# Modes of action for biological control of Botrytis cinerea by antagonistic bacteria

Rana Haidar, Marc Fermaud, Carlos Calvo Garrido, Jean Roudet, Alain Deschamps

► To cite this version:

Rana Haidar, Marc Fermaud, Carlos Calvo Garrido, Jean Roudet, Alain Deschamps. Modes of action for biological control of Botrytis cinerea by antagonistic bacteria. Phytopathologia Mediterranea, 2016, 55 (3), pp.13-34. 10.14601/Phytopathol_Mediterr-18079. hal-01602491

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REVIEW

# Modes of action for biological control of *Botrytis cinerea* by antagonistic bacteria

RANA HAIDAR<fnref n="1" />,<fnref n="2" />, MARC FERMAUD<fnref n="1" />, CARLOS CALVO-GARRIDO<fnref n="1" />, JEAN ROUDET<fnref n="1" /> and ALAIN DESCHAMPS<fnref n="1" />

**Summary.** The role of beneficial bacteria in biocontrol of plant diseases, particularly those caused by the necrotrophic fungus *Botrytis cinerea*, has been investigated by testing many bacteria under laboratory and field conditions. Bacteria may protect plants against *B. cinerea* by direct antagonistic interactions between biocontrol agents and this pathogen, as well as indirect effects through the induction of host resistance. This review focuses on various bacteria that act as biological control agents (BCAs) of *B. cinerea* and their associated mechanisms. The modes of action (MoAs) include: i) synthesis of anti-fungal metabolites, such as antibiotics, cell wall-degrading enzymes and volatile organic compounds (VOCs); ii) competition for nutrients and/or a niche; and iii) induction of host resistance. The challenge for development of BCAs is to reduce the variability of efficiency and to prove persistence under a large range of conditions. We discuss the advantages and drawbacks of MoA for future applications of bacteria in the field and in post-harvest storage, as well as combination of different MoAs as a strategy to achieve a more regular efficacy.

**Key words:** biocontrol, grey mold, antibiosis, competition, induced systemic resistance.

## Introduction

*Botrytis cinerea* is a major necrotrophic fungal pathogen causing grey mold, a serious disease affecting a large number of economically important agricultural and horticultural crops. This pathogen leads to considerable yield and quality losses in field production and postharvest storage worldwide (Jarvis, 1977; Williamson *et al.*, 2007; Sharma *et al.*, 2009). In spite of the availability of various anti-*Botrytis* fungicides, their use in pre- and/or post-harvest conditions is not considered as sustainable because of their potential adverse effects on human and environmental health (Komárek *et al.*, 2010) and the appearance of resistant strains (Leroux, 2004; Walker *et al.*, 2013; Hahn, 2014; Romanazzi and Feliziani, 2014). Because of its high genetic variability,

short life cycle, and prolific reproduction, *B. cinerea* is considered a high-risk pathogen for the development of fungicide resistance (Brent and Hollomon, 1998; Leroux *et al.*, 2002). This resistance has been detected in many countries and on numerous crops (Leroux, 2004; Zhao *et al.*, 2010; Walker *et al.*, 2013; Hahn, 2014). Development of complementary and alternative methods to chemical control, such as the use of non-pathogenic microorganisms (or their secretions) as biological control agents (BCAs), before and/or after harvest, is increasingly considered as a promising and attractive alternative.

In recent decades, there has been continued and rigorous research worldwide with a greater impetus to explore a wide range of bacteria possessing antagonistic properties against *B. cinerea* (Elmer and Reglinski, 2006; Compant *et al.*, 2013). However, in the majority of these studies, the efficacy of the BCAs was evaluated under controlled conditions, and the fact that most of them fail in the field is now widely

<footnote n="1">¹ SAVE, Bordeaux Science Agro, INRA, 33882, Villenave d'Ornon, France</footnote>

<footnote n="2">² Tichreen University, Faculty of Science, Biology Department, PO Box 2231, Latakia, Syrian Arab Republic</footnote>

<footnote>Corresponding author: R. Haidar
E-mail: haidarrana@gmail.com</footnote>
known. Despite the large number of scientific papers published on this topic, the number of efficient bacteria commercialized for use as microbial fungicides against *B. cinerea* in the pre- and/or postharvest stages remains limited (Nicot *et al.*, 2011; Romanazzi *et al.*, 2016) (Table 1).

Specific attention is given to the mechanisms by which beneficial bacteria antagonize plant pathogen activity. It is important to know the precise mode(s) of action (MoAs) of a BCA to develop and implement an adapted control strategy in the field. Therefore, understanding the MoAs may help to improve the regularity of biocontrol efficiency by using the BCA in an active form and with the appropriate inoculum quantity. This knowledge should help to devise successful and reproducible biological control methods under pre- and/or post-harvest conditions. Furthermore, the MoA may be among the key parameters for product development, and may also be relevant for marketing purposes (Köhl *et al.*, 2011).

Interest in different MoAs exhibited by bacterial BCAs against *B. cinerea* has increased in the last decades. Early studies conducted by Blakeman and co-workers have reported the important role of the competition for nutrients by bacteria in the inhibition of the conidial germination of *B. cinerea* (Sztejnberg and Blakeman, 1973; Brodie and Blakeman, 1975). In a recent review about the biological control of grapevine pathogens (Compant *et al.*, 2013), the authors indicated only a few examples of beneficial

bacterial strains for control of gray mold with their potential MoAs. Recently, several reviews have presented data on the use of plant growth promoting rhizobacteria (PGPR) as soil inoculants for the biocontrol of plant diseases. Some of these studies have taken account of the mechanisms of the biocontrol bacteria against a wide range of pathogens (Bloemberg and Lugtenberg, 2001; Labuschagne *et al.*, 2010; Karunaratne, 2011; Pan and Bhagat, 2011; Bhattacharyya and Jha, 2012; Glick, 2012; Gupta *et al.*, 2015). These reviews addressed the biological control of *B. cinerea* and also included other plant pathogens.

To our knowledge, there have been two reviews dealing with the mechanisms involved in the biological suppression of *B. cinerea* (Elad, 1996; Elad and Stewart, 2004). The authors discussed the involvement of different MoAs in the biological suppression of *B. cinerea*, not only by bacteria but also using other microorganisms. In the present, we have restricted the scope to the different MoAs of bacterial antagonists against *B. cinerea* because of the huge breadth of the subject matter dealing with this important plant pathogen. We give a special emphasis to the different mechanisms by which beneficial bacteria might inhibit *B. cinerea*. We report various examples of bacteria that exert direct and/or indirect effects on the pathogen in a wide range of crop pathosystems. The diversity and function of the inhibiting bacteria cited in the literature were examined, which is helpful for

<table><caption>**Table 1.** Commercially available biopesticides based on bacteria, for control of gray mold on different crops.</caption><thead><tr><td>®Trade name</td><td>Bacterial strain</td><td>Company (and/or country)</td></tr></thead><tbody><tr><td>Pantovital®</td><td>Pantoea agglomerans</td><td>IRTA (Spain)</td></tr><tr><td>Serenade Max®</td><td>Bacillus subtilis</td><td>Bayer, formerly BASF (Germany)</td></tr><tr><td>Bio-save®</td><td>Pseudomonas syringae</td><td>Jet harvest solutions (USA)</td></tr><tr><td>Amylo-X®</td><td>Bacillus amyloliquefaciens</td><td>Biogard CBC (Italy)</td></tr><tr><td>Double Nickel 55WDG/LC™</td><td>Bacillus amyloliquefaciens</td><td>Certis (USA)</td></tr><tr><td>Companion®</td><td>Bacillus subtilis GB03</td><td>Growth products (USA)</td></tr><tr><td>Botokira Wettable Powder®</td><td>Bacillus subtilis IK-1080</td><td>(Idemitsu Kosan Inc., Japan)</td></tr><tr><td>Bio Arc®</td><td>Bacillus megaterium</td><td>Sphere Bio-Arc PVTLtd (India)</td></tr><tr><td>Mycostop®</td><td>Streptomyces griseoviridis Strain K61</td><td>Verdera Oy (Finland)</td></tr><tr><td>Actinovate®</td><td>Streptomyces lydicus WYEC 108</td><td>Novozymes (Denmark)</td></tr></tbody></table>
future research aiming to develop new bacterial bio-control agents against *B. cinerea*.

## Competition for space and nutrients

Competition occurs when biocontrol agents and pathogens require the same resources (Campbell, 1989). Efficient competition for the available nutrients and for niches represents an important mechanism accounting for bacterial protection of plants against various pathogens (Stockwell et al., 1998; Punja and Utkhede, 2003; Pal and McSpadden-Gardener, 2006; Cabrefiga et al., 2007; Lugtenberg and Kamilova, 2009). The suppression of *B. cinerea* infection through competition for niches and nutrients has been shown to be efficient in several *in situ* and *in vivo* studies (Table 2). *Botrytis cinerea* is highly susceptible to competition because external nutrients are required for conidial germination (Elad, 1996), germ tube growth and the successful completion of infection (Redmond et al., 1987; Elad and Stewart, 2004). Furthermore, this mechanism was suggested to be one of the major mechanisms in the control of post-harvest diseases (Duffy, 2001; Spadaro and Gullino, 2004; Sharma et al., 2009; Jamalizadeh et al., 2011). The ability of antagonists to rapidly colonize

fruit wounds, prior to colonization by *B. cinerea*, is an important feature of post-harvest biocontrol agents that reduce grey mold incidence and/or severity in fruits (Nunes et al., 2001, 2002; Sharma et al., 2009; Jamalizadeh et al., 2011).

Given the limited nutritional resources at the leaf surface (Andrews, 1992; Huang and Erickson, 2005), the efficiency of phylosphere colonization with nutrient uptake by bacteria is a key feature for successful antagonism by exhausting the available substrates and thus, reducing *Botrytis* infection. For instance, in greenhouse conditions, *Pseudomonas putida* Cha94 and *Bacillus amyloliquefaciens* BL3 controlled *B. cinerea* infection on pepper by colonizing the floral and foliar canopies (Park et al., 1999). Similarly, in bean and tomato plants, the severity of the *B. cinerea* infection and sporulation was reduced by several saprophytic bacterial isolates including *Xanthomonas maltophilia*, *Bacillus pumilus*, *Lactobacillus* sp. and *Pseudomonas* sp. (Elad et al., 1994). The effect of these isolates was due to competition on detached leaves for the available nutrients. Similarly, competition for nutrients by bacteria near spores may result in increased inhibition of germination of *B. cinerea* spores on leaf surfaces (Sztejnberg and Blakeman, 1973; Brodie and Blakeman, 1975).

<table><caption>**Table 2.** Examples of bacteria providing biocontrol of *Botrytis cinerea*, through competition for space and/or nutrients.</caption><thead><tr><td>Antagonistic bacteria</td><td>Plant/fruit/ conditions</td><td>Mechanism of inhibition</td><td>Reference</td></tr></thead><tbody><tr><td>Pantoea agglomerans LRC 954, Pseudomonas fluorescens LRC 1788</td><td>Lentil seedling</td><td>Competition for space and nutrients</td><td>Huang and Erickson, 2005</td></tr><tr><td>Erwinia herbicola (Pantoea agglomerans)</td><td>Apple juice</td><td>Competition for nutrients</td><td>Bryk et al., 1998</td></tr><tr><td>Pseudomonas putida Cha94, B. amyloliquefaciens BL3</td><td>Pepper</td><td>Competition for space and nutrients</td><td>Park et al., 1999</td></tr><tr><td>Rahnella aquatilis</td><td>Apple fruit / in vitro</td><td>Competition for nutrients</td><td>Calvo et al., 2007</td></tr><tr><td>Pantoea agglomerans</td><td>Apple fruit</td><td>Competition for space and nutrients</td><td>Nunes et al., 2002</td></tr><tr><td>Bacillus sp.(isolate UYBC38)</td><td>In vitro / in situ (grape, peach, and apple)</td><td>Competition for nutrients</td><td>Rabosto et al., 2006</td></tr><tr><td>Paenibacillus polymyxa</td><td>Strawberry fruit pulp suspension</td><td>Competition for nutrients</td><td>Helbig, 2001</td></tr><tr><td>Xanthomonas maltophilia, Bacillus pumilus, Lactobacillus sp., Pseudomonas sp.</td><td>Bean and tomato</td><td>Competition for nutrients</td><td>Elad et al., 1994</td></tr></tbody></table>
In the rhizosphere, good establishment of introduced bacteria is also considered a critical factor that can limit their biocontrol efficacy (Lugtenberg and Kamilova, 2009). For example, the suppression of *B. cinerea* symptoms on lentil seedlings was demonstrated following rhizosphere colonization by *Pan-toea agglomerans* LRC 954 and *Pseudomonas fluorescens* LRC 1788 (Huang and Erickson, 2002, 2005).

Although conidial germination of *B. cinerea* depends significantly on nutrient availability, interference with the subsequent infection process may also occur by utilizing antagonistic bacteria that use the available nutrients more efficiently than the pathogen. The application of competitive bacteria on floral organs, harbouring pollen grains, stigma exudates, and dead and/or senescent petals, deprives *B. cinerea* of important nutrient sources. Moreover, the exclusion of *B. cinerea* from an infection site by bacteria colonizing rapidly in this area might be a suitable method for protecting wounded host tissues, notably wounded fruits during maturation and post-harvest storage.

However, under post-harvest conditions, and regarding the rapid colonization potential of *B. cinerea*, growth and viability potential of introduced bacteria are required to enhance their efficacy and to ensure durable effects against the pathogen. It is important to note here that more BCA examples, involving this MoA, are found in yeasts. The capacity of yeasts to grow rapidly, with the rapid depletion of available nutrients, is associated with formation of biofilms covering wounds, and their tolerance to lower relative humidity levels make them highly efficient competing BCAs under postharvest conditions (Wilson and Wisnieswski, 1994; Liu et al., 2013; Spadaro and Droby, 2016). In general, yeasts produce fewer antibiotics than bacteria, so this may be an advantage for using yeasts as BCAs under post-harvest conditions (Wisniewski and Wilson, 1992; Liu et al., 2013).

## Antibiosis

Antibiosis is a commonly assumed mechanism in biocontrol activity of bacteria on leaf surfaces, in the rhizosphere and in fruit wounds (Ghaouth et al., 2004). According to Thomashow et al. (1997), antibiotics encompass a chemically heterogeneous group of organic, low molecular weight compounds produced by microorganisms, that are deleterious to the growth or metabolic activities of other microorganisms.

Antibiotic-producing microorganisms have often been considered first in screening for BCAs, using *in vitro* tests performed on agar and/or in liquid culture. Many literature reports support the great potential of bacteria that produce antifungal metabolites to control *B. cinerea* both *in vitro* and on a wide range of host plant species. The potential antifungal metabolites of numerous and various bacterial genera and species (*Bacillus*, *Paenibacillus*, *Pseudomonads*, *Burkholderia* and *Streptomyces*) are illustrated in Table 3.

Extensive research programmes show that *Bacillus* is among the most effective bacterial genera for control of plant diseases (Nagorska et al., 2007; Ongena and Jacques, 2008; Raaijmakers et al., 2010; Kumar et al., 2011). *Paenibacillus*, originally included within *Bacillus*, has been reclassified as a separate genus (Ash et al., 1993). It is widely accepted that the *Bacillus* and *Paenibacillus* species produce various antifungal substances, including lipopeptide antibiotics, anti-fungal proteins, volatile compounds, lytic enzymes, other antibiotics and plant defense-related enzymes (Selim et al., 2005; Huang and Chen, 2008; Ongena and Jacques, 2008; Raza et al., 2008; Govindasamy et al., 2010; Malfanova et al., 2012; Zhang et al., 2013; Zhao et al., 2013). Although most of the secondary metabolites produced by *Bacillus* spp. are peptide antibiotics (Ongena and Jacques, 2008; Pérez-García et al., 2011; Falardeau et al., 2013), the production of many non-peptide antibiotics has been reported for several *Bacillus* spp. (Baruzzi et al., 2011; Hamdache et al., 2012).

Among these antimicrobial compounds, the cyclic lipopeptides (LPs) are well studied in the context of the biological control of *B. cinerea* by bacteria. The involvement of these compounds in the protective action of the *Bacillus* spp. against *B. cinerea* under pre- and post-harvest conditions has been widely reported. *Bacillus subtilis* is a key species, and it inhibits the growth of a wide range of plant pathogens by producing a great variety of powerful antifungal metabolites, including lipopeptides (Stein, 2005; Ongena et al., 2005). The cyclic lipopeptides belong to three major families, i.e., surfactin, iturin and fengycin (or plipastatin). They have been highlighted for their potential use in biotechnological and biopharmaceutical applications because of their surfactant properties (Ongena and Jacques, 2008). Surfactin shows surfactant activity and bactericidal effects and also hemolytic, antiviral and antimycoplasma
<table><caption>**Table 3.** Overview of literature reporting inhibition of *Botrytis cinerea* by antifungal metabolites produced by bacterial species.</caption><thead><tr><th>Antagonistic bacteria</th><th>Plant/fruit/ conditions</th><th>Antifungal metabolites</th><th>Effect</th><th>Reference</th></tr></thead><tbody><tr><td><i>Pseudomonas cepacia</i> strain LT412</td><td>Apple and pears</td><td>Pyrrolnitrin</td><td>Mycelial growth inhibition and disease reduction</td><td>Janisiewicz and Roitman, 1988</td></tr><tr><td><i>Pseudomonas corrugata</i> strain p94</td><td>Tomato leaf</td><td>Secondary metabolites (bacteriocin)</td><td>Disease reduction</td><td>Guo et al., 2007</td></tr><tr><td><i>Pseudomonas fluorescens</i>, <i>Bacillus</i> sp.</td><td>Grapes</td><td>Not identified</td><td>Disease reduction</td><td>Krol, 1998</td></tr><tr><td><i>Pseudomonas fluorescens</i> strain Pf1TZ</td><td><i>In vitro</i> / <i>in planta</i> (vine vitroplants)</td><td>Not identified</td><td>Mycelial growth inhibition, and disease reduction</td><td>Kilani-Feki et al., 2010</td></tr><tr><td><i>Pseudomonas sp.</i>, <i>Pseudomonas fluorescens</i>, <i>Pseudomonas putida</i><br><i>Pseudomonas aeruginosa</i></td><td><i>In vitro</i></td><td>Not identified</td><td>Mycelial growth inhibition</td><td>Trotel-Aziz et al., 2006; Kruijt et al., 2009; Kumar et al., 2011; Patkowska and Konopiński, 2014; Hernández-León et al., 2015</td></tr><tr><td><i>Burkholderia phytofirmans</i> PsJN</td><td><i>In vitro</i> / grapevine plantlets</td><td>Not identified</td><td>Mycelial growth inhibition and disease reduction, plant growth promotion</td><td>Ait Barka et al., 2000; Ait Barka et al., 2002</td></tr><tr><td><i>Burkholderia gladioli</i></td><td><i>In vitro</i> / <i>in situ</i> (leaves of strawberry)</td><td>Not identified</td><td>Inhibition of conidial germination</td><td>Walker et al., 1996; Walker et al., 2001</td></tr><tr><td><i>Burkholderia cepacia</i> Cs5</td><td><i>Vitis vinifera</i> plants</td><td>Didecyl-phthalate and two methyl-alkyl-quinolones</td><td>disease reduction</td><td>Kilani-Feki and Jaoua, 2011</td></tr><tr><td><i>Bacillus</i> sp. PB10, <i>Bacillus licheniformis</i>, <i>Bacillus megaterium</i>, <i>Bacillus subtilis</i>, <i>Bacillus sp.</i></td><td><i>In vitro</i> / cut leaves of tomato</td><td>Not identified</td><td>Mycelial growth inhibition and disease reduction</td><td>Enya et al., 2007</td></tr><tr><td><i>Bacillus licheniformis</i>, <i>Bacillus megaterium</i></td><td><i>In vitro</i> / perilla plants</td><td>Not identified</td><td>Inhibition of mycelial growth and conidial germination, disease reduction</td><td>Son et al., 2002</td></tr><tr><td><i>Bacillus licheniformis</i> N1</td><td>tomato plants</td><td>Not identified</td><td>Disease reduction and plant growth promotion</td><td>Lee et al., 2006</td></tr><tr><td><i>Bacillus subtilis</i> CL27, <i>B. pumilus</i> CL45</td><td><i>In vitro</i></td><td>Antibiotic appeared not to be peptide</td><td>Mycelial growth and sporulation inhibition</td><td>Leifert et al., 1995</td></tr><tr><td><i>Bacillus subtilis</i> CL27</td><td><i>Astilbe</i> seedlings</td><td>Antibiotic appeared not to be peptide</td><td>Disease reduction</td><td>Leifert et al., 1995</td></tr></tbody></table>

(Continued)
<table><caption>Table 3. (Continued).</caption><thead><tr><th>Antagonistic bacteria</th><th>Plant/fruit/ conditions</th><th>Antifungal metabolites</th><th>Effect</th><th>Reference</th></tr></thead><tbody><tr><td>Bacillus subtilis<br>Brevibacterium linens</td><td>In vitro / tomato fruit</td><td>Antifungal compounds, including surfactins</td><td>Mycelial growth inhibition and disease reduction</td><td>On et al., 2015</td></tr><tr><td>Bacillus subtilis CU12</td><td>In vitro</td><td>Antimicrobial compound (acyclic dimer of 3-hydroxypropionaldehyde (HPA))</td><td>Mycelial growth inhibition</td><td>Wise et al., 2012</td></tr><tr><td>Bacillus subtilis GA1</td><td>Apple fruits</td><td>Fengycins</td><td>Mycelial growth inhibition and disease reduction</td><td>Touré et al., 2004</td></tr><tr><td>Bacillus subtilis BBG100</td><td>In vitro</td><td>Mycelial growth inhibition</td><td>Mycosubtilin and surfactin</td><td>Leclère et al., 2005</td></tr><tr><td>Bacillus subtilis M4</td><td>Apple fruits</td><td>Cyclolipoproteides like fengycins</td><td>Disease reduction</td><td>Ongena et al., 2005</td></tr><tr><td>Bacillus subtilis B-916</td><td>In vitro</td><td>Antifungal protein: Bacisubin</td><td>Mycelial growth inhibition</td><td>Liu et al., 2007</td></tr><tr><td>Bacillus atrophaeus CAB-1</td><td>In vitro</td><td>Lipoproteides: fengycin (C15-C17), a type of unidentified lipopeptide, and antifungal protein (putative phage-related pre-neck appendage protein)</td><td>Mycelial growth inhibition</td><td>Zhang et al., 2013</td></tr><tr><td>Bacillus sp. BS061</td><td>In vitro / tomato seedlings</td><td>Not identified</td><td>Mycelial growth inhibition and disease reduction</td><td>Kim et al., 2013</td></tr><tr><td>Bacillus amyloliquefaciens strain TF28</td><td>In vitro</td><td>Crude lipopeptides (iturin A)</td><td>Mycelial growth inhibition</td><td>Zhang et al., 2012</td></tr><tr><td>Bacillus amyloliquefaciens CNU114001</td><td>In vitro / tomato plants</td><td>Lipoprotein: iturin</td><td>Spore germ tube elongation inhibition and disease reduction</td><td>Ji et al., 2013</td></tr><tr><td>Bacillus amyloliquefaciens IMAUB1034</td><td>In vitro / tomato plants</td><td>Antifungal cyclic lipopeptide (analogue of the surfactin)</td><td>Mycelial growth inhibition and disease reduction</td><td>Wang et al., 2012</td></tr><tr><td>Brevibacillus brevis</td><td>In vitro / leaf sections of Chinese cabbage</td><td>Antibiotic (gramicidin S)</td><td>Germination and mycelial growth inhibition</td><td>Edwards and Seddon, 2001</td></tr><tr><td>Paeribacillus polymyxa, Brevibacillus brevis</td><td>In vitro / strawberry fruits and plants</td><td>Peptide polymyxin B (P. polymyxa) and gramicidin S (B. brevis)</td><td>Germination and mycelial growth inhibition, disease reduction</td><td>Haggag, 2008; Haggag et al., 2013</td></tr></tbody></table>

(Continued)
<table><caption>Table 3. (Continued)</caption><thead><tr><th>Antagonistic bacteria</th><th>Plant/fruit/ conditions</th><th>Antifungal metabolites</th><th>Effect</th><th>Reference</th></tr></thead><tbody><tr><td>Bacillus subtilis TS 01 and ZR 02, Bacillus subtilis, Bacillus sp., Bacillus lentimorbus, Bacillus megaterium, Bacillus subtilis, B. subtilis, Bacillus subtilis CPA-8, B. amyloliquefaciens, Bacillus sp.; Bacillus sp.</td><td>In vitro</td><td>Not identified</td><td>Mycelial growth inhibition</td><td>Todorova and Kozhuharova, 2009; Walker et al., 1998; Ferreira, 1990; Yánez-Mendizábal et al., 2011; Hamdache et al., 2012; Cui et al., 2014; Patkowska and Konopiński, 2014</td></tr><tr><td>Streptomyces sp., Micromonospora sp.</td><td>In vitro / in vitro grapevine leaves and plantlets</td><td>Not identified</td><td>Morphological changes and severe structural alterations of the fungal mycelium</td><td>Loqman et al., 2009</td></tr><tr><td>Streptomyces araujoniae ASBV-IT</td><td>Strawberry pseudofruits</td><td>Multiantibiotic complex: macrotetralides class (including monactin, dinactin, trinactin, and tetranactin) and the cyclodepsipeptide valinomycin</td><td>Conidia germination inhibition and disease reduction</td><td>Silva et al., 2014</td></tr><tr><td>Streptomyces barakatei Streptomyces beta-vulgaris Streptomyces hygroscopicus, Streptomyces rimosus, Streptomyces anulatus</td><td>In vitro / grapevine leaves</td><td>Secondary metabolites</td><td>Mycelial growth inhibition and disease reduction</td><td>Lebrihi et al., 2009 a,b</td></tr><tr><td>Streptomyces S37</td><td>In vitro, grapevine plantlets</td><td>streptochlorin, nigericin and piericidin A1.</td><td>Mycelial growth inhibition and disease reduction</td><td>Liu et al., 2004; Ilic et al., 2007; Xin and Ming-shan, 2008; Svetlana et al., 2008; Postolaky et al., 2012; Couillerot et al., 2014</td></tr><tr><td>Xenorhabdus nematophila TB</td><td>In vitro, detachedtomatofruits</td><td>Antimicrobialmetabolites</td><td>Mycelial growth inhibition and disease reduction</td><td>Couillerot et al., 2014</td></tr></tbody></table>
activities, but no marked fungitoxicity. Iturin and
fengycin are cyclopeptides with antifungal activity
(Leclère et al., 2005; Ongena and Jacques, 2008; Raai-
jmakers et al., 2010), particularly against pathogenic
fungi and yeasts, but no antibacterial activity (Onge-
na and Jaques, 2008). In addition to these three main
lipopeptide families, other lipopeptides, including
kurstakin, maltacines, and polymyxins, have been
identified in various *Bacillus* spp. (Raaijmakers et
al., 2010; Borriss, 2015). The antimicrobial activity
of lipopeptides is due to their ability to bind to li-
pid membrane bilayers in the pathogen cells, which
causes permeability changes and/or structure dam-
age. While fengycin and iturin produce pores in the
plasmamembrane, surfactin is reported to dissolve
this structure (Henry et al., 2011; Patel et al., 2011;
Pérez-García et al., 2011).

Previous studies demonstrated the important
role of lipopeptides, not only in disease suppression
(Thomashow and Weller, 1988; Keel et al., 1989) but
also in the ability of the *Bacillus* species to colonize
plant rhizospheres (Cao et al., 2011; Fan et al., 2011).
Furthermore, these compounds play a key role in
the beneficial interaction between *Bacillus* spp. and
plants by acting as elicitors, stimulating plant im-
mune-related responses and then suppressing dis-
eases (Ongena and Jacques, 2008; Jourdan et al., 2009;
Cawoy et al., 2014).

Pseudomonads possess many traits that make them well suited as biocontrol and growth-promoting agents. These include i) effective competition and colonization of plant surfaces, ii) the capacity to utilize seed and root exudates, and iii) rapid growth *in vitro* and the production of a wide range of bioactive metabolites, such as antibiotics, siderophores, volatiles compounds, and growth-promoting substances (Weller, 1988, 2007).

Many compounds produced by *Burkholderia* sp. exhibit antifungal activity, including lipopeptides, cepaciamides A and B, cepacidines, siderophores, altericidin, pyrrolnitrin, glidobactins and volatile compounds (Schmidt *et al.*, 2009; Tenorio-Salgado *et al.*, 2013).

Many species of actinomycetes are also of great
interest, particularly Streptomycetes, because nearly
two thirds of all known antibiotics are produced by
*Streptomyces* (Taechowisan *et al.*, 2012; Lucas *et al.*,
2013; Mahajan and Balachandran, 2015). The genus
includes well-known antifungal BCAs that inhibit
various pathogenic fungi (El-Tarabily *et al.*, 2000; Er-

rakhi *et al.*, 2007; Loqman *et al.*, 2009; Hamedi and
Mohammadipanah, 2015).

The multiple antifungal activities of several anti-
biotics, their direct action on pathogens and the pos-
sible use of the antibiotic-rich bacterial culture su-
pernatants makes antibiosis one of the most illustrat-
ed bacterial MoAs. There have been comprehensive
overviews of the antibiotics produced by beneficial
bacteria used as BCAs, their potential functions
and their role in biocontrol (Raaijmakers et al., 2002;
Raaijmakers and Mazzola, 2012). However, little in-
formation is available about bacterial production of
antimicrobial compounds under field conditions.
The impact of several abiotic parameters on this pro-
duction, notably for lipopeptides, was reported by
considering oxygen availability, temperature and ni-
trate sources (Tampakaki et al., 2009; Pretorius et al.,
2015). The potential wash-off by rainfall of the active
compound(s) may also occur in the field. Antibiosis is
considered an important MoA in the biological con-
trol of post-harvest diseases, but as discussed above,
a BCA that does not produce antibiotics, or produces
only a small amount of the compounds, might be
more suitable under such conditions. Moreover, the
resistance of the fungal pathogen, particularly B. ci-
nerea, against antibiotics produced by bacterial con-
trol agents has already been reported (Schoonbeek et
al., 2002; Duffy et al., 2003; Bardin et al., 2015). Further
investigations should address the environmental im-
pacts and biosafety issues of antibiotic compounds
produced by bacteria used as BCAs.

Production of lytic enzymes and the
interference with pathogen activity and
growth

In addition to antibiotics, some biocontrol bacte-
rial strains produce other metabolites, such as en-
zymes that can interfere with the metabolic activi-
ties and/or growth of pathogens (Elad, 1996; Duffy
et al., 2003; Compant et al., 2005). Some enzymes
can degrade or detoxify virulence factors of plant
pathogens. For example, oxalic acid contributes to
the virulence of *B. cinerea* (Germeier et al., 1994). The
degradation of oxalate by a bacterial strain closely
related to *Cupriavidus campinensis* (named “oxB”) re-
duced *B. cinerea* pathogenicity of *Arabidopsis thaliana*
and crop plants (Schoonbeek et al., 2007). The pro-
duction of extracellular cell wall-degrading enzymes
is associated with the biocontrol abilities of bacteria.
Lytic enzymes, such as chitinases, glucanases, cel-
lulases, proteases, and lipases that degrade cell wall
components of pathogenic fungi, contribute to direct
suppression of plant pathogens (Neeraja *et al.*, 2010;
Maksimov *et al.*, 2011; Bouizgarne, 2013). Halophilic
bacteria, such as *Virgibacillus marismortui*, *B. subtilis*,
*B. pumilus*, *B. licheniformis*, *Terribacillus halophilus*,
*Halomonas elongata*, *lanococcus rifietoensis*, *Staphylococ-
cus equorum* and *Staphylococcus* sp., moderately sup-
pressed growth of *B. cinerea* *in vitro*, by producing
extracellular antifungal hydrolytic enzymes, includ-
ing chitinase, β-1,3-glucanase, cellulase and protease
(Essghaier *et al.*, 2009). With respect to the control of
*B. cinerea* by *Bacillus cereus*, the effect of strain IO8
was mediated by chitinase production (Hammami
*et al.*, 2013). However, for another strain, B-02, anti-
fungal activity was caused by effects on DNA syn-
thesis, mitochondrial membrane potential and the
reactive oxygen quantity in the pathogen hyphae
(Li *et al.*, 2012). Similarly, the role of chitinases was
also revealed, in *Bacillus thuringiensis* UM96, to ac-
count for the protection of *Medicago truncatula* from
*B. cinerea* infection (Martínez-Absalón *et al.*, 2014).
Joo (2005) demonstrated antifungal activity of puri-
fied chitinase from *Streptomyces halstedii* AJ-7 against
various red pepper fungal pathogens, including *B.
cinerea*.

Two strains of *Pseudomonas* sp. (B194 and B224) and two strains of *Erwinia herbicola* lysed *B. cinerea* germ tubes and hyphae (Bryk *et al.*, 1998, 2004). *Seratia plymuthica* and *S. marcescens* produced lytic enzymes, such as chitinases and proteases, and inhibited growth of pathogens, including *B. cinerea*, *Fusarium oxysporum*, *Sclerotinia sclerotiorum* and *Rhizoctonia solani* (Frankowski *et al.*, 2001; Kamensky *et al.*, 2003; Ningaraju, 2006).

Another MoA is the inhibition of hydrolytic en-
zyme synthesis in *B. cinerea* during the first phase
of the host-pathogen interaction, which is crucial
for infection success (Baarlen *et al.*, 2007; Nakajima
and Akutsu, 2014; Nafisi *et al.*, 2015). Decreases in
the activities of pectin lyase and polygalacturonase
produced by *B. cinerea* were observed during bio-
logical control on vines by *Bacillus circulans* (Paul
*et al.*, 1997). Similarly, bacterial metabolites, such as
siderophores, may control *B. cinerea* by inhibiting
portions of pathogen virulence factors. As an exam-
ple, enterochelin, from the enterobacterium *Rahnella
aquatilis* BNM, inhibited pathogen production of
polygalacturonase and laccase (Sansone *et al.*, 2011;

Sanz Ferramola *et al.*, 2013). Thus, the inhibition of
key *B. cinerea* enzymes enhances the inhibitory ac-
tivities of the biocontrol agents (Table 4).

The *B. cinerea* antagonist enzymatic activity by the BCA rapidly takes place at the host organ surface before and/or at the pathogen's arrival. *Botrytis cinerea* is a fast-germinating pathogen which rapidly enters the host when there are wounds and/or when the host tissues are highly susceptible (Nair and Allen, 1993; Ciliberti *et al.*, 2015). This MoA may be less efficient in the field because *B. cinerea* remains for only a short period at host surfaces before infection occurs. Compared to bacteria, yeasts also secrete lytic enzymes, and this is known to play an important role in their biocontrol activity (Wisniewski *et al.*, 1991; Zimand *et al.*, 1996; Kapat *et al.*, 1998; Spadaro and Droby, 2016). Spadaro and Droby (2016) provided more information about the enzymes produced by yeasts, and their biocontrol roles in yeasts in post-harvest situations. Although lytic enzymes might be effective against a wide spectrum of phytopathogens, their non-specificity may result in suppression of beneficial microorganisms existing in particular environments (Pretorius *et al.*, 2015).

Pathogen growth inhibition can also be achieved
indirectly by changing the growth conditions on
plant surfaces, to make them unsuitable for success-
ful infection. For example, *B. pumilus* NCIMB 13374
and *P. fluorescens* NCIMB 13373 inhibited *B. cinerea*
growth in strawberries by increasing the pH from
6 to approx. 8. These bacterial strains also produce
antifungal compounds (Swadling and Jeffries, 1998).

Production of volatile organic compounds

In recent years, the effects of bacterial volatile
organic compounds on plants and pathogens have
been increasingly studied. VOCs may be one of the
key mechanisms for achieving successful biological
control of plant pathogens (Ryu *et al.*, 2004; Effmert
*et al.*, 2012). The emission of complex blends of VOCs
by bacteria was reported in many studies (Kai *et al.*,
2009; Effmert *et al.*, 2012). Volatile bacterial substanc-
es, identified by gas chromatography combined with
mass spectrometry (GC-MS), may vary in number
and type, depending on the bacterial isolate (Wan
*et al.*, 2008; Kai *et al.*, 2009; Arrebola *et al.*, 2010). For
example, more than 120 different VOCs were identi-
fied from 26 *Streptomyces* spp., comprising alkanes,
<table><caption>**Table 4.** Overview of literature reporting inhibition of Botrytis cinerea by bacteria producing enzymes and/or interfering with the activity and growth of this pathogen.</caption><thead><tr><th>Antagonistic bacteria</th><th>Plant/fruit/ conditions</th><th>Enzymes</th><th>Anti-fungal effects</th><th>Reference</th></tr></thead><tbody><tr><td>Bacillus cereus IO8</td><td>In vitro / tomato plants</td><td>Chitinase</td><td>Mycelial growth inhibition and disease incidence reduction</td><td>Hammami et al., 2013</td></tr><tr><td>Virgibacillus marismortui, Bacillus subtilis, Bacillus pumilus, Bacillus licheniformis, Terribacillus halophilus, Halomonas elongata, lanococcus rifioensis, Staphylococcus equorum, Staphylococcus sp.</td><td>In vitro / strawberry fruits</td><td>Chitinase, β-1, 3-glucanase, cellulase and protease</td><td>Mycelial growth inhibition and disease incidence reduction</td><td>Essghaier et al., 2009</td></tr><tr><td>Serratia plymuthica IC14</td><td>In vitro / cucumber leaves</td><td>Endochitinase</td><td>Mycelial growth inhibition and disease incidence reduction</td><td>Kamensky et al., 2003</td></tr><tr><td>Serratia plymuthica HRO-C48</td><td>In vitro</td><td>Endochitinase (E.C. 3.2.1.14), CHIT60, one N-acetyl β-1,4-D-hexosaminidase (E.C. 3.2.1.52), CHIT100</td><td>Spore germination and germ tube elongation inhibition</td><td>Frankowski et al., 2001</td></tr><tr><td>Pseudomonas sp. (B194 and B224) Erwinia herbicola (Pantoea agglomerans)</td><td>Apple juice</td><td>Not identified</td><td>Inhibition of germination and germ tube elongation</td><td>Bryk et al., 1998, 2004</td></tr><tr><td>Bacillus thuringiensis UM96</td><td>In vitro /in vitroMedicago truncatula plants</td><td>Chitinases</td><td>Mycelial growth inhibition and disease reduction</td><td>Martínez-Absalón et al., 2014</td></tr><tr><td>Bacillus cereus B-02</td><td>In vitro</td><td>Possible presence of bacteriocins, toxins and other metabolites</td><td>Spore Germination and hyphal growth inhibition; changes in cell morphology such as distortion, shrinking and swelling</td><td>Li et al., 2012</td></tr><tr><td>Rahnella aquatilis BNM</td><td>In vitro / apple</td><td>Interference with activity and growth of the pathogen</td><td>Mycelial growth inhibition and disease reduction</td><td>Sansone et al., 2011; Sanz Ferramola et al., 2013</td></tr><tr><td>Bacillus circulans G1 070</td><td>In vitro / grapevine vitroplants</td><td>Interference with activity and growth of the pathogen</td><td>Mycelial growth inhibition and disease reduction</td><td>Paul et al., 1997</td></tr><tr><td>Bacillus pumilus NCIMB 13374 Pseudomonas fluorescens NCIMB 13373</td><td>In vitro / strawberries</td><td>Interference with activity and growth of the pathogen</td><td>Conidial germination inhibition and disease reduction</td><td>Swadling and Jeffries, 1998</td></tr></tbody></table>

(Continued)
<table><caption>Table 4. (Continued).</caption><thead><tr><th>Antagonistic bacteria</th><th>Plant/fruit/ conditions</th><th>Enzymes</th><th>Anti-fungal effects</th><th>Reference</th></tr></thead><tbody><tr><td>Gluconobacter cerinus</td><td>In vitro</td><td>Interference with activity and growth of the pathogen</td><td>Mycelial growth inhibition</td><td>Guzzon et al., 2014</td></tr><tr><td>OxB related to Cupriavidus sp. Bacillus pumilus B-30087</td><td>A. Arabidopsis thaliana, cucumber, grapevine, tomato</td><td>Degradation of virulence factors</td><td>Disease reduction</td><td>Schoonbeek et al., 2007</td></tr><tr><td>Streptomyces halstedii A]-7</td><td>In vitro</td><td>Chitinase</td><td>Mycelial growth inhibition</td><td>Joo, 2005</td></tr><tr><td>Bacillus subtilis</td><td>grapevine leaf</td><td>N-acetyl glucosaminidase (Nagase), the chitin-1,4-chitobiosidase (Biase) and endochitinase</td><td>Disease reduction</td><td>Machia et al., 2015</td></tr></tbody></table>

alkenes, alcohols, esters, ketones, sulfur compounds, and terpenoids (Schöller et al., 2002). Bacterial VOCs inhibit or prevent fungal growth, impair fungal spores and hyphae, and/or promote plant growth (Kai et al., 2007; Kai et al., 2009; Weisskopf, 2013). However, the VOCs from one bacterial strain do not cause the same inhibitory effect, or to the same degree, on different fungi. The responses may depend on the specific fungus-bacterial combination (Kai et al., 2009).

VOCs derived from the *Streptomyces* species can prevent growth of *B. cinerea*. For example, VOCs from *Streptomyces platensis* F-1 (phenylethyl alcohol and (+)-epi-bicyclesesquiphellandrene) suppressed pathogen growth. In addition to the reduction of *Botrytis* fruit rot in strawberry, they also decreased the level of leaf blight in rice and oilseed rape (Wan et al., 2008). In tomato fruit, the inhibitory effects of VOCs on growth of *B. cinerea* from VOCs produced by *Streptomyces globisporus* JK-1 grown on autoclaved wheat seeds has been demonstrated (Li et al., 2010).

Similarly, within the genera *Bacillus* and *Paenibacillus*, the potential role of different VOCs to inhibit *B. cinerea* infection has been demonstrated, but only as one of the MoAs of these bacteria (Berrada et al., 2012; Zhang et al., 2013). Different degrees of inhibitory effects of VOCS from *Paenibacillus polymyxa* and *Bacillus sp.* (B. subtilis BLO2, B. pumilus BSH-4 and ZB13) were observed *in vitro* on S. sclerotiorum, B. cinerea and *Cercospora kikuchii* (Liu et al., 2008). Similarly, Chen et al. (2008) showed the antagonistic effects of these compounds generated by *B. subtilis*, on mycelial growth and the conidial germination of *B. cinerea*. For *B. cereus* (strain C1L), the protective effect against *B. cinerea* in tobacco was achieved by inoculating *B. cereus* into the soil, or by drenching the soil with dimethyl disulfide (Huang et al., 2012). The volatiles 1-octen-3-ol, benzothiazol, and citronellol, produced by *P. polymyxa* (strain BMP-11), inhibited mycelial *in vitro* growth of eight fungal pathogens, including *B. cinerea* (Zhao et al., 2011).

In addition to their strong antimicrobial inhibitory potential, bacteria emit VOCs which can promote plant growth, enhance plant tolerance to abiotic stress, and elicit induced systemic resistance (ISR) (Ryu et al., 2003, 2004; Bhattacharyya et al., 2015; Kanchiswamy et al., 2015; Liu and Zhang, 2015). Because *B. cinerea* was recently considered an endophyte (Van Kan et al., 2014), a potential antagonistic interaction might take place between the VOCs pro-
duced by endophytic bacteria and the pathogen dur-
ing this endophytic stage. Furthermore, compared
with diffusible compounds, VOCs may facilitate
interactions between physically separated microor-
ganisms. VOC-producing bacteria are well suited
to control fungal decay under post-harvest storage
conditions in controlled environment, as biofumi-
gants, although safety issues associated with these
biochemicals need to be evaluated.

Most studies concerning VOC effects on gray
mold have been performed under controlled condi-
tions, and thus, their advantages and drawbacks for
field applications must be considered. The impacts
of environmental parameters, particularly air move-
ments, may be of major importance. Strong air cur-
rents could significantly decrease the concentration
of produced VOCs, and limit their efficacy. Further-
more, adding nutrients into the soil, such as carbon
sources, may promote bacterial production of VOCs
(Fiddaman et al., 1994). A possible drawback of this
MoA is the inhibitory effects of certain VOCs at high
concentrations on plant growth (Bailly and Weis-
skopf, 2012).

Induction of host plant resistance

In addition to these direct interactions, some
plant growth-promoting bacteria are indirectly ef-
fective against plant pathogens by enhancing plant
defense mechanisms, which is induced systemic
resistance (ISR). This corresponds to a state of de-
fense in the whole plant, preparing it to respond
more quickly and intensely to a pathogen attack
(Van Loon et al., 1998; Bloemberg and Lugtenberg,
2001). ISR involves the jasmonic acid (JA) and eth-
ylene (ET) signaling pathways (Pieterse et al., 1996;
Glick, 2012). Several examples of bacterial strains
eliciting resistance against B. cinerea were reported in
different cultivated plant species. The induction of
systemic resistance in grapevine using beneficial mi-
croorganisms was reviewed by Complant et al. (2013).

Several compounds produced by bacteria, in-
cluding volatiles, siderophores, flagellin, and lipo-
peptides are known to elicit e ISR against B. cinerea in
many plant species (Ongena et al., 2005; Ongena and
Jacques, 2008; Wu et al., 2015). As for P. aeruginosa,
the strain 7NSK2 produces a siderophore, pyochelin,
and the antibiotic pyocyanin, which trigger the ISR
in both tomato and bean against B. cinerea (De Meyer
and Hofte, 1997; De Meyer et al., 1999; Audenaert et

al., 2002). While the LPS, siderophores, and flagellin
produced by *P. putida* WCS358 induce resistance in
*Arabidopsis*, LPS and siderophores were the only ac-
tive elicitors in bean and tomato to induce systemic
resistance (ISR) (Meziane et al., 2005). Cawoy et al.
(2014) found that adding 10 µM of pure surfactin re-
duced *B. cinerea* development on tobacco leaves. The
ability of mycosubtilin (iturin family) from *B. subti-
lis* to trigger immune responses against *B. cinerea* in
grapevine was reported by Farace et al. (2015). The
ability of several bacteria, including *Micromonospora*,
*Saccharothrix algeriensis* and *P. fluorescens*, has recent-
ly been shown to induce plant systemic resistance
and then reduce *B. cinerea* infections (Muzammil et
al., 2014; Gruau et al., 2015; Martínez-Hidalgo et al.,
2015).

Considering field development in the future,
different factors may influence induced resistance
*in natura*, including the plant genotype, the plant
growth stage, the environment and plant nutrition
(Heil, 2001; Walter *et al.*, 2013). For *B. cinerea*, the
importance of this MoA on grape berries is limited
to the early fruit stages. Stilbenic phytoalexins are
induced mostly in the early growth stages in grape
berries, which progressively lose their potential for
stilbene synthesis towards fruit maturity (PR pro-
teins are, however, possibly induced in such matur-
ing tissues). Furthermore, although hypersensitive
responses (HR) were reported to be effective against
many pathogens, host cell death induced by *B. ci-
nerea* in Arabidopsis promotes growth of the fungus
(Govrin and Levine, 2000). Furthermore, this MoA
is also associated with metabolic costs and energety
trade-offs within host plants. These costs can include
allocation from plant growth and development to-
wards defense, as well as ecological costs, such as
negative effects on symbiotic interactions (Walters
and Heil, 2007; Walters *et al.*, 2013). However, more
research must be carried out to provide better un-
derstanding of the effects of biotic and abiotic fac-
tors on plant induction for plant health, growth, and
productivity.

Discussion and future prospects

We have described the different MoAs of bacte-
rial BCAs against *B. cinerea*, and some advantages
and drawbacks of each have been cited for each
MoA. It is important to emphasize that the lines be-
tween the different MoAs may be indistinct. Most
bacterial biocontrol strains produce antibiotic com-
pounds and enzymes by which they interfere with
pathogen growth and metabolic activity. Meanwhile,
many lipopeptides and VOCs also induce host plant
resistance. In terms of future field applications, some
MoAs are likely to be of prime importance depend-
ing on *B. cinerea* epidemiology. A key example is the
competition MoA for suppressing *B. cinerea* at host
floral phenological stages, which are highly sus-
ceptible to the pathogen because of the abundant
nutrient sources (pollen grains, floral exudates, se-
nescing petals). Another example of a potentially
very well-suited MoA in relation to the pathogen
infection pathway is the production of VOCs under
post-harvest storage conditions. However, favour-
ing one specific MoA at a precise epidemiological
stage, even if perfectly adapted, may not be suffi-
cient for insuring biocontrol efficacy. The bacterial
BCAs must survive and multiply at host surfaces to
interfere with the pathogen. For *B. cinerea*, we con-
sider the pathogen to be of minor epidemiological
importance in the biological phase within the soil, so
bacterial BCA survival and development should be,
above all, assured at the surface of aerial host organs
(leaves, flowers, fruits). Abiotic parameters related
to temperature and relative humidity directly affect
the survival of microorganisms, including microbial
BCAs on the surfaces of host plants (Kredics et al.,
2003). In general, bacteria are more sensitive to moist-
ture and water availability than fungi. Bacteria may
exhibit poor ability to survive in hot, dry summer
conditions, with the exception of spore-forming bac-
teria (*Bacillales*) which survive as spores and resume
active forms under favorable conditions. Natural
bacterial populations on fruit surfaces are generally
less than those of yeasts (Barata el al., 2012). Repeat-
ed sprays as field treatments seem to be required
for bacterial BCAs. Some bacteria penetrate through
host stomata or via injuries in leaves and stems, and
then live also as endophytes without being disturbed
by external abiotic and/or biotic stresses. From this
point of view, bacteria in the *Bacillale*, notably *Bacil-
lus* spp., have been considered as BCAs of interest
because of their potential endophytic aptitude (Ba-
con and Hinton, 2002). This is also why BCA bacte-
rial strains isolated from the host plant to be pro-
tected are likely to be well-adapted for survival and
development on and within that host. It should be
noted that recent molecular methods are likely to be
of prime importance for detection and quantification

of specific BCA strains in particular environments.
These technologies are also well-suited for assessing
location, proliferation and/or survival of BCAs on
and within host plants (Narayanasamy, 2013; Soto-
Muñoz et al., 2014).

Biological control with microorganisms presents inherent variability in efficacy, notably depending on the different MoAs and the host plants and organisms involved. Furthermore, the effects of external abiotic factors under field conditions represent another important source of variability in efficacy. Therefore, biological control of disease in the field, based on only one MoA, may result in moderate and highly variable efficacy and be of little significance from a commercial point of view. The combination of different MoAs is widely considered as a strategy to overcome the variability in the biocontrol of fruit pathogens, including *B. cinerea* (Elmer and Reglinski, 2006; Romanazzi et al., 2016). Furthermore, there may be risk of loss of biocontrol durability, which has been little studied but is likely to be important in the near future. As for chemicals, a combination of different MoAs, particularly for synthetic fungicides, is a key strategy for reducing risks of development of resistance to pesticides. Thus, a similar strategy may be promoted in biocontrol (Bardin et al., 2015).

When dealing with bacteria, a combination of dif-
ferent MoAs can be achieved in different ways. First,
one species showing more than one MoA (Lahlali et
al., 2013) can be used. Production of several differ-
ent antibiotic molecules by the one species may also
be considered here. For example, B. subtilis strains
produce several cyclic lipopeptides (Falardeau et al.,
2013). Second, two bacterial strains (from the same
or from different species) can be used together, each
presenting a different MoA (Magnin-Robert et al.,
2013). Third, different MoAs can be used at differ-
ent times, depending the stage of the particular grey
mold epidemic. Specific MoAs may be more appro-
priate at key epidemiological stages than others.

From practical perspectives, combinations with
non-bacterial microorganisms and/or with natural
products may also be worth considering, either in
joint applications or focusing according to key epi-
demiological stages. Yeast and yeast-like fungi are
complementary to bacteria by surviving in different
conditions, or by being more efficient as saprophytic
competitors (Nunes et al., 2002). Natural products
may improve the dispersion, persistence and sur-
vival on plant phylloplanes, but others also elicit
natural plant defenses and/or increase the efficacy
of biocontrol treatments by contributing to pathogen
growth suppression (Teixidó et al., 2001). Combin-
tions with fungicide treatments can also be effective
in conventional agriculture; however, compatibility
with phytosanitary products can be another impor-
tant consideration, as well as in combination with
natural products (Francesco and Mari, 2014).

Some examples of MoA combinations have been
considered in the literature. However, few combined
field biocontrol programmes with trusted efficacy are
yet to become available. Therefore, more applied re-
search, focused on such combinations, is necessary,,
especially in the field, combining commercialized
products and also products in developmental stages
for different crops. Public institutions and extension
services may adapt combinational strategies to local
conditions. Nonetheless, the private sector should
also be involved in developing such strategies and
supporting public research to improve the variable,
and sometimes poor, efficacy of some of the biocon-
trol products already available. In parallel, new in-
vivo and field screenings, associated with studies on
the effects of BCAs on the more general microbiota,
and on the particular MoAs of each effective strain,
will allow for the design of new combinations and
timings to be tested in the field. This should be based
on a priori knowledge of complimentary MoAs, and
this approach is likely to become more widely used
and purposeful thanks to new tools provided by
next generation sequencing technologies (Massart et
al., 2015).

Another constraint for biological control with
bacteria is the necessity for adequate registration
systems prior to commercialization. Most of the
regulations are following the OECD guidance docu-
ments, particularly the #67 published in 2012. The
EU through the EFSA assessment procedure inte-
grates BCAs as "biopesticides" entering in the com-
mon registration process for all pesticides (Regula-
tion EC 1107/2009). BCAs are considered as low risk
according to their sustainable use, and can be includ-
ed in safe integrated pest management (IPM) prac-
tices (Villaverde et al., 2014). Regulations in the USA
are more specific for biopesticides, depending on the
Environmental Protection Agency (EPA). Registra-
tion is a long and difficult process that is common
for every biological control product, limiting the
development of bacterial or fungal BCAs. This has
been discussed in several publications, where the

constraints related to different modes of action are
also outlined (Spadaro and Gullino, 2010; Walters
et al., 2012; Bashan et al., 2014; OECD report, 2014).
Nonetheless, there is a growing perspective for more
appropriate consideration of the properties and haz-
ards of BCAs, which may lead to simpler evaluations
of the human and environmental safety of biocontrol
products in the future (Sundh, 2014).

In conclusion, bacterial antagonists represent an
important biocontrol option against diseases caused
by *B. cinerea*, due to their rapid development and
the variety of antifungal and defense elicitor com-
pounds they produce. Some MoAs are likely to be
more suitable than others to concrete conditions in
pre- and/or postharvest stages. However, combina-
tion of different MoAs, and combination with other
control strategies, is likely to provide opportunities
to reduce variability in biocontrol efficacy. Basic and
applied research is therefore needed to design more
refined application programmes, which will provide
reliable solutions for plant protection and produc-
tion in conventional and organic agriculture in the
near future.

Acknowledgments

The authors are grateful to the Syrian government
for a Ph.D. grant to Rana Haidar and for financial
support through the CASDAR project (V1302). We
also thank the Conseil Interprofessionnel du Vin de
Bordeaux for financial support. We are also grateful
to the Agreenskills postdoctoral fellowship program
for Carlos Calvo-Garrido and for financial support
from the European Union. The manuscript was ed-
ited for English with the Elsevier web shop service
before submission.

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Accepted for publication: June 13, 2016

Published online: July 28, 2016