increase ratio of about 1:10 is usually obtained. Two in- 

 creases may be necessary to get from soil storage to a 

 large-scale production of inoculum. After about 72 h, asco- 

 spores are present in the cultures and can be seen readily 

 on the lids of the petri dishes at 100 x magnification. Plates 

 are usually 72 h old when they are used to inoculate plants. 



Plants for inoculation are grown in metal utility carts filled 

 with a 1:1 mixture of soil and peat. We plant on about 4-cm 

 centers and inoculate when the plants have 7 to 10 fully ex- 

 panded leaves, which takes about 30 days in the greenhouse. 

 Because vigorously growing plants are necessary to get 

 susceptible host-plant responses to infection (55), we grow 

 our plants under metal haiide lamps that provide the 20,000 

 lux at plant height needed for true symptom expression. 



Plants are placed in an inoculation-incubation chamber (54) 

 that is maintained at 20 D C in the dark with saturated relative 

 humidity. Sporulating cultures are inverted and suspended 

 about 20 cm above the tops of the plants. The plates are 

 spaced on about 15-cm centers and must be rearranged fre- 

 quently to ensure uniform inoculations. Lateral dispersal of 

 falling spores is minimal. Cultures are allowed to drop 

 spores until a spore density of 10/cm 2 on trap slides is 

 reached. At this time, usually 1 Vi to 4 h, plates are removed, 

 and the plants are sprayed with a fine mist of distilled water 

 until leaf surfaces are wet. Plants are retained in the moist 

 chamber for 30 to 48 h and are allowed to dry slowly before 

 being returned under the lights in the greenhouse. When the 

 plants are returned to the greenhouse, the light intensity 

 must be maintained at the minimum 20,000 lux because 

 susceptible symptom expression does not occur under low- 

 light, slow-growth conditions. 



Symptom development is rapid with this disease. Often 

 chlorotic flecks are visible when the plants are removed 

 from the moist chamber. Resistance is determined by the 

 size and type of lesions at about 10 days after inoculation. 

 We recommend a 1 to 5 scale for rating disease response. 

 The classes are 1 = no spots; 2 = very small black spots; 



3 = large black or small tan spots without chlorotic halos; 



4 = small tan spots with halos and large tan spots without 

 halos; and 5 = large tan spots with pronounced halos and 

 coalesced spots forming blotched leaf areas. Plants rated 1 

 and 2 are considered to be resistant. Heavy inoculations 

 should be avoided because spots do not develop normally, 

 and leaflets tend to desiccate and abscise. A susceptible 

 check cultivar is useful in estimating the frequency of es- 

 capes as well as the suitability of environmental conditions. 

 A resistant check should be used to compare results be- 

 tween tests. 



Downy Mildew Resistance 

 Laboratory Methods 



D. L Stuteville 



Kansas State University, Manhattan 



Physiological races of Peronospora trifoliorum d By. occur; 

 therefore, a diverse collection of conidia (sporangia) is rec- 

 ommended for screening. Conidia from field plants usually 

 are contaminated with bacteria and fungi and germinate 

 poorly. Producing inoculum for tests on seedlings in the lab- 

 oratory is preferable. To inhibit contaminants, add 50 ^g 

 nystatin and 10 ^g tetracycline/ml of inoculum (83). The fol- 

 lowing schedule permits using conidia from infected seed- 

 lings in a test as inoculum for the next test. We prefer, how- 

 ever, to include separate flats seeded thickly with suscep- 

 tible alfalfa from each test to produce inoculum for the next 

 test. Conidium germination is greatest when plants are har- 

 vested and placed immediately in water 12 to 16 h after they 

 are covered to induce sporulation. Conidium viability de- 

 clines rapidly if harvest is delayed or if conidia are exposed 

 to dry air for more than a few seconds (23). A low percentage 

 of conidia, however, will survive for a few weeks on diseased 

 seedlings stored at -20°C. Conidia will remain viable for 

 many years in liquid nitrogen (7). 



To prepare inoculum, excise seedlings or leaves with sporu- 

 lation and place immediately into a jar containing chlorine- 

 free water (22). Shake the jar vigorously to dislodge conidia 

 and pour the spore suspension through a tea strainer to 

 remove plant debris. To inoculate, spray the spore suspen- 

 sion onto seedlings to point of runoff. Uniform infection 

 requires at least 25,000 viable conidia/ml of inoculum. Pre- 

 vent plants from drying for 12 h by placing them under an 

 airtight cover. The same covering procedure is used to 

 induce sporulation, which requires darkness and nearly 

 100 percent relative humidity (23). 



Plants are maintained in growth chambers at 20°C and 

 5,000 to 10,000 lux of continuous fluorescent lighting 

 throughout, except for designated dark periods. We use the 

 following schedule routinely as it requires little attention on 

 weekends. 



Day 1 



(Friday) 



Plant seeds in 1.3-cm-deep rows in flats of fine sand. We 



space two to three seeds/cm within rows 2.5 cm apart. 



Days 3 to 5 



Sprinkle water on flats twice daily to settle the sand around 



the seedlings to insure an even emergence. 



Day 5 



(Tuesday) 



Seedlings are emerged, and cotyledons are expanded. Spray 



on inoculum, cover plants, and turn off lights for at least 



12 h (overnight). 



23 



