Biofilms are matrix-enclosed bacterial populations that are adherent to each other and to surfaces and/or interfaces and are mainly composed of polysaccharides, proteins, lipids, and extracellular DNA.
1. Introduction
Biofilms are matrix-enclosed bacterial populations that are adherent to each other and to surfaces and/or interfaces and are mainly composed of polysaccharides, proteins, lipids, and extracellular DNA [
1,
2,
3]. During biofilm formation, the cells transit from a motile to a sessile lifestyle by interacting with a surface and starting to produce an extracellular matrix that holds them together and attaches them to the surface [
2]. Therefore, the cells forming biofilms are referred to as sessile cells, which differ from their non-encased free-swimming counterparts, the planktonic cells [
4]. Recent studies indicate that biofilms represent the main mechanism of active bacterial life due to their dominance in all habitats throughout the world [
5,
6]. Compared to the planktonic lifestyle, the biofilm lifestyle confers several benefits to the integrating cells, such as protection against antimicrobial agents and predators, tolerance towards changing environmental conditions, and colonization aptitudes [
3,
7,
8].
Bacteria form biofilms in artificial and natural environments, including the soil, internal and external tissues of all living organisms, rocks, and water, among others [
5]. Many different bacteria form biofilms, but the
Pseudomonas genus is among the most studied for several reasons: (1) it harbors species with the ability to colonize a wide variety of environments due to the high metabolic and physiologic versatility found in this group of microorganisms, (2) it has ecological relevance due to its interactions with living organisms, and (3) it has potential biotechnological applications [
9]. The
Pseudomonas aeruginosa species, a ubiquitous bacterium that can also act as an opportunistic human pathogen, has long been used as a model bacterium within the
Pseudomonas genus for the study of biofilm formation and pathogenesis due to its relevance in the clinical environment [
10]. The extracellular matrix of
P. aeruginosa has been studied in-depth and, to date, is known to contain three exopolysaccharides: alginate, polysaccharide synthesis loci (Psl), and pellicle loci (Pel) [
11]. The role of these exopolysaccharides in the biofilm architecture of
P. aeruginosa and the impact of their production in the clinical setting, such as protection against antibiotic treatments and host defenses, have been explored in several studies [
12,
13,
14,
15,
16,
17,
18,
19,
20]. Although
P. aeruginosa produces infections in humans, there are also some examples in which this bacterium can act as a pathogen for plants [
21,
22]. However, the biological significance of alginate, Psl, and Pel exopolysaccharides in a nonclinical context has not been studied.
Bacteria belonging to the
Pseudomonas genus are common inhabitants of plant surfaces [
23,
24]. The role played by
Pseudomonas in the agricultural industry is remarkable as several economically important activities are derived from their interaction with plants. Among these activities, there are harmful diseases that involve severe economic losses and beneficial activities such as plant growth stimulation, the promotion of plant health and nutrient availability in soils, and induction of plant immune defenses [
25,
26]. Pathogenic plant-associated
Pseudomonas are predominantly present on the phyllosphere. The phyllosphere is an extreme and unstable habitat as it is exposed to highly variable nutrient and water availability, temperatures, and ultraviolet (UV) radiation. Therefore, the microbial populations associated with the phyllosphere must be adapted to these continuously fluctuating conditions [
23,
27,
28]. The extracellular matrix of epiphytic bacteria contributes to the fitness [
29,
30,
31], protection [
8,
32], and hydration of the cells [
33], allowing cells to cope with these ever-changing conditions. Conversely, beneficial plant-associated
Pseudomonas usually prevail in the rhizosphere. Compared to the phyllosphere, the environmental fluctuations that take place on the rhizosphere are weak and buffered [
34]. Nevertheless, the rhizosphere is not considered a uniform and stable environment as the conditions can change abruptly in extremely short distance ranges [
35,
36]. Biofilm formation by beneficial plant-associated
Pseudomonas plays advantageous roles for both the plant and bacteria [
27,
37]. On the one hand, they can increase plant yield by improving mineral uptake and phytohormone production, inducing the competitive suppression of pathogens and triggering plant-induced systemic resistance [
38]. On the other hand, these biofilms allow the attachment of the cells to a nutrient source and confer protection against plant defenses and environmental fluctuations [
27,
37]. Furthermore, the biofilms produced by rhizospheric bacteria enhance soil aggregation, which improves the water-holding capacity, fertility, and porosity of the soils, leading to an increase in agricultural productivity [
39,
40,
41,
42].
Some of the biofilm components, mainly exopolysaccharides, that are required for biofilm formation and pathogenesis in P. aeruginosa find their equivalents in pathogenic and beneficial plant-interacting Pseudomonas.
2. Ecological Significance of Biofilm Formation by Plant-Interacting Bacteria
Plant-associated bacteria develop a biofilm lifestyle during their interactions with plants [
27,
37]. Depending on whether biofilms are formed by pathogenic or beneficial individuals, the ecological outcome resulting from the interaction can be completely different. In the context of pathogenic plant-associated bacteria, the role of different components involved in biofilm formation has been studied. For instance, the biofilms formed by
Erwinia amylovora, the causal agent of fire blight disease in different plant species of the
Rosaceae family, and specifically the amylovoran and levan exopolysaccharides, physically blocked the vascular system of plants [
43,
44,
45]. A mutant of
Ralstonia solanacearum, the causal agent of bacterial wilt disease, in the
lecM gene, which encodes a lectin, showed reduced biofilm formation in vitro and colonization of the intercellular spaces of tomato leaves and was impaired in virulence [
46]. The
gumB mutant of
Xanthomonas citri, which produces canker disease in citrus plants, was unable to produce the polysaccharide xantan and exhibited reduced biofilm formation, survival and symptom development on lemon leaves [
30]. Similarly,
Xylella fastidiosa, which causes economically important diseases in several host plants, produced exopolysaccharides that played roles in the virulence of this bacterium, as these are required for bacterial movement within plants and plant-to plant transmission through insects [
47,
48].
Notably, the
Pseudomonas syringae complex harbors most of the phytopathogens within the
Pseudomonas genus [
49,
50]. In particular, the species
P. syringae is one of the most ubiquitous bacterial participants of the phyllosphere [
51]. This ubiquity, together with the fact that it can infect almost all important agricultural crops [
25,
52], has made it a model for the study of plant–bacteria interactions.
P. syringae possesses a great diversity of virulence factors that engage in plant infection, as well as adaptation mechanisms that improve bacterial survival over the plant surface. Generally,
P. syringae produces a type III secretion system (T3SS), effector proteins, motility appendages, phytotoxins, multidrug efflux pumps, extracellular polysaccharides, cell wall-degrading enzymes, and ice nucleation activity [
53]. Copper- and UV radiation-resistance genes, as well as exopolysaccharide production, play fundamental roles in
P. syringae fitness and survival [
31,
50,
54,
55,
56,
57,
58]. In
P. syringae pv. syringae (Pss), biofilm formation has been proven to influence the transition between pathogenic and epiphytic lifestyles in plants [
29,
31,
59].
In the context of beneficial plant-associated bacteria,
Bacillus subtilis, a Gram-positive bacterium that acts as a biocontrol agent of several plant pathogens, requires the production of extracellular matrix components involved in biofilm formation, such as those encoded by
tapA-sipW-tasA and
epsA-O operons, for the colonization of the plant roots and for conferring plant protection [
60].
Pseudomonas fluorescens, an important rhizobacterium that promotes plant health and nutrition, requires biofilm formation for the colonization of plant surfaces [
61]. A cellulose exopolysaccharide mutant in the
P. fluorescens SBW25 strain was compromised in the colonization of the rhizosphere and the phyllosphere of sugar beet compared to the wild-type strain [
61]. In general, the
P. fluorescens species and some closely related species that belong to the
P. fluorescens complex are among the most studied bacteria within soil communities, because they frequently show agricultural, biotechnological, and ecological interest, mostly due to their beneficial plant features [
62]. In particular, the
P. fluorescens and
Pseudomonas chlororaphis species stand out because of their potential use as biocontrol agents as they frequently contribute to plant health by exerting antagonist activities against pathogens [
63,
64,
65]. Phenotypes linked to biofilm formation have also been observed to favor bacteria–plant root interactions and biocontrol activity of
P. chlororaphis and
Pseudomonas putida species [
66,
67,
68,
69,
70]. Usually, biocontrol agents can form biofilms, and increasing evidence strongly suggests that biofilm-forming ability should be considered in assessing their potential beneficial performance [
71].
3. Main Exopolysaccharides Produced by Plant-Associated Pseudomonas
Among all the exopolysaccharides that are produced by plant-associated
Pseudomonas [
72], those that have been mainly studied are alginate, cellulose, and Psl (). A description of their functions in biofilm formation and architecture and their ecological significance during pathogenic and beneficial plant–bacteria interactions are listed below.
Table 1. Main exopolysaccharides produced by different Pseudomonas spp. strains that are involved in biofilm formation.
Strain |
Clusters Encoding the Main Exopolysaccharides Described in Pseudomonas 1 |
|
alg |
wss |
psl |
pel |
P. aeruginosa PAO1 |
+ 2 |
- 2 |
+ |
+ |
P. aeruginosa PA14 |
+ |
- |
- |
+ |
P. syringae pv. syringae B728a |
+ |
- |
+ |
- |
P. syringae pv. tomato DC3000 |
+ |
+ |
+ |
- |
P. savastanoi pv. phaseolicola 1448A |
+ |
+ |
+ |
- |
P. syringae pv. syringae UMAF0158 |
+ |
+ |
+ |
- |
P. fluorescens SBW25 |
+ |
+ |
+ |
- |
P. fluorescens Pf0-1 |
+ |
- |
- |
- |
P. fluorescens F113 |
+ |
- |
- |
- |
P. chlororaphis PCL1606 |
+ |
- |
+ |
- |
P. chlororaphis O6 |
+ |
- |
+ |
- |
P. chlororaphis subsp. aureofaciens 30–84 |
+ |
- |
+ |
- |
P. putida KT2440 |
+ |
- 3 |
- |
- |