Methodological Aspects of Multiplex Terminal Restriction Fragment Length Polymorphism-Technique to Describe the Genetic Diversity of Soil Bacteria, Archaea and Fungi.

The molecular fingerprinting methods used to evaluate soil microbial diversity could also be used as effective biosensors for the purposes of monitoring ecological soil status. The biodiversity of microorganisms is a relevant index of soil activity and there is a necessity to develop tools to generate reliable results for an emerging approach in the field of environmental control using microbial diversity biosensors. This work reports a method under development for determining soil microbial diversity using high efficiency Multiplex PCR-Terminal Restriction Fragment Length Polymorphism (M-T-RFLP) for the simultaneous detection of bacteria, archaea and fungi. Three different primer sets were used in the reaction and the analytical conditions were optimized. Optimal analytical conditions were achieved using 0.5 µM of primer for bacteria and 1 µM for archaea and fungi, 4 ng of soil DNA template, and HaeIII restriction enzyme. Comparative tests using the proposed analytical approach and a single analysis of each microorganism group were carried out to indicate that both genetic profiles were similar. The Jaccard similarity coefficient between single and multiplexing approach ranged from 0.773 to 0.850 for bacteria and fungi, and 0.208 to 0.905 for archaea. In conclusion, the multiplexing and pooling approaches significantly reduced the costs and time required to perform the analyses, while maintaining a proper effectiveness.

The Status of Soil Microbiome as Affected by the Application of Phosphorus Biofertilizer: Fertilizer Enriched with Beneficial Bacterial Strains.

Regarding the unfavourable changes in agroecosystems resulting from the excessive application of mineral fertilizers, biopreparations containing live microorganisms are gaining increasing attention. We assumed that the application of phosphorus mineral fertilizer enriched with strains of beneficial microorganisms contribute to favourable changes in enzymatic activity and in the genetic and functional diversity of microbial populations inhabiting degraded soils. Therefore, in field experiments conditions, the effects of phosphorus fertilizer enriched with bacterial strains on the status of soil microbiome in two chemically degraded soil types (Brunic Arenosol - BA and Abruptic Luvisol - AL) were investigated. The field experiments included treatments with an optimal dose of phosphorus fertilizer (without microorganisms - FC), optimal dose of phosphorus fertilizer enriched with microorganisms including Paenibacillus polymyxa strain CHT114AB, Bacillus amyloliquefaciens strain AF75BB and Bacillus sp. strain CZP4/4 (FA100) and a dose of phosphorus fertilizer reduced by 40% and enriched with the above-mentioned bacteria (FA60). The analyzes performed included: the determination of the activity of the soil enzymes (protease, urease, acid phosphomonoesterase, ß-glucosidase), the assessment of the functional diversity of microorganisms with the application of BIOLOGTM plates and the characterization of the genetic diversity of bacteria, archaea and fungi with multiplex terminal restriction fragment length polymorphism and next generation sequencing. The obtained results indicated that the application of phosphorus fertilizer enriched with microorganisms improved enzymatic activity, and the genetic and functional diversity of the soil microbial communities, however these effects were dependent on the soil type.

The influence of ecological and conventional plant production systems on soil microbial quality under hops (Humulus lupulus).

The knowledge about microorganisms-activity and diversity under hop production is still limited. We assumed that, different systems of hop production (within the same soil and climatic conditions) significantly influence on the composition of soil microbial populations and its functional activity (metabolic potential). Therefore, we compared a set of soil microbial properties in the field experiment of two hop production systems (a) ecological based on the use of probiotic preparations and organic fertilization (b) conventional-with the use of chemical pesticides and mineral fertilizers. Soil analyses included following microbial properties: The total number microorganisms, a bunch of soil enzyme activities, the catabolic potential was also assessed following Biolog EcoPlates®. Moreover, the abundance of ammonia-oxidizing archaea (AOA) was characterized by terminal restriction fragment length polymorphism analysis (T-RFLP) of PCR ammonia monooxygenase α-subunit (amoA) gene products. Conventional and ecological systems of hop production were able to affect soil microbial state in different seasonal manner. Favorable effect on soil microbial activity met under ecological, was more probably due to livestock-based manure and fermented plant extracts application. No negative influence on conventional hopyard soil was revealed. Both type of production fulfilled fertilizing demands. Under ecological production it was due to livestock-based manure fertilizers and fermented plant extracts application.

The Use of Interactions Between Microorganisms in Strawberry Cultivation (Fragaria x ananassa Duch.).

As the market indicates a growing interest in organically grown fruit, there is a need for biostimulants to counter the adverse effects of pathogenic fungi and fungal-like-pathogens. Four microbial pathogens (Botrytis cinerea, Verticillium sp., Phytophthora sp., and Colletotrichum sp.) which are the most often causes of strawberry diseases were selected. Five kinds of biostimulants (C1, C2, C3, C4, and C5) containing bacterial consortia were developed to combat the pathogens. The antagonistic effect of selected microorganisms against strawberry pathogens was observed. The effectiveness of various beneficial bacteria in combating fungal pathogens of cv. Honeoye strawberries was compared and the impact of their activity on fruit quality was assessed. The most significant effect on the strawberry firmness was found for the C2 consortium, which provided the strawberries infected with the pathogens group (MIX: B. cinerea, Verticillium sp., Phytophthora sp., and Colletotrichum sp.) with a 140% increase in maximum load in a puncture test compared to the positive control (C0). Strawberries contaminated with Phytophthora sp. after the application of Consortium C4 (C4) showed the largest increase (127%) in soluble solid content (SSC) when compared to the C0. Fruit contaminated with Colletotrichum sp. and B. cinerea after the application of C2 and Consortium 5 (C5), respectively, had the highest levels of anthocyanins and total phenolic content, when compared to C0. The largest increase, which reached as high as 25%, in D-galacturonic acid content was observed for the group of pathogens after Consortium 1 (C1) application. The extraction of strawberry pectin allowed for the study of the rheological properties of pectin solutions; on this basis, strawberry pectin from the control (NC) was distinguished as it showed the highest viscosity (0.137-0.415 Pas). Taking into account the individual effects of bacteria on strawberry pathogenic fungi and fungal-like-pathogens, it is possible to reduce the adverse effects of fungal disease and to improve the properties of strawberries by selecting the appropriate bacterial consortium. Interactions between microorganisms are often complex and not fully understood, which suggests the need for further research in this direction.

Immunocytochemical studies on the distribution of arabinogalactan proteins (AGPs) as a response to fungal infection in Malus x domestica fruit.

Arabinogalactan proteins (AGPs) are cell components implicated in plant-microbe interactions. Despite the significance of AGPs in response to stress factors, their distribution during development of fungal disease in fruit is unknown. In our work, in situ analysis of AGP arrangement in fruit inoculated with Penicillium spinulosum during the consecutive days of infection development was carried out. For immunolocalization of AGPs, samples were incubated with JIM13, MAC207, LM2, and LM14 antibodies recognizing the AGP carbohydrate moieties. To analyse cell walls without proper action of AGP, an experiment with ß-glucosyl Yariv reagent specifically binding AGPs was performed. The results showed an increase of signal fluorescence in the fruit after 16 days of fungal disease. Higher amounts of the examined epitopes were observed in the infection-altered sites of the fruit, in close vicinity to a surface filled by fungal spores. The results indicate that the Yariv reagent treatment induced progress of the fungal disease. Changes in the AGP presence during the fungal disease confirmed their involvement in defence against pathogen attack in fruit.

A method for early detection and identification of fungal contamination of building materials using e-nose.

The aim of the study was to develop a method for early detection and identification of fungal contamination of building materials using an electronic nose. Therefore, the laboratory experiments based on the analysis of the air in the vicinity of fungal isolates potentially found in the building materials were performed. The results revealed that the employed gas sensors array consisting of MOS-type sensors enables the detection of the differences among the examined samples of fungi and distinguishing between the non-contaminated and contaminated samples, shortly after fungal contamination occurs. Electronic nose readouts were analysed using Principal Component Analysis and the results were verified with standard chromatographic analysis by means of SPME-GC/MS method, which proved that gas sensors array can be applied for early detection of fungal contamination.

Fast and Accurate Microplate Method (Biolog MT2) for Detection of Fusarium Fungicides Resistance/Sensitivity.

The need for finding fungicides against Fusarium is a key step in the chemical plant protection and using appropriate chemical agents. Existing, conventional methods of evaluation of Fusarium isolates resistance to fungicides are costly, time-consuming and potentially environmentally harmful due to usage of high amounts of potentially toxic chemicals. Therefore, the development of fast, accurate and effective detection methods for Fusarium resistance to fungicides is urgently required. MT2 microplates (Biolog(TM)) method is traditionally used for bacteria identification and the evaluation of their ability to utilize different carbon substrates. However, to the best of our knowledge, there is no reports concerning the use of this technical tool to determine fungicides resistance of the Fusarium isolates. For this reason, the objectives of this study are to develop a fast method for Fusarium resistance to fungicides detection and to validate the effectiveness approach between both traditional hole-plate and MT2 microplates assays. In presented study MT2 microplate-based assay was evaluated for potential use as an alternative resistance detection method. This was carried out using three commercially available fungicides, containing following active substances: triazoles (tebuconazole), benzimidazoles (carbendazim) and strobilurins (azoxystrobin), in six concentrations (0, 0.0005, 0.005, 0.05, 0.1, 0.2%), for nine selected Fusarium isolates. In this study, the particular concentrations of each fungicides was loaded into MT2 microplate wells. The wells were inoculated with the Fusarium mycelium suspended in PM4-IF inoculating fluid. Before inoculation the suspension was standardized for each isolates into 75% of transmittance. Traditional hole-plate method was used as a control assay. The fungicides concentrations in control method were the following: 0, 0.0005, 0.005, 0.05, 0.5, 1, 2, 5, 10, 25, and 50%. Strong relationships between MT2 microplate and traditional hole-plate methods were observed regarding to the detection of Fusarium resistance to various fungicides and their concentrations. The tebuconazole was most potent, providing increased efficiency in the growth inhibition of all tested isolates. Almost all among tested isolates were resistant to azoxystrobin-based fungicide. Overall, the MT2 microplates method was effective and timesaving, alternative method for determining Fusarium resistance/sensitivity to fungicides, compering to traditional hole-plate approach.

The application of the Biolog EcoPlate approach in ecotoxicological evaluation of dairy sewage sludge.

An increasing amount of sewage sludge requires reasonable management, whereas its storage might be environmentally hazardous. Due to the organic matter and nutrient presence in sediments, it may be used as organic fertilizer. However, beyond the valuable contests, sewage sludge can also contain toxic or dangerous ingredients like heavy metals. Therefore, there is a need to develop methods for rapid assessment of sediment ecotoxicity that will determine its possible applicability in agriculture. The Biolog® EcoPlate enables the metabolic profile diversity evaluation of microbial populations in environmental samples, which reflects the state of their activity. It is regarded as a modern technology that by means of biological properties allows quick characterization of the ecological status of environmental samples, such as sewage sludge.