Use of ITS nuclear sequences from Phelipanche aegyptiaca as a direct tool to detect single seeds of broomrape species in the soil

Abstract

Broomrape (Phelipanche and Orobanche spp.) are obligate holoparasites that attack roots of almost all economically-important crops in semiarid regions of the world. Broomrape seeds are extremely small (dust-like seeds), averaging 200 to 300 μm in size and because of the miniscule seed size it is difficult to detect and confirm via conventional methods. In this study our aim was to develop a PCR-based assay specific for broomrape soil-borne seeds and sensitive enough to detect a single or few broomrape seeds in a soil sample. For this purpose, we used complementary polymerase chain reaction (PCR) primers based upon unique sequences in the internal transcribed spacer (ITS) regions of the nuclear ribosomal DNA of Phelipanche aegyptiaca. Genomic DNA was extracted from soil samples artificially infested with broomrape seeds or tissue of Phelipanche aegyptiaca Pers., Orobanche cumana Wallr. and Phelipanche crenata Forsk. and subjected to PCR analysis. Using ITS-350 primers, a specific PCR product (350 bp) was amplified and detected in all samples containing broomrape species, but was not detected in soil sample free of broomrape seeds or tissues. Additionally, the PCR-based assay was sensitive enough to detect even a single broomrape seed in the soil. As expected the universal internal control primers amplified a PCR product (555 bp) of genomic DNA extracted from soil samples with or without broomrape tissues or seeds. This diagnostic method is simple, reliable and rapid and could help for assessment of broomrape seed contamination in a crop field.