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General Discussion
The newly developed selective medium BCT (a selective medium for bacterial canker of tomato) proved to be superior to all other previously known semiselective media for Cmm in selectivity, in detection sensitivity and in allowing a fast growth of a very wide range of Cmm strains without exceptions. In addition, the new selective medium appears to be very promising for a sensitive detection of other subspecies of Clavibacter michiganensis, too.
Also the newly adapted PCR primers were significantly superior in specificity to the published ones tested and allowed amplifications of all 76 tested Cmm strains without exception. Even some so-called avirulent Cmm strains were amplified.
A Bio-PCR protocol for a highly sensitive detection of Clavibacter michiganensis subsp.
michiganensis (Cmm), the causal agent of bacterial canker of tomato (Solanum lycopersicum / Lycopersicon esculentum) was developed. The protocol is based on the enrichment of viable cells of the target bacterium by plating seed extracts or plant homogenates on the newly developed selective medium. Bacterial upgrowth was directly used as template for PCR detection using new sets of Cmm-specific primers. This Bio-PCR assay allows a sensitive detection of very few Cmm cells in plant homogenates and seed extracts, even when populations of saprophytic bacteria are very high. Furthermore, the protocol allows very fast and early detection of Cmm (within 4 days). In contrast, false negative or significantly delayed results were obtained using earlier recommended semiselective media. The new Bio-PCR protocol improves reliability and sensitivity, and also reduces the time for Cmm-detection significantly, and further additional tests for identifying Cmm are no longer necessary. The protocol could be useful for issuing seed-health certifications and for testing asymptomatic tomato plants on latent infection by Cmm.
Using this new Bio-PCR protocol for testing of tomato seed lots may allow detection of only one infected seed within 10,000 seeds. This high sensitivity in detecting infected seeds was not possible before, and this may be the reason of further distribution of the pathogen via infested seeds in recent years, although the seed had been tested and certified as pathogen free in the past.
Other advantages of the new Bio-PCR for Cmm are:
- the assay needs less time for detection (detection is possible within 4 days), - the effect of PCR inhibitors that exist in plant and seed extracts is eliminated,
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- also the impact of saprophytic bacteria that are found in plant extracts is minimized, - additional tests for identifying the target bacterium are not necessary,
- no need for DNA extraction,
- even non-recognizable small Cmm colonies in the bacterial upgrowth which cannot be distinguished due to the potentially high recovering number of saprophytes are deteced.
Compared with all previous reports, seed treatment methods achieved by this study allow a radical and absolute eradication of Cmm from seed lots without any significant impact on seed germination. The treatment by soaking dry tomato seeds in chemical solutions has the potential to be more effective than the acid seed extraction recommended by EPPO. The EPPO method relies on treating fresh seeds with acid during seed extraction from the tomato pulp, however without a defined standard acid extraction method. Soaking dry seeds in acids allows absorption of acids by soaked dry seeds and finally allows eradicating the internal population of the pathogen that may exist under the hard seed coat. Therefore, soaking dry seeds can be carried out within very short time (30-120 minutes), but an additional seed drying process is required after the treatments. This method allows external seed surface disinfection and internal eradicating of the bacterial population. The internal Cmm-population may be able to survive during seed storage for many years and can be a potential danger later on, when these seeds are placed in a seed bed for germination.
Our surveys on the occurrence of bacterial canker of tomato in the Mediterranean Syrian provinces and discussions with Syrian plant protection inspectors and growers, as well as our observations on the occurrence of Cmm in many new locations in Germany and Austria where this disease was unknown before, indicated that seed transmission was responsible for introducing the pathogen into these locations, although tested and healthy certified seeds were used in these locations. Our assumption was confirmed when we found out that the protocols applied for detecting Cmm in seeds, cannot detect low Cmm populations in seeds and may, therefore, often reveal false negative results.
The European directions for issues of Seed-Health Certifications require that seeds must be obtained from plants that did not develop any disease symptoms and that either seeds were extracted by acids or were tested according to an internationally approved testing method. Our comments on such regulations are:
Our investigations revealed that healthy appearing plants may nevertheless harbour vi-able Cmm populations in low concentration. Thus, we proved that the incubation peri-od could last five or six months, depending on the inoculum dose, the plant age during
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infection and the weather temperatures. We observed that even tomato plants grown from highly infected seeds did not develop symptoms during the first five months.
These results were confimed by field observations when the new plants showed dis-ease symptoms at an age of approximately five months (three months after transplant-ing two month-old plantlets).
Since no standardized and effective acid seed extraction method is recommended, seed companies process their seed lots differently. Therefore, this weak rule cannot be an alternative for a scrutinized seed testing protocol in order to certify seeds as pathogen free.
The recommended seed testing protocols that are based on plating assays of seed ex-tracts on the old semiselective media often revealed false negative results.
Since such weaknesses of seed health testing are internationally known (Olivier, 2009), alternative solutions are very urgently required. For the time being it is suggested to require fulfilling of all criteria to certify seeds as pathogen free, this means:
- seeds must be obtained from plants without any disease symptoms, - seed extraction by acids is obligatory,
- seeds must be tested for Cmm infestation as thoroughly as possible.
We hope that this thesis can help to understand the present weaknesses of tomato seed health tests regarding Cmm and that it will be possible in the future to provide pathogen free seed to the grower which can be obtained by effective seed treatments as well as by highly sensitive and reliable detection methods.