growth chamber or greenhouse in 21- x 62-cm wooden flats 

 with 3.8 cm on center spacing, approximately 128 seedlings 

 per flat. When 4 to 5 weeks old, the seedlings are placed in a 

 2.4- x 2.4- x 1.8-m dark incubation chamber similar to the 

 one described by Leath and Hill (54), except that moisture is 

 provided by an air pressure mist atomizer operated once 

 daily for 5 min. The chamber holds 18 flats. Chamber tem- 

 perature is maintained at 22°C. Plants are sprayed with tap- 

 water mist and then inoculated with a dry dust suspension 

 of 0.45 g of urediospores in 8.9 g of talcum powder. A pres- 

 sure pump at 12 to 15 psi is used on a small jar duster for 

 inoculation. Counts with a hemacytometer verify that 

 approximately 100 million spores settle in the area of the 

 chamber in the dust suspension. The seedlings are removed 

 from the incubation chamber after 48 h. 



The most desirable time for rating the diseased plants is 16 

 to 20 days after inoculation. The most severely infected leaf- 

 let of each plant is scored for development of pustule size. 

 Scoring is on a scale from 1 to 5. Class 1 = no symptom 

 development; 2 = leaf flecks, possibly with a few small 

 closed pustules; 3 = a few flecks and closed pustules plus 

 several small open pustules; 4 = many small open pus- 

 tules; and 5 = many medium to large open pustules. Score 

 classes 1 and 2 are considered resistant since these plant 

 types inhibit pustule development. In score classes 3 and 4, 

 some pustule development is inhibited, whereas the plant 

 types in score class 5 promote complete pustule develop- 

 ment and abundant spore production. Percentage resistant 

 plants or ASI of cultivars can be used for comparisons with 

 check cultivars. 



Yellow Leaf Blotch Resistance 



M. D. Rumbaugh 



USDA-ARS, Utah State University, Logan 



Methods used at South Dakota State University, Brookings 



Greenhouse techniques are used to evaluate for resistance 

 to yellow leaf blotch caused by Leptotrochila medicaginis 

 (Fckl.) Schuepp (77). Seeds of entries to be evaluated are 

 planted in a nonsteamed loam soil-sand mixture (2:1) in 7.6- 

 x 7.6-cm greenhouse plant bands, 30 to a wooden flat. 

 Seedlings subsequently are thinned to one per band, and 

 plants are allowed to grow several months at 20° to 25° C 

 with several prunings to promote branching. A minimum of 

 100 plants per cultivar is recommended, divided among at 

 least four replications. 



Inoculum consists of naturally diseased leaves (40 to 50) 

 collected from the field and placed between 10- x 15-cm 

 sheets of plasticized fiber glass window screening. The 

 leaves between the screens are held outdoors from early 

 summer to late fall until apothecia develop and mature on 



them (76). For inoculations, the diseased leaves, still be- 

 tween the screens, are wetted and the screens are arranged 

 close together over plants to be inoculated. This arrange- 

 ment may involve as few as 12 and as many as 35 flats at 

 one time within a frame tightly covered with clear, 3-mil 

 polyethylene sheeting. Protection from direct sunlight is 

 provided, and greenhouse temperatures are held at 20° to 

 25°C. The diseased leaves between the screens are held 

 over the plants for 48 h, and the plastic sheeting is removed 

 24 h later. 



Plants are scored for disease symptoms between the sec- 

 ond and third week after uncovering them. Because of pos- 

 sible nonuniform inoculations, they are scored for symptom 

 type, not for abundance of infected leaves. Leaves are rated 

 on a scale from 1 to 5: 1 = no evident infection; 2 = small, 

 dark circular spots with no chlorosis; 3 = small, dark circu- 

 lar spots with chlorosis; 4 = one or more chlorotic leaf sec- 

 tors extending from the leaflet edge to the midrib, with 

 incipient pycnidia; and 5 = most to entire leaflet chlorotic 

 with robust pycnidia and leaf curling. Resistance can be 

 expressed as percentage resistant plants or as ASI. Lesion- 

 type scores 1, 2, and 3 together are considered resistant; 4 

 and 5 are susceptible. 



Alfalfa Seed Chalcid Resistance 



M. W. Nielson 



Forage Insects Research Laboratory, Tucson, Ariz. 



Evaluations of seed chalcid (Bruchophagus roddi Gussa- 

 kovsky) resistance can be conducted either in the field or in 

 the greenhouse. Field evaluation of cultivars begins by 

 establishing plants in rows spaced 1 m apart, with 50 to 

 60 cm between plants within rows. Six replications are usu- 

 ally used. Fields are managed so that plants are setting 

 seed during August when alfalfa seed chalcids reach maxi- 

 mum populations. Samples of 25 racemes, with fully devel- 

 oped pods, are taken from each plant and are placed in a 

 pint-sized ice cream carton. Adult chalcids are allowed to 

 emerge. Statistical comparisons of cultivars are made on 

 the basis of the mean number of adults per 25 racemes per 

 plant (65). 



Greenhouse evaluations are made by caging 10 adult chal- 

 cids on a raceme. From 6 to 8 racemes are caged per plant. 

 The adult chalcids are reared from infested seed screenings 

 held in storage at 10°C. After 2 weeks, the caged racemes 

 are removed and placed in rearing cartons. The number of 

 emerged adult chalcids are counted after another 2 weeks 

 (60). As with field procedures, cultivars are compared by 

 mean number of emerged adults per plant; however, culti- 

 vars also are rated according to percent resistant plants. 

 Resistant plants are those that average no more than one 

 adult per raceme. 



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