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Frontiers in Microbiology 2024The rhizosphere microbiome is critical to plant health and resistance. PGPR are well known as plant-beneficial bacteria and generally regulate nutrient utilization as...
INTRODUCTION
The rhizosphere microbiome is critical to plant health and resistance. PGPR are well known as plant-beneficial bacteria and generally regulate nutrient utilization as well as plant responses to environmental stimuli. In our previous work, one typical PGPR strain, IRHB3, isolated from the soybean rhizosphere, had positive impacts on soil-borne disease suppression and growth promotion in the greenhouse, but its biocontrol mechanism and application in the field are not unclear.
METHODS
In the current study, IRHB3 was introduced into field soil, and its effects on the local rhizosphere microbiome, disease resistance, and soybean growth were comprehensively analyzed through high-throughput sequencing and physiological and molecular methods.
RESULTS AND DISCUSSION
We found that IRHB3 significantly increased the richness of the bacterial community but not the structure of the soybean rhizosphere. Functional bacteria related to phosphorus solubilization and nitrogen fixation, such as , , , , and , were recruited in rich abundance by IRHB3 to the soybean rhizosphere as compared to those without IRHB3. In addition, the IRHB3 supplement obviously maintained the homeostasis of the rhizosphere microbiome that was disturbed by , resulting in a lower disease index of root rot when compared with . Furthermore, JA-mediated induced resistance was rapidly activated by IRHB3 following and expression, and meanwhile, a set of nodulation genes, , , and , were also considerably induced by IRHB3 to improve nitrogen fixation ability and promote soybean yield, even when plants were infected by . Thus, IRHB3 tends to synergistically interact with local rhizosphere microbes to promote host growth and induce host resistance in the field.
PubMed: 38380096
DOI: 10.3389/fmicb.2024.1328863 -
Microbiological Research Oct 2020Three morphological mutants (M71a, M71b, M71c) of the antagonist Pseudomonas chlororaphis M71, naturally arose during a biocontrol trial against the phytopathogenic...
Three morphological mutants (M71a, M71b, M71c) of the antagonist Pseudomonas chlororaphis M71, naturally arose during a biocontrol trial against the phytopathogenic fungus Fusarium oxysporum f.sp. radicis-lycopersisci. In this study, the three mutants were investigated to elucidate their role in the biocontrol of plant pathogens. M71a and M71b phenotypes were generated by a mutation in the two-component system GacS/GacA. The mutation determined an increase in siderophore production and an impaired ability to release proteases, to swarm, to produce phenazine and AHLs and to colonize tomato roots. In vitro antagonistic activity against different plant pathogens was partially reduced in M71a, while M71b resulted effective only against Pythium ultimum. Biocontrol efficacy against Fusarium oxysporum f.sp. radicis-lycopersisci, was partially reduced in M71a and completely lost in M71b. M71c phenotype was impaired in swarming motility, did not produce biofilms and its antagonistic activity was similar to the parental M71 strain. M71c showed an enhanced ability to colonize tomato roots, on which its progeny in part reverted to the M71 parental phenotype. Volatile organic compounds (VOCs) emitted by all four strains, inhibited the growth of Clavibacter michiganensis subsp. michiganensis and Seiridium cardinale in vitro. Real-time screening of VOCs by PTR-MS combined with GC-MS analysis, showed that methanethiol was the main component of the blend produced by all four M71 strains. However, the emissions of hydrogen cyanide, dimethyl disulfide, 1,3-butadiene and acetone were significantly affected by the three different mutations. These findings highlight that the simultaneous presence of different M71 phenotypes may improve, through the integration of different mechanisms, the ecological fitness and biocontrol efficacy of P. chlororaphis M71.
Topics: Bacterial Proteins; Biological Control Agents; Fusarium; Solanum lycopersicum; Mutation; Pest Control, Biological; Phenazines; Phenotype; Plant Diseases; Plant Roots; Pseudomonas chlororaphis; Siderophores; Volatile Organic Compounds
PubMed: 32535393
DOI: 10.1016/j.micres.2020.126517 -
Microbial Biotechnology Jan 2024Rhamnolipids (RL) are biosurfactants naturally produced by the opportunistic pathogen Pseudomonas aeruginosa. Currently, RL are commercialized for various applications...
Rhamnolipids (RL) are biosurfactants naturally produced by the opportunistic pathogen Pseudomonas aeruginosa. Currently, RL are commercialized for various applications and produced by Pseudomonas putida due to the health risks associated with their large-scale production by P. aeruginosa. In this work, we show that RL containing one or two rhamnose moieties (mono-RL or di-RL, respectively) can be produced by the innocuous soil-bacterium Pseudomonas chlororaphis subsp chlororaphis ATCC 9446 at titres up to 66 mg/L (about 86% of the production of P. aeruginosa PAO1 in the same culture conditions). The production of RL depends on the expression of P. aeruginosa PAO1 genes encoding the enzymes RhlA, RhlB and RhlC. These genes were introduced in a plasmid, together with a transcriptional regulator (rhlR) forming part of the same operon, with and without RhlC. We show that the activation of rhlAB by RhlR depends on its interaction with P. chlororaphis endogenous acyl-homoserine lactones, which are synthetized by either PhzI or CsaI autoinducer synthases (producing 3-hydroxy-hexanoyl homoserine lactone, 3OH-C6-HSL, or 3-oxo-hexanoyl homoserine lactone, 3O-C6-HSL, respectively). P. chlororaphis transcriptional regulator couple with 3OH-C6-HSL is the primary activator of gene expression for phenazine-1-carboxylic acid (PCA) and phenazine-1-carboxamide (PCN) production in this soil bacterium. We show that RhlR coupled with 3OH-C6-HSL or 3O-C6-HSL promotes RL production and increases the production of PCA in P. chlororaphis. However, PhzR/3OH-C6-HSL or CsaR/3O-C6-HSL cannot activate the expression of the rhlAB operon to produce mono-RL. These results reveal a complex regulatory interaction between RhlR and P. chlororaphis quorum-sensing signals and highlight the biotechnology potential of P. chlororaphis ATCC 9446 expressing P. aeruginosa rhlAB-R or rhlAB-R-C for the industrial production of RL.
Topics: Pseudomonas chlororaphis; Acyl-Butyrolactones; Pseudomonas aeruginosa; Soil; Bacterial Proteins; Glycolipids; Pseudomonas; 4-Butyrolactone
PubMed: 38041625
DOI: 10.1111/1751-7915.14377 -
Bio-protocol Jan 2018Increased antibiotic resistance of plants and human pathogens and continuous use of chemical fertilizers has pushed microbiologists to explore new microbial sources as...
Increased antibiotic resistance of plants and human pathogens and continuous use of chemical fertilizers has pushed microbiologists to explore new microbial sources as potential antagonists. In this study, eight strains of and , have been isolated from different plant sources and screened for their antagonistic and plant growth promoting potential ( Shahid , 2017 ). All strains were compared with reference strain PB-St2 and their secondary metabolites were isolated by the use of solvent partitioning and subjected to LC/ESI/MS for confirmation of compounds. The ESI-mass spectra obtained were used to characterize the surfactants ionization behavior and [M + H] and [M + Na] ions were monitored for phenazines, derivatives of lahorenoic acid and cyclic lipopeptide (WLIP). LC-MS and HPLC methods were developed to see the elution of dominant metabolites in a single run to avoid the labor and separate methods of detection for all compounds. The method was found suitable and distinctively separated the compounds at different retention times in gradient flow. This method can be helpful to explore the metabolome of sp. overall and in identification and quantification of strain specific metabolites.
PubMed: 34179247
DOI: 10.21769/BioProtoc.2702 -
Frontiers in Microbiology 2022Currently, plant growth-promoting rhizobacteria (PGPR) microbial inoculants are heavily used in agricultural production among which sp. and sp. are two excellent...
Currently, plant growth-promoting rhizobacteria (PGPR) microbial inoculants are heavily used in agricultural production among which sp. and sp. are two excellent inoculum strains, which are widely used in plant growth promotion and disease control. However, few studies have been conducted on the combined use of the two bacteria. The aim of this study was to investigate the effects of co-inoculation of these two bacteria on soybean [ (L.) ] growth and physiological indexes and further study the effect of microbial inoculants on native soil bacterial communities and plant endophyte microbiota, especially microorganisms in rhizosphere and root. A pot experiment was conducted and four treatments were designed: group without any strain inoculant (CK); group inoculated with H1 inoculant (J); group inoculated with Y1 inoculant (Y) and group inoculated with equal volume of H1 inoculant and Y1 inoculant (H). Compared with CK, the three inoculant groups J, Y, and H exhibited improved soybean growth and physiological indexes, and group H was the most significant ( < 0.05). In terms of rhizosphere bacterial community structure, the relative abundance of native (9.31%) was higher in the H group than in the J (6.07%), Y (3.40%), and CK (5.69%) groups, which has potential value of disease suppression. Besides, compared with bacterial communities of the other three groups in soybean roots, group H increased the abundance of beneficial bacterial community for the contents of , , and significantly increased ( < 0.05). In conclusion, we found that the composite inoculum of H1 and Y1 could effectively promote soybean growth, increase yield and improve the beneficial bacterial community in root and rhizosphere and have certain value for soil improvement.
PubMed: 36699592
DOI: 10.3389/fmicb.2022.1079348 -
Microorganisms Mar 2022Rhizosphere colonization by phytobeneficial Pseudomonas spp. is pivotal in triggering their positive effects on plant health. Many Pseudomonas spp. Determinants,...
Rhizosphere colonization by phytobeneficial Pseudomonas spp. is pivotal in triggering their positive effects on plant health. Many Pseudomonas spp. Determinants, involved in rhizosphere colonization, have already been deciphered. However, few studies have explored the role played by specific plant genes in rhizosphere colonization by these bacteria. Using isogenic Arabidopsis thaliana mutants, we studied the effect of 20 distinct plant genes on rhizosphere colonization by two phenazine-producing P. chlororaphis strains of biocontrol interest, differing in their colonization abilities: DTR133, a strong rhizosphere colonizer and ToZa7, which displays lower rhizocompetence. The investigated plant mutations were related to root exudation, immunity, and root system architecture. Mutations in smb and shv3, both involved in root architecture, were shown to positively affect rhizosphere colonization by ToZa7, but not DTR133. While these strains were not promoting plant growth in wild-type plants, increased plant biomass was measured in inoculated plants lacking fez, wrky70, cbp60g, pft1 and rlp30, genes mostly involved in plant immunity. These results point to an interplay between plant genotype, plant growth and rhizosphere colonization by phytobeneficial Pseudomonas spp. Some of the studied genes could become targets for plant breeding programs to improve plant-beneficial Pseudomonas rhizocompetence and biocontrol efficiency in the field.
PubMed: 35336236
DOI: 10.3390/microorganisms10030660 -
Scientific Reports Aug 2021Trans-2,3-dihydro-3-hydroxyanthranilic acid (DHHA) is a cyclic β-amino acid used for the synthesis of non-natural peptides and chiral materials. And it is an...
Trans-2,3-dihydro-3-hydroxyanthranilic acid (DHHA) is a cyclic β-amino acid used for the synthesis of non-natural peptides and chiral materials. And it is an intermediate product of phenazine production in Pseudomonas spp. Lzh-T5 is a P. chlororaphis strain isolated from tomato rhizosphere found in China. It can synthesize three antifungal phenazine compounds. Disruption the phzF gene of P. chlororaphis Lzh-T5 results in DHHA accumulation. Several strategies were used to improve production of DHHA: enhancing the shikimate pathway by overexpression, knocking out negative regulatory genes, and adding metal ions to the medium. In this study, three regulatory genes (psrA, pykF, and rpeA) were disrupted in the genome of P. chlororaphis Lzh-T5, yielding 5.52 g/L of DHHA. When six key genes selected from the shikimate, pentose phosphate, and gluconeogenesis pathways were overexpressed, the yield of DHHA increased to 7.89 g/L. Lastly, a different concentration of Fe was added to the medium for DHHA fermentation. This genetically engineered strain increased the DHHA production to 10.45 g/L. According to our result, P. chlororaphis Lzh-T5 could be modified as a microbial factory to produce DHHA. This study laid a good foundation for the future industrial production and application of DHHA.
Topics: 3-Hydroxyanthranilic Acid; Culture Media; Fermentation; Ferric Compounds; Gene Knockdown Techniques; Genes, Bacterial; Genes, Regulator; Phenazines; Pseudomonas chlororaphis
PubMed: 34385485
DOI: 10.1038/s41598-021-94674-8 -
Developing a CRISPR-assisted base-editing system for genome engineering of Pseudomonas chlororaphis.Microbial Biotechnology Sep 2022Pseudomonas chlororaphis is a non-pathogenic, plant growth-promoting rhizobacterium that secretes phenazine compounds with broad-spectrum antibiotic activity. Currently...
Pseudomonas chlororaphis is a non-pathogenic, plant growth-promoting rhizobacterium that secretes phenazine compounds with broad-spectrum antibiotic activity. Currently available genome-editing methods for P. chlororaphis are based on homologous recombination (HR)-dependent allelic exchange, which requires both exogenous DNA repair proteins (e.g. λ-Red-like systems) and endogenous functions (e.g. RecA) for HR and/or providing donor DNA templates. In general, these procedures are time-consuming, laborious and inefficient. Here, we established a CRISPR-assisted base-editing (CBE) system based on the fusion of a rat cytidine deaminase (rAPOBEC1), enhanced-specificity Cas9 nickase (eSpCas9pp ) and uracil DNA glycosylase inhibitor (UGI). This CBE system converts C:G into T:A without DNA strands breaks or any donor DNA template. By engineering a premature STOP codon in target spacers, the hmgA and phzO genes of P. chlororaphis were successfully interrupted at high efficiency. The phzO-inactivated strain obtained by base editing exhibited identical phenotypic features as compared with a mutant obtained by HR-based allelic exchange. The use of this CBE system was extended to other P. chlororaphis strains (subspecies LX24 and HT66) and also to P. fluorescens 10586, with an equally high editing efficiency. The wide applicability of this CBE method will accelerate bacterial physiology research and metabolic engineering of non-traditional bacterial hosts.
Topics: Animals; CRISPR-Cas Systems; DNA; Deoxyribonuclease I; Gene Editing; Genome, Bacterial; Pseudomonas chlororaphis; Rats
PubMed: 35575623
DOI: 10.1111/1751-7915.14075 -
PeerJ 2021strain PA23 is a biocontrol agent that is able to protect canola against the pathogenic fungus . This bacterium secretes a number of metabolites that contribute to...
BACKGROUND
strain PA23 is a biocontrol agent that is able to protect canola against the pathogenic fungus . This bacterium secretes a number of metabolites that contribute to fungal antagonism, including pyrrolnitrin (PRN), phenazine (PHZ), hydrogen cyanide (HCN) and degradative enzymes. In order to be successful, a biocontrol agent must be able to persist in the environment and avoid the threat of grazing predators. The focus of the current study was to investigate whether PA23 is able to resist grazing by the protozoan predator (Ac) and to define the role of bacterial metabolites in the PA23-Ac interaction.
METHODS
Ac was co-cultured with PA23 WT and a panel of derivative strains for a period of 15 days, and bacteria and amoebae were enumerated on days 1, 5, 10 and 15. Ac was subsequently incubated in the presence of purified PRN, PHZ, and KCN and viability was assessed at 24, 48 and 72 h. Chemotactic assays were conducted to assess whether PA23 compounds exhibit repellent or attractant properties towards Ac. Finally, PA23 grown in the presence and absence of amoebae was subject to phenotypic characterization and gene expression analyses.
RESULTS
PRN, PHZ and HCN were found to contribute to PA23 toxicity towards Ac trophozoites, either by killing or inducing cyst formation. This is the first report of PHZ-mediated toxicity towards amoebae. In chemotaxis assays, amoebae preferentially migrated towards regulatory mutants devoid of extracellular metabolite production as well as a PRN mutant, indicating this antibiotic has repellent properties. Co-culturing of bacteria with amoebae led to elevated expression of the PA23 / quorum-sensing (QS) genes and and , which are under QS control. PHZ and PRN levels were similarly increased in Ac co-cultures, suggesting that PA23 can respond to predator cues and upregulate expression of toxins accordingly.
CONCLUSIONS
PA23 compounds including PRN, PHZ and HCN exhibited both toxic and repellent effects on Ac. Co-culturing of bacteria and amoebae lead to changes in bacterial gene expression and secondary metabolite production, suggesting that PA23 can sense the presence of these would-be predators and adjust its physiology in response.
PubMed: 33552738
DOI: 10.7717/peerj.10756 -
Biology Jan 2022DAHP synthase catalyzes the first step in the shikimate pathway, deriving the biosynthesis of aromatic amino acids (Trp, Phe and Tyr), phenazine-1-carboxamide, folic...
DAHP synthase catalyzes the first step in the shikimate pathway, deriving the biosynthesis of aromatic amino acids (Trp, Phe and Tyr), phenazine-1-carboxamide, folic acid, and ubiquinone in . In this study, we identified and characterized one DAHP synthase encoding gene , which differs from the reported DAHP synthase encoding genes , and in . . PhzC accounts for approximately 90% of the total DAHP synthase activities in . HT66 and plays the most critical role in four DAHP synthases in the shikimate pathway. Inactivation of resulted in the reduction of PCN production by more than 90%, while the absence of genes , and reduced PCN yield by less than 15%, and the production of PCN was restored after the complementation of gene . Moreover, the results showed that in . HT66 is not sensitive to feedback inhibition. This study demonstrated that gene is essential for PCN biosynthesis. The expression level of both and genes are not inhibited in feedback by PCN production due to the absence of a loop region required for allosteric control reaction. This study highlighted the importance of PhzC and applying . for shikimate pathway-derived high-value biological production.
PubMed: 35053084
DOI: 10.3390/biology11010086