PCR Primers Targeting Fungal P450nor Gene for Characterization of N2o Producing Fungi.

We designed PCR primers targeting fungal P450nor with an aim of using them as tools to survey, identify and characterize N₂O producing fungi in different ecosystems. Preliminary priming tests results show that a total of four primers with amplicon size 149 – 411 bp designed specifically for Fusarium oxysporum produced amplicons of expected size in five isolates of F. oxysporum and four isolates of Gibberella fujikuroi species complex. The DNA sequences of all isolates that positively amplified with these primer pairs matched with F. oxysporum cyp55A1 gene for cytochrome P450nor accession D14517.1 in the NCBI database at levels between 75 and 100%. Additionally, phylogenetic analysis with Maximum Composite Likelihood indicated formation of one cluster with a bootstrap value of at least 83. Similarly, specific primers designed for Aspergillus oryzae with amplicon size 160 – 335 bp produced positive results for A. oryzae, A. terreus and A. sydowii; but no amplification in other species of Aspergillus (A. fumigatus, A. versicolor and Aspergillus spp.) with N₂O-producing activity. Although the A. oryzae primer pairs were designed with the A. oryzae gene (CYP55A5) sequence, the sequence for A. sydowii matched with A. terreus NIH2624 cytochrome P450 55A3 at 97% similarity level. Thus, indicating that these primers could be optimized for other species of Aspergillus. These specific primers for F. oxysporum and A. oryzae acted specifically on F. oxysporum and A. oryzae, respectively, when used on soil DNA, thus confirming their specificity and potential use as markers for identification of N₂O producing Fusarium and Aspergillus. Additionally, they showed that soil systems harbor N₂O producing Fusarium and Aspergillus, and will ultimately be used to determine community composition, diversity and abundance of these genera in different soil systems.