Search Results (22)

The secondary metabolite 2,4-diacetylphloroglucinol (2,4-DAPG), which is produced by Pseudomonas bacteria, is a potent antimicrobial agent with well-documented properties that suppress phytopathogens. However, its broader ecological impact on soil microbial communities is not understood. Through a combination of controlled microcosm and field trials, we have demonstrated that the effects of 2,4-DAPG are highly context-dependent. Laboratory exposure (10 mg kg⁻¹) altered the abundance of 8.53% of bacterial and 6.91% of fungal amplicon sequence variants, and simplified the bacterial co-occurrence networks (reduced number of nodes and links). In contrast, field conditions amplified bacterial sensitivity (the Shannon index decreased from 4.77 to 4.17, p < 0.05) but maintained fungal stability (Shannon index varied from 3.93 to 3.97, p > 0.05); these conditions affected a smaller proportion of fungal ASVs (4.23%). Taxonomic analysis revealed consistent suppression of fungi of the Mucoromycota (e.g., Mortierella) and context-dependent shifts in bacteria, with an enrichment of Bacillota (e.g., Bacillus, Paenibacillus) in the laboratory but not in the field. Enzymatic responses revealed a dose-dependent activation of the C-cycle, with up to 7.4-fold increases in the laboratory and up to a 10.5-fold increase in the field. P- and N- cycles showed more complex dynamics, with acid phosphatase activity increasing 3.8-fold in laboratory conditions and recovering from initial suppression to an increase of 144% in field conditions, while N-acetylglucosaminidase activity increased and L-leucine aminopeptidase decreased under laboratory conditions. Our results suggest that the response of microorganisms to 2,4-DAPG in natural soils is reduced, probably due to functional redundancy and pre-adaptation to abiotic stresses. This difference between laboratory and field studies warns against extrapolating data from controlled experiments to predict outcomes in agricultural ecosystems, and emphasizes the need for a context-specific evaluation of biocontrol agents. Full article

Common bean (Phaseolus vulgaris L.) is a critical protein-rich legume supporting food and nutritional security globally. However, Fusarium wilt, caused by Fusarium solani, remains a major constraint to production, with yield losses reaching up to 84%. While biocontrol strategies have been explored, most microbial agents are sourced from mesophilic environments and show limited effectiveness under abiotic stress. Here, we report the isolation and characterization of extremophilic Bacillus spp. from the hypersaline Lake Bogoria, Kenya, and their biocontrol potential against F. solani. From 30 isolates obtained via serial dilution, 9 exhibited antagonistic activity in vitro, with mycelial inhibition ranging from 1.07–1.93 cm 16S rRNA sequencing revealed taxonomic diversity within the Bacillus genus, including unique extremotolerant strains. Molecular screening identified genes associated with the biosynthesis of antifungal metabolites such as 2,4-diacetylphloroglucinol, pyrrolnitrin, and hydrogen cyanide. Enzyme assays confirmed substantial production of chitinase (1.33–3160 U/mL) and chitosanase (10.62–28.33 mm), supporting a cell wall-targeted antagonism mechanism. In planta assays with the lead isolate (B7) significantly reduced disease incidence (8–35%) and wilt severity (1–5 affected plants), while enhancing root colonization under pathogen pressure. These findings demonstrate that extremophile-derived Bacillus spp. possess robust antifungal traits and highlight their potential as climate-resilient biocontrol agents for sustainable bean production in arid and semi-arid agroecosystems. Full article

Pseudomonas bacteria are renowned for their remarkable capacity to synthesize antibiotics, namely mupirocin, gluconic acid, pyrrolnitrin, and 2,4-diacetylphloroglucinol (DAPG). While these substances are extensively employed in agricultural biotechnology to safeguard plants against harmful bacteria and fungi, their potential for human medicine and healthcare remains highly promising for common science. However, the challenge of obtaining stable producers that yield higher quantities of these antibiotics continues to be a pertinent concern in modern biotechnology. Although the interest in antibiotics of Pseudomonas bacteria has persisted over the past century, many uncertainties still surround the regulation of the biosynthetic pathways of these compounds. Thus, the present review comprehensively studies the genetic organization and regulation of the biosynthesis of these antibiotics and provides a comprehensive summary of the genetic organization of antibiotic biosynthesis pathways in pseudomonas strains, appealing to both molecular biologists and biotechnologists. In addition, attention is also paid to the application of antibiotics in plant protection. Full article

Boeremia exigua var. exigua is a recurrent pathogen causing root rot in industrial chicory. Currently, there is no chemical or varietal control for this disease, and thus, management strategies need to be developed. This study determined the biocontrol effect of strains of Pseudomonas protegens bacteria with antimicrobial compounds on the fungus B. exigua var. exigua under in vitro, in vivo, and field conditions. In addition, root colonization by these bacteria was estimated by the phlD-specific PCR-based dilution end point assay. Eighteen isolates of Pseudomonas spp were evaluated, and the strains that showed the greatest in vitro inhibition of fungal mycelial growth (mm), Ca10A and ChB7, were selected. Inoculation with the strain ChB7 showed less severity (necrotic area) under in vivo conditions (root trials) compared with the control inoculated with the pathogen (p ≤ 0.05). The molecular analysis revealed that the root colonization of plants grown in pots was equal to or greater than 70%. Similar levels were observed in the field trials conducted at the Selva Negra and Canteras experimental stations (2015–2016 season), with values ranging from 85.7 to 70.5% and from 75.0 to 79.5%, respectively. Regarding yield (ton ha⁻¹), values were higher in the treatments inoculated with strains Ca10A and ChB7 (p ≤ 0.05) at both experimental sites, while a lower incidence and severity of root rot were observed at Selva Negra. These results suggest that the Chilean strains of P. protegens are a promising tool for the control of root diseases in industrial chicory. Full article

Winter oilseed rape (OSR) is becoming an increasingly popular crop in rotations as it provides a cash crop and reduces the incidence of take-all fungal disease (caused by Gaeumannomyces graminis) in subsequent wheat production. The exact mechanism of this inhibition of fungal pathogens is not fully understood; however, the selective recruitment of bacterial groups with the ability to suppress pathogen growth and reproduction is thought to play a role. Here we examine the effect of tillage practice on the proliferation of microbes that possess the phlD gene involved in the production of the antifungal compound 2,4-diacetylphloroglucinol (2,4-DAPG), in the rhizospheres of both winter oilseed rape and winter wheat grown in rotation over a two-year period. The results showed that conservation strip tillage led to a significantly greater phlD gene copy number, both in the soil and in the roots, of oilseed rape and wheat crops, whereas crop rotation of oilseed rape and wheat did not increase the phlD gene copy number in winter wheat. Full article

phlD is a novel kind of polyketide synthase involved in the biosynthesis of non-volatile metabolite phloroglucinol by iteratively condensing and cyclizing three molecules of malonyl-CoA as substrate. Phloroglucinol or 2,4-diacetylphloroglucinol (DAPG) is an ecologically important rhizospheric antibiotic produced by pseudomonads; it exhibits broad spectrum anti-bacterial and anti-fungal properties, leading to disease suppression in the rhizosphere. Additionally, DAPG triggers systemic resistance in plants, stimulates root exudation, as well as induces phyto-enhancing activities in other rhizobacteria. Here, we report the cloning and analysis of the phlD gene from soil-borne gram-negative bacteria—Pseudomonas. The full-length phlD gene (from 1078 nucleotides) was successfully cloned and the structural details of the PHLD protein were analyzed in-depth via a three-dimensional topology and a refined three-dimensional model for the PHLD protein was predicted. Additionally, the stereochemical properties of the PHLD protein were analyzed by the Ramachandran plot, based on which, 94.3% of residues fell in the favored region and 5.7% in the allowed region. The generated model was validated by secondary structure prediction using PDBsum. The present study aimed to clone and characterize the DAPG-producing phlD gene to be deployed in the development of broad-spectrum biopesticides for the biocontrol of rhizospheric pathogens. Full article

The dimorphic fungus Candida albicans is one of the most important opportunistic pathogens for humankind. The use of fungicides against Candida could be associated with sub-inhibitory effects, which are referred to as fungal stress responses and are undesirable for the host. In this work, we investigated the antifungal action of 2,4-diacetylphloroglucinol (2,4-DAPG) against Candida albicans ATCC 10231 with a focus on their biofilm-forming ability. We found that 2,4-DAPG was able to reduce the ability of Candida cells to form biofilms, but complete inhibition and eradication effects were not achieved. Furthermore, C. albicans cells in the adherent state were characterized by reduced susceptibility to 2,4-DAPG compared to planktonic cells. The investigation of the mechanisms that could explain the antibiofilm action of 2,4-DAPG revealed a reduction in the cell`s surface hydrophobicity and the inhibition of the yeast-to-hyphae transition. The inhibition of the Candida cells filamentation was accompanied by an increase in the expression of the NRG1 gene, which is a negative regulator of hyphal development. In addition, we microscopically visualized the treated biofilms and revealed numerous channels that were decorated with particles and localized on the hyphae. We assumed that these hyphal structures could be associated with the secretion of aspartyl proteases (Sap). The performed assessments revealed an increase in the activity of Sap, which was accompanied by an increase in the expression of the sap2 and sap4 genes. The antifungal action of 2,4-DAPG is known to be associated with affecting the permeability of cellular structures, which leads to H⁺ATPase malfunction and the disruption of mitochondrial respiration. The subsequent cytosol acidification and generation of ROS trigger the inhibition of Candida filamentation and activation of Sap production. The introduction of antioxidant Trolox simultaneously with 2,4-DAPG leads to a reduction in Sap production. Collectively, the obtained data indicate new aspects of the interaction of fungal cells with 2,4-DAPG, an antimicrobial metabolite of Pseudomonas spp. Full article

There is currently no effective treatment against Alzheimer’s disease (AD), although many strategies have been applied to reduce beta-amyloid (Aβ) levels. Here, we investigated 2,4-diacetylphloroglucinol (DAPG) effects on Aβ levels and mechanisms of action. DAPG was the most effective phloroglucinol derivative for reducing Aβ levels, without being toxic, in various models including HEK293 cells overexpressing Swedish mutant amyloid precursor protein (APP) (293sw), primary astrocytes isolated from APPsw/PS1dE9 transgenic mice, and after intrahippocampal injection of DAPG in APPsw/PS1dE9 transgenic mice. DAPG-mediated Aβ reduction was associated with increased soluble APPα (sAPPα) levels mediated by a disintegrin and metalloproteinase domain-containing protein 10 (ADAM10) but not ADAM17. ADAM10 inhibition in DAPG-treated cells prevented the effects on sAPPα but only partly on intracellular and secreted Aβ. To identify regulators of sAPPα and Aβ secretion, various inhibitors of intracellular trafficking were administered with DAPG. Brefeldin A (BFA) reversed DAPG-mediated changes in Aβ secretion in 293sw cells, whereas golgicide A (GCA) and BFA were effective in primary astrocytes, indicating a cell type-specific regulation of the trafficking. Moreover, GCA or BFA effects on sAPPα, but not Aβ, levels in primary astrocytes resembled those of ADAM10 inhibition, indicating at least partly independent trafficking pathways for sAPPα and Aβ. In conclusion, DAPG might be a promising drug candidate against AD regulating ADAM10 and intracellular trafficking, but optimizing DAPG ability to cross the BBB will be needed. Full article

Antibiosis has been proposed to contribute to the beneficial bacteria-mediated biocontrol against pea Aphanomyces root rot caused by the oomycete pathogen Aphanomyces euteiches. However, the antibiotics required for disease suppression remain unknown. In this study, we found that the wild type strains of Pseudomonas protegens Pf-5 and Pseudomonas fluorescens 2P24, but not their mutants that lack 2,4-diacetylphloroglucinol, strongly inhibited A. euteiches on culture plates. Purified 2,4-diacetylphloroglucinol compound caused extensive hyphal branching and stunted hyphal growth of A. euteiches. Using a GFP-based transcriptional reporter assay, we found that expression of the 2,4-diacetylphloroglucinol biosynthesis gene phlA_Pf-5 is activated by germinating pea seeds. The 2,4-diacetylphloroglucinol producing Pf-5 derivative, but not its 2,4-diacetylphloroglucinol non-producing mutant, reduced disease severity caused by A. euteiches on pea plants in greenhouse conditions. This is the first report that 2,4-diacetylphloroglucinol produced by strains of Pseudomonas species plays an important role in the biocontrol of pea Aphanomyces root rot. Full article

Microbial bio-products are becoming an appealing and viable alternative to chemical pesticides for effective management of crop diseases. These bio-products are known to have potential to minimize agrochemical applications without losing crop yield and also restore soil fertility and productivity. In this study, the inhibitory efficacy of 2,4-diacetylphloroglucinol (DAPG) produced by Pseudomonas fluorescens VSMKU3054 against Ralstonia solanacearum was assessed. Biochemical and functional characterization study revealed that P. fluorescens produced hydrogen cyanide (HCN), siderophore, indole acetic acid (IAA) and hydrolytic enzymes such as amylase, protease, cellulase and chitinase, and had the ability to solubilize phosphate. The presence of the key antimicrobial encoding gene in the biosynthesis of 2,4-diacetylphloroglucinol (DAPG) was identified by PCR. The maximum growth and antimicrobial activity of P. fluorescens was observed in king’s B medium at pH 7, 37 °C and 36 h of growth. Glucose and tryptone were found to be the most suitable carbon and nitrogen sources, respectively. DAPG was separated by silica column chromatography and identified by various methods such as UV-Vis, FT-IR, GC-MS and NMR spectroscopy. When R. solanacearum cells were exposed to DAPG at 90 µg/mL, the cell viability was decreased, reactive oxygen species (ROS) were increased and chromosomal DNA was damaged. Application of P. fluorescens and DAPG significantly reduced the bacterial wilt incidence. In addition, P. fluorescens was also found effective in promoting the growth of tomato seedlings. It is concluded that the indigenous isolate P. fluorescens VSMKU3054 could be used as a suitable biocontrol agent against bacterial wilt disease of tomato. Full article

Soil microorganisms that inhabit extreme environments have unique metabolic capacities and/or physical structures that allow them to survive in oligotrophic conditions. The bioprospecting of unknown bacteria in the context of current advances in genome mining is fundamental for the discovery of natural products with novel properties or applications. In this study, the plant growth-promoting and biocontrol traits of a Pseudomonas isolated from soil associated with plants from the Atacama Desert were characterized by whole-genome sequencing and in vitro assays. A high-quality genome draft of Pseudomonas sp. isolate C3 was obtained. An automated biosynthetic gene cluster analysis using antiSMASH 6.0 revealed the presence of a cluster of genes for the biosynthesis, regulation, and transport of the metabolite 2,4-diacetylphloroglucinol, which showed a high protein sequence identity (>89%) with a validated orthologous gene cluster from another Pseudomonas. In addition, via an in vitro assay, the biocontrol activity of Pseudomonas sp. isolate C3 against Botrytis cinerea, Monilinia fructicola, Phytium sp., Alternaria sp., Geotrichum candidum, and Fusarium oxysporum was corroborated. Finally, through KofamKOALA, the presence of genes involved in different metabolic pathways of plant growth-promoting traits was identified, which was corroborated by in vitro assays. This study provides information obtained from genomic analyses and culture tools on a bacterial isolate from the Atacama Desert characterized by plant growth-promoting capacities and biocontrol activity. Full article

Pseudomonas protegens (strain DSMZ 13134) is a biocontrol agent with promising antagonistic activity hinging on antibiosis against the fungal forest pathogens Heterobasidion spp. Here, by using High-Performance Liquid Chromatography coupled to Mass Spectrometry (HPLC-MS), we assessed whether monocultures of P. protegens (strain DSMZ 13134) produce the three major determinants of biocontrol activity known for the genus Pseudomonas: 2,4-diacetylphloroglucinol (2,4-DAPG), pyoluteorin (PLT), and pyrrolnitrin (PRN). At the tested culture conditions, we observed the production of PLT at concentrations ranging from 0.01 to 10.21 mg/L and 2,4-DAPG at a concentration not exceeding 0.5 mg/L. Variations of culture conditions involving culture medium, incubation temperature, and incubation period had no consistent influence on PLT production by the bacterium. Assays using culture medium amended with PLT at the same concentration of that present in cell-free filtrate of the bacterium, i.e., 3.77 mg/L, previously documented as effective against Heterobasidion spp., showed a remarkable activity of PLT against genotypes of all the four Heterobasidion species present in Europe, including the non-native invasive H. irregulare. However, such antifungal activity decreased over time, and this may be a constraint for using this molecule as a pesticide against Heterobasidion spp. When the bacterium was co-cultured in liquid medium with genotypes of the different Heterobasidion species, an increased production of PLT was observed at 4 °C, suggesting the bacterium may perform better as a PLT producer in field applications under similar environmental conditions, i.e., at low temperatures. Our results demonstrated the role of PLT in the inhibition of Heterobasidion spp., although all lines of evidence suggest that antibiosis does not rely on a single constitutively produced metabolite, but rather on a plethora of secondary metabolites. Findings presented in this study will help to optimize treatments based on Pseudomonas protegens (strain DSMZ 13134) against Heterobasidion spp. Full article

The 2,4-Diacetylphloroglucinol (2,4-DAPG) and phenazine (PCA)-producing Pseudomonas inhibit wheat pathogens’ development, but the relationship between communities of pathogens and genotypes of these bacteria has been little studied. Relationships between wheat crown fungi associated with the presence of 2,4-DAPG and PCA-producing pseudomonads were evaluated in four commercial wheat crops located in the La Araucanía and Los Lagos Regions of Chile, during two crops seasons. Portions of the base of the first internode of the culm collected during the grain-filling stage were cultured in an artificial medium for fungal isolation, while roots of the same wheat plants and from plants collected previous harvest, and also used to assess yield and plant height, were used for the detection of 2,4-DAPG and PCA-producing Pseudomonas spp. using PCR with specific primers. Genera Phaeosphaeria, Fusarium, Rhizoctonia, and Microdochium were repeatedly isolated (52.6%, 22.1%, 7.8%, and 4.9%, respectively) and the genetic composition of 2,4-DAPG and PCA-producing Pseudomonas spp. varied between fields and sampling periods. Genetic groups A, B, D, K, L, and P associated with the phlD gene were detected. The presence of 2,4-DAPG-producing bacteria benefited crop health, relating their existence with increasing yield and plant height, and the reduction in the incidence and severity of disease caused by pathogenic microorganisms on the first internode of wheat culms. Full article

Kiwifruit worldwide suffers from the devastating diseases of bacterial canker caused by Pseudomonas syringae pv. actinidiae (Psa) and gray mold caused by Botrytis cinerea. Here, an endophytic bacterium XL17 isolated from a rape crown gall was screened out for its potent antagonistic activities against Psa and B. cinerea. Strain XL17 and its cell-free culture filtrate (CF) inhibited the growth of Psa and B. cinerea, Psa-associated leaf necrosis, and B. cinerea-associated kiwifruit necrosis. Electron microscopy showed that XL17 CF could damage the cell structures of Psa and B. cinerea. Genome-based taxonomy revealed that strain XL17 belongs to Pseudomonas bijieensis within the P. corrugata subgroup of the P. fluorescens species complex. Among the P. corrugata subgroup containing 31 genomospecies, the presence of the phl operon responsible for the biosynthesis of the phenolic polyketide 2,4-diacetylphloroglucinol (DAPG) and the absence of the lipopeptide/quorum sensing island can serve as the genetic marker for the determination of a plant-protection life style. HPLC detected DAPG in extracts from XL17 CF. MALDI-TOF-MS analysis revealed that strain XL17 produced cyclic lipopeptides of the viscosin family and orfamide family. Together, phenotypic, genomic, and metabolic analyses identified that P. bijieensis XL17 producing DAPG and cyclic lipopeptides can be used to control bacterial canker and gray mold pathogens of kiwifruit. Full article

The impact of inoculated plant growth-promoting rhizobacteria (PGPR) on its host physiology and nutrition depends on inoculum level. Whether the impact of the inoculated PGPR on the indigenous rhizosphere microbiota also varies with the PGPR inoculum level is unclear. Here, we tested this issue using the PGPR Azospirillum lipoferum CRT1—maize model system, where the initial seed inoculation is known to enhance maize growth and germination, and impacts the maize rhizomicrobiota, including microbial functional groups modulating plant growth. A. lipoferum CRT1 was added to the seeds at standard (10^5–6 cells.seed⁻¹) or reduced (10^4–5 cells.seed⁻¹) inoculation levels, in three fields. The effect of the two PGPR formulations was assessed on maize growth and on the nifH (nitrogen fixation), acdS (ACC deaminase activity) and phlD (2,4-diacetylphloroglucinol production) microbial functional groups. The size of the three functional groups was monitored by qPCR at the six-leaf stage and the flowering stage, and the diversity of the nifH and acdS functional groups (as well as the bacterial community) were estimated by MiSeq metabarcoding at the six-leaf stage. The results showed that the benefits of the reduced inoculant formulation were significant in two out of three fields, but different (often lower) than those of the standard formulation. The effects of formulations on the size of the three functional groups differed, and depended on field site and functional group. The reduced formulation had an impact on the diversity of nifH and acdS groups at one site, whereas the standard formulation had an impact at the two other sites. Inoculation significantly impacted the total bacterial community in the three fields, but only with the reduced formulation. In conclusion, the reduced inoculant formulation impacted the indigenous rhizosphere microbiota differently, but not less efficiently, than the standard formulation. Full article