Development of specific molecular markers to distinguish and quantify broomrape species in a soil sample from infected field

Abstract

Broomrapes (Orobanche and Phelipanche) are obligate holoparasites that cause heavy damage to numerous crops, reducing the yield and its quality. The parasite develops in the soil and exerts the greatest damage prior to its emergence; therefore the majority of field loss may occur before diagnosis of infection. Because of the parasite tiny seed size (200 to 300 m) and dormancy for several decades in the field, it is very difficult to diagnose the parasite by conventional methods. Therefore, to restrict the parasite seeds spread and contamination to other commercial fields, development of DNA-based molecular markers to identify and quantify broomrape species in a soil sample is much needed. In this study, we developed a specific molecular marker (RbcL-M) based on rbcL (large subunit of the ribulose-bisphosphate carboxylase) gene from Orobanche crenata to differentiate between Orobanche crenata and Orobanche cumana. Likewise, a specific marker (ITS100) based upon unique sequences in the internal transcribed spacer (ITS) regions of the nuclear ribosomal DNA of Phelipanche aegyptiaca to quantify three species of the parasite (P. aegyptiaca, O. crenata and O. cumana) in a soil sample was developed. Genomic DNA was extracted from soil samples artificially infested with broomrape seeds or tissue of P. aegyptiaca, O. cumana and O. crenata and subjected to PCR analysis. RbcL-M marker successfully amplified a PCR product (1300bp) when O. crenata seeds or tissues (collected from several locations in Israel) were added to the soil samples. The same marker amplified a PCR product (1000bp) when O. cumana seeds or tissues were added to the soil samples. RbcL-M marker did not amplify soil samples with seeds or tissues of P. aegyptiaca or any soil-borne DNA. Furthermore, using ITS-100 marker and Real-Time PCR analysis, allowed quantitative diagnostic of the parasite in a soil sample from infected sunflower field. As expected the universal internal control primer (UCP-555) amplified a PCR product (555bp) when genomic DNA extracted from soil samples with or without broomrape tissues. The development of an efficient, simple and robust molecular marker to detect and distinguish between broomrape species, has a significant insights on assessment the level of infestation and planning eradication program to the parasite in a field crop.