
Microorganisms, including bacteria, cyanobacteria, and fungi, specifically interact with
plant tissues through quorum sensing (QS). The latter exists widely in all kinds of microor-
ganisms and is a communication channel for microorganisms [86] and allows individual
microbe within colonies to coordinate with each other. In one experiment on Arabidopsis, B.
subtilis 6051 formed a stable, extensive biofilm to protect plants from Pseudomonas syringae
attacks [87]. Furthermore, both the bacterium Azotobacter chroococcum and the fungus Tricho-
derma viride [88] can form biofilms to protect various crops against pathogens [89]. Strains
of B. velezensis QST713 can also develop a biofilm on inert surfaces to inhibit the growth of T.
aggressivum, which causes green mold disease [90]. The mechanism of biofilm formation is
a powerful tool that helps plants to protect themselves from pathogenic attacks. However,
previous knowledge of BCAs and plant species interactions is required before utilization,
as BCAs interact specifically with plant species, and thus, the adopted techniques should
be species-specific.
3.1.4. Competition for Nutrients and Space

Within BCAs species, competition is defined as the niche overlap of BCAs and
pathogens, resulting in simultaneous demand for the same resource [91]. Competition for
nutrients (carbohydrates, nitrogen, and oxygen) and space is often suggested as a potential
mechanism of action in biological control systems [92]. Fungi, in particular, use the com-
petitive saprophytic ability (CSA), which was originally used to describe the ability of the
fungus to colonize dead organic substrates through competition [93]. A fungus with high
CSA also exhibits adaptation abilities including the production of antimicrobial substances
which are inhibitory to the pathogen growth through both mycoparasitism and the rapid
adaptation to environmental triggers (temperature and water availability) [93,94]. Many
plant fungal pathogens, including B. cinerea, and S. sclerotiorum, can be effectively managed
through the mechanism of CSA [95]. Both B. cinerea and S. sclerotiorum are necrotrophic
fungal pathogens [96,97]. B. cinerea conidia and S. sclerotiorum ascospores require external
nutrients from plant wounds, senescent plant organs, and/or pollen grains for germination,
hyphal development, creation of infection cushions, and infection of plant tissues. Numer-
ous studies highlighted that fungal saprophytes such as Candida oleophila, Clolostachys rosea,
Epicoccum purpurascens, Metschnikowia fructicola, Pichia guilliermondii, Rhodotorula glutinis,
Trichoderma harzianum, and Ulocladium atrum are excellent competitors and are therefore
effective BCAs of B. cinerea and S. sclerotiorum [98–104]. Competition for both nutrients
and space is the key physical mechanism used by some BCAs to prevent the development
of pathogens.
BCAs decrease the population level of pathogens by depleting food sources as, gen-
erally, BCAs are more efficient in nutrient uptake than pathogens [105]. BCAs also use
competition for space. Certain microorganisms such as yeasts and bacteria form extracellu-
lar polysaccharide capsules that facilitate the adhesion to the fruit surface [106]. Various
BCAs limit the amount of nutrients available and reduce the pathogen spore germina-
tion percentage [107,108], which subsequently reduces germ-tube growth, infection, and
necrosis, and then prevents the expansion of the pathogen [108].
Successful biological controls require colonization by the added biocontrol agents
under nutrient deficiency. These BCAs reduced the available nutrients in the wound site
and make nutrients inaccessible for the pathogens to germinate, grow, and infect their
hosts [109,110]. Iron, for example, is an essential micronutrient that is present in a high per-
centage of soils. This element, however, has low solubility in soil with a pH > 6, making its
acquisition hard for microorganisms [111]. Iron bioavailability has thus become a limiting
factor that may lead to competition among organisms. In these conditions, BCAs produce
siderophores which are iron-chelating compounds that bind to iron, thus making it inac-
cessible to pathogens. This iron sequestering mechanism causes inhibition of both growth
and metabolic activity of soil-borne pathogens. One siderophore compound in particular,

rhodotorulic acid produced by Rhodotorula glutinis, improved the control of apple blue
rot caused by P. expansum [112]. In vitro experiments showed that Pseudomonas aeruginosa
produced siderophores, in addition to several metabolites with a broad spectrum of antago-
nistic activities against Fusarium oxysporium f. sp. ciceri, F. udum, and Aspergillus niger [113].
Additionally, Bacillus species including B. subtilis and fluorescent pseudomonads have
been effective in rhizosphere colonization [110,114]. B. subtilis produced siderophores
that played an important role in the control of F. oxysporum [115]. In avocado flowers,
for example, B. subtilis B246 effectively adhered to conidia and hyphae of stem-end rot
pathogens and caused cell degradation, thus preventing pathogens from colonizing the
flowers [116,117]. This competition mechanism is described in various biocontrol studies
with various antagonists such as Pantoea agglomerans [118], Serratia plymuthica [118], and
Aureobasidium pullulans [119]. These BCAs were proven to be effective against post-harvest
pathogens of fruits [120]. Especially for the control of post-harvest diseases, the use of
microorganisms that compete with pathogens for nutrients may be a preferable option
to antibiotic-producing microorganisms to avoid food contamination and a build-up of
antibiotic resistance within the pathogen population. Generally, for a successful control
through competing mechanisms, both the pathogen and the antagonist must have the same
requirement for a specific nutrient or resource. The effectiveness of these controls entails
that BCAs are present in sufficient quantities at the correct time and location. Overall,
the key effects of competition include (i) a reduction of pathogen potential by nutrient
competition, (ii) an increase of saprotrophic competition for initial resources in substrate col-
onization, and (iii) a reduction of the actual amount of the pathogen in either the dormant
survival or pathogenic growth phases. Those mechanisms involved in BCA controls can
physically protect key crops from devastating pathogens while respecting the environment.
Effective use of the correct BCAs is, therefore, an important component of a successful
disease management program.
3.1.5. Parasitism
Parasitism is an important biological control mechanism exhibited by antagonists
to directly target pathogens that cause soil-borne and foliar diseases [121]. Parasitism
or predation occurs when an antagonist feeds on or within the pathogen. It causes the
immediate destruction of affected pathogens [122]. Some BCAs use parasitism mechanisms
by producing propagules which are aggregates of BCA around or inside the pathogen. The
density and the distance between a pathogen and its nearest BCA propagule are important
components in effectively controlling pathogens. The denser BCAs are to each other, the
more effective they are at controlling pathogens. Many pathogens share the same BCA
propagule as their nearest BCA neighbor, and this interaction between BCAs and pathogens
helps tremendously in the parasitism process [123]. Some pathogens are resistant to BCAs
if optimum environmental conditions are not fulfilled such as adequate temperature and
space. BCAs are also influenced by their quality and quantity when introduced to a tar-
geted ecosystem. Pathogen resistance can be determined by measuring the proportion of
pathogen propagules that remain infective as a function of the amount of BCAs introduced
to the system [124]. Consequently, efforts should be devoted to improving the parasitism
process for successful controls. Some of these measures include the use of mixtures of
BCAs, optimal timing of antagonist application, integrated biological and chemical controls,
and optimization of environmental conditions [124]. The consumption of one fungus by
another is called mycoparasitism [125]. Mycoparasitism specifically utilizes fungal cell-
wall-degrading enzymes to access the cells [126]. Trichoderma, for example, can effectively
eliminate phytopathogenic fungi through the suppression of other microorganisms at the
same site, making Trichoderma the dominant organism in the infested site [127–129]. Para-
sitism between yeasts and fungus was also studied. Cytological damage and protuberances
in the cell wall and degeneration of the cytoplasm were observed in vivo culture of both
Candida saitoana yeast cells with B. cinerea mycelium [125]. In 1991, Wisniewski et al. [125]
observed a strong in vitro adhesion of the antagonistic yeast Pichia guilliermondii cells to
B. cinerea mycelium [130]. Mechanisms of action determining the success of biocontrol
are complex, which, to some extent, may explain the limited effectiveness of biocontrol
against plant diseases in field crops. In this case, the screening of these BCAs can be
achieved by phenotype-based screenings by evaluating pathogen growth by dual culture
assay or by volatile antifungal compounds (VOCs) or by marker-based screening: secre-
tion of antimicrobial metabolites (antibiosis via bacterial supernatant), the secretion of
cell-wall-degrading or screening bacteria with specific inhibitory mechanisms [76,131].
3.2. Indirect Mode of Action

Indirect mechanisms include the induction of resistance by stimulating plant defense
reactions and stimulating plant growth and soil fertilization [74,132]. BCAs can initiate
plant systemic resistance, which results in an accumulation of structural barriers and elicita-
tion of many biochemical and molecular defense responses in the host. This action requires
a signalization of the pathway of phytohormones, phytoalexins, and defense enzymes
such as phenylalanine ammonia-lyase, chitinase, PR-proteins, and phenolic compounds
(Figure 3) [74,133]. Plant defense signaling molecules include salicylic acid involved in
defense against biotrophic pathogens and systemic acquired resistance, as well as jasmonic
acid and ethylene, both of which are generally considered necessary for defense against
necrotrophic pathogens and are beneficial in plant–microbe interactions [134,135]. Inocula-
tion of the plants with specific PGPRs elicits a phenomenon referred to as induced systemic
resistance (ISR; Van Loon et al. [136]). ISR provides plants with the ability to withstand
attacks by pathogens, which, without bacterial pre-inoculation, would be potentially lethal.
Salicylic acid, in response to BCA treatment, activates a hypersensitive response leading
to localized programmed cell death that limits pathogen spread, together with the expres-
sion of pathogenesis-related genes, which exhibit antimicrobial properties and provide
protection against stress [75]. Ethylene is produced when plants are exposed to abiotic or
biotic stressors. The 1-aminocyclopropane-1-carboxylate (ACC) synthase is the immediate
precursor of ethylene [137]. An increase in the levels of this phytohormone in plants leads
to senescence, chlorosis, and abscission [138]. BCAs with ACC deaminase activity can
prevent the increase of phytohormone levels in the plant through ACC hydrolysis into
α-ketobutyrate and ammonia products, which can be easily assimilated by plants [138]. The
application of BCAs may provide tolerance to stress conditions such as (flooding, drought,
temperature, and various contaminants), thus allowing plant survival, even under critical
conditions [137]. BCAs could also act by stimulating plant growth through the production
of plant growth regulators such as gibberellic acid (GA3), indole-3-acetic acid (IAA), cy-
tokinins, and abscisic acid that have a close relation with plant nutrient availability [133].
The contribution to improving plant nutrition could be manifested in a variety of ways,
such as the availability of iron (Fe), siderophore production, phosphate solubilization (P),
nitrogen (N) fixation, etc. [132].
3.2.1. Induced Resistance and Priming

Plant defense against biotic threats is known to be activated by beneficial microor-
ganisms [80], through a variety of mechanisms, giving plants resistance to diseases [139].
However, the molecular processes through which plants with an activated defense respond
to biotic stresses more quickly are still unknown [80]. The defense network of plants is
made up of various components that are triggered in response to pathogen attacks. To
perform efficiently, this network necessitates a significant commitment at the cellular level,
including genetic reprogramming. This includes the stimulation of defense-related genes in
plants, such as pathogen resistance genes (PR proteins), as well as the expression of genes
encoding specific metabolites or proteins that are involved in the defense setup of the plant
system (Figure 3) [140]. For example, the activation of multiple defense responses in the
biocontrol activity of several examined BCAs to combat grapevine bunch rot caused by B.
cinerea and Aspergillus carbonarius is indicated by transcriptomic analysis of genes encoding
pathogenesis-related proteins PR2, PR3, PR4, and PR5 [141]. Beneficial microbes cause early
plant ISR events such as increased expression of pathogenesis-related PR genes, increased
activity of defense-related substances such as phenylalanine ammonia-lyase, polyphenol
oxidase, peroxidase, β-1,3, glucanase, chitinase, and the accumulation of reactive oxygen
species [142,143].
Figure 3. Schematic model of signal transduction events triggered by microbes. Microbes may
produce microbe-associated molecular patterns (MAMPs) or damage-associated molecular patterns
(DAMPs), such as flagellin or chitin, which are perceived by pattern-recognition receptors (PRRs), or
other elicitors, such as volatile organic compounds (VOCs), or siderophores, which are perceived by
receptors. The activated receptors may then trigger different signaling cascades, acting as a precursor
for the biosynthesis of phytohormones that trigger defensive pathways. The kinase cascade may also
phosphorylate transcription factors that modulate the expression of early and late response genes.
Abbreviations: 3-indole acetic acid (IAA); abscisic acid (ABA); brassinosteroid (BR); cytokinin (CYT);
enhanced disease susceptibility (EDS); ethylene (ET); exopolysaccharides (EPS); gibberellic acid
(GA); induced systemic resistance (ISR), jasmonic acid (JA); lipopolysaccharides (LPS); nonexpressor
of pathogenesis-related genes (NPR); pathogenesis-related protein (PR); peptidyl arginine deiminase
(PAD), salicylic acid (SA); senescence-associated gene (SAG), systemic acquired resistance (SAR).
Induced resistance offers the possibility of long-term and broad-spectrum disease

control using the natural resistance of plants. The resulting resistance is usually broad
and long-lasting but rarely complete, with most inducing agents only lowering the disease
prevalence by 20% to 85% [144]. Systemic acquired resistance (SAR) and ISR are two
forms of induced resistance that have been characterized based on distinctions in signaling
pathways and efficacy spectra [144]. ISR and SAR are two types of plant systemic resistance
induced by non-pathogenic and pathogenic microorganisms, respectively [139,145,146].
They frequently overlap with interaction occurring between the two pathways [147]. ISR
has emerged as a major method by which bacteria and fungi in the rhizosphere prepare
the entire plant body for increased defense against a variety of diseases and insect her-
bivores [147]. Plants use long-distance systemic signaling to protect distal tissue after
ISR activation, inducing quick and powerful immune responses against pathogen inva-
sions [139]. BCAs can therefore suppress pests and diseases by activating the plant immune
system [148]. Various BCAs have proven the ability to produce ISR in the past. Ben-
eficial bacteria such as Bacillus spp. and Pseudomonas spp. can help plants to develop
broad-spectrum disease resistance by stimulating defense responses [149,150]. Bacillus amy-
loliquefaciens, Bacillus atrophaeus, Bacillus cereus, Pseudomonas fluorescens, and other bacteria
are efficient against fungal, bacterial, and viral invasion by ISR [139]. Trichoderma spp. and
arbuscular mycorrhizal fungi (AMFs) have long been considered to be widespread potential
BCAs [151,152]. A meta-analysis was conducted on papers published between 2010 and
2021 that looked at cross-talk in the tomato–Trichoderma–B. cinerea system. The analysis
was carried out on 15 publications, starting with a collection of 40 papers. Trichoderma’s
role in the control of grey mold in tomato leaves (decrease in disease intensity, severity,
and occurrence, as well as modulation of resistance genes in the host) was highlighted
in the research [151]. Defense priming, or AMF-induced resistance, is becoming more
widely recognized as AMF’s ability to induce systemic resistance to insect herbivores and
diseases [152].
The induction of a distinct physiological state known as “priming” occurs when plants
are infected with necrotizing pathogens or when helpful microorganisms colonize the roots
of plants. The different cellular defense mechanisms that are triggered during the attack
by pathogens or insects, or in reaction to abiotic stress, are activated faster, stronger, or
both in primed plants [153]. Plants often switch to a primed state of heightened defense
when they detect prospective opponents, invading pathogens, wound signals, or abiotic
stress, and some natural or manufactured compounds. The communication appears to vary
depending on the considered beneficial microorganism and the elicited plant species [154].
Here, we presented the molecular mechanisms by which some BCAs, especially Trichoderma,
confer plant protection [63]. Trichoderma is a genus of filamentous fungus that colonizes the
root surface and play an important role in stimulating plant growth. However, the main
proteins and chemical pathways that control this stimulation are still unknown [155]. Sev-
eral Trichoderma spp. can interfere with signaling networks in their host plants to improve
disease resistance and stress tolerance, in addition to their ability to directly antagonize
plant pathogens and boost plant growth [63]. Trichoderma isolates use different strategies
to boost the defense pathways of the plant host, depending on their origin and applica-
tion place. Whilst the phyllosphere Trichoderma isolate (BHUF4) used the SAR channel
to elicit the defense response in the host plant under Colletotrichum truncatum challenge,
the rhizospheric Trichoderma strain (T16A) used the ISR pathway [140]. Trichoderma adapts
to different interactions including inter- and cross-kingdom interactions [63]. For exam-
ple, during the mycoparasitism between Trichoderma species and the phytopathogenic
fungal infections, several signaling cascades are activated [156]. Important processes in
Trichoderma mycoparasitism are adapted such as the development of infection structures
called appressoria during mycoparasitism, the generation of hydrolytic enzymes, antimi-
crobial metabolites, and the induction of systemic resistance in plants. All these processes
rely on signaling pathways that are activated by the binding of host-derived ligands to
receptors [157].
3.2.2. Implication of Phytohormones

Several low-molecular-weight compounds known as phytohormones, such as SA, JA,
and ET, influence the immune response in plants. To trigger the plant immune response
against pathogen and parasite attacks, BCAs use a variety of sophisticated molecular
processes [80]. SAR is one of the most well-studied mechanisms, which provides long-
term protection against a wide range of microorganisms [158]. SAR confers long-term
resistance to (hemi) biotrophic pathogens and pests and is mediated by SA. It is linked to
the activation of pathogenesis-related (PR-) genes. Rhizobacteria ISR is largely effective
against necrotrophic diseases and herbivorous insects and is regulated by JA and ET. It is
not related to changes in PR-gene expression [147,159]. The transmission of ISR signaling
was formerly thought to be dependent on JA and ET, but not on SA. The involvement
of both the SA and JA/ET signaling pathways, as well as the regulatory roles of small
RNA in ISR, has been revised in the previous decade [139]. T. harzianum T22 improves
plant direct resistance against stink bug (Nezara viridula) feeding attack by increasing JA
marker gene transcript levels early. Tomato plants were shown to respond to N. viridula at
the molecular level. N. viridula feeding activates the JA defensive signaling pathway, as
evidenced by an increase in ToLOX D expression after 8 h of stink bug feeding. ToPIN2 was
also highly elevated 8 h after herbivore feeding, most likely because of the JA-cascade being
activated. Upregulation of ToPIN2 may play a role in the lowered growth rate of stink bug
nymphs [160]. Beneficial root endophytes, such as Trichoderma and Glomus spp., have been
proven in numerous studies to minimize endoparasitic nematode infections by eliciting the
plant immune system [80,161,162]. When attacked by root-knot nematodes (RKNs), tomato
plants treated with T. harzianum had their SA-signaling pathway and ET biosynthesis
activated, which helped in controlling the infection. Monitoring the expression of the
genes PR-1/PR-5 and JERF3/ACO, which are indicators of the SA- and JA/ET-dependent
signaling pathways, respectively, revealed this effect. Five days after nematode inoculation,
roots of plants pre-treated with T. harzianum-strains showed an over-expression of PR-1,
PR-5, and ACO genes. In T. harzianum-colonized plants challenged with nematodes, JERF3
gene expression remained unchanged [161]. Plants are primed against RKNs through BCA
contact with roots. BCA-mediated immunity appears to be dependent on SA-mediated
SAR and is linked to both the activation and inhibition of chitinase and glucanase enzyme
activities, as well as the inhibition of the plant antioxidant enzyme system [80].
Hence, the screening of such mechanisms can be based on phenotype-based screenings
by measuring plant growth and root colonization traits, a reduction in disease severity, and
alleviating abiotic stress. It can also be based on marker-based screening, either by using
specific medium culture for BCAs screening or by searching the presence of genes responsi-
ble for these mechanisms [76]. Some other techniques require a high-throughput tool, such
as induced systemic response markers, expression of pathogenesis-related) proteins at the
transcriptomic level, and the production of reactive oxygen species (ROS) [76,79]. BCAs
with multiple modes of action are highly demanded. The nature of the mode of action of
selected BCA is among the data requirements for the registration of BCA-based products.
4. Biological Control: Facing Reality

The modern application of biological control was introduced at the end of the nine-
teenth century; however, related techniques were in use for at least 2000 years [34]. Biologi-
cal control is composed of four different types, including conservation, natural, classical,
and augmentative controls [163]. The method is widely considered an attractive, eco-
friendly alternative for pest management [164].
Although the strategy offers a promising alternative to synthetic pesticides in the
control of pests and plant disease, the method faces many challenges. Serious ecological
consequences such as outbreaks could be associated with the introduction of non-native
living species that could become invasive and cause significant deleterious impacts on the
environment [164]. Moreover, the use of BCAs has not always been successful, probably
due to changes in environmental conditions. Trichoderma sp., for example, showed its
predatory behavior only under limited nutrient conditions. It has been reported that
the Trichoderma spp. do not attack R. solani in the presence of compost, which gives the
availability of cellulose as nutrients for the agent [164].
Even if the evidence about the effectiveness of biopesticide is reported to be satisfactory,
the availability of such products is still not well established in the market. Additionally,
the commercialization of plant-based biopesticide highly depends on the availability of
plant sources in large quantities and cultivation. Moreover, a plant could have many active
substances, which makes biopesticide formulation a very challenging task [165]. Several
other issues are related to the use of BCAs, including the shelf life of biological pesticides
which are known to have a high rate of biodegradability. Furthermore, pesticide-based
microbes may not be efficient on all pests in the field; they only control a small portion of
pests [165].
4.1. Biopesticide from Lab to the Field

The screening of potential strains is part of specialized laboratories’ mission to develop
biopesticide products that are highly efficient against plant pathogens in agriculture [166].
These strains are generally selected based on their effectiveness against pathogens, host
range, availability, ease for mass production, formulation, and application by farmers. The
strains that had the lowest LC50 and LT50 are tested under greenhouse and later in con-
trolled field experiments. The formulation and field efficacy trials are conducted by partners
from the private sector and accredited laboratories, who conduct the required eco and mam-
malian toxicity tests and quality assurance for later commercialization of biopesticide [167].
However, field bioassays are required to confirm the efficacy of selected products.
The biopesticides are classified by governments so that the authorities can regulate
their use through authorizations. The main focus of this regulation is the environment,
human safety, and the reliability of the product. The efficacy and labeling of the biopes-
ticide also should meet the requirement set up by the authorities and EU for their safe
handling [168]. The regulation portfolio of biopesticide registration is normally a modified
version of the conventional chemical pesticides, with risk assessment. That includes tox-
icological and ecotoxicological evaluations, their mode of action, and host range. These
requirements can be challenging for regulators. One of the challenges is identifying the
appropriate biopesticides while at the same time ensuring safety and consistency standards
that are acceptable for commercialization [168].
4.2. Limited Number of Registered Products

An increase in agricultural productivity has occurred in the latest decades due to
population expansion and, as a result, the demand for food [169]. To date, the consumption
of chemical fertilizers and pesticides has led to ecological imbalance and the contamination
of natural resources [170]. Sustainable agriculture is the solution to problems arising from
decades of uncontrolled use of chemical-based agronomic techniques to boost crop yields.
As a result, an eco-friendly substitute for chemical fertilizers and pesticides is a necessity.
With their diverse and unique capabilities, microbial agents represent an appealing and
realistic option to substitute chemicals [171].
BCAs influence the interactions between plants, pathogens, and their environments,
resulting in biological and physical pathways that affect pathogen fitness, plant health, and
ecological function. These interconnections create a panorama of compromises between
natural and social functions of biological control, therefore requiring a full assessment of
its benefits and costs from societal and farmer perspectives for their long-term develop-
ment and deployment [15]. BCAs have shown significant results against a wide range
of phytopathogenic fungi, oomycetes, bacteria, and weeds. They constitute a promis-
ing alternative to synthetic pesticides, although their application in agriculture remains
remarkably scarce.
Commercially accessible biocontrol solutions that control plant disease, unlike insect
biocontrol, are a novel potential. The first bacterium, A. radiobacter strain K 84, was enlisted
by the United States Environmental Protection Agency (EPA) in 1979 to combat crown
gall disease in plants. A decade later, the EPA approved the first fungus, T. harzianum
ATCC 20476, to manage plant diseases. Currently, 14 bacteria and 12 fungi strains have
been recorded by EPA that aid in treating plant disease [172]. The plurality of these
BCAs was commercialized as one or more products on the market. Commercialization
technology is still in its beginning phases (Figure 4). Most of the EPA-registered species
(64%) were recorded in the last decade, while the remaining 36% were documented within
the last five years. For these products to reach the market, several technological challenges
were surmounted. Table 1 lists some commercially available biocontrol products on the
market [173,174].
To date, BCAs have a commercial value of less than 5% of the whole crop protection
industry [175], despite their well-known documentation. The limited number of licensed
products for biocontrol of plant diseases is significantly linked to the low technology
transfer, implying that the agricultural sector, mainly in developing countries, has yet to
recognize its economic potential. Besides that, for promising microbial agent candidates,
sufficient knowledge of organism-specific research methods for mass manufacturing and
conceptualization is often lacking, making mass production of the entire microbe in in vivo
conditions an expensive and time-consuming task. The cost of this approach is a major
constraint, as its production and licensing are too expensive when compared to chemical
agents [174,176]. Furthermore, ensuring BCAs reach right area at the right time and with
sufficient density to be effective, as well as keeping them permanently there, represent one
of the most challenging aspects of their use. Since biocontrol involves the introduction
of non-native living organisms, serious ecological impacts may be associated with them.
Non-native species, for instance, may become invasive and have harmful environmental
consequences if they spread beyond the area where they were introduced.
Figure 4. The potential market of biopesticide.
Moreover, some BCAs exhibit efficacy under in vivo circumstances in the laboratory, but
ecological restrictions hamper their performance under real full-scale conditions [177–179],
making them economically non-viable with chemical pesticides. Given the increasing
number of microorganisms used for biological control, few are recorded against plant
diseases in the EU, according to the European project ENDURE (European Network for
Durable Exploitation of Crop Protection Strategies). Several studies found that when certain
BCAs were introduced under commercial field circumstances, they were less effective or
altogether ineffective, despite their high performance in controlled conditions [45,180–182].
For example, many Pseudomonas BCAs showed a good performance in trials but cannot be
translated into consistent, efficient disease control in various field settings [182]. For the
antagonist, Candida oleophila strains, a significant difference in enzymatic activity existed
between in vivo and in vitro applications [15,183].
Despite the fact that BCAs are genetically stable, their use has not been very successful
due to ongoing climate change. As a result, it is critical to choose agents that are effective
across a variety of environmental situations (soil texture, temperature, humidity, radiation).
A small change in temperature amends the microbial concentration. The virulence of
the agents is decreased when they are exposed to UV rays from the sun. Some BCAs
only exhibit predatory behavior when nutrients are scarce rather than in normal growth
conditions. For example, Trichoderma spp. does not directly attack the R. solani when fresh
bark compost is introduced. The availability of cellulose itself is a reason for this. When
lower amounts of cellulose, genes governing chitinase synthesis, in Trichoderma spp. are
activated, the enzyme for parasitic activity is then produced [164,184].
Since BCAs have a limited shelf life, their viability must be managed. For instance,
Trichoderma viride is viable for four months, while Pseudomonas fluorescens can only survive
for three months [164,185]. Bacillus is thought to be a microbiological factory that generates
a large set of antimicrobial substances and is found in about 85% of commercially available
BCAs [175,186]. Although basic fermentation and formulation processes can create this type
of formula, the commercial applicability of such procedures is limited. This is due to a lack
of acceptable methods for large-scale manufacturing and appropriate technology to address

the problem of short product shelf life. Maintaining a high spore density is important to
achieve optimal sporulation efficiency, which is a viable solution to this problem. As a
result, the entire production process must be streamlined in generating an efficient and
high-quality product [187,188].
The evolvability of target pathogens is negatively correlated with the endurance of
BCAs [189]. BCAs targeting pathogens with higher genetic variation from sexual repro-
duction, large effective population size, and long dispersal ability are expected to have a
shorter durability [190]. Continuous commercial use of the same BCAs might lead to a
strong selection of infections, resulting in the emergence of new pathogen populations that
can evade or attenuate the negative effects of the BCAs [15,45]. Moreover, because it is
difficult to estimate the specificity of the age and stage of targeted pathogens, the time of
application of BCAs must be established [174,185].
Another aspect limiting the amount of BCAs registered is growing skepticism regard-
ing their efficacy. Many farmers, particularly in underdeveloped countries, are hesitant to
adopt the practice due to technical difficulties, environmental concerns, or economic ap-
peal [45,75,191]. They choose which plant disease management technologies to implement,
and their viewpoints on biological control are influenced by economic, technological, and
practical considerations. Examples of the latter include effectiveness, profit, availability,
and convenience. To entice farmers to use biological control, technology must be simple
to assess and prepare, whilst providing economic advantages over alternative options in
terms of cost, demand, and supply efficiency.
Some farmers prefer formulations in granules that can be stored and easily applied
by machines over those that require more expensive equipment or cold storage. Farmers
are discouraged from using the biological control strategy due to a lack of awareness of
technological features and a limited number of successful technologies. In this scenario,
training and field demonstrations are crucial for exchanging information between tech-
nology developers and end-users. Biological management has a direct impact on farmers’
expenses and revenue, but it also has an indirect impact on their economic benefits through
its effect on the ecological function and sustainability of farmland [15]. On the other side, it
became evident that current regulation and registration procedures for microorganisms,
which are mostly based on those for chemical pesticides, are not well suited. This has
contributed to the slow implementation of BCAs [176]. To summarize, the full potential
of BCAs was not realized, mostly because research is only confined to the laboratory, and
thus, little emphasis has been dedicated to developing commercial formulations of BCAs.
Furthermore, because of a lack of information on how to use what has been commercially
produced, farmers have not used it effectively. As a result, there is a need to strengthen the
notion of biological control extension to popularize the concept.
Table 1. Biopesticides, their trade names and targets [18,172–174,182,192–197].
Target Disease and/or Target

Biopesticides Active Agents Trade Name Crop Manufacturer/Distributor
Organism Pathogen
A. Bacteria biopesticides
Agrobacterium radiobacter strain k84 Galltrol Agrobacterium tumeifaciens Ornamentals, Fruits, Nuts AgBioChem, Los Molinos, CA, USA
Cherries, cucurbits, grapes, leafy
Foliar pathogens, rots, Fire blight,
Bacillus subtilis QST 713 Serenade vegetables, peppers, potatoes, AgraQuest, Davis, CA, USA
and blights
tomatoes, and walnuts
Root-knot nematode, Remiform Cereals, millets, pulses, oilseeds,
nematode Cyst nematode, fibre crops, sugar crops, forage
Bacillus firmus NCIM 2637 Bionemagon —-
Burrowing nematode, Lesion crops, plantation crops, vegetables,
nematode fruits, etc.
Crop seeds, including seeds for
Fusarium, Pythium, Rhizoctonia, Growth products, White Plains, NY,
Bacillus subtilis GB03 Companion, Kodiak cotton, peanuts, soybeans, wheat,
Aspergillus, and others USA
barley, peas, and beans
Damping-off Becker Underwood, Ames, Iowa,
Cotton, beans, barley, wheat, corn,
Bacillus subtilis MBI 600 Subtilex; Histick N/T Fusarium, Rhizoctonia, Alternaria, and USA; Premier Horticulture,
peas, peanuts, and soybeans
Aspergillus Quakertown, PA, USA
Bacillus subtilis var. amyloliquefaciens Shade and forest tree seedlings,
Taegro Rhizoctonia and Fusarium Earth BioSciences, Salem, OR, USA
strain FZB24 ornamentals, and shrubs
Ecoguard; Novozymes Biofungicide Ornamental plants and ornamental Novozymes Biologicals, Davis, CA,
Bacillus licheniformis strain SB3086 Foliar pathogens and blights
GreenRelief turf USA
GB34 Concentrate Biological
Bacillus pumilus strain GB34 Rhizoctonia, Fusarium Soybean Gustafson, Inc, Plano, TX, USA
Fungicide
Bio-Dart, Biolep, Halt, Taciobio-Btk,
Bacillus thuringiensis Lepidopteran pests Stored grains, fiber, and food crops —-
Tacibio, Technar
Bacillus thuringiensis tenebrionis Novodor, Trident Colorado potato beetle Potato —-
Almonds, blueberry, citrus crops,
Aphids, leafhopper, lygus, stink
Burkholderia spp. strain A396 Venerate cucurbits, fruiting, vegetables, —-
bug, thrips
grapes
Table 1. Cont.

Organism Pathogen
Armyworms, Aphids, Asian Citrus Blueberry, citrus crops, cucurbits,
Chromobacterium subtsugae Grandevo WDG Psyllid, Mites, Spotted Wing fruiting vegetables, grapes, leafy —-
Drosophila, Thrips, Whiteflies. greens
Pythium, Rhizoctonia solani, Fusarium Vegetables and ornamentals in EcoSoil Systems, San Diego, CA,
Pseudomonas chlororaphis strain 63–28 AtEze
oxysporum greenhouses USA
Psuedomonas fluoroscens Fruit crop, tomato, potato Plant Health Technologies,
Frostban Fire blight, bunch rot
strain A506 Burlington, CO, USA
Rhizoctonia solani, Sclerotinia
homeocarpa, Colletotrichum
Vegetables and ornamentals in EcoSoil system, Canyon Lake, TX,
Pseudomonas aureofaciens strain TX-1 Bio–Ject, Spot Less graminicola, Pythium
greenhouses, golf course turf USA
aphanadermatum, Michrodochium
nivale
Frost Technology Corporation, St
Fruit crops, almond, potato, and Croix Falls, WI, USA;
Pseudomonas fluorescens A506 BlighBan A506; Frostban Frost damage, fire blight, bunch rot
tomato crops Plant Health Technologies,
Burlington, CO, USA
B. Fungi and Yeast Biopesticides
Ampelomyces quisqualis Fruit, vegetable, and ornamental

AQ10 BioFungicide Powdery mildew Ecogen, Grand Junction, CO, USA
isolate M-10 crops
Arizona Cotton Research and
Aspergillus flavus AF36 Aspergillus flavus AF36 Aspergillus flavus Cotton Protection Council, Phoenix AZ,
USA
Circle One Global, Cuthbert, GA,
Aspergillus flavus NRRL21, 882 Afla-guard Aspergillus flavus Peanut
USA
Biosoft, ATEC Beauveria,
Larvo-Guard, Biorin, Biolarvex, Coffeeberry borer, diamondback
Coffee berries, canola, mustard,
Beauveria spp. Biogrubex, Biowonder, Veera, moth, thrips, grasshoppers, —-
cruciferous vegetables, and others
Phalada 101B, Bioguard, Bio-power, whiteflies, aphids, codling moth
Myco-Jaal
Table 1. Cont.

Organism Pathogen
Rice blast, durian, and black Pepper
Rice, black pepper, citrus,
Chaetomium globosum Ketomium rot, citrus rot, strawberry rot, —-
strawberry, tomato, corn, and others
anthracnose, and others
PROPHYTA Biologischer
Coniothyrium minitans Sclerotinia sclerotiorum and Sclerotinia Pflanzenschutz GmbH, Wismar,
Contans WG; Intercept Agricultural soil
CON/M/91-08 minor Germany; Technology Sciences
Group, Sacramento, CA, USA
Vegetables, herbs, spices, turf,
Kemira Agro Oy, Helsinki, Finland;
Gliocladium catenulatum strain JI446 Prima stop soil guard Soil-borne pathogens ornamentals, tree, and shrub
RegWest Co., Holland, MI, USA
seedlings
Thermo Trilogy Corporation,
Gliocladium virens GL-21 SoilGard Soil-borne pathogens Ornamentals, vegetables, cotton
WALTHAM, MA, USA
Clubroot Certis USA L.L.C., Columbia, MD,
Gliocladium virens SoilGard 12G Canola and crucifer vegetable crops
Plasmodiophora brassicae USA
Meta-Guard, Biomet, Biomagic, Coleoptera and Lepidoptera,
Cotton, vegetable, field crops, and
Metarhizium anisopliae Meta, Biomet, Sun Agro Meta, termites, mosquitoes, leafhoppers, —-
others
BioMagic, ABTEC, Verticillium beetles, grubs
Plant Products Co., Leamington,
Pseudozyma flocculosa strain Roses and cucumbers in ON, Canada; Technology Sciences
Sporodex L. Powdery mildew
PF-A22UL greenhouses Group, Washington
DC, USA
Clubroot Natural Industries Inc., Houston,
Streptomyces lydicus Actinovate AG Canola and crucifer vegetable crops
Plasmodiophora brassicae TX, USA
Streptomyces griseoviridis strainK61 Mycostop Soil-borne pathogens Ornamentals, tree seedlings Kemira Oy, Helsinki, Finland
Wounds in ornamental, shade, and BINAB Bio-Innovation AB,
Trichoderma harzianum ATCC 20, 476 Binab T Tree wound pathogens
forest trees Helsingborg, Sweden
Tomato wilt
Trichoderma album 2.5% Bio Zeid Tomato —-
Fusarium oxysporum
Table 1. Cont.

Organism Pathogen
Trichoderma harzianum T39 Trichodex Botrytis cinerea Most of the food crops Bio works, Victor, NY, USA
Trichoderma harzianum T22 Root shield, plant shield Soil-borne pathogens Greenhouse nurseries Bio works, Victor, NY, USA
Verisoft, ABTEC, Verticillium,
Vert-Guard, Bioline, Biosappex,
Whitefly, coffee green bug,
Verticillium lecanii Versitile, Ecocil, Phalada 107 V, Coffee crops and others —-
homopteran pests
Biovert Rich, ROM Verlac, ROM
Gurbkill, Sun Agro Verti, Bio-Catch
C. Postharvest biofungicides
Pome fruit, citrus, strawberry, stone

Candida oleophila Aspire Botrytis, Penicillium, Monilinia Ecogen, Grand Junction, CO, USA
fruit
Various fruits, vegetables, flowers,
Candida oleophila isolate I-182 Aspire Postharvest diseases Ecogen, Grand Junction, CO, USA
ornamentals, other plants
Cryptococcus albidus YieldPlus Botrytis, Penicillium, Mucor Pome fruit, citrus Lallemand, Bellville, South Africa
IRTA/Sipcam-Inagra, Valencia
Candida sake Candifruit Penicillium, Botrytis, Rhizopus Pome fruit
Spain
Pome fruit, citrus, strawberry, Jet Harvest Solutions, Longwood,
Pseudomonas syringae Biosave Penicillium, Botrytis, Mucor
cherry, potato FL, USA
South Africa
Bacillus subtilis Avogreen Cercospora, Colletotrichum Avocado
Lesaffre
Candida oleophila Nexy Botrytis, Penicillium Pome fruit
Belgium
Aureobasisium pullulans BoniProtect Botrytis, Penicillium, Monilinia Pome fruit Bio-ferm, Herzogenburg, Austria
IRTA/Sipcam-Inagra, Valencia
Pantoea agglomerans Pantovital Botrytis, Penicillium, Monilinia Citrus, pome fruit
Spain
Botrytis, Penicillium, Rhizopus, Table grape, pome fruit, strawberry,
Metschnikowia fructicola Shemer Bayer/Koppert, The Netherlands
Aspergillus stone fruit, sweet potato
4.3. Legislative Procedures

Although the number of biocontrol products used in plant disease management is
growing, they still account for just 1% of agricultural control measures, while synthetic
fungicides account for 15% of total chemicals used in agriculture [172].
The commercialization of biofungicides is a multi-step process with several restrictions.
BCAs, like synthetic pesticides, are submitted to risk assessments before being approved
for commercialization. The European Regulation (EC) No 1107/2009 specifies rules for
the marketing of plant protection products based on a risk assessment. Regulation (EC)
No 540/2011 establishes a list of authorized microorganisms for biocontrol usage in Europe.
However, current risk assessment approaches are not entirely adapted to the extremely
difficult task of assessing the safety and dangers of such living substances. Eventually,
instructions on how to conduct BCA risk assessments are required [198].
Regulation EC1107/2009 took effect in June 2011 to prevent the approval of substances
that present unacceptable risks to human/animal health and the environment. As a re-
sult of the revised registration standards, some active compounds will not be reapproved
due to their classification (cut-off criteria). When a substance meets one of the following
environmental requirements, it cannot be registered: persistent organic pollutant (POP);
persistent, bioaccumulative, and toxic (PBT); and/or highly persistent, very bioaccumula-
tive (vPvB). These toxicological criteria will also be used to eliminate substances classified
as carcinogens (C1A and C1B), mutagens (M1A and M1B), or reproductive toxins (R1A and
R1B) [199].
Regulation EC 1107/2009 (2011)-Article 4 (3) claims that a plant protection product
shall meet the following requirements: (a) it shall be sufficiently effective; (b) it shall have
no immediate or delayed harmful effect on human health or animal health, directly or
through drinking water, food, feed or air, or consequences in the workplace or through
other indirect effects or on groundwater; (c) it shall not have any unacceptable effects on
plants or plant products; (d) it shall not cause unnecessary suffering and pain to vertebrates;
and (e) it shall have no unacceptable effects on the environment, having particular regard to
the following considerations: (i) its fate and distribution in the environment; (ii) its impact
on non-target species; (iii) its impact on biodiversity and the ecosystem [199].
Finally, commercial authorizations for biopesticides (and synthetic pesticides) are
awarded after a laborious process [9,199] requiring a set of tests, such as toxicological and en-
vironmental investigations, as well as efficiency. In many circumstances, toxicological stud-
ies do not exist or are too expensive and time-consuming for local manufacturers [168,197].
Nonetheless, the present trend toward lessening the usage of synthetic pesticides and
easing the regulatory process for low-risk compounds may allow BCAs to be produced
and utilized globally [200].
5. Factors Affecting the Success/Failure of Biological Control of Plant Pathogens

Despite decades of research, the importance of biological control in plant health
management has remained relatively minor in the face of rising demand for alternatives
to chemical control. In laboratory conditions (in vitro or planta), reports of a substantial
effect were significantly more common than in field trials. In comparison to chemicals, it is
widely understood that moving from the controlled conditions of a laboratory experiment
to the harsh conditions encountered in the field has proven more difficult for biopesticides.
Whilst the BCA field efficacy can match or exceed that of chemical pesticides, this can
vary over time and from one area to another [201]. In other words, the antagonist that
controls or suppresses pathogens in the laboratory is rarely effective in the field. This
is due to the antagonist, the pathogen, the host, the environment, and all the intricate
interactions that occur between them. The host, for example, passes through a series of
evolutions or mutations, changing its physical, biological, and chemical features. The
antagonist’s efficacy is also determined by pathogenic features. The antagonist may change
in response to changes in environmental conditions, population, and the presence of
microbial colonizers in the biological system [32]. For example, although Trichoderma is
by far the most widely studied fungal BCA, its widespread use has been impeded by
its unpredictability in the field. Understanding how Trichoderma interacts with plants,
other microorganisms, and the environment is critical for developing and implementing
Trichoderma-based agricultural production and protection strategies [63].
5.1. Effect of the Plant on Biocontrol Activity

The outcome of biological control can be heavily influenced by plant species and
genotypes [202]. Umer et al. [32] widely documented the plant effect on biocontrol activity.
The plant itself serves a dual purpose in biocontrol [32]. The degree of rhizosphere colo-
nization and antibiotic production by antagonists, as well as the development of induced
resistance by plants, can all be influenced by plant genotype. The plant susceptibility to
some nematode species, for example, can influence the effectiveness of control; good hosts
will require more suppression than bad hosts [202]. The plant also serves as a place of action
for pathogens and antagonists, where they interact. Pathogen and antagonist interactions
are influenced by the host exudate excretion, ion, and water absorption, gaseous exchange,
and surface temperature [32]. When plant genes are expressed, they have an impact on the
microbial community on the surface of the plant and its surrounding space.
5.2. Effect of the Pathogen on Biocontrol Activity

Pathogen behavior is one of the most significant factors to consider when choosing
BCAs; due to genetic variability and ecological fitness diversity, each pathogen has a unique
behavior interaction with the host. It is important to highlight that pathogen behavior
differs from that of the antagonist due to both its pathogenicity and susceptibility to
antagonist action [32]. The persistence of a plant protection control efficacy in space and
time is referred to as its durability. It depends on two factors: (i) the selection pressure
exerted on plant-pathogen populations and (ii) the pathogen’s ability to adapt to the
control strategy. In other words, regardless of the complexity of their mode of action, plant
pathogens can show a wide range of sensitivity to BCAs (including very low sensitivity).
Certain pathogens can adapt themselves to the selection pressure exerted by BCAs in a few
generations [203]. Biological control is frequently thought to have a longer lifespan than
chemical control. This assumption may not always be justified, according to research on
pest management in agricultural systems. Resistance of several pests to one or more Bt
toxins, as well as the emergence of resistance of the codling moth Cydia pomonella to the C.
pomonella granulovirus, have been documented.
In contrast to pest control, the persistence of biological control of plant diseases has
received little attention, and no scientific papers demonstrating the loss of efficacy of
BCAs against plant pathogens in practice have been reported yet. The success of BCAs
against plant diseases may not be durable, as pathogen populations may develop resistance
similar to that seen with single-mode chemical fungicides. Variation in susceptibility
to the mode of action within the pathogen population, leading to changes in pathogen
populations toward less sensitive strains due to selection pressure, and the fitness of the
selected strains in the environment under conditions without selection pressure are all
important factors in the deterioration of effectiveness [203]. For successful biocontrol, a
weak link in the pathogen life cycle needs to be identified as an opportunity window. The
antagonist should penetrate the opportunity window and disrupt the disease cycle. For
some unspecialized necrotrophic pathogens, for example, the window of opportunity is
to prevent the uptake of nutrients necessary for growth. When the antagonist competes
for nutrients and prevents the saprophytic phase from establishing, this phase will be
suppressed. The loss of endogenous nutrients from the pathogen spore may limit or
prevent germination when antagonistic organisms are present in sufficient quantities in
the area surrounding the pathogen spores. The synthesis of enzymes or antibiotics by the
antagonist may also be effective against these diseases [32].
5.3. Effect of the Biocontrol Agent on Biocontrol Activity

The ability of BCAs to adapt to local biotic and abiotic environmental conditions is
the main reason behind their diminished efficiency in the field. Therefore, the spatial
distribution pattern of BCAs in the rhizosphere should be studied to fully understand this
phenomenon [69]. Additionally, more suitable native strains of BCAs should be collected
and screened to achieve relevant biocontrol results [67]. The efficiency of BCAs is closely
linked to their modes of action [204] and frequently involves tradeoffs with other natural
qualities of the agents, such as specificity and environmental persistence [31]. The most
commonly observed hurdles in biocontrol include the microbial strain intrinsic biological
properties (such as its “ecological competence”), as well as the quality of the formulated
preparations utilized in the field or inadequate application time or method [201]. When
an ideal relationship prevails, the biocontrol agent will be more effective. Therefore, for
successful biocontrols, the application of the antagonist at the right time is critical. When the
antagonist is applied before the pathogen’s establishment, the biocontrol will accomplish its
goals [32]. Furthermore, to achieve optimal disease control, it is critical to understand how
BCAs work. Understanding the mode of action is also necessary for identifying potential
risks to humans or the environment, as well as risks of resistance development against
the BCA [35]. Fluorescent pseudomonads have many features that make them ideal BCAs.
These characteristics include (i) efficient colonization of roots, tubers, hypocotyls, and other
parts of the plant; (ii) the ability to use a variety of organic substrates commonly found in
root and seed exudates; (iii) ease of cultivation in the laboratory, (iv) production of a variety
of secondary metabolites; and (v) compatibility with commonly used pesticides and other
biocontrol agents [28].
5.4. Effect of the Environment on Biocontrol Activity

Soil biological and environmental factors have a significant impact on the success
of biocontrol programs. Subsequently, soil biology research should identify the numer-
ous significant characteristics of various organisms, particularly in the plant rhizosphere,
by recognizing their relative contribution to the biocontrol process. Similarly, ecological
research should look into all biotic and abiotic elements that influence the BCAs as vi-
tal components of plant health [205]. Biological control of postharvest diseases (causing
losses up to 55%) through BCAs is a potentially sustainable control method. However,
in comparison to synthetic chemical pesticides, there is a significant lack of reliability in
field conditions. The biocontrol activity of BCAs has been improved by combining BCAs
application to nutrient additives, salts, edible coatings, or physical treatments, but with
only limited success. Complex microbial populations inhabit the fruit surface, which is
frequently resilient. It may be hard to build a BCA in such an environment. Integrating
the role of microbial communities in the formation of a BCA which is compatible with
other soil microbiome is a viable option for managing BCAs dependability in realistic
conditions. A microbial BCA is a complex metabolic phenotype that can be broken down
into many processes, all of which are maintained by a complex network function in the
soil. Combining BCAs application with a suitable complex biocontrol mix that includes
beneficial helper strains, essential macro, micronutrients, and biocontrol prebiotics may
aid the establishment of BCAs in the epiphytic microbial network. At the same time, it
could be able to attain biocontrol efficacy and consistency on par with synthetic chemical
pesticides. In addition, based on the literature, the time of beneficial microbial application
has been examined [206]. Sare et al. [206] proposed, for example, that shifting treatment
at the blooming stage (to establish a “path dependency”) be investigated for future fruit
and vegetable postharvest disease management. Other plant organs, such as seeds, could
benefit from this application moment shift [206]. In many cases, although plant-parasitic
nematodes (PPNs) had demonstrated potential susceptibility in laboratory or field plots,
satisfactory success was not attained [207]. Econem, a bionematicide containing in vitro
produced Pasteuria sp., was found to be ineffective in the management of a PPN, Belono-
laimus longicaudatus Rau, on golf course grass. In one of eight field experiments, the product
only reduced B. longicaudatus populations on a single sample [208]. Timper et al. [202]
highlighted examples of how agricultural practices can help or hinder the biological control
of PPNs and other soil-borne pests. The effectiveness of nematode antagonists can be
improved by supplying additional food sources, such as when organic amendments are
provided to the soil. Some organic additions, such as manures and plants that contain
allelopathic substances, can, however, be harmful to nematode antagonists. Crop rotation,
fallow periods, tillage, and pesticide sprays are all examples of production practices that
can disrupt antagonistic organism populations directly. By diminishing antagonists’ pri-
mary nematode host, these measures can have an indirect effect [202]. BCAs are being more
widely utilized to combat a variety of PPN pests, and they are a safer alternative to toxic
chemical nematicides. Despite this, due to their lack of efficacy, uneven field performance,
and/or negative economic circumstances, they have been confined to a limited sector of the
pesticide industry. A holistic understanding of soil biological and ecological components
can improve the efficacy and biocontrol success including improved sampling, a better
understanding of BCAs interactions with soil biota and ecology, cost-effective BCAs use,
genetic manipulation for better PPNs control, grower acceptance and awareness of BCAs
techniques, and commercial application [205].
6. Conclusions
The available data suggest that the assumption that biological control is always more
sustainable than chemical control is not always valid. However, concluding the existence of
unique features related to the plant pathogen or the BCA that could explain the BCA’s lack
of effectiveness in practice is insufficient. Briefly, to ensure BCAs’ long-term efficacy against
target plant diseases, it is critical to understand how their efficacy may be compromised.
This knowledge will lead to the identification of risk factors that will promote the selection
of plant pathogen strains that are resistant to BCAs. It will also lead to the identification of
BCAs with a lower threat of failure efficacy, i.e., BCAs with modes of action that do not favor
the selection of resistant isolates in natural plant pathogen populations [203]. Manipulation
of the environment, the use of mixtures of beneficial organisms, physiological and genetic
enhancement of the biocontrol mechanisms, formulation manipulation, and the integration
of biocontrol with other alternative methods that provide additive effects can all help to
improve the efficiency of these biocontrol products. These BCAs could be used effectively
in sustainable agriculture to boost plant growth [28]. To ensure effective biocontrol, it is
critical to choose agents that are effective in a variety of situations, including soil texture,
wetness, temperature extremes, and competition [31]. In addition to the aforementioned
factors, the need for scientists to publish negative data, as well as their ability to do so,
appears to be critical to the success of biological control. The researchers will be able to
determine biopesticides weaknesses such as lack of efficacy, uneven field performance,
and/or unfavorable economic considerations to tackling them in the future [209].
Author Contributions: Conceptualization, R.L. and E.A.B.; methodology, S.E., N.R., H.E.H., J.K.,
Z.B., R.L. and E.A.B.; software, E.A.B., S.E., N.R., Q.E., H.E.H. and Z.B.; validation, R.L. and E.A.B.;
data curation, S.E., N.R., J.K., H.E.H., Z.B. and R.L., resources, Z.B. and R.L.; writing—original draft
preparation, S.E., N.R., J.K., H.E.H., Z.B. and R.L.; writing—review and editing, R.L., Z.B. and E.A.B.;
supervision, R.L. and E.A.B.; project administration, E.A.B. and R.L. All authors have read and agreed
to the published version of the manuscript.
Funding: This Research was financially supported by MESRSI under the SIRAM project of PRIMA
Call Section 2.
Institutional Review Board Statement: Not applicable.
Informed Consent Statement: Not applicable.
Data Availability Statement: The data used for the analyses in this study are available within
the article.
Acknowledgments: This research was financially supported by Department of Plant Protection, Ecole
Nationale d’Agriculture de Meknes, and Unité de Recherche Résistance Induite et Bio-Protection des
Plantes-EA 4707, Université de Reims Cham-pagne-Ardenne.
Conflicts of Interest: The authors declare no conflict of interest.
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microorganisms

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Microorganisms 2022, 10, 596. https://doi.org/10.3390/microorganisms10030596 https://www.mdpi.com/journal/microorganisms

2. Rhizosphere as a Potential Reservoir of Biopesticides

2.1. Beneficial Bacteria

2.2. Fungi and Yeasts

3. BCAs Modes of Action

Figure 2. Possible modes of action of biological control agents.

3.1. Direct Mode of Action

3.1.1. Involvement of Lytic Enzymes

3.1.2. Antimicrobial Molecules

3.1.3. Biofilm Formation

3.1.4. Competition for Nutrients and Space

and metabolic activity of soil-borne pathogens. One siderophore compound in particular,

3.2. Indirect Mode of Action

3.2.1. Induced Resistance and Priming

Induced resistance offers the possibility of long-term and broad-spectrum disease

3.2.2. Implication of Phytohormones

4. Biological Control: Facing Reality

4.1. Biopesticide from Lab to the Field

4.2. Limited Number of Registered Products

Figure 4. The potential market of biopesticide.

of acceptable methods for large-scale manufacturing and appropriate technology to address

Table 1. Biopesticides, their trade names and targets [18,172–174,182,192–197].

Target Disease and/or Target

Target Disease and/or Target

B. Fungi and Yeast Biopesticides

Ampelomyces quisqualis Fruit, vegetable, and ornamental

Target Disease and/or Target

Target Disease and/or Target

Pome fruit, citrus, strawberry, stone

4.3. Legislative Procedures

5. Factors Affecting the Success/Failure of Biological Control of Plant Pathogens

5.1. Effect of the Plant on Biocontrol Activity

5.2. Effect of the Pathogen on Biocontrol Activity

5.3. Effect of the Biocontrol Agent on Biocontrol Activity

5.4. Effect of the Environment on Biocontrol Activity

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