This study has carried out for distinguishing of Leptosphaeria maculans and L. biglobosa (aggressive and non-aggressive groups respectively) using colony growth, ability for pycnidium and pigment production in four mediums and temperatures of Forty nine isolates (Zaman Mirabadi et al., 2010) (totally 784 treatments) in three replication and three times. The result of analysis variation has shown that significant difference between isolates considering colony growth in the different mediums and temperature treatments. The maximum and the minimum colony growth was observed in 30⁰c –v8 juice Agar (48.05 mm) medium and 15⁰c-Czapek Dox Agar (17.01 mm) respectively after 12 days. The greatest and lowest pigment production in the different temperature was observed in PDA and MEA (except in 30⁰c). The minimum and maximum pigment production was observed in 15⁰c and 30⁰c respectively. The percentage of presence yellow-orange and brown pigment was variable in the different treatments. Brown pigment in V8 and CDA was dominated while yellow- orange was seen in PDA and MEA. 93.78 % isolates produced pigment in Czapek Dox Broth. The maximum pycnidium production was observed in V8 and 20⁰c. According to the obtained results, Dasht-Naz, Eslamabad, Fazelabad, Kohi khail and bykar ayesh were belonged to aggressive species (L.maculans) and Talokola, Lala, Khalkhail and mustard isolate too were located as L.biglobosa.
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