Investigations for improving the detection of Clavibacter michiganensis ssp. michiganensis in tomato seedlings

Bacterial canker caused by Clavibacter michiganensis ssp. michiganensis is one of the most serious tomato diseases, inducing substantial economic losses worldwide. It is considered a quarantine organism in the European Union and in several other countries. There is neither an efficient suppression, nor are there any resistant tomato varieties.

Bacterial canker of tomato is a parasitic vascular wilt disease, the bacterium is located in xylem vessels and spreads at temperatures ranging between 26° and 28°C.

Proving the evidence of Clavibacter michiganensis ssp. michiganensis is not always possible, especially on tomato seedlings. This slow-growing pathogen is difficult to be detected, with symptoms only appearing as plants approach maturity. As obviously a very low level of bacteria is always difficult to be detected with common ELISA tests, in these tests for detection improvement the intention was to raise bacteria concentration at least to the threshold of common ELISA tests.

At the beginning, tomato seedlings were inoculated with different bacteria dilutions. The plants were 22 to 30 cm high on average. They were inoculated by stabbing the stem at the height of approximately 10 cm. Following this procedure, leaves and stems were tested separately. Leave results were always negative even with control dilution which was highest in these tests (8 × 10⁸ cfu/ml), but stem results were all positive. Therefore, for following tests only stems of tomato seedlings were used.

The experiments were based on 3 different methods, carried out alone or in combination, to enrich bacteria concentration in plant sap. Methods were as follows: incubating at 20° and 26°C, filtering and centrifuging. A direct test on plant sap from artificially inoculated plants served as control. Samples for detection with ELISA test were taken exactly on days 3, 5 and 7 after inoculation. When using the lowest bacteria dilution of 1,2 × 10 cfu/ml, only 3 days after infection the following variants showed positive results in ELISA testing: plant sap incubated at 20° (16 hours), filtrate of plant sap also incubated at 20° (16 hours), filtrate + centrifugate at 20° (16 hours) as well as pure centrifugate incubated at 20° (16 hours). Thus, bacteria reproduction was increased and made possibly existing bacteria in tomato seedlings more sensitive to ELISA testing.

In addition, Clavibacter michiganensis ssp. michiganensis was successfully detected directly from young plants of about 30 cm height by PCR. A favourable method to test tomato seedlings is represented by the ELISA testing method.