For export there is a need to determine the absence of Clavibacter michiganensis subsp. sepodonicus (Cms) and Ralstonia solanacearum (Rsol) on tomato and pepper seed. Naktuinbouw Laboratories have developed new tests to support the sector in this regard.
For all these tests we have chosen the fast, reliable and sensitive Seed-Extract PCR (ZE-PCR) method. We also developed and validated a protocol of Clavibacter michiganensis subsp. michiganensis (Cmm) on pepper. In addition to ZE-PCR, we have also developed and validated a plating assay for detection of Cmm.
ZE-PCR is a technique for rapid DNA screening for the presence of pathogens
Plating assays consist of a serial tests which is time consuming, but the ZE-PCR (Seed Extract PCR) has a fast pre-screening. We extract DNA from the seeds immediately upon arrival and do a PCR directly on it.
Next to the speed, another advantage is that ZE-PCR has less interference from overgrowth by other micro-organisms on the seeds, as it can be the case with plating on selective media where we look at the growth of pathogens. The disadvantage is that we cannot distinguish between live and dead pathogens, therefore it is only a pre-screening.
Cmm, Cms, Rsol for tomato or pepper
The protocols for the Q pathogens have been approved by the NVWA.
Rsol is an important pathogen, but it is unlikely that this pathogen is truly seed-transmittable. Cms is known as a pathogen of potato, but for tomato this is much less clear. Seed transmission of Cms via tomato seed is therefore controversial and the question is whether Cms contaminated tomato seed is the actual way of spreading.
The test will therefore mainly be requested for export purposes.
Hypochlorite treatment and ZE-PCR are incompatible
By treating seeds with hypochlorite, DNA breaks down. This greatly reduces the sensitivity of ZE-PCR. This means that the chance of false negative results increases, which means that infected batches can be exported incorrectly. Treatment of seed with HCl and trisodium phosphate has no effect on the sensitivity of ZE-PCR.
• Cmm on pepper seeds
- Method: Plating test
- Sample: 10,000 or 30,000 untreated seeds
- Basic test rate: € 226,60 or € 281,00
- Rate PCR follow-up test: € 67,00
- Duration of the basic and follow-up test: 35 days
- Risk of unassessable results when seed contains many live fungi and bacteria.
- Prescribed ISHI method and status within GSPP.
- Distinguishing between dead and living DNA.
• Combi Cmm, Cms, Rsol on tomato or pepper seeds
- Method: Seed Extract PCR
- Sample: 10,000 untreated seeds
- Rate basic test: € 219,00
- Duration basic test: 4 days
- Not suitable for batches treated with hypochlorite / bleach
- Pre-screening method and no distinction between dead and living DNA (under review at ISHI)
*For basic rates, see the 2021 price list.