Functional diversity investigation of bacterial communities in distinct soil types with RISA after preculturing (RISA-APC) method

Microorganisms play a leading role in soil development and preservation; moreover, they could indicate the soil health and conditions. In this study, we analyzed the bacterial composition of three different soil types with a newly developed RISA-APC method. A novelty of this method is the pre-culturing step: this preculturing of the bacterial communities were performed on solid media supplemented with different carbon sources (e.g.: carboxy-methyl cellulose, xylan, chitin, starch, tributyrin, casein and protocatechuic acid). For the investigation of heavy metal tolerant bacteria, a preculturing on YEG media containing CuSO4 or CdCl2 were used. The mini-colonies developed after a short incubation time was investigated. This RISA-APC method proved to be a useful tool for the comparison of different soil types, and for the examination of changes in the soil bacterial community structure. It was clearly shown that the most diverse functional diversity values occurred in the forest soil and the less diverse bacterial community was detected in sandy soil samples

Effect of pesticides on the growth and secreted chymotrypsin-like activity of a biocontrol strain of bacillus amyloliquefaciens

Biocontrol is an alternative approach to reduce the harmful effects of pathogen species in the agriculture. Pesticide-tolerant biocontrol agents are preferred in the integrated pest management because they can be applied together with different fungicides, herbicides and insecticides. A potent biocontrol agent, Bacillus amyloliquefaciens SZMC 22206 strain was isolated and studied previously. It was revealed, that the extracellular chymotrypsin-like protease and fengycin secretion of the strain resulted its antagonistic effect. The aim of our present study was to analyse the effect of different pesticides on the growth and activity of the extracellular chymotrypsin-like proteases of this Bacillus strain. The tested pesticides were a fungicide (carbendazim), and three frequently used herbicides (linuron, chlortoluron and 2,4-dichlorophenoxyacetic acid). The tested Bacillus strain was able to grow in the presence of the pesticides, but the activities of the extracellular chymotrypsin-like proteases were significantly reduced in some cases

Production of extracellular proteases by human pathogenic Trichoderma longibrachiatum strains

Species belonging to the filamentous fungal genus Trichoderma are well known as potential candidates for the biological control of plant pathogenic fungi and as cellulase producers of biotechnological importance. Several data were published in the last decade also about the clinical importance of this genus, indicating that Trichoderma strains may be potential opportunistic pathogens in immunocompromised patients. However, there is a lack of information about the potential virulence factors of clinical Trichoderma strains. This study was designed to examine the extracellular proteolytic enzymes of six clinical T. longibrachiatum isolates. Supernatants from induced liquid cultures of the examined strains were screened for proteolytic enzyme activities with 11 different chromogenic p-nitroaniline substrates. The production of trypsin-like, chymotrypsin-like and chymoelastase-like protease activities cleaving N-Benzoyl-L-Phe-L-Val-L-Arg-p-nitroanilide, N-Succinyl-L-Ala-L-Ala-L-Pro-L-Phe-p-nitroanilide,and N-Succinyl-L- Ala-L-Ala-L-Pro-L-Leu-p-nitroanilide, respectively, was common among the strains examined. Separation of trypsin- and chymotrypsin-like activities by column chromatography revealed, that both systems are complex consisting of several isoenzymes. The pH-dependence of these two protease systems was also studied. Based on the results, the different isoenzymes seem to have different optimal pH values. Extracellular proteolytic enzymes may be involved in the pathogenecity of Trichoderma strains as facultative human pathogens

Extracellular enzyme systems of antagonistic Bacillus strains isolated from tomato rhizosphere

Chitinolytic, proteolytic and lipolytic enzymes could be important in the biological control of soil borne plant-pathogenic microorganisms by antagonistic microbes and in this way besides antibiotic production, the secretion of certain extracellular enzymes also could have great importance in the effectiveness of biocontrol bacilli. Some of these enzymes could directly promote the inhibition processes, while others could help the competition of the bacilli against other rhizosphere microbes. In case of five Bacillus strains, which showed excellent antagonistic effects against phytopathogenic fungi and bacteria, the secretion of some components of the protease, chitinase, cellulase, lipase and β-1,3-glucanase enzyme systems were investigated under inductive and non-inductive circumstances. Enzyme activities were measured both with classical methods and with other experimental approaches based on chromogenic enzyme substrates. The best biocontrol strains constitutively secreted chymotrypsin-like proteases and/or trypsin-like proteases, and lipases. On the contrary, the chitinase components were only secreted in chitin containing media. Cellulases and β-1,3- glucanases were produced only at very low level either in inductive media

Methods for the investigation of cytotoxicity and genotoxicity of air samples

Air pollution means the presence of a mixture of solid particles and gases in the air. This significantly harms human health and the environment. Though in Europe, there is a substantial improvement in air quality in the last decades, the concentrations of different air pollutants are still too high. The Department of Microbiology started to take part in a project (TÁMOP-4.2.2.A11/1/KONV-2012-0047) in which a multidisciplinary research action targets practical aspects (e.g. detection) of air pollution. In the frame of this, cytotoxicity and genotoxicity investigations are in progress with filters derived from various air sampling sites. Experiments were started to work out methodologically solid sample processing protocol. Gentle sample pre-processing method applied, which ensure, that besides bulk of the toxic compounds get to solution from the filter, the samples become germ free. The filter extracts are processed in Eppendorf-tubes in a high frequency Eppendorf-tube shaker, with sterile glass beads. An important task is the removal of the heat and radiation resistant Bacillus spores which are present in substantial amount on the surface of the air filters. Instead of heat or radiation treatments – which other ways could cause undesired chemical reactions with the toxic compounds – the extracts are centrifuged through a nitrocellulose membrane (0.22 micron pore size) containing spin column. With the germfree extracts citotoxicity investigations are performed with Pseudomonas putida test strain in a microplate-based cultivation system. The growth of the Pseudomonas cultures is followed with turbidimetry with microtiter plate photometer. Genotoxicity investigations are carried out with the same extracts applied in Ames tests with the TA98 and TA 1535 Salmonella typhimurium tester strains