1.0 cm of moist soil and watered. Direct seeding can be 

 used; however, less uniform stands often result. 



Plant stands are counted 2 weeks after emergence, and a 

 fine sprinkler can or mist atomizer is used to sprinkle or 

 spray inoculate an aqueous suspension of stem nematodes 

 onto the seedlings. A minimum inoculation rate of 100 

 nematodes per seedling can be used, but a heavier rate of 

 200 nematodes per seedlings is recommended. Best results 

 are obtained when the plants and soil are prevented from 

 drying rapidly after inoculation. This can be accomplished 

 by either scheduling greenhouse inoculations late in the 

 day or by turning growth chamber lights off for 12 h after 

 inoculation. Although these procedures are usually ade- 

 quate, in areas of low humidity covering the plants with wet 

 cheesecloth and a plastic cover to slow drying may be 

 advantageous. To ensure against escapes, a second inocu- 

 lation with 200 nematodes per seedling should be made 



2 weeks after the initial inoculation. 



Individual plants are rated 12 weeks after the second inocu- 

 lation according to swelling and distortion of the lower 

 stems and crown buds caused by stem nematode attack. 

 Plants are rated on a 1 to 5 scale: 1 = no swelling and dis- 

 tortion, 2 = slight swelling but no distinct symptom, 



3 = moderate swelling and distortion, 4 = severe swelling 

 and distortion, and 5 = severe swelling, severe necrosis, or 

 death. 



The percentage of resistant plants for each cultivar is ob- 

 tained by considering those plants rated 1 and 2 as resist- 

 ant, dividing by the number of plants present at the 2-week 

 stand count, and multiplying the quotient by 100 (table 3). 

 Individual plant ratings may also be used to obtain an ASI 

 for each cultivar. A minimum of 100 plants per cultivar is 

 recommended in each test with at least five replications per 

 test. Resistant and susceptible check cultivars are included 

 in each test. 



Nematodes for stem nematode studies can be obtained in 

 two ways. The most productive way is to maintain monox- 

 enic cultures of nematodes in the laboratory. This is done 

 by culturing the nematode on alfalfa tissue growing on a 

 nutrient medium (21). Nematodes are extracted from alfalfa 

 tissue by placing the infected plant tissue and medium on a 

 20-mesh sieve screen covered by two-ply facial tissue. The 

 screen is set in a pan of water so the water barely covers the 

 plant tissue. The nematodes settle in the water below the 

 screen, and the water is collected daily. Most of the nema- 

 todes are extracted within 48 h. The nematodes are stored 

 in a refrigerator at 3°C until used. Nematodes will settle in 

 standing water and can be concentrated by siphoning off 

 the upper water. The nematode concentration per milliliter 

 can be determined by counting nematodes in three or four 



1-ml samples using a stereomicroscope. Nematodes should 

 be used as soon as possible after extracting to avoid lower- 

 ing their infectivity. 



Table 3.— Evaluation of 7 alfalfa cultivars for stem nema- 

 tode resistance 16 weeks after planting and 

 inoculation 



Cultivar 



Stand count, 

 2 weeks 



Plants rated 

 1 and 2 1 



Resistant 

 plants 2 



Ranger 



(check) 98 



Vernal 94 



Saranac 98 



DuPuits 97 



Lahontan 



(check) 98 



Washoe 86 



Apalachee 96 



Number 



10 



11 



39 

 52 



60 

 51 

 84 



Percent 



10.2 

 11.7 

 39.8 

 53.6 



61.2 

 59.3 

 87.5 



'Plants rated 1 to 5: 1 = no swelling and distor- 

 tion; 5 = severe swelling, necrosis and distor- 

 tion, or plant death. 



Percentage resistant plants = (number plants 

 rated 1 and 2 divided by 2-week stand count) x 

 100. 



A second way to obtain nematodes is to extract them di- 

 rectly from infected plant material collected from the green- 

 house or field using these procedures. Seasonal fluctua- 

 tions in nematode numbers, however, can cause field col- 

 lections to be disappointing. Not only is finding large num- 

 bers of D. dipsaci in the field sometimes difficult, but also 

 contamination with other types of nematodes may occur. 

 Field symptoms are most apparent in early spring when 

 new growth begins and again in the fall following the last 

 cutting. Consequently, one can expect to have the greatest 

 success in collecting large numbers of nematodes in the 

 field during those periods. 



31 



