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

Abstract

AbstractBroomrapes (OrobancheandPhelipanche) 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 μm) 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 onrbcL(large subunit of the ribulose-bisphosphate carboxylase) gene fromOrobanche crenatato differentiate betweenOrobanche crenataandOrobanche cumana.Likewise, a specific marker (ITS100) based upon unique sequences in the internal transcribed spacer (ITS) regions of the nuclear ribosomal DNA ofPhelipanche aegyptiacato quantify three species of the parasite (P. aegyptiaca, O. crenataandO. cumana) in a soil sample was developed. Genomic DNA was extracted from soil samples artificially infested with broomrape seeds or tissue ofP. aegyptiaca, O. cumanaandO. crenataand subjected to PCR analysis. RbcL-M marker successfully amplified a PCR product (1300bp) whenO. crenataseeds or tissues (collected from several locations in Israel) were added to the soil samples. The same marker amplified a PCR product (1000bp) whenO. cumanaseeds or tissues were added to the soil samples. RbcL-M marker did not amplify soil samples with seeds or tissues ofP. aegyptiacaor 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.