Molecular identification of Pythium aphanidermatum isolates by using semi-specific primers

Abstract

Identification of Pythium aphanidermatum is difficult and time-consuming due to its morphological and biological characteristics. in the current study semi-specific polymerase chain reaction (PCR) was developed for identification of P. aphanidermatum. Fourteen isolates collected from different areas of Iran were studied. On the basis of the fragment obtained in the random amplified polymorphic DNA (RAPD), after purification, cloning and sequencing, the primers were designed. The designed primers could not distinguish P. aphanidermatum isolates from P. deliense isolate. Then, new primers were designed based on differences of the sequence of fragments obtained from the semi-specific PCR. In order to generate the highest specificity and efficiency, annealing temperature and extension time were optimized for the primer pairs. Six other Pythium species as well as isolates of Phytophthora drechsleri, Rhizoctonia solani and Fusarium solani were used to assess the designed primers. Results showed that the primers designed amplified the 656bp and 618pb fragments in all isolates of P. aphanidermatum and P. deliense whereas no amplification was observed in other isolates. The optimal annealing temperature and the best extension time were considered to be 66 °C and 60 sec., respectively. So, the primers designed are able to identify the isolates of Pythium aphanidermatum and to separate them from other fungi causing root rot of sugar beet and other species of Pythium except P. deliense. * Corresponding Author: Nazanin Allaghehband Zadeh  n.alaghebandzadeh@srbiau.ac.ir