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United States Department of Agriculture

Agricultural Research Service

Title: A Pcr Assay for Identification of Clavibacter Michiganensis Subsp. Insidiosus

Author
item Samac, Deborah

Submitted to: International Seed Testing Association Plant Disease Committee Seed Health
Publication Type: Proceedings
Publication Acceptance Date: August 30, 1999
Publication Date: N/A

Interpretive Summary: Bacteria that cause plant diseases can be spread between locations in a number of ways. Some bacteria are able to invade the seeds of plants and can be carried with the seeds to new locations. When the frequency of seed infestation is high, seed may need to be tested and infested seed lots discarded to limit the spread of the disease. One of the diseases of alfalfa, bacterial wilt, is found at a low frequency in seed. A test was developed to identify the bacterium causing the disease and was used to measure the frequency of seed infestation. For the test, alfalfa seed are ground and suspended in a nutrient broth. The broth is diluted and used to inoculate agar plates. Bacterial colonies growing on the plates are identified using a rapid and specific test based on amplifying a diagnostic part of the bacterial DNA. Alfalfa plants were grown and infected with the bacterium and seeds produced from infected plants. Only 6.25 to 7.7% of diseased plants transmitted the bacterium to seed. From these plants, 2.5 to 8.7% of seeds contained the bacterium. Considering all infected plants, the rate of seed transmission was low, approximately 0.13%. Diseased plants produced fewer seed than healthy plants. Seed produced in fields where the frequency of diseased plants is low should have very low numbers of infected seed. The test could be used to assess health of alfalfa seed lots. The rates of seed infection will be of interest to seed producers as well as regulators concerned with seed health issues.

Technical Abstract: Clavibacter michiganensis subsp. insidiosus (Cmi) causes bacterial wilt of alfalfa and is found in most alfalfa-growing areas in North America. Bacteria multiply in xylem vessels and cause wilting, stunting, and yellowing although symptoms may not be evident until the second or third year after infection. Bacteria persist for years in dried plant material and can be recovered from seed obtained from diseased plants. In pure culture, Cmi grows slowly as fluidal pale yellow colonies that often develop a dark blue pigment. However, non-fluidal and non-pigmented pathogenic isolates can be recovered from diseased plants, and epiphytic bacteria with similar colony characteristics are often associated with alfalfa seed. A PCR assay, based on amplification of a multicopy insertion element from bacteria added directly to the reaction, was developed to identify Cmi isolated from plants and seed. Seed obtained from diseased plants were tested for the presence of Cmi by grinding and plating seed on a semi-selective agar medium. Only 6.3-7.7% of diseased plants transmitted Cmi to seed. In seed lots from these plants, 2.5-8.7% of seeds contained Cmi. PCR assays using seed grindate were not successful in detecting the bacterium.

Last Modified: 7/28/2014
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