•Bacillus group are well known as producers of a wide array of antimicrobials.•Most important molecules are non-ribosomally synthesized peptides and lipopeptides.•Very strong antimicrobial activity ...was confirmed through numerous studies.•Bacillus lipopeptides have strong impact through direct antibiosis on plant pathogens.
Bacteria from the Bacillus group are microorganisms that inhabit a large number of different habitats. They are well known as producers of a wide array of antagonistic compounds of different structures, having between 5 to 8% of the total genome devoted to biosynthesis of secondary metabolites. Most important bioactive molecules from the genus Bacillus are non-ribosomally synthesized peptides and lipopeptides, polyketide compounds, bacteriocins and siderophores. Lipopeptides from Bacillus have very complex mechanisms of biosynthesis catalyzed by non-ribosomal peptide synthetases (NRPSs), large enzyme complexes with modular structure, with each module being in charge for the incorporation of a particular amino acid. In general, they have a broad spectrum of antagonistic activity against plant pathogenic bacteria, fungi and viruses. Most important molecules from this group, circular lipopeptides from surfactin, iturin and fengycin families affect the target cells on the membrane level. Bacillus strains exhibit their biocontrol capacity predominantly through inhibitory activity on the growth of plant pathogens, as well as inducing systemic resistance in plants and competing for ecological niches with plant pathogens. Our previous studies showed the presence of multiple biosynthetic operons for synthesis of non-ribosomal lipopeptides in the collection of natural isolates of Bacillus, with many strains having more than one of them. Several strains of Bacillus sp. that we have recently characterized showed very strong antibacterial and antifungal activity against phytopathogens. The PCR analysis showed the presence of biosynthetic operons for iturin, bacillomycin, fengycin and surfactin in tested strains. Measurement of the kinetics of production of antimicrobial substances showed that, in most cases, synthesis started at the beginning of exponential phase of growth, reaching the maximum of antimicrobial activity at the beginning of the stationary growth phase and stayed at this level for the whole duration of observed period. Preparations of cell-free supernatants of tested strains were active against many fungal and bacterial pathogens, in vitro and in vivo. Mass spectrometry and HPTLC bioautography analysis of purified compounds confirmed the presence of lipopeptides of mentioned families, hence confirming the biocontrol capacity of Bacillus isolates.
New information has come to light about the biological activity of propolis and the quality of natural products which requires a rapid and reliable assessment method such as High Performance ...Thin-Layer Chromatography (HPTLC) fingerprinting. This study investigates chromatographic and chemometric approaches for determining the antimicrobial activity of propolis of Serbian origin against various bacterial species. A linear multivariate calibration technique, using Partial Least Squares, was used to extract the relevant information from the chromatographic fingerprints, i.e. to indicate peaks which represent phenolic compounds that are potentially responsible for the antimicrobial capacity of the samples. In addition, direct bioautography was performed to localize the antibacterial activity on chromatograms. The biological activity of the propolis samples against various bacterial species was determined by a minimum inhibitory concentration assay, confirming their affiliation with the European poplar type of propolis and revealing the existence of two types (blue and orange) according to botanical origin. The strongest antibacterial activity was exhibited by sample 26 against Staphylococcus aureus, with a MIC value of 0.5 mg/mL, and Listeria monocytogenes, with a MIC as low as 0.1 mg/mL, which was also the lowest effective concentration observed in our study. Generally, the orange type of propolis shows higher antimicrobial activity compared to the blue type. PLS modelling was performed on the HPTLC data set and the resulting models might qualitatively indicate compounds that play an important role in the activity exhibited by the propolis samples. The most relevant peaks influencing the antimicrobial activity of propolis against all bacterial strains were phenolic compounds at RF values of 0.37, 0.40, 0.45, 0.51, 0.60 and 0.70. The knowledge gained through this study could be important for attributing the antimicrobial activity of propolis to specific chemical compounds, as well as the verification of HPTLC fingerprinting as a reliable method for the identification of compounds that are potentially responsible for antimicrobial activity. This is the first report on the activity of Serbian propolis as determined by several combined methods, including the modelling of antimicrobial activity by HPTLC fingerprinting.
In this study the efficacy of two different methods for extracting lipopeptides produced by five
strains-ethyl acetate extraction, and acid precipitation followed by methanol extraction-was ...investigated using mass spectrometry. High performance thin layer chromatography (HPTLC) was also used for the simultaneous separation of complex mixtures of lipopeptide extracts and for the determination of antimicrobial activity of their components. The mass spectra clearly showed well-resolved groups of peaks corresponding to different lipopeptide families (kurstakins, iturins, surfactins, and fengycins). The ethyl acetate extracts produced the most favorable results. The extracts of SS-12.6, SS-13.1, and SS-38.4 showed the highest inhibition zones. An iturin analog is responsible for the inhibition of
and
phytopathogenic strains. HPTLC bioautography effectively identified the active compounds from a mixture of lipopeptide extracts, proving
its potential for use in direct detection and determination of antimicrobials. In the test of potential synergism among individual extracts used in different mixtures, stronger antimicrobial effects were not observed. Biochemical and phylogenetic analysis clustered isolates SS-12.6, SS-13.1, SS-27.2, and SS-38.4 together with
, while SS-10.7 was more closely related to
.
•The potential applications of sixteen essential oils as biocontrol agents.•Most oils were dominated with monoterpenes.•Fungi isolated from herbal drugs were inhibited in vitro and in situ.•Savory, ...oregano, thyme and rose oils proved to be the best inhibitors of fungi.•The mixture of thyme and oregano oils exhibited excelling antifungal activity.
The development of protective products with natural origin as alternatives to synthetic fungicides is currently in the spotlight. Qualitative and quantitative chemical analysis of 16 selected essential oils was determined. Then, antifungal properties determined by in vitro microdilution method against 21 fungi isolated from herbal drugs were evaluated. All tested oils showed some antifungal activity against all fungi used. Savory, thyme and oregano oils, characterized by the presence of phenol such as carvacrol and thymol, and rose oil containing mainly monoterpene alcohols (citronellol and geraniol) proved to be the most effective inhibitor of all fungi tested. Also, combination of particular oils showed reduction of the MIC values when combined, commendatory mixtures for potential application in practice. Moreover, the reduction of the total number of fungi, in situ, using selected essential oils was determined.
pv. aptata is a member of the sugar beet pathobiome and the causative agent of leaf spot disease. Like many pathogenic bacteria,
relies on the secretion of toxins, which manipulate host-pathogen ...interactions, to establish and maintain an infection. This study analyzes the secretome of six pathogenic
pv. aptata strains with different defined virulence capacities in order to identify common and strain-specific features, and correlate the secretome with disease outcome. All strains show a high type III secretion system (T3SS) and type VI secretion system (T6SS) activity under apoplast-like conditions mimicking the infection. Surprisingly, we found that low pathogenic strains show a higher secretion of most T3SS substrates, whereas a distinct subgroup of four effectors was exclusively secreted in medium and high pathogenic strains. Similarly, we detected two T6SS secretion patterns: while one set of proteins was highly secreted in all strains, another subset consisting of known T6SS substrates and previously uncharacterized proteins was exclusively secreted in medium and high virulence strains. Taken together, our data show that
pathogenicity is correlated with the repertoire and fine-tuning of effector secretion and indicate distinct strategies for establishing virulence of
pv. aptata in plants.
•A total of 14 different plant resins from Serbia were analyzed.•Phenolic composition and antimicrobial activity were determined.•Populus species were recognized as the origin of two subtypes of ...Serbian propolis.•Phenolics were responsible for the antibacterial activity of plant resin samples.•Synergism in antibiosis was detected among several pure phenolic compounds.
Extensive employment of plant resins and propolis, as an antiseptic agents dating from ancient times in numerous cultures indicating that it may have antimicrobial and other biological properties. Resins from deciduous trees from the Populus and Salix genera, several fruit trees from the genus Prunus and a few other species were analyzed for their phenolic composition and antibacterial activity. Phenolic profiling of the plant resins was performed by high-performance thin-layer chromatography (HPTLC) and ultra-high-performance liquid chromatography (UHPLC) coupled with hybrid mass spectrometry. Antimicrobial activity against seven bacterial species was determined by minimum inhibitory concentration (MIC) assays and bioautography. The synergism, additivism, and antagonism of phenolic compounds were used to define the nature and type of interactions. Populus spp. showed higher amounts of p-hydroxybenzoic acid, p-coumaric acid, caffeic acid, chrysin, apigenin, quercetin, pinocembrin, pinobanksin and galangin, which confirmed the botanical origin of the orange and blue types of propolis. In addition, Gram-positive bacteria exhibited high susceptibility to poplar samples while being resistant to samples from other origins. Cherry bud samples had high amounts of naringenin and showed strong activity against Bacillus subtilis and Listeria monocytogenes. The combinations of tested phenolics showed mainly additive or indifferent effects.
Bacillus
species are among the most researched and frequently applied biocontrol agents. To estimate their potential as environmentally friendly microbial-based products, reliable and rapid plant ...colonization monitoring methods are essential. We evaluated repetitive element-based (rep) and Random Amplified Polymorphic DNA (RAPD) PCR (Polymerase Chain Reaction) genotyping in a diversity assessment of 251 strains from bulk soil, straw, and manure samples across Serbia, highlighting their discriminative force and the presence of unique bands. RAPD 272, OPG 5, and (GTG)
5
primers were most potent in revealing the high diversity of a sizable environmental
Bacillus
spp. collection. RAPD 272 also amplified a unique band for a proven biocontrol strain, opening the possibility of Sequence Characterized Amplified Region (SCAR) marker design. That will enable colonization studies using the SCAR marker for its specific detection. This study provides a guide for primer selection for diversity and monitoring studies of environmental
Bacillus
spp. isolates.
Comprehensive analysis of phenolic profiles of botanically different subtypes of Turkish propolis samples were performed using UHPLC–LTQ/Orbitrap/MS/MS method, and additionally total phenolic (TPC) ...and total flavonoid contents (TFC) as well as their antioxidative activities were evaluated by spectrophotometry. Antimicrobial activity of Turkish propolis against oral cavity bacteria from the genus Streptococcus (S. pyogenes, S. sanguinis, S. mutans) and Candida albicans ATCC 10231 was determined by diffusion and microdilution methods. Extensive fingerprint analysis of Turkish propolis revealed the presence of fifty one phenolic compounds, with fifteen quantified which confirm their affiliation to the two subtypes of the European propolis. All analysed samples have shown antimicrobial potential against all tested bacteria, with S. pyogenes being the most sensitive one. Turkish propolis, especially its orange subtype, can be considered as the high-quality product due to its rich phenolic and flavonoid content, strong antioxidative and antimicrobial activities. Turkish propolis could be, therefore, a good raw material for food and pharmaceutical industry.
•Phenolic profiling of three subtypes of Turkish poplar type propolis was studied.•Quality control parameters of three subtypes of propolis were investigated.•O-subtype propolis had higher total phenolic and flavonoid contents than B- subtype.•O- subtype of propolis showed higher antioxidative and antimicrobial activities.
European plum (
Prunus domestica
L.) is a significant commercial crop in Serbia in terms of total fruit production, and is traditionally processed into slivovitz brandy. The brown rot disease caused ...by
Monilinia laxa
drastically reduces plum yield almost every year. Fungal communities associated with leaves and fruits of four local Serbian plum cultivars (Požegača, Ranka, Čačanska Lepotica and Čačanska Rodna) were investigated in two phenological stages during early (May) and late (July) fruit maturation. Alpha diversity indices showed that fungal communities were heterogeneous and Beta diversity indicated that autochthonous fungal communities depended upon seasonal changes and the cultivars themselves. The phylum Ascomycota was the most abundant in all samples, with relative abundance (RA) between 46% in the Požegača cultivar (May) and 89% in the Lepotica cultivar (July). The most abundant genus for all plum cultivars in May was
Aureobasidium
, with RA from 19.27 to 33.69%, followed by
Cryptococcus
, with 4.8 to 48.80%. In July, besides
Cryptococcus
, different genera (
Metschnikowia
,
Fusarium
, and
Hanseniaspora
) were dominant on particular cultivars. Among all cultivable fungi, molecular identification of eleven
M. laxa
isolates from four plum cultivars was performed simultaneously. Bacterial isolates from the plum phyllosphere were tested for their potential antifungal activity against indigenous
M. laxa
isolates. The most potent antagonist P4/16_1, which significantly reduced mycelial growth of
M. laxa
, was identified as
Pseudomonas synxantha
. Further characterization of P4/16_1 revealed the production of volatile organic compounds and phenazine-1-carboxylic acid (PCA). Crude benzene extract of PCA exhibited 57–63% mycelial growth inhibition of
M. laxa
. LC/MS analysis of the crude extract confirmed the presence of phenazine derivatives amongst other compounds. Scanning electron microscopy revealed morpho-physiological changes in the hyphae of
M. laxa
isolates caused by the cell culture and the
P. synxantha
P4/16_1 crude benzene extract. This is the first report of antagonistic activity of
P. synxantha
against
M. laxa
induced by diffusible and volatile antifungal compounds, and it appears to be a promising candidate for further investigation for potential use as a biocontrol agent against brown rot-causing fungi.
Compost green mould and dry bubble are the most devastating diseases of the cultivated white button mushroom. In search of efficient disease control, previously and newly isolated Bacillus spp. ...strains obtained from mushroom substrates were examined for antagonistic activity against Trichoderma aggressivum f. europaeum T77, Trichoderma harzianum T54 and Lecanicillium fungicola var. fungicola Ša2V6in vitro. All 33 tested Bacillus spp. strains inhibited the growth of pathogens. The strains with the strongest antagonistic activity were selected for in vivo experiments in which the efficacy of strains in pathogen suppression and their effect on mushroom yield were evaluated, in comparison with a commercial biofungicide based on Bacillus velezensis QST 713 and a fungicide based on prochloraz-manganese. In mushroom growing room trials with T. aggressivum f. europaeum, the most effective in pathogen suppression were (in decreasing order) prochloraz-Mn, Bacillus amyloliquefaciens B-241, Bacillus velezensis QST 713, Bacillus pumilus B-138 and B. subtilis B-233. The commercial prochloraz-Mn fungicide was the best at suppression of L. fungicola var. fungicola, while B. amyloliquefaciens B-241 was the most successful out of the three tested Bacillus spp. strains. In all in vivo experiments, B. amyloliquefaciens B-241 showed similar results in suppression of pathogens as the commercial biofungicide B. velezensis QST 713. Regarding the impact on mushroom productivity in trials with both pathogens, no statistically significant differences were noted when the treatments were compared with inoculated and uninoculated controls. Strain B. amyloliquefaciens B-241 showed the greatest potential for biocontrol of both compost green mould and dry bubble disease, which makes it a good candidate for further trials at commercial scale.
•Bacillus spp. reduced the growth of Trichoderma spp. and Lecanicillium spp. in vitro.•B. amyloliquefaciens B-241 efficiently suppressed both pathogens in vivo.•Tested Bacillus spp. strains had no negative impact on A. bisporus growth.•B. amyloliquefaciens B-241 has potential for biocontrol of green mould and dry bubble.
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