Total fungal elicitor preparation

Two pathotypes of Ascochyta rabiei with the following strain/code number; FUM 1003/ASR003 (Pathotype No. 3) and FUM 1006/ASR009 (Pathotype No. 6) were obtained from the Microorganisms Collection of Ferdowsi University of Mashhad (WDCM 1207), Iran. They had been classified in our previous studies by (Shokouhifar et al. 2003b). The fungal pathotypes were cultured on PDA (Potato Dextrose Agar, Merck, Germany) to prepare fresh mycelium. Two plugs of the fresh mycelium were cut and transferred to 100 ml PDB medium (Potato Dextrose Broth, Merck, Germany) in 500 ml Erlenmeyer flask and incubated at 24 ± 2 °C with 120 rpm for 10 days. The Fungal mycelium were collected and washed for three times by distilled water and transferred to 100 ml of 200 mM phosphate buffer (pH = 7.00). Collected mycelium were homogenized using a homogenizer apparatus (Heidolph DIAX 900, Germany) at 4 °C for 5 min. Mycelium debris were precipitated by 15 min centrifugation in 10,000 rpm at 4 °C. Supernatant were aliquot into 10 ml tubes and stored at − 80 °C.