Greenhouse

Container .......Permanent sand benches for direct seeding, plastic pots, or Conetainers (Container Nursery, Canby, OR); method should permit deep vertical tap root growth to 25 to 35 cm

Media ...........Fertilized sand or UC soil mixture (50% silt; 50% peat moss)

Temp/Light ......21 to 27°C; 14 to 16 hour day length

No. of Plants .... 35 to 50 plants per rep

No. of Reps .....3 minimum

Culture .........Control insect populations; supplemental fertilizer (0-10-10) added once a month with water applied from below the crown.

Other ...........Inoculate seed with Rhizobium meliloti Dang; seed directly into sand bench or into Conetainer; place cones in the sand (permits water and nutrient control and avoids excess water at crown)

INOCULUM CULTURE

Source.......Pure cultures are isolated from alfalfa stems or crowns onto acidified potato dextrose agar or dilute V8 juice agar amended with streptomycin (30 pmm); radial growth is slow (2 mm per day); incubate infected leaves or stems containing pycnidia in a moist petri dish for 48 hours and streak pycnidiospores onto plates

Storage..........Pure cultures on V8JA slants at 4°C; transfer pycnidiospores (not mycelium) to maintain pycnidial type

Storage Life ....6 to 10 weeks; renew by pycnidiospore transfer to fresh medium (avoid mycelium)

 INOCULATION PROCEDURE

Age of Plant:  Earliest stage for reproducible infection using CA isolates is two-week regrowth on plants cut at first flowering (30 to 45 days)

Type of Inoc.: Pycnidiospore suspension from 10 to 14 day old V8JA plates that have been  flooded with spores

Concentration .. 10⁶ spores per mL +0.01% Tween 20 (wetting agent); blended water agar may increase adherence

Foliar............Spray foliage until just before runoff, allow foliage to dry prior to misting

Crown ..............Inject spores (10⁶ per mL) into upper tap root with PS #19 hypodermic needle with side orifice (Pupper and Sons, Inc., New Hyde Park, NY) or pour inoculum on scalpel wounds to prevent escapes

 INCUBATION

 Location .........For leaf spot inoculate in mist chamber or greenhouse with mist system for 48 to 72 hours at 20 to 22°C and 16 hour daylength

 Spacing ..........2.5 to 5 cm apart

 Length ...........Rate foliar lesions at 14 to 21 days after inoculation; rate crown rot at 2 to 6 months

 RATING

 Foliar

1 Resistant ......Free of symptoms

2 Resistant ......Lesions on leaves and petioles but none on stems

3 Resistant ......Lesions on leaves and up to 25% of stem tissue

4 Susceptible ....Lesions on leaves and 25% to 60% of stem tissue

5 Susceptible ....Lesions on leaves and 60% to 100 % of stem tissue

 Root and crown

0 Resistant ......No necrotic tissue at inoculation site

1 Resistant ......Local superficial lesion

2 Resistant ......Necrotic tissue (reddish flecks) with 1 to 5 mm lesion

3 Susceptible ....Necrotic tissue 5 to 10 mm laterally and 10 to 20 mm down from inoculation site

4 Susceptible ....Necrotic tissue 10 to 20 mm laterally and 20 to 30 mm down from inoculation site

5 Susceptible ... Necrotic throughout, dead plant

 *Seed of UC 129 is available from D.C. Erwin. Values for resistant standards are the totals of all resistant categories.

DISTRIBUTION AND SEVERITY OF STAGONOSPORA CROWN ROT

Stagonospora stem and crown rot, Stagonospora meliloti (Lasch) Petr.

SOURCES OF INOCULUM

 Name .....Ann Martinsen
Address ..Dept. of Plant Pathology, Univ. of CA
Davis, CA 95616
Phone ....(916) 752-7043

 Name .....D. C. Erwin, and R. A. Khan
Address ..Dept. of Plant Pathology, Univ. of CA
Riverside, CA 92521
Phone ....(714) 787-4128

SCIENTISTS WITH EXPERTISE

 Name .............. D. G. Gilchrist
Address ............ Dept. of Plant Pathology, Univ. of CA
Davis, CA 95616

 Phone .............. (916)752-6938

  Name ............. .. D. C. Erwin
Address .......... .. Dept. of Plant Pathology, Univ. of CA
Riverside, CA 92521
Phone ............... (714) 787-4128

 Name .....J.A.G. Irwin
Address...Dept. of Botany
University of Queensland
St. Lucia, Australia 4067

Greenhouse selections made at UC Davis and evaluated at three field locations in CA ranked similarly under all conditions.

CULTURE OPTIONS AND RANGE OF CONDITIONS

Pure cultures are grown on V8 Juice Agar at 22 to 24°C for pycnidial formation, and at 10 to 16°C for mycelial growth. Streaking of pycnidial spores or pouring a spore suspension on V8 plates will produce a "lawn" of pycnidia in 10 to 14 days. Light enhances pycnidial production.

HELPFUL INFORMATION

Temperatures may vary 21 to 27°C but higher temperatures reduce severity. All seedling plants tested in CA have been highly resistant to infection prior to emergence of the first flower buds. Reliable rating of genotypes requires regrowth following the first flowering. Foliar and stem infection is highly correlated with crown and root infection.

ALTERNATIVE METHODS

Since foliar inoculation is easier, faster, reliable and closer to natural conditions it is the method of choice, but crown inoculation also can be done by uprooting young plants after cutting the foliage, rinsing the roots with water, soaking the roots and crowns in a spore suspension then replanting. The incubation period for this method is two to three months.

REFERENCES

1. Erwin, D.C., R.A. Khan, O.K. Ribeiro, and W.F. Lehman. 1987. Growth sporulation and pathogenicity of Stagonospora meliloti and selection for resistance to crown and leaf spot of alfalfa. Plant Dis. 71: 181 - 185.

2. Erwin, D.C., R.A. Khan, and W.F. Lehman. 1987. Registration of UC 129 alfalfa germplasm with moderate resistance to Stagonospora leaf spot and root rot. Crop Sci. 27:820.

3. Graham, J.H., F.I. Frosheiser, D.L. Stuteville, and D.C. Erwin, eds. 1979. In A compendium of alfalfa diseases. American Phytopath. Soc. St. Paul, MN 65 pp.

4. Stuteville, D.L., and D.C. Erwin. 1990. In A compendium of alfalfa diseases. Amer. Phytopath. Soc. St. Paul, MN, 2nd ed. 84 pp.