DETECTION OF CLAVIBACTER MICHIGANENSIS SUBSP. MICHIGANENSIS FROM TOMATO PLANTS AND SEEDS USING ELISA, IF AND PCR WITH COMMERCIAL AND OWN PRIMERS

Bacterial canker of tomato, caused by bacterium Clavibacter michiganensis subsp. michiganensis (Cmm) is a well-known disease resulting in serious losses to both greenhouse and field tomato crops in areas where tomatoes are planted. Cmm was detected and isolated from various parts of strongly infected plants of 10 indeterminate tomato cultivars. The most sensitive technique was optimized PCR with Dreier’s and laboratory-generated PCR primers from the Cmm tomatinase gene. All samples taken from base, stem and petiole of tomato plants were positive in PCR with both primer pairs, and also using IIF and PTA-ELISA in contrast to the samples taken from leaves. Using IIF and PTA-ELISA with PAbs of Neogen Europe Ltd. (UK), Cmm was reliably detected to dilution 1:1000 and 1:100 respectively. Seeds of 4 indeterminate and 4 determinate cultivars were consecutively artificially infected by the mixture of the two reference Cmm strains (BCCM/LMG 7333 and 5727) soaking 48 hours in inoculum (10⁸ CFU/ml). Isolation of bacterial DNA from crushed seeds was performed by DNeasy Plant Mini Kit (Qiagen) according to the manufacturer’s protocol with subsequent PCR with two primer sets (Dreier’s and own pairs) about 24 hours, one week and one month after inoculation. Detection of Cmm by PCR with both commercial and our own primer sets was comparable.