Search

Background: Bacteria produce antimicrobial compounds to compete for nutrients and space in particular habitat. Bacteria of the genus Pseudomonas have gained relevance in recent years due to their ability to produce bacteriocins and a wide range of secondary metabolites of medical and agricultural importance, such as 2,4-diacetylphloroglucinol (DAPG), ribosomal peptide synthetase (RPS), pyrrolnitrin (PRN) and pyoluteorin (PLT), which have exhibited antimicrobial properties. Objective: The present research focused on establishing an expression system for the heterologous production of the bacteriocin EMM1 from Pseudomonas protegens in a soluble, stable and biologically active form in order to evaluate the performance of this protein in the treatment of emerging drug-resistant bacteria of clinical interest. Methodology: The 840 bp bacteriocin EMM1 gene was obtained from Pseudomonas protegens strain, cloned into the expression vector pET28b(+) and subsequently expressed in Escherichia coli Gold BL21 (DE3). The amino acid tag MASKEEDN (PFT) was added at the N-terminal end of the bacteriocin to improve its solubility and stability in order to achieve its purification by immobilized metal affinity chromatography (IMAC). The antimicrobial activity was evaluated by inhibition zones and plate microdilution on reference and clinical strains and the hemolytic activity on human blood cells was determined. Results: This recombinant protein named EMM1-PFT showed unique characteristics with respect to its size, stability and oligomeric state. Bactericidal activity was observed on reference strains E. coli ATCC 3922, E. coli ATCC 25922, Staphylococcus aureus ATCC 25923, clinical isolates of Pseudomonas aeruginosa and Yersinia enterocolitica and low hemolytic activity on human erythrocytes. Conclusions: A new recombinant protein was obtained that was partially characterized and active against clinical strains. These advances allow us to suggest further studies to understand its mechanism of action, synergistic activity with antibiotics and improvement of the spectrum of antibacterial activity through protein engineering. EMM1-PFT is a new alternative for the eradication of emerging MDR bacteria. [ABSTRACT FROM AUTHOR]

Aims This study investigates the traits of three plant growth-promoting (PGP) and antagonistic bacteria, Pseudomonas protegens MP12, Bacillus sp. 3R4, and Bacillus sp. T22, to assess their potential application as biocontrol agents by using the ecofriendly and low-cost substrate Corn Steep Liquor (CSL) medium. Analyses of antagonism through volatile organic compounds (VOCs) production, biofilm formation, and growth performance were carried out. Methods and results Dual antagonism assay showed that all strains displayed significant antagonistic activity against Botrytis cinerea through VOCs. Gas chromatography demonstrated that strains in the CSL exhibited higher VOCs production than nutrient medium. Moreover, enhanced biofilm formation analysed by Calgary Biofilm Device, growth, and biomass were noted in CSL cultures. Pseudomonas protegens MP12, which showed higher cell concentration and biomass yield, was selected for freeze-drying treatments. Storage cell viability assays evidenced that it can be effectively preserved for nearly 7 months at 4°C. Conclusion The results here obtained showed that CLS medium enhanced VOCs production, biofilm formation, growth, and biomass of the antagonistic bacteria of the three strains. Eventually, the more effective strain P. protegens MP12 can be stored for nearly 7 months at 4°C. [ABSTRACT FROM AUTHOR]

Abstract The rhizosphere bacterium Pseudomonas protegens FD6, which is associated with the production of antibiotics such as pyoluteorin (PLT) and 2,4-diacetylphloroglucinol (2,4-DAPG), has strong antagonistic effects on phytopathogens. Hfq is a conserved RNA chaperone involved in the regulation of stress tolerance, antibiotic production, and bacterial virulence. Here, we determined the regulatory effects of Hfq on biocontrol traits in P. protegens FD6. Mutation of hfq in FD6 reduced the growth rate, swimming and swarming ability, and production of proteases, siderophores, and hydrogen cyanide (HCN). Transmission electron microscopy revealed that disruption of hfq led to the loss of flagella. Furthermore, Hfq exerted a suppressive effect on biofilm formation and PLT production while not affecting antagonistic activity and control effect against tomato bacterial wilt. However, Hfq positively regulated the production of 2,4-DAPG and activated the phlD expression. Additionally, mutation of hfq abolished the function of the type VI secretion system (T6SS) due to a decrease in the expression of T6SS-related genes. Overall, these results suggest that Hfq plays a pleiotropic role in modulating the expression of biocontrol trait-related genes in P. protegens FD6.

The rhizosphere bacterium Pseudomonas protegens FD6, which is associated with the production of antibiotics such as pyoluteorin (PLT) and 2,4-diacetylphloroglucinol (2,4-DAPG), has strong antagonistic effects on phytopathogens. Hfq is a conserved RNA chaperone involved in the regulation of stress tolerance, antibiotic production, and bacterial virulence. Here, we determined the regulatory effects of Hfq on biocontrol traits in P. protegens FD6. Mutation of hfq in FD6 reduced the growth rate, swimming and swarming ability, and production of proteases, siderophores, and hydrogen cyanide (HCN). Transmission electron microscopy revealed that disruption of hfq led to the loss of flagella. Furthermore, Hfq exerted a suppressive effect on biofilm formation and PLT production while not affecting antagonistic activity and control effect against tomato bacterial wilt. However, Hfq positively regulated the production of 2,4-DAPG and activated the phlD expression. Additionally, mutation of hfq abolished the function of the type VI secretion system (T6SS) due to a decrease in the expression of T6SS-related genes. Overall, these results suggest that Hfq plays a pleiotropic role in modulating the expression of biocontrol trait-related genes in P. protegens FD6. [ABSTRACT FROM AUTHOR]

The relationships between plants and bacteria are essential in agroecosystems and bioinoculant development. The leaf endophytic Pseudomonas protegens E1BL2 was previously isolated from giant Jala maize, which is a native Zea mays landrace of Nayarit, Mexico. Using different Mexican maize landraces, this work evaluated the strain's plant growth promotion and biocontrol against eight phytopathogenic fungi in vitro and greenhouse conditions. Also, a plant field trial was conducted on irrigated fields using the hybrid maize Supremo. The grain productivity in this assay increased compared with the control treatment. The genome analysis of P. protegens E1BL2 showed putative genes involved in metabolite synthesis that facilitated its beneficial roles in plant health and environmental adaptation (bdhA, acoR, trpE, speE, potA); siderophores (ptaA, pchC); and extracellular enzymes relevant for PGPB mechanisms (cel3, chi14), protection against oxidative stress (hscA, htpG), nitrogen metabolism (nirD, nit1, hmpA), inductors of plant-induced systemic resistance (ISR) (flaA, flaG, rffA, rfaP), fungal biocontrol (phlD, prtD, prnD, hcnA-1), pest control (vgrG-1, higB-2, aprE, pslA, ppkA), and the establishment of plant-bacteria symbiosis (pgaA, pgaB, pgaC, exbD). Our findings suggest that P. protegens E1BL2 significantly promotes maize growth and offers biocontrol benefits, which highlights its potential as a bioinoculant. [ABSTRACT FROM AUTHOR]

Pseudomonas protegens can serve as an agricultural biocontrol agent. P. protegens often encounters hyperosmotic stress during industrial production and fi eld application. The ability of P. protegens to withstand hyperosmotic stress is important for its application as a biocontrol agent. AlgU is a global regulator responsible for stress response and biocontrol ability. However, the specific regulatory role of AlgU in the hyperosmotic adaptation of P. protegens is poorly understood. In this study, we found that the AlgU mutation disrupted the hyperosmotic tolerance of P. protegens. Many genes and metabolites related to cell envelope formation were significantly downregulated in ΔalgU compared with that in the wild-type (WT) strain under hyperosmotic conditions, and we found that the algU mutation caused membrane integrity to be compromised and increased membrane permeability. Further experiments revealed that the cell envelope integrity protein TolA, which is regulated by AlgU, contributes to cell membrane stability and osmotic tolerance in P. protegens. In addition, several genes related to oxidative stress response were significantly downregulated in ΔalgU, and higher levels of intracellular reactive oxygen species were found in ΔalgU. Furthermore, we found that the synthesis of N-acetyl glutaminyl glutamine amide is directly regulated by AlgU and contributes to the hyperosmotic adaptation of P. protegens. This study revealed the mechanisms of AlgU's participation in osmotic tolerance in P. protegens, and it provides potential molecular targets for research on the hyperosmotic adaptation of P. protegens. [ABSTRACT FROM AUTHOR]

Wheat (Triticum aestivum L.), a staple grain consumed worldwide, is heavily affected by Fusarium species, which cause harmful diseases that threaten its production. The present study was conducted to investigate the biocontrol activity of monocultures of Pseudomonas protegens ML15 and Trichoderma koningiopsis Tr21 as well as their co-culture, as a sustainable strategy to combat Fusarium cerealis. The cell-free supernatant and cell-free extract of co-culture demonstrated increased inhibitory effects against F. cerealis, compared to the monocultures. GC-MS analysis revealed that cell-free extract of P. protegens ML15, T. koningiopsis Tr21, and co-culture contained different bioactive metabolites. Pyrrolo[1,2-a]pyrazine-1,4-dione derivatives were major compounds in the cell-free extract of co-culture. Further analysis using NMR and HPLC, revealed that co-culture produced higher concentrations of pyoluteorin, 2,4-diacetylphloroglucinol, and 2,4-di-tert-butylphenol, compared to their respective monocultures. In vivo plant experiments indicated that co-culture treatment reduced F. cerealis infection and improved wheat development. Our findings highlight the exciting potential of co-culturing P. protegens ML15 and T. koningiopsis Tr21 as a formidable biocontrol duo, particularly effective against notorious fungal plant pathogens like Fusarium. This innovative approach holds promise for revolutionizing agricultural practices, offering sustainable solutions to combat crop diseases, and ensuring global food security.

Soil and rhizosphere bacteria act as a rich source of secondary metabolites, effectively fighting against a diverse array of pathogens. Certain Pseudomonas species harbor biosynthetic gene clusters for producing both pyoluteorin and 2,4-diacetylphloroglucinol (2,4-DAPG), which are polyketides that exhibit highly similar antimicrobial spectrum against bacteria and fungi or oomycete. A complex cross talk exists between pyoluteorin and 2,4-DAPG biosynthesis, and production of 2,4-DAPG was strongly repressed by pyoluteorin, yet the underlying mechanism is still elusive. In this study, we find that the TetR family transcription factor PhlH is involved in the cross talk between pyoluteorin and 2,4-DAPG biosynthesis. PhlH binds to a palindromic sequence within the promoter of phlG (PphlG), which encodes a C-C bond hydrolase responsible for degrading 2,4-DAPG. As a signaling molecule, pyoluteorin disrupts the PhlH-PphlG complex by binding to PhlH, leading to decreased levels of 2,4-DAPG. Proteomics data suggest that pyoluteorin regulates multiple physiological processes including fatty acid biosynthesis and transportation of taurine, siderophore, and amino acids. Our work not only reveals a novel mechanism of cross talk between pyoluteorin and 2,4-DAPG biosynthesis, but also highlights pyoluteorin's role as a messenger in the complex communication network of Pseudomonas. IMPORTANCE Antibiosis serves as a crucial defense mechanism for microbes against invasive bacteria and resource competition. These bacteria typically orchestrate the production of multiple antibiotics in a coordinated fashion, wherein the synthesis of one antibiotic inhibits the generation of another. This strategic coordination allows the bacterium to focus its resources on producing the most advantageous antibiotic under specific circumstances. However, the underlying mechanisms of distinct antibiotic production in bacterial cells remain largely elusive. In this study, we reveal that the TetR family transcription factor PhlH detects the secondary metabolite pyoluteorin and mediates the cross talk between pyoluteorin and 2,4-DAPG biosynthesis in the biocontrol strain Pseudomonas protegens Pf-5. These findings hold promise for future research, potentially informing the manipulation of these systems to enhance the effectiveness of biocontrol agents. [ABSTRACT FROM AUTHOR]

Gray mold, caused by Botrytis cinerea is a major cause of post-harvest rot of fresh fruits and vegetables. The utilization of selected microorganisms as biocontrol agents is a promising alternative to effectively control gray mold on tomatoes. The current study was conducted to explore potential biocontrol mechanisms of the Pseudomonas strain to control infections on post-harvest tomatoes. Among the 8 tested bacterial isolates, Pseudomonas protegens ML15 demonstrated antagonistic activity to Botrytis cinerea. Moreover, P. protegens ML15 exhibited the production of siderophores, hydrogen cyanide, ammonia, exopolysaccharides, lipase, biosurfactant, 2,4-diacetylphloroglucinol, and several other antifungal compounds, such as 1-tetradecanol, cyclododecane, 2,4-di-tert-butylphenol, and 2-methyl-1-hexadecanol. A comprehensive genomic analysis of P. protegens ML15 unravels 18 distinct genetic regions with the potential for biosynthesizing secondary metabolites, known for their pivotal role in biocontrol responses against plant pathogens. In vivo, experiments showed that both culture suspension and cell-free supernatant of P. protegens ML15 significantly reduced fungal growth (53.0 ± 0.63%) and mitigated disease development (52.8 ± 1.5%) in cherry tomatoes at four days post-B. cinerea inoculation. During the infection, the application of P. protegens ML15 resulted in the augmentation of total antioxidant, phenolic content, and ascorbic acids content. Thus, our results suggested that P. protegens ML15's role as a biocontrol agent against B. cinerea-induced postharvest tomato decay achieved through the secretion of antifungal substances, induction of tomato defense responses, and inhibition of mycelial growth of B. cinerea. These findings provide a significant contribution to the ongoing search for alternative, eco-friendly methods of controlling gray mold in fresh products. The utilization of P. protegens ML15 as a biocontrol agent could help to reduce the reliance on chemical fungicides and promote sustainable agriculture practices. [ABSTRACT FROM AUTHOR]

The high incidence of the boring nematode R. similis in banana cultivation has prompted the continuous use of nematicides, as the only effective solution, but not very sustainable in the face of the microbiota and soil fertility; As a biological alternative, the biocontrol activity of PGPRs has a suppressive effect against large nematological populations. As a study target, the effect of bacterial bioformulates on the control of R. similis and enhancer on the development of banana seedlings cultivar Williams was evaluated. Five study factors were used: Three bioformulates made with different components, which were inoculated in combination with 4 strains PGPRs Acinetobacter calcoaceticus BMR 2-12, Serratia marcescens PM 3-8, Pseudomonas protegens CHA0 and Enterobacter asburiae PM 3-14; Nematicide and Without PGPRs for their biocontrol and promoter evaluations in the presence of the nematode applied in initial populations between 10,000-15,000 plus four repopulations of 8,000-10,000 in banana seedlings. The biocontrol factor of Bioformulated-1 with CFU/mL loads of 9.70X109 obtained greater effectiveness in the presence of R. similis, reducing to 1, 000/100 g of root, counteracting its incidence of damage to 12.28 %. Likewise, the effect of the Bioformulated products improved various plant growth parameters, involving an increase in plant height with 42.74 and 42.42 cm (Bio-1 and 3); greater number of leaves of 5.80 and 5.40 (Bio-1 and 2); root length with 35.64 cm (Bio-3), revealing to have high benefits towards sustainable agriculture. [ABSTRACT FROM AUTHOR]

Gray mold, caused by Botrytis cinerea is a major cause of post-harvest rot of fresh fruits and vegetables. The utilization of selected microorganisms as biocontrol agents is a promising alternative to effectively control gray mold on tomatoes. The current study was conducted to explore potential biocontrol mechanisms of the Pseudomonas strain to control infections on post-harvest tomatoes. Among the 8 tested bacterial isolates, Pseudomonas protegens ML15 demonstrated antagonistic activity to Botrytis cinerea. Moreover, P. protegens ML15 exhibited the production of siderophores, hydrogen cyanide, ammonia, exopolysaccharides, lipase, biosurfactant, 2,4-diacetylphloroglucinol, and several other antifungal compounds, such as 1-tetradecanol, cyclododecane, 2,4-di-tert-butylphenol, and 2-methyl-1-hexadecanol. A comprehensive genomic analysis of P. protegens ML15 unravels 18 distinct genetic regions with the potential for biosynthesizing secondary metabolites, known for their pivotal role in biocontrol responses against plant pathogens. In vivo, experiments showed that both culture suspension and cell-free supernatant of P. protegens ML15 significantly reduced fungal growth (53.0 ± 0.63%) and mitigated disease development (52.8 ± 1.5%) in cherry tomatoes at four days post-B. cinerea inoculation. During the infection, the application of P. protegens ML15 resulted in the augmentation of total antioxidant, phenolic content, and ascorbic acids content. Thus, our results suggested that P. protegens ML15’s role as a biocontrol agent against B. cinerea-induced postharvest tomato decay achieved through the secretion of antifungal substances, induction of tomato defense responses, and inhibition of mycelial growth of B. cinerea. These findings provide a significant contribution to the ongoing search for alternative, eco-friendly methods of controlling gray mold in fresh products. The utilization of P. protegens ML15 as a biocontrol agent could help to reduce the reliance on chemical fungicides and promote sustainable agriculture practices.

Two novel P. protegens bacteriophages PseuP_222 and Pseu_224 and their host P. protegens CEMTC 4060 were isolated from the same sample (Inya river, Siberia). Both phages have siphovirus morphology and belong to lambdoid phages. Comparative genome analysis revealed a low nucleotide and amino acid sequence similarity of PseuP_222 and PseuP_224 between themselves, and between them and other lambdoid phages. Bioinformatics analysis indicated that PseuP_222 and PseuP_224 are members of a genetically diverse group of phages of environmental Pseudomonas spp.; this group is distant from a large group of P. aeruginosa phages. In phylogenetic trees, the positioning of the terminase large subunits, major capsid proteins, tail tape measure proteins, and CI-like repressors of PseuP_222 and PseuP_224 were remote and changed relative to those of the Escherichia lambda phage and lambdoid phages of Pseudomonas spp. However, the nucleoid-associated protein NdpA/YejK and P5-like structural protein from both phages showed high similarity and were not found in lambda phage and other lambdoid phages of Pseudomonas spp. Substantial divergences of the PseuP_222 and PseuP_224 genomes and proteomes indicated that the evolutionary history of these phages was mostly independent and they probably began to use one host only recently. [ABSTRACT FROM AUTHOR]

Summary: The antagonistic bacterium Pseudomonas protegens secretes the cyclic lipopeptide (CLiP) orfamide A, which triggers a Ca2+ signal causing rapid deflagellation of the microalga Chlamydomonas reinhardtii.We performed chemical synthesis of orfamide A derivatives and used an aequorin reporter line to measure their Ca2+ responses. Immobilization of algae was studied using a modulator and mutants of transient receptor potential (TRP)‐type channels.By investigating targeted synthetic orfamide A derivatives, we found that N‐terminal amino acids of the linear part and the terminal fatty acid region are important for the specificity of the Ca2+‐signal causing deflagellation. Molecular editing indicates that at least two distinct Ca2+‐signaling pathways are triggered. One is involved in deflagellation (Thr3 change, fatty acid tail shortened by 4C), whereas the other still causes an increase in cytosolic Ca2+ in the algal cells, but does not cause substantial deflagellation (Leu1 change, fatty acid hydroxylation, fatty acid changes by 2C). Using mutants, we define four TRP‐type channels that are involved in orfamide A signaling; only one (ADF1) responds additionally to low pH.These results suggest that the linear part of the CLiP plays one major role in Ca2+ signaling, and that orfamide A uses a network of algal TRP‐type channels for deflagellation. [ABSTRACT FROM AUTHOR]

Mixed species biofilms exhibit increased tolerance to numerous stresses compared to single species biofilms. The aim of this study was to examine the effect of grazing by the heterotrophic protist, Tetrahymena pyriformis, on a mixed species biofilm consisting of Pseudomonas aeruginosa, Pseudomonas protegens, and Klebsiella pneumoniae. Protozoan grazing significantly reduced the single species K. pneumoniae biofilm, and the single species P. protegens biofilm was also sensitive to grazing. In contrast, P. aeruginosa biofilms were resistant to predation. This resistance protected the otherwise sensitive members of the mixed species biofilm consortium. Rhamnolipids produced by P. aeruginosa were shown to be the primary toxic factor for T. pyriformis. However, a rhamnolipid-deficient mutant of P. aeruginosa (P. aeruginosa DrhlAB) maintained grazing resistance in the biofilm, suggesting the presence of at least one additional protective mechanism. P. aeruginosa with a deleted gene encoding the type III secretion system also resisted grazing. A transposon library was generated in the DrhlAB mutant to identify the additional factor involved in community biofilm protection. Results indicated that the Pseudomonas Quinolone Signal (PQS), a quorum sensing signaling molecule, was likely responsible for this effect. We confirmed this observation by showing that double mutants of DrhlAB and genes in the PQS biosynthetic operon lost grazing protection. We also showed that PQS was directly toxic to T. pyriformis. This study demonstrates that residing in a mixed species biofilm can be an advantageous strategy for grazing sensitive bacterial species, as P. aeruginosa confers community protection from protozoan grazing through multiple mechanisms. [ABSTRACT FROM AUTHOR]

Pseudomonas protegens is an important biocontrol agent with the ability to suppress plant pathogens and promote plant growth. P. protegens' ability to endure hyperosmotic stress is crucial to its effectiveness as a biocontrol agent. This study elucidated potassium's role and mechanism of action in enabling the hyperosmotic tolerance of P. protegens. Potassium was observed to significantly improve the growth of P. protegens under hyperosmotic conditions. Four functionally redundant potassium transporters, KdpA1, KdpA2, TrkH, and Kup, were identified in P. protegens , of which KdpA2 and TrkH were particularly important for its growth under hyperosmotic conditions. Potassium enhanced the biofilm formation and cell membrane stability of P. protegens under hyperosmotic conditions. In addition, we revealed that K+ stimulates the expression of several genes related to DNA damage repair in P. protegens under hyperosmotic conditions. Further experiments revealed that the DNA repair-related recG induced by potassium contributes to P. protegens ' hyperosmotic tolerance. We also found that the sigma factor RpoN participates in the hyperosmotic adaptation of P. protegens. Furthermore, we revealed that the opuCABCD operon, whose expression is induced by potassium through RpoN, serves as the key pathway through which betaine, choline, and carnitine improve the hyperosmotic tolerance of P. protegens. [ABSTRACT FROM AUTHOR]

Pseudomonas protegens Pf-5 is an effective biocontrol agent that protects many crops against pathogens, including the fungal pathogen Botrytis cinerea causing gray mold disease in Cannabis sativa crops. Previous studies have demonstrated the important role of antibiotics pyoluteorin (PLT) and 2,4-diacetylphloroglucinol (DAPG) in Pf-5-mediated biocontrol. To assess the potential involvement of PLT and DAPG in the biocontrol exerted by Pf-5 against B. cinerea in the phyllosphere of C. sativa, two knockout Pf-5 mutants were generated by in-frame deletion of genes pltD or phlA, required for the synthesis of PLT or DAPG respectively, using a two-step allelic exchange method. Additionally, two complemented mutants were constructed by introducing a multicopy plasmid carrying the deleted gene into each deletion mutant. In vitro confrontation assays revealed that deletion mutant ΔpltD inhibited B. cinerea growth significantly less than wild-type Pf-5, supporting antifungal activity of PLT. However, deletion mutant ΔphlA inhibited mycelial growth significantly more than the wild-type, hypothetically due to a coregulation of PLT and DAPG biosynthesis pathways. Both complemented mutants recovered in vitro inhibition levels similar to that of the wild-type. In subsequent growth chamber inoculation trials, characterization of gray mold disease symptoms on infected cannabis plants revealed that both ΔpltD and ΔphlA significantly lost a part of their biocontrol capabilities, achieving only 10 and 19% disease reduction respectively, compared to 40% achieved by inoculation with the wild-type. Finally, both complemented mutants recovered biocontrol capabilities in planta similar to that of the wild-type. These results indicate that intact biosynthesis pathways for production of PLT and DAPG are required for the optimal antagonistic activity of P. protegens Pf-5 against B. cinerea in the cannabis phyllosphere. [ABSTRACT FROM AUTHOR]

Pseudomonas protegens Pf-5 is an effective biocontrol agent that protects many crops against pathogens, including the fungal pathogen Botrytis cinerea causing gray mold disease in Cannabis sativa crops. Previous studies have demonstrated the important role of antibiotics pyoluteorin (PLT) and 2,4-diacetylphloroglucinol (DAPG) in Pf-5-mediated biocontrol. To assess the potential involvement of PLT and DAPG in the biocontrol exerted by Pf-5 against B. cinerea in the phyllosphere of C. sativa, two knockout Pf-5 mutants were generated by in-frame deletion of genes pltD or phlA, required for the synthesis of PLT or DAPG respectively, using a two-step allelic exchange method. Additionally, two complemented mutants were constructed by introducing a multicopy plasmid carrying the deleted gene into each deletion mutant. In vitro confrontation assays revealed that deletion mutant ∆pltD inhibited B. cinerea growth significantly less than wild-type Pf-5, supporting antifungal activity of PLT. However, deletion mutant ∆phlA inhibited mycelial growth significantly more than the wild-type, hypothetically due to a co-regulation of PLT and DAPG biosynthesis pathways. Both complemented mutants recovered in vitro inhibition levels similar to that of the wild-type. In subsequent growth chamber inoculation trials, characterization of gray mold disease symptoms on infected cannabis plants revealed that both ∆pltD and ∆phlA significantly lost a part of their biocontrol capabilities, achieving only 10 and 19% disease reduction respectively, compared to 40% achieved by inoculation with the wild-type. Finally, both complemented mutants recovered biocontrol capabilities in planta similar to that of the wild-type. These results indicate that intact biosynthesis pathways for production of PLT and DAPG are required for the optimal antagonistic activity of P. protegens Pf-5 against B. cinerea in the cannabis phyllosphere.

Two novel P. protegens bacteriophages PseuP_222 and Pseu_224 and their host P. protegens CEMTC 4060 were isolated from the same sample (Inya river, Siberia). Both phages have siphovirus morphology and belong to lambdoid phages. Comparative genome analysis revealed a low nucleotide and amino acid sequence similarity of PseuP_222 and PseuP_224 between themselves, and between them and other lambdoid phages. Bioinformatics analysis indicated that PseuP_222 and PseuP_224 are members of a genetically diverse group of phages of environmental Pseudomonas spp.; this group is distant from a large group of P. aeruginosa phages. In phylogenetic trees, the positioning of the terminase large subunits, major capsid proteins, tail tape measure proteins, and CI-like repressors of PseuP_222 and PseuP_224 were remote and changed relative to those of the Escherichia lambda phage and lambdoid phages of Pseudomonas spp. However, the nucleoid-associated protein NdpA/YejK and P5-like structural protein from both phages showed high similarity and were not found in lambda phage and other lambdoid phages of Pseudomonas spp. Substantial divergences of the PseuP_222 and PseuP_224 genomes and proteomes indicated that the evolutionary history of these phages was mostly independent and they probably began to use one host only recently.

The rhizospheric bacterium Pseudomonas protegens Pf-5 can colonize the seed and root surfaces of plants, and can protect them from pathogen infection. Secondary metabolites, including lipopeptides and polyketides produced by Pf-5, are involved in its biocontrol activity. We isolated a crude extract from Pf-5. It exhibited significant surface activity and strong antibacterial activity against Pantoea ananatis DZ-12, which causes maize brown rot on leaves. HPLC analysis combined with activity tests showed that the polyketide pyoluteorin in the crude extract participated in the suppression of DZ-12 growth, and that the lipopeptide orfamide A was the major biosurfactant in the crude extract. Further studies indicated that the pyoluteorin in the crude extract significantly suppressed the biofilm formation of DZ-12, and it induced the accumulation of reactive oxygen species in DZ-12 cells. Scanning electron microscopy and transmission electron microscopy observation revealed that the crude extract severely damaged the pathogen cells and caused cytoplasmic extravasations and hollowing of the cells. The pathogenicity of DZ-12 on maize leaves was significantly reduced by the crude extract from Pf-5 in a dose-dependent manner. The polyketide pyoluteorin had strong antibacterial activity against DZ-12, and it has the potential for development as an antimicrobial agent. [ABSTRACT FROM AUTHOR]

Two polyurethanases PueA and PueB from Pseudomonas protegens Pf‐5 have been reported to have hydrolytic activity against synthetic p‐nitrophenyl palmitate of lipase substrate, and PueA may play a more effective role in this activity. However, it is still unknown whether PueA and PueB play similar parts in the lipase activity against natural acylglycerols and achieve the extracellular secretion via their cognate ABC exporter AprDEF. In this study, we investigated these questions through the construction of four markerless deletion mutants in Pf5139 (Δupp derivative of Pf‐5), two heterologous co‐expression strains and their three control strains in lipase‐free Escherichia coli BL21(DE3), and detected their lipase activities by the tributyrin plate assay and the liquid culture assay. The results showed that PueA and PueB, classified as subfamily I.3 lipases, are major extracellular lipases involved in the uptake of oil in Pf‐5, and PueA plays a leading role in extracellular lipase activity. In addition, the extracellular secretion of PueA and PueB can be partly mediated via AprDEF in Pf‐5 and BL21(DE3). Finally, PueA and PueB are also able to achieve the extracellular secretion without the assistance of AprDEF in Pf‐5 and BL21(DE3). [ABSTRACT FROM AUTHOR]

Many Pseudomonas protegens strains produce the antibiotics pyoluteorin (PLT) and 2,4-diacetylphloroglucinol (2,4-DAPG), both of which have antimicrobial properties. The biosynthesis of these metabolites is typically controlled by multiple regulatory factors. Virulence factor regulator (Vfr) is a multifunctional DNA-binding regulator that modulates 2,4-DAPG biosynthesis in P. protegens FD6. However, the mechanism by which Vfr regulates this process remains unclear. In the present study, chromatin immunoprecipitation of FLAG-tagged Vfr and nucleotide sequencing analysis were used to identify 847 putative Vfr binding sites in P. protegens FD6. The consensus P. protegens Vfr binding site predicted from nucleotide sequence alignment is TCACA. The qPCR data showed that Vfr positively regulates the expression of phlF and phlG, and the expression of these genes was characterized in detail. The purified recombinant Vfr bound to an approximately 240-bp fragment within the phlF and phlG upstream regions that harbor putative Vfr consensus sequences. Using electrophoretic mobility shift assays, we localized Vfr binding to a 25-bp fragment that contains part of the Vfr binding region. Vfr binding was eliminated by mutating the TACG and CACA sequences in phlF and phlG, respectively. Taken together, our results show that Vfr directly regulates the expression of the 2,4-DAPG operon by binding to the upstream regions of both the phlF and phlG genes. However, unlike other Vfr-targeted genes, Vfr binding to P. protegens FD6 does not require an intact binding consensus motif. Furthermore, we demonstrated that vfr expression is autoregulated in this bacterium. These results provide novel insights into the regulatory role of Vfr in the biocontrol agent P. protegens.

Background: Zinc is an essential micronutrient required for optimum plant growth. Zinc-solubilizing bacteria convert applied inorganic zinc to available forms that could be used by plants. Research design: In present study, experiments were conducted to isolate, characterize, and evaluate Zn solubilization potential of different bacteria. Results: Among 10 isolated strains, Pseudomonas protegens (RY2, MF351762) was found to be the most promising strain having zinc-solubilizing potential on 4 different insoluble zinc sources. In quantitative assay, Zn solubilization by RY2 was significantly higher than other strains at different incubation time. P. protegens RY2 was selected (based on zinc solubilizing and plant growth promoting activities like P solubilization and ACC deaminase) for plant experiments. Meanwhile, available Zn release rate in soil was determined at day 10 of incubation. Chickpea seeds were inoculated with RY2 strain and ZnO is used as zinc source. Growth parameters and quantifying zinc content of shoot and root using atomic absorption spectrophotometer were determined. Enhanced shoot and root dry weights and lengths were observed in chickpea plants compared to control. Maximum increase of 44%, 67%, and 75% in T2 (Soil + RY2), T5 (Soil + ZnO + RY2), and T7 (Soil + manure + ZnO + RY2), respectively, was found in shoot length compared to control (T1). Conclusion: The study indicated that zinc-solubilizing RY2 strain possesses potential for enhanced Zn in soil so it would allow reduced inorganic Zn application. [ABSTRACT FROM AUTHOR]

In order to meet the requirements of soil inoculants for release rate and biodegradability, oxidized sodium alginate (OSA) and starch to form a composite material used to encapsulate Pseudomonas protegens SN15-2. The results indicated that OSA microcapsules show higher degradation and release rate than alginate microcapsules because of the lower molecular weight of OSA. Meanwhile, increasing the proportion of starch within the maximum concentration range of 4% can further improve the release rate and degradability of the microcapsules. This encapsulation strategy has broad application prospects to meet the needs of soil inoculant production. [ABSTRACT FROM AUTHOR]

Bacterial biofilms are important medically, environmentally and industrially and there is a need to understand the processes that govern functional synergy and dynamics of species within biofilm communities. Here, we have used a model, mixed-species biofilm community comprised of Pseudomonas aeruginosa PAO1, Pseudomonas protegens Pf-5 and Klebsiella pneumoniae KP1. This biofilm community displays higher biomass and increased resilience to antimicrobial stress conditions such as sodium dodecyl sulfate and tobramycin, compared to monospecies biofilm populations. P. aeruginosa is present at low proportions in the community and yet, it plays a critical role in community function, suggesting it acts as a keystone species in this community. To determine the factors that regulate community composition, we focused on P. aeruginosa because of its pronounced impact on community structure and function. Specifically, we evaluated the role of the N-acyl homoserine lactone (AHL) dependent quorum sensing (QS) system of P. aeruginosa PAO1, which regulates group behaviors including biofilm formation and the production of effector molecules. We found that mixed species biofilms containing P. aeruginosa QS mutants had significantly altered proportions of K. pneumoniae and P. protegens populations compared to mixed species biofilms with the wild type P. aeruginosa. Similarly, inactivation of QS effector genes, e.g. rhlA and pvdR, also governed the relative species proportions. While the absence of QS did not alter the proportions of the two species in dual species biofilms of P. aeruginosa and K. pneumoniae, it resulted in significantly lower proportions of P. aeruginosa in dual species biofilms with P. protegens. These observations suggest that QS plays an important role in modulating community biofilm structure and physiology and affects interspecific interactions.

Bacterial biofilms are important medically, environmentally and industrially and there is a need to understand the processes that govern functional synergy and dynamics of species within biofilm communities. Here, we have used a model, mixed-species biofilm community comprised of Pseudomonas aeruginosa PAO1, Pseudomonas protegens Pf-5 and Klebsiella pneumoniae KP1. This biofilm community displays higher biomass and increased resilience to antimicrobial stress conditions such as sodium dodecyl sulfate and tobramycin, compared to monospecies biofilm populations. P. aeruginosa is present at low proportions in the community and yet, it plays a critical role in community function, suggesting it acts as a keystone species in this community. To determine the factors that regulate community composition, we focused on P. aeruginosa because of its pronounced impact on community structure and function. Specifically, we evaluated the role of the N-acyl homoserine lactone (AHL) dependent quorum sensing (QS) system of P. aeruginosa PAO1, which regulates group behaviors including biofilm formation and the production of effector molecules. We found that mixed species biofilms containing P. aeruginosa QS mutants had significantly altered proportions of K. pneumoniae and P. protegens populations compared to mixed species biofilms with the wild type P. aeruginosa. Similarly, inactivation of QS effector genes, e.g. rhlA and pvdR , also governed the relative species proportions. While the absence of QS did not alter the proportions of the two species in dual species biofilms of P. aeruginosa and K. pneumoniae , it resulted in significantly lower proportions of P. aeruginosa in dual species biofilms with P. protegens. These observations suggest that QS plays an important role in modulating community biofilm structure and physiology and affects interspecific interactions. [ABSTRACT FROM AUTHOR]

Microbial nitrogen fixation presents a viable alternative to chemical fertilizers, yet the limited colonization and specificity of naturally occurring nitrogen-fixing microorganisms present significant challenges to their widespread application. In this study, we identified a nitrogen fixation gene cluster (VNnif) in Vibrio natriegens (VN) and tested its nitrogenase activity through the acetylene reduction assay. We investigated the potential utilization of nitrogenase by incorporating the nitrogenase gene cluster from VN into plant growth-promoting rhizosphere bacteria Pseudomonas protegens CHA0 and enhancing its activity to 48.16 nmol C 2 H 2 /mg/h through promoter replacement and cluster rearrangement. The engineered strain CHA0-PVNnif was found to positively impact the growth of Arabidopsis thaliana col-0 and Triticum aestivum L. (wheat). This study expanded the role of plant growth-promoting rhizobacteria (PGPR) and provided a research foundation for enhancing nitrogenase activity.

Seed treatments with antagonistic bacteria could reduce the severity of crown and root rot diseases in wheat crops. The objective of this study was to evaluate the potential antagonistic activity of a bacterial consortium of three Chilean strains of Pseudomonas protegens against the wheat crown and root rot pathogens Gaeumannomyces graminis var. tritici, Rhizoctonia cerealis, and Fusarium culmorum. Two field experiments were carried out on artificially infested soil during two consecutive seasons (2016–2017 and 2017–2018) in an Andisol soil of southern Chile. Control treatments (not inoculated with fungi) were also included. Each treatment included a seed treatment of spring wheat cv. Pantera-INIA with and without the bacterial consortium. Both phytosanitary damage (incidence and severity) and agronomic components were evaluated. Bacterial populations with the phlD+ gene in the wheat plant rhizosphere during anthesis state (Z6) were also quantified. In both seasons, infection severity decreased by an average of 16.8% in seeds treated with P. protegens consortium, while yield components such as spikes m−1 and number of grains per spike increased. The use of antagonistic bacteria resulted in a total yield increase only during the first experimental season (P < 0.05). In general, accumulated rainfall influenced the antagonistic effect of the consortium of P. protegens strains, accounting for the differences observed between the two seasons. The results suggest that this P. protegens consortium applied on seeds can promote plant growth and protect wheat crops against crown and root rot pathogens in Southern Chile under field conditions.

The application value of Pseudomonas protegens in agriculture has been widely studied, but the manufacturing processes used to produce P. protegens limits its usefulness as a biological control agent because of its poor shelf life due to low stress resistance during processing. We combined hyperosmotic cultivation and fluidized-bed drying to prepare much more stable P. protegens preparations. During drying and cryogenic storage, the viability of hyperosmotic cells was significantly improved compared to that of cells prepared by previous methods. Addition of skim milk provided an excellent protective effect for both hyperosmotic and standard cells. In two field trials, when the concentration of the P. protegens preparation was 0.333% w/v, the biocontrol efficacy against tomato bacterial wilt was more than 80%, which was significantly higher than that of the control provided by the antibiotics zhongshengmycin and streptomycin sulfate. The results show that P. protegens products can be successfully prepared by combination of hyperosmotic cultivation and fluidized-bed drying. [ABSTRACT FROM AUTHOR]

Copyright of Acta Agriculturae Zhejiangensis is the property of Acta Agriculturae Zhejiangensis Editorial Office and its content may not be copied or emailed to multiple sites or posted to a listserv without the copyright holder's express written permission. However, users may print, download, or email articles for individual use. This abstract may be abridged. No warranty is given about the accuracy of the copy. Users should refer to the original published version of the material for the full abstract. (Copyright applies to all Abstracts.)

Seed treatments with antagonistic bacteria could reduce the severity of crown and root rot diseases in wheat crops. The objective of this study was to evaluate the potential antagonistic activity of a bacterial consortium of three Chilean strains of Pseudomonas protegens against the wheat crown and root rot pathogens Gaeumannomyces graminis var. tritici , Rhizoctonia cerealis , and Fusarium culmorum. Two field experiments were carried out on artificially infested soil during two consecutive seasons (2016–2017 and 2017–2018) in an Andisol soil of southern Chile. Control treatments (not inoculated with fungi) were also included. Each treatment included a seed treatment of spring wheat cv. Pantera-INIA with and without the bacterial consortium. Both phytosanitary damage (incidence and severity) and agronomic components were evaluated. Bacterial populations with the phlD + gene in the wheat plant rhizosphere during anthesis state (Z6) were also quantified. In both seasons, infection severity decreased by an average of 16.8% in seeds treated with P. protegens consortium, while yield components such as spikes m−1 and number of grains per spike increased. The use of antagonistic bacteria resulted in a total yield increase only during the first experimental season (P < 0.05). In general, accumulated rainfall influenced the antagonistic effect of the consortium of P. protegens strains, accounting for the differences observed between the two seasons. The results suggest that this P. protegens consortium applied on seeds can promote plant growth and protect wheat crops against crown and root rot pathogens in Southern Chile under field conditions. [ABSTRACT FROM AUTHOR]

Aims: This study investigated the adaptation strategies of Pseudomonas protegens to hyperosmotic growth environments (HGE). Methods and Results: The current study combined transcriptomics and proteomics to provide an overview of the molecular mechanism of P. protegens adaptation to HGE. The results revealed that HGE exerted prominent impact on the synthesis of proteins involved in multiple cellular functions. Comparison of the differentially accumulated proteins at both transcriptomics and proteomics levels indicated the existence of complex post‐transcriptional modification during P. protegens hyperosmotic adaptation. Conclusions: During HGE adaptation, the cells form a complex self‐protection mechanism. Significance and Impact of the Study: The results of the current study can help researchers to gain insights regarding the mechanism of P. protegens adaptation to HGE. These findings provide information for the application of stress methods and facilitate its broad commercial utilization. [ABSTRACT FROM AUTHOR]

Pseudomonas protegens is a gram-negative bacterium with an excellent biological control effect. Compared to standard cells growing in NaCl-free media, the ability of the hyperosmotic cells (450 mM NaCl) to resist high temperatures and freezing was significantly improved. It is of great significance to apply the P. protegens to elaborate on the hyperosmotic adaptation mechanism. RNA-seq was used to sequence P. protegens cultured with 0 mM and 450 mM NaCl. Comparative transcriptomic analyses of the different treatments were performed using gene ontology and the Kyoto encyclopedia of genes and genome. The results revealed that hyperosmotic stress had prominent impacts on the genes involving in multiple cellular functions. The hyperosmotic environment significantly affected carbohydrate, energy, and amino acid metabolism, as well as membrane system and cell motility. Our findings indicated that P. protegens adopted a series of approaches, including the high cytoplasmic concentrations of potassium ions and the uptake or synthesis of osmoprotectants, for surviving hyperosmotic stress. Among these, trehalose and proline synthesis appeared to be an important method to withstand prolonged hyperosmotic stress in P. protegens. These data provide crucial resource that may determine specific responses to the hyperosmotic environment in P. protegens. [ABSTRACT FROM AUTHOR]

Pseudomonas protegens (formerly Pseudomonas fluorescens) FD6, which was isolated from the canola rhizosphere, is a biocontrol agent that protects against the fungal pathogens Botrytis cinerea and Monilinia fructicola. The complete sequence of the 6.7-Mb Pseudomonas protegens FD6 genome was determined. Genomic analysis of strain FD6 led to the identification of twelve putative gene clusters of secondary metabolites, including the antibiotics 2,4-diacetylphloroglucinol (2,4-DAPG), pyoluteorin (PLT) and pyrrolnitrin (PRN). To assess the role of 2,4-DAPG and PLT in the biocontrol activity of P. protegens FD6, two mutants of P. protegens FD6, including strains ΔphlC and ΔpltD, were constructed using homologous recombination technology. The results showed that no significant differences were observed in the antagonistic action of the ΔphlC mutant compared with wild-type FD6. However, the ΔpltD mutant no longer exhibited antifungal activity. HPLC analysis indicated that ΔphlC mutant could not biosynthesize 2,4-DAPG, whereas PLT production in the ΔphlC mutant was significantly increased by 67-fold compared with wild-type FD6. Biosynthesis of PLT in the ΔpltD mutant was not detected, whereas the level of 2,4-DAPG was vastly decreased over sixteenfold compared with strain FD6. The major antagonistic activity produced by P. protegens FD6 corresponded to 2,4-diacetylphloroglucinol and pyoluteorin. There is an inverse interaction between 2,4-DAPG and PLT. [ABSTRACT FROM AUTHOR]

Pseudomonas protegens H78 produces multiple secondary metabolites, including antibiotics and iron carriers. The guanosine pentaphosphate or tetraphosphate ((p)ppGpp)-mediated stringent response is utilized by bacteria to survive during nutritional starvation and other stresses. RelA/SpoT homologues are responsible for the biosynthesis and degradation of the alarmone (p)ppGpp. Here, we investigated the global effect of relA/spoT dual deletion on the transcriptomic profiles, physiology, and metabolism of P. protegens H78 grown to mid- to late log phase. Transcriptomic profiling revealed that relA/spoT deletion globally upregulated the expression of genes involved in DNA replication, transcription, and translation; amino acid metabolism; carbohydrate and energy metabolism; ion transport and metabolism; and secretion systems. Bacterial growth was partially increased, while the cell survival rate was significantly reduced by relA/spoT deletion in H78. The utilization of some nutritional elements (C, P, S, and N) was downregulated due to relA/spoT deletion. In contrast, relA/spoT mutation globally inhibited the expression of secondary metabolic gene clusters (plt, phl, prn, ofa, fit, pch, pvd, and has). Correspondingly, antibiotic and iron carrier biosynthesis, iron utilization, and antibiotic resistance were significantly downregulated by the relA/spoT mutation. This work highlights that the (p)ppGpp-mediated stringent response regulatory system plays an important role in inhibiting primary metabolism and activating secondary metabolism in P. protegens. [ABSTRACT FROM AUTHOR]

Objectives: Developing a counterselective system for efficient markerless gene deletions in biocontrol strain P. protegens Pf-5. Results: We successfully implemented a markerless deletion of upp in Pf-5 to obtain the 5-FU resistant strain Pf5139. With this strain, we performed markerless gene deletions for each component of Gac/Rsm system and a 17 kb DNA fragment with the deletion ratio of 20 to 50%, and efficiently constructed a strain with triple deletions based on the suicide plasmid pJQ200UPP. In addition, there is no obvious connection between the deleted fragment length and the deletion ratio. Conclusion: The upp-based counterselective system in this study is efficient and valuable for markerless gene deletions in Pf-5, indicating that it has great potential in the study of gene function and in the application of genome reduction for Pseudomonas strains. [ABSTRACT FROM AUTHOR]

In our previous studies, Bacillus megaterium KU143, Microbacterium testaceum KU313, and Pseudomonas protegens AS15 have been shown to be antagonistic to Aspergillus flavus in stored rice grains. In this study, the biocontrol activities of these strains were evaluated against Aspergillus candidus, Aspergillus fumigatus, Penicillium fellutanum, and Penicillium islandicum, which are predominant in stored rice grains. In vitro and in vivo antifungal activities of the bacterial strains were evaluated against the fungi on media and rice grains, respectively. The antifungal activities of the volatiles produced by the strains against fungal development and population were also tested using I-plates. In in vitro tests, the strains produced secondary metabolites capable of reducing conidial germination, germ-tube elongation, and mycelial growth of all the tested fungi. In in vivo tests, the strains significantly inhibited the fungal growth in rice grains. Additionally, in I-plate tests, strains KU143 and AS15 produced volatiles that significantly inhibited not only mycelial growth, sporulation, and conidial germination of the fungi on media but also fungal populations on rice grains. GC-MS analysis of the volatiles by strains KU143 and AS15 identified 12 and 17 compounds, respectively. Among these, the antifungal compound, 5-methyl-2-phenyl-1H-indole, was produced by strain KU143 and the antimicrobial compounds, 2-butyl 1-octanal, dimethyl disulfide, 2-isopropyl-5-methyl-1-heptanol, and 4-trifluoroacetoxyhexadecane, were produced by strain AS15. These results suggest that the tested strains producing extracellular metabolites and/or volatiles may have a broad spectrum of antifungal activities against the grain fungi. In particular, B. megaterium KU143 and P. protegens AS15 may be potential biocontrol agents against Aspergillus and Penicillium spp. during rice grain storage.

The Heterobasidion annosum species complex includes major fungal pathogens of conifers worldwide. State-of-the-art preventative stump treatments with urea or with commercial formulations of the fungal biological control agent Phlebiopsis gigantea (i.e., Rotstop®) may become no longer available or are not approved for use in many areas of Europe infested by the three native Heterobasidion species and by the North American invasive H. irregulare, making the development of new treatments timely. The efficacy of Proradix® (based on Pseudomonas protegens strain DSMZ 13134), the cell-free filtrate (CFF) of the same bacterium, a strain of P. gigantea (MUT 6212) collected in the invasion area of H. irregulare in Italy, Rotstop®, and urea was comparatively investigated on a total of 542 stumps of Abies alba, Picea abies, Pinus pinea, and P. sylvestris in forest stands infested by the host-associated Heterobasidion species. Additionally, 139 logs of P. pinea were also treated. Results support the good performances of Rotstop®, and especially of urea against the native Heterobasidion species on stumps of their preferential hosts and, for the first time, towards the invasive North American H. irregulare on stumps of P. pinea. In some experiments, the effectiveness of Proradix® and of the strain of P. gigantea was weak, whereas the CFF of P. protegens strain DSMZ 13134 performed as a valid alternative to urea and Rotstop®. The mechanism of action of this treatment hinges on antibiosis; therefore, further improvements could be possible by identifying the active molecules and/or by optimizing their production. Generally, the performance of the tested treatments is not correlated with the stump size.

Antibiotic biosynthesis by microorganisms is commonly regulated through autoinduction, which allows producers to quickly amplify the production of antibiotics in response to environmental cues. Antibiotic autoinduction generally involves one pathway-specific transcriptional regulator that perceives an antibiotic as a signal and then directly stimulates transcription of the antibiotic biosynthesis genes. Pyoluteorin is an autoregulated antibiotic produced by some Pseudomonas spp. including the soil bacterium Pseudomonas protegens Pf-5. In this study, we show that PltR, a known pathway-specific transcriptional activator of pyoluteorin biosynthesis genes, is necessary but not sufficient for pyoluteorin autoinduction in Pf-5. We found that pyoluteorin is perceived as an inducer by PltZ, a second pathway-specific transcriptional regulator that directly represses the expression of genes encoding a transporter in the pyoluteorin gene cluster. Mutation of pltZ abolished the autoinducing effect of pyoluteorin on the transcription of pyoluteorin biosynthesis genes. Overall, our results support an alternative mechanism of antibiotic autoinduction by which the two pathway-specific transcriptional regulators PltR and PltZ coordinate the autoinduction of pyoluteorin in Pf-5. Possible mechanisms by which PltR and PltZ mediate the autoinduction of pyoluteorin are discussed.

This study included eight bacterial isolates originating from the apple phyllosphere or soil environment that were previously selected using the pear fruitlet test (Mikiciński 2017). Identification of these isolates based on phenotypic assays and DNA analysis showed that five of them belonged to species for which an antagonistic activity against Erwinia amylovora and the protective capacity of apple and pear against fire blight were not previously demonstrated. These were L16 identified as Pseudomonas vancouverensis, 3 M as Pseudomonas chlororaphis subsp. aureofaciens, 35 M – Pseudomonas congelans, 43 M – Enterobacter ludwigii, and 59 M – Pseudomonas protegens. Investigation of the biotic relationships between the tested strains and E. amylovora showed that 3 M, 35 M and 59 M inhibited the growth of the pathogen on five out of six media used (NAS, KB, LB, R2A, NAG), but 43 M did not do so on any of these media. Strain L16 did not inhibit the growth of the pathogen on LB or R2A medium. In contrast, all strains grown on medium 925 stimulated the growth of the pathogen, which showed no growth without co-cultivation with these strains. The experiments on apple trees and detached apple branches showed the ability of the tested bacteria to protect flowers at medium to high levels, depending on the experiment (55–93%). In some cases, this protection was even higher than that of the copper product used for comparison. In studies assessing the bacterial ability to protect shoots of M.26, the highest efficacy was observed for strains 35 M (96%) and 43 M (93%) but on 'Gala Must' all tested strains showed 100% of efficacy. [ABSTRACT FROM AUTHOR]

There is an intimate relationship between microorganisms and the formation and stability of soil microaggregates, realized by the immobilization and occlusion of microorganisms. Little is known about the initial aggregate formation phase and the role of microorganisms in this process under the impact of environmental changes such as wetting and drying. We investigated this initial aggregate formation process of montmorillonite and goethite in combination with two bacterial strains, Pseudomonas protegens strain CHA0 and Gordonia alkanivorans strain MoAcy 2, in the presence or absence of stress conditions in form of wetting and drying cycles for up to eight days. Montmorillonite and goethite formed microaggregates instantaneously, the size of these aggregates being enhanced in the presence of microorganisms, resulting in up to twofold larger aggregates. This increase in aggregate size was strain-dependent. However, the aggregates that developed during the first 48 h broke into smaller structures later on. A microscopic analysis of the microaggregates revealed that notably the larger microaggregates harbored bacteria and that microaggregates had a sheltering effect on living cells, especially when exposed to desiccation stress. Additionally, aggregate formation was analyzed in the presence of a Pseudomonas protegens mutant strain (CHA211) with increased production capability of extracellular polymeric substances (EPS). About fivefold higher survival rates of culturable cells were observed after desiccation for this EPS overproducing mutant strain in comparison to the wild-type. Our results hint at an aggregate formation process characterized by a rapid occlusion of mineral compounds, and, after the addition of microorganisms, the bacterial colonization of small microaggregates, leading to an increase in aggregate size. The further development of the aggregate size distribution varied depending on the presence of microbial taxa and was modulated by environmental conditions like desiccation events. • The presence of bacterial cells increases the size of microaggregates. • Desiccation stress induces the formation of larger microaggregates. • Microaggregates support bacterial survival upon desiccation stress. • Extracellular polymeric substances support bacterial survival upon desiccation stress. [ABSTRACT FROM AUTHOR]

Abstract Bacteria isolated from different environments can be exploited for biocontrol purposes by the identification of the molecules involved in the antifungal activity. The present study was aimed at investigating antifungal protein compounds purified from a previously identified plant growth promoting bacterium, Pseudomonas protegens N isolated from agricultural land in northern Algeria. Therefore, a novel protein was purified by chromatographic and ultrafiltration steps and its antifungal activity together with growth-inhibition mechanism was evaluated against different fungi by plate-based assays. In addition, stereomicroscopy and transmission electron microscopy (TEM) was performed to explore the inhibition activity of the compound on spore germination processes. The protein, showing a molecular mass of about 100 kDa under native conditions, was revealed to be in the surface-membrane fraction and displayed an efficient activity against a variety of phytopathogenic fungi, being Alternaria the best target towards which it exhibited a marked fungicidal action and inhibition of spore germination. Moreover, the compound was able to significantly decrease fungal infection on tomato fruits producing also morphological aberrations on conidia. The obtained results suggested that the isolated compound could represent a promising agent for eco-friendly management of plant pathogens in agriculture. [ABSTRACT FROM AUTHOR]

Extracts rich in saponins from Quillaja saponaria Mol. and populations of rhizobacteria from the genus Pseudomonas, which produce antimicrobial compounds, have been associated with reduction of the fungus Gaeumannomyces graminis var. tritici, the causal agent of take-all disease, which is responsible for severe loss of wheat (Triticum aestivum L.) crops worldwide. However, there is a limited background on the interaction between these bacteria and natural triterpenoids. The aim of this research was to assess the effect of saponin rich extract on Pseudomonas protegens strains 2,4-diacetylphloroglucinol-producers under in vitro and in plant conditions and determining the synergistic effect to be used together to control G. graminis var. tritici in wheat plants. We determined that 8% and 90% of saponins rich Q. saponaria extracts have a differential effect on P. protegens according to their purity (P ≤ 0.05). On wheat seedlings, quillaja extract with 90% of saponins did not affect the three antagonistic bacterial strain populations assessed, but affected biofilm formation at saponins concentration of 7360 mg L-1. Pseudomonas protegens strains had a variable antagonist activity in wheat plants, and controlled the fungus when were combined with different concentrations of pure Q. saponaria extract, with the concentration of 1840 ppm reducing the take-all disease in 32.5% with respect to the control inoculated with G. graminis var. tritici (P ≤ 0.05). However, no synergistic effects when the plant extract was combined with the bacterial strains were observed. These results showed the promising and complexity of combining bacterial and plant extract to develop a biopesticide, which could control this fungal disease. [ABSTRACT FROM AUTHOR]

The formation of C-C bonds by using CoA independent acyltransferases may have significant impact for novel methods for biotechnology. We report the identification of Pseudomonas strains with CoA-independent acyltransferase activity as well as the heterologous expression of the enzyme in E. coli. The cloning strategies and selected expression studies are discussed. The recombinant acyltransferases were characterized with regard to thermal and storage stability, pH,- and co-solvent tolerance. Moreover, the impact of bivalent metals, inhibitors, and other additives was tested. Careful selection of expression and working conditions led to obtain recombinant acyltransferase form Pseudomonas protegens with up to 11 U mL−1 activity. [ABSTRACT FROM AUTHOR]

Nanoparticles are widely used as antimicrobial compounds. At sub-lethal concentrations, they may also stress microbes, potentially inducing antibiosis. Here we assess whether nanoparticles can serve as an enhancer of antibiosis in beneficial microbes. Several host-associated bacteria can suppress pathogens, providing therefore a first line of defense against diseases. In the present study, we assessed whether nanoparticles stimulate the antifungal activity of Pseudomonas protegens CHA0, a model plant-associated bacterium, against the ascomycete yeast Candida albicans . We synthesized and characterized four of the most common nanoparticles, namely Ag, SiO 2 , TiO 2, and ZnO, with an average size of 25, 11, 25 and 35 nm, respectively. The dose-dependent effect of these nanoparticles on the growth of Pseudomonas protegens CHA0 was assessed. Ag, SiO 2 , TiO 2, and ZnO nanoparticles inhibited the growth of Pseudomonas protegens by 100, 22, 15 and 15%, respectively at a concentration of 250 μg/mL. We then selected sub-lethal dose (500 ng/mL) and assessed whether the same nanoparticles stimulated the production of antifungal compounds inhibiting C . albicans . Incubating the bacteria in the presence of nanoparticles led to a four-fold increase in antifungal activity. We finally show that nanoparticles induce the expression of the prn operon, responsible for the production of antifungal compound pyrrolnitrin, within hours after nanoparticle exposure. This study shows that nanoparticle application may be a valuable tool to stimulate the antifungal activity of fluorescent pseudomonads, potentially assisting the development of future sustainable disease control strategies. [ABSTRACT FROM AUTHOR]

In our previous studies, Bacillus megaterium KU143, Microbacterium testaceum KU313, and Pseudomonas protegens AS15 have been shown to be antagonistic to Aspergillus flavus in stored rice grains. In this study, the biocontrol activities of these strains were evaluated against Aspergillus candidus, Aspergillus fumigatus, Penicillium fellutanum, and Penicillium islandicum, which are predominant in stored rice grains. In vitro and in vivo antifungal activities of the bacterial strains were evaluated against the fungi on media and rice grains, respectively. The antifungal activities of the volatiles produced by the strains against fungal development and population were also tested using l-plates. In in vitro tests, the strains produced secondary metabolites capable of reducing conidial germination, germ-tube elongation, and mycelial growth of all the tested fungi. In in vivo tests, the strains significantly inhibited the fungal growth in rice grains. Additionally, in l-plate tests, strains KU143 and AS15 produced volatiles that significantly inhibited not only mycelial growth, sporulation, and conidial germination of the fungi on media but also fungal populations on rice grains. GC-MS analysis of the volatiles by strains KU143 and AS15 identified 12 and 17 compounds, respectively. Among these, the antifungal compound, 5-methyl-2-phenyl-l H-indole, was produced by strain KU143 and the antimicrobial compounds, 2-butyl 1-octanal, dimethyl disulfide, 2-isopropyl-5-methyl-1-heptanol, and 4-trifluoroacetoxyhexadecane, were produced by strain AS15. These results suggest that the tested strains producing extracellular metabolites and/or volatiles may have a broad spectrum of antifungal activities against the grain fungi. In particular, B. megaterium KU143 and P. protegens AS15 may be potential biocontrol agents against Aspergillus and Penicillium spp. during rice grain storage. [ABSTRACT FROM AUTHOR]