Evaluation of optimal reference genes for the normalization by qPCR in viable but nonculturable state in Xanthomonas campestris pv. campestris

Abstract

AbstractXanthomonas campestris pv. campestris (Xcc), the pathogen of black rot of crucifers, could be induced into the viable but nonculturable (VBNC) state under stress, such as being induced by low concentration of Cu2+ and acid condition induction. The main objective of current study was to select the optimal reference genes for data normalization by qPCR in the VBNC state of Xcc. In this study, Xcc was induced into VBNC state by Cu2+ with an initial concentration of OD600 = 0.18 and OD600 = 0.45, and all viable bacterial cells lost the culturability and entered into the VBNC state at 24 h and 48 h respectively. The concentration of VBNC cells was 3.3 × 107 cells/ml and 1.5 × 107 cells/ml respectively. Eight candidate reference genes (gyrB, pbpA, gapA, rpoB, tufA, 16S rRNA, ugpC and recA) were selected to assess the expression stability in the VBNC state of Xcc by four algorithms (geNorm, NormFinder, delta‐Ct and BestKeeper), while RefFinder was used to integrate the output from the four algorithms in fine. According to the algorithm analysis and validation of clpX expression analysis, the most suitable reference gene this study identified was the combination of ugpC and pbpA, and the least suitable gene was 16S rRNA in the Xcc VBNC state. In this study, we identified the most stable internal controls under Cu2+‐inducing conditions for calibration qRT‐PCR analyses of Xanthomonas campestris pv. campestris and will be helpful to explore the stress resistance mechanism in this bacterium.