# Should the biofilm mode of life be taken into consideration for microbial biocontrol agents?

Caroline Pandin, Dominique Le Coq, Alexis Canette, Stéphane Aymerich, Romain Briandet

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Caroline Pandin, Dominique Le Coq, Alexis Canette, Stéphane Aymerich, Romain Briandet. Should the biofilm mode of life be taken into consideration for microbial biocontrol agents?. Microbial Biotechnology, 2017, 10 (4), 10.1111/1751-7915.12693. [hal-01605120](https://hal.science/hal-01605120)

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Should the biofilm mode of life be taken into
consideration for microbial biocontrol agents?

Caroline Pandin,<fnref n="1" /> Dominique Le Coq,<fnref n="1" />,<fnref n="2" />

Alexis Canette,<fnref n="1" /> Stéphane Aymerich<fnref n="1" /> and

Romain Briandet<fnref n="1" />*

Summary

Almost one-third of crop yields are lost every year due to microbial alterations and diseases. The main control strategy to limit these losses is the use of an array of chemicals active against spoilage and unwanted pathogenic microorganisms. Their massive use has led to extensive environmental pollution, human poisoning and a variety of diseases. An emerging alternative to this chemical approach is the use of microbial biocontrol agents. Biopesticides have been used with success in several fields, but a better understanding of their mode of action is necessary to better control their activity and increase their use. Very few studies have considered that biofilms are the preferred mode of life of microorganisms in the target agricultural bio-topes. Increasing evidence shows that the spatial organization of microbial communities on crop sur-faces may drive important bioprotection mechanisms. The aim of this review is to summarize the evidence of biofilm formation by biocontrol agents on crops and discuss how this surface-associated mode of life may influence their biology and interactions with other microorganisms and the host and, finally, their overall beneficial activity.

Introduction

Approximately 30% of crop yields are lost every year
worldwide, mostly due to diseases caused by pests,

weeds or pathogenic microorganisms (Teng and Krupa,
1980; Teng, 1987; Oerke, 1999, 2006; Savary et al.,
2012). The microbiological control of agricultural products
along the food chain is still mainly ensured by the exten-
sive use of chemical pesticides, preservatives and syn-
thetic drugs (Horrigan et al., 2002). Environmental
pollution and associated human diseases caused by this
excessive use of chemicals during last century has led
many agencies and governments worldwide to support an
alternative route, where agriculture can be productive and
economically viable, while still addressing societal
and environmental concerns (Anonymous, 1999; Hazell
and Wood, 2008; Aktar et al., 2009). Biological protection
strategies are used and encouraged from farm to forks to
prevent pathogen contaminations and livestock or crop
diseases (Pal and McSpadden Gardener, 2006; Sundh
and Melin, 2010; Jordan et al., 2014). Biological control,
or ‘biocontrol’, consists in the removal of the harmful activ-
ity of one organism via one or more organisms or natural
products extracted from microorganisms, plants, animals
or minerals (Pal and McSpadden Gardener, 2006).

The relationship between survival, persistence and vir-
ulence of pathogenic microorganisms with their biofilm
mode of life have been clearly established since the early
1980s (Costerton et al., 1978; Lam et al., 1980). Accord-
ing to the National Institute of Health, 80% of human
infections involves microbial biofilms (NIH, 2002). Biofilm-
associated infections have also been reported in agricul-
tural settings, e.g., in crops and animal diseases (Davey
and O'toole, 2000; Prigent-Combaret et al., 2012; Li
et al., 2015). Indeed, the sessile mode is the preferential
lifestyle of microorganisms, regardless of their biotope
(Davey and O'toole, 2000; Morris and Monier, 2003). A
biofilm can be described as a spatially structured commu-
nity of microorganisms, generally embedded in an extra-
cellular matrix, and adhering to a living or inert surface
(Costerton et al., 1999; O'Toole et al., 2000). Biofilm for-
mation is generally favoured in harsh environmental con-
ditions, such as low nutritive or toxic media (Rendueles
and Ghigo, 2015) and most bacteria can form biofilms in
various environments (Morris and Monier, 2003; Aparna
and Yadav, 2008). *Staphylococcus aureus* and *Pseu-
domonas aeruginosa* are two opportunistic pathogenic

Received 15 July, 2016; revised 19 January, 2017; accepted 20
January, 2017. *For correspondence. E-mail romain.briandet@
inra.fr; Tel. +33 1 74 07 16 98.

Microbial Biotechnology (2017) 00(00), 000–000

doi:10.1111/1751-7915.12693

<footnote n="1">¹Micalis Institute, INRA, AgroParisTech, Université Paris-Saclay, 78350 Jouy-en-Josas, France.</footnote>

<footnote n="2">²Micalis Institute, INRA, AgroParisTech, CNRS,
Université Paris-Saclay, 78350 Jouy-en-Josas, France.</footnote>
bacteria that cause a diverse set of diseases and are the most highly used model bacteria for biofilm studies. They can colonize the human nasopharynx and form biofilms when specific environmental conditions are met, causing invasive diseases, such as chronic pneumonia. These infections are difficult to treat because of the persistence of biofilms and their high resistance to antimicrobials (Blanchette and Orihuela, 2012; Ding et al., 2016a). Bacteria can colonize and form biofilms on stems, leaves and the rhizosphere of plants, as well as soil particles, mushrooms or organic compost (Figs 1A and 2) (Ramey et al., 2004; Prigent-Combaret et al., 2012). For example, *Dickeya dadantii*, the causal agent of soft rot disease in a wide range of plant species, can colonize and form biofilms on chicory leaves, causing disease due to the production of degradative enzymes (Prigent-Combaret et al., 2012; Pandin et al., 2016).

Although less explored, the formation of biofilms by moulds, yeast and algae, alone or in combination, in a variety of biotopes has also been reported (Morris and Monier, 2003; Aparna and Yadav, 2008; Zarnowski et al., 2014; He et al., 2016; Rajendran and Hu, 2016; Sheppard and Howell, 2016). *Aspergillus fumigatus*, a human pathogen, is a filamentous fungus that can form structured biofilms. The cohesive cement of the fungal biostructure is a polymeric extracellular matrix that protects the hyphae from the host immune system, similar to bacterial biofilms (Breitenbach et al., 2016; Mitchell et al., 2016; Sheppard and Howell, 2016; Shirazi et al., 2016). *Fusarium oxysporum* f. sp. *cucumerinum*, the pathogen responsible for cucumber Fusarium wilt, can also grow inter- and intracellularly, allowing the rapid colonization of the plant and biofilm formation (Li et al., 2015). Until recently, efforts in biofilm research have

<figure><img src="image_2.png" /><figcaption>**Fig. 1.** Biofilm formation on crops and *in vitro*: (A): On crops: The first step involves deposition on the substratum (1) followed by adhesion (2) to the support through cell wall decorations and extracellular appendages. Once attached, a proliferation phase (3) and the diversification of cell types initiate the spatial organization of the biostructure, leading to biofilm maturation (4). Biofilm ageing or environmental conditions unfavourable for the maintenance of the biofilm results in regulated dispersion of the biofilm (5), disseminating free cells and cell clusters that will start a new biofilm cycle on a new surface.</figcaption></figure>

B–D. *In vitro*: Structural diversity of three biocontrol agents as observed *in vitro* (24 h of axenic culture in microplates at 25°C) by confocal laser scanning microscopy (Leica SP8); (B) *Bacillus amyloliquefaciens* FZB42 expressing a green fluorescent protein (GFP), forming flat undifferentiated architecture, (C) *Bacillus amyloliquefaciens* SQR9 expressing a GFP and (D) *Bacillus subtilis* QST 713 (labelled in green with syto 9, Invitrogen, France) forming differentiated 3D biostructures.
<figure><img src="image_3.png" /><figcaption>**Fig. 2.** Microbial biofilms on the carpophore and culture compost of *Agaricus bisporus*.</figcaption></figure>

A–C. Confocal laser scanning microscopy of *Agaricus bisporus* carpophore (red autofluorescent hyphae), harbouring *Bacillus amyloliquefaciens* FZB42 expressing GFP and forming (A) clusters, (B) biofilm features and (C) bundles. *Agaricus bisporus* carpophores were immersed under axenic conditions in TSB (Tryptone Soy Broth, Sigma-Aldrich, France) inoculated with *Bacillus amyloliquefaciens* FZB42 (GFP tagged) and incubated for 48 h at 17°C. Observations were performed using a Leica SP8 (Leica Microsystems, Danaher, Germany).

D–G. Scanning electron microscopy of natural biofilms formed on *Agaricus bisporus* carpophore and compost protected with *Bacillus subtilis* QST 713, a biocontrol agent used at the French Mushroom Centre (Distré, France). Samples were fixed in 0.10 M cacodylate buffer containing 2.5% (v/v) glutaraldehyde (pH 7.4) and post-fixed in 1% osmium tetroxide. Samples were then dehydrated with increasing concentrations of ethanol at room temperature (50–100%). After drying, samples were mounted on grids, sputter-coated in argon plasma with platinum (Polaron SC7640, Elexience, France) and observed using a FE-SEM S4500 (Hitachi, Japan). (D) *Pseudomonas*-like bacteria with extracellular material, (E) *Bacillus*-like bacteria, (F) fungi hyphae with extracellular material, (G) bacterial microcolony.

focused mainly on the medical field and essentially
towards their eradication. With the emergence of biocon-
trol in agriculture, many microbiological products have

been developed and are used in fields (Borriss, 2015).
The main way of action of most of these commercial
products is the antagonistic effect of antimicrobial
molecules secreted by the biocontrol agent (Chowdhury et al., 2015; Mora et al., 2015). However, recent research in this field has made it possible to consider other major biological processes, including biofilm formation of biocontrol agents in crops (Bais et al., 2004; Bogino et al., 2013; De la Fuente et al., 2013).

## The formation of biofilms by microbial biocontrol agents

*Evidence of biofilm formation on crops by biocontrol agents*

There is ongoing research to identify new biocontrol agents from environmental isolates and numerous biocontrol products have been developed and put on the agricultural market, mostly in Europe and North America (Borriss, 2015). Various products are in use and are effective on a wide range of plants. These include biofungicides, bactericides and biofertilizers based on *Bacillus subtilis* QST 713 or *Bacillus amyloliquefaciens* FZB42 (Borriss, 2015). These biocontrol products have an antagonistic effect towards unwanted microbes due to their secretion of antimicrobials, such as surfactin, fengycin or iturin (Ongena et al., 2005; Ongena and Jacques, 2008; Cawoy et al., 2014, 2015; Saravanakumar et al., 2016). However, their precise mechanisms of action in fields are still unknown. Few studies have focused on the determinants of effective bioprotection. The surface colonization step and biofilm formation by biocontrol agents are highlighted in the publications cited in Table 1. These reports demonstrate that many biocontrol agents can form biofilms on crops and in the rhizosphere. It has also been shown that biofilm formation by biopesticides can be stimulated by plant root exudates (Espinosa-Urgel et al., 2002; Timmusk et al., 2005; Hagag and Timmusk, 2008; Khezri et al., 2011; Chen et al., 2013; Sang and Kim, 2014; Zhang et al., 2015), or by exposure of the microorganisms to antimicrobial products or stress (Bais et al., 2004; Selin et al., 2010; Fan et al., 2011; Xu et al., 2014; Chi et al., 2015; Wu et al., 2015; Zhou et al., 2016), but only a few studies have focused on biocontrol mechanisms that may be related to the properties of the mature biofilm itself, rather than the secretion of antimicrobials. *Bacillus* are ubiquitous spore forming bacteria predominantly found in soil. They are frequently used as biocontrol agents because they can sporulate and be stored for long periods (Branda et al., 2004; Borriss, 2015). *Bacillus amyloliquefaciens* FZB42 forms biofilms with little spatial organization *in vitro* (Fig. 1B), but exhibits a strong swarming capacity allowing a rapid surface colonization. For example, this strain can form biofilms on the fruiting body of *Agaricus bisporus* by forming bacterial clusters surrounded by extracellular matrix in contact with the mycelium of the

carpophore (Fig. 2A and B), as well as cell bundles (Fig. 2C). Fan et al. (2011) reported the induction of biofilm formation of *B. amyloliquefaciens* FZB42 by root exudates of maize and surfactin. Similarly, surfactin triggers biofilm formation by *B. subtilis* UMAF6614 on the melon phylloplane (Zeriouh et al., 2014). Root exudate of cucumber also drives the chemotaxis of *Bacillus amyloliquefaciens* SQR9 and induces the production of bacil-lomycin D that triggers biofilm formation in the rhizosphere (Xu et al., 2014). Similarly, stem lesions of rice induce the production of GltB, leading to the produc-tion of bacillomycin L and surfactin, both involved in the biofilm formation of *B. subtilis* Bs916 (Zhou et al., 2016). Other biocontrol agents, such as endophytes, can also form biofilms. For example, some bacteria of the genus *Paenibacillus* form biofilms in wheat seeds and protect them from the invasion of *Fusarium graminearum* (Díaz Herrera et al., 2016).

Another family of biocontrol agents consists of the Gram-negative *Pseudomonas*, ubiquitous bacteria found in many plant rhizospheres (Table 1) (Espinosa-Urgel et al., 2002; Steddom et al., 2002; Matilla et al., 2010; Selin et al., 2010). Biofilm formation by *Pseudomonas putida* 06909 on citrus roots is induced by exudates of the phytopathogen *Phytophthora parasitica*. The bacteria colonize the mycelium of the fungi by feeding on its exudates and then form a protective biofilm on the citrus roots, which prevents new growth of the pathogen (Steddom et al., 2002; Ahn et al., 2007).

Living in a biofilm profoundly alters microbial properties relative to the planktonic mode of life (Whiteley et al., 2001; Shemesh et al., 2007; Vlamakis et al., 2008, 2013; Bridier et al., 2011b). Ongoing research is currently deciphering the molecular mechanisms involved in biofilm formation and their repercussions on biocontrol efficacy.

*Molecular mechanisms involved in biofilm formation of biocontrol agents*

Until recently, few studies in the biocontrol field have considered that the preferred lifestyle of microorganisms in the environment is the biofilm mode of life. The main features associated with biofilm formation are a diversification of cell types and increased tolerance to the fluctuation of environmental factors, boosting microbial persistence in the environment (Vlamakis et al., 2008, 2013; Flemming et al., 2016). Bacteria and fungi can form biofilms on crops (as illustrated by the cultivated mushroom microbiota in Fig. 2), and in both cases, biofilm formation is composed of five major steps described in Fig. 1A (Costerton et al., 1999; O’Toole et al., 2000; Bianciotto et al., 2001; Davies, 2003; Triveni et al., 2012; Vlamakis et al., 2013; Pu et al., 2014; Haagensen
<table><caption>**Table 1.** Biocontrol agent reported to form biofilms and the described associated biocontrol mechanisms.</caption><thead><tr><td>Biocontrol strain</td><td>Host/Location</td><td>Biofilm induction</td><td>Biocontrol mechanism</td><td>References</td></tr></thead><tbody><tr><td>Bacillus atrophaeus 176s</td><td>Lettuce, sugar beet, tomato</td><td>Surfactin triggers biofilm formation</td><td>Induced systemic resistance (ISR) antimicrobial-producing biofilm (fengycin, surfactin)</td><td>(Aleti et al., 2016)</td></tr><tr><td>Bacillus subtilis</td><td>Wheat seeds</td><td>Root exudates, death or lysis of cortex cells</td><td>Biofilm formation, antimicrobial, volatile compounds decrease mycelial growth</td><td>(Khezri et al., 2011)</td></tr><tr><td>Bacillus subtilis 3610</td><td>Tomato roots</td><td>Root exudates induce matrix</td><td>Antimicrobial-producing biofilm (surfactin)</td><td>(Chen et al., 2013)</td></tr><tr><td>Bacillus subtilis 6051</td><td>Arabidopsis thaliana</td><td>Surfactin triggers biofilm formation</td><td>Antimicrobial-producing biofilm (surfactin)</td><td>(Bais et al., 2004)</td></tr><tr><td>Bacillus subtilis Bs916</td><td>Rice stem</td><td>Stem lesions induce GltB production triggering bacillomycin L and, surfactin production involved in biofilm formation</td><td>Antimicrobial-producing biofilm (fengycin)</td><td>(Zhou et al., 2016)</td></tr><tr><td>Bacillus subtilis UMAF6614</td><td>Melon phylloplane</td><td>Surfactin triggers biofilm formation</td><td>Antimicrobial-producing biofilm (bacillomycin, fengycin)</td><td>(Zeriouh et al., 2014)</td></tr><tr><td>Bacillus amyloliquefaciens SQR9</td><td>Cucumber roots</td><td>Root exudates induce chemotaxis and enhance bacillomycin D production</td><td>Antimicrobial-producing biofilm (bacillomycin)</td><td>(Xu et al., 2014)</td></tr><tr><td>Bacillus amyloliquefaciens SQR9</td><td>Maize roots</td><td>Root exudates induce the expression of genes related to extracellular matrix production</td><td>Promote plant growth</td><td>(Zhang et al., 2015)</td></tr><tr><td>Bacillus amyloliquefaciens SQY 162</td><td>Tobacco roots</td><td>Pectin enhances surfactin production, increasing biofilm biomass</td><td>May trigger induced systemic resistance (ISR) antimicrobial-producing biofilm (surfactin)</td><td>(Wu et al., 2015)</td></tr><tr><td>Bacillus amyloliquefaciens FZB42</td><td>Maize roots</td><td>Root exudates and surfactin trigger biofilm formation</td><td>Likely not linked with the production of antibiotic or biofilm formation</td><td>(Fan et al., 2011)</td></tr><tr><td>Paenibacillus polymyxa</td><td>Arabidopsis thaliana</td><td>Root exudates induce matrix synthesis</td><td>Niche exclusion and mechanical protection</td><td>(Timmusk et al., 2005)</td></tr><tr><td>Paenibacillus polymyxa A26</td><td>Wheat seeds</td><td>Not mentioned</td><td>Niche exclusion of pathogens</td><td>(Abd El Daim et al., 2015)</td></tr><tr><td>Paenibacillus polymyxa B5</td><td>Arabidopsis thaliana</td><td>Root exudates</td><td>Niche exclusion of pathogens</td><td>(Haggag and Timmusk, 2008)</td></tr><tr><td>Pseudomonas corrugata CCR04 and CCR80</td><td>Pepper roots</td><td>Root exudates</td><td>Competitive colonization, such as swimming and swarming activities, biofilm formation, antimicrobial activity</td><td>(Sang and Kim, 2014)</td></tr><tr><td>Pseudomonas chlororaphis PA23</td><td>Canola roots</td><td>Phenazine enhances biofilm formation</td><td>Antimicrobial-producing biofilm (pyrrolnitrin)</td><td>(Selin et al., 2010)</td></tr><tr><td>Pseudomonas putida 06909</td><td>Citrus roots</td><td>Phytophthora exudates as attractants and growth substrates for bacteria</td><td>Biofilm formation and mycelial colonization of the pathogen Phytophthora</td><td>(Steddom et al., 2002; Ahn et al., 2007)</td></tr><tr><td>Pseudomonas putida KT2440</td><td>Corn roots Arabidopsis thaliana</td><td>Root exudates</td><td>Promote plant growth and induced systemic resistance (ISR)</td><td>(Espinosa-Urgel et al., 2002; Matilla et al., 2010)</td></tr><tr><td>Pichia kudriavzevii</td><td>Pear fruit</td><td>Oxidative stress</td><td>Greater activation of the antioxidant system in the biofilm form</td><td>(Chi et al., 2015)</td></tr><tr><td>Kloeckera apiculate</td><td>Citrus fruit</td><td>Phenylethanol promotes filamentous adhesion and biofilm formation</td><td>Niche exclusion and mechanical protection</td><td>(Pu et al., 2014)</td></tr></tbody></table>

et al., 2015; Li et al., 2015; Gulati and Nobile, 2016; Sheppard and Howell, 2016). *Bacillus subtilis* is the most highly documented bacterial model currently used to study the regulatory molecular mechanisms that govern biofilm formation. One specificity of the biofilm mode of life is the diversification of cell types. The presence of several bacterial subpopulations within the biofilm of *B. subtilis* has been clearly demonstrated, suggesting the spatiotemporal regulation of gene expression within such 3D structures (Vlamakis et al., 2008, 2013). Matrix-producing cells, surfactin-producing cells, flagellated motile cells and sporulated cells coexist in the same

community (Fig. 1A) and are spatially and temporally organized, differentially expressing specific sets of genes (Vlamakis et al., 2008; van Gestel et al., 2015; Mielich-Süss and Lopez, 2015; Wang et al., 2016). Indeed, the combination of surfactin- and matrix-producing cells enables the organization of cells into bundles (Fig. 2C). These interfacial microbial cables allow bacteria to visit surrounding spaces to increase the biofilm surface area for nutrient and oxygen intake (van Gestel et al., 2015). Several genes involved in the phenotypic heterogeneity have been identified and extensively analysed in this species. For example, *hag*, encoding a flagellar protein
and expressed by a subpopulation of motile cells; tasA,
eps, blsA expressed by matrix-producing cells; sfrA,
involved in the production of surfactin lipopeptide; sigF,
involved in cell sporulation; swr, involved in swarming
motility; and the com genes, involved in genetic compe-
tence (Kearns et al., 2004; Verhamme et al., 2007;
López and Kolter, 2010; Vlamakis et al., 2013; van Ges-
tel et al., 2015; Mielich-Süss and Lopez, 2015). All these
genes are directly or indirectly regulated by various regu-
lators (e.g. Spo0A, DegU, ComA, SinI, SinR, AbrB),
which can thus play a role in the regulation of plant bio-
protection by B. subtilis (López and Kolter, 2010; Vla-
makis et al., 2013; Cairns et al., 2014; Mielich-Süss and
Lopez, 2015; Romero et al., 2016). Indeed, a mutation in
a gene coding for a positive regulator (e.g. SinI) will
decrease plant colonization and protection by diminish-
ing attachment of cells to the roots, while mutations in a
gene coding for a repressor (e.g. SinR, AbrB) will
increase plant protection by an increased numbers of
root-attached cells and the formation of hyper-robust
biofilms (Chen et al., 2013).

Major components of biofilm structure that ensure its
cohesion are the extracellular polymeric substances
(EPS) that are mostly composed of water and extracellu-
lar biopolymers (polysaccharides, proteins, DNA, lipids)
(Flemming and Wingender, 2010). Many microbial EPS
have a backbone composed of various biomolecules
forming gels with various cohesive and viscoelastic prop-
erties. Trapping a high amount of water is important for
microbial survival against desiccation on plant surfaces
(Abdian and Zorreguieta, 2016). This organic slime also
protects their inhabitants from the action of environmen-
tal pollutants and toxic compounds (Sutherland, 2001;
Sheppard and Howell, 2016). Another important compo-
nent of the biofilm structure are amyloid fibres formed by
the protein TasA. These filaments bind cells together,
leading to formation of complex structures in biofilms that
can hold and concentrate molecules (e.g. quorum sens-
ing signalling molecules), and may also form aggregates
to defend cells within the biofilm (de Jong et al., 2009;
Romero et al., 2010; Flemming et al., 2016).

Several studies have recently highlighted various
physiological behaviours of *Bacillus* within biofilm com-
munities, demonstrating the high level of complexity of
their interactions (Mitri *et al.*, 2011; Houry *et al.*, 2012;
Liu *et al.*, 2015; Prindle *et al.*, 2015; Flemming *et al.*,
2016). Prindle *et al.* (2015) described a new function for
ion channels in biofilms in which they conduct electrical
signals *via* spatial propagation of potassium waves
which depolarize adjoining cells and coordinate the state
of the exterior and interior cells of the biofilm. In addition,
Liu *et al.* (2015) discovered a ‘collective oscillation’ phe-
nomenon involved in toxic chemical tolerance, based on
metabolic codependency between exterior and interior

cells of the biofilm, and consisting of cyclic pauses dur-
ing biofilm growth which increase the availability of nutri-
ents in the deepest layers. Houry et al. (2012) also
demonstrated that motile bacilli, expressing a bacteri-
cide, can kill a heterologous biofilm population and then
occupy the newly created space (Houry et al., 2012).
Altogether, these cellular traits show the complexity of
living associated with a surface in a spatially organized
microbial community. They also give an overview of the
protection that biofilms can provide to their inhabitants
on plant surfaces. Those basic insights into biofilm
development and interaction might pave our way
towards various applications in the field of crop
protection.

Biofilm-specific properties that should be considered
in biocontrol mechanisms

Only a few published studies have considered the possi-
bility of interspecies and microbial–host interactions in
spatially organized plurimicrobial biofilms involved in
agricultural biocontrol (De la Fuente et al., 2013; Triveni
et al., 2015) (Table 1). The biofilm-associated properties
to be considered can be divided into five classes
(Fig. 3): (i) antagonism by niche exclusion orchestrated
by spatial and nutritive competition (Timmusk et al.,
2005; Haggag and Timmusk, 2008; Pu et al., 2014; Abd
El Daim et al., 2015), (ii) microbial communication, e.g.
cooperation/interference (Hogan et al., 2004; Audrain
et al., 2015; Chen et al., 2015), (iii) production of antimi-
crobials by biofilm cells (Bais et al., 2004; Selin et al.,
2010; Chen et al., 2013; Sang and Kim, 2014; Xu
et al., 2014; Zeriouh et al., 2014; Wu et al., 2015; Zhou
et al., 2016), (iv) stress tolerance (Timmusk et al., 2005;
Harriott and Noverr, 2009; Pu et al., 2014) and (v) direct
effects on plant physiology, e.g. the induction of plant
defences (Wu et al., 2015) and/or stimulation of plant
growth (Espinosa-Urgel et al., 2002; Zhang et al., 2015).
This new vision could significantly change our under-
standing of the interactions involved in biocontrol by con-
sidering them in terms of spatial/nutritive competition
(Habimana et al., 2011), tolerance/resistance (Bridier
et al., 2011a) or their physiology, as microorganisms in a
biofilm differ greatly from their planktonic homologues
(Stewart and Franklin, 2008). These local processes are
described in the following sections, using illustrative
examples from other fields, if they have not been
explored yet in the microbial biocontrol area.

Spatial and nutritive competition

The spatial organization of biocontrol agent biofilms on
crop surfaces varies depending on their genetic potential
and the environmental conditions. For example, the
<figure><img src="image_4.png" /><figcaption>**Fig. 3.** Proposed mechanisms of plant interactions with biocontrol agents and pathogenic strains. (IDR: induced disease resistance).</figcaption></figure>

biofilms of *Bacillus* biocontrol agents display wide archi-
tectural diversity between strains. *In vitro*, biofilms of
*B. amyloliquefaciens* SQR9 and *B. subtilis* QST 713
exhibit the classical thick and highly organized 3D struc-
ture of bacilli (Fig. 1C and D). In contrast, *B. amylolique-
faciens* FZB42 forms only thin structures of a few cell
layers (Fig. 1B). However, this strain outcompetes the
other two due to its swarming activity, leading to rapid
coverage of the entire surface. This ability to rapidly col-
onize a niche (Fig. 3) has been described previously as
a potential biocontrol mechanism and could be called
upon for the strain *B. amyloliquefaciens* FZB42 (Tim-
musk *et al.*, 2005; Haggag and Timmusk, 2008; Fan
*et al.*, 2011; Abd El Daim *et al.*, 2015). For *Paenibacillus
polymyxa*, root exudates of plants induce invasive root
colonization and biofilm formation that invades sites that
could be potentially occupied by pathogens, thus pre-
venting them from settling onto the surface by forming a
protective biofilm (Timmusk *et al.*, 2005; Haggag and
Timmusk, 2008; Abd El Daim *et al.*, 2015). In an orga-
nized, 3D community, nutrients may be consumed faster
than they can diffuse throughout the matrix (Breugel-
mans *et al.*, 2008; Stewart and Franklin, 2008). Growth
and survival in such a dense community is frequently
associated with spatial competition. Habimana *et al.*
(2011) explained the inhibition of *Listeria monocytogenes*

by *Lactococcus lactis* on surfaces by considering the 3D
race between the two species. Using a simplified individ-
ual-based model approach, they demonstrated that the
differences in the growth parameters (lag phase and
growth rate) of the two species could explain the
observed inhibition of the pathogenic cells. *Lactococcus
lactis* cells rapidly formed layers on the mixed commu-
nity and completely saturated the interface in contact
with the nutrient, limiting nutrient access to the pathogen.
This example illustrates that part of a biofilm population
can be starved within the bulk of the biostructure, even
in a very rich environment. In addition, Liu *et al.* (2016)
underlined that the specific interactions between species,
such as strong or weak cooperation, exploitation or com-
petition, contribute mostly to the spatial organization of
biofilms, as these interactions create fitness effects in
multispecies biofilms. Taking spatial organization and
interspecies interactions within multispecies biofilms into
account could increase our understanding of the interac-
tions that take place in agrosystems that use biocontrol
agents.

Antibiosis

The production of secondary metabolites by selected
organisms is one of the best described mechanisms of
agricultural microbial biocontrol (Ongena et al., 2005; Ongena and Jacques, 2008; Khezri et al., 2011; Cawoy et al., 2014, 2015; Chen et al., 2015; Aleti et al., 2016; Raza et al., 2016; Saravanakumar et al., 2016). *Bacillus* genomes contain many genes involved in the production of secondary metabolites, recently compiled in an exhaustive classification of known and putative antimicrobial compounds (Zhao and Kuipers, 2016). Indeed, 4–5% of the genome of *B. subtilis* is allocated to the production of antibiotics and 8.5% of the genome of *B. amyloliquefaciens* FZB42 is allocated to the production of secondary metabolites with antimicrobial properties (Stein, 2005; Chen et al., 2009; Zhao and Kuipers, 2016). Many exhibit interesting antibacterial properties (e.g. difficidin), antifungal properties (e.g. bacillomycin D, fengycin and surfactin), or both (e.g. bacilysin) (Ongena and Jacques, 2008; Chen et al., 2009; Guo et al., 2014; Guo et al., 2015; Chowdhury et al., 2015; Luo et al., 2015; Kröber et al., 2016). Most of the studies that have analysed the profile of antimicrobial production have relied on experiments using planktonic laboratory cultures. However, in *B. amyloliquefaciens* FZB42, the genes involved in bacilysin synthesis are overexpressed in biofilms, suggesting that the bacteria have a stronger antagonistic effect in their sessile mode of life (Fig. 3) (Kröber et al., 2016). Similarly, in *B. subtilis*, the regulator NtdR controls the expression of the *ntdABC* operon, encoding enzymes involved in the biosynthesis of the antibiotic kanosamine (Inaoka et al., 2004; Vetter et al., 2013). A global transcriptomic study that compared gene expression of *B. subtilis* in various modes of life showed that this operon is strongly overexpressed in biofilms (Nicolas et al., 2012), suggesting the possible involvement of kanosamine in interspecies interactions in plurimicrobial biofilms. Volatile organic compounds (VOCs) can also trigger antimicrobial activity (Khezri et al., 2011; Audrain et al., 2015; Raza et al., 2016). Raza et al. (2016) demonstrated that VOCs of *B. amyloliquefaciens* SQR9 inhibited the growth of *Ralstonia solanacearum* on agar medium or in soil. Altogether, these studies show that secondary metabolites with antimicrobial activity can be overproduced (or simply produced) in the biofilm lifestyle, improving antagonistic biocontrol activity. The presence of EPS or amyloid fibres in biofilms can also locally concentrate these molecules and prevent their dilution into the ambient aqueous environment, and thus presumably increase the virulence of biocontrol agents against pathogens in agrosystems (Bianciotto et al., 2001; Romero et al., 2010; Xu et al., 2014; Flemming et al., 2016). Previous studies highlighted effects of antimicrobials secreted by one producer on crop protection. Santhanam et al. (2015) have also shown that in certain cases, a consortium of different antimicrobial producers is required for optimal plant bioprotection.

Microbial communication

Biofilms are dense, spatially organized communities of microorganisms with extensive forms of social life. They can use specific signalling molecules (autoinducers) that allow them to sense and communicate with the local surrounding populations (Fuqua et al., 1994). This quorum sensing (QS) is involved in various biological processes, such as swarming, stress tolerance (pH, antimicrobials, etc.), the production of secondary metabolites, horizontal gene transfer, colonization, biofilm maturation and the synthesis of virulence factors (Fuqua et al., 1994; Von Bodman et al., 2003). These signalling pathways are widely used in bacteria–bacteria and bacteria–eukaryote associations to regulate and coordinate their interactions. For example, N-acylhomoserine lactones (AHL) in Gram-negative bacteria, oligopeptides in Gram-positive bacteria and gamma-butyrolactones in species of the genus *Streptomyces* are autoinducers (Danhorn and Fuqua, 2007). In *Pseudomonas aeruginosa*, QS controls the expression of many bacterial functions. The LasI-LasR QS system, with the autoinducer synthase LasI and the signal receptor LasR, is involved in biofilm maturation and the organization of its 3D structure. A lasI mutant can initiate biofilm formation but is unable to form a mature biofilm, suggesting that LasI is involved in the late stages of biofilm development (Davies et al., 1998; De Kievit et al., 2001). Kavanaugh and Horswill (2016) demonstrated that the *Staphylococci* QS system, agr, is involved in biofilm disruption and dispersal.

In the field of biocontrol, it was shown that the protec-
tive activity of *Pseudomonas fluorescens* 2P24 on wheat
was mainly controlled by the Pcol-PcoR QS system that
governs biofilm formation, and not directly by the produc-
tion of antimicrobial metabolites (Wei and Zhang, 2006).
Such social behaviour has been shown to also govern
intermicrobial and interkingdom interactions, such as
communication interference represented in Fig. 3 or
cooperative communication (Zhang and Dong, 2004;
Kalia, 2013). For example, *P. aeruginosa* secretes
3-oxo-C12-HSL that affects the growth of *C. albicans*
hyphae and inhibits its biofilm formation (Hogan *et al*.,
2004). Amyloid fibres of the matrix form aggregates that
can act as QS inhibitors by binding QS signalling mole-
cules, and thus locally concentrate these molecules that
can reach a 1000-fold higher concentration in the matrix
than in planktonic cell environments (Charlton *et al*.,
2000; Hense *et al*., 2007; Romero *et al*., 2010; Flem-
ming *et al*., 2016). Other types of molecules can quench
or degrade QS signalling molecules of another species
(Zhang and Dong, 2004). Indeed, AHL-lactonase of
*Bacillus thuringiensis* hinders the accumulation of AHL of
*Erwinia carotovora*, thus decreasing the virulence of this
bacterium on potatoes (Dong *et al*., 2004). In *Bacillus*,
the lipopeptide surfactin, in addition to its antibiotic prop-
erties, can act like a signalling molecule to promote bio-
film formation of the other relative *Bacillus* (López *et al*.,
2009; Aleti *et al*., 2016). Volatile organic compounds
emitted by prokaryotic and eukaryotic microbes are also
part of their communication repertoire and can trigger
global reprogramming of gene expression of their per-
ceivers (Farag *et al*., 2013; Audrain *et al*., 2015; Raza
*et al*., 2016). For example, acetic acid emitted by *B. sub-
tilis* biofilms can promote biofilm formation of other physi-
cally separated *B. subtilis* cells and thus act as an
important pathway of cell–cell communication (Audrain
*et al*., 2015; Chen *et al*., 2015). These communication
phenomena specific to biofilms, or amplified in biofilms,
could be used to improve biocontrol in agrosystems.

Stress tolerance: adaptability properties and matrix as a
protective shield

Bacteria in biofilms exhibit specific physiologies associ-
ated with increased tolerance/resistance of the overall
community to harsh conditions (Costerton et al., 1999;
Whiteley et al., 2001; Shemesh et al., 2007; Bridier
et al., 2011b). Physiological differences between sessile
and planktonic cells are mostly related to differential pat-
terns of gene expression associated with the two modes
of life (Whiteley et al., 2001; Shemesh et al., 2007).
Transcriptomic analysis of *Streptococcus mutans*, a bac-
terium associated with tooth decay, showed that 12% of
the genome was differentially expressed in biofilm com-
munities relative to their single-cell homologues. The dif-
ferentially expressed genes coding for known functions
are involved in transport, signalling, metabolism, protein
and antimicrobial synthesis (Shemesh et al., 2007). Mark
et al. (2005) evaluated the influence of exudates of two
varieties of sugar beets on the transcriptomic profile of
*Pseudomonas aeruginosa* PAO1. They showed that the
expression of 516 genes was altered in response to one
exudates and 451 to the other, and 134 genes
responded to both. They found that genes coding for the
synthesis of alginate, a major component of the biofilm
matrix, were upregulated. These results suggest that
*P. aeruginosa* PAO1 forms a biofilm in response to
sugar beet exudates. They also showed that the tran-
scriptomic profile of *Pseudomonas aeruginosa* PAO1 in
response to exudates is variety dependent. Similarly,
Matilla et al. (2007) compared the transcriptome profiles
of *Pseudomonas putida* KT2440 in the planktonic expo-
nential growth phase, the planktonic stationary growth
phase, the sessile form, in sand microcosms and in the
rhizosphere. They showed that transcriptomic profile of
the planktonic mode of life in the stationary growth
phase was the most different from that of the rhizo-
sphere, whereas that of the biofilm lifestyle was more

comparable. Indeed, they found that the gene involved
in the synthesis of alginate was upregulated in the rhizo-
sphere (Matilla et al., 2007; : additional data file). These
'omics' studies confirm the presence of biofilm formation
in the rhizosphere or in response to plant exudates.
These techniques should be increasingly considered in
the study of microbial interactions in agrosystems and
extended to metagenomics, metaproteomics and meta-
transcriptomic approaches as successfully performed in
other fields (Blottière et al., 2013; Kaul et al., 2016).

Other cellular variations can occur during biofilm for-
mation. In the early stages, *Pseudomonas aeruginosa*
shows enhanced genetic diversification. Resulting phe-
notypes vary from cells involved in accelerated biofilm
formation to those with enhanced dispersion properties.
In the first case, biofilms exhibited pronounced spatial
differentiation leading to rough and wrinkled colonies on
agar. In the second case, the biofilms showed little spa-
tial organization resulting in small and flat colonies
(Boles et al., 2004). These differences illustrate the
genetic plasticity of cells within a biofilm that enables
them to cope with harsh environmental conditions. Ste-
wart and Franklin (2008) also reported the existence of
nutrient and oxygen gradients within biofilms creating a
stratification of local microenvironments associated with
a diversification of cell physiologies (Fig. 3). Population
heterogeneity can generate multiple regulatory pathways
leading, for example, to the phenomenon of competence
in a subpopulation of cells, which coupled with the spa-
tial proximity, facilitates horizontal gene transfer between
biofilm cells. This can include the acquisition of plasmids
carrying antimicrobial resistance genes (Witte, 2000;
Abraham, 2011; Kung and Almeida, 2014; Liu et al.,
2016). This diversification of cell types in biofilms
strongly suggests that the biofilm lifestyle of biocontrol
agents enables them to better adapt to, and resist, the
hostile conditions encountered in agrosystems (the
so-called insurance effects in Boles et al., 2004) than
their planktonic counterparts.

Biofilms are ubiquitous and subject to harsh condi-
tions, such as the presence of antimicrobials or desicca-
tion. Stewart (2015) recently performed a meta-analysis
of the literature from which he proposed that biofilm tol-
erance to antimicrobials depends neither on the size or
chemistry of the antimicrobials nor the composition of
the microbial biofilm or the material to which it adheres
(Stewart, 2015). Based on his analysis, biofilm-asso-
ciated tolerance is primarily related to the nature and
composition of the biofilm matrix. Indeed, the composi-
tion of the matrix creates various meshes within the bio-
film leading to a diffusion–reaction limitation that can
reduce antimicrobial penetration and local biodisponibility
(Fig. 3) (Stewart et al., 2001; Stewart and Franklin,
2008; Flemming and Wingender, 2010; Bridier et al.,
2011b; Stewart, 2015). Stewart (2015) also stressed that the presence of the matrix and the associated 3D organization renders slow-growth populations less sensitive to certain stresses than their planktonic counterparts.

The matrix also plays a central role in interspecies and interkingdom interactions. *Staphylococcus aureus* and *Candida albicans* are often associated in human diseases, where they form a polymicrobial biofilm (Harriott and Noverr, 2009; Lindsay and Hogan, 2014). This association allows *S. aureus* to resist vancomycin, an antibiotic that is usually efficient against the planktonic form of *S. aureus*. The biofilm of *C. albicans* serves as the backbone of *S. aureus* microcolonies that form on their surface with the matrix of *C. albicans* covering and protecting the cells of *S. aureus* from the action of the antibiotic (Harriott and Noverr, 2009). Other reports have described the protection of *Staphylococcus epidermidis* by *C. albicans* (Adam et al., 2002) or of *S. aureus* by *B. subtilis* (Sanchez-Vizuete et al., 2015). In the latter case, the authors identified a single gene of the *B. subtilis* NDmed whose disruption suppressed the protective effect. This gene (*ypqP*) is likely involved in the production of matrix exopolysaccharides (Sanchez-Vizuete et al., 2015). Non-polysaccharidic components of the matrix can also contribute to the matrix shield; the amphiphilic protein BlsA produced by *B. subtilis* prevents the penetration of biocides by forming a hydrophobic ‘raincoat’ layer at the biofilm-air interface (Epstein et al., 2011; Kobayashi and Iwano, 2012). Biocontrol agents likely benefit from this protective shield on crop surfaces to avoid invasion by aggressive detrimental flora and protect crops.

## The direct response of crops to biocontrol agents

Plants can develop natural defence systems against pathogenic microorganisms during their interactions with their environment (biotic and abiotic) (Pieterse and Wees, 2015). Several induced diseases resistance (IDR) mechanisms have been described, including induced systemic resistance (ISR) that is an innate defence mechanism of the plant (Choudhary and Johri, 2009; Pieterse and Wees, 2015) elicited by various environmental stimuli, such as VOCs and QS signalling molecules (Farag et al., 2006; Choudhary and Johri, 2009; Matilla et al., 2010; Wu et al., 2015; Aleti et al., 2016). Various stimuli, such as VOCs, QS signals and phytohormones produced and concentrated in the biofilm matrix, can stimulate plant growth, analogous to ISR (Fig. 3) (Espinosa-Urgel et al., 2002; Farag et al., 2006; Han et al., 2006; Spaepen, 2015; Zhang et al., 2015; Díaz Herrera et al., 2016; Ding et al., 2016b). Thus, VOCs, originating from various sources, can induce ISR and plant growth (Yi et al., 2009; Farag et al., 2013;

Audrain et al., 2015; Raza et al., 2016; Sharifi and Ryu, 2016). These host responses can also be induced by products coming from plant growth-promoting rhizobacteria that have already colonized the root surface, or endophyte colonization of its host (Whipps, 2001; Farag et al., 2006; Borriss, 2015; Díaz Herrera et al., 2016). The plant growth-promoting rhizobacteria *B. subtilis* GB03 and *B. amyloliquefaciens* IN937a can produce 2,3-butanediol and acetoin on plant roots and promote both plant growth and ISR by eliciting these phenomena (Ryu et al., 2003, 2004; Farag et al., 2006). Han et al. (2006) also showed that the surface area of tobacco leaves increased when they were exposed to 2,3-butanediol secreted by *Pseudomonas chlororaphis* O6 or exposed to the strain itself, even without physical contact. Phytohormones (auxins, cytokinins, gibberellins, abscisic acid and ethylene) are elicitors, which can be produced by bacteria and play a role in promoting plant growth (Spaepen, 2015; Zhang et al., 2015). The auxin, indole-3-acetic acid, is produced by *B. amyloliquefaciens* SQR9 and *B. amyloliquefaciens* FZB42 biofilms and promotes the growth of maize and *Lemna minor* (Chen et al., 2007; Idris et al., 2007; Zhang et al., 2015). Endophytes can promote growth of wheat and protect it from *Fusarium graminearum* (Díaz Herrera et al., 2016).

## Perspectives for sustainable agroecological approaches

Biocontrol mechanisms triggered by biological control agents in agriculture are not yet well understood, and even unknown in certain cases. A single biocontrol agent can use a combination of various biocontrol processes, best described for the strain *B. amyloliquefaciens* FZB42. The use of this bacilli can lead to antagonism, spatial and nutritional competition, antimicrobial production, the stimulation of plant growth and the induction of plant resistance (Timmusk et al., 2005; Haggag and Timmusk, 2008; Babalola, 2010; Fan et al., 2011; Xu et al., 2011; Kröber et al., 2014; Chowdhury et al., 2015; Kröber et al., 2016; Abd El Daim et al., 2015). The biofilm mode of life is still poorly taken into account in biocontrol, although it clearly plays a role in agrosystems and governs some of the observed beneficial effects. It would be informative, in the near future, to include phenotypic screening of the ability of strains to form biofilms as a rapid selection criterion of biocontrol agents. Several high-throughput screening tests that could be used for this application are described in the literature (Azeredo et al., 2016). Better genetic knowledge of the various cell functions in biofilms will also open doors to selection criteria based on the presence of specific genes involved in important and specific biocontrol functions (Kaul et al., 2016).
Invoking biofilm formation as a determinant of biocon-
trol efficacy could be a new attractive strategy to better
control its beneficial effects. This could be achieved, for
example, using natural biofilm promoting molecules that
trigger the biocontrol agent QS response. In the case of
*B. subtilis*, this could be surfactin, VOCs, specific micro-
bial exopolysaccharides or crop extracts (Bais *et al.*,
2004; Chen *et al.*, 2013; Chen *et al.*, 2015; Audrain *et al.*,
2015; Zhou *et al.*, 2016). This effect could also be
obtained by adding a second strain (or more) with the
ability to stimulate biofilm formation by the initial biocon-
trol agent, for example through VOC synthesis (Filoche
*et al.*, 2004; Audrain *et al.*, 2015; Chen *et al.*, 2015; Fig-
ueiredo *et al.*, 2016). Attention should be paid, in this
case, to select strains without antagonistic activity against
each other or the beneficial action of the biocontrol agent,
as previously reported for certain cocktails in the literature
(Xu *et al.*, 2011). The benefit of the biofilm mode of life
for biocontrol agents could also be obtained using dedi-
cated formulations, as suggested in other areas; for
example, the development of new formulas grown as bio-
films to orally administer probiotics (e.g. beads of agar or
alginates) is under consideration (Rieu *et al.*, 2014). Simi-
larly, a system using bacteria-containing polymersomes,
which permits rapid biofilm growth, has been developed
for bioremediation to reduce the toxicity of environmental
selenium contamination (Barlow *et al.*, 2016).

Increasing evidence, based on available data from the
agrosystem and biofilm fields, strongly suggests that the
combination of features associated with the 3D biofilm
mode of life should be considered when evaluating the
performance of biocontrol organisms.

Acknowledgements

This project was supported by the Région Ile-de-France,
DIM ASTREA. We thank the MIMA2 imaging platform
and J. Deschamps (INRA) for assistance in microscopy.
We thank R. Borriss and R. Zhang for providing the
*Bacillus amyloliquefaciens* FZB42 and SQR9 strains
respectively. Finally, we warmly thank R. Védie and T.
Rousseau from the French Mushroom Centre (Centre
Technique du Champignon, Distré) for helpful discus-
sions, and Alex Edelman and Associates for English
revision of the manuscript.

Conflict of interest

None declared.

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