Search Results (8)

Fungal phytopathogens represent a large and economically significant challenge to food production worldwide. Thus, the application of biocontrol agents can be an alternative. In the present study, we carried out biological, metabolomic, and genetic analyses of three endophytic isolates from nodules of Chamaecytisus albus, classified as Pseudomonas chlororaphis acting as antifungal agents. The efficiency of production of their diffusible and volatile antifungal compounds (VOCs) was verified in antagonistic assays with the use of soil-borne phytopathogens: B. cinerea, F. oxysporum, and S. sclerotiorum. Diffusible metabolites were identified using chromatographic and spectrometric analyses (HPTLC, GC-MS, and LC-MS/MS). The phzF, phzO, and prnC genes in the genomes of bacterial strains were confirmed by PCR. In turn, the plant growth promotion (PGP) properties (production of HCN, auxins, siderophores, and hydrolytic enzymes, phosphate solubilization) of pseudomonads were bioassayed. The data analysis showed that all tested strains have broad-range antifungal activity with varying degrees of antagonism. The most abundant bioactive compounds were phenazine derivatives: phenazine-1-carboxylic acid (PCA), 2-hydroxy-phenazine, and diketopiperazine derivatives as well as ortho-dialkyl-aromatic acids, pyrrolnitrin, siderophores, and HCN. The results indicate that the tested P. chlororaphis isolates exhibit characteristics of biocontrol organisms; therefore, they have potential to be used in sustainable agriculture and as commercial postharvest fungicides to be used in fruits and vegetables. Full article

This study aimed to develop the eco-friendly production of bioactive 1-hydroxyphenazine (HP) through fermentation using an industrial processing by-product of cassava as the main carbon/nitrogen source. Cassava starch processing by-product (CSPB) was screened as a suitable substrate for fermentation to produce HP with a high yield. Mixing CSPB with a minor amount of tryptic soy broth (TSB) at a ratio of 8/2 and with 0.05% K₂HPO₄ and 0.05% FeSO₄ was effective in HP production by Pseudomonas aeruginosa TUN03. HP was also further scaled up through production on a bioreactor system, which achieved a higher level yield (36.5 µg/mL) in a shorter fermentation time (10 h) compared to its production in the flask (20.23 µg/mL after 3 days). In anti-fungal activity tests against various Fusarium phytopathogens, HP exhibited the most significant effect on Fusarium oxysporum F10. It could inhibit the mycelial growth of this fungus, with an inhibition rate of 68.7% and anti-spore germination activity of up to 98.4%. The results of the docking study indicate that HP effectively interacted with the protein 1TRY targeting anti-F. oxysporum, with all obtained docking parameters in the accepted range. This study supports the novel use of CSPB as the carbon/nitrogen source for P. aeruginosa fermentation to produce HP, a F. oxysporum anti-fungal agent reported here for the first time. Full article

Previous research has indicated that Pseudomonas aurantiaca ST-TJ4 possesses a notable antagonistic impact on Phytophthora cinnamomi and holds promising potential for biocontrol. In this study, a combination of a single-factor experiment, a Plackett–Burman design and a response surface approach was employed to investigate the optimal formula of ST-TJ4 fermentation medium. Furthermore, the stability of ST-TJ4 fermentation filtrate and its biocontrol effect on Ph. cinnamomi in vivo were also evaluated. The results revealed that the optimal culture conditions for ST-TJ4 involved the use of 20.59 g/L of glucose and 18.76 g/L of yeast extract powder. Following optimization, the fermentation filtrate of ST-TJ4 exhibited an inhibition rate of 76.5%, representing a 15% increase compared to previous levels. Additionally, phzA, phzB, phzD, phzE, phzF and phzO genes involved in the synthesis of phenazine-1-carboxylic acid (PCA) and 2-hydroxyphenazine (2-OH-PHZ) were also upregulated. The ST-TJ4 fermentation filtrate demonstrated strong alkali resistance, weak acid resistance and favorable temperature and UV light stability. Furthermore, in vitro inoculation experiments confirmed that optimizing the fermentation medium reduced Ps. cinnamomi’s ability to infect the leaves of Rhododendron pulchrum. Full article

In green chemistry, filamentous fungi are regarded as a kind of robust microorganism for the biotransformation of natural products. Nonetheless, the screening of microorganisms is crucial for the effective biotransformation of natural products, such as phenazine compounds. The precursor metabolite of most phenazine derivatives in Pseudomonas spp. is phenazine-1-carboxylic acid (PCA), the key constituent of shenqinmycin, widely used to control rice sheath blight in southern China. In this study, a new fungus strain Aspergillus sclerotiorum was isolated, which can efficiently convert PCA into 3-hydroxy-phenazine 1-carboxylic acid (3-OH-PCA). Moreover, an effective whole cells biotransformation system was designed by screening optimal reaction conditions and carbon sources. Hence, Aspergillus sclerotiorum exhibited desirable adaptation by the consumption of different carbon sources and maximum whole-cell biomass (10.6 g/L DCW) was obtained as a biocatalyst from glucose. Optimal conditions for whole-cell biocatalysis of PCA were evaluated, including a PCA concentration of 1120 mg/L, a pH of 7.0, a temperature of 25 °C, a rotation rate of 200 rpm, and dry cell weight of 15 g/L for 60 h; thus, 1060 mg/L of 3-OH-PCA was obtained and the conversion efficiency of PCA was 94%. Hence, the results of the repeated batch mood revealed that the biotransformation efficiency of fungus pellets reduced with each subsequent cycle, but remained stable in all five cycles with the provision of a glucose supplement. These findings present the prospect of using filamentous fungi for the whole-cell biocatalysis of phenazine in enormous amounts and the efficient production of 3-OH-PCA. Moreover, these results laid the foundation for further research to disclose the genetic-based mechanism of the strain responsible for PCA biotransformation. Full article

The development of MFC using electroactive industrial microorganisms has seen a surge of interest because of the co-generation for bioproduct and electricity production. Vibrio natriegens as a promising next-generation industrial microorganism chassis and its application for microbial fuel cells (MFC) was first studied. Mediated electron transfer was found in V. natriegens MFC (VMFC), but V. natriegens cannot secrete sufficient electron mediators to transfer electrons to the anode. All seven electron mediators supplemented are capable of improving the electronic transfer efficiency of VMFC. The media and carbon sources switching study reveals that VMFCs have excellent bioelectricity generation performance with feedstock flexibility and high salt-tolerance. Among them, 1% glycerol as the sole carbon source produced the highest power density of 111.9 ± 6.7 mW/cm². The insight of the endogenous electronic mediators found that phenazine-1-carboxamide, phenazine-1-carboxylic acid, and 1-hydroxyphenazine are synthesized by V. natriegens via the shikimate pathway and the phenazine synthesis and modification pathways. This work provides the first proof for emerging industrial biotechnology chassis V. natriegens as a novel high salt-tolerant and feedstock flexibility electroactive microorganism for MFC, and giving insight into the endogenous electron mediator biosynthesis of VMFC, paving the way for the application of V. natriegens in MFC and even microbial electrofermentation (EF). Full article

Pseudomonas fluorescens 9 and Bacillus subtilis 54, proposed as biofungicides to control Macrophomina phaseolina, a dangerous pathogen of soybean and other crops, were grown in vitro to evaluate their ability to produce metabolites with antifungal activity. The aim of the manuscript was to identify the natural compounds responsible for their antifungal activity. Only the culture filtrates of P. fluorescens 9 showed strong antifungal activity against M. phaseolina. Its organic extract contained phenazine and mesaconic acid (1 and 2), whose antifungal activity was tested against M. phaseolina, as well as Cercospora nicotianae and Colletotrichum truncatum, other pathogens of soybean; however, only compound 1 exhibited activity. The antifungal activity of compound 1 was compared to phenazine-1-carboxylic acid (PCA, 3), 2-hydroxyphenazine (2-OH P, 4), and various semisynthetic phenazine nitro derivatives in order to perform a structure–activity relationship (SAR) study. PCA and phenazine exhibited the same percentage of growth inhibition in M. phaseolina and C. truncatum, whereas PCA (3) showed lower activity against C. nicotianae than phenazine. 2-Hydroxyphenazine (4) showed no antifungal activity against M. phaseolina. The results of the SAR study showed that electron attractor (COOH and NO₂) or repulsor (OH) groups significantly affect the antifungal growth, as well as their α- or β-location on the phenazine ring. Both PCA and phenazine could be proposed as biopesticides to control the soybean pathogens M. phaseolina, C. nicotianae, and C. truncatum, and these results should prompt an investigation of their large-scale production and their suitable formulation for greenhouse and field applications. Full article

Pseudomonas chlororaphis PA23 was isolated from the rhizosphere of soybeans and identified as a biocontrol bacterium against Sclerotinia sclerotiorum, a fungal plant pathogen. This bacterium produces a number of secondary metabolites, including phenazine-1-carboxylic acid, 2-hydroxyphenazine, pyrrolnitrin (PRN), hydrogen cyanide, proteases, lipases and siderophores. It also synthesizes and accumulates polyhydroxyalkanoate (PHA) polymers as carbon and energy storage compounds under nutrient-limited conditions. Pseudomonads like P. chlororaphis metabolize glucose via the Entner-Doudoroff and Pentose Phosphate pathways, which provide precursors for phenazine production. Mutants defective in phenazine (PHZ; PA23-63), PRN (PA23-8), or both (PA23-63-1) accumulated higher concentrations of PHAs than the wild-type strain (PA23) when cultured in Ramsay’s Minimal Medium with glucose or octanoic acid as the carbon source. Expression levels of six pha genes, phaC1, phaZ, phaC2, phaD, phaF, and phaI, were compared with wild type PA23 by quantitative real time polymerase chain reaction (qPCR). The qPCR studies indicated that there was no change in levels of transcription of the PHA synthase genes phaC1 and phaC2 in the phz^- (PA23-63) and phz^- prn^- (PA23-63-1) mutants in glucose medium. There was a significant increase in expression of phaC2 in octanoate medium. Transcription of phaD, phaF and phaI increased significantly in the phz^- prn^- (PA23-63-1) mutant. Mutations in regulatory genes like gacS, rpoS, and relA/spoT, which affect PHZ and PRN production, also resulted in altered gene expression. The expression of phaC1, phaC2, phaF, and phaI genes was down-regulated significantly in gacS and rpoS mutants. Thus, it appears that PHZ, PRN, and PHA production is regulated by common mechanisms. Higher PHA production in the phz^- (PA23-63), prn- (PA23-8), and phz^- prn^- (PA23-63-1) mutants in octanoic medium could be correlated with higher expression of phaC2. Further, the greater PHA production observed in the phz^- and prn^- mutants was not due to increased transcription of PHA synthase genes in glucose medium, but due to more accessibility of carbon substrates and reducing power, which were otherwise used for the synthesis of PHZ and PRN. Full article

A fast and efficient approach was established to identify bacteria possessing the potential to biosynthesize phenazines, which are of special interest regarding their antimicrobial activities. Sequences of phzE genes, which are part of the phenazine biosynthetic pathway, were used to design one universal primer system and to analyze the ability of bacteria to produce phenazine. Diverse bacteria from different marine habitats and belonging to six major phylogenetic lines were investigated. Bacteria exhibiting phzE gene fragments affiliated to Firmicutes, Alpha- and Gammaproteobacteria, and Actinobacteria. Thus, these are the first primers for amplifying gene fragments from Firmicutes and Alphaproteobacteria. The genetic potential for phenazine production was shown for four type strains belonging to the genera Streptomyces and Pseudomonas as well as for 13 environmental isolates from marine habitats. For the first time, the genetic ability of phenazine biosynthesis was verified by analyzing the metabolite pattern of all PCR-positive strains via HPLC-UV/MS. Phenazine production was demonstrated for the type strains known to produce endophenazines, 2-hydroxy-phenazine, phenazine-1-carboxylic acid, phenazine-1,6-dicarboxylic acid, and chlororaphin as well as for members of marine Actinobacteria. Interestingly, a number of unidentified phenazines possibly represent new phenazine structures. Full article