In laboratory assay, the diversity of bacteria and microscopic fungi and the community-level physiological profiling (CLPP) of microorganisms were observed after the addition of bio-sludge (40 t.ha-1) from a biogas station and addition of bio-fertlizers-AZOTER (10 dm-3 .ha-1) to the arable soil with PCR-DGGE and BIOLOG ® method (Eco Plates). The differences were recorded in the microbial diversity (bacteria and microscopic fungi) among variants according to the Shannon index. The differences in community of microscopic fungi were markedly higher among the soil samples with the additions of both bio-fertilizers compared to control soil samples. The occurrence of individual OTUs (operational taxonomic units) bacteria and microscopic fungi were different after 105 days of incubation from the status after the 1 st day of incubation. The community metabolic diversity (CMD) was influenced by the incubation time (105 days) as well, but not by application of bio-fertilizers. We observed a significant decrease (LSD test, P <0.05) in community metabolic diversity (CMD) and average metabolic response (AMR) of microorganisms in samples collected on the 105 th day of the experiment compared to samples collected on the 1 st day of the experiment in all tested samples.
MATERIAL AND METHODS Sample collection Actinomycetes were isolated from soil (Haplic Fluvisol) located near Nitra (Slovakia-latitude 48° 31' 35.97'' N, longitude 18° 11' 46.82'' E), one compost, one vermicompost and soil amended with compost or vermicompost. Vermicompost and compost were applied to the soil at a total dose of 20 Mg.ha −1. The applied compost was prepared by mixing straw (11% by dry weightdw), hay (3% by dw), silage (32% by dw), sheep manure (9% by dw), cow manure (34% by dw) and soil from the subsoil (11% by dw). Vermicompost was prepared by mixing the compost (41% by dw), cow manure (20% by dw), peat and forest litter (21% by dw), soil (10% by dw), a compound of grass, straw and hay (6% by dw) and the fill vermin (2% by dw). The fill vermin (older vermicompost) containing earthworms and cocoons of earthworms (70 earthworms dm −3) was added to the compost. The basic chemical and microbial characteristics of soil samples and both composts (Table 1) were determined according to the defined values: oxidizable carbon (Cox) by the Tiurin method, total nitrogen (Nt) by the Kjeldahl distillation method, pH (H2O) and microbial biomass carbon (Cmic), which was determined by the fumigation-extraction method of Vance et al. (1987). Plant diseases caused by pathogenic fungi are a severe problem for agriculture, and some organic fertilizers have shown a suppressive capacity due to the antagonistic action of microbial communities inhabiting those substrates. The purpose of this work was to isolate and identify actinomycetes from soil, compost and vermicompost able to antagonize phytopathogens. In total, out of 352 actinomycetes successfully recovered on Pochon medium and of which were selected representative 22 mycelium forming isolates, which were subjected to morphological and genotypic characterization. Genetic characterization based on 16S rDNA fragment sequencing revealed that, all the actinomycete isolates belong to the genus Streptomyces. The antifungal activity of isolates was tested against eight phytopathogenic fungi, and they were most activated against Rhizoctonia solani, Alternaria tenuissima, Aspergillus niger and Penicillium expansum. Isolates 51VK13 (Streptomyces sampsonii) and 12VK13 (Streptomyces flavovariabilis) isolated from soil amended with vermicompost showed the greatest antagonistic activity. Their bioactive potential was also confirmed by presence of genes for nonribosomal peptide synthetase (NRPS) and polyketide synthase types I and II (PKS-I and PKS-II). It can, therefore, be concluded that soil amended with organic fertilizers such as compost, and in particular vermicompost, provides a lower incidence of phytopathogens, especially fungi. Isolated actinomycetes have also shown high potential for the production of bioactive compounds useful in the control of plant diseases. ARTICLE INFO
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, β, 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 β-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.
The object of this study was to find and characterise streptomycete isolates with antimicrobial activity accomplished by the agar plug method against phytopathogenic species. The test-microorganisms were selected according to their importance in agriculture. All of them belong to phytopathogenic species which reduce yields of agriculturally important plants worldwide. A total number of four phytopathogenic bacteria (Xanthomonas campestris (CCM 22), Pseudomonas syringae (CCM 2868), Erwinia amylovora (CCM 1114), Clavibacter michiganensis subsp. sepedonicus (CCM 7014)) and four phytopathogenic fungi (Botrytis cinerea, Fusarium poae, Alternaria tenuissima, Alternaria arborescens) were used for this experiment. Overall twenty streptomycete isolates showed antimicrobial activity against at least two of the test-microorganisms. These active isolates were subsequently characterized. Streptomycete isolates were observed for morphological characteristics, such as morphology and colour of aerial and substrate mycelium, colour of diffusible pigments, production of melanoid pigments on peptone-yeast extract iron agar and sporophore and spore chain morphology following the International Streptomyces Project. Genes coding polyketide synthase (PKS-I) and nonribosomal peptide synthetase (NRPS), which are considered to responsible for the synthesis of large number of biologically active compounds, like antibiotics and antifungals, that are produced by Streptomyces species largely, were screened. The occurrence of these genes in the genome of our isolates was in accordance with results from antimicrobial activity analysis. Classification to genus Streptomyces were confirmed by DNA sequencing.
The aim of the study was to observe changes in the diversity of bacterial community in maize rhizosphere influenced by organic and mineral fertilization. Four variants of fertilization were tested -vermicompost (VC) at recommended annual dose 40t*ha -1 , doubled annual dose of VC, recommended dose of ammonium saltpeter with dolomite (LAD 27) and combination of VC and LAD 27. Experiment was conducted with potted maize plants in controlled conditions of greenhouse during 74 days. Using PCR-DGGE method, we investigated differences in total bacteria community as well as in community of ammonia oxidizing bacteria. Based on occurrence of operative taxonomic units (OTU) we found differences in bacterial species spectra among fertilization variants. The highest Shannon´s biodiversity index was observed in variant with VC addition in dose 80 t*ha -1 .The fertilizers effect on diversity of ammonia oxidizing bacteria was not significant however in each variant with vermicompost addition was the occurrence of new specific OTU observed. This OTU was identified as Nitrosospira sp. It was proven that some bacterial species introduced to soil with vermicompost addition can survive for at least 74 days and these bacteria can influence basic functions of soil microbiocenosis in nitrogen cycle. AbstraktCieľom štúdie bolo sledovať zmeny diverzity bakteriálneho spoločenstva v rizosfére kukurice vplyvom organického a minerálneho hnojenia. Boli testované štyri varianty hnojeniavermikompost (VC) v odporúčanej ročnej dávke 40t*ha -1 , dvojitá ročná dávka VC, odporúčaná dávka liadku amónneho s dolomitom (LAD 27) a kombinácia VC a LAD 27. Experiment bol realizovaný na nádobách s rastlinami kukurice v kontrolovaných podmienkach skleníka počas 74 dní. Využitím metódy PCR -DGGE sme skúmali rozdiely v celkovej komunite baktérií ako aj komunite nitrifikačných baktérií. Na základe prítomnosti operatívnych taxonomických jednotiek (OTU) sme zistili rozdiely v spektrách bakteriálnych druhov v závislosti od variantov hnojenia. Najvyšší Shannonov index biodiverzity bol zaznamenaný vo variante s prídavkom VC v dávke 80 t*ha -1 . Vplyv hnojenia na diverzitu nitrifikačných baktérií nebol preukazný, avšak v každom variante s prídavkom vermikompostu bol zaznamenaný výskyt nového špecifického OTU. Tento OTU bol identifikovaný ako Nitrosospira sp. Dokázalo sa, že niektoré druhy baktérií dodané do pôdy prídavkom vermikompostu sú schopné prežívať po dobu najmenej 74 dní a môžu ovplyvňovať základné funkcie pôdnej mikrobiocenózy v cykle dusíka.
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