Anthracnose Resistance 



S. A. Ostazeski and J. H. Elgin, Jr. 



Beltsville Agricultural Research Center, Beltsville, Md. 



Alfalfa seedlings and mature plants react similarly when 

 infected by the anthracnose fungus (Collectotrichum trifolii 

 Bain). This simplifies the evaluations of resistance. Seedling 

 evaluation procedures were first described by Ostazeski 

 and others (68). These procedures have since been refined 

 and used to develop resistant populations (13) and cultivars 

 (14). 



Until recently, only one race of the anthracnose fungus was 

 known to occur. Since 1978, however, race 2 has been iso- 

 lated from scattered fields of previously anthracnose- 

 resistant alfalfa in North Carolina, Maryland, and Virginia 

 (70, 71,89). The cultivars 'Arc' and 'Saranac' serve to differen- 

 tiate the two races. Race 1 is virulent on 'Saranac' only, 

 whereas race 2 is equally virulent on both cultivars. 'Sara- 

 nac AR' is resistant to both races. 



Both races are cultured identically. The inoculum source is 

 obtained from isolates collected periodically from infected 

 plants in the field. These can be increased on such culture 

 media as oatmeal agar, potato dextrose agar, V-8 juice agar, 

 or lima bean agar. Oatmeal agar is preferable. Optimum 

 temperature for growth and sporulation in culture for most 

 isolates is 25°C. Alternating on-off cycles of fluorescent 

 light during incubation can enhance sporulation. Maximum 

 sporulation is obtained from 7- to 10-day-old plates which 

 were seeded with fresh spore suspensions. Poor inocula- 

 tions often result when plate cultures older than 10 days are 

 used for inoculum (88). Spores are harvested by flooding 

 plate cultures with about 10 ml of water supplemented with 

 two drops of Tween 20/L Spore concentration is adjusted to 

 1 x 10 6 spores/ml and is applied to run off with an atomizer, 

 sprayer, or air brush. About 50 ml of inoculum are applied to 

 each 30- by 60-cm flat of seedlings. 



A dry inoculum method also has been developed (9). Dis- 

 eased plants from susceptible cultivars grown under con- 

 trolled conditions are dried and ground. This inoculum is 

 applied to seedlings as dust at a rate of 10 g/flat. Dry inocu- 

 lum can be stored at least 10 months at - 20°C without loss 

 of virulence (69). Dry inoculum should not be collected from 

 field epiphytotics because it probably contains other patho- 

 gens, especially Rhizoctonia. 



Seeds are sown in steamed soil in sterilized flats. Plant 

 spacing is 3.8 cm between rows and 2.0 cm within rows. 

 Before and after inoculation, seedlings are grown in a 

 growth chamber at 23°C, with 16-h daylength and 17.2 klux 

 light. 



About 14 days after seeding, the seedlings are inoculated 

 and moved to a dark walk-in incubation chamber similar to 



that described by Leath and Hill (54) and operated at approx- 

 imately 23°C. Our chamber is humidified with a two-nozzle, 

 compressed air-powered atomizer and operates 1 min of 

 every hour. 



As an alternate, a moist chamber can be fashioned directly 

 over the plants with a wooden or pipe framework covered 

 with polyethylene sheeting. More uniform humidity for 

 infection is attained if plants are in a dark enclosure. Tem- 

 perature should be 20 to 25°C. High humidity can be main- 

 tained with one or more humidifiers. Using such a setup on 

 a bench in a darkened growth chamber required us to oper- 

 ate the humidifiers 2 consecutive minutes each half hour. 

 Inoculated seedlings are held in the moist chamber for 48 h. 



Plants are scored about 2 weeks after inoculation. Scoring 

 is from 1 (resistant) to 5 (dead) and corresponds to lesion 

 type (68). Type 1 stems have no lesions or only small, water- 

 soaked spots. Type 2 is a long, narrow lesion with few, if 

 any, acervuli and no sporulation. Type 3 lesions are long, 

 wide, not girdling, with acervuli usually present. Type 4 

 lesions are large, coalescing, and sporulating eventually 

 girdling and killing the stems. In cultivar evaluations, plants 

 with lesion types 1 and 2 can be classified as resistant. 

 Percentages of resistant plants can be compared among 

 cultivars by relating each cultivar to the standard resistant 

 check. An ASI also can be used to compare cultivars. 



Before initiating new programs on anthracnose resistance, 

 other less conventional evaluation methods should be con- 

 sidered. Specifically, Graham and others (26) have described 

 the agar plate method for selecting alfalfa resistant to 

 C. trifolii. Cornmeal agar, in plates, is used to grow the fun- 

 gus, for germinating seed, and for growing resistant seed- 

 lings until they can be transplanted to soil. The authors 

 claim large populations can be screened in a minimum of 

 time and space as effectively as with other methods. 



Morrison (58) described a seedling box test for evaluating 

 alfalfa for resistance to anthracnose in which plastic shoe 

 boxes are used to grow, inoculate, and incubate seedlings. 

 Resistance could be evaluated within 3 weeks of sowing 

 seed. 



Common Leaf Spot Resistance 



Laboratory and Greenhouse Methods 



K. T. Leath and R. R. Hill, Jr. 



U.S. Regional Pasture Research Laboratory, University 



Park, Pa. 



Cultures of the fungus Pseudopeziza medicaginis (Lib.) 

 Sacc. are stored under refrigeration on oatmeal agar (36 g of 

 Difco oatmeal agar preparation plus 7 g of agar/L of distilled 

 water). These cultures are flooded with distilled water, and 



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