Biological Control 144 (2020) 104240

Contents lists available at ScienceDirect

Biological Control
journal homepage: www.elsevier.com/locate/ybcon

Screening for novel biocontrol agents applicable in plant disease T

management – A review
Katrijn Raymaekersa,b,1, Lisa Poneta,b,1, Dominique Holtappelsc, Barbara Berckmansa,b,
Bruno P.A. Cammuea,b,⁎
a
Centre of Microbial and Plant Genetics, KU Leuven, Heverlee, Belgium
b
VIB Center for Plant Systems Biology, Ghent, Belgium
c
Laboratory of Gene Technology, KU Leuven, Heverlee, Belgium

ARTICLE INFO ABSTRACT

Keywords: In current plant disease control, the divergence from chemical pesticide use has been fueled by the public
Biological control concern about toxicity and environmental impact of these products, the development of pesticide-resistant pa-
Antagonism thogens, a rising ban of existing pesticides and, a decreased registration of new ones. Due its high potential as
Induced resistance alternative/complement to these pesticides, biological disease control is now generally recognized and constitute
Marker-based screening
an important tool in integrated pest management (IPM). However, one of the factors hampering the large-scale
Phenotype-based screening
implementation of biocontrol is the lack of efficient, commercially available biocontrol agents (BCAs). The
Microbial plant diseases
identification of novel BCAs thus forms a critical step in the development of commercial biocontrol products and
requires rapid and robust screening methods suitable to evaluate high numbers of candidate BCAs. In this re-
view, we present an overview and discussion of the screening systems reported to select novel BCAs for bio-
control of microbial plant diseases. Distinction is made between screenings for BCAs with direct antagonistic
effect on the pathogen (via e.g. parasitism, antibiosis, or competition) and BCAs that exert their biocontrol
activity indirectly by induction in the plant of an induced systemic resistance (ISR) to the pathogen. For both
types, we further discriminate between phenotype- and marker-based approaches, which evaluate directly the
intended phenotype (disease reduction) or the expression of a marker predictive for this phenotype, respectively.
Finally, we discuss the importance of the type and origin of candidate BCA collections as a significant de-
terminant of the screening success.

1. Introduction
In view of a further growing world population, estimated to reach
up to 9.7 billion people by 2050 (Godfray et al., 2010; United Nations,
2019), a significant increase in food supply is required. According to the
Food and Agriculture Organization of the United Nations (FAO), a
minimal raise of 50% of agricultural food production by that time is
needed in a scenario of modest economic growth (FAO, 2018, 2017),
while in other models even higher required increases up to 110%
(Savary et al., 2012; Tilman et al., 2011) are postulated. To cope with
this growing food demand, and given the limited availability of
additional agricultural area, most attention is given to raise the yield
per area and decrease yield losses (FAO, 2017; Mauser et al., 2015;
Pradhan et al., 2015). Regarding the latter, actual crop losses in the
field due to diseases or pests are estimated to globally range from 17 to
30 % for the five major crops being wheat, rice, maize, potato and
soybean (Savary et al., 2019). Therefore, further increasing the effi-
ciency of plant disease control is expected to significantly contribute to
the globally increased food demand (FAO, 2009; Valin et al., 2014).
Current plant disease management comprises in first instance non-
chemical measures, which are predominantly preventive and include
mainly cultural practices, such as the use of disease-resistant plant

Abbreviations: BCA, biocontrol agent; BTH, benzo(1,2,3)thiadiazole-7-carbothioic acid; ET, ethylene; INA, 2,6-dichloro-isonicotinic acid; IPM, integrated pest
management; ISR, induced systemic resistance; JA, jasmonic acid; MTP, microtiter plate; NCI, N-cyanomethyl-2-chloroisonicotinamide; PGPR, plant-growth-pro-
moting rhizobacterium; PR, pathogenesis-related; PR1, pathogenesis-related protein 1; ROS, reactive oxygen species; SA, salicylic acid; SAR, systemic acquired
resistance
⁎
Corresponding author: Centre of Microbial and Plant Genetics, Katholieke Universiteit Leuven, Kasteelpark Arenberg 20, 3001 Heverlee, Belgium.
E-mail address: bruno.cammue@kuleuven.vib.be (B.P.A. Cammue).
1
Katrijn Raymaekers and Lisa Ponet contributed equally to this work.

https://doi.org/10.1016/j.biocontrol.2020.104240
Received 30 September 2019; Received in revised form 21 February 2020; Accepted 26 February 2020
Available online 08 March 2020
1049-9644/ © 2020 Elsevier Inc. All rights reserved.
K. Raymaekers, et al.
cultivars and crop rotation (Barzman et al., 2015; Collinge et al., 2019).
Chemical pesticides, on the other hand, are used both for preventive as
well as for curative disease management (Collinge et al., 2019;
Lamichhane et al., 2016; Strange and Scott, 2005). They play a crucial
role in current agriculture regarding increased crop yield and quality,
improved food safety (e.g. regarding reduced microbial toxin content),
and enhanced shelf-life (Cooper and Dobson, 2007; Kawasaki and
Lichtenberg, 2015). Together with enhanced application of fertilizers,
irrigation, and selective breeding, the increased use of pesticides, re-
ported for example in the period 1960–1990 of up to 20-fold, led to a
doubling of crop production in that same period (Oerke, 2006), a trend
which keeps on going till today (FAO, 2019a). Despite their un-
doubtedly positive contribution to an efficient control of plant diseases
and pests, concerns about the negative effects of chemical pesticides on
human health, as well as about their negative impact on environment
including contamination of soil and water and toxicity to beneficial
organisms are raising (Carvalho, 2006; Damalas et al., 2011; Geiger
et al., 2010; Kim et al., 2017). In addition, intensive use of pesticides
resulted in the development of resistant pests and pathogens (Ash,
2018; Borel, 2017), as exemplified by neonicotinoid-resistant insects
and fungi insensitive to broad-spectrum strobilurin or azole fungicides
(Bass et al., 2015; Ma and Michailides, 2005; Price et al., 2015). This
led to a need for even higher application dosages and the urge for al-
ternative pesticides with different mode of action (Syed Ab Rahman
et al., 2018; van den Bosch et al., 2018). The availability and variety of
such novel pesticides are, however, currently limited and insufficient to
counteract the problem of increasing resistance development (Bruce
et al., 2017; Pertot et al., 2017). Moreover, the accessibility of efficient
pesticides is further declining due to an increasing legislation strin-
gency (Bruce et al., 2017). The introduction of directives such as 91/
414/EEC (European Parliament, 2009), for example, led to the with-
drawal of approximately 750 of 1,000 products during the period of
1993 and 2011 from the list of legally authorized active pesticide
substances, while only 180 new products were registered (Chapman,
2014).
Complementary to their more stringent rules on pesticides use, the
EU promotes low-pesticide-input disease management such as the
Integrated Pest Management (IPM) program implemented through
Directive 2009/128/EC (European Parliament, 2009). The FAO defines
IPM as: “The careful consideration of all available pest control techni-
ques and subsequent integration of appropriate measures that dis-
courage the development of pest populations and keep pesticides and
other interventions to levels that are economically justified and reduce
or minimize risks to human health and the environment. IPM empha-
sizes the growth of a healthy crop with the least possible disruption to
agro-ecosystems and encourages natural pest control mechanisms”
(FAO, 2019b). As such, the EU aims at a more sustainable way of plant
protection (Barzman et al., 2015; European Parliament, 2009; Robin
and Marchand, 2019) with a focus on prevention and biocontrol of
diseases and pests. As a consequence, the pathogen population, though
not fully eradicated, is intended to be kept low enough to avoid sig-
nificant symptom development (Stenberg, 2017). In its most stringent
definition, biocontrol means the use of naturally occurring (micro-)or-
ganisms to control plant diseases or pests (Eilenberg et al., 2001; Fravel,
2005). So-called biocontrol organisms are widely found in nature and
include bacteria, fungi, viruses, yeasts, and protozoans and can control
plant diseases either directly or indirectly (Köhl et al., 2019). The
former implies a direct antagonistic effect of the biocontrol organism on
the pathogen, which can be achieved by parasitism, antibiosis, or
competition for nutrients or infection sites. In the indirect way of dis-
ease control, the biocontrol organism induces plant-mediated responses
allowing the plant to react faster and more efficiently upon subsequent
pathogen attack (Vos et al., 2015). In a broader definition, biocontrol
has been suggested to also include the use of non-living agents of bio-
logical origin (Timmusk et al., 2017; Vinale et al., 2008), a definition
which is also supported by the International Biocontrol Manufacturers
2
Biological Control 144 (2020) 104240
Association (IBMA, 2019). In this review, we will apply an even more
extended definition of biocontrol thereby covering the use of biological
agents (e.g. organisms or their secreted compounds) and compounds
that trigger the plant’s own defense, and term them all as biocontrol
agents (BCAs).
The high potential of BCAs as plant protection tools in IPM is cur-
rently limited by different factors. Research on BCAs, including both
their identification and further unraveling of their mode of action, has
mainly focused on a single crop - disease/pest system in a bottom-up
approach, rather than using a more holistic top-down one (Barratt et al.,
2018; Mathys et al., 2012). Another hurdle is the stringent registration
procedure (including environmental risk assessments), characterized by
high costs (reported over $250 million per case) and long application
procedures (often more than ten years), restricting the commercial
implementation of newly identified BCAs (Holtappels et al., 2019).
Therefore, improvement of the entire process of identification, devel-
opment and, implementation of new BCA-based products is necessary
(Barratt et al., 2018; Berg, 2009; Lamichhane et al., 2017; Le Mire et al.,
2016; Regnault-Roger, 2012; Robin and Marchand, 2019). Interesting
reviews in that context have covered specific parts of the screening
process for BCAs, such as the criteria to consider during a screening for
commercial antagonists (Köhl et al., 2011), screenings for bacterial
BCAs against soilborne fungal pathogens (Pliego et al., 2011) and for
non-living BCAs for resistance induction in plants (Bektas and Eulgem,
2015).
The focus of the current review paper is to give a broader and up-
dated overview and discussion of different reported screening systems
for the identification of BCAs with direct and/or indirect biocontrol
activity against microbial diseases, thereby discussing the need for new
high-throughput screening systems (Lamichhane et al., 2017; Nicot
et al., 2012; Pliego et al., 2011). Selecting a suitable screening method
is in first instance determined by the desired biocontrol mechanism of
the BCA and the currently available scientific tools. Therefore, this re-
view is divided into two main parts covering screenings for BCAs ex-
hibiting direct and indirect biocontrol activity, respectively, and further
discriminating between phenotype- and marker-based approaches.
2. Screening for BCAs exhibiting direct biocontrol activity
Biocontrol of diseases by BCAs through direct pathogen antagonism
is mainly based on four mechanisms. The first one, parasitism, only
applies to living BCAs as it involves the interaction of two organisms, in
which the parasite (here the BCA) lives in or on the host (here the plant
pathogen), resulting in antagonistic effects on the latter. Such interac-
tions can include parasitism of viruses (bacteriophages) on bacteria
(Buttimer et al., 2017), of bacteria on fungi (mycophagy) (Purin and
Rillig, 2008), and of fungi on fungi (mycoparasitism) (Barnett, 1963).
The latter includes the well-known parasitism of plant-pathogenic fungi
by Trichoderma species (Benítez et al., 2004; Druzhinina et al., 2011;
Mukherjee et al., 2012). A second mechanism relies on BCA excretion of
antimicrobial compounds, including cell wall-degrading enzymes, such
as chitinases and glucanases; murein hydrolases; or a plethora of vo-
latile or non-volatile antibiotic metabolites, such as pyrones, buteno-
lides, iturin, terpenoids, siderophores, and peptaibols (Cao et al., 2018;
Mutawila et al., 2016; Vinale et al., 2008; Vollmer et al., 2008). A third
mechanism involves the secretion of enzymes by BCAs that interfere
with pathogenicity factors, such as pectinases and cutinases, thereby
reducing pathogen virulence (Bertagnolli et al., 1996; Elad and Kapat,
1999; Kapat et al., 1998; Zimand, 1996). Finally, BCAs can antagonize
pathogens through competition for nutrients or space on the plant
surfaces, leading to a reduced infection pressure of the pathogen
(Benítez et al., 2004). When screening for BCAs exhibiting such direct
biocontrol mechanisms, it is not always possible to distinguish between
these four mechanisms. In Table 1, an overview of different, reported
screenings for such BCAs is given, thereby discriminating in general
between phenotype- and marker-based screenings.
 Table 1
Examples of reported screenings for BCAs with direct biocontrol activity. Distinction is made based on screen type being phenotype-based (A), marker-based (C) and combined phenotype/marker-based (B) screenings.
Details given concern the primary screening used to make the first BCA selections. Only screenings on 10 or more candidate BCAs are considered here. NA: not applicable.
Test pathosystem Screened candidate BCAs Test setup Reference
K. Raymaekers, et al.

Target pathogen(s) Screening parameter Type Origin Size Medium Format

A. Phenotype-based screenings

Rhizoctonia solani pathogen growth Trichoderma spp. soil from sugar beet field diseased by R. solani 16 solid plate (Anees et al., 2010)
(+volatiles)
Sclerotinia sclerotiorum pathogen growth plant growth-promoting rhizosphere of wheat, maize, clover and 19 solid plate (Sun et al., 2017)
rhizobacteria (+culture alfalfa
filtrate)
Xanthomonas campestris pathogen growth Paenibacillus spp. Brassica hosts or soil 24 solid plate (Ghazalibiglar et al.,
2016)
X. campestris, Erwinia carotovora, Pseudomonas pathogen growth Bacillus spp. rhizosphere of cereals 25 solid plate (Földes et al., 2000)
syringea, Alternaria alternata, Botrytis allii, B.
cinerea, Colletotrichum lindemuthianum, Drechslera
sorokiniana, Fusarium oxysporum
Erwinia amylovora pathogen growth epiphytic bacteria and yeast leaf and blossom surface of healthy pear trees 22 solid plate (Sharifazizi et al.,
2017)
Ralstonia solanacearum pathogen growth endophytic bacteria healthy tomato stems 41 solid plate (Nawangsih et al.,
2011)
P. syringae, Xanthomonas arboricola, X. fragariae pathogen growth lactid acid bacteria collection of the Institute of Food and 55 solid plate (Daranas et al.,
Agricultural Technology and Center for 2019)
Innovation and Development of Plant Health,
Universitat de Girona, Girona, Spain
E. amylovora pathogen growth bacteria soil and flowers of fire blight host plants 61 solid plate (Ait Bahadou et al.,

3
2018)
Agrobacterium tumefaciens pathogen growth bacteria plant tumors or the rhizosphere of galled 70 solid plate (Tolba and Soliman,
plants 2013)
Penicillium digitatum pathogen growth yeasts citrus leaves, fruits and citrus-growing soils 71 solid plate (Liu et al., 2017)
Pythium ultimum pathogen growth actinomycete strains rhizosphere-associated and non-rhizosphere- 82 solid plate (Crawford et al.,
associated soils 1993)
Fusarium graminearum, F. culmorum pathogen growth endophytic fungi, yeast and inner tissues of wheat plants 86 solid plate (Comby et al., 2017)
bacteria
Geotrichum citri-aurantii pathogen growth yeasts citrus leaves, flowers, fruits and citrus- 95 solid plate (Ferraz et al., 2016)
growing soils
Phytophthora infestans pathogen growth bacteria rhizosphere or phyloplane samples 95 solid plate (Tomar et al., 2014)
Penicillium italicum pathogen growth yeasts citrus leaves, flowers, fruits and citrus- 97 solid plate (da Cunha et al.,
growing soils 2018)
R. solanacearum pathogen growth rhizobacteria rhizosphere of healthy and diseased tomato 110 solid plate (Huang et al., 2013)
plants
R. solanacearum pathogen growth rhizobacteria rhizopshere of potato, tomato, pepper, coffee 118 solid plate (Lemessa and Zeller,
and maize plants 2007)
F. oxysporum, F. graminearum, F. semitecum, F. solani, pathogen growth Pseudomonas spp. soil and rhizosphere of healthy crop plants 128 solid plate (Agaras et al., 2015)
F. verticilloides
Colletotrichum truncatum, C. graminicola,
Macrophomina phaseolina, Phomopsis sp.,
Cercospora sojina
Phytophthora capsici pathogen growth endophytic bacteria roots of black pepper from farms with P. 129 solid plate (Toh et al., 2016)
capsici infection history
Fusarium guttiforme, Chalara paradoxa pathogen growth plant extracts 63 plant species 131 solid plate (Sales et al., 2016)
F. oxysporum, X. campestris pathogen growth bacteria, actinobacteria and agro-industrial waste-based composts 135 solid plate (Suárez-Estrella
fungi et al., 2013)
(continued on next page)
Biological Control 144 (2020) 104240
 Table 1 (continued)

Test pathosystem Screened candidate BCAs Test setup Reference

Target pathogen(s) Screening parameter Type Origin Size Medium Format

K. Raymaekers, et al.

R. solanacearum pathogen growth endophytic and roots, stems and rhizosphere of various crops 149 solid plate (Ramesh and
rhizobacteria (+culture Phadke, 2012)
filtrate)
S. sclerotiorum, P. infestans, R. solani pathogen growth bacteria phylloplane and rhizosphere of potato and 200 solid plate (Daayf et al., 2003)
canola
Verticillium dahliae pathogen growth fungi endorhiza, rhizosphere and bulk soil of cotton 373 solid plate (Zheng et al., 2011)
plants
V. dahliae pathogen growth endorhizosphere bacteria root tips of tomato plants grown in solarized 435 solid plate (Tjamos et al., 2004)
soils
R. solani, F. oxysporum pathogen growth bacteria suppressive and non-suppressive soils 1,788 solid plate (Adesina et al.,
2007)
V. dahlia, R. solani pathogen growth endophytic and ectophytic rhizosphere, phyllosphere, endorhiza and 2,648 solid plate (Berg et al., 2005)
bacteria endosphere of potato plants
Dothistroma septosporum metabolic activity of the Trichoderma spp. and collection of the Bio-Protection Research 12 solid plate (McDougal et al.,
pathogen bacteria Centre, Lincoln University, Lincoln, New 2011)
Zealand
R. solani pathogen growth volatiles of Trichoderma and various soils and plant materials 84 solid plate (Dennis and
Gliocladium spp. Webster, 1971)
S. sclerotiorum pathogen growth volatiles of bacteria various, unspecified biological materials 197 solid plate (Fernando et al.,
including canola 2005)
Venturia inaequalis germination and germtube bacteria and yeast soil and different apple tissues in abandoned, 931 solid plate (Burr et al., 1996)
growth commercial or research orchards
F. solani, M. phaseolina, R. solani pathogen growth Rhizobia and Bradyrhizobia various institutional collections 21 (1) solid (1) plate (Omar and Abd-
spp. (2) liquid (2) flask Alla, 1998)

4
Plasmodiophora brassicae resting spore germination bacteria rhizosphere of tuber mustard 124 liquid tube (Luo et al., 2018)
Magnaporthe grisea conidial germination and culture filtrate of fungi and collection of the Korean Research Institute of 1,000 liquid GelBond (Oh and Lee, 2000)
appressorium formation actinomycetes Bioscience and Biotechnology, Taejon, Korea
Phaeomoniella chlamydospora (1) pathogen growth bacteria (+volatiles) grapevine wood tissue or grape berry surface 46 (1) solid (1) plate (Haidar et al.,
(2) stem necrosis of Bordeaux vineyards (2) plant (2) unspecified 2016b)
(grapevine): stem
cuttings
R. solani, Sclerotinia homoeocarpa (1) pathogen growth endophytic bacteria seeds, shoots and roots of 14 genotypes of the 190 (1) solid (1) plate (Shehata et al.,
(2) chlorosis severity corn grass family (2) plant (creeping (2) tube 2016)
bentgrass): adult
plants
P. digitatum pathogen growth yeasts fruits and leaves of citrus plants 10 plant (lemon): fruits tray (Perez et al., 2017)
Septoria tritici leaf necrosis endophytic fungi healthy leaves of wheat plants in areas of high 25 plant (wheat): pot (Latz et al., 2020)
S. tritici disease pressure seedlings
(1 + 2) B. cinerea (1) lesion development bacteria grapevine wood tissue or grape berry surface 46 plant (grapevine): (1) plate (Haidar et al.,
(2 + 3) Neofusicoccum parvum (2) rot severity (1) leaves (2) box 2016a)
(3) canker formation and (2) berries (3) unspecified
stem necrosis (3) stem cuttings
P. digitatum pathogen growth yeasts and Bacillus spp. fruit surface of papaya and citrus varieties 152 plant (orange): fruits box (Abraham et al.,
2010)
Plasmopara viticola downy mildew severity endophytic bactera healthy grapevine leaves 239 plant (grapevine): plate (Zhang et al., 2017)
leaves
R. solanacearum wilt incidence and severity rhizobacteria rhizosphere and rhizoplane of tomato and 298 plant (tomato): pot (Santiago et al.,
eucalyptus seedlings 2015)
F. oxysporum wilt severity Bacillus spp. roots of healthy and diseased cucumber plants 400 plant (cucumber): flask (Li et al., 2012)
seedlings
Pythium irregular, P. torulosum, P. graminicola root rot severity bacteria wheat roots 600 plant (wheat): tube (Milus and
seedlings Rothrock, 1997)
Biological Control 144 (2020) 104240

(continued on next page)

 Table 1 (continued)

Test pathosystem Screened candidate BCAs Test setup Reference

Target pathogen(s) Screening parameter Type Origin Size Medium Format

K. Raymaekers, et al.

B. Combined (1) phenotype- and (2) marker-based screenings

(1) V. dahliae, S. sclerotiorum, R. solani (1) pathogen growth rhizobacteria rhizosphere of strawberry, potato and oilseed 60 (1) solid plate (Berg et al., 2001)
(2) NA (2) protease, chitinase and β- rape (2) solid and liquid
1,3- glucanase production
(1) Pythium myriotylum (1) pathogen growth rhizobacteria rhizosphere of ginger 100 (1) solid plate (Dinesh et al., 2015)
(2) NA (2) α-amylase, cellulase, (2) solid and liquid
pectinase, protease and
hydrogen cyanide
production
(1) F. graminearum (1) pathogen growth bacteria different parts of wheat tissues 966 solid plate (Wang et al., 2015)
(2) NA inhibition
(2) cellulase, chitinase,
glucanase, protease and
siderophore production
(1) Monilinia fructicola (1) pathogen growth bacteria collection of the Plant Bacteriology 1,219 (1) solid plate (Mota et al., 2017)
(2) NA (2) ammonia, amylase, Laboratory, Universidade Federal de Pelotas, (1) solid and liquid
lipase, protease and Brazil
chitinase production

C. Marker-based screenings

NA protease, cellulase, chitinase, bacteria bulk soil, rhizosphere, endosphere, surface 935 solid plate (Tokpah et al.,
glucanase and siderophore and interior of stems, phyllopshere and 2016)

5
production endosphere from diseased and healthy rice
NA oxalic acid degradation bacteria rhizosphere of broad-leaved dock and rape >42 solid plate (Schoonbeek et al.,
2007)
NA siderophore production bacteria rhizosphere of pepper, tomato and rubber > 97 solid plate (Yu et al., 2011)
trees
NA root colonization rhizobacteria rhizosphere and roots of tomato and cucumber mixture plant (tomato): tube (Kamilova et al.,
seedlings 2005)
NA root colonization rhizobacteria rhizosphere and roots of paprika, eggplant, mixture plant (tomato): tube (Validov et al.,
corn, camel thorn, wormwood, cucumber, seedlings 2007)
tomato and avocado
Biological Control 144 (2020) 104240
K. Raymaekers, et al. Biological Control 144 (2020) 104240

2.1. Phenotype-based screening for BCAs with direct biocontrol activity
In phenotype-based screenings, the intended, direct antagonistic
effect on the pathogen is measured through evaluation of either pa-
thogen growth, its ability to form specific infectious structures (e.g.
appressoria), or its pathogenic effect on the plant (e.g. disease intensity)
(Table 1).
The so-called “dual culture assay” is one of the most reported direct
screening methods for BCAs with direct biocontrol activity since it is
easy to perform and broadly applicable for identification of bacterial,
yeast, fungal, oomycete, and viral BCAs. The assay consists in general of
co-cultivating the pathogen and the BCA on (semi-)solid medium, and
evaluating the antagonistic capacity of the BCA by measuring the zone
of pathogen growth inhibition (Pliego et al., 2011; Ramesh and Phadke,
2012; Sales et al., 2016). This type of screening has been preferentially
performed for larger library screenings of up to 2,500 BCAs (Adesina
et al., 2007; Berg et al., 2005; Mota et al., 2017; Wang et al., 2015).
Two different setups can be discriminated. In a first one, both the BCA
and pathogen are spotted at different locations on the agar plate, either
as droplets or agar plugs containing the pathogen or BCA inocula
(bacterial cells, fungal spores, or mycelial fragments). In case of an
antagonistic BCA, growth of the pathogen colonies is inhibited in the
proximity of the outgrowing BCA colonies (Fig. 1A) (e.g. Comby et al.,
2017; Toh et al., 2016; Wang et al., 2015). As an alternative to spot
applications, the pathogen inoculum can be evenly distributed over the
medium either by adding it to or spraying it on the medium before and
after medium solidification, respectively. Subsequently, the BCA in-
oculum can be spotted onto the medium and pathogen growth inhibi-
tion zones can be measured around antagonistic BCA colonies (Fig. 1B)
(e.g. Huang et al., 2013; Shehata et al., 2016). A comparable setup
(Fig. 1B) has been shown to be applicable for viral BCAs like
bacteriophages. More specifically, the host range of a phage, being the
bacteria allowing successful infection by the phage and production of
virulent virions (Hyman and Abedon, 2010), can be tested using a dual
culture assay as a critical factor for evaluating its applicability in
practice (Hyman, 2019). A dual culture spot assay has, for example,
been applied to determine the infectivity of Pantoea agglomerans and
Pseudomonas syringae pv. porri phages (Adriaenssens et al., 2011;
Rombouts et al., 2016). It should be noted that in general large
screenings for viral BCAs are less reported in scientific literature.
The typical setup of dual culture assays, as described in general
above, can be adapted for specific purposes. In case of large growth rate
differences between the BCAs and the pathogen, for example, this setup
becomes difficult to score, as was also addressed by McDougal et al.
(2011). In their screening for BCAs acting against the fungus Dothris-
troma septosporum causing pine needle blight, antagonistic BCA activity
was evaluated by identifying the loss of GFP expression in a GFP-la-
beled D. septosporum strain, rather than by measuring growth inhibi-
tion. Another adaptation of the dual culture assay has to be considered
when screening specifically for BCAs that produce volatile anti-
microbial metabolites. To ensure that the observed inhibition of the
pathogen relies exclusively on excreted volatile BCA compounds, both
the BCA and pathogen need to be grown in one volume but physically
separated. The latter can be obtained, for example, (i) by simply sealing
two agar base plates, inoculated with the potential BCA and the test
pathogen, respectively (Fig. 1C) (Dennis and Webster, 1971; Humphris
et al., 2002; Stinson et al., 2003); (ii) by creating a medium-free zone
between the BCA and pathogen growing areas on an agar plate (Fig. 1D)
(Atmosukarto et al., 2005; Mercier and Jiménez, 2004; Strobel et al.,
2001); or (iii) by using specific Petri dishes with separate compartments
(Fig. 1E) (Ezra and Strobel, 2004; Fernando et al., 2005; Fialho et al.,
2010; Fialho et al., 2011). In all cases, the volume of the common

Fig. 1. Phenotype-based screening setups for direct biocontrol mechanisms. In a dual culture setup, both the candidate BCAs (blue) and the pathogen (orange) are
spotted on the solid agar medium (A) or the pathogen can be evenly distributed over the medium on which the candidate BCAs are subsequently spot-inoculated (B).
Adaptations of the dual culture assay to screen for BCAs producing antimicrobial volatiles include sealing two plates with the pathogen and BCA, respectively (C),
creating a medium–free zone between the pathogen and the BCA (D), or using special plates with separated compartments (E). Besides assays on solid medium (A-E),
screening for BCAs with direct antagonistic activity can also be performed in liquid medium (F) facilitating automated handling on robotized platforms, or on plant
parts (G).

6
K. Raymaekers, et al.
growth space of BCA and pathogen determines the dilution of the vo-
latile inhibitory compounds and as such the sensitivity of the assay.
Screenings for direct inhibition mechanisms using assays in liquid
medium have also been reported (Broekaert et al., 1990; Omar and
Abd-Alla, 1998). In this type of assay, the pathogen and BCAs are in-
oculated in the medium, either simultaneously or consecutively (de-
pending on differences in growth rate) and inhibition of pathogen
growth is evaluated. When using non-living BCAs, pathogen growth can
easily be observed by measuring the optical density of the culture, and/
or by microscopic evaluation of pathogen development (Broekaert
et al., 1990). Moreover, performing such assays in standard microtiter
plates (MTPs) (Fig. 1F) allows screening of large BCA libraries in a high-
throughput manner, especially when using robotized platforms. Suc-
cessful BCA-screening in liquid medium is exemplified by the study of
Oh and Lee (2000). They evaluated the antagonistic effect of 1,000
culture filtrates of different fungi and actinomycetes on conidial ger-
mination and appressorium formation of Magnaporthe grisea using mi-
croscopical analysis. This resulted in the identification of five culture
filtrates with biocontrol potential of which three were further validated
for control of rice blast in greenhouse trials. Screening in liquid media
becomes more complex when living BCAs are used, as discrimination
has to be made between growth of the BCA and the pathogen. In a
screening for bacterial BCAs against the fungal pathogens Fusarium
solani, Macrophominia phaseolina and Rhizoctonia solani, Omar and Abd-
Alla (1998) co-cultured the BCAs and pathogens in 25 ml flasks and
subsequently analyzed the inhibitory effects by measuring the dry
weight of the outgrowing fungal mycelia. Out of 22 screened bacteria,
three were found to exert antagonistic activity against the three test
pathogens, which was further confirmed by disease assays in different
crops (okra, soybean, and sunflower). Screening of phages in liquid
medium has not yet been reported for biocontrol applications. For
medical purposes, on the other hand, this has been reported to be
feasible. Xie et al. (2018) developed a liquid dual culture assay to test
the host range and virulence of 15 Salmonella phages against a panel of
20 bacterial strains in a 96-well MTP format.
Although BCA screenings for direct antagonistic effects are most
commonly performed in vitro, also assays on plant material have been
reported (e.g., Abraham et al., 2010; Comby et al., 2017; Perez et al.,
2017; Zhang et al., 2017). Despite some practical drawbacks, these in
vivo assays have the advantage (i) to test the antagonistic potential of
the BCA under more natural conditions; and (ii) to be applicable for
screenings against pathogens difficult to grow in vitro, such as obligate
biotrophs which require living host cells for their proliferation. In
general, these types of screenings are performed on those plant parts on
which pathogen infections occur and disease symptoms are observed
(Fig. 1F). For example, Abraham et al. (2010) tested the biocontrol
potential of 60 yeast and 92 Bacillus isolates against the postharvest
pathogen Penicillium digitatum on oranges. Ten yeast and ten bacterial
isolates were found capable of reducing the infected surface area by
>50%.
Successful in planta screening for BCAs antagonistic to biotrophic
pathogens has been demonstrated by Zhang et al. (2017). In this study
grape leaf discs were used to screen 239 bacterial grapevine endophytes
for their antagonistic effect against the biotrophic pathogen Plasmopara
viticola, causing downy mildew, resulting in the selection of two isolates
further demonstrated to protect grapevines against this disease in field
trials.
Such in planta assays are also often used as a second screening step
after a classical dual culture assay such as reported by Perez et al.
(2017) for biocontrol of P. digitatum disease in lemon. In a similar setup,
they demonstrated that ten yeasts previously identified in an in vitro
dual culture screening for antagonistic activity against P. digitatum,
Penicillium italicum and Penicillium citri (Perez et al., 2016), also exerted
their antagonistic activity in vivo. A similar combined approach was
applied to identify BCAs antagonistic to the fungal pathogen Fusarium
graminearum (Comby et al., 2017), the causal agent of the important
7
Biological Control 144 (2020) 104240
Fusarium head blight disease of cereals and producer of several my-
cotoxins threatening the health of human and animal consumers of
cereals (Windels, 2000). In this study 22 out of 86 screened wheat
endophytes were found to exert antagonistic activity in a dual culture
assay against F. graminearum, of which three were subsequently con-
firmed for their biocontrol activity of Fusarium head blight on detached
wheat spikelets. In general, such concatenation of different screening
methods thus combines the advantages of the different approaches in-
cluding high-throughput handling in vitro versus in vivo screening under
natural conditions.
2.2. Marker-based screening for BCAs with direct biocontrol activity
In contrast to the above-mentioned approaches, in marker-based
screenings the inhibitory effect of a BCA is evaluated through the
analysis of markers which are closely linked to this antagonism. They
include secreted antimicrobial metabolites or lytic enzymes (e.g. cell
wall-degrading chitinases and glucanases) or the occurrence of com-
petitive root colonization by the BCA. Though less reported than the
phenotype-based screenings, marker-based methods can be useful for
high-throughput screenings or for BCAs with a specific inhibitory me-
chanism. The biocontrol potential of such identified BCAs obviously
requires confirmation of the final intended phenotype in in vivo disease
assays.
Obviously, crucial in marker-based screenings of BCAs is the choice
of a proper marker closely linked to the desired phenotype, being here a
direct antagonism of the pathogen. In most of these studies, different
types of lytic enzymes or antimicrobial metabolites were analyzed as
markers. Tokpah et al. (2016), for example, screened 935 bacterial
isolates for their biocontrol potential against the major fungal rice pa-
thogen Magnaporthe grisea causing blast disease. In this approach, the
bacteria were evaluated for their specific secretion of chitinases, cel-
lulases, glucanases, proteases, and siderophores when grown on solid
substrates specifically allowing determination of these compounds. As
such, 30 bacteria were selected and further confirmed for their ability
to protect rice against blast disease in greenhouse experiments, in-
dicating a high correlation between the marker-based screening and the
desired biocontrol phenotype. In another study, Schoonbeek et al.
(2007) specifically analyzed the degradation of oxalic acid, a patho-
genicity factor of important fungal pathogens such as Sclerotinia scler-
otiorum and Botrytis cinerea. By using agar plates with Ca-oxalate as sole
carbon source, they were able to isolate 42 oxalic acid-degrading bac-
teria. Four isolates that gave medium to strong protection against S.
sclerotiorum and B. cinerea on the model plant Arabidopsis thaliana were
further characterized and their biocontrol ability was confirmed in
cucumber, tomato and grapevine. This study clearly indicates the po-
tential of oxalic acid-degrading enzymes as a single marker for
screening of potential BCAs against S. sclerotiorum and B. cinerea.
Finally, marker-based screenings were also applied for selection of
BCAs with a direct antagonistic activity based on competition with the
pathogen. In the study by Kamilova et al. (2005) plant root colonization
was used as a marker for the competitive potential of a BCA. Therefore,
a crude mixture of rhizosphere bacteria originating from two different
rhizosphere samples was inoculated on sterile seedlings of cucumber or
tomato, aiming at enriching for enhanced competitive root tip coloni-
zers. In practice, this was done by isolating the strains that succeeded in
reaching the root tip seven days after seedling inoculation and subse-
quently using them to inoculate fresh, sterile seedlings. After three
enrichment cycles, 16 randomly chosen isolates were analyzed in
competitive root tip colonization assays on tomato against P. fluorescens
WCS365, which was considered at that time as the best competitive
tomato root tip colonizer. The five unique strains that showed a better
competitive root tip colonization compared to P. fluorescens WCS365
were further characterized and the biocontrol potential of four of these
selected strains could be confirmed in tomato plants against one of the
most destructive necrotrophic pathogens, Fusarium oxysporum f.sp.
K. Raymaekers, et al.
radicis‐lycopersici, causing crown and root rots. A similar marker-based
screening approach was further used on a larger scale by Validov et al.
(2007) who used bacteria from 17 different rhizosphere samples to
enrich for enhanced root tip colonizers, ultimately resulting in the
identification of seven new F. oxysporum f.sp. radices-lycopersici BCAs.
3. Screening for BCAs with indirect biocontrol activity
In contrast to acting via direct pathogen antagonism, BCAs can also
exert their biocontrol activity indirectly via enhancing host defense
mechanisms. In plants, two main types of induced resistance can be
distinguished, being systemic acquired resistance (SAR) and induced
systemic resistance (ISR). Both immune responses are triggered locally
by a pathogen or beneficial organisms, respectively, and offer the plant
systemic protection against a broad spectrum of pathogens (Pieterse
et al., 2014).
In 1901, both Beauverie and Ray independently discovered that
plants previously infected by B. cinerea showed less susceptibility to
secondary infections (Beauverie, 1901; Ray, 1901). This phenomenon
was later termed “systemic acquired resistance” (SAR) by Ross (1961)
when studying pathogen-induced resistance in tobacco. Since then, a lot
of knowledge on the underlying mechanisms of SAR accumulated, for
which we refer to excellent, recent reviews by Klessig et al. (2018) and
Shine et al. (2019). In the current concept, pathogen infection and
subsequent activation of a local immune response lead to the accu-
mulation of the hormone salicylic acid (SA), including in systemic tis-
sues (Malamy et al., 1990; Métraux et al., 1991; Rasmussen et al., 1991;
Uknes et al., 1993). This SA accumulation and signaling are essential
for the establishment of SAR (Vernooij et al., 1994). SAR is further
characterized by the expression of pathogenesis-related (PR) proteins
many of which possess antimicrobial activity (van Loon et al., 2006).
Interestingly, in tobacco it was shown that also exogenous application
of SA and related compounds like benzoic acid and acetylsalicylic acid
could induce SAR (against Tobacco Mosaic Virus) and cause the accu-
mulation of PR proteins (White, 1979). These findings were a major
breakthrough regarding potential applications in crop disease control
leading to the identification of more potent functional analogs of SA
including 3-allyloxy 1,2-benzisothiazole-1,1-dioxide (Probenazole)
(Watanabe et al., 1977), 2,6-dichloro-isonicotinic acid (INA) (Kunz
et al., 1988; Métraux et al., 1991), benzo(1,2,3)thiadiazole-7-car-
bothioic acid (BTH) (Kunz et al., 1997; Schurter et al., 1993), N-(3-
chloro-4-methylphenyl)-4-methyl-1,2,3-thiadiazole-5-carboxamide
(Tiadinil) (Tsubata et al., 2006), N-cyanomethyl-2-chlor-
oisonicotinamide (NCI) (Yoshida et al., 1990; Yoshida et al., 1989), and
Isotianil (Ogawa et al., 2011). However, being mostly identified by
agrochemical companies like Meiji Seika Kaisha Ltd. (Probenazole),
Ciba-Geigy (now Syngenta; INA and BTH), Nihon Nohyaku Co., Ltd.
(Tiadinil and NCI), and Bayer AG (now Bayer CropScience AG; Isotianil)
details concerning the screening methods used to identify these syn-
thetic plant defense elicitors are not available; consequently they could
not be further discussed in this review.
Induced systemic resistance was later discovered by three in-
dependent research groups (Alström, 1991; van Peer et al., 1991; Wei
et al., 1991). They observed that colonization of plant roots by selected
strains of plant-growth-promoting rhizobacteria (PGPRs) could trigger
enhanced disease resistance, also in systemic (non-colonized) plant
parts, and named this immune response “(rhizobacteria-) induced sys-
temic resistance”. Subsequently, numerous PGPRs, mainly Pseudo-
monas, Serratia, and Bacillus species (De Vleesschauwer and Höfte,
2009; Kloepper et al., 2004; van Loon and Bakker, 2006) but also fungi
including nonpathogenic F. oxysporum, Trichoderma spp., Piriformospora
indica, and symbiotic arbuscular mycorrhizal fungi (Alabouvette et al.,
2009; Cameron et al., 2013; Franken, 2012; Jung et al., 2012; Shoresh
et al., 2010; Vinale et al., 2008) were reported to induce ISR in both
monocots and dicots. Originally it was assumed that, in contrast to SAR,
ISR is not mediated by SA, but rather by jasmonic acid (JA) and
8
Biological Control 144 (2020) 104240
ethylene (ET) as central regulators (Pieterse et al., 1998). However,
increasing evidence is accumulating that several bacteria and fungi
induce a SA-dependent ISR that resembles pathogen-induced SAR me-
chanisms (Mathys et al., 2012; Pieterse et al., 2014; Van der Ent et al.,
2009). It is thus not always straightforward to make a difference be-
tween SAR and ISR. Therefore, on the “1st International Symposium of
Induced Resistance to Plant Diseases” in 2000, it was unanimously
agreed to use the more general term “induced resistance”
(Hammerschmidt et al., 2001; Tuzun, 2006), albeit that both SAR and
ISR are still more frequently used in their original definitions. In order
to avoid any confusion, in this review we will further use the term ISR
to indicate a systemic resistance induced in plants triggered by non-
pathogenic BCAs and non-living ones.
Compared to BCAs with a direct pathogen inhibition activity, the
use of ISR-inducing BCAs provides some advantages in view of their
application in crop protection. First of all, since these BCAs do not exert
their biocontrol effect via a direct antimicrobial activity, their appli-
cation is not expected to have direct negative impact on beneficial
microbes in the plant environment (Köhl et al., 2019). Secondly, it has
been postulated that the use of such BCAs would put significantly less
selection pressure on the pathogen to become resistant, due to the di-
versity of defense mechanisms induced in the plant and the lack of
direct interaction of the BCA with the pathogen (Bardin et al., 2015;
Romanazzi et al., 2016). For example, in Asia the synthetic ISR-inducer
Probenazole has widely been used (as Oryzemate®) to control rice blast
caused by Magnaporthe grisea. Despite its extensive use since 1975, no
development of resistance in the blast fungus has been reported (Iwata,
2001). Finally, the systemic activity of these BCAs offers additional
advantages. Indeed, it allows also the protection of specific plant parts
that are either difficult to reach through spraying or irrigation, or that
develop after treatment. Moreover, application of these BCAs can be
restricted to plant parts not intended for consumption, thereby avoiding
problems with legally restricted residues (European Parliament, 2005)
and an important concern for consumers (Hughner et al., 2007).
Therefore, in view of identification of novel BCAs, it remains interesting
to also screen for ISR-inducing BCAs.
Similarly to our overview of screenings for BCAs with direct inter-
action (see Section 2.1), distinction will be further made between
phenotype-based and marker-based screenings. Obviously, screening
for such indirect biocontrol mechanisms is more challenging as it im-
plies a third partner in the interaction, being the plant. Consequently,
fewer screenings for ISR-inducing BCAs are reported as compared to
those with direct antagonistic activity, as reflected in Table 2.
3.1. Phenotype-based screening for indirect mechanisms
Phenotype-based screenings for induced resistance are focusing on a
reduction of disease severity as targeted phenotype. However, with
respect to the cultivation of the test plants, application of BCAs and
pathogens, and evaluation of disease development, such assays are in
general labor-, space- and time-consuming. For this reason high-
throughput screenings are less obvious, which is reflected in general by
the relatively low number of BCAs that were reportedly evaluated
within such screens (Table 2) (Akram et al., 2013; Alström, 1991).
Moreover, regarding both the assay setup and evaluation of results,
attention has to be paid to the possible contribution of direct BCA-pa-
thogen interaction (see Section 2) on the observed biocontrol activity.
To increase the throughput of these screenings, they can be minia-
turized by growing plants in vitro in standard MTPs. It should be noted,
however, that not all pathosystems are suited for such in vitro condi-
tions and miniaturization, hence the number of examples remains
limited (Han et al., 2012; Schreiber et al., 2011, 2008). Schreiber et al.
(2008), for example, developed a rapid high-throughput assay using the
A. thaliana - P. syringae pathosystem to screen a collection of 200 che-
mical compounds from the Library of AcTive Compounds (LATCA li-
brary UC Riverside, U.S.A.) for potential BCAs. In practice, A. thaliana
 K. Raymaekers, et al.

Table 2
Examples of reported screenings for BCAs with indirect biocontrol activity. Distinction is made based on screen type being phenotype-based (A) and marker-based (B). Details given concern the primary screening used to
make the first BCA selections. Only screenings on 10 or more candidate BCAs are considered here. 12 and 96 MTP: 12- and 96-well microtiter plate, NA: not applicable.
Test pathosystem Screened candidate BCAs Test setup Reference

Host Pathogen Screening parameter Type Origin Size Plant Medium Format
material

A. Phenotype-based screenings

Tomato F. oxysporum wilt severity Bacillus spp. Institue of Agricultural Sciences and Department of 14 whole soil pot (Akram et al.,
Microbiology and Molecular Genetics, University of the plants 2013)
Punjab, Pakistan
cucumber Colletotrichum lesion development plant-growth-promoting Esso Chemical Ag Biologicals, Saskatoon, Canada 94 whole soil pot (Wei et al., 1991)
orbiculare rhizobacteria plants
tomato P. syringae lesion development rhizobacteria and rhizosphere and rhizoplane of tomato 500 whole soil pot (Silva et al., 2003)
actinomycetes plants
tobacco Pectobacterium soft rot symptoms oligotrophic bacteria rhizosphere of various soils 252 whole solid 12 MTP (Han et al., 2012)

9
carotovarum plants
Arabidopsis thaliana P. syringae cotyledon bleaching synthetic compounds LATCA collection 200 whole liquid 96 MTP (Schreiber et al.,
plants 2008)
A. thaliana F. graminearum dark spot formation on natural compounds TimTec NP280 Natural Product Collection 80 whole liquid 96 MTP (Schreiber et al.,
cotyledons plants 2011)

B. Marker-based screenings

tobacco NA ROS production natural compounds ICSN (Institut de Chimie des Substances Naturelles) library 1,600 cells liquid 96 MTP (Zahid et al.,
2017)
A. thaliana P. syringae cell death synthetic and natural The Spectrum Collection and RIKEN Natural Products 2,677 cells liquid 96 MTP (Noutoshi et al.,
compounds Depository 2012a)
A. thaliana P. syringae cell death synthetic and natural The Spectrum Collection 1,909 cells liquid 96 MTP (Noutoshi et al.,
compounds 2012b)
A. thaliana P. syringae cell death synthetic compounds DIVERSet NovaCore NQ612 library 10,000 cells liquid 96 MTP (Noutoshi et al.,
2012c)
parsley Phytophthora sojae coumarin derivatives synthetic and natural Sigma 30 cells liquid flask (Siegrist et al.,
(elicitor) production compounds 1998)
CaBP22-333-Promotor::GUS A. NA marker pCaBP22- synthetic and natural Microsource Spectrum Collection, Sigma TimTec 42,000 whole liquid 96 MTP (Knoth et al.,
333
thaliana ::GUS compounds MyriaScreen Collection, Chembridge Nova Core library plants 2009)
and Chembridge DIVERSet library
Biological Control 144 (2020) 104240
K. Raymaekers, et al.
seedlings were grown in liquid medium in 96-well MTPs, facilitating
the uniform addition of both compounds and pathogen, and the mi-
croscopic analysis of cotyledon bleaching. The latter was proven to be
associated with growth of the bacteria within the seedling tissues. The
screening resulted in the successful identification of a group of sulfo-
namide compounds that efficiently protected A. thaliana plants against
P. syringae. As the BCA and pathogen are co-cultivated in this setup, a
direct antagonism of the sulfonamide compounds on P. syringae could
not be excluded and additional experiments were performed to prove
that these compounds did not protect the plant by inhibiting bacterial
growth in the liquid assay (Schreiber et al., 2008). Based on this
screening success, a similar assay using a fungal pathogen (F. grami-
nearum) was developed and used to screen 80 compounds from the
TimTec NP280 Natural Product Collection (TimTec LLC, Newark, DE,
U.S.A.) for their capacity to induce plant resistance by microscopic
analysis of disease symptoms (Schreiber et al., 2011). Two hit com-
pounds, sulfamethoxazole and donaxina, were identified and their ISR-
inducing activity in wheat was confirmed in soil assays against the
important Fusarium head blight disease (Parry et al., 1995) caused by F.
graminearum (Schreiber et al., 2011). The data obtained by Schreiber
et al. (2011) demonstrate that BCAs identified in a fast (nine days) in
vitro screening on a model pathosystem can lead to valuable BCAs in-
ducing ISR in economically important crops.
Research by Han et al. (2012, 2006) indicated that such in vitro
screenings could also be applied for the isolation of living ISR-inducing
BCAs. Indeed, they developed a three-week screening assay in which
tobacco plants were grown in vitro in 12-well MTPs on solid medium. A
collection of 252 oligotrophic bacteria was added onto the roots while
leaves were subsequently infected with the rot-causing pathogen Pec-
tobacterium carotovorum. Soft rot disease was rated based on the number
of symptomatic leaves, which led to the identification of two ISR-in-
ducing bacteria (Han et al., 2012). In these studies, also a link between
the root colonization capacity and resistance-inducing potential of the
bacteria was postulated. This was in agreement with the observations
by Lugtenberg and Kamilova (2009) that efficient root colonization is
essential for most PGPRs to exert their beneficial effects, including in-
duction of ISR. Similarly, Silva et al. (2003) used a root colonization
bioassay to evaluate PGPRs and actinomycetes, isolated from the to-
mato plant rhizosphere, that were previously identified as potent ISR-
inducing BCAs in a phenotype-based screening on tomato against P.
syringae pv. tomato. All 28 isolates were found to be good root coloni-
zers, in contrast to isolates lacking biocontrol activity. The authors
therefore suggested their root colonization bioassay as a reliable and
efficient tool to select for PGPRs with ISR-inducing capacity.
3.2. Marker-based screening for indirect mechanisms
Although it is possible to phenotypically screen for novel ISR-in-
ducing BCAs, the main drawback remains the relatively low size of
potential BCA collections that can be evaluated (e.g. up to 500 in the
study by Silva et al. (2003)). One major reason is that the analysis of the
observed phenotypical parameters, being symptom development or
eventual root colonization, is still analyzed in a non-automated macro-/
microscopical way. Therefore, when aiming at screening larger libraries
of potential ISR-inducing BCAs, automated analysis of a marker asso-
ciated with the intended resistance phenotype is recommended. This is
illustrated by the fact that such marker-based screenings were indeed
reported for the analysis of large collections of up to 42,000 BCAs
(Knoth et al., 2009; Noutoshi et al., 2012c). Obviously, BCAs identified
in such marker-based screenings always need to be validated in plant
assays regarding the intended ISR phenotype.
In a search for adequate ISR markers, researchers first evaluated
known processes that are in general induced in the plant upon inter-
action with a pathogen or microorganism. One of the first induced plant
mechanisms in such interactions is the production of reactive oxygen
species (ROS) or the oxidative burst. The latter was used by Zahid et al.
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Biological Control 144 (2020) 104240
(2017) as a marker to develop a screening tool applicable in three-days-
old tobacco BY-2-suspension-cultured cells to screen 1,600 plant-de-
rived compounds selected from the ICSN chemical library (Institut de
Chimie des Substances Naturelles, CNRS, Gif-sur-Yvette, France). De-
tection of ROS was performed in MTPs by automated fluorescence de-
tection using the H2DCF-DA assay (Gerber and Dubery, 2004). This
enabled Zahid et al. (2017) to select 16 ROS-inducing compounds in a
high-throughput manner, including the steroid holaphyllamine (HPA)
further confirmed in planta (A. thaliana) to trigger ISR against P. syr-
ingae pv. tomato DC3000.
Another suitable marker is pathogen-induced plant cell death.
Proof-of-concept for using this marker was given in a setup of A.
thaliana cell suspensions challenged with the bacterial pathogen P.
syringae pv. tomato DC3000 (Noutoshi et al., 2012c). Reduction of pa-
thogen-induced cell death, measured using an automated Evans blue
dye assay (Gaff and Okong’o-ogola, 1971; Yao et al., 2018), could be
obtained by treatment with known resistance-inducing compounds
such as SA and Tiadinil (Bektas and Eulgem, 2015; Yasuda et al., 2004).
This assay was subsequently used to screen 10,000 small organic
compounds of the DIVERSet library (DIVERSet NovaCore NQ612,
Chembridge, USA). Several hit compounds, including imprimatins,
were further demonstrated to protect A. thaliana plants against infec-
tion by P. syringae pv. tomato DC3000 (Noutoshi et al., 2012c). The
same screening method was further used to screen additional libraries
such as 1,920 compounds of the Spectrum Collection (MicroSource
Discovery Systems Inc., Gaylordsville, CT, USA) (Noutoshi et al.,
2012a,b) and 768 compounds of the RIKEN library (Natural Products
Depository, RIKEN ASI, Saitama, Japan) (Kato et al., 2012; Noutoshi
et al., 2012a). Seven sulfonamide compounds were identified and de-
monstrated to act as ISR-inducing compounds in disease assays with P.
syringae on A. thaliana. Unlike SA and its analogs, these compounds do
not induce PR1 gene expression in absence of the pathogen, and unlike
imprimatins, they do not inhibit SA glucosyl transferases, showing the
potential of the screening tool to identify compounds that induce the
same phenotype (ISR), albeit via different signaling mechanisms
(Noutoshi et al., 2012a–c).
Another example of a possible marker is the induction of phytoa-
lexin production, known for long as an induced defense mechanism in
plants (Piasecka et al., 2015; Smith, 1996) and to be associated with ISR
(Mathys et al., 2012). Production of phytoalexins was evaluated by
Siegrist et al. (1998) through measurement of their fluorescence in
parsley cell suspensions triggered by an elicitor from the cell wall of the
oomycete pathogen Phytophthora sojae. Proof-of-principle for this
screening tool was obtained with the known resistance-inducer BTH
(Bokshi et al., 2003; Godard et al., 1999; Gorlach et al., 1996). Sub-
sequent small-scale screening of 30 potential BCAs resulted in the se-
lection of saccharin, which was further demonstrated to induce ISR in
cucumber, tobacco and bean plants against both fungal and viral dis-
eases (Siegrist et al., 1998). As this assay only takes three days, it can in
principle easily be elaborated for high-throughput screening of larger
BCA libraries.
While in previous examples mainly cell suspensions were used,
high-throughput screenings on whole plants are possible as well, for
example by using differentially expressed genes as markers. They can
be detected at transcriptomic level (e.g. by qRT-PCR) or at protein level
by coupling their promotor to a gene encoding a reporter protein which
can easily be detected via automated enzymatic, luminescent, or
fluorescent assays, making them powerful tools in high-throughput
screenings (Boulin et al., 2006; Naylor, 1999). For example, Knoth et al.
(2009) used CaBP22 as a marker gene, which is a member of the “Late/
sustained Up-regulation in Response to Hyaloperonospora parasitica
(LURP)” genes in A. thaliana, whose expression is induced by the oo-
mycete pathogen Hyaloperonospora arabidopsidis (Eulgem et al., 2004).
Using an A. thaliana pCaBP22::GUS reporter line, they screened a
42,000 compound library composed of 2,000 compounds of the Spec-
trum Collection (MicroSource Discovery Systems Inc., Gaylordsville,
K. Raymaekers, et al.
CT, USA), 10,000 compounds of the TimTec MyriaScreen Collection
(Sigma TimTec MyriaScreen, Merck KGaA, Darmstadt, Germany),
10,000 compounds of Chembridge Nova Core (Chembridge, USA) and
20,000 compounds of the Diverset library (Chembridge, USA)). This led
to the identification of 114 inducers of the marker gene including 3,5-
dichloroanthranilic acid, subsequently demonstrated to be an inducer
of resistance in A. thaliana against H. arabidopsidis and P. syringae pv.
tomato DC3000. This is the largest screening for ISR-inducing com-
pounds found in literature, and suggests the applicability of this type of
marker-based screenings for high-throughput purposes. Remarkably on
the other side, this successful screening by Knoth et al. (2009) pub-
lished already a decade ago, still remains the only reported example of
a marker gene-based screening for ISR-inducing BCAs.
4. Screening sources for BCAs
Once the desired type of BCAs (direct versus indirect) and an ap-
propriate screening system (phenotype- versus marker-based) have
been defined (see previous sections), the choice of a library for BCA
screening is the next critical determinant for the success of the BCA
identification. Therefore, for both non-living BCAs and biocontrol or-
ganisms, the acquisition or assembly of a library of candidate BCAs will
be discussed further.
Screens for non-living BCAs are most of the time performed with
one or more commercially or publicly available libraries. These can
consist of synthetic compounds (Noutoshi et al., 2012c; Schreiber et al.,
2008), natural products (Schreiber et al., 2011; Zahid et al., 2017), or a
combination of both (Knoth et al., 2009; Noutoshi et al., 2012a, 2012b;
Siegrist et al., 1998). According to Dejonghe and Russinova (2017), two
types of small-molecule libraries can be distinguished: large, combi-
natorial libraries and smaller, more focused collections. Combinatorial
libraries are constructed by combining different chemical building
blocks to generate large numbers of small molecules covering all pos-
sible combinations, resulting in a structurally diverse collection. Ex-
amples of such large combinatorial libraries are the ChemBridge DI-
VERSet library (100,000 compounds) (ChemBridge, 2019) and the
Sigma TimTec MyriaScreen Diversity Collection (10,000 compounds)
(Sigma-Aldrich, 2019; TimTec, 2019). When choosing this type of li-
brary, the aim is clearly on providing chemical diversity (Dejonghe and
Russinova, 2017). Focused libraries and natural product libraries, on
the other hand, are in general constructed by selecting molecules that
are bioactive in a given biological system and consequently these col-
lections are often smaller. Examples of such small, focused libraries
reportedly used in BCA screenings are the LATCA collection (3,580
compounds) (The Cutler Lab, 2008), the MicroSource Spectrum Col-
lection (2,560 compounds) (MicroSource Discovery Systems, n.d) and
the TimTec Natural Product Library (800 compounds) (TimTec, 2019).
The molecules contained in these types of collections can originate from
one or different large libraries. The LATCA collection (“Library of An-
notated Compounds for Arabidopsis”), for example, contains approxi-
mately 3,600 active molecules selected for their ability to inhibit hy-
pocotyl growth in etiolated A. thaliana seedlings and was assembled
starting from different large libraries including the Chembridge DI-
VERSet library and MicroSource Spectrum Collection (Dejonghe and
Russinova, 2017). Inversely, existing small libraries can be combined to
larger ones as was done by Knoth et al. (2009) to generate a collection
of 42,000 small molecules.
Also for living BCAs, diverse microorganism collections are avail-
able. Daranas et al. (2019), for example, made use of the culture col-
lection of the Institute of Food and Agricultural Technology and Center
for Innovation and Development of Plant Health (Universitat de Girona,
Girona, Spain); the bacteria tested in the screening by Mota et al.
(2017) belonged to the collection of the Plant Bacteriology Laboratory
(Laboratório de Bacteriologica Vegetal, Universidade Federal de Pe-
lotas, Pelotas, Brazil). Besides these in-house collections, also publicly
available ones such as the BCCM/MUCL Agro-food and Environmental
11
Biological Control 144 (2020) 104240
Fungal Collection (Université Catholique de Louvain, Louvain-la-
Neuve, Belgium) present a valuable source for identifying novel BCAs.
In recent years, efforts have been made, both in an academic as well as
in an industrial context, to construct large collections of plant-asso-
ciated bacteria and fungi derived from different plant parts of eco-
nomical important crops including potato, tomato, rice, sugarcane, etc.,
as summarized by Finkel et al. (2017). These strain collections can serve
as the starting point for the discovery of potential biocontrol organisms.
However, the majority of the reported screenings for biocontrol
organisms rather starts with the isolation of microorganisms from
specific sites to build up more customized libraries (for examples see
Table 1 and 2). Such sampling from niches representative for the in-
tended region of final agricultural application is recommended to ob-
tain more environmentally adapted BCAs and as such maximize their
survival and biocontrol potential. As biocontrol organisms and patho-
gens are viewed as competitors, their interaction is usually considered a
win-lose relationship. Sampling BCAs from sites where the BCA is
dominant and diseases are consequently suppressed may therefore be a
first option (Huang et al., 2013). Indeed, several studies reported the
isolation of BCAs with potent biocontrol activity from the rhizosphere
or plant parts of healthy plants (Agaras et al., 2015; Latz et al., 2020;
Nawangsih et al., 2011; Sharifazizi et al., 2017). In this respect, disease
suppressive soils are generally considered valuable sources for isolating
naturally occurring BCAs against soilborne diseases (Adesina et al.,
2007; Krause et al., 2003; Pliego et al., 2011). For example, Adesina
et al. (2007) isolated bacterial strains from agricultural soils both with
and without known disease-suppression and evaluated them for their in
vitro antagonistic activity against R. solani and F. oxysporum. Though
biocontrol organisms could be identified from all soils, the overall
proportion of antagonists was highest in three of the four tested sup-
pressive soils (between 1.5 and 2.5 times higher).
On the other hand, collections of microorganisms isolated from
diseased plants have also been shown to contain very potent BCAs (Ait
Bahadou et al., 2018; Anees et al., 2010; Tolba and Soliman, 2013).
Huang et al. (2013) hypothesized that such BCAs must have developed
specific survival strategies to cope with the prevalence of pathogens
and therefore make them competitive BCA candidates. To test this
hypothesis, bacteria isolated from the rhizosphere of healthy and dis-
eased tomato plants were compared for their capacity to biocontrol
bacterial wilt caused by Ralstonia solanacearum. It was demonstrated
that the rhizosphere of both healthy and diseased plants harbored
several BCAs that antagonize R. solanacearum in vitro and reduce disease
severity and promote plant growth under greenhouse conditions. In-
terestingly, BCAs from the diseased-plant rhizosphere showed higher
root-colonizing and biocontrol capacities than BCAs from the healthy-
plant rhizosphere, suggested to result from their adaptations to a pa-
thogen-prevalent environment (Huang et al., 2013). The theory that
diseased plants or soils are good sources of potential BCAs is also
supported by the study of Anees et al. (2010). In this study, Trichoderma
strains were isolated from both the healthy and diseased area of a sugar
beet field containing patches infested with R. solani and tested for their
in vitro and in vivo antagonistic activity against this pathogen. As the
Trichoderma strains found in the diseased area were better antagonists
than those found in the healthy area, they concluded that the presence
of R. solani increases the number of antagonistic Trichoderma strains in
the surrounding soil (Anees et al., 2010). The same is true for the iso-
lation of viral BCAs, for which searches for bacteriophages are generally
performed on locations where the host pathogen is found (Hyman,
2019).
Interestingly also the microenvironment from which the micro-
organisms are isolated apparently determines the hit rate of finding
novel BCAs. Berg et al. (2005), for example, revealed a high diversity in
the microbial composition of bacterial communities of field-grown
potato plants isolated from different ectophytic (rhizosphere and
phyllopshere) and endophytic (endorhiza and endosphere) micro-
environments. Remarkably, when screening for antagonistic BCAs, they
K. Raymaekers, et al.
also demonstrated significant differences between all four communities
in the obtained hit rates. Indeed, when testing them for in vitro in-
hibitory activity against the fungal pathogens Verticillium dahlia and R.
solani, the highest proportion of BCAs antagonistic to both pathogens
was found in the endorhiza and (to a lesser extent) in the rhizosphere. A
plausible explanation is that both test pathogens are soilborne and will
predominantly invade the plant via the roots (Berg et al., 2005). Un-
fortunately, no leaf pathogens were included in the antagonistic assays
to further support this hypothesis.
The study of Berg et al. (2005) also highlights the potential of a
specific group of microorganisms as potential BCAs, being endophytes.
They are known to grow asymptomatically, inter- or intracellularly,
systemically or locally within a plant host for at least a part of their life
cycle (Card et al., 2016; De Silva et al., 2019; Hardoim et al., 2015;
Hyde and Soytong, 2008) and are found in association with the ma-
jority of plant species in natural and managed ecosystems (Card et al.,
2016). Endophytes can provide beneficial effects for their host in-
cluding plant growth promotion, removal of contaminants from the
soil, and increased tolerance to abiotic and biotic stresses (Azevedo
et al., 2000; Card et al., 2016; Schulz et al., 2002). Regarding the latter,
endophytes (including their bioactive secretions) recently received in-
creased attention as potential BCAs (Card et al., 2016; Collinge et al.,
2019; De Silva et al., 2019) as reflected by the large number of reported
endophyte screenings for novel BCAs (Burr et al., 1996; Comby et al.,
2017; Ghazalibiglar et al., 2016; Latz et al., 2020; Li et al., 2012;
Nawangsih et al., 2011; Ramesh and Phadke, 2012; Shehata et al.,
2016; Toh et al., 2016; Tokpah et al., 2016; Wang et al., 2015; Zheng
et al., 2011), some of which already led to commercialization (as re-
viewed in detail by De Silva et al. (2019)).
5. Discussion
Although BCAs are generally recognized as important tools for more
sustainable disease management and represent valuable alternatives/
complements to classical pesticides, they still make up only a small
percentage (less than 10%) of the overall global crop protection market
(Phillips McDougall, 2018). One of the reasons for this low share is the
lack of commercially available BCAs and as such the need for more
extended biocontrol research (Lamichhane et al., 2017). One critical
step in the development of novel commercial BCA-based products is the
screening for appropriate candidates. As it is estimated that less than
1% of newly identified BCAs are developed into successful commercial
products (Bailey and Falk, 2011; Glare et al., 2012), high numbers of
candidate BCAs should be evaluated, further demonstrating the need
for rapid and robust high-throughput screening methods. Therefore the
objective of this review was to give a comprehensive overview of the
different screening systems currently reported, to discuss their eventual
limitations, and to identify opportunities for the further development of
advanced high-throughput systems.
Ideally, the biocontrol activity of candidate BCAs is evaluated using
a phenotype-based screening under field conditions representative for
the intended agricultural applications (Köhl et al., 2019). However,
screening a large collection of candidates in the field and/or green-
house is too time- and space-consuming and expensive. Moreover,
variable abiotic and biotic parameters in the field restrain reproducible
results and may complicate the analysis and the mutual comparison of a
large number of candidates (Pliego et al., 2011). In order to enable
large-scale screening for BCAs, a simple, rapid, and reproducible pri-
mary screening is needed that can be used to select a reduced amount of
promising candidates for further analysis in field trials (Agaras et al.,
2015). According to Köhl et al. (2011) in this primary screening step,
the partners of the tripartite system (plant, pathogen, and BCA) are
optimally interacting under controlled conditions that are re-
presentative for the targeted epidemiological stage and environmental
conditions in the crop. From all the reported phenotype-based screen-
ings for direct pathogen inhibition listed in Table 1, only four use a
12
Biological Control 144 (2020) 104240
whole-plant approach in their primary screening. Latz et al. (2020) used
wheat seedlings to screen for BCAs with the potential to control Septoria
tritici in planta. However, this screening can be considered small-scale as
only 25 endophytes were analyzed. In the screening of Li et al. (2012), a
gnotobiotic system was used to avoid field-soil variables and to make
the bioassay more controllable and reproducible, but consequently, the
sterile conditions make this assay less representative. Santiago et al.
(2015) used a whole-plant bioassay to identify potential BCAs con-
trolling eucalyptus bacterial wilt caused by R. solanacearum under
greenhouse conditions favorable for disease development. Tomato was
used as a model as this plant species is highly susceptible to R. sola-
nacearum and allows fast plant and disease development. The use of
such a model-system was proven to be a valuable strategy to speed up
whole plant bioassays but still only a relatively low amount of isolates
(298) could be screened. In the last screening (Milus and Rothrock,
1997) a miniaturized setup was used with wheat seedlings grown in
tubes which allowed the screening of 600 bacterial isolates for BCAs
that can control Pythium root rot.
Another strategy to simplify a plant-based primary bioassay is to use
plant organs instead of whole plants as this requires less space and time.
In this respect, a multi-organ approach was applied by Haidar et al.
(2016a) to screen bacterial isolates from grapevines for their direct
inhibitory effect on B. cinerea and Neofusicoccum parvum, more speci-
fically using leaf disks, detached berries, and cuttings. This study re-
vealed the type of plant organ as an important determinant for the
screening output as clear differences in the inhibitory activity of the
tested strains were observed among the three plant organs used. Multi-
organ screening is therefore recommended, especially for screening
against pathogens able to infect different plant organs, such as B. ci-
nerea. In other cases, such as for postharvest pathogens, the choice of
plant organ is more straightforward. For example, Perez et al. (2017)
and Abraham et al. (2010) both used citrus fruits to identify antagonists
of the postharvest fungus P. digitatum. Finally, regarding the need of in
vivo plant material for screening for BCAs against obligate biotrophic
pathogens, plant parts are also reportedly used, as exemplified by the
study of Zhang et al. (2017) on grapevine leaf disks for identification of
antagonists of the downy mildew pathogen P. viticola.
From the current review, however, it is clear that the majority of the
phenotype-based screenings for direct biocontrol are not plant-based
but uses an in vitro dual culture assay as a primary selection step. In this
type of assay, potential BCAs are detected in an easy, fast, and re-
producible way, which allows screening of relatively large collections
(Köhl et al., 2019), reportedly up to 2,648 (Berg et al., 2005). Because
of its high versatility, this type of assays allows the screening of dif-
ferent types of microorganisms (including bacteriophages (e.g.
Rombouts et al., 2016), bacteria (e.g. Berg et al., 2005), yeasts (e.g. da
Cunha et al., 2018), and fungi (e.g. Zheng et al., 2011)), cell-free cul-
ture filtrates (e.g. Ramesh and Phadke, 2012), and plant extracts (Sales
et al., 2016) as potential BCAs. Moreover, such setup can easily be
adapted for specific purposes such as screening for volatile BCAs (e.g.
Stinson et al., 2003; Atmosukarto et al., 2005; Fialho et al., 2011). Si-
milarly, this approach is also often used to screen libraries of candidate
BCAs against more than one pathogen (e.g. Agaras et al., 2015; Comby
et al., 2017; Daranas et al., 2019; Földes et al., 2000; Jensen et al.,
1998; Suárez-Estrella et al., 2013), which is not achieved in the ma-
jority of the plant-based methods (Pliego et al., 2011). This multi-pa-
thogen focus allows identification of a generalist BCA or a combination
of specific BCAs with activity against multiple pathogens, which will
facilitate the more widespread use of BCAs (Glare et al., 2012). On the
other hand, viral BCAs like bacteriophages have shown to be subjected
to great evolutionary fluxes as viruses in general are known to re-
combine and mutate at high frequencies (Gómez and Buckling, 2011;
Scanlan et al., 2011). This gives the potential to select for phages with
extended host ranges. An example is illustrated by the Appelman’s
protocol (Burrowes et al., 2019), in which a mixture of phages was used
in different dilutions to infect various strains of one bacterial species or
K. Raymaekers, et al.
different bacterial species. Due to recombination events between the
phages in the cocktail, hybrid phages were created with a presumably
broader host range (Burrowes et al., 2019).
In general, primary selection of potential BCAs via in vitro dual
culture assays has proven to be a valuable strategy to identify, on a
higher throughput scale, BCAs with further confirmed in vivo biocontrol
activity (e.g. Sharifazizi et al., 2017; Wang et al., 2015; Huang et al.,
2013; Ramesh and Phadke, 2012). However, this assay also implies
important limitations. First of all, screening for BCAs in a dual culture
assay is obviously restricted to those with direct biocontrol activity
against the pathogen. Moreover, further bias occurs on BCAs that in-
hibit pathogen growth (via parasitism or the production of antibiotics),
while overlooking BCAs that act by niche competition with the pa-
thogen or interacts with its virulence factors. Therefore, it is impossible
to evaluate the full spectrum of potential biocontrol mechanisms ex-
pressed by an individual BCA in this type of assays, limiting the op-
portunities to find promising novel BCAs (Köhl et al., 2019, 2011;
Pliego et al., 2011). Secondly, a correlation between the in vitro and in
vivo biocontrol activity of potential BCAs could not be found in all re-
ported dual assays studies (e.g. Burr et al., 1996; Daayf et al., 2003;
Lemessa and Zeller, 2007). A reason might be that the production of
antibiotics by the potential BCAs often depends on specific environ-
mental and nutritional conditions (Köhl et al., 2019, 2011; Pliego et al.,
2011). Regarding the latter, it appears that no single growth medium
can support the expression of the full range of antibiotics produced by a
specific organism (Pliego et al., 2011; Lemessa and Zeller, 2007; Daayf
et al., 2003). Altogether, it can be concluded that the wide-spread in
vitro dual culture assay is a valuable screening method for identification
of potential BCAs, but with limitations for specific applications and for
the exploration of the full biocontrol potential of the candidate BCAs
(Köhl et al., 2019, 2011; Pliego et al., 2011).
On the other hand, it is not possible to omit the plant as third
partner of the tripartite biocontrol interaction in screenings for BCAs
exerting indirect biocontrol activity (Shoresh et al., 2010). Such
bioassays, however, are time-, space- and labor-intensive, especially
when making use of whole plants, and therefore difficult to use in au-
tomated screenings. Moreover, when specifically aiming at ISR-indu-
cing BCAs, the BCA and pathogen need to be physically separated in the
bioassay to exclude potential direct effects, making the bioassay even
more complex (Köhl et al., 2019). This probably explains the limited
number of ISR-inducing BCAs reportedly identified using a phenotype-
based whole plant screening in soil (Akram et al., 2013; Wei et al.,
1991). Different adaptations to these assays have been proposed to cope
with these limitations. Firstly, miniaturization of these phenotype-
based assays to in vitro assays in 12-well (Han et al., 2012, 2006) and
96-well (Schreiber et al., 2011, 2008) MTPs has simplified them in
terms of time, plant material, and growth facilities, and also allowed
high-throughput screening of larger libraries. Secondly, the use of
markers specifically linked to the ISR phenotype have been demon-
strated to be a valuable strategy to make screenings for ISR-inducing
BCAs more straightforward and allow high numbers (up to 42,000) of
candidate BCAs to be screened (Knoth et al., 2009; Noutoshi et al.,
2012c,a,b; Siegrist et al., 1998; Zahid et al., 2017). Moreover, for all the
reported marker-based screenings a high predictive value of the marker
for the final phenotype could be observed through in planta disease
assays (Knoth et al., 2009; Noutoshi et al., 2012c, 2012a, 2012b;
Siegrist et al., 1998; Zahid et al., 2017). Important to note here is that
almost all these reported large-scale marker-based screenings (Knoth
et al., 2009; Noutoshi et al., 2012c,a,b; Zahid et al., 2017) were per-
formed on libraries containing non-living BCAs, and it remains unclear
whether they would also be applicable and with similar efficiency to
screen for living ISR-inducing BCAs. Also strikingly, though marker-
based screenings were successful for screening (non-living) BCAs with
indirect biocontrol activity, they appear rather rare for identification of
antagonistic BCAs and are mostly limited to in vitro detection of BCA-
produced hydrolytic enzymes and secondary metabolites on selective
13
Biological Control 144 (2020) 104240
media (Schoonbeek et al., 2007; Tokpah et al., 2016; Yu et al., 2011). A
plausible explanation is that these marker-based assays do not provide
clear additional advantages over the commonly used in vitro dual cul-
ture assay.
To conclude, when screening for BCAs several considerations should
be made. A first one is which type(s) of BCAs are envisaged, being living
BCAs, non-living natural BCAs, and/or non-living synthetic BCAs. This
choice will have a major impact on (i) the screening setup and com-
plexity; (ii) technical and financial aspects of BCA mass production,
formulation, and delivery; (iii) the application method; (iv) persis-
tence/survival in the field; (v) compatibility with existing disease
control practices; and (vi) regulatory aspects for release or application
in the field. A second important consideration is which type of bio-
control mechanism is preferred, being a direct or indirect mode of ac-
tion, or a combination of both. Further, the choice for phenotype-
versus marker-based screening very much depends on the desired type
of biocontrol mechanism, the size of the test library, the intended level
of throughput of the screening, and the availability of a marker with
high predictive value for the intended biocontrol phenotype. It should
however be kept in mind that any screening method will be somehow
biased and thus selective, meaning that not all potential BCAs will be
picked up when screening a candidate BCA collection for biocontrol
activity. Typical examples are the widely used dual culture assay, se-
lective for BCAs with direct antagonistic activity, and whole plant as-
says in which the application of candidate BCAs and pathogen are
physically separated, selective for ISR-inducing BCAs. Some screening
systems reported here are, intentionally or not, suited to detect both
direct and indirect biocontrol activity (e.g. Haidar et al., 2016a; Latz
et al., 2020; Milus and Rothrock, 1997; Santiago et al., 2015; Schreiber
et al., 2008). It is anyhow expected that further intensification of efforts
and efficiency in screenings for novel BCAs, acting through a direct
and/or indirect biocontrol mechanism, will result in the requested in-
creased supply of potent BCAs applicable in more sustainable and low-
pesticide disease control management programs such as IPM.
CRediT authorship contribution statement
Katrijn Raymaekers: Conceptualization, Investigation, Writing -
original draft, Writing - review & editing, Visualization. Lisa Ponet:
Conceptualization, Investigation, Writing - original draft, Writing - re-
view & editing, Visualization, Funding acquisition. Dominique
Holtappels: Investigation, Writing - original draft, Writing - review &
editing, Funding acquisition. Barbara Berckmans: Conceptualization,
Writing - review & editing, Funding acquisition. Bruno P.A. Cammue:
Conceptualization, Writing - original draft, Writing - review & editing,
Supervision, Project administration, Funding acquisition.
Acknowledgements
This research was funded by predoctoral scholarships from FWO-
strategic basic research to LP (1S84519N) and DH (1S02520N); BB is
supported through funding from the European Union’s Horizon 2020
research and innovation programme under the Marie Skłodowska-Curie
grant agreement No. 840819; BC acknowledges FWO for funding of his
FWO Research Project G.OC25.18 N and FWO-SBO project S006017N.
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