The pitaya canker disease is a serious fungal disease caused by Neoscytalidium dimidiatum. In recent years, the outbreak of the pitaya canker disease in the world's pitaya producing areas has seriously affected the pitaya industry development. In order to study the expression level and function of genes responding to stress in N. dimidiatum, it is necessary to screen the reference genes that express stably when the pathogen grows under different cultural conditions. The N. dimidiatum strain LJ02 mycelia cultured in potato dextrose liquid medium（PDW）were taken as the control group, and the mycelia cultured in LB medium, and the mycelia cultured in PDW medium supplemented with hydrogen peroxide, pyrazolyl ester and difenoconazole, respectively, were taken as the treatment groups. The expression stabilities of 18 candidate reference genes（CYT1, SDH2_1, TBCC, SUI1, RPL19, PPH, ATP5B, UBE2_16, RPL13, UBE2_2, PRS17, SUCLA2, ATP5A, TUB1_2, ACT1, EFTU, EF1A and GAPDH）were evaluated by real-time quantitative PCR（RT-qPCR）and stability evaluation software（geNorm, NormFinder, Bestkeeper and RefFinder）. Based on the results of RT-qPCR and software analysis, it was found that the Ct values of 18 candidate genes were within 14.80-24.66 under different culture conditions, with moderate expression levels. The reference gene with the highest stability was CYT1, followed by SUI1, while GAPDH and ACT1 were with the worst expression stability. Using at least two reference genes may improve analysis accuracy of RT-qPCR. At this time, SUCLA2 and ATP5A is the most stable reference gene combination. The results provide a theoretical basis for gene expression analysis in the biological process of N. dimidiatum.
