|Specific TaqMan assay for the detection of Acidovorax valerianellae on the cotyledons of corn salad (Valerianella locusta).
Sven Berendsen: Rijk Zwaan Breeding B.V.
<div><span>The causal agent of the bacterial black spot disease on corn salad (<em>Valerianella locusta</em>) are the Gram negative bacteria <em>Acidovorax valerianellae </em>(Av). This bacterium has been proven to be soil and seed transmittable. The primary way to prevent corn salad infection is to start with Av free soil and seeds. ISHI-Veg has described a method for the detection of Av on corn salad seeds in which bacteria on symptomatic corn salad cotyledons from a grow-out test are confirmed by PCR. The described PCR is time consuming and lacks an internal amplification control (IAC). Therefore, a new TaqMan PCR assay was designed based on the amplification region of the ISHI-PCR. The TaqMan was validated and compared with the described ISHI-PCR utilizing a collection of Av isolates. Data reveals that the TaqMan PCR functions as good as the described PCR. To turn the reaction into a Real Time PCR duplex, we added the IAC specific TaqMan for corn salad non-pathogen bacterium <em>Clavibacter michiganensis </em>subsp.<em> tesselarius</em> (Cmt) to this assay. Av suspected and negative cotyledons were grinded in 0.85% sterile saline that was spiked with a known concentration of Cmt. Samples were processed as described by ISHI-Veg method and the crude DNA samples were tested by ISHI-PCR and the new Real-Time PCR. Based on our findings the new Real-Time PCR is an improved alternative for the confirmation of Av suspected corn salad cotyledons, and will be evaluated within ISHI-Veg.</span></div>