## 5 Mode of Action  
### Direct Inhibition of Soil-Borne Plant Pathogens  
Phloroglucinol derivatives have been shown to inhibit the growth of numerous bacterial, fungal and oomycete soil-borne pathogens, including the fungal ascomycetes _Gaeuamomyces tritici_ and _Thielavopsis basicola_, the oomycete _Pythium ultimum_, the Gram-negative bacterium _Pectobacterium atrospictium_ and the Gram-positive bacterium _Clavibacter michiganensis_ subsp. _michiganensis_[10; 12; 13; 99; 100]. Plant pathogens and even different isolates of the same pathogen display differential sensitivity to DAPG [13; 87]. Phloroglucinol derivatives produced by plant-beneficial _Pseudomonas_ spp. accumulate in the rhizosphere [13; 86; 101; 102; 103] at concentrations up to several micrograms per gram of root. DAPG is, however, rapidly degraded in the rhizosphere [103], suggesting that root-associated microcolonies of plant-beneficial _Pseudomonas_ spp. actively maintaining DAPG production is required. Plant-associated _Pseudomonas_ spp. only colonize a small portion of the root surface [104], which means that, locally, DAPG concentration is likely to be sufficient to inhibit soil-borne plant pathogens. Most reports indicate that, among phloroglucinol derivatives, DAPG is more active than MAPG, whereas DAPG and MAPG are more active than phloroglucinol [105; 106]. Like other antibiotics, the antimicrobial activity of phloroglucinol derivatives is strongly influenced by the pH and is higher at lower pH [105]. This could originate from the fact that at high pH, DAPG is likely to be deprotonated, which could impair its capacity to cross biological membranes.  
Several studies have examined the effect of phloroglucind derivatives on the physiology of oomycetes and fungi. Exposure of the oomycete _Pythium ultimum_ to DAPG led to the inhibition of zoosporogenesis, zoospor motility and zoospore germination [105], several processes crucial for pathogenesis. The various structures produced by _P. ultimum_ during its life cycle greatly differ in their sensitivity to DAPG, with zoospores being the most sensitive. Indeed, exposure to DAPG at a concentration as low as 3.2 ng mL\({}^{-1}\) disintegrated most zoospores [105]. By contrast, vegetative mycelium could tolerate DAPG concentration in the range of 10-20 ug mL\({}^{-1}\), suggesting several tolerance mechanisms. Microscopy studies of the hyphal tips of _P. ultimum_ revealed that exposure to DAPG provoked several structural changes, including alteration of the plasma membrane, vacuolization and cell content disintegration [105]. DAPG also inhibits zoosporogenesis and zoospores motility in _Plasmopara viticola_ and _Aphanomyces cochilioides_ in a dose-dependent manner [106]. The main mode of action of DAPG likely resides in its capacity to act as a proton ionophore, dissipating the proton gradient across the mitochondrial membrane [107; 108; 109], which lead to the loss of mitochondrial function and the inhibition of growth. Long exposure to DAPG can generate oxidative stress with the production of superoxide and hydrogen peroxide [110], which is similar to the effect that long exposure to other uncoupling agents produces [109].  
DAPG exhibits high antimicrobial activity against several bacteria, including the Gram-positive model _Bacillus subtilis_ and the Gram-negative plant pathogen _Pseudomonas syringae_[13]. Conversely, it is quite inefficient against _Pseudomonas aeruginosa_ and _Pseudomonas fluorescens_ strains [13; 111]. This discrepancy could originate from differential membrane permeability and/or the presence of detoxifying efflux pumps. It has been proposed that DAPG targets the bacterial envelop [111], but little is known about the mode of action of DAPG towards bacteria. At subinhibitory concentrations, DAPG has been shown to reduce biofilm and spore formation in _B. subtilis_[112].