INTERACTIONS/ASSOCIATIONS OF MICROBES IN SOIL
7.3.1 PLANT-MICROBE BIOFILM ASSOCIA TIONS
Microbial cells within the soil release numerous enzymes. Quorum Sensing (QS) molecules, and various growth factors that promote the growth of other cells around them (Foster and Bell, 2012). Microbes can associate with plants at different sites and exhibit different types of the interaction effect. The association of microbes with plant tissues mostly occurs at the rhizosphere region (root), phyllosphere region (leaf), and vasculatures wherein microbes invade the host tissues. The association at these regions can prove to be beneficial, symbiotic, or pathogenic. Some of the bacterial species also exhibit a biocontrol effect and provide protection against other invading pathogens. Some of the organisms invading different parts of plant tissues, and their infection sites have been summarized in Table 7.1 (modified from Lakshmanan et al., 2012). The table consists of Grampositive bacteria as well as Gram-negative bacteria, both of which equally associate with a wide variety of plants forming biofilm, but vastly differ in their interaction effects.
TABLE 7.1 Some of the Plant-Microbial Biofilms with Their Interaction Effects and Sites of Association
‘Molecular Dialogue’ is the term used to refer to the exchange of chemical signals during plant-bacterial symbiosis. The most classical case is the infection of root hair of leguminous plants with nitrogen-fixing bacteria
Rhizobium at the rhizosphere. Rhizobia may survive under different lifestyles; soil, root hair adherence, and infection of root hair, or it can also gl ow within the root nodules of legumes where they fix nitrogen (Downie, 2010). Plant Growth Promoting Rhizobacteria (PGPR) employs a range of mechanisms, either alone or in combination, to successfully colonize the root system (Compant et al., 2010). Plant -Rhizobium interaction begins with the adherence of single Rhizobium cells to the meristematic cells of the root leading to nodulation. Root exudates from the legumes, mainly flavonoids act as inducing factors for the expression of Nod genes of Rhizobia (Cooper, 2007). Nod factors cause infection of root hair (deformation), root curling, and nodule initiation, which are most important for symbiotic associations. Symbiotic association is dependent on four main types of surface polysaccharides (Fujishige et al., 2006). Adhesion, active nodule formation, and biofilm development is mainly due to the release of EPS and Lipopolysaccharides (LPS) (Sorroche et al., 2012). Flavonoids, along with LPS and EPS, contribute significantly to nodule activity (Cooper, 2007). Biofilm formation is independent of chemical signals from plants. Plant microbial association in the rhizosphere region has been shown pictorially (Figure 7.3).
FIGURE 7.3 Plant-microbe association in the rhizosphere region of the soil.
The first step during biofilm formation is the attachment of rhizobia (Rhizobium, Bradyrhizobium, Mesorhizobium, Sinorhizobium, and other related genera) to host roots causing infection and nodulation. The second binding step begins with the synthesis of bacterial cellulose fibrils that result in an irreversible binding and foimation of bacterial aggregates onto the host surface. Nodulation genes (nodDABC) occur in both alpha and beta types of rhizobia that nodulate legumes (Giraud et al., 2007). Hence biofilm formation is believed to be conserved by evolution with the significant role of a Nod factor gene (Fujishige et ah, 2008).
7.3. 7.1.2 Plant-Azospirillum Interactions
Biofilm lifestyle is often crucial for the survival of bacteria and for the establishment of specific symbiosis with actinorhizal host plants or nonspecific root colonization (Danhom and Fuqua, 2007; Rodriguez-Navarro et ah, 2007). Azospirillum brasileuse although attaches with wheat roots; it generally does not exhibit species-specificity. Attachment begins with a weak, reversible, and nonspecific binding. The association occurs due to the bacterial surface proteins, capsular polysaccharides, and adhesion component of flagella (Zhu et ah, 2002). The first step of attachment and aggregation of A. brasileuse is due to the outer membrane of bacteria. Biofilm assembly and disassembly is attributed to several QS molecules along with mechanical and nutritional stress (Karatan and Watnick, 2009). QS are vital in regulating bacterial colonization both in the rhizosphere and the rhizoplane (Soto et ah, 2006; Compant et ah, 2010). Biofilm formation is controlled by the production of TV-Acyl-homoserine lactones (AHLs). However, there are also reports that AHL’s are not always required for plant colonization (Muller et ah, 2009; Compant et ah, 2010). Bacteria residing within the biofilm function due to intercellular communication facilitated by bacterial products that diffuse from cells (Watnick and Kolter, 2000).
Receptor proteins of cellular pathways are mediated by secondary messenger, cyclic-di-Guanosine Monophosphate (c-di-GMP), which controls flagellar motor speed (Boehm et ah, 2010). c-di-GMP, in few species like Vibrio cholerae and ShewaneUa oneidensis increases biosynthesis of EPS to facilitate cell adhesion (Beyhan et ah, 2006; Thonnaim et ah, 2006; Krasteva et ah, 2010).
A. brasileuse under aerobic conditions produces considerable amounts of Nitric Oxide (NO), which influences lateral root formation in host plants (Creus et ah, 2005; Molina-Favero et ah, 2008). NO also stimulates biofilm formation by controlling the levels of c-di-GMP (Plate and Marietta, 2012). Production of NO in A. brasileuse Sp245 derived from denitrification is a key regulatory step in biofilm formation (Di Palma et ah, 2013). NO also triggers the disassembly of Pseudomonas aeruginosa
biofilms as shown by (Barraud et al., 2006, 2009) through upstream c-di- GMP signaling pathway. Thus NO is a regulatory molecule that can cause biofilm formation or its dispersion.
184.108.40.206.3 Roleof Plant Growth PromotingRhizobacteria (PGPR)
Bacteria that colonize roots and promote plant growth are known as PGPR. PGPR activity is exhibited by bacteria that belong to genera like Azospirillum, Azotobacter, Bacillus, Clostridium, Enterobacter, Gluco- nacetobacter, Pseudomonas, and Serratia. Gram-positive bacterium like Bacillus subtilis is associated with roots of many crop plants. Studies have demonstrated that B. subtilis promotes plant growth through secretion of cytokinins and other volatiles. These volatiles help the plant to overcome salt stress through its effect on high-affinity ion transporter HKT1 (Beauregard et al., 2013). The overall effect of PGPRs on the plant has been represented pictorially (Figure 7.4).
FIGURE 7.4 Different roles of PGPRs in the rhizosphere region.
Bacillus subtilis form biofilm on roots (Danhom et al., 2007). The extracellular matrix in Bacillus is composed of two major components, an EPS and protein tasAthat polymerize into amyloid-like fibers. EPS production is regulated by epsA-0 operon and tasA under the control of transcriptional regulator SpoOA, whose activity, in turn, depends on its phosphorylation (Beauregard et al., 2013). Biofilm initiation in B. subtilis is regulated by SpoOA through various environmental signals. abrB and sinR pathways of anti-repression directly or indirectly control 15-gene eps operon that is required for biosynthesis of EPS, the tapA-sipWtasA operon, bslA gene encoding a hydrophobic biofilm coat protein and also motility genes such as hag, lytA, and lytF (Ma et al., 2017). It is the abrB and sinR pathways that are critical for biofilm formation (Cairns et al., 2014).
220.127.116.11.2 Plant-Pseudomonas Interactions
Pseudomonas fluorescens is a Gram-negative bacterium that colonizes the roots and the endophytic tissues at the root surface level forming biofihns (Kiely et al., 2006). AHLs are important mediators for surface attachment in Gram-negative P fluorescens. Biocontrol effect is exhibited at rhizoplane level where they form discontinued colonies in the grooves of epidermal cells. Root and seed exudates from plants are used effectively by bacteria for their growth. P fluorescens acts non-specifically to protect plants from soil phytopathogens (Couillerot et al., 2009). Competitive inhibition could also serve as a mechanism that can offer an advantage to host plants. Biofilm formation regulated through QS signals in P fluorescens allows the organism to create a niche, where it produces antifungal compounds, mainly phenazine antibiotic, which protects the wheat rhizosphere.
18.104.22.168.3 Plant-Erwinia Interactions
Bacterial adhesins, which may be monomeric or complex protein structures are responsible for cell and surface attachment and include the pili and fimbriae along with multiple appendages (Pizarro-Cerda et al., 2006; Kim et al., 2008). Envinia amylovora, a Gram-negative plant pathogen causes fire blight disease. This organism is highly virulent and rapidly disseminates through vasculatures of rosaceous species like apple and pear trees (Koczan et al., 2011). The most important requisites for biofilm formation by E. amylovora are amylovoran (an EPS and pathogenicity factor) and levan (Koczan et al., 2009). The former protects cells from host-elicited immune responses and the latter is a known virulence factor. E. amylovora encodes cell surface structures that are necessary for initial cell attachment along with peritrichous flagella and a type III secretion apparatus; however, the role of other surface appendages remains unknown (Koczan et al., 2011).
7.3.2 MICROBE-MICROBE BIOFILM ASSOCIA TIONS
Soil biofilm communities are cohabited by diversified ecosystem with high levels of synergy among most species (Ren et al., 2015). Multispecies biofilm showed more protective mechanism than individual microbial biofilm as reported by Lee et al. (2014). This shows that there is a high level of synergy and cooperative social behavior among the mixed communities.
Seeds of peanut are infected by fungus Aspergillus uiger that causes crown root disease. Biological control of peanut seed from this fungus gained widespread interest owing to environmental concerns due to the use of chemical pesticides. Several studies have also revealed the use of PGPR’s in the biological control of pests. One such organism is the bacterium Ряеш'£ясг7/н.у polymyxa found in the rhizosphere of wheat. This plant beneficial bacterium forms biofilm communities around the roots and the biofilm intum utilizes root exudates secreted by plants. Biocontrol effect could be due to antibiotic compounds produced by the plants (Haggag et al., 2008). Antagonistic effect of Paenibacillus can also be exhibited by competing with the pathogen for colonization sites on the roots by making it unavailable for the pathogen (Timmusk et al., 2005).
Biofilm communities are important in an agricultural setting (Powers et al., 2015). Interspecific interactions in a mixed-species biofilm are mediated via AHL’s that act as costimulatory signals. Biofilm communities of B. subtilis and P. protegens show antagonistic interactions as demonstrated by Powers et al. (2015). The active molecule involved in the inhibitory mechanism is 2-4-diacetylphloroglucinol (DAPG) produced by Pseudomonas. DAPG inhibits both spomlation as well as biofilm formation by B. subtilis.
B. subtilis also acts as a biocontrol agent by preventing plant infection by bacterial pathogens through the secretion of AiiA enzyme, a lactonase enzyme. This enzyme functions as an inactivator of acylhomo- serine-lactone molecules released by plant pathogens (Chen et al., 2013). B. subtilis acts as a bio controller by secreting surfactins that act as an antimicrobial agent against the plant pathogen Pseudomonas syringae. B. subtilis also provides protection against other plant pathogens like Envinia and Xantliomonas (Bais et al., 2006). It also enhances plant protection by inducing systemic resistance (SR) in plants for protection against an array of pathogens. Fungal-bacterial interactions play a dynamic role depending on the species, strain, and environment, but they can also demonstrate endosymbiotic, synergistic or antagonistic behavior. For example, the plant fungal pathogen Rhizopus microsporus exhibits endosymbiotic relationship with Gram-negative bacteria, Burkholderia rhizoxinica and Burkholderia endofungorum. The fungus, in turn, uses bacteria to produce rhizoxin, which causes rice seedling blight (Dixon and Hall, 2015).
Thus the commercial use of PGPR’s as replacements to chemical pesticides and supplements helps to control various plant diseases (Shivasakthi et al., 2014) and is a rapidly expanding area of research. Late blight disease of tomato is one such example where Phytopthora infestans infects tomato crops. In a study by Kumar et al. (2015), a combination of Trichoderma harzianum OTPB3, B. subtilis OTPB1, and P. putida OTfl were used to induce SR. These organisms control the disease through the production of Indole Acetic Acid (IAA) and Gibberellins (G3) (Chowdappa et al., 2013).