CONIDIA AS A SUBSTRATE FOR INTERNAL TRANSCRIBED SPACER-BASED PCR IDENTIFICATION OF MEMBERS OF THE LEPTOSPHAERIA-MACULANS SPECIES COMPLEX

The blackleg disease of oilseed rape is caused by an ascomycete specie s complex termed Lepiosphaeria maculans (anamorph Phoma lingam). L. ma culans isolates collected worldwide were gathered in the International Blackleg of Crucifers Network (IBCN) collection. Representative IBCN isolates, along with one FI nigrificans isolate, were further analyzed using polymerase chain reaction (PCR) amplification of the internal t ranscribed spacer (ITS) region. ITS size polymorphism discriminated th ree groups: (i) P. nigrificans, (ii) Tox(+) and 'Lepidium' isolates, a nd (iii) NA1, NA2, NA3, 'Thlaspi', and 'Erysimum' isolates. Digestion of the ITS region with 19 selected endonucleases showed restriction si te polymorphism between the different subgroups: digestion with RsaI c ould discriminate Tox(+) from 'Lepidium' isolates, whereas digestion w ith four enzymes, i.e., HaeIII, EcoRII, RsaI, and AluI, was needed to discriminate between NA1, NA2, NA3,'Thlaspi', and 'Erysimum' isolates. No restriction site pelymorphism was observed between isolates within the 'Thlaspi', Tox(+), NA1, and NA2 subgroups. Direct amplification o f the ITS region could be achieved using intact conidia, collected eit her in axenic cultures or on leaf lesions, with only a 4-min 95 degree s C denaturation step prior to PCR reaction. A routine identification protocol requiring no DNA extraction and a sequential use of a few res triction enzymes following PCR has been used successfully for large-sc ale identification of French field isolates.