Identification of seeds of Phelipanche ramosa, Phelipanche mutelii and Orobanche cumana in the soils from different agricultural regions in Bulgaria by molecular markers

Abstract

Broomrapes are root holoparasites from Orobanchaceae family parasitizing other flowering plants. Several of the most aggressive broomrape species are widely spread in Bulgaria often causing serious yield losses of important crops. It is important to be able to detect the presence of broomrape seeds in the soil. In the present study, we combined a proven PCR-based assay for detection of broomrape seeds with methods to distinguish species based on nuclear ITS sequences to identify broomrape seeds isolated from soil samples and to study the population structure and the intraspecific variations within the three species. Fifty-six soil samples from 23 different regions in Bulgaria were studied. Based on molecular and bioinformatics analyses we found that 22 samples contained seeds of Orobanche cernua var. cumana and six samples, seeds of Phelipanche ramosa. Phylogenic and bioinformatics analyses surprisingly revealed that the isolated sequences from supposedly P. mutelii seeds diverge from those annotated in NCBI by other authors on 16 different nucleotide positions and formed two distant branches of the phylogenic tree. On the other hand, the isolated sequences were almost identical with P. rosmarina. Tajimas’ D-test revealed that O. cumana populations are currently stable. Regarding the Phelipanche representatives, based on the D-test we can hypothesize that P. mutelii/P. rosmarina populations are in a period of active expansion, while P. ramosa populations are contracting. All isolated sequences were deposited in NCBI Genbank database.