|1362-1||Technical optimization of SIP (Stable Isotope Probing) for Description of Microbial Community Associated with the Rhizosphere of Sugarcane|
|Autores:||Diogo Paes da Costa (ESALQ-USP - Escola Superior de Agricultura) ; Ademir Durrer Bigaton (ESALQ-USP - Escola Superior de Agricultura) ; Thiago Gumieri (ESALQ-USP - Escola Superior de Agricultura) ; Fernando Dini Andreote (ESALQ-USP - Escola Superior de Agricultura) |
The SIP (Stable Isotope Probing) methodology is based on the incorporation of substrates enriched with stable isotopes that serve as cell biomarkers for characterization of phylogenetic and metabolic aspects of indigenous microbial groups in diverse environments. In this way, the SIP allows the characterization of functional genes related to metabolism of carbon by microorganisms associated with rhizosphere systems. So, if you can draw a parallel correlation between carbon cycling and microbial diversity in soil-plant-atmosphere system, as well as his progressive distribution in trophic levels through microbial feedback. Observing this system, the aim of this study was to perform a technical optimization of SIP to characterize future microbial communities associated with the rhizosphere of sugarcane. The experiment was conducted with the variety RB86-7515 cultivated in pots, under closed systems with chromatographic control of levels of CO2 (390 and 700 ppm), kept in a greenhouse acclimatized. After 3 months of cultivation was carried out enrichment of systems with 1000 mg of 13CO2, keeping the negative controls. The samplings were made after 2, 4 and 8 days. Rhizospheric soil DNA was extracted and subjected to ultracentrifugation methodology in the cesium trifluoroacetate solution (CsTFA) at a speed of 64,000 rpm, adding 500 ng of DNA in each centrifuge tube (2.2 mL). The DNA was recovered through the Beckman system of fractionation, determining the densities and their refractive indices. SIP methodology has provided 25 fractions per sample, separating the 12C-DNA and 13C-DNA in the ranges of density between respectively 1.5-1.6 g/mL and 1.63-1.65 g/mL, respectively. These values vary depending on the content of G+C present in the genomes, reflecting the specificity of certain groups of microorganisms with the environment and with the available carbon sources. In this case, the microorganisms have incorporated 13C from the root exudates, being possible the detection of 13C-DNA in fractions obtained from plants sampled to 8 days after the beginning of enrichment for both atmospheric concentrations. The results indicate that the technique of SIP is a tool able to characterize potential microbial groups associated with the rhizosphere of sugarcane and can correlate functional genes with the process of cycling of C and changes of atmospheric levels of CO2.
Palavras-chave: Stable Isotope Probing, Sugarcane, Microbial feedback, Levels of CO2, Microbial diversity