Morphological and molecular characterization of Clavibacter michiganensis subsp. michiganensis causing bacterial canker in tomatoes

Bacterial canker is one of the emerging diseases of tomatoes caused by Clavibacter michiganensis subsp michiganensis (Cmm) in the Northern hill region of India. The present investigation was conducted to estimate the presence and spread of Cmm in the newer areas, to isolate and identify the cause of bacterial canker through conventional, serological and molecular diagnostic tools. 25 tomato plant samples along with the infected fruits collected from different tomato growing regions of the Uttarakhand and Himachal Pradesh were analyzed, and the bacterium was isolated from the seed of the infected tomato fruits. The present study revealed overall mean Cmm incidence of 19.57% and overall disease severity of 16.11% in the Northern hill region of India. The isolated bacterial strains were characterized culturally in semi-selective media (D2ANX and SCM), morphologically (SEM) and molecularly (16S rRNA and pat-1 genes). A total of 10 samples produced yellow colored colonies on D2ANX medium and 9 samples produced grey colored colonies on the SCM medium suggesting them to be of Clavibacter michiganensis subsp michiganensis (Cmm). Amongst the 10 isolates collected three isolates were serologically identified as Cmm through the DAS - ELISA technique. However with PCR reactions we found that only 2 strains out of 10 were of Cmm and the sequence of the most virulent Cmm isolate was submitted at NCBI with the accession number MH321815. These findings suggest that Cmm is present in the tomato seed, which can serve as a potential means of spread of a quarantine pest in the relatively newer areas of Northern hill regions of India.

Automated summary

Tomato bacterial canker caused by Clavibacter michiganensis subsp michiganensis (Cmm) is an emerging disease in the Northern hill region of India. This study investigated the presence and spread of Cmm, and identified the cause of bacterial canker using conventional, serological, and molecular diagnostic methods. The results showed a Cmm incidence of 19.57% and disease severity of 16.11% in the region. Tomato seeds were found to be a potential means of quarantine pest spread.