Abstract
Broomrapes are holoparasitic plants spreading through seeds. Each plant produces hundreds of thousands of seeds which remain viable in the soils for decades. To limit their spread, drastic measures are being taken and the contamination of a commercial seed lot by a single broomrape seed can lead to its rejection. Considering that broomrapes species identification from a single seed is extremely difficult even for trained botanists and that among all the described species, only a few are really noxious for the crops, numerous seed lots are rejected because of the contamination by seeds of non-noxious broomrape species. The aim of this study was to develop and evaluate a High Resolution Melting assay identifying the eight most noxious and common broomrape species (Phelipanche aegyptiaca, Orobanche cernua, O. crenata, O. cumana, O. foetida, O. hederae, O. minor, and P. ramosa) from a single seed. Based on trnL and rbcL plastidial genes amplification, the designed assay successfully identifies O. cumana, O. cernua, O. crenata, O. minor, O. hederae, and O. foetida; P. ramosa, and P. aegyptiaca can be differentiated from other species but not from each other. Tested on 50 seed lots, obtained results perfectly matched identifications performed by sequencing. Through the analysis of common seed lots by different analysts, the reproducibility of the assay was evaluated at 90%. Despite an original sample preparation process it was not possible to extract enough DNA from some seeds (10% of the samples). The described assay fulfills its objectives and allows an accurate identification of the targeted broomrape species. It can be used to identify contaminants in commercial seed lots or for any other purpose. The assay might be extended to vegetative material.
Highlights
Broomrapes (Orobanche and Phelipanche spp.) are angiosperms in the Orobanchaceae which have evolved into obligate root holoparasitic plants (Joel, 2009)
Broomrape seeds (P. aegyptiaca, O. cernua, O. crenata, O. cumana, O. foetida, O. hederae, O. minor, and P. ramosa) were either obtained from international collections or collected during field sampling by GEVES, Syngenta, Terres Inovia, or University of Nantes
For lots number 7, 12, 15, 31, and 33, respectively, harvested in 2013, 2011, 2014, 1994, and 1987, it was not possible to obtain any amplification. For these five last seed lots, single seed extraction was performed on two more seeds with the similar results
Summary
Broomrapes (Orobanche and Phelipanche spp.) are angiosperms in the Orobanchaceae which have evolved into obligate root holoparasitic plants (Joel, 2009). One single broomrape plant can produce hundreds of thousands of extremely small seeds, between 200 and 300 μm, each weighing around 5 μg and composed of only 200 to 300 cells (Joel, 1987). They are dispersed mainly by wind and water, and remain viable in the soils for many. Infection of the host plant can lead in reduction of crop yields up 100% This makes broomrapes one of the most devastating parasitic weeds in the Mediterranean and western Asian regions and in many other parts of the world (Parker, 2009). Among these noxious parasitic species are the closely related Phelipanche ramosa L. and P. aegyptiaca Pers. (synonym Orobanche ramosa and O. aegyptiaca) (Joel, 2009), O. cumana Wallr., O. cernua Loefl., O. crenata Forsk., O. foetida Poir., and O. minor Sm., while O. hederae Vaucher ex Duby has no agronomical impact but is extremely common
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