Lepto Leaf Spot Resistance Leptosphaerulina briosiana (

Container..........	Flats or tool carts, 31x62x7.5cm
Medium............	Not critical
Temp/Light........	18 to 24°C; daylength not critical light,intensity >1000 Mmol m^-2sec^-1
No. of Plants ....	20 to 25 per replication
No. of Reps.......	4 minimum
Other ...............	Inoculate with Rhizobium meliloti Dang or fertilize as needed; insect control, none within 1 week of inoculation.

Source..............	Infected leaves
Storage.............	6 months; longer on silica gel
Temperature......	4°C

Age of Plant .....	6 to 8 weeks or 4 to 5 trifoliates
Type of Inoc.......	Sporulating V-8 juice agar plate cultures, 3 to 10 days old, 19 to 23°C, cw fluorescence >20mmol m²sec^-1
Method ............	Cultures inverted 30 to 60 cm above plants, approximately one culture per 900 cm² of plant material, in place until 10 spores per cm² collected in trap slides; culture plates should be relocated every 15 to 30 min to ensure uniform coverage; plants are sprayed with water when plates are removed.
Length .............	2 to 4 hours, variable
Conditions ........	Saturated R.H., 20+1°C, dark

Duration............	48 hours at 100% RH, 20°C
Location............	Move from moist conditions to green house after leaves have dried slowly out of direct sunlight.
Measurement ....	Type and size of leaf spot, usually 10 to 14 days after inoculation.

5 Susceptible ..Spots >3mm, with tan center, halo, spots coalesced, leaf withered

Lepto leaf spot, Leptosphaerulina briosiana (Poll.) Graham & Luttrell
Click on the map above for a larger version. Se e also the KEY.

Name ............K.T. Leath
Address.........USDA-ARS
U.S. Regional Pasture Research Lab
University Park, PA 16802
Phone ...........814-863-0945

Light is critical for spore production. Fluorescent or near UV is as effective as natural daylight. Some glass petri dishes do not pass sufficient light below 340 nm wavelength for good sporulation. Cultures can be started either by placing pieces of agar containing fungal hyphae onto agar surface or by spreading a spore suspension, (prepared by scraping surface of mature culture in water), over agar plate surface. The latter is the quicker method, but if plates contain bacterial contaminants they will not be usable. When cultures are ready to use, ascospores discharged onto petri dish lid are visible. These can serve as contaminant free source for subsequent cultures.

Vigorous plants are essential for expression of susceptible response. Light intensity after inoculation must exceed 1000 mole m^-2sec^-1. Supplemental light (metal halide or other) is necessary during winter at some locations to produce vigorous plants. Use of lightweight potting mix is best if plants are to be pulled during scoring.

Temperatures from 15 to 25°C are probably usable. Light during infection not required. Lower temperatures slow infection but seldom cause failure; too high temperatures or drying leaf surfaces will result in failure.

Plants may be cut back at scoring and regrowth used for different disease evaluation. Isolation of fungus is usually done by direct transfer of spores from sporulating pycnidia produced on leaf tissue. Infection does not kill stems or plants.

Field evaluations may be possible but field infections are rarely of sufficient purity, uniformity, and severity to facilitate satisfactory selection. Inoculations have been made by spraying spores onto leaves. Cultures are scraped in water, comminuted, and filtered through cheesecloth to remove large particles. This method often results in a light inoculation.

1. Leath, K.T. 1971. Quality of light required for sporulation by Leptosphaerulina. Phytopathology 61:70-72.

2. Leath, K.T., and R.R. Hill, Jr. 1974. Large incubation chamber suited for use in selection for disease resistance. Crop Sci. 14:901-903.

3. Leath, K.T., and R.R. Hill, Jr. 1974. Leptosphaerulina briosiana on alfalfa: relation of lesion size to leaf age and light intensity. Phytopathology 64:243-245.