Standard Tests to Characterize Alfalfa Bacterial Wilt Resistance March 1991

Clavibacter michiganense subsp. insidiosum (McCull)Davis et al
(syns. Corynebacterium insidiosum (McCull.) H.L. Jones).
Cheryl Fox and Judy Thies

PLANT CULTURE

Greenhouse

Container ..............	Bench or flat deep enough to allow root development
Media ...................	Sand or soil mixture
Temp/Light ............	24 to 30°C; 16+ hour daylength
No. of Plants .........	50 to 70 per replication
No. of Reps ...........	3 minimum
Other ....................	Inoculate with Rhizobium meliloti Dang; promote good growth; spray and fertilize as necessary

INOCULUM CULTURE

Source .................	Infected root tissue
Storage.................	Ground up, washed roots
Temperature .........	-10°C
Storage Life ..........	Up to several years if frozen

INOCULATION PROCEDURE

Age of Plant...........	8 weeks old
Concentration ........	50g ground root per lL H2O
Inoc. Time .............	20 to 30 min
Method .................	Bare root soak
Type of Inoc...........	Bacterial water suspension

INCUBATION

Location................	Transplant to field (June)
Plant Counts..........	Count 2 to 4 weeks after establishment
Cultural .................	Maintain vigorous growth
Spacing ................	0.15 x 1.0 m
Age at Rating ........	5 months (3 months in field)

RATING

Plants are removed from the field and the tap root sectioned for rating.
0 Resistant ...........	Root clean and white
1 Resistant ...........	Very small yellow-brown spots visible in stele
2 Susceptible ........	Discoloration affecting up to one-third of stele
3 Susceptible ........	Nearly entire stele discolored, cortex white
4 Susceptible ........	Discoloration throughout slele and cortex, plant alive
5 Susceptible ........	Plant dead (based on plant count)

CHECK CULTIVARS

	Approximate Expected Resistance (%)	Acceptable Range of Reaction (%)
Resistant
Vernal**	42	30-50
Susceptible
Narragansett**	1	0-5
Sonora**	1	0-5
Values for resistant standards are totals of 0's and l's.

DISTRIBUTION AND SEVERITY OF
BACTERIAL WILT

Bacterial wilt, Clavibacter michiganense subsp. insidiosum (McCull) Davis et al

SOURCE OF INOCULUM

Name ...................	Judy Thies
Address ................	Department of Plant Pathology 495 Borlaug Hall 1991 Buford Circle University of MN St. Paul, MN 55108
Phone ..................	612-625-8240

SCIENTIST WITH EXPERTISE

Name ...................	Don Barnes
Address ................	University of Minnesota USDA/ARS 411 Borlaug Hall 1991 Buford Circle University of MN St. Paul, MN 55108
Phone ..................	612-625-4780

RACES

There are no known races of Clavibacter michiganense.

CULTURE OPTIONS
AND RANGE OF CONDITIONS

Pure cultures of Clavibacter michiganense can be grown (2), however pure cultures often appear to be less virulent than ground root inoculum.

PLANT GROWTH OPTIONS
AND RANGE OF CONDITIONS

Best results are obtained when the plants are grown under optimum conditions in the field and in the greenhouse. It is important to transplant healthy plants with well developed roots to assure good transplant survival and uniform tests.

NOCULATION CONDITIONS
AND RANGE OF CONDITIONS

Roots must not be allowed to dry out between pulling and inoculating. After inoculation, plant tops are trimmed to within 5 cm of the crown and roots to 10 to 12 cm. Several bundles can be wrapped together in paper or cloth towels to keep them moist until transplanting.

HELPFUL INFORMATION

Plants may be stored in 1 to 2 cm water at 2 to 4°C for up to several days prior to transplanting. A tobacco transplanter or modified vegetable transplanter works well for trans planting. Plants are undercut at 15 cm and root seclioned for rating. A carrot or beet lifter also works well for removing the plants from the ground. Plants may be rated at any time between 12 to 16 weeks after transplanting. Ratings may be expressed as an Average Severity Index (ASI) or as a percentage adjusted to the long time average of Vernal (42%). The percentage of resistant plants adjusted to Vernal is very useful in comparing cultivars tested in different years.

ALTERNATE METHODS

The root soak field evaluation method is most effective in determining resistance in alfalfa. However a combination of root soak and cotyledon wounding has proved effective for screening large numbers of seedlings in the greenhouse (1,2,3,5).

REFERENCES

1. Barnes, D.K., C.H. Hanson, F.I. Frosheiser, and L.J.Elling. 1971. Recurrent selection for bacterial wilt resistance in alfalfa. Crop Sci. 11:545-54.

2. Frosheiser, F.I., and D.K. Barnes. In Standard tests to characterize pest resistance in alfalfa cultivars. 1984. USDA. Misc. Pub. No. 1434. pp. 20.

3. Frosheiser, F.I. Alfalfa cotyledon inoculation with bacterial wilt inoculum prepared from infected alfalfa roots. Phytopalhology 56:566-567.

4. Kernkamp, M.F., and G. Hemerick. 1952. A deep freeze method of maintaining virulent inoculum of the alfalfa wilt bacterium, Corynebacterium insidiosum.Phytopathology. 42:13.

5. Krietlow, K.W. Infecting seven-day-old alfalfa seedlings with wilt bacteria through wounded cotyledons.Phytopathology. 53:800-803