To explore the presence and expression of the nifH gene of diazotrophic endophytes in sugarcane (Saccharum spp. hybrids) cv. NiF8, we conducted pot experiments for two seasons in a glasshouse from 10 June to 17 September (high temperature and long days) in 2005 and 1 September to 9 December (low temperature and short days) in 2006. The expression of nifH genes in the stems and roots of sugarcane plants at 50 (or 59) and 100 days after transplanting was investigated by reverse transcription (RT)-PCR and by examining the nifH nucleotide sequence diversity. N 2 fixation was assessed by the 15 N-dilution method. The nifH RNA sequences in the stems and roots of the first experiment were similar to those of Bradyrhizobium sp. and Azorhizobium caulinodans. In the second experiment, nifH expression from these bacteria was not detected in the stems. nifH gene expression in the stems was in accordance with 18-24% N derived from air in the high-temperature season and negligible in the low-temperature season. Both the proliferation of N 2 fixers and expression of the nifH gene indicated that bacteria similar to rhizobia may contribute to N 2 fixation in sugarcane under our experimental conditions.

False-positive results due to DNA contamination in PCR reagents have become a big problem in the amplification of small amounts of DNA. Recently, it was revealed that PCR reagents were contaminated with the nifH (dinitrogenase reductase) gene. We found that the PCR primers supplied by some manufacturers contained nifH gene and nifH-like DNA. This contamination resulted in false-positive results when searching for nifH genes in environmental samples. The sequences of the contaminating DNA appeared to be widely varied in the phylogenetic analysis of nifH. For this reason, great care should be taken when analyzing trace amounts of nucleotides.