DIFFERENTIATION OF THE PHYTOPATHOGENIC FUNGI INVOLVED IN SEPTORIA LEAF SPOT COMPLEX BY CLASSICAL AND MOLECULAR METHODS
SPASIMIRA NEDYALKOVA1, SLAVTCHO SLAVOV2, PETYA CHRISTOVA2,
ZORNITSA STOYANOVA3, ROSSITZA RODEVA3*
1 – Field Crop Institute – Agricultural Academy, 6200 Chirpan, Bulgaria
2 – AgroBioInstitute – Agricultural Academy, 1164 Sofia, Bulgaria
3 – Institute of Plant Physiology and Genetics – Bulgarian Academy of Sciences, 1113 Sofia, Bulgaria
*Corresponding author: r.rodeva@abv.bg
Keywords: Parastagonospora nodorum, Parastagonospora avenae f. sp. triticea, Zymoseptoria tritici, Triticum durum, Triticum aestivum, ITS sequencing
Abstract: Three fungal species (Parastagonospora nodorum, Parastagonospora avenae f. sp. triticea and Zymoseptoria tritici) are involved in septoria leaf spot complex. The successful identification of the pathogens by symptoms, morphological and cultural characteristics is difficult, especially the differentiation between the first two species. Internal transcribed spacer (ITS) is the most commonly sequenced DNA region in molecular studies of fungi and is recommended as a universal barcode for this group of eukaryotic organisms. The purpose of present investigation was to confirm the affiliation of some isolates from this complex to the corresponding pathogenic species through the application of classical and molecular methods. The typical symptoms caused by the respective fungi were described and illustrated. Details of the morphological and cultural characteristics of the isolates were provided. In vitro test for mycelial interactions between isolates of P. nodorum and P. avenae f. sp. triticea was carried out by the dual culture method. The ITS regions of rDNA of 12 monoconidial isolates obtained from Triticum durum and T. aestivum were sequenced and analyzed by alignment to ITS sequences of standards in GenBank (NCBI database). Seven of isolates showed 99% similarities with P. nodorum in the ITS region of nuclear rDNA. Two other isolates were 100% identical to Phaeosphaeria avenaria f. sp. triticea (old synonym of the sexual morph of P. avenae f. sp. triticea). One isolate was referred to P. avenae, which determined its phylogenetic distance from the other isolates of the same group. The sequences of two other isolates overlapped 100% with Z. tritici standards in NCBI. Phylogenetic analysis showed distribution of isolates in 2 groups – P. nodorum/P. avenae involved in one group and Z. tritici in another, which confirmed the genetic relatedness along with the phenotypical similarity of the first two species. The obtained results proved that combining classical and molecular methods would be necessary for the correct differentiation of phytopathogenic fungi included in the septоria leaf spot complex.