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Development of a PCR-based method for the detection of Alternaria radicina, A. alternatia, and/or A. dauci in carrot material

Specific primers for detection and identifica-tion of the Alternaria species on carrot seeds and roots were designed and shown to be highly sensitive and able to differentiate between the three Alternaria species occurring on carrot, i.e. A. radicina, A. alternata and A. dauci. The A. alternata primers cross-reacted with A. longipes and A. lini, however these species do not occur on carrot.

A. alternata and A. radicina could be detected in DNA isolated from carrot material (seeds and infected root material) applying the specific primers, even at low infection levels. A positi-ve correlation was found between the percentage of seed infection established by the blotter method and the intensity of the amplified, specific product. Based on a small-scale ring test with 5 participating laboratories, the PCR method was found not suited for application on carrot material, in particular seeds. The difficulties experienced are related to problems with the DNA isolation and extraction procedure, likely as the result of interfering oily substances in the seeds. For analysis of carrot root material, the PCR assay developed seems to be useful.

This PCR-based assay needs to be further optimised. Nonetheless, when the problems with interfering components can be solved, this assay will speed up the analysis time and the sensitivity for Alternaria detection. A faster detection method then the methods currently in use is requested by seed companies and inspection services, because an analysis time of 7-14 days may hamper trade. The method developed so far has been published and members of the International Seed Health Initiative (seed companies and inspection services) and the International Seed Testing Association have shown interest in the method. It is likely that seed companies and inspection services will test it, and jointly optimise the method for future use.

Reported by

Plant Research International
Droevendaalsesteeg 1
Wageningen 6708 PB
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