Changes in soil microbial community and activity caused by application of dimethachlor and linuron.

Soil microorganisms and their activities are essential for maintaining soil health and fertility. Microorganisms can be negatively affected by application of herbicides. Although effects of herbicides on microorganisms are widely studied, there is a lack of information for chloroacetamide herbicide dimethachlor. Thus, dimethachlor and well known linuron were applied to silty-loam luvisol and their effects on microorganisms were evaluated during112 days long laboratory assay. Dimethachlor and linuron were applied in doses 1.0 kg ha-1 and 0.8 kg ha-1 corresponding to 3.33 mg kg-1 and 2.66 mg kg-1 respectively. Also 100-fold doses were used for magnification of impacts. Linuron in 100-fold dose caused minor increase of respiration, temporal increase of soil microbial biomass, decrease of soil dehydrogenase activity, and altered microbial community. Dimethachlor in 100-fold dose significantly increased respiration; microbial biomass and decreased soil enzymatic activities. Microbial composition changed significantly, Proteobacteria abundance, particularly Pseudomonas and Achromobacter genera increased from 7 to 28th day. In-silico prediction of microbial gene expression by PICRUSt2 software revealed increased expression of genes related to xenobiotic degradation pathways. Evaluated characteristics of microbial community and activity were not affected by herbicides in recommended doses and the responsible use of both herbicides will not harm soil microbial community.

Influence of Nitrogen Nutrition on Fatty Acids in Oilseed Rape (Brassica napus L.).

The aim of the study was to evaluate the effect of nitrogen nutrition on the content of fatty acids and selected qualitative parameters (nitrogenous substances, ash, crude fiber) in winter oilseed rape (Brassica napus L.). The experiment was carried out at the Víglas-Pstrusa Research and Breeding Station in 2008-2009 and 2009-2010 by complete block design with four repetitions. Nitrogen fertilization was applied at four levels, plus an untreated control (after agrochemical soil analysis) by DAN 27 (Dolomite Ammonium Nitrate): 100, 120, 140, and 160 kg/ha N. Application date was in BBCH scale phase 59-60. The fatty acid contents (MUFA-monosaturated fatty acids; PUFA-polyunsaturated fatty acids) were determined by gas chromatography in the extracted fat, which is determined by extraction method. Within the result evaluation, statistically significant increases in the contents of linoleic and linolenic acids were recorded in all variants treated by nitrogen fertilizer, which is positive in terms of the use of rapeseed oil for food and energy purposes. The statistically significant decrease of oleic acid after the application of nitrogen fertilizers is negative for industry use of rapeseed oil.

Effects of sulfonylurea herbicides chlorsulfuron and sulfosulfuron on enzymatic activities and microbial communities in two agricultural soils.

Sulfonylurea herbicides are widely used for weed control in agriculture, and they are suspected to alter microbial communities and activities in the soil. This study investigates the impact of two sulfonylurea herbicides chlorsulfuron and sulfosulfuron on microbial community and activity in two different soils taken from two sites in west part of the Slovak Republic. The soil from the Malanta site was silt-loam luvisol with pH(H2O) 5.78 while the soil from the Stefanov site was sandy-loam regosol with pH(H2O) 8.25. These soils were not treated by sulfonylurea herbicides at least for 2 years prior to the study. In laboratory assay, the herbicides were applied to soil in their maximal recommended doses 26 and 25 g per hectare of chlorsulfuron and sulfosulfuron, respectively. Their effect was evaluated on the 3rd, 7th, 14th, 28th, 56th, and 112th day after application to soil. Illumina high-throughput amplicon sequencing of the 16S rRNA gene and ITS region was used to monitor changes on prokaryotic and fungal community composition. Enzymatic activity was evaluated using 11 substrates. Physiological profile of microbial community was analyzed using Biolog© ecoplates. Significant changes in enzymatic activity caused by the application of herbicides were found during the first 28 days. The application of herbicides altered the activity of cellobiohydrolase, arylsulphatase, dehydrogenase, phosphatase, and FDA hydrolase. Chlorsulfuron caused a more varying response of enzymatic activity than sulfosulfuron, and observed changes were not the same for both soils. In Malanta soil, chlorsulfuron decreased dehydrogenase activity while it was increased in the Stefanov soil. Phosphatase activity was decreased in both soils on 7th and 14th day. There were only minor changes in prokaryotic or fungal community or physiological profiles regarding pesticide application. Differences between soils and incubation time explained most of the variability in these parameters. Diversity indices, physiological parameters, and enzymatic activity decreased over time. The results have shown that chlorsulfuron and sulfosulfuron can affect the function and activity of the soil microbial community without significant change in its composition.

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.

Rapid identification of Streptomyces tetracycline producers by MALDI-TOF mass spectrometry.

The main objective of this study was using matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) for assembling of DSM (German Collection of Microorganisms) Streptomyces spectral database and identification of wild Streptomyces cultures, which were clustered by MALDI-TOF Biotyper OC software as well as for teracycline detection by observing of obtained spectra using flexAnalysis software. Production of tetracycline was confirmed by thin-layer chromatography. Presence of tetracycline mass spectrum was verified by several tetracycline producers (Streptomyces aureofaciens LMG 5968, S. aureofaciens 84/25, and S. aureofaciens BMK) and by pure tetracycline mass. Our results showed that it is possible to use MALDI-TOF MS for identification of tetracycline producers within Streptomyces genera by several easy steps. The purpose of this study was to establish cheap and quick detection of tetracycline producers.

Streptomyces globosus DK15 and Streptomyces ederensis ST13 as new producers of factumycin and tetrangomycin antibiotics.

Fifty seven soil-borne actinomycete strains were assessed for the antibiotic production. Two of the most active isolates, designed as Streptomyces ST-13 and DK-15 exhibited a broad range of antimicrobial activity and therefore they were selected for HPLC fractionation against the most suppressed bacteria Staphylococcus aureus (ST-13) and Chromobacterium violaceum (DK-15). LC/MS analysis of extracts showed the presence of polyketides factumycin (DK15) and tetrangomycin (ST13). The taxonomic position of the antibiotic-producing actinomycetes was determined using a polyphasic approach. Phenotypic characterization and 16S rRNA gene sequence analysis of the isolates matched those described for members of the genus Streptomyces. DK-15 strain exhibited the highest 16S rRNA gene sequence similarity to Streptomyces globosus DSM-40815 (T) and Streptomyces toxytricini DSM-40178 (T) and ST-13 strain to Streptomyces ederensis DSM-40741 (T) and Streptomyces phaeochromogenes DSM-40073 (T). For the proper identification, MALDI-TOF/MS profile of whole-cell proteins led to the identification of S. globosus DK-15 (accession number: KX527570) and S. ederensis ST13 (accession number: KX527568). To our knowledge, there is no report about the production of these antibiotics by S.globosus and S. ederensis, thus isolates DK15 and ST13 identified as S. globosus DK-15 and S.ederensis ST-13 can be considered as new sources of these unique antibacterial metabolites.

Antimicrobial and enzymatic activity of actinomycetes isolated from soils of coastal islands.

Microbiological investigation of unexplored ecosystems is crucial for discovering of antibiotic producing actinomycetes. The present study was conducted to determine antimicrobial activity and identify the most active strains. Actinomycetes were isolated using the spread plate technique following by serial dilution of samples on starch casein agar. The screening method consists of primary and secondary testing. The most active isolates were identified based on molecular and cultural methods. 42 out of 66 isolates displayed antimicrobial potential. 63% exhibited antibacterial activity, 16% antifungal activity, and 16% displayed both activities. Identified isolates, Streptomyces scabrisporus, Streptomyces sparsogenes, Streptomyces misakiensis, Streptomyces cirratus, Streptomyces lincolnensis, Streptomyces endophyticus, Streptomyces chartreusis, and Streptomyces alboniger showed a broad spectrum of enzymatic activities. The results indicated that these isolates may serve as antibiotic and enzyme-producing microbes.

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.

Characterization of Antimycins - Producing Streptomycete Strain VY46 Isolated from Slovak Soil

ABSTRACT The strain no. VY46 was isolated from agricultural soil of Slovak republic and tested for potential antimicrobial activity against various human pathogens. On the basis of results, strain VY46 significantly inhibited growth of yeast Candida albicans and therefore was used for further characterization. In order to explore the potential bioactivities, extract of the fermented broth culture was prepared with organic solvent extraction method. The ethylacetate extract was subjected to HPLC fractionation against Candida albicans and followed by LC/MS analysis for potential production of anticandidal substances. The analysis resulted in the identification of two antimycins antibiotics, which may be responsible for important anticandidal activity of the strain. On the basis of liquid chromatography and mass spectrometry the antibiotics were identified as Urauchimycin A and Kitamycin A. According tothe results from cultural, morphological, physiological, biochemical and 16S rRNA gene sequence methods, the strain was identified as Streptomyces albidoflavus. In addition, neighbor-joining phylogenetic tree confirmed the relationships of this strain to other members of Streptomyces genera.

Effect of Dursban 480 EC (chlorpyrifos) and Talstar 10 EC (bifenthrin) on the physiological and genetic diversity of microorganisms in soil.

This investigation was undertaken to determine the impact of the insecticides Dursban 480 EC (with organophosphate compound chlorpyrifos as the active ingredient) and Talstar 10 EC (with pyrethroid bifenthrin as the active ingredient) on the respiration activity and microbial diversity in a sandy loam luvisol soil. The insecticides were applied in two doses: the maximum recommended dose for field application (15 mg kg(-1) for Dursban 480 EC and 6 mg kg(-1) for Talstar 10 EC) and a 100-fold higher dose for extrapolation of their effect. Bacterial and fungal genetic diversity was analysed in soil samples using PCR DGGE and the functional diversity (catabolic potential) was studied using BIOLOG EcoPlates at 1, 3, 7, 14, 28, 56 and 112 days after insecticide application. Five bacterial groups (α, ß, γ proteobacteria, firmibacteria and actinomycetes) and five groups of fungi or fungus-like microorganisms (Ascomycota, Basidiomycota, Chytridiomycota, Oomycota and Zygomycota) were analysed using specific primer sets. This approach provides high resolution of the analysis covering majority of microorganisms in the soil. Only the high-dose Dursban 480 EC significantly changed the community of microorganisms. We observed its negative effect on α- and γ-proteobacteria, as the number of OTUs (operational taxonomic units) decreased until the end of incubation. In the ß-proteobacteria group, initial increase of OTUs was followed by strong decrease. Diversity in the firmibacteria, actinomycetes and Zygomycota groups was minimally disturbed by the insecticide application. Dursban 480 EC, however, both positively and negatively affected certain species. Among negatively affected species Sphingomonas, Flavobacterium or Penicillium were detected, but Achromobacter, Luteibacter or Aspergillus were supported by applied insecticide. The analysis of BIOLOG plates using AWCD values indicated a significant increase in metabolic potential of microorganisms in the soil after the high-dose Dursban application. Analysis of respiration demonstrated high microbial activity after insecticide treatments; thus, microbial degradation was relatively fast. The half-life of the active insecticide compounds were estimated within the range of 25 to 27 days for Talstar and 6 to 11 days for Dursban and higher doses stimulated degradation. The recommended dose levels of both insecticides can be considered as safe for microbial community in the soil.

Keratinophilic fungi isolated from soils of long-term fold-grazed, degraded pastures in national parks of Slovakia.

A total of 939 isolates of 11 genera representing 15 species of keratinophilic fungi were isolated and identified from the soils of three long-term fold-grazed pastures in national parks of Slovakia (Pod Ploskou, Strungový príslop, and Pod Keckou) and one non-fold-grazed pasture in sierra Stolicke vrchy (Diel) using the hair-baiting technique. Keratinophilic fungi were present in all soil samples with a prevalence of Trichophyton ajelloi and Paecilomyces lilacinus. These fungi were more abundant in soil from fold-grazed pasture (Strungový príslop) compared to non-fold-grazed pasture (Diel). The occurrence of the other keratinophilic fungi was substantially lower, likely because of low pH in some soils.

Influence of benomyl and prometryn on the soil microbial activities and community structures in pasture grasslands of Slovakia.

The effects of pesticides (a herbicide and a fungicide) on the microbial community structure and their activity were analyzed in soil from four alpine pasture grasslands in Slovakia. Specifically, the effects of the herbicide, Gesagard (prometryn active ingredient), and fungicide, Fundazol 50 WP (benomyl active ingredient), on the microbial respiration activity (CO2 production), the numbers of selective microbial physiological groups (CFU.g(-1)) and the structure (relative abundance) of soil microbial communities [(phospholipid fatty acid (PLFA)] were analyzed under controlled laboratory conditions. All treatments including the treatments with pesticides increased (statistically significantly) the production of CO2 in all fields during 21 days of incubation and posed a statistically insignificant negative influence on the numbers of the observed physiological groups of microorganisms. The significantly negative influence was evaluated only in the numbers of two physiological groups; spores of bacteria utilizing organic nitrogen and bacteria, and their spores utilizing inorganic nitrogen. A shift in the microbial composition was evident when the PLFA patterns of samples from different sites and treatments were compared by the Principal Component Analysis (PCA). According to the second component PCA 2 (15.95 %) the locations were grouped into two clusters. The first one involved the Donovaly and Dubakovo sites and the second one contained the Velka Fatra and Mala Fatra locations. The PLFA composition of the soils showed important changes after the treatment with pesticides according to PCA 1 (66.06 %). Other treatments had not had a significant effect on the soil microbial community with the exception of the population of fungi. The lower relative abundance (significant effect) of Gram-positive bacteria, actinomycetes and general group of bacteria were determined in samples treated by the herbicide Gesagard. The application of fungicide Fundazol decreased (statistically significantly) the relative abundance of actinomycetes and general group of bacteria and paradoxically increased the population of fungi.