Antifungal activity of lemon, eucalyptus, thyme, oregano, sage and lavender essential oils against Aspergillus niger and Aspergillus tubingensis isolated from grapes

Today, it is very important to find out the protection of products of natural origin as an alternative to synthetic fungicides. The promising alternative is the use of the essential oils (EOs). Essential oils from plants have great potential as a new source of fungicide to control the pathogenic fungi.The main objective of this study was evaluation of the antifungal activity of lemon (Citrus lemon L.), eucalyptus (Eucalyptus globulus LABILL.), thyme (Thymus vulgaris L.), oregano (Origanum vulgare L.) sage (Salvia officinalis L.) and lavender (Lavandula angustifolia MILLER.) EOs against Aspergillus niger and Aspergillus tubingensis isolated from grapes and their ability to affect the growth. It was tested by using the vapor contact with them. At first both tested isolates were identified by using PCR method. Sequence data of 18S rRNA supported the assignment of these isolates to the genus Aspergillus and species A. niger (ITS region: KT824061; RPB2: KT824060) and A. tubingensis (ITS region: KT824062; RPB2: KT824059). Second, EO antifungal activity was evaluated. The effect of the EO volatile phase was confirmed to inhibit growth of A. niger and A tubingensis. EOs were diluted in DMSO (dimethyl sulfoxide) final volume of 100 μL. Only 50 μL this solution was distributed on a round sterile filter paper (1 x 1 cm) by micropipette, and the paper was placed in the center of the lid of Petri dishes. Dishes were kept in an inverted position. The essential oils with the most significant activity were determined by method of graded concentration of oils - minimum inhibitory doses (MIDs). The most effective tested EOs were oregano and thyme oils, which totally inhibited growth of tested isolates for all days of incubation at 0.625 μL.cm-3 (in air) with MFDs 0.125 μL.cm-3 (in air). Lavender EO was less active aginst tested strains (MIDs 0.313 μL.cm-3). The results showed that the tested EOs had antifungal activity, except lemon and eucalyptus. Sage EO was the only one which decelerated the radial growth of colony of both tested strains after all days of cultivation in comparison with a control sets. Our study provides the support that essential oils can be used to control plant pathogens such as A. niger and A. tubingensis

Antimicrobial activity of actinomycetes and characterization of actinomycin-producing strain KRG-1 isolated from Karoo, South Africa

In the present study we reported the antimicrobial activity of actinomycetes isolated from aridic soil sample collected in Karoo, South Africa. Eighty-six actinomycete strains were isolated and purified, out of them thirty-four morphologically different strains were tested for antimicrobial activity. Among 35 isolates, 10 (28.57%)showed both antibacterial and antifungal activity. The ethyl acetate extract of strain KRG-1 showed the strongest antimicrobial activity and therefore was selected for further investigation. The almost complete nucleotide sequence of the 16S rRNA gene as well as distinctive matrix-assisted laser desorption/ionization-time-of-flight/mass spectrometry (MALDI-TOF/MS) profile of whole-cell proteins acquired for strain KRG-1 led to the identification of Streptomyces antibioticus KRG-1 (GenBank accession number: KX827270). The ethyl acetate extract of KRG-1 was fractionated by HPLC method against the most suppressed bacterium Staphylococcus aureus (Newman). LC//MS analysis led to the identification of the active peak that exhibited UV-VIS maxima at 442 nm and the ESI-HRMS spectrum showing the prominent ion clusters for [M-H2O+H]+ at m/z 635.3109 and for [M+Na]+ at m/z 1269.6148. This information could be assigned to chromopeptide lactone antibiotic - actinomycin. Our results suggest that unexplored soils could be an interesting source for exploring antibacterial secondary metabolites

Characteristics of microbial biomass carbon and respiration activities in arable soil and pasture grassland soil

The aim of our work was to survey and state the representative values and range of the microbial biomass carbon (Cmic) and microbial respiration activities in arable soils of three types (Chernozem, Luvisol, Planosol) and in pasture grassland soil (Cambisol). In arable soils no significant differences between the soil types were found concerning the content of Cmic. Way of soil use significantly influenced Cmic with higher values on pasture grassland soil. In Cambisol, a relatively strong relationships between Cmic and Corg (r = 0.919) as well as between Cmic and Ntot (r = 0.922) were determined. The basal respiration (BR) was no affected neither by a soil type nor by a way of soil use. On all monitored sites microbes responded positively to the addition of glucose (potential respiration - PR), but the strongest response was observed on the soil types with good mineralisation ability, such as Chernozems and Luvisols. On pasture grassland soil, the substrate availability index (PR/BR) with values of 2.32 was observed, which is lower compared to 7.60 of arable soil. This fact to indicate that pasture grassland soil had more easy decomposable organic compounds than arable soils

Využitie MALDI-TOF hmotnostnej spektrometrie na rýchlu identifikáciu druhov Beauveria bassiana a Beauveria pseudobassiana

Species of the genus Beauveria are entomopathogenic fungi used in biological control to protect against insect pests. Identification of species is based on observation of morphological, phylogenetic and genetic differences. Currently, MALDI-TOF (Matrix Assisted Laser Desorption/Ionization-Time of Flight) mass spectrometry is often used in microbial diagnostic but identification of Beauveria species has not been tested before. The aim of this work was the possibilities of this method and to compare it with genetic analysis. Twenty strains of B. bassiana and B. pseudobassiana isolated from insect cadavers, mycoinsecticides and soil samples were used. Sequences of ITS (Internal Transcribed Spacer) region were used for genetic identification of strains. Sequences and results from protein analysis of isolates were compared with the reference strains of B. bassiana and B. pseudobassiana. Results of MALDI Biotyper softvare identification were correct for all strains. Identification was reliable with high log score (>2.1) for most of strains. Score value less than 1 was observed only in a single case - strain GHA isolated from mycoinsecticide Botanigard.The result of both analyses was also dendrogram (phylogenetic tree) which showed high degree of result consistence. MALDI-TOF identification of fungi from Beauveria genus is usable for fast and reliable identification of entomopathogenic fungi isolates acquired from environment and results can be utilized in conservation biocontrol strategies in agriculture and forest ecosystems.Rod Beauveria patrí medzi entomopatogénne huby využívané v biologickej regulácii hmyzích škodcov v poľnohospodárstve. Identifikácia druhov je založená na pozorovaní morfologických, fylogenetických a genetických rozdielov. V súčasnosti sa do popredia dostáva aj MALDI-TOF (Matrix Assisted Laser Desorption/Ionization-Time of Flight) hmotnostná spektrometria, ktorá v prípade rodu Beauveria nebola doposiaľ testovaná. Cieľom našej práce bolo zhodnotiť možnosť využívan ia tejto metódy na základe porovnania s genetickou analýzou. Použitých bolo 20 kmeňov B. bassiana a B. pseudobassiana, získaných izoláciou z kadáverov hmyzu, komerčne dostupných prípravkov a pôdnych vzoriek. Na genetickú identifikáciu kmeňov bol použitý región ITS (Internal Transcribed Spacer). Výsledné sekvencie a analýzy proteínových spektier vzoriek boli porovnávané s referenčnými kmeňmi B. bassiana a B.pseudobassiana. Výsledky identifikácie s využitím softvéru MALDI Biotyper boli správne pre všetky analyzované kmene. Identifikácia bola spoľahlivá s vysokým log skóre (>2.1) pre väčšinu kmeňov. Len v jedinom prípade – kmeň GHA, izolovaný z mykoinsekticídu Botanigard, bolo skóre nižšie ako 1. Výsledkom oboch analýz bol dendrogram (fylogenetický strom), ktorý poukázal na zhodnú klasifikáciu s referenčnými kmeňmi. MALDI-TOF identifikáciu húb z rodu Beauveria je možné využiť na rýchlu a spoľahlivú diagnostiku izolátov entomopatogénnych húb získaných z prostredia a výsledky využiť v systémoch biologickej ochrany lesných a poľnohospodárskych ekosystémov

Isolation, antimicrobial activity of myxobacterial crude extracts and identification of the most potent strains

Broad spectrum antimicrobial agents are urgently needed to fight frequently occurring multidrug-resistant pathogens. Myxobacteria have been regarded as “microbe factories” for active secondary metabolites, and therefore, this study was performed to isolate two bacteriolytic genera of myxobacteria, Myxococcus sp. and Corallococcus sp., from 10 soil/sand samples using two conventional methods followed by purification with the aim of determining the antimicrobial activity of methanol extracts against 11 test microorganisms (four Gram-positive, four Gram-negative, two yeasts and one fungus). Out of thirty-nine directly observed strains, 23 were purified and analyzed for antimicrobial activities. Based on the broth microdilution method, a total of 19 crude extracts showed antimicrobial activity. The range of inhibited wells was more important in the case of anti-Gram-positive-bacterial activity in comparison with the anti-Gram-negative-bacterial and antifungal activity. In light of the established degree and range of antimicrobial activity, two of the most active isolates (BNEM1 and SFEC2) were selected for further characterization. Morphological parameters and a sequence similarity search by BLAST revealed that they showed 99% sequence similarity to Myxococcus xanthus − BNEM1 (accession no. KX669224) and Corallococcus coralloides - SFEC2 (accession no. KX669225). As these isolates had antimicrobial activity, they could be considered for use in the development of antibiotics for pharmaceutical use

Effect of fertilization on biological activity of community of soil streptomycetes

The search for new natural mechanisms to inhibit the growth of phytopathogenic microorganisms has become widely widespread. Therefore, the main objective of the present study was determination of antimicrobial activities of actinomycetes isolated from agricultural soil, which was fertilized mainly by organic fertilizers, against 8 selected phytopathogenic strains. Among the actinomycetes, Streptomyces species have been extensively studied, because they have been recognized as an important source of secondary metabolites, which can suppress the growth of undesirable pests in crops. The results indicated that the richest source of Streptomyces colonies was soil fertilized with compost (103 x104 CFU*g-1 dry soil). On the basis of morphological signs, total of 65 isolates were selected and examined for antimicrobial activities. Isolates exhibited the best activity against Gram negative bacterium Clavibacter michiganensis subsp. sepedonicus, disease agent of “ring rot” of potatoes and against fungus Fusarium poae, disease agent of Fusarium head blight of wheat. Twelve isolates exhibited promising broad-spectrum activity against tested organisms. On the basis of results, six of them were selected for further screening. Comparison of polyphasic studies with available literature led to identification of biological active strains S. olivochromogenes (13SC11), S. avermitilis (13SC2), S. rishiriensis (13SC13), S. globisporus (13SC19), S. sampsonii (13SPC10) and S. avidinii (13SPC4). After quantification analysis of various enzymes, tested isolates produced alkaline phosphatase, leucinearylamidase, valinearylamidase, acid phosphatase, naphtol-AS-BI-phosphohydrolase, glucosidase in high values (>40 nmol) and were positive for nitrate reduction, hydrolysis of gelatin, urease, and esculin. These isolates can be used in the development of new biopesticides anf biofertilizers with antibacterial and antifungal effect

Effect of fertilization on biological activity of community of soil streptomycetes

The search for new natural mechanisms to inhibit the growth of phytopathogenic microorganisms has become widely widespread. Therefore, the main objective of the present study was determination of antimicrobial activities of actinomycetes isolated from agricultural soil, which was fertilized mainly by organic fertilizers, against 8 selected phytopathogenic strains. Among the actinomycetes, Streptomyces species have been extensively studied, because they have been recognized as an important source of secondary metabolites, which can suppress the growth of undesirable pests in crops. The results indicated that the richest source of Streptomyces colonies was soil fertilized with compost (103 x104 CFU*g-1 dry soil). On the basis of morphological signs, total of 65 isolates were selected and examined for antimicrobial activities. Isolates exhibited the best activity against Gram negative bacterium Clavibacter michiganensis subsp. sepedonicus, disease agent of “ring rot” of potatoes and against fungus Fusarium poae, disease agent of Fusarium head blight of wheat. Twelve isolates exhibited promising broad-spectrum activity against tested organisms. On the basis of results, six of them were selected for further screening. Comparison of polyphasic studies with available literature led to identification of biological active strains S. olivochromogenes (13SC11), S. avermitilis (13SC2), S. rishiriensis (13SC13), S. globisporus (13SC19), S. sampsonii (13SPC10) and S. avidinii (13SPC4). After quantification analysis of various enzymes, tested isolates produced alkaline phosphatase, leucinearylamidase, valinearylamidase, acid phosphatase, naphtol-AS-BI-phosphohydrolase, glucosidase in high values (>40 nmol) and were positive for nitrate reduction, hydrolysis of gelatin, urease, and esculin. These isolates can be used in the development of new biopesticides anf biofertilizers with antibacterial and antifungal effect.Hľadanie nových prirodzených mechanizmov na potlačenie rastu fytopatogénnych mikroorganizmov sa stalo veľmi rozšíreným. Z tohto dôvodu je hlavným cieľom tejto štúdie detekcia antimikrobiálnej aktivity aktinomycét izolovaných z poľnohospodársky využívanej pôdy hnojenej najmä organickými hnojivami, voči 8 vybraným fytopatogénnym mikroorganizmom. Spomedzi aktinomycét, sme sa v tejto štúdii zamerali na streptomycéty, pretože patria medzi významných producentov sekundárnych metabolitov, ktoré môžu potlačiť rast neželaných škodcov úrody. Výsledky ukázali, že najbohatším zdrojom kolónií streptomycét bola pôda hnojená kompostom (103 x104 KTJ*g-1 sušiny). Na základe zistených morfologických znakov sme na štúdium antimikrobiálnej aktivity vybrali 65 izolátov. Izoláty vykazovali najlepší účinok voči baktérii Clavibacter michiganensis subsp. sepedonicus, pôvodcu choroby “kružkovatosti” zamiakov a voči hube Fusarium poae, pôvodcu choroby fuzáriovej sneti pšenice. 12 izolátov vykazovalo širokospektrálne účinky voči testovaným organizmom, 6 z nich bolo vybraných na ďalšie analýzy. Porovnanie ich polyfázických znakov s dostupnou literatúrou viedlo k identifikácii produkčných druhov S. olivochromogenes (13SC11), S. avermitilis (13SC2), S. rishiriensis (13SC13), S. globisporus (13SC19), S. sampsonii (13SPC10) a S. avidinii (13SPC4). Tieto izoláty sa vyznačovali tiež vysokou produkciou alkalickej fosfatázy, leucínarylamidázy, valínarylamidázy, kyslej fosfatázy, naftol-AS-BI-fosfohydrolázy a glukozídazy (> 40 nmol) a boli pozitívne pri redukcii dusičnanov, hydrolýzy želatíny, produkcii ureázy a eskulínu. Všetky tieto izoláty môžu byť použité pri vývoji nových biopesticídov a biohnojív s antifungálnymi a antibakteriálnymi účinkami

Soil myxobacteria as a potential source of polyketide-peptide substances.

Myxobacteria, a group of antimicrobial producing bacteria, have been successfully cultured and characterized from ten soil samples collected from different parts of Slovakia. A total of 79 myxobacteria belonging to four genera (Myxococcus, Corallococcus, Sorangium, and Polyangium) were isolated based on aspects of their life cycle. Twenty-five of them were purified, fermented, and screened for antimicrobial activities against 11 test microorganisms. Results indicated that crude extracts showed more significant activities against Gram-positive than against Gram-negative bacteria or fungi. Based on a higher degree and broader range of antimicrobial production, the two most potential extracts (K9-5, V3-1) were selected for HPLC fractionation against Micrococcus luteus and Staphylococcus aureus and LC/MS analysis of potential antibiotic metabolites. The analysis resulted in the identification of polyketide-peptide antibiotics, namely corallopyronin A and B (K9-5) and myxalamid B and C (V3-1), which were responsible for important Gram-positive activity in the observed strains. A sequence similarity search through BLAST revealed that these strains showed the highest sequence similarity to Corallococcus coralloides (K9-5, NCBI accession number KX256198) and Myxococcus xanthus (V3-1, NCBI accession number KX256197). Although screening of myxobacteria is laborious, due to difficulties in isolating cultures, this research represented the first report covering the isolation and cultivation of this challenging bacterial group from Slovakian soils as well as the screening of their antimicrobial activity, cultural identification, and secondary metabolite identification