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FRONTIERS IN PLANT SCIENCE


PLANT PATHOGEN INTERACTIONS

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Articles

EDITED BY

JESÚS MERCADO-BLANCO



Institute for Sustainable Agriculture, Spanish National Research Council, Spain

REVIEWED BY

LINDA THOMASHOW



Wheat Health, Genetics, and Quality Research, Agricultural Research Service,
United States Department of Agriculture, United States

GIANFRANCO ROMANAZZI



Marche Polytechnic University, Italy

CLARA PLIEGO PRIETO



Andalusian Institute for Research and Training in Agriculture, Fisheries, Food
and Ecological Production (IFAPA), Spain

The editor and reviewers' affiliations are the latest provided on their Loop
research profiles and may not reflect their situation at the time of review.

TABLE OF CONTENTS

 * * Abstract
   * Introduction: Microbial Biological Control Agents
   * Interaction Via Plant Metabolism: Induced Resistance and Priming
   * Indirect Interaction With Pathogens: Competition
   * Direct Interaction With Pathogens
   * Life Is More Complex: More Modes of Action and Mixed Modes of Action
   * Novel Biocontrol Approaches and Mode of Action
   * Synthesis and Future
   * Summary Points
   * Future Issues
   * Author Contributions
   * Funding
   * Conflict of Interest Statement
   * Acknowledgments
   * References




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Open Supplemental Data


REVIEW ARTICLE

Front. Plant Sci., 19 July 2019 | https://doi.org/10.3389/fpls.2019.00845


MODE OF ACTION OF MICROBIAL BIOLOGICAL CONTROL AGENTS AGAINST PLANT DISEASES:
RELEVANCE BEYOND EFFICACY

Jürgen Köhl1*, Rogier Kolnaar2 and Willem J. Ravensberg3
 * 1Wageningen Plant Research, Wageningen University & Research, Wageningen,
   Netherlands
 * 2Linge Agroconsultancy B.V., Oosterhout, Netherlands
 * 3Koppert Biological Systems, Berkel en Rodenrijs, Netherlands

Microbial biological control agents (MBCAs) are applied to crops for biological
control of plant pathogens where they act via a range of modes of action. Some
MBCAs interact with plants by inducing resistance or priming plants without any
direct interaction with the targeted pathogen. Other MBCAs act via nutrient
competition or other mechanisms modulating the growth conditions for the
pathogen. Antagonists acting through hyperparasitism and antibiosis are directly
interfering with the pathogen. Such interactions are highly regulated cascades
of metabolic events, often combining different modes of action. Compounds
involved such as signaling compounds, enzymes and other interfering metabolites
are produced in situ at low concentrations during interaction. The potential of
microorganisms to produce such a compound in vitro does not necessarily
correlate with their in situ antagonism. Understanding the mode of action of
MBCAs is essential to achieve optimum disease control. Also understanding the
mode of action is important to be able to characterize possible risks for humans
or the environment and risks for resistance development against the MBCA.
Preferences for certain modes of action for an envisaged application of a MBCA
also have impact on the screening methods used to select new microbials.
Screening of MBCAs in bioassays on plants or plant tissues has the advantage
that MBCAs with multiple modes of action and their combinations potentially can
be detected whereas simplified assays on nutrient media strongly bias the
selection toward in vitro production of antimicrobial metabolites which may not
be responsible for in situ antagonism. Risks assessments for MBCAs are relevant
if they contain antimicrobial metabolites at effective concentration in the
product. However, in most cases antimicrobial metabolites are produced by
antagonists directly on the spot where the targeted organism is harmful. Such
ubiquitous metabolites involved in natural, complex, highly regulated
interactions between microbial cells and/or plants are not relevant for risk
assessments. Currently, risks of microbial metabolites involved in antagonistic
modes of action are often assessed similar to assessments of single molecule
fungicides. The nature of the mode of action of antagonists requires a
rethinking of data requirements for the registration of MBCAs.




INTRODUCTION: MICROBIAL BIOLOGICAL CONTROL AGENTS

Biological control of plant diseases is the suppression of populations of plant
pathogens by living organisms (Heimpel and Mills, 2017). Amongst beneficial
microorganisms isolates can be selected which are highly effective against
pathogens and can be multiplied on artificial media. Application of such
selected and mass produced antagonists in high densities once or several times
during a growing season is called “augmentative biological control” (Eilenberg
et al., 2001; Heimpel and Mills, 2017; van Lenteren et al., 2018). For
commercial augmentative biological control of diseases, growers use MBCAs
containing living microorganisms, that are registered plant protection products
produced by biocontrol companies. In some cases, antimicrobial metabolites
produced by selected microbial organisms are included in the product, and some
products even contain only antimicrobial metabolites without living cells of the
antagonist (Glare et al., 2012). Legally speaking these compounds are considered
chemical actives in the EU. Also mycoviruses and bacteriophages can be potential
MBCAs against plant pathogens. In Australia, Brazil, Canada, Europe, Japan, New
Zealand, and United States a total of 101 MBCAs has been registered in 2017 for
disease control (van Lenteren et al., 2018).

Microbial biological control agents protect crops from damage by diseases via
different modes of action (Figure 1). They may induce resistance or prime
enhanced resistance against infections by a pathogen in plant tissues without
direct antagonistic interaction with the pathogen (Pieterse et al., 2014;
Conrath et al., 2015). Another indirect interaction with pathogens is
competition for nutrients and space (Spadaro and Droby, 2016). MBCAs may also
interact directly with the pathogen by hyperparasitism or antibiosis.
Hyperparasites invade and kill mycelium, spores, and resting structures of
fungal pathogens and cells of bacterial pathogens (Ghorbanpour et al., 2018).
Production of antimicrobial secondary metabolites with inhibiting effects
against pathogens is another direct mode of action (Raaijmakers and Mazzola,
2012). Low amounts of in situ secreted secondary metabolites support antagonists
to gain a competitive advantage. In some cases, biocontrol agents have been
selected which secrete already efficient secondary metabolites into the growth
media during mass production that are applied together with or without living
cells of antagonists in the biological control product.


FIGURE 1

Figure 1. Microbial biological control agent (MBCA) temporally interacting in
situ with the targeted pathogen activating different modes of action in cascades
of events.




Pathogen populations thus can be limited by antagonistic microorganisms in very
different ways. The nature of the mode(s) of action does not only determine how
a pathogen population is affected by the antagonist. Also the characteristics of
the MBCA depend on the exploited mode of action. Possible risks for humans or
the environment, risks for resistance development against the biocontrol agent,
its pathogen specificity and its dependency on environmental conditions and crop
physiology may differ between different modes of action. Preferences for certain
modes of action for an envisaged application of a biocontrol agent will also
have impact on the screening methods used to select new antagonists (Köhl et
al., 2011).

The objective of this paper is to review modes of action of microorganisms used
to control plant diseases, building on recent detailed reviews on mechanisms of
microorganisms antagonistic to plant pathogens (Compant et al., 2005; Lugtenberg
and Kamilova, 2009; Raaijmakers and Mazzola, 2012; Pieterse et al., 2014;
Conrath et al., 2015; Massart et al., 2015b; Spadaro and Droby, 2016; Karlsson
et al., 2017; Mauch-Mani et al., 2017; Ghorbanpour et al., 2018; and various
reviews on specific biocontrol microorganisms listed by Lugtenberg et al., 2013)
with emphasis on implications for screening techniques, risk assessments and
practical use. This review does not include biological control of invertebrates
(van Lenteren et al., 2018) or weeds nor the incorporation of genes into
antagonists or plants to enhance biocontrol efficiency and the risk assessment
of such genetically modified organisms potentially used in biological control of
plant diseases (Timms-Wilson et al., 2005).


INTERACTION VIA PLANT METABOLISM: INDUCED RESISTANCE AND PRIMING

Plants defend themselves with a broad variety of physical and chemical
mechanisms against pathogens. Enhancing resistance is one of the most potential
agronomic strategies to prevent biotic losses in crops. Constitutive mechanisms
such as cuticles are complemented by inducible resistance mechanisms. Induced
plant defense mechanisms are triggered by stimuli recognized by specific
recognition receptors. Typical recognized stimuli are PAMPs which induce defense
pathways in the plant to increase host resistance against the recognized
attacking pathogen. Resistance can be induced locally in the attacked tissue or
spread via signaling through the plant or even to neighboring plants resulting
in SAR. This type of induced resistance is a direct reaction to the stimulus of
necrotizing pathogens (Conrath et al., 2015). Another type of induced resistance
is ISR where the enhanced defensive capacity of the whole plant to multiple
pathogens is induced by beneficial microbes (Conrath et al., 2015). Both types
of induced resistance decrease again in absence of the stimulus. Different to
induction of resistance, priming of plants by stimuli leads to the sensitization
for enhanced defense not only in the presence of the stimulus but also to a long
lasting system of faster or stronger defense mechanisms in the future
(Mauch-Mani et al., 2017). Even transgenerational priming has been reported
(Conrath et al., 2015).

Resistance inducing stimuli produced by microorganisms are called MAMPs. These
specific molecular signatures are recognized by corresponding extracellular
PRRs. After signal reception early responding molecular mechanisms can be
measured in the plant cells within a few minutes (Boller and Felix, 2009). The
typical pathways of plants induced by MAMPs resulting in ISR have been studied
in details and recently been reviewed by Pieterse et al. (2014). For priming,
Conrath et al. (2015) and Mauch-Mani et al. (2017) recently reviewed compounds
that are induced during the priming phase and molecular mechanisms that are
present in plants in the post-challenge primed state.

Induced defense mechanisms involve the production of reactive oxygen species,
phytoalexins, phenolic compounds, or pathogenesis-related proteins or the
formation of physical barriers like modifications of cell walls and cuticles by
the induced plant (Wiesel et al., 2014). These metabolic activities are
energy-dependent so that long-lasting stimuli will result in energy costs for
the plant to maintain induced defense mechanisms active. In contrast to directly
induced resistance, priming of defense allows plants to react to stimuli later
in a fast and robust manner with lower energy costs (Mauch-Mani et al., 2017).
Stimuli inducing resistance and priming may be released from specifically
selected MBCAs. However, plants are exposed also to stimuli from other origins,
e.g., from pathogenic fungi or bacteria, herbivores, or abiotic stresses
(Mauch-Mani et al., 2017). It is thus likely that crops grown in the field
environment are frequently exposed to such stimuli inducing certain resistance
levels so that applications of resistance inducing MBCAs may not result in
additional induction of the resistance.

Plant defense reactions stimulated by MBCAs depend upon the plant genotype and
are not considered in toxicological and eco-toxicological risk assessments of
the MBCAs. Besides the general assessment of potential pathogenicity of the
MBCAs for humans, animals, or plants, the released stimuli as acting mechanisms
may be assessed for their toxicological and eco-toxicological risks. There is a
broad range of potential MAMPs acting as stimuli, formerly called elicitors,
belonging to very different groups of compounds (Boller and Felix, 2009). In
their review, various pairs of MAMPS and the related PRRs are discussed but it
is stated that many more MAMPs must exist in nature which have not been
identified yet. The most studied MAMPs are the bacterial proteins flagellin and
elongation factor EF-Tu. For both proteins the receptor systems are known and
reactions are induced by subnanomolar concentrations. Other examples for MAMPs
with known receptors are glucan, chitin, xylan, e.g., produced by Phytophthora
megasperma and Trichoderma viride. Boller and Felix (2009) list many other MAMPs
with still unknown receptors such as proteinaceous MAMPs from bacteria, e.g.,
superoxide dismutase and 23-amino-acid peptide, or from oomycetes, e.g., pep13
trans-glutaminase, sterol-binding elicitins, and cellulose-binding lectins;
lipophylic MAMPs, e.g., ergosterol and arachidonic acid; oligosaccharide MAMPs,
e.g., N-glycosylated yeast peptides, cell wall components from glucan and chitin
and peptidoglycans; and lipooligosaccharides produced by gram-negative bacteria.
Common to all signaling compounds is that they induce reactions in the plant
cells when present at millimolar to subnanomolar levels (Boller and Felix,
2009).

Also siderophores produced by iron-competing bacteria (see below), antibiotics,
such as DAPG and pyocyanin, biosurfactants and VOCs, such as 2R,3R-butanediol
produced by B. subtilis GB03 (130) and a C13 volatile emitted by Paenibacillus
polymyxa can act as elicitors inducing ISR (Pieterse et al., 2014). Mild viruses
can also function as elicitors in plants. This principle is used for the
biological control of Pepino mosaic virus in tomato which relies on mild
variants of the Pepino mosaic virus (Schenk et al., 2010; European Food Safety
Authority [EFSA], 2017). Products containing one or a combination of two mild
virus isolates are commercially available.

There is very limited information on the background amounts of MAMPs in the
environment or quantitative data on amounts produced by MBCAs after release in
the environment. Persistent compounds that may be present at higher
concentrations in the environment cannot have a signaling function in the
interplay between microorganisms and plants that is strictly regulated in order
to minimize resource expenditure and fine-tune signaling cascades (Mhlongo et
al., 2018). It can thus be assumed that microorganisms including MBCAs release
low amounts of such signaling compounds and that the compounds are exposed to
rapid microbial and abiotic degradation processes so that concentrations in the
environment generally are very low. It is expected that new approaches in
“signalomics” based on metabolomics and metametabolomics will highlight the
complex chemical communication within microbiomes and between microbiomes and
plants including the contribution of signaling by released MBCAs to the
continuous chemical cross-talk between organisms in the environment (Mhlongo et
al., 2018).

There is no selection pressure on the pathogen via resistance inducing MBCAs
themselves or by released signaling compounds because they do not interact
directly with the pathogen. The development of resistance against induction of
resistance is thus not likely (Romanazzi et al., 2016). However, the induced
resistance itself causes selection pressure on the pathogen and pathogens may
overcome induced resistance mechanisms (Bardin et al., 2015). This risk depends
much on the evolutionary potential of the pathogen. Pathogens with mixed
reproduction system, high potential for genotype flow, large effective
population sizes and high mutation rates break plant resistance easier compared
to pathogens with strict asexual reproduction, low potential for gene flow,
small effective population sizes, and low mutation rates (McDonald and Linde,
2002).

Screening for MBCAs with high potential in induction of resistance is complex
because the mode of action depends on a sequence of events from establishment of
the MBCA, release of signaling compounds, induction of a cascade of metabolic
events to induce plant defense mechanisms and the response of the pathogen to
this defense mechanism. The final outcome of the biocontrol mechanism depends
thus on the growing conditions for the MBCA on the plant, the physiology of the
plant, the genetics of the chosen cultivar and the conditions for pathogen
germination and infection. The final outcome of interactions of potential MBCAs
with the plant and of the induced plant with the pathogen has to be quantified
under standardized conditions, preferably under a range of environmental
conditions and using different representative host genotypes. Screening systems
can be simplified to allow high throughput screening by focusing in a first
screening cycle on the selection of MAMPs with potential to induce known
pathways in target host plants or even in cells of a model plant. For example,
the expression of 28 genes involved in complementary plant defense mechanisms
was measured in leaves of apple seedlings treated with potential resistance
inducers (Dugé De Bernonville et al., 2014). Results correlated strongly with
efficacies achieved with the tested inducers against Erwinia amylovora in trials
under controlled conditions. In a comparable study, Badosa et al. (2017) used
measurements of hydrogen peroxide production in tobacco cells in a first
screening followed by measurements of expression of defense genes in tomato in a
second screening. Selected inducers were effective in pear plants against E.
amylovora.

In conclusion, using MBCAs for disease control through induction of resistance
or priming relies on a complex sequence of events where the MBCA initially has
to establish on the host, followed by the release of specific inducers which are
recognized by specific receptors by the plant and thereafter triggering pathways
within the host plant resulting in the onset of defense reactions or priming to
make the plant ready for later challenges by pathogens. The first part of this
sequence of events depends on the MBCA, the second part on the genetics and
physiological status of the plant. MBCAs have to be selected in complex
bioassays (Table 1) that are not standardized (Romanazzi et al., 2016). MAMPs
released by the MBCA may be recognized by a specific plant species or a broader
range of plant species so that one or several host pathogen combinations may be
suppressed or controlled by the MBCA. The biocontrol effect depends also on the
active colonization of the plant surface by the MBCA, which thus need
appropriate ecological competence, and on the physiology of the plant, which
needs the potential to develop a sufficiently high level of resistance. The MBCA
induces defense reactions of the plant through a broad variety of signaling
compounds which are low molecular weight compounds, in some cases breakdown
products of cell walls. They are commonly produced by microorganisms at very low
millimolar to subnanomolar concentrations for chemical communication between
microorganisms and with plants. These processes are ubiquitous in the
environment and common wherever different microorganisms coexist with plants. It
can thus be assumed that the production of signaling compounds by applied MBCAs
will pose very low toxicological or eco-toxicological risks (Table 2) and
generally do not warrant a risk assessment.


TABLE 1

Table 1. Modes of action in relation to development and use of microbial
biological control agents.



TABLE 2

Table 2. Modes of action in relation to risk assessment and registration of
microbial biological control agents.





INDIRECT INTERACTION WITH PATHOGENS: COMPETITION

Germination and growth of plant pathogens depend on nutrient uptake. Obligate
biotrophic pathogens use exclusively nutrients from infected living host cells
and do not depend on any exogenous nutrient sources in the environment outside
the host plant (Agrios, 2005). The majority of plant pathogens exploit nutrient
sources in a much less specific way by degrading dead organic plant matter.
Necrotrophic plant pathogenic bacteria, fungi and oomycetes kill and
subsequently invade tissues of host plants and utilize the available nutrients
as primary colonizers of these killed tissues. Once necrosis has been induced by
the pathogen, non-pathogenic microorganisms with saprophytic life style can
potentially also colonize necrotic tissues so that a saprophytic competitive
substrate colonization between different populations is common resulting in
competition for nutrients and space. The principle competitive advantage of
necrotrophic pathogens is that they are the first colonizers directly after they
incited cell death. Non-pathogenic saprophytic endophytes being latently present
in attacked host tissue may have a similar competitive advantage and may play an
important role in competitive substrate colonization, e.g., of leaf lesions
caused by necrotrophic pathogens.

The interaction with the host by killing and invading host tissue leading to
damage in the infected crop is the most recognized stage of the life cycle of
necrotrophic pathogens. However, most pathogen populations have another life
style during significant parts of their life cycle when they live as saprophytes
on necrotic plant tissues in soil, on crop residues, residues of non-hosts and
on plant surfaces. During this stage pathogen populations survive, grow and
spread independently of the host. The biology of such pathogens differs between
species, some can develop completely independent from the host, other pathogen
species will complete their life cycle only in the presence of the host during
specific stages of their life cycle. Common for all necrotrophic pathogens
during their saprophytic stage is that they depend on exogenous nutrients
available in the environment, e.g., in colonized necrotic residues of host and
non-host tissues. Successful host infection of most fungal necrotrophic
pathogens also depends on exogenous nutrients during spore germination and
formation of infection structures on host tissues (Chou and Preece, 1968;
Fokkema et al., 1983). Also bacterial pathogens often depend on exogenous
nutrients for multiplication to reach population levels sufficiently high to
attack host tissues.

This dependency on exogenous nutrients during significant parts of their life
cycle makes non-biotrophic pathogens vulnerable to nutrient competition (Köhl
and Fokkema, 1998). Consequently, highly competitive microorganisms are
potential candidates for biological control using competition for nutrients and
space as mode of action. To exploit this mode of action in disease control,
detailed knowledge on the epidemiology is essential to identify stages where
limitations of nutrients and space will affect pathogen development. Typical
situations are the presence of free nutrients in wounds of fruits which
stimulate infection by various fruit pathogens, the presence of senescent floral
tissues stimulating flower infection by Botrytis cinerea and the presence of
dead host tissues on which the primary inoculum of pathogens is produced (Köhl
et al., 1995; Calvo-Garrido et al., 2014; Spadaro and Droby, 2016). Potential
competitive MBCAs must be able to occupy such niches, to survive and to consume
rapidly nutrient sources essential for pathogen infection such as sugars, pollen
and plant exudates on plant surfaces and in plant residues so that outcompeted
pathogens will not be able to infect the host. The pathogen population will
decline but will not be killed by the antagonist.

An example of use of this efficient mode of action is wound protection of fruits
from pathogen invasion by fast colonizing yeasts (Spadaro and Droby, 2016).
Yeasts as single cell organisms are able to multiply rapidly under favorable
conditions in wounds of fruits which are rich in nutrient supply. They can
consume a broad range of carbohydrates such as disaccharides and monosaccharides
but also various nitrogen sources (Spadaro et al., 2010). Spadaro and Droby
(2016) reviewed competition processes between antagonistic Pichia guilliermondii
and pathogenic Penicillium digitatum, P. expansum, B. cinerea, or Colletotrichum
spp. in wounds of different fruits and Aureobasidium pullulans and P. expansum
in apple wounds. Competition for carbohydrates in the carbohydrate rich wound
environment in combination with competition for the limited amounts of nitrogen
sources such as amino acids play the key roles in the antagonistic interactions
(for references: see Spadaro and Droby, 2016).

Besides carbohydrates and nitrogen sources, restricted iron availability due to
the low solubility of Fe3+ ions can be a limiting factor for microbial growth.
Many microorganisms can produce a variety of low-molecular-weight siderophores
with a high affinity for ferric iron (van Loon, 2000). Microbial strains with
the ability to produce high amounts of siderophores with high affinity to iron
play an important role in disease suppression and can be selected for biological
control through competition for iron with pathogens that produce lesser amounts
of siderophores with lower affinity for iron (Bakker et al., 1993; van Loon,
2000; Whipps, 2001; Lugtenberg and Kamilova, 2009). This mechanism has been
investigated in particular for isolates of Pseudomonas spp. and it has been
demonstrated that siderophore mediated iron competition result in reduced
pathogen populations in rhizospheres (Raaijmakers et al., 1995). Iron
competition is also the mode of action of several fungal antagonists. For
example, Trichoderma asperellum producing iron-binding siderophores controls
Fusarium wilt (Segarra et al., 2010). The yeast Metschnikowia pulcherrima
transforms pulcherriminic acid and iron ions to the red pigment pulcherrimin.
This process leads to iron depletion in media inhibiting development of B.
cinerea, A. alternata, and P. expansum (Saravanakumar et al., 2008).

Microbial biological control agents can also be targeted at the saprophytic
stage of necrotrophic pathogens to outcompete the pathogen so that primary
inoculum production on necrotic plant tissues is reduced or infections routes
via senesced tissues to healthy tissues are blocked (Köhl and Fokkema, 1998).
Examples are the application of Microsphaeropsis ochracea controlling Venturia
inaequalis in apple (Carisse et al., 2000), Clonostachys rosea controlling B.
cinerea in roses (Morandi et al., 2003), and Ulocladium atrum controlling
Botrytis spp. in various crops. In cyclamen, colonization of senesced leaves by
the pathogen is an essential step toward mycelial infections of attached healthy
tissues. The antagonist can outcompete B. cinerea on senesced cyclamen leaves so
that this infection route is blocked, resulting in disease control efficacy as
obtained with conventional fungicides (Köhl et al., 1998). Resource capture by
antagonists with resource competition as sole mechanism depends very much on the
level of available nutrients and timing and distribution of the antagonist at
the starting point of the interaction with the pathogen. A spatially explicit
model has been developed by Kessel et al. (2005) which describes spatial and
temporal competitive substrate colonization by U. atrum and B. cinerea under
different simulated conditions. Such models can be applied to better understand
effects of timing, densities and distributions on the outcome of competitive
substrate colonization and thus to optimize biological control.

Competitive antagonists are usually screened for efficacy in bioassay systems
under controlled conditions, e.g., on wounded fruit, seedlings, or necrotic host
tissues. Since rapid growth and substrate colonization are of key importance
during competition, these assays should be completed with screening assays for
selection of antagonist with superior ecological competence (Köhl et al., 2011).
If the most limiting nutrient source is already known for the envisaged
antagonist, more simplified high throughput systems may be applied, e.g.,
competition assays with candidate antagonists and pathogen on nutrient media
with limiting amounts of the identified nutrient source. Several methods have
been applied to understand better the underlying processes during nutrient
competition. Phenotypic microarray plates with different carbon and nitrogen
sources can give first insights in the potential for competition between
pathogen and antagonist using a nutritional similarity index. To unravel the
mode of action of A. pullulans strains against Monilinia laxa in wound
protection of peaches, Di Francesco et al. (2017) incubated A. pullulans and M.
laxa in in vitro assays in peach juice. HPLC analysis of the growth medium
revealed that specifically depletion of asparagine as nitrogen source restricts
growth of M. laxa. In similar studies with a different strain of A. pullulans
selected for protection of apple fruit from P. expansum, Janisiewicz et al.
(2000) found that depletion of aspartic acid serine and glutamic acid in apple
juice restricted the pathogen. Filonow (1998) used radiolabeled glucose to study
its utilization by antagonistic yeasts and B. cinerea.

Competitive antagonists may modulate growth conditions for the pathogen in the
targeted niche not only through nutrient depletion but also by other mechanisms.
Application of Bacillus brevis resulted in fast drying of leaf surfaces and
reduced B. cinerea by 68% similar to the application of a standard fungicide in
Chinese cabbage (Seddon and Edwards, 1993). Modulating leaf wetness periods by
antagonists, e.g., via secretion of biosurfactants, may be a powerful mode of
action for prevention of leaf diseases and diseases in stored products without
any direct interaction between pathogen and antagonist. A. pullulans strains
antagonistic to Erwinia amylovora causing fire blight in pome fruit strongly
reduce growth of the bacterial pathogen through shifting the pH of the medium
down to pH 4.0 (Kunz, 2006). Acidifying of the growth substrate may be an
additional mode of action supporting antagonists during competition with
bacterial pathogens.

Pathogen populations are continuously exposed to environmental stresses such as
extreme temperatures, drought, limiting nutrient availability, and sub-optimal
pH values so that they are selected and adapted to environmental stresses common
in their micro-habitat. Modulations of environmental stresses resulting in
reduced nutrient availability by highly competitive MBCAs or in moderate changes
of the pH or shortening of leaf wetness periods as demonstrated for A. pullulans
(Kunz, 2006) and Bacillus brevis (Seddon and Edwards, 1993), respectively, will
not add significant additional selection pressure on pathogen populations so
that a build-up of resistance cannot be expected. Biological control using
nutrient competition as mode of action works through the local and temporal
increase of highly competitive biocontrol strains during defined critical
development stages of the pathogens life cycles. Applied antagonists modulate
growth conditions in the targeted niche making condition less favorable for
pathogen development without any direct interaction with the pathogen. The
antagonists produce enzymes to degrade complex organic matter, simple
carbohydrates or amino acids or produce siderophores in case of competition for
iron. These principle processes are basic for the ubiquitous saprophytic
activities of microorganisms during utilization and decomposition of organic
matter of plants or microorganisms, which are the fundamental processes to
maintain nutrient cycling and plant growth in ecosystems. Different from plant
pathogens, saprophytic fungi cannot colonize whole plants or fruits abundantly
or cause spoilage. Consequently, it can be concluded that exploiting such common
and essential processes in biological control will not cause environmental
risks.

In conclusion, antagonists with nutrient competition as mode of action can be
selected using adequate bioassays (Table 1). They often have a broader host
range since modulation of environmental conditions in a micro-niche potentially
affects various less competitive pathogens. Active metabolism and growth are
essential for niche colonization and nutrient depletion. Thus, the efficacy of
MBCAs strongly depend on their ecological competences. The risk for development
of resistance against competition by pathogens can be considered as very low.
Nutrient competition acts through enzyme activities and other mechanisms to bind
limiting nutrients. Since these processes are ubiquitous in the environment and
common wherever saprophytic microorganisms competitively colonize micro-niches,
toxicological and eco-toxicological risks of adding nutrient competing
antagonists to ecosystems can be considered as very low (Table 2), and do not
generally warrant a risk assessment.


DIRECT INTERACTION WITH PATHOGENS


HYPERPARASITISM

Parasitism is the direct competitive interaction between two organisms in which
one organism is gaining nutrients from the other. If the host is also a
parasite, e.g., a plant pathogen, the interaction is defined as hyperparasitism.
This kind of interaction is often observed between fungi. For bacteria,
hyperparasitism rarely has been reported. Bdellovibrio bacteriovorus is a
predatory bacterium which has the unusual property to use cytoplasm of other
Gram-negative bacteria as nutrients (McNeely et al., 2017). In initial research
on biological control, B. bacteriovorus was tested in liquid cocultures with
phytopathogenic bacteria belonging to the Burkholderia cepacia complex. Specific
strains of B. bacteriovorus predated a broad host panel of the pathogen complex.
Predation by B. bacteriovorus strains of other plant pathogenic bacteria such as
Agrobacterium tumefaciens, Xanthomonas vesicatoria, X. campestris pv.
campestris, Erwinia carotovora pv. carotovora, Pseudomonas syringae pv.
glycinea, P. syringae pv. tomato, P. marginalis, and Erwinia herbicola was
confirmed in similar tests (McNeely et al., 2017).

In biotrophic mycoparasitism, the hyperparasite depends on the living host
fungus and gains nutrients from the host cells via haustoria without killing the
host. Host and mycoparasitic fungus interact in a stable and balanced way
(Jeffries, 1995). These often species–specific interactions may be important
components in disease suppressiveness in ecosystems but are hardly to be
exploited for commercial augmentative biocontrol because mass production of the
hyperparasite depends on living host mycelium as substrate. Hyperparasites with
a necrotrophic life style gain nutrients from dead host cells but also from
other commonly available organic matter which allow mass production on
artificial media making this group of hyperparasites much more favorable for
commercial use as MBCA compared to biotrophic hyperparasites. Necrotrophic
hyperparasites invade host spores or hyphal cells after killing such cells. Main
mechanisms of parasitism is the excretion of CWDEs combined in some cases with
excretion of secondary metabolites in close contact with the host cell leading
to openings in the cell wall and subsequent disorganization of the cytoplasm.
Cell wall degradation is typically caused by a range of chitinases,
β-1,3-glucanases and proteases or, in case of hyperparasites of oomycota,
cellulases. Such a necrotrophic hyperparasitism with invasion of killed host
cells is frequently observed by microscopy and electron microscopy. The assumed
nutrient transfer from the dead host cell to the invading fungus often has not
been proven because it is technically difficult to investigate such processes,
especially in the in situ situation in the field (Jeffries, 1995).

For some pathogen groups, researchers thoroughly investigated the phenomenon of
hyperparasitism and found many antagonistic fungal species. For example, 30
hyperparasitic species against Rhizoctonia solani belonging to 16 genera have
been reported by Jeffries (1995). Obligate biotrophic pathogens have been of
particular interest for biocontrol using hyperparasites. Hijmegen and Buchenauer
(1984) report on eight hyperparasitic species of powdery mildews. Zheng et al.
(2017) report on approximately 30 fungal species which show hyperparasitism
against rust pathogens, including Cladosporium uredinicola against Puccinia
violae (Traquair et al., 1984) and Alternaria alternata against Puccinia
striiformis f. sp. tritici (Zheng et al., 2017). In bioassays on rust-inoculated
wheat seedlings, A. alternata germ tubes contacted with and penetrated into
urediniospores of the pathogen at 24 hpi, and caused complete urediniospore
collapse at 36–48 hpi.

The most studied mycoparasites are belonging to the genera Trichoderma and
Clonostachys. Antagonistic isolates of these genera vary in host range and
individual strains mostly have a range of plant pathogenic hosts. They produce
structures for attachment and infection, and kill their hosts by CWDEs, often in
combination with antimicrobial secondary metabolites (Harman et al., 2004;
Harman, 2006; Mukherjee et al., 2012; Karlsson et al., 2017; Nygren et al.,
2018). These lytic enzymes are not constitutive but their production is
triggered by complex signaling after recognition of the host. Surface compounds
such as lectins from the host cell wall, surface properties and diffusible
host-released secondary metabolites play important roles in the recognition and
signaling pathways such as MAPK cascades, cAMP pathway and G-protein signaling
(Karlsson et al., 2017). Recognition of the fungal host then leads to
transcriptional reprogramming and expression of the “molecular weapons” involved
in host attack and lysis, including certain CWDEs. Mycoparasitism-related gene
families in Trichoderma such as ech42 and prb1 are upregulated during
mycoparasitism. As result of the initial activities of CWDEs, oligosaccharides
and oligopeptides are released by the host that are then recognized by
Trichoderma receptors and thereby act as inducers (Karlsson et al., 2017). This
attack by a necrotrophic mycoparasite results in further increase of
permeability and degradation of host cell walls and death of the host. A
synergistic transcription of various genes involved in cell wall degradation was
also reported for Trichoderma atroviride in interaction with B. cinerea and
Phytophthora capsici (Reithner et al., 2011).

Screening for hyperparasitic strains often is done by using host structures as
baits, especially if such structures are large for easy handling and
observations. Sclerotia, e.g., of Sclerotinia sclerotiorum, microsclerotia,
e.g., of R. solani, individual urediniospores or pustules of rust pathogens, and
individual conidia or pustules of powdery mildew have been exposed to potential
antagonist candidates and macroscopical and microscopical observations were made
to find strains which invade the host structures, often accompanied with
discoloration of these structures. Such studies generally are completed by
assessments of the viability of invaded host structures, e.g., Zheng et al.
(2017) confirmed that the viability of urediniospores from A. alternata treated
pustules was only 25% whereas 80% of spores from untreated rust pustules were
viable.

Alternative screening of candidate antagonists for their activity of fungal
CWDEs under in vitro conditions seems to be less adequate because activity
levels of single enzymes in situations without interaction with the hosts will
not be representative for the highly regulated interplay between antagonist and
pathogen. In these interactions, different enzymes are secreted in subsequent
events, regulated by signaling by different secondary metabolites (Karlsson et
al., 2017). Furthermore, isolates selected for high constitutive enzyme
production may not be strong competitors in competitive environments because
they continuously invest into formation of metabolites which are needed only for
their function in the particular situations of antagonism in close contact with
the host. Due to this high complexity of a hyperparasitism, which often is a
cascade of events, all depending on each other and leading to ultimate cell
death only after activating the whole cascade, screening assays should not focus
in a simplified way on single events, such as formation of a single enzyme, but
should measure the final results of the entire cascade of events.

Enzymes such as CWDEs are complex proteins consisting of several 100 or 1000
amino acids with the function to catalyze the conversion of specific substrates
into specific products. Functioning of enzymes depends not only on amino acid
sequences but also on their complex tertiary structures (Iyer and
Ananthanarayan, 2008). Unfolding of these structure or disordered polypeptides
lead to enzyme denaturation and irreversible loss of the enzymatic activity.
Enzymes are sensitive to physical denaturation, e.g., by heat or cold
temperatures, chemical denaturation by various factors from acids to chelating
agents and to microbial denaturation, e.g., by proteases. The generally high
sensitivity of enzymes to denaturation is a main obstacle in technological
processes so that enzyme stabilization during production and application is
common in technological applications. Proteases, cellulases, lipases, amylases,
and other enzymes are produced at industrial scales by microorganisms and are
commonly used in paper processing, food manufacture, medical device cleaning,
ethanol manufacture, as well as many common household cleaning processes such as
laundry and dishwashing (Anonymous, 2002). Enzymes used for such technical
applications have been tested through many years and it has been proven that
enzymes have a very safe toxicological profile with a good record of
occupational health and safety for the consumer. Studies revealed that enzymes
seem unlikely to be dangerous to the aquatic environment due to their ready
biodegradability and the low effects on aquatic organisms observed (Anonymous,
2002).

Cell wall-degrading enzymes are commonly produced in the environment by
microorganisms during decomposition of organic matter originating from dead
plant tissues and dead microorganisms including dead fungal hyphae, and
continuously play an essential role in nutrient cycling in all ecosystems. Given
this background activity of enzymatic CWDEs in natural ecosystems, application
of hyperparasites in biological control will not significantly increase cell
wall degrading activities in the environment. Hyperparasites produce low amounts
of fungal CWDEs during short time periods locally in micro-niches when they
interact with their hosts. The produced low amounts of chitinases,
β-1,3-glucanases and proteases present in the environment very locally during
short time periods are substrate-specific and highly sensitive to denaturation
in the environment with its usually high microbial activity combined with
chemical and physical factors enhancing enzyme denaturation. In conclusions
relevant toxicological and ecotoxicological risks of hyperparasite applications
can be considered as very low because activities are highly specific, production
is restricted in time and space and rapid denaturation is common.

The development of resistance by a plant pathogen against hyperparasitism by a
biological control agents has not yet been reported. Pathogens can develop
resting structures such as endospores, chlamydospores, and melanised sclerotia
with high resistance against hyperparasitism by naturally occurring antagonistic
microorganisms (Bardin et al., 2015). Pathogens can also repress synthesis of
enzymes needed by the antagonist for hyperparasitic interactions. A considerable
variation in susceptibility of S. sclerotiorum to the commercially applied
hyperparasite Coniothyrium minitans has been observed in different regions in
France (Nicot et al., 2016). Sclerotia produced by the various strains of S.
sclerotiorum differed in average thickness and thickness of their melanised
cortical tissue. However, both morphological traits did not correlate with
susceptibility to hyperparasitism by C. minitans (Nicot et al., 2018). With the
background of continuous selection pressure by hyperparasites present in the
natural microbiome it is not likely that a temporal increase of this pressure by
an antagonist application will enhance resistance of the pathogen.

In conclusion, antagonists with hyperparasitism as mode of action can be
selected using adequate bioassays (Table 1). They generally have a narrow host
range and their activity depends on environmental conditions because their
antagonistic activity depends on active growth. The risk for development of
resistance against hyperparasites by pathogens can be considered as low.
Hyperparasitism acts through CWDEs which production is highly regulated by
signaling from the potential host pathogen. Since these enzymes, ubiquitously
produced in all ecosystems, are highly substrate specific and highly susceptible
to rapid degradation, toxicological and eco-toxicological risks can be
considered as very low (Table 2) and do not warrant a risk assessment.


ANTIBIOSIS BY ANTIMICROBIAL METABOLITES

Antimicrobial metabolites are secondary metabolites belonging to heterogeneous
groups of organic, low-molecular weight compounds produced by microorganisms
that are deleterious to the growth or metabolic activities of other
microorganisms (Thomashow et al., 1997). They are produced and released to the
environment in small quantities by many microorganisms. Huge numbers of known
antibiotics are produced by actinomycetes (8700 different antibiotics), bacteria
(2900) and fungi (4900) (Bérdy, 2005). Less than 1% of microscopically counted
bacteria can be cultured on usual culture media (Amann et al., 1995).
Approximately one-third of the bacterial divisions have no cultured
representatives and are known only through rRNA sequences (Clardy et al., 2006).
It thus can be assumed that the majority of antibiotics produced in situ in the
environment is still unknown (Raaijmakers and Mazzola, 2012). Microbial genome
analysis revealed huge numbers of cryptic antibiotic gene clusters encoding
still unknown antibiotics. Antimicrobial metabolites are often considered as the
most potent mode of action of microorganisms against competitors allowing
antibiotic producing microorganisms competitive advantages in resource-limited
environments (Raaijmakers and Mazzola, 2012). Production of antimicrobial
metabolites, mostly with broad-spectrum activity, has been reported for
biocontrol bacteria belonging to Agrobacterium, Bacillus, Pantoea, Pseudomonas,
Serratia, Stenotrophomonas, Streptomyces, and many other genera. In Bacillus,
especially lipopeptides such as iturin, surfactin, and fengycin have been
investigated (Ongena and Jacques, 2008), in Pseudomonas many antibiotic
metabolites such as DAPG, pyrrolnitrin and phenazine have been studied
(Raaijmakers and Mazzola, 2012). Many antibiotics are produced only when a
microbial population reaches certain thresholds. This quorum-sensing phenomenon
is well described for phenazine-producing Pseudomonas. Genomic information
reveals that also these genera have the potential to produce many still unknown
secondary metabolites with possible antimicrobial activity. Also fungal
antagonists can produce antimicrobial compounds. For Trichoderma and closely
related Clonostachys (former Gliocladium), 6-PAP, gliovirin, gliotoxin, viridin
and many more compounds with antimicrobial activity have been investigated
(Ghorbanpour et al., 2018). Microorganisms producing antimicrobial metabolites
with the potential to interfere with antibiotics in human and veterinary
medicine must be excluded from use as MBCAs (Anonymous, 2013a).

The inhibitory effect of secondary metabolites on spore germination or hyphal
growth of pathogens can be quantified in vitro on nutrient media testing the
effects of the antagonistic microorganisms cultured in dual cultures, their
metabolites as present in supernatants of cultures of these microorganisms or
the purified concentrations of the metabolite. In vitro assays are used since
the early beginning of scientific research on microbial antagonists, e.g., by
Dennis and Webster (1971a, b). Studying inhibitory effects of potential
antagonists on agar or in liquid media in dual cultures has several advantages.
Assays are fast, resource efficient, highly reproducible and effects are easily
to be quantified by measuring colony sizes or percentages of germinated spores.
The resulting inhibition zones visualize clearly biocontrol effects and are
often used to explain the principles of biocontrol. These advantages may also
have led to a bias in biocontrol research. Screening of new antagonists often
starts with using in vitro assays which are very suitable to detect in vitro
antagonists which act via antimicrobial metabolites in the artificial
environment. This leads to an overestimation of the importance of this mode of
action in comparison to other mechanisms which cannot be detected in such in
vitro assays. As a biased result, in self-fulfilling prophecy, in vitro assays
may confirm the importance of in vitro antibiosis in biocontrol by
systematically excluding other modes of action.

The main disadvantage of in vitro dual cultures is that production of secondary
metabolites depends on nutrient concentration and composition of the chosen
medium. Common nutrient media are approximately 100 times richer in nutrients
compared to the rhizosphere, and bulk soils are even much less rich in
nutrients, supporting even 10–1000 times less bacteria than the rhizosphere
(Lugtenberg et al., 2017). Consequently, amounts of secondary metabolites in in
vitro systems are much higher than reached in natural habitats. Furthermore,
agar media or liquid media are ideal for diffusion of the antibiotic compounds
which is not the case in habitats such as soil or leaf surfaces. Several studies
demonstrated that in vitro antagonism does not predict antagonism in complex
assays including host plants which simulate the natural habitat situation under
controlled or even in field situations (Knudsen et al., 1997). An example is the
screening of Trichoderma isolates for their potential to control R. solani. Köhl
(1989) tested 256 isolates belonging to T. viride, T. hamatum, T. harzianum, or
T. koningii in dual cultures with R. solani and in pot experiments with lambs
lettuce seeds planted in R. solani infested soil. Dual cultures on yeast
dextrose agar revealed 192 antagonistic isolates. For these isolates, the
average efficacy in reduction of damping off in the pot experiments was 61.2%.
For the remaining 64 isolates, showing no in vitro antagonism, the average
efficacy in pot experiments was similar with 59.7%. This example demonstrates
that in vitro antagonism depends on the chosen conditions and by far does not
explain the antagonistic potential of isolates. Also recent transcriptomic
studies confirm that in vitro produced metabolites may not be expressed or play
a minor role in situ (Koch et al., 2018).

Antibiosis observed on agar plates historically resulted in the development of
pharmaceutical antibiotics. With similar expectations, results of agar plates
often are translated to the control of plant pathogens in the field situation
with antimicrobial metabolites seen as sole mode of action against competitors.
There is very limited information on measured antimicrobial effects of
antagonists in situ compared to the large number of publications of in vitro
effects. Transcriptome analyses of microbial activities in soil confirms that
antimicrobial metabolites are produced in soil. Raaijmakers and Mazzola (2012)
listed results of various authors who quantified different antibiotics produced
in situ in soils by bacterial strains introduced at high densities. Production
of 5 ng to 5 μg per gram of soil or plant tissue were reported depending on
experimental conditions, strains used and type of produced antibiotic with
exceptional higher values up to 180 μg per gram for a Bacillus subtilis isolate.
Antibiotic concentration may be higher in certain microniches, but an important
fraction of the antibiotics may be bound to the producing cells and may not
diffuse in the habitat (Raaijmakers and Mazzola, 2012). Antibiotics are not
stable in the soil environment. Arseneault and Filion (2017) report on half-life
of antibiotics produced by biocontrol strains in soil ranging between 0.25 and 5
days depending on biocontrol strain, antibiotic and experimental conditions.
Such short life spans can be due to microbial decomposition but also to chemical
and physical inactivation. Information on in situ concentration of antimicrobial
metabolites produced by MBCAs against plant disease and their life span is
hardly to be quantified and therefore often missing and not included in risk
assessments on non-target effects (Mudgal et al., 2013).

Despite the low concentrations, the inhomogeneous distribution and short
lifespan of antimicrobial compounds produced by biocontrol strains in situ,
studies with mutants of biocontrol strains disrupted in specific antibiotic
synthesis demonstrated that antibiotic metabolites play an important role in
microbial interactions in soil and plant surfaces (Handelsman and Stabb, 1996;
Raaijmakers and Mazzola, 2012). There is increasing evidence that antimicrobial
metabolites have important functions for the producing microorganisms at
subinhibitory concentrations. In other words: such compounds are characterized
as being antibiotic because of their effect on microorganisms at high
concentration under in vitro conditions although their function in the natural
habitat is very different at the prevailing lower concentrations. Arseneault and
Filion (2017) discuss modulation of gene expression by low antibiotic
concentrations instead of inciting of cell death at high concentrations.
Antibiotics at low concentrations can be involved in signaling and microbial
community interactions, communication with plants, and regulation of biofilm
formation. Raaijmakers and Mazzola (2012) discussed a range of functions of
antimicrobial metabolites at low concentrations: there is evidence that
antimicrobials including lipopetides protect bacteria from grazing by
bacteriovorus nematodes such as Caenorhabditis elegans. Also volatile antibiotic
compounds may play a role in long-distance interactions amongst soil organisms
including bacterial predators. Lipopeptides of Bacillus and Pseudomonas are
involved in the surface attachment of bacterial cells and biofilm formation by
activating signaling cascades finally resulting in the formation of
extracellular matrices which protect microorganisms from adverse environmental
stresses. Some antibiotics, especially lipopeptides support the mobility of
bacteria, most likely via changing the viscosity of the colonized surfaces.
Surface-active antibiotics allow bacteria to move to nutrient rich locations and
also change the water dynamics on leaf surfaces which indirectly affects
pathogen development. Other groups of antibiotics influence the nutritional
status of plants. For example, DAPG-producing Pseudomonas upregulates the
nitrogen fixation by plant growth-promoting Azospirillum brasilense, and
redox-active antibiotics support mobilization of limiting nutrients such as
manganese and iron.

Screening of new antagonists acting through antimicrobial metabolites needs to
address the insights in ecological functioning of such compounds. Efficient
antagonists produce antimicrobial metabolites in situ in microniches at
sufficiently high concentrations to gain advantage over competitors or at low
concentration to fulfill various functions like signaling or nutrient
mobilizations, thus functions different from antibiosis. As for most other modes
of action, the design of adequate bioassays is essential which combine
interactions between potential antagonist, pathogen, plant and are conducted
under representative environmental conditions regarding soil environment and
microclimate. The often applied in vitro screening by far does not mimic the
real conditions under which antagonists should be active. However, screening
under in vitro conditions for strong producers of potential antimicrobial
compounds is the first method if the exploitation of the metabolites is
envisaged. Antimicrobial metabolites can be produced by selected isolates of
antagonistic bacteria or fungi in bioreactors in fermentation processes
optimized for high yield of the preferred metabolite. Commercial biological
control products may contain microbial metabolites as active ingredient together
with the producing microbial antagonist so that after application the direct
effect of the metabolite is combined with the potential production of additional
metabolite in situ. Other products may contain only the produced metabolites,
possibly in combination with remains of dead cells of the producing antagonist.
Such a use of microbial metabolites is strictly speaking outside the scientific
definition of biological control which is defined as the use of living
beneficial organisms to suppress populations of plant pathogens (Heimpel and
Mills, 2017), but in a broader definition, use of metabolites is also considered
as biological control (Glare et al., 2012).

Several reports demonstrate variability within pathogen populations in their
sensitivity to antimicrobial secondary metabolites. Selected isolates of
Pseudomonas spp. produce DAPG with antimicrobial activity against several plant
pathogens. A high diversity in sensitivity to DAPG between isolates for
Gaeumannomyces graminis var. tritici has been reported by Mazzola et al. (1995)
and for B. cinerea by Schouten et al. (2008). Isolates of B. cinerea also differ
in sensitivity to pyrrolnitrin (Ajouz et al., 2011). These examples indicate
that selection pressure by broad use of biological control agents with a single
antimicrobial secondary metabolite as mode of action may result in the selection
of less sensitive pathogen strains so that the efficacy of the MBCA will not be
durable. For B. cinerea, a pathogen with high potential to develop resistance
against chemical fungicides through adaptation, adaptation to antimicrobial
compounds produced by MBCAs has been found (Li and Leifert, 1994). A similar
adaptation to pyrrolnitrin, produced by P. chlororaphis, was developed by
strains of B. cinerea in in vitro assays with increasing concentrations of the
antimicrobial compound in agar growth media (Ajouz et al., 2010). Interestingly,
the build-up of resistance resulted in reduced fitness of the strains so that
such strains will not persist in absence of selection pressure by pyrrolnitrin.
Pathogen strains with higher resistance against antimicrobial compound produced
by MBCAs are able to excrete such compounds, e.g., by ABC transporters, degrade
the antimicrobial compounds or interfere with the biosynthesis of the compounds
by antagonists (Bardin et al., 2015). Since selection pressure depends on dose
and exposure duration, the risk for building up resistance is lower if the
antimicrobial compounds are produced by the antagonist in situ only during
direct interaction with the pathogen, often even at subinhibitory
concentrations, compared to situations were formulated antimicrobial compounds
produced by antagonists already during fermentation are applied at higher dose
to the entire crop.

Risk assessments are required for registration of MBCAs as plant protection
products for antimicrobial metabolites which are considered as relevant
(Anonymous, 2011). Plant pathogenic microorganisms potentially producing
mycotoxins and human and animal pathogens potentially producing neurotoxins are
excluded from use in biological control. Other secondary metabolites with proven
antimicrobial activity which are produced by MBCAs in bioreactors and applied as
formulated bioactive compounds included in the end product in amounts effective
in disease control (Glare et al., 2012) are relevant metabolites which need to
be assessed for potential toxicological and eco-toxicological risks. In addition
to the risk assessment performed for the MBCA, a “chemical” risk assessment may
be needed for relevant metabolites if they are stable, active without the
microorganism, produced at relevant concentrations and present in the MBCA. If
such metabolites potentially are produced in vitro, but not present in the MBCA
or only at low concentration, they are not relevant for risk assessment
(Sachana, 2018). However, for the majority of MBCAs, antimicrobial metabolites
are produced at low concentrations in situ in microniches with low nutrient
availability. Concentrations are subinhibitory if modes of action different from
antibiosis are exploited (Raaijmakers and Mazzola, 2012). In other situations,
metabolite production may be locally and temporally above a minimal inhibitory
concentration resulting in inhibition or killing of the targeted pathogen. Such
an antibiosis will be restricted in time because of the short life span of
antimicrobial metabolites in the environment. Furthermore, the producing
antagonist populations will drop after application (Scheepmaker and van de
Kassteele, 2011). There is a continuum of microbial activity including
production of a great variety of secondary metabolites in the natural
environment. Rough estimation of population densities show that even at the
moment of application of a MBCA its contribution to the total microbial activity
in a given niche is far below 1% (Koch et al., 2018; Lugtenberg, 2018).
Unlimited growth of applied saprophytic microorganisms, often a fear of
regulators, will not occur in the environment where saprophytic microbial
populations are regulated by competitive exploitation of limited resources.
Thus, applications of MBCAs with potential in situ production of antimicrobial
metabolites will not add relevant toxicological or eco-toxicological risks to
the cropping system.

In conclusion, antagonists with antimicrobial metabolites as mode of action can
be selected using adequate bioassays if in situ production by living antagonists
is envisaged or in vitro if the application of the formulated metabolites is
envisaged (Table 1). They generally have a broad host range and their activity
depends on environmental conditions if their antagonistic activity depends on in
situ production, thus on active growth. The risk for development of resistance
against antimicrobial metabolites by pathogens can be considered as low in cases
where metabolites are produced in situ. In cases where a single formulated
microbial metabolite is applied on crops, the risk of development of resistance
will be, depending on the genetics of the targeted pathogen and the stability of
the metabolite in the environment, comparable to risk for chemical active
substances. Because of the low concentrations of in situ produced antimicrobial
metabolites in microniches with low nutrient availability in combination with
the typically short lifespans of the metabolites in the environment and the
presence of antimicrobial metabolites produced by indigenous microorganisms,
toxicological and eco-toxicological risks can be considered as low. If
formulated metabolites are applied, their toxicological and eco-toxicological
risks are determined by their toxicological profile, the applied concentration
and their stability in the environment (Table 2).


LIFE IS MORE COMPLEX: MORE MODES OF ACTION AND MIXED MODES OF ACTION

The research on mode of action of MBCAs usually focuses on induced resistance
and priming, competition, hyperparasitism, and antibiosis, but more modes of
action are known. For example, fungal viruses in the family Hypoviridae are used
to induce hypovirulence in Cryphonectria parasitica, the causing agent of
chestnut blight (Milgroom and Cortesi, 2004; Double et al., 2018). Other
antagonists act via the inactivation of enzymes involved in pathogen infections
(Elad, 2000, see below) or the enzymatic degradation of pathogen structures such
as a lectin needed by the rice blast pathogen Magnaporthe oryzae for spore
attachment on the host leaf surface which can be degraded by a specifically
selected isolate of Chryseobacterium sp. (Ikeda et al., 2013). It can be
expected that employing multi-omics will identify many still undetected ways of
interactions between microorganisms. It is also known that secondary metabolites
and other compounds produced by MBCAs can act through different modes of action.
For example, DAPG can have a direct effect as antimicrobial metabolite against
the pathogen but also acts as MAMP (Pieterse et al., 2014). Thus, both
antibiosis and induced resistance act simultaneously and an artificial
separation between the in situ effect of DAPG on a single mode of action is
hardly possible. Another example is the production of iron-binding siderophores
for nutrient competition with the pathogen that are also recognized by the
plants as MAMPs inducing resistance (Höfte and Bakker, 2007).

The systematic discrimination of the modes of action of MBCAs is a scientific
exercise to unravel how MBCAs act. This information is important for optimizing
the use of MBCAs but also asked for registration where the mode of action has to
be indicated (Anonymous, 2013a). However, nature of microbial interactions is
more complex and does not fit into such pragmatic categories of scientists,
regulators, and risk managers. In many cases where the mode of action
intensively has been studied for a single biocontrol strain, results confirm
that antagonistic interactions are driven by more than one mode of action.
Separation into different modes of action is also not always clear and seems to
be artificial. For example, Trichoderma spp. produce hydrolytic enzymes that
permeabilize and degrade the fungal cell wall as one of the key steps in the
successful attack of the fungal hosts (Karlsson et al., 2017). The increased
permeability of the cell wall is facilitating the subsequent entry of secondary
antimicrobial metabolites.

Isolate T39 of Trichoderma harzianum, originally selected for the control of B.
cinerea, also controls the foliar pathogens Pseudoperonospora cubensis, S.
sclerotiorum, and Sphaerotheca fusca (Elad, 2000). Isolates of antagonistic
Trichoderma spp. are generally known to produce antimicrobial metabolites and to
act via hyperparasitism (Harman et al., 2004). Detailed studies on T. harzianum
T39 revealed that no antimicrobial metabolites are interfering with the targeted
pathogens. The isolate is able to produce chitinases but Elad (2000) found no
correlation between the ability of this strain or other, non-antagonistic
strains of T. harzianum with their biocontrol activity. T. harzianum T39
produces several proteases in situ on bean leaves which restrain enzymes of B.
cinerea. The proteases reduced the activities of the pathogen enzymes exo- and
endopolygalacturonase, pectin methyl esterase, pectate lyase, chitinase,
β-1,3-glucanase, and cutinase, that are essential for the pathogen during host
infection. In experiments with protease inhibitors the biocontrol effect was
fully or partially nullified. The biocontrol effect of T. harzianum T39 can thus
partly be explained by the production of enzymes which suppress pathogen
enzymes. The other proven modes of action of T. harzianum T39 were nutrient
competition, ISR and locally induced resistance. Elad (2000) concluded that
various modes of action are responsible for the control of biotrophic and
necrotrophic foliar pathogens by T. harzianum T39 and he assumed that multiple
mechanisms are also involved in other biocontrol systems, but in most cases only
part of the possible mechanisms have been elucidated.

Pseudozyma flocculosa is an efficient antagonist of Erysiphales (Bélanger et
al., 2012) that does not penetrate powdery mildew cells but cause a rapid cell
death. P. flocculosa can produce 6-methyl-9-heptadecanoic acid and the
glycolipid flocculosin. Since there was no evidence for induced resistance in
treated plants and nutrient competition seemed to be unlikely in antagonism
against a biotrophic pathogen, it was concluded that antibiosis is the sole mode
of action. However, gene expression studies revealed that there was no
significant increase in expression of the relevant genes at any time during the
antagonistic process so that other modes of action must be responsible (Bélanger
et al., 2012). There is now increasing evidence that competition for the
micronutrients Zn and Mn plays a role during the dedicated tritrophic
interaction: powdery mildew takes up these elements from the host plant and P.
flocculosa draws these elements then from the pathogen.

Both examples of in depth investigations of the mode of action of MBCAs
illustrate that tritrophic interactions between host, pathogen and MBCA are
complex and often different from what is initially expected (Elad, 2000;
Bélanger et al., 2012). New, rather unexpected (combinations of) mechanisms may
be revealed by future analysis of the increasing genomic and transcriptomic
information. Current examples are studies on gene expression of Clonostachys
rosea (Nygren et al., 2018) and the genome analysis of Metschnikowia fructicola
(Piombo et al., 2018). The examples also illustrate that antagonists evolve a
great variety of (combinations of) mechanisms to interact with other
microorganism rather than rely on using a “single molecule approach” similar to
the approach of using synthetic fungicides. Such a highly regulated in situ
production of various ubiquitous mechanisms commonly used in the microbial
interplay in the environment makes the use of MBCAs a particular safe and
sustainable technology.

Because of the ubiquitous character of in situ modes of action specific risk
assessments are not relevant. Because of the complexity of the cascades of
physiological events the indication of the principal (single) mode of action as
data requirement of Commission Regulation 283 (Anonymous, 2013a; see Box 1) is
impossible.



Box 1. What are the data requirements and the uniform principles concerning the
mode of action of the microorganism against plant diseases in the EU?

The most important data requirements related to the mode of action of active
substances are set out in Commission Regulation (EU) No. 283/2013 (Anonymous,
2013a).

“The principal mode of action shall be indicated. (…)” If “the micro-organism
produces a toxin with a residual effect on the target organism (…), the mode of
action of this toxin shall be described.”

“If the plant protection action is known to be due to the residual effect of a
toxin/metabolite (…), a dossier for the toxin/metabolite has to be submitted
(…)”

“Any available information on the mechanism (…)” and “(…) the influence of the
produced metabolites on the micro-organism’s mode of action shall be provided.”

The data requirements for plant protection products (preparations) are set out
in Commission Regulation (EU) No. 284/2013 (Anonymous, 2013b).

“(…) the pest controlling action (fungitoxic, fungistatic action, nutrient
competition, etc.) must be stated.

It must also be stated whether or not the product is translocated in plants and,
where relevant, if such translocation is apoplastic, symplastic or both.”

The uniform principles for evaluation and authorisation of plant protection
products are set out in Commission Regulation (EU) No. 546/2011 (Anonymous,
2011).

The micro-organism in the plant protection product should ideally function as a
cell factory working directly on the spot where the target organism is harmful.
(…)

“Micro-organisms may produce a range of different metabolites (e.g., bacterial
toxins or mycotoxins) (…)” that “(…) may be involved in the mode of action of
the plant protection product. The characterization and identification of
relevant metabolites must be assessed and the toxicity of these metabolites must
be addressed. (…)

The mode of action of the micro-organism shall be evaluated in as much detail as
appropriate. The possible role of metabolites/toxins for the mode of action
shall be evaluated and (…) the minimal effective concentration (…) shall be
established. (…) Aspects to be considered in the evaluation, are:

(a) antibiosis;

(b) induction of plant resistance;

(c) interference with the virulence of a pathogenic target organism;

(d) endophytic growth;

(e) root colonization;

(f) competition of ecological niche (e.g., nutrients, habitats);

(g) parasitization;

(h) invertebrate pathogenicity.”

Mode of action is taken into account at evaluation of the degree of adverse
effects on the treated crop, operator exposure, viable residues, fate, and
behavior in the environment and at risk assessment of birds, mammals, aquatic
organisms, bees, arthropods other than bees and earthworms and nitrogen and
carbon mineralization in the soil.




NOVEL BIOCONTROL APPROACHES AND MODE OF ACTION

Microbial biological control agents interact with the plant, the targeted
pathogen and the resident microflora. Studies on the interactions with the
resident microflora have been hampered in the past because of limitations of
available methods. This changed drastically with arrival of Next Generation
Sequencing (NGS) methods such as metagenomics and metatranscriptomics allowing
to identify the composition and functions of the microbiome (Massart et al.,
2015a, 2015b). Further steps by adding information of metametabolomics and
signalomics (Mhlongo et al., 2018) will complete the picture on interactions
between introduced MBCAs and resident microbiota. As a result, a holistic in
depth understanding on MBCA-microbiota interactions will support better timing,
formulation and application of MBCAs and prevent failures. It is expected that
three new developments will have significant impact on biological control of
plant diseases. First, functional analysis will allow a “prebiotic approach”
(Massart et al., 2015a). Application of specific compounds or complex substrates
will modulate indigenous microbiota compositions with the aim to enhance
microbial suppression of plant pathogens (Mazzola and Freilich, 2017). Such a
manipulation of resident microbiota toward disease suppression may be comparable
to conservation biological control applied in insect pest control, e.g., via
improving nutrient availability for beneficial insect populations by planting
flower strips. Simple or complex substrates applied for such a prebiotic
approach may not be considered as plant protection products. A second approach
will be the selection and application of “helper” strains (Massart et al.,
2015a) which have no biocontrol properties on their own but support MBCAs in
establishment, survival and antagonistic activity in situ. A third expectation
is that core microbiomes will be designed (Gopal et al., 2013; Massart et al.,
2015a; Syed Ab Rahman et al., 2018) consisting of different strains of
biological control species combining various modes of action. Gopal et al.
(2013) stated that the transfer of tailor-made core-microbiomes will become a
viable strategy for plant disease management.

The ecological considerations supporting the idea of assembled consortia are
sound (Table 1). However, practical considerations may hamper their
introduction. Validation and optimization of in silico-designed consortia under
ranges of relevant environmental conditions will be complex and will need
substantial resources. In a commercial setting, development of mass production,
down streaming and storage procedures separately for each individual consortium
member will need substantially more resources and investments compared to
production of single strain MBCAs (Table 1). Registration of assembled consortia
as plant protection products will add further difficulties. Regulations in the
EU demand the risk assessment of each active ingredient before the product can
be registered. In case of assembled consortia, costs will thus increase
substantially. In this context, strategies to develop helper strains or to shape
the indigenous microbiota may clearly have advantages above the use of assembled
core consortia (Table 2). On the other hand, an adapted legislation for novel
disease control systems would benefit society as a whole as well as the
environment.


SYNTHESIS AND FUTURE

Microbial biological control agents use a broad arsenal of modes of action which
are used wherever microorganisms interact, communicate, and regulate their
co-existence between microbial cells and between microorganisms and plants. The
exploitation of different modes of action has different advantages and
disadvantages in relation to the development of commercial MBCAs by industries
and their practical use by growers (Table 1), but also regarding the perception
of possible toxicological and ecotoxicological risks for producers, users,
consumers, and the environment (Table 2). Studies on mode of action of
well-documented antagonists show that antagonism generally is not based on a
single action of a certain mode of action, but on a sequence of events with the
use of different modes of action over time. During such cascades of
physiological events signals often are the result of the earlier used modes of
action, e.g., cell wall degradation products after use of CWDEs (Karlsson et
al., 2017).

For the development of specific biocontrol products, certain modes of action may
be preferred. In such cases, screening of new MBCAs can be very focused, e.g.,
on selection of suitable MAMPS inducing resistance or priming the host plant,
utilization of a specific nutrient element or substrates, or the selection for a
potential antibiotic metabolite. This screening strategy may be powerful if new
strains are being selected superior to an existing, well characterized
antagonist or for further strain improvement within an existing antagonist
strain. However, in most other cases, selection procedures should be preferred
that allow the selection of new combinations of known and still unknown modes of
action which are produced directly at the site of interaction. This view on
preferable biocontrol mechanisms is expressed also in Commission Regulation (EU)
546/2011 (Anonymous, 2011) which states “that micro-organism in the plant
protection product should ideally function as a cell factory working directly on
the spot where the target organism is harmful.” For this objective, the overall
effect on pathogen and disease development has to be assessed rather than the
expression of a single expected main mechanism of action. The key challenge for
screening projects is thus the development of suitable robust bioassays which
combine the interactions between pathogen, host, and antagonist under controlled
conditions. Depending on disease characteristics and host, the design of such
assays can be troublesome and challenging, especially if “difficult biology” has
to be combined with the cost-effective testing of large numbers of candidate
antagonists. Attractive alternative routes via in vitro tests should not be used
to avoid biased selection with emphasis on one mode of action, thus excluding
many other powerful modes of action or combination thereof, which may be even
ineffective at all if evaluated on their own.

The efficacy of biological control agents against plant diseases may not be
durable because pathogen populations may develop resistance comparable to the
frequently observed build-up of resistance against chemical fungicides with a
single mode of action. Important factors for an erosion of effectiveness are
variation in susceptibility to the mode of action within the pathogen
population, selection pressure resulting in shifts within pathogen populations
toward less susceptible strains and the fitness of the selected strains in the
environment under conditions without selection pressure (Bardin et al., 2015).
For the choice of a certain mode of action, the kind of selection pressure needs
to be considered in respect to the pathogen’s evolutionary potential which
determine the ability to adapt to the selection pressure via selection within a
population with sufficient variation in susceptibility. Variation in
susceptibility of pathogens has been found for some pathogens such as S.
sclerotiorum and G. graminis var. tritici (Mazzola et al., 1995; Schouten et
al., 2008; Nicot et al., 2016). However, development of resistance has not
reported yet for commercially used biological control products for control of
plant diseases (Nicot et al., 2011). The risk of resistance development in MBCAs
used in sustainable IPM systems is also low because IPM combines a variety of
measures to prevent damage by diseases without relying on a single control
method.

The build-up of resistance is a serious problem in single molecule-single mode
of action chemical fungicides which shorten their economic life span. For MBCAs
the principle modes of action exhibit much less selection pressure on pathogens
additional to the always present selection pressure during natural competitive
interactions of organisms. Furthermore, it is common that a combination of
different modes of action are active and each mode of action is based on
multiple actors, e.g., different CWDEs, a set of different MAMPs or different
antimicrobial metabolites with sometimes very different signaling functions. For
MBCAs it thus can be concluded that build-up of resistance is much less likely
compared to the build-up of resistance against chemical plant protection
products. Only exceptional uses of MBCAs such as the use of in vitro produced
highly concentrated and purified secondary metabolites or the use of genetically
modified MBCAs with extraordinarily high expression of a single antimicrobial
metabolite may result in selection pressures comparable to single site
fungicides.

Knowledge of the mode of action of the microorganisms is required and has also
to be considered in the context of other potential risks before a MBCA can be
approved for use as plant protection product. Risk assessments of MBCAs are
regulated in the EU by Regulation (EC) No. 1107/2009 (Anonymous, 2009) and by
Commission Regulation (EC) No. 546/2011 (Anonymous, 2011) regarding the uniform
principles for evaluation and authorisation of plant protection products
containing microorganisms, by Commission Regulation (EU) No. 283/2013 in its
Part B on microorganisms including viruses (Anonymous, 2013a) setting out the
data requirements for active substances and by Commission Regulation (EU) No.
284/2013 in its Part B on microorganisms including viruses (Anonymous, 2013b)
setting out the data requirements for plant protection products (Box 1).

The regulations focus strongly on the possible risks of secondary metabolites
and toxins potentially produced by microorganisms. Several groups of fungi are
known to produce mycotoxins, several groups of bacteria are known to produce
toxins including the botulinum-neurotoxin (BoNT). Microorganisms producing such
mycotoxins or toxins in relevant amounts are excluded from the use in biological
control because of the potential risks for humans and animals. MBCAs may produce
other secondary metabolites as sole mode of action, or – in the majority of
cases – as component of a cascade of different secondary metabolites in
combination or alternation with other metabolites such as CWDEs or MAMPS. The
function of the produced metabolites often is not antibiosis but to fulfill
other functions including signaling at subinhibitory concentrations. Secondary
metabolite production is highly regulated and restricted to micro niches and in
time. Such metabolites are rapidly degraded and thus have short life spans in
the environment.

Only for MBCAs which produce potential antimicrobial metabolites in vitro or
during the mass production fermentation process and contain such metabolites in
the formulated end product at effective concentration, thorough risk assessment
is indicated and the minimal effective concentration against the target and
representative non-target organisms can be established. However, in all other
cases, such metabolites are not relevant for a risk assessment. Furthermore,
reliable quantification of temporal metabolite concentrations in microniches in
the in situ situation can hardly to be achieved. The perception of risks caused
by antimicrobial metabolites in biological control may be more a result of the
broad use of in vitro studies on antibiosis in biocontrol research rather than
the result of studies on on-site production of such metabolites in the
environment. In vitro antagonism can easily be visualized through inhibition
zones on culture media. The similar method is used for the screening of
pharmaceutical antibiotics that aims at the development of products containing
single molecules for medical treatments. Communication on biocontrol research
based on in vitro assays, showing inhibition zones, may create a wrong view on
the nature of biocontrol control resulting in the fear of the use of antibiotics
in crop protection. Since results of in vitro assays generally do not correlate
with results obtained in bioassays or with crops (Koch et al., 2018), there are
no reasons to rely on such artificial systems in studies on antagonist
screenings and in research on the function of microbiomes. Biocontrol research
unraveling the mechanisms in the much more complex in situ situations may reduce
the unjustified fears for microbial metabolites produced by MBCAs.

In conclusion, MBCAs are functioning directly on the spot where the targeted
organism is harmful (Anonymous, 2011), generally combining different modes of
action to highly regulated cascades of events. Current thinking on how to
consider the mode of action during the risk assessment and registration
procedure of MBCAs focuses on a single mode of action and potential risks of in
vitro produced metabolites, very similar to the risk assessment of synthetic
fungicides with a single compound as active ingredient. A rethinking is needed
considering that the effectiveness of MBCAs in most cases is based on natural,
complex, highly regulated interactions between microbial cells and plants on
site but are not the results of a single action of a single metabolite.
Toxicological and ecotoxicological risks of such complex processes of
interaction can be considered as very low. Moreover, humans and other organisms
have been and still are exposed to such processes in evolutionary terms and
adverse effects are not known. Since an antimicrobial action of a single
metabolite is not relevant in many cases, the existing EU regulations may
require such a rethinking in the registration of MBCAs as long as antimicrobial
metabolites are not present in the formulated MBCA at relevant concentrations.


SUMMARY POINTS

1. Microbial biological control agents use a great variety of mechanisms to
protect plants from pathogens.

2. Important modes of action strengthen the resistance of the plant, e.g.,
induced resistance or priming, or modulate the local growth conditions for
pathogen development, e.g., nutrient competition, but do not interfere directly
with the pathogen.

3. Hyperparasitism and secondary metabolites are directly affecting the targeted
pathogen via highly regulated cascades of physiological events but not by a
single constitutively produced metabolite.

4. Secondary metabolites produced in vitro may have antimicrobial activity at
high concentration but low amounts are produced in situ very locally during
interaction and metabolites have short life spans, often with functions such as
signaling, very different from antibiosis.

5. During the cascades of events a range of different compounds with different
modes of action are used to outcompete the pathogen. Such events of signaling
and interaction are common wherever microorganisms interact.

6. The highly regulated in situ production of ubiquitous mechanisms commonly
used in the microbial interplay makes the use of MBCAs a safe and sustainable
technology.

7. In situ produced compounds such as MAMPs, enzymes or secondary metabolites
are not relevant for risk assessments so that detailed toxicology and
ecotoxicological studies of these compounds are not relevant, and should not be
required.

8. The fear of antimicrobial metabolites produced by MBCAs after their release
is not based on real risks but fed by the wrong perception on how biocontrol
acts if studied under in vitro conditions.

9. If antimicrobial metabolites are the active ingredient in the formulation of
the biocontrol product, risk assessment of such metabolites is relevant.


FUTURE ISSUES

1. Better screening assays for finding the next generation of MBCAs are needed
to measure the overall effect of the interplay of different modes of action.

2. Multi-omics will help to further understand the complex events during
microbial interactions in the environment.

3. Current EU regulations on registration of MBCAs should allow a science-based
differentiation between the majority of compounds involved in modes of action to
be considered as safe and not relevant for detailed risk assessment and the
limited number of cases relevant for risk assessments where secondary
metabolites are present as active ingredients in MBCAs formulations at high
concentrations.


AUTHOR CONTRIBUTIONS

JK conceived and designed the research. JK, RK, and WR contributed to the
manuscript and revised it critically for important intellectual content. All
authors approved the final version of the manuscript.


FUNDING

This study was funded by the Dutch Ministry of Agriculture, Nature and Food
Quality (Programme Durable Plant Production, TKI-project 1406-130 project) with
contributions from Artemis, ‘s-Gravenzande, Netherlands, the International
Biocontrol Manufacturers’ Association (IBMA), Brussels, Belgium, and Linge
Agroconsultancy B.V., Oosterhout, Netherlands.


CONFLICT OF INTEREST STATEMENT

RK was employed by company Linge Agroconsultancy b.v. and WR was employed by
company Koppert Biological Systems. JK declares no competing interests.


ACKNOWLEDGMENTS

We thank the members of the greenTEAM of the Board for the Authorisation of
Plant Protection Products and Biocides (CTGB), Ede, Netherlands, for their
valuable comments on earlier versions of the manuscript.


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Keywords: biological control, plant diseases, mode of action, antagonist, risk
assessment, screening

Citation: Köhl J, Kolnaar R and Ravensberg WJ (2019) Mode of Action of Microbial
Biological Control Agents Against Plant Diseases: Relevance Beyond Efficacy.
Front. Plant Sci. 10:845. doi: 10.3389/fpls.2019.00845

Received: 18 February 2019; Accepted: 12 June 2019;
Published: 19 July 2019.

Edited by:

Jesús Mercado-Blanco, Instituto de Agricultura Sostenible (IAS), Spain

Reviewed by:

Linda Thomashow, Agricultural Research Service (USDA), United States
Gianfranco Romanazzi, Marche Polytechnic University, Italy
Clara Pliego Prieto, Andalusian Institute for Research and Training in
Agriculture, Fisheries, Food and Ecological Production (IFAPA), Spain

Copyright © 2019 Köhl, Kolnaar and Ravensberg. This is an open-access article
distributed under the terms of the Creative Commons Attribution License (CC BY).
The use, distribution or reproduction in other forums is permitted, provided the
original author(s) and the copyright owner(s) are credited and that the original
publication in this journal is cited, in accordance with accepted academic
practice. No use, distribution or reproduction is permitted which does not
comply with these terms.

*Correspondence: Jürgen Köhl, jurgen.kohl@wur.nl



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