Detection and Identification of Clavibacter michiganensis subsp insidiosus inducing Alfalfa Wilt Disease Using Specific PCR and Physiological Methods in Iran

Alfalfa.(Medicago saliva) is the most important forage crop and the first tamed forage crop worldwide. Iran is the geographical center of alfalfa and this crop has been planted by ancient Persians. Hamedan province of Iran is a suitable niche for alfalfa growth but many diseases including alfalfa bacterial wilt disease cause economic crop losses. Bacterial wilt caused by Clavibacter michiganensis subsp. insidiosus is an important disease of alfalfa worldwide. This pathogen spreads through the xylem and causes wilt. Detection and identification of the pathogen, especially in symptomless plant material, is difficult and often requires planting on specific media. A sensitive polymerase chain reaction (PCR)-based identification method has been developed for Clavibacter michiganensis subsp. insidiosus. Here, we investigated the presence of alfalfa bacterial wilt disease in Hamedam. Wilted plants were collected from the main alfalfa growing areas of Hamedan province. Plant samples were disinfected and grinded in phosphate buffer. An aliquot of the bacterial suspension was stroked on nutrient agar medium containing 5% yeast extract. Pathogenicities of the strains were confirmed on alfalfa plants under greenhouse condition. Bacterial strains caused wilting and chlorosis,after 40 days on tested plants. The phenotypic features of the strains were characterized and the PCR assay confirmed the identity of bacteria as Clavibacter michiganensis subsp. insidiosus strains.