Gilroy, California, USA
March 22, 2012
Source: Eurofins STA Laboratories newsletter
By Jordan Hendrickson, CA Bacteriology Leader
Tomato bacterial canker caused by Clavibacter michiganensis subsp. michiganensis (Cmm) is a very devastating and problematic seed- borne disease, especially for greenhouse tomato production. It was first identified in Grand Rapids, Michigan in 1909 and has now been identified in almost all significant tomato production areas worldwide. The seed industry has been testing tomato seed for contamination by the pathogen for many years now, and recently there has been a significant improvement in the seed assay for Cmm.
This pathogen, when seed- borne, is usually at very low infection levels and not evenly spread throughout the seed lot. This makes detection of Cmm in seed problematic and has thus been a concern for the tomato seed industry. Outbreaks of the disease continue to occur almost every year in spite of widespread testing of seed lots by seed companies.
Thanks to the efforts of seed pathologists associated with the International Seed Health Initiative (ISHI), a new seed assay has been developed and promoted for Cmm detection. The basic seed assay that has been used for years is still the building block for this new assay, but important improvements have been made to make the assay more reliable and sensitive. Historically, seed assays for bacterial pathogens have relied on soaking the seed in a sterile buffered solution (sometimes after crushing or grinding the seed) and then plating diluted portions of that seed wash onto semi-selective media in petri plates and then observing those plates over several days, watching for the development of “typical” colonies of the pathogen. Once these colonies are identified, they are used to inoculate healthy tomato seedlings in what is referred to as a “pathogenicity” test. If the bacterial colony identified on the semi-selective media is Cmm, that seedling will develop symptoms of bacterial canker, thus completing the identification of the pathogen in the seed.
There are several weak points in the above general procedure. Tomato seed is often treated with anti-bacterial agents (Hydrochloric Acid, sodium hypochlorite, etc.) immediately after the seed is harvested and thus there is little or no active bacteria on the surface of the seed. A simple seed soak prior to a seed assay thus may not yield any bacterial cells to detect. Crushing or grinding the seed prior to soaking can help but often chemicals or saprophytic bacteria are released that can be inhibitory to Cmm. The semi-selective media used can often allow many other organisms to grow which out compete the Cmm, a notoriously slow growing pathogen in culture. Relying on “typical” colony formation is an issue since Cmm does not always form “typical” colonies. Atypical colonies can be over looked and thus not selected for pathogenicity tests.
So what does this new canker assay (known as ISHI version 4) offer that helps to resolve the issues above? First of all, it recommends a gentler extraction process known as “stomaching” that does not fully crush or grind the seed but is beyond just soaking. The semi-selective media have been changed so that less saprophytes and competing organisms develop. The Cmm colonies tend to be more typical on these new media. There is a “spiking” step that helps assure that Cmm can be recovered and is not being repressed by the media or other organisms. Also, this spiking step also helps to assure us that there is no cross contamination between samples and the positive control that is used. This step involves adding a known quantity of a “marked” strain of Cmm to an extra aliquot of the sample that can be recovered and identified – thus indicating that target pathogen is not being repressed in any way and that what is found in the actual sample is not the positive control that is being used. Lastly, it recommends a PCR test on recovered bacterial colonies that will help identify them as true Cmm. The pathogenicity test has not been eliminated in favor of the PCR test; in fact, it is still an integral part of the procedure.
ESTA staff has been intimately involved with the discussions at ISHI about the development of this new seed assay and have participated in the validation testing. We are confident that this new protocol brings a higher level of confidence to the testing of tomato seed for seed- borne Cmm and that as this new assay is put to wider use, the seed industry and commercial tomato producers will greatly benefit. At ESTA, we have now converted our Cmm testing to this new assay and no longer offer the older method. The downside to this is that this new protocol is more time consuming, has costlier inputs and reduces the number of samples we can process in a set time with our current resources. Thus, the cost of the test has had to increase – but the conversion to this new protocol will give our clients greater confidence in the quality of their tomato seed lots.
For additional information about tomato bacterial canker and Cmm, please visit our website at www.eurofinsus.com/stalabs/knowledge-center where you will find a downloadable PDF that was developed by the American Seed Trade Association.