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Bacillus amyloliquefaciens Q-426 with strong antifungal activity was isolated from the compost samples in Dalian of China. Four kinds of diketopiperazines were extracted from the strain including cyclo(Pro-Phe) (cPP), which had cyclo(L-Pro-L-Phe), cyclo(D-Phe-L-Pro), cyclo(D-Pro-D-Phe) and cyclo(L-Pro-D-Phe) isomers. Results showed that (1) cPP production reached the maximum at 12 h and then maintained the constant yield; (2) cPP maintained a stable production in a wide temperature range of 31–42°C; (3) cPP production were quite different for various carbon sources because of the different growth rate of the strain; (4) cPP maintained a higher level yield in neutral and weakly alkaline environment (pH 6–10) than acid (pH 3–5) environment. Moreover, cPP played a negative effect on the synthesis of antifungal substance when the bacteria maintained a good growth, and the previous studies found that cPP showed a positive response to biosensors which were used to detect signal molecules. Based on these studies, diketopiperazines were suspected as the signal molecule of B. amyloliquefaciens Q-426. [ABSTRACT FROM AUTHOR]