Bacterial community investigations with RISA-after preculturing (RISA-APC) method for estimating the functional diversity in different soil types

Microorganisms play a key role in soil development and preservation; moreover, they could indicate the soil health and conditions. The relatively fast and dynamic changes in the bacterial communities could be good indicators for the soil quality and also show the influence of the different environmental factors. There are different works, investigating the connection between the bacterial community structure changes and soil quality. In this study, we provide a possible solution for getting a broad a picture about the functional structure of bacterial communities in the soil. We named it RISA-APC method (ribosomal RNA (rRNA) intergenic spacer analysis, after preculturing). The essence of the technique: preculturing the microbes of the soil samples in distinct media and thereafter performs a molecular diversity analysis of the developed microbial communities. The region of the rRNA gene cluster between the small (16S) and large (23S) subunits in bacteria is called the internal transcribed spacer region (ITS). The ITS length polymorphism could be visualized with gelelectrophoresis after applying PCR with suitable RISA primers, and the resulted mixture of fragments is characteristic, such as a barcode and indicates the composition and diversity of the investigated bacterial community. The RISA analysis of DNA samples, extracted from mini-colonies appearing after preculturing of aliquots of the soil samples on solid media, solidified with agarose, supplemented with different carbon sources, could supply us information about the functional diversity of the bacterial and fungal communities. In our presented investigations we analyzed three soil types deriving from wheat field, forest and sandy soils with RISA-APC. The carbon sources were: carboxy-methyl cellulose, xylane, chitin, starch, tributyrine, casein and protocatechuic acid. For the investigation of the heavy metal tolerant microorganisms, we used CuSO4 or CdCl2 supplemented YEG media. Our RISA-APC method clearly correlated, as regards the complexity of RISA-fingerprints, with the expected basic taxonomical complexity of the soil types and with the carbon search used for preculturing. The RISA=APC method developed in our study 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, despite the fact that this approach does not provide bases for a precise species or genus identification. Furthermore, this method could be combined with various statistical methods to analyze these correlations in detail.