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ACS Synthetic Biology Mar 2024The advancement of metabolic engineering and synthetic biology has promoted in-depth research on the nonmodel microbial metabolism, and the potential of nonmodel...
The advancement of metabolic engineering and synthetic biology has promoted in-depth research on the nonmodel microbial metabolism, and the potential of nonmodel organisms in industrial biotechnology is becoming increasingly evident. The nonmodel organism is a safe plant growth promoting bacterium for the production of phenazine compounds; however, its application is seriously hindered due to the lack of an effective gene expression precise regulation toolkit. In this study, we constructed a library of 108 promoter-5'-UTR (PUTR) and characterized them through fluorescent protein detection. Then, 6 PUTRs with stable low, intermediate, and high intensities were further characterized by report genes encoding β-galactosidase from K12 and encoding PCA monooxygenase from GP72 and thus developed as a static gene expression regulation system. Furthermore, the stable and high-intensity expressed PUTR was fused with the LacO operator to construct an IPTG-induced plasmid, and a self-induced plasmid was constructed employing the high-intensity PUTR regulated by cell density, resulting in a dynamic gene expression regulation system. In summary, this study established two sets of static and dynamic regulatory systems for , providing an effective toolkit for fine-tuning gene expression and reprograming the metabolism flux.
Topics: Pseudomonas chlororaphis; Metabolic Engineering; Gene Expression Regulation, Bacterial; Promoter Regions, Genetic; Bacterial Proteins
PubMed: 38377538
DOI: 10.1021/acssynbio.3c00714 -
Frontiers in Microbiology 2023Morel mushroom ( spp.) is a rare edible and medicinal fungus distributed worldwide. It is highly desired by the majority of consumers. Bacterial diseases have been...
Morel mushroom ( spp.) is a rare edible and medicinal fungus distributed worldwide. It is highly desired by the majority of consumers. Bacterial diseases have been commonly observed during artificial cultivation of . Bacterial pathogens spread rapidly and cause a wide range of infections, severely affecting the yield and quality of . In this study, two strains of bacterial pathogens, named M-B and M-5, were isolated, cultured, and purified from the tissues of the infected . Koch's postulates were used to determine the pathogenicity of bacteria affecting , and the pathogens were identified through morphological observation, physiological and biochemical analyses, and 16S rRNA gene sequence analysis. Subsequently, the effect of temperature on the growth of pathogenic bacteria, the inhibitory effect of the bacteria on on plates, and the changes in mycelial morphology of mycelium were analyzed when mycelium was double-cultured with pathogenic bacteria on plates. The results revealed that M-B was subsp. and M-5 was . Strain M-B started to multiply at 10-15°C, and strain M-5 started at 15-20°C. On the plates, the pathogenic bacteria also produced significant inhibition of mycelium, and the observation of mycelial morphology under the scanning electron microscopy revealed that the inhibited mycelium underwent obvious drying and crumpling, and the healthy mycelium were more plump. Thus, this study clarified the pathogens, optimal growth environment, and characteristics of bacterial diseases, thereby providing valuable basic data for the disease prevention and control of production.
PubMed: 38029130
DOI: 10.3389/fmicb.2023.1231353 -
Phytopathology Jun 2024Mixtures of fungicides with different modes of action are commonly used as disease and resistance management tools, but little is known of mixtures of natural and...
Mixtures of fungicides with different modes of action are commonly used as disease and resistance management tools, but little is known of mixtures of natural and synthetic products. In this study, mixtures of metabolites from the rhizobacterium Pseudomonas chlororaphis strain ASF009 formulated as Howler EVO with below label rates (50 µg/ml) of conventional sterol demethylation inhibitor (DMI) fungicides were investigated for control of anthracnose of cherry (Prunus avium) caused by Colletotrichum siamense. Howler mixed with metconazole or propiconazole synergistically reduced disease severity through lesion growth. Realtime PCR showed that difenoconazole, flutriafol, metconazole, and propiconazole induced the expression of DMI target genes CsCYP51A and CsCYP51B in C. siamense. The addition of Howler completely suppressed the DMI fungicide-induced expression of both CYP51 genes. We hypothesize that the downregulation of DMI fungicide-induced expression of the DMI target genes may, at least in part, explain the synergism observed in detached fruit assays.
PubMed: 38857059
DOI: 10.1094/PHYTO-03-24-0090-R -
New Pseudomonas Bacterial Strains: Biological Activity and Characteristic Properties of Metabolites.Microorganisms Jul 2023This paper investigates the antagonistic and plant growth promotion activity of the new indigenous bacteria antagonist strains BZR 245-F and sp. BZR 523-2. It was...
This paper investigates the antagonistic and plant growth promotion activity of the new indigenous bacteria antagonist strains BZR 245-F and sp. BZR 523-2. It was found that on the 10th day of cultivation, BZR 245-F and BZR 523-2 exhibit an antagonistic activity against at the level of 59.6% and 15.1% and against var. at the level of 50.2% and 8.9%, respectively. Furthermore, the BZR 523-2 strain stimulated the growth of winter wheat seedlings more actively than the BZR 245-F strain. When processing seeds of winter wheat, sp. BZR 523-2 indicators were higher than in the control: plant height increased by 10.3%, and root length increased by 18.6%. The complex characteristic properties of the metabolite were studied by bioautography and HPLC-MS. Bioautography proved the antifungal activity of phenazine nature compounds synthesized by the new bacterial strains. We qualitatively and quantitatively analyzed them by HPLC-MS analysis of the strain sample metabolites. In the BZR 245-F sample, we found more phenazine compounds of various types. Their total phenazine concentration in the BZR 245-F was more than five times greater than in the BZR 523-2. We defined crucial differences in the quantitative content of the other metabolites. Despite the difference between new indigenous bacteria antagonist strains, they can be used as producers of effective biopesticides for sustainable agriculture management.
PubMed: 37630503
DOI: 10.3390/microorganisms11081943 -
Materials (Basel, Switzerland) Jul 2023Fibrous materials composed of core-sheath fibers from poly(ethylene oxide) (PEO), beeswax (BW) and 5-nitro-8-hydroxyquinoline (NQ) were prepared via the...
Fibrous materials composed of core-sheath fibers from poly(ethylene oxide) (PEO), beeswax (BW) and 5-nitro-8-hydroxyquinoline (NQ) were prepared via the self-organization of PEO and BW during the single-spinneret electrospinning of a homogeneous blend solution of the partners. Additionally, the application of the same approach enabled the preparation of fibrous materials composed of core-double sheath fibers from PEO, poly(L-lactide) (PLA) and NQ or 5-chloro-7-iodo-8-hydroxyquinoline (CQ), as well as from PEO, poly(ε-caprolactone) (PCL) and NQ. The consecutive selective extraction of BW and of the polyester with hexane and tetrahydrofuran, respectively, evidenced that core-double sheath fibers from PEO/polyester/BW/drug consisted of a PEO core, a polyester inner sheath and a BW outer sheath. In order to evaluate the possibility of the application of fibrous materials from PEO/BW/NQ, PEO/PLA/BW/NQ, PEO/PCL/BW/NQ and PEO/PLA/BW/CQ for plant protection, microbiological studies were performed using both phytopathogenic microorganisms (, and ) and beneficial microorganisms (, and ). It was found that the fibrous materials had anti-bacterial and anti-fungal activity against both phytopathogenic and beneficial microorganisms. This is the first report on the activity of fibrous materials loaded with 8-hydroxyquinoline derivatives not only against phytopathogenic but also against beneficial microorganisms that are of importance in agriculture.
PubMed: 37445197
DOI: 10.3390/ma16134882 -
Microbial Ecology Jul 2023The interaction of plants with bacteria and the long-term success of their adaptation to challenging environments depend upon critical traits that include nutrient...
The interaction of plants with bacteria and the long-term success of their adaptation to challenging environments depend upon critical traits that include nutrient solubilization, remodeling of root architecture, and modulation of host hormonal status. To examine whether bacterial promotion of phosphate solubilization, root branching and the host auxin response may account for plant growth, we isolated and characterized ten bacterial strains based on their high capability to solubilize calcium phosphate. All strains could be grouped into six Pseudomonas species, namely P. brassicae, P. baetica, P. laurylsulfatiphila, P. chlororaphis, P. lurida, and P. extremorientalis via 16S rRNA molecular analyses. A Solibacillus isronensis strain was also identified, which remained neutral when interacting with Arabidopsis roots, and thus could be used as inoculation control. The interaction of Arabidopsis seedlings with bacterial streaks from pure cultures in vitro indicated that their phytostimulation properties largely differ, since P. brassicae and P. laurylsulfatiphila strongly increased shoot and root biomass, whereas the other species did not. Most bacterial isolates, except P. chlororaphis promoted lateral root formation, and P. lurida and P. chlororaphis strongly enhanced expression of the auxin-inducible gene construct DR5:GUS in roots, but the most bioactive probiotic bacterium P. brassicae could not enhance the auxin response. Inoculation with P. brassicae and P. lurida improved shoot and root growth in medium supplemented with calcium phosphate as the sole Pi source. Collectively, our data indicate the differential responses of Arabidopsis seedlings to inoculation with several Pseudomonas species and highlight the potential of P. brassicae to manage phosphate nutrition and plant growth in a more eco-friendly manner.
Topics: Arabidopsis; Pseudomonas; Seedlings; Phosphates; RNA, Ribosomal, 16S; Plant Roots; Indoleacetic Acids; Bacteria
PubMed: 35867140
DOI: 10.1007/s00248-022-02080-y -
Journal of Applied Microbiology May 2024We aimed to develop an effective bacterial combination that can combat Fusarium oxysporum infection in watermelon using in vitro and pot experiments.
AIMS
We aimed to develop an effective bacterial combination that can combat Fusarium oxysporum infection in watermelon using in vitro and pot experiments.
METHODS AND RESULTS
In total, 53 strains of Bacillus and 4 strains of Pseudomonas were screened. Pseudomonas strains P3 and P4 and Bacillus strains XY-2-3, XY-13, and GJ-1-15 exhibited good antagonistic effects against F. oxysporum. P3 and P4 were identified as Pseudomonas chlororaphis and Pseudomonas fluorescens, respectively. XY-2-3 and GJ-1-15 were identified as B. velezensis, and XY-13 was identified as Bacillus amyloliquefaciens. The three Bacillus strains were antifungal, promoted the growth of watermelon seedlings and had genes to synthesize antagonistic metabolites such as bacilysin, surfactin, yndj, fengycin, iturin, and bacillomycin D. Combinations of Bacillus and Pseudomonas strains, namely, XY-2-3 + P4, GJ-1-15 + P4, XY-13 + P3, and XY-13 + P4, exhibited a good compatibility. These four combinations exhibited antagonistic effects against 11 pathogenic fungi, including various strains of F. oxysporum, Fusarium solani, and Rhizoctonia. Inoculation of these bacterial combinations significantly reduced the incidence of Fusarium wilt in watermelon, promoted plant growth, and improved soil nutrient availability. XY-13 + P4 was the most effective combination against Fusarium wilt in watermelon with the inhibition rate of 78.17%. The number of leaves; aboveground fresh and dry weights; chlorophyll, soil total nitrogen, and soil available phosphorus content increased by 26.8%, 72.12%, 60.47%, 16.97%, 20.16%, and 16.50%, respectively, after XY-13 + P4 inoculation compared with the uninoculated control. Moreover, total root length, root surface area, and root volume of watermelon seedlings were the highest after XY-13 + P3 inoculation, exhibiting increases by 265.83%, 316.79%, and 390.99%, respectively, compared with the uninoculated control.
CONCLUSIONS
XY-13 + P4 was the best bacterial combination for controlling Fusarium wilt in watermelon, promoting the growth of watermelon seedlings, and improving soil nutrient availability.
Topics: Fusarium; Citrullus; Plant Diseases; Bacillus; Disease Resistance; Pseudomonas; Antibiosis; Pseudomonas fluorescens; Seedlings; Antifungal Agents
PubMed: 38632051
DOI: 10.1093/jambio/lxae074 -
Biology Sep 2023Phenazine compounds are widely used in agricultural control and the medicine industry due to their high inhibitory activity against pathogens and antitumor activity. The...
Phenazine compounds are widely used in agricultural control and the medicine industry due to their high inhibitory activity against pathogens and antitumor activity. The green and sustainable method of synthesizing phenazine compounds through microbial fermentation often requires a complex culture medium containing tryptone and yeast extract, and its cost is relatively high, which greatly limits the large-scale industrial production of phenazine compounds by fermentation. The aim of this study was to develop a cost-effective minimal medium for the efficient synthesis of phenazine compounds by . Through testing the minimum medium commonly used by , an ME medium for with a high production of phenazine compounds was obtained. Then, the components of the ME medium and the other medium were compared and replaced to verify the beneficial promoting effect of Fe and NH on phenazine compounds. A cost-effective general defined medium (GDM) using glycerol as the sole carbon source was obtained by optimizing the composition of the ME medium. Using the GDM, the production of phenazine compounds by reached 1073.5 mg/L, which was 1.3 times that achieved using a complex medium, while the cost of the GDM was only 10% that of a complex medium (e.g., the KB medium). Finally, by engineering the glycerol metabolic pathway, the titer of phenazine-1-carboxylic acid reached the highest level achieved using a minimum medium so far. This work demonstrates how we systematically analyzed and optimized the composition of the medium and integrated a metabolic engineering method to obtain the most cost-effective fermentation strategy.
PubMed: 37887002
DOI: 10.3390/biology12101292 -
Letters in Applied Microbiology Jul 2023The use of microbial consortia has become a promising alternative for the management of various diseases. In this study, 18 artificial consortia were designed,...
The use of microbial consortia has become a promising alternative for the management of various diseases. In this study, 18 artificial consortia were designed, consisting of five bacteria, five fungi, and a mixture of five fungi and five bacteria; from a collection of microorganisms isolated from the rhizosphere of cape gooseberry plants grown in two soils potentially suppressive against Fusarium oxysporum. When evaluated under greenhouse conditions for their biocontrol activity on cape gooseberry plants, one consortium was selected for their high efficacy (over 90%) in the control of vascular wilt caused by F. oxysporum f. sp. physali. This was constituted by 10 microorganisms, the bacteria Paenibacillus peoriae, Bacillus subtilis, Lysinibacillus sp., B. simplex, and Pseudomonas chlororaphis; and the fungi Beauveria bassiana, Scopulariopsis brevicaulis, Trichoderma gamsii, T. ghanense, and T. lignicola. On the other hand, four of the consortia evaluated in the presence of the pathogen mitigated the deleterious effect produced by the pathogen on plant growth, expressing higher dry weights, both in the aerial and root parts. This work represents the first report on using these mixtures of microorganisms to control vascular wilt produced by F. oxysporum. However, further studies are needed to determine their activity in cape gooseberry fields.
Topics: Fusarium; Microbial Consortia; Ribes; Physalis; Bacillus subtilis; Fungi; Plant Diseases
PubMed: 37348479
DOI: 10.1093/lambio/ovad072 -
Journal of Economic Entomology Feb 2024Pseudomonas fluorescens group, such as Pseudomonas protegens and Pseudomonas chlororaphis, can be utilized as insect-killing agents. Most insecticidal Pseudomonas...
Pseudomonas fluorescens group, such as Pseudomonas protegens and Pseudomonas chlororaphis, can be utilized as insect-killing agents. Most insecticidal Pseudomonas described so far have high toxicity for insects of the order Lepidoptera. In this study, Pseudomonas strain PcR3-3 was isolated from the willow root. It showed a high mortality for the coleopteran species Plagiodera versicolora (Coleoptera: Chrysomelidae), but not for the lepidopteran Helicoverpa armigera. Strain PcR3-3 displayed high colonization ability in the P. versicolora compared with P. chlororaphis PCL1391, indicating that the insecticidal activities correlated with the colonization ability of Pseudomonas strain in the host. Phylogenetic analysis of the genome revealed that PcR3-3 belonged to P. chlororaphis subsp. aureofaciens. Numerous insecticidal protein-encoding genes, typical biosynthetic gene clusters for some insecticidal metabolite and type VI secretion system, known to be involved in insect pathogenicity, were present in the P. chlororaphis PcR3-3 genome. However, the insecticidal toxin Fit-encoding gene which commonly presents in P. chlororaphis, was not found in the P. chlororaphis PcR3-3 genome. Furthermore, there are some divergent insecticidal genes between P. chlororaphis PcR3-3 and P. chlororaphis PCL1391. This finding implies that P. chlororaphis PcR3-3 is a promising biocontrol agent for pest management applications. The P. chlororaphis-P. versicolora association can be used as a model system to study the interaction between Pseudomonas and coleopteran insects.
Topics: Animals; Pseudomonas chlororaphis; Insecticides; Phylogeny; Genomics; Insecta; Coleoptera
PubMed: 38146627
DOI: 10.1093/jee/toad232