Identification of cucumber damping-off based on morphological and molecular characterizations in Rafsanjan

Cucumber damping-off is a limiting factor in commercial greenhouse production. To determine the causal agents of disease, sampling and fungal isolation were performed during 2009 and 2011. A total of 23 isolates of Pythium sp. were obtained using selective media. Their pathogenicity was evaluated on cucumber seedlings under greenhouse conditions. All isolates were pathogenic. Morphological and molecular identifications of the isolates were performed. P. aphanidermatum was identified based on microscopic characteristics. The complete sequences of ribosomal DNA internal transcribed spacers regions of selected isolates were determined and submitted to GenBank. The GenBank-BLAST homology search revealed P. aphanidermatum as the most similar sequence (> 99% identity) with GenBank entry AB355599. PCR primers were designed for the identification of P. aphanidermatum in which had a unique ITS sequence. The PCR primer combination Paph54F/ITS2 was species-specific with no cross-reaction to other Pythium species or to soil borne cucumber fungal pathogens tested. The detection limit was as little as 200 fg pure genomic DNA. P. aphanidermatum could be detected in lesions of infected cucumber stem tissue. No signals were obtained from healthy cucumber tissue DNA.