Day 6 



(Wednesday, a.m.) 



Uncover plants, turn on lights, and rogue any plants emerged 



since inoculation. 



Days 7 to 10 



Continue roguing newly emerged plants. 



Day 11 



(Monday, p.m.) 



Cover plants again and turn off lights for 12 to 16 h (over- 

 night) to induce sporulation. Do not water plants just before 

 covering because the fungus will not sporulate in free water. 

 Day 12 

 (Tuesday) 

 Evaluate plants. 



Evaluation of cultivars is based on the percentage of 

 mildew-free plants compared with standard resistant check 

 cultivar('Saranac') included in each flat. We have noted con- 

 siderable variability in the virulence of some of our P. 

 trifoliorum isolates, however, at least 15 percent of the 

 'Saranac' plants are resistant to any isolate we have tested. 

 A minimum of four50-plant replications is recommended for 

 each plant line tested. 



Field Methods 



D. K. Barnes 



University of Minnesota, St. Paul 



Spaced plants in the field also can be used to evaluate lev- 

 els of downy mildew resistance in alfalfa cultivars (73). Field 

 epiphytotics are usually observed on succulent vegetative 

 growth during early spring or late fall. 



In the field, individual plants can be classified according to 

 degree of resistance. A 4-class scale has been used at Min- 

 nesota and Utah (1 = no symptoms; 2 = small, usually 

 nonsporulating lesions on one or two leaves; 3 = sporulat- 

 ing lesions on 10 to 25 pet of the leaves; and 4 = general 

 infection over the entire plant). Plants classified as 1 or 2 are 

 considered resistant. Cultivars can be compared statisti- 

 cally with susceptible and resistant check cultivars by 

 either ASI or percentage of resistant plants. 



isolated readily from diseased roots on acidified potato dex- 

 trose agar. Stock cultures are maintained in sterile soil in 

 culture tubes. The fungus remains viable and retains its 

 pathogenicity for long periods (several years) when main- 

 tained in this manner even though the soil becomes dry. 



Inoculum is increased by growing the fungus in nutrient 

 broth prepared by dissolving 2 g NaN0 3 , 1 g KH 2 PO„ 0.5 g 

 MgS04.7H 2 0, 0.5 g KCI, 0.01 g FeSCv7H 2 0, 0.5 g yeast 

 extract, and 15 g sucrose in 1 L of distilled water and auto- 

 claving for 20 min at 121 °C. Potato broth may be substi- 

 tuted for nutrient broth. Wash and slice 300 g potatoes and 

 boil in water for 15 to 20 min. Strain through four layers of 

 cheesecloth and add water to make 1 L. Autoclave for 20 

 min at 121 °. Add a small amount of infested soil from stock 

 cultures to the broth and incubate on a shaker for 4 days at 

 room temperature (21 °). At higher temperatures the incuba- 

 tion period may be shortened. Abundant microconidia and 

 minimum mycelial fragments are produced. The spore sus- 

 pension is diluted with water to a concentration of 1.5 x 10 6 

 spores/ml. If facilities for estimating the spore concentra- 

 tion are lacking, the concentrated inoculum may be diluted 

 1:20 to obtain the proper concentration. 



Alfalfa seedlings are grown in sand in greenhouse benches. 

 They are inoculated with Rhizobium meliloti and fertilized 

 and watered to promote optimum growth. The plants are 

 lifted when 10 to 12 weeks old, the roots are washed in tap 

 water, and about 50 plants for each plot are tied in a bundle. 

 The roots are kept in tap water until all plants for all plots in 

 a replicate are prepared. Then the roots are immersed in the 

 inoculum for 20 to 30 min. After inoculation, the tops are 

 trimmed to about 4 cm from the crown and the roots are 

 trimmed to about 12 cm. The bundles are arranged into the 

 planting order, and five are wrapped together in paper towel- 

 ing and set upright in a container with 1 to 2 cm of water to 

 keep the roots wet until transplanting. If not planted imme- 

 diately, inoculated plants may be stored at 3° to 4°C for sev- 

 eral days. The plants are transplanted about mid-June, using 

 a modified tobacco transplanter, into the field in single-row 

 plots, spaced about 20 cm apart in rows spaced about 1 m 

 apart. The number of established plants is recorded 12 to 14 

 days after transplanting. Many of the susceptible plants are 

 dead within 5 to 6 weeks. 



Fusarium Wilt Resistance 



F. I. Frosheiser and D. K. Barnes 

 University of Minnesota, St. Paul 



The method developed to evaluate resistance to fusarium 

 wilt is similar to that used for bacterial wilt, except that lab- 

 oratory cultures of the pathogen are used as inoculum 

 instead of infected host tissue (25). Fusarium oxysporum 

 Schlecht f. sp. medicaginis (Weimor) Snyd. & Hans, can be 



The surviving plants are evaluated about 3 months after 

 planting. They are undercut and lifted, and each taproot is 

 sectioned and rated for disease severity. The ratings are 

 based on a to 5 scale: = no discoloration in the root; 



1 = small dark strands in the stele; 2 = small dark-brown 

 arcs or rings in cross section of the stele; 3 = larger dark- 

 brown areas, arcs or rings, or partial dark-brown ring in 

 outer stele; 4 = entire outer stele dark brown, plant alive; 

 5 = plant dead (calculated as loss in stand count from 



2 weeks after transplanting). Plants rated and 1 are con- 



24 



