Verticillium Wilt Resistance 



R. N. Peaden 



irrigated Agriculture Research and Extension Center, 



Prosser, Wash. 



Laboratory cultures of the alfalfa strain of Verticillium albo- 

 atrum Reinke & Berth, are used in root-soak techniques for 

 the greenhouse, growth chamber, or field. This organism is 

 isolated from infected leaves or stems that show distinct 

 symptoms. The best stems are still green and show a yellow 

 streak or partially chlorotic leaves. The tissue is surface 

 sterilized in 70 percent alcohol for 5 to 15 s (depending on 

 the thickness of plant material), rinsed in sterile distilled 

 water, blotted on sterile filter paper, placed on water agar in 

 petri dishes, and incubated at 20° to 25°C for 2 to 5 days. 

 Transfer is then made to prune agar (85) and the cultures are 

 incubated for 10 days at 20° to 25°. Conidia are floated off 

 the dish in sterile distilled water to produce 7 to 10 ml of 

 inoculum. If a counting devise is available, the concentra- 

 tion is adjusted to approximately 8 x 10 6 conidia per ml. (A 

 hemacytometer works very well.) 



Stock cultures may be maintained on silica gel (74). Difco 

 Czapek Dox broth can be used to produce large quantities 

 of Verticillium inoculum more efficiently if a shaker is avail- 

 able. A small amount of fungus is transferred from prune 

 agar to 50 ml of sterile Czapek broth in a 125-ml flask. It is 

 placed on a shaker at 120 to 130 r/min at 20° to 25°C, for 7 to 

 10 days. The contents are then filtered through two layers of 

 cheesecloth and centrifuged at 3,800 x G for 20 min. The 

 supernatant is discarded and the pellet resuspended in ster- 

 ile distilled water and diluted to a conidial concentration of 

 approximately 8 x 10 6 /ml. Inoculum can be stored at 5° for 

 a week or more. Each flask yields 200 to 400 ml of dilute 

 inoculum. The centrifuge procedure, though not absolutely 

 necessary, produces a cleaner inoculum with a higher per- 

 centage of conidia than that strained through cheesecloth. 



Roots of 4- to 12-week-old plants are washed; cut to a conve- 

 nient length or injured, if already short enough; and soaked 

 in the inoculum for about 5 min (time is not critical). Top 

 growth of older (>8 weeks) plants is trimmed. Twenty-five 

 plants per entry per replication are transplanted into flats or 

 pots in the greenhouse or growth chamber at 20° to 25°C 

 and plants can be rated for disease severity in 3 to 5 weeks. 

 Fresh inoculum usually is used for each replication. Inocu- 

 lated plants transplanted into the field require the full grow- 

 ing season for best results. Rating is on a scale of 1 to 5 

 with 1 = no symptoms; 2 = 1 or 2 leaflets showing slight 

 mottling but no distinct symptoms; 3 = distinct symptoms; 

 4 = severe symptoms; 5 = dead plants. Stunting is not 

 considered in the rating. Plants rated 1 and 2 are classified 

 as resistant, and results can be expressed as percentage 

 resistant plants or all data can be used to compute an ASI. 

 Susceptible checks, such as 'Vernal' or 'Saranac', normally 



show less than 5 percent escapes. 'Vertus' contains approx- 

 imately 45 percent resistant plants. 



Rust Resistance 



J. E. McMurtrey III and J. H. Elgin, Jr. 



Beltsville Agricultural Research Center, Beltsville, Md. 



Laboratory methods are most suitable for evaluating rust 

 (Uromyces striatus Schroet.) resistance in alfalfa although 

 several researchers have successfully screened and evalu- 

 ated germplasm in the field (11,15). One laboratory method, 

 the detached leaflet-petri dish technique, was described 

 earlier (39) and is useful for evaluating small numbers of 

 plants. For larger numbers of plants, however, the following 

 techniques are recommended. 



To minimize the possibility of culturing a single physiolog- 

 ical race of rust, urediospores should be collected from dis- 

 eased plants in late summer and fall from a number of field 

 locations (39). Initial collection may be done by applying 

 light suction to a cyclone spore collector similar to that 

 used with Puccinia in cereal rust (8). Flexible tubing is at- 

 tached to the collector providing an opening for oral suction, 

 and the bottom half of a 33- x 8-mm gelatin capsule is used 

 as a receptacle to retain the spores. The capsule is capped 

 with the top half for storage. Urediospores can be collected 

 and stored dry at -8°C for 12 months with little loss of viabil- 

 ity. Alternate methods of collecting urediospores are by 

 brushing, tapping, or shaking diseased leaflets over funnels 

 or pans. Spores also may be stored in envelopes or other 

 suitable containers. 



The inoculum usually does not have to be increased unless 

 small numbers of plants (< 50) are to be tested. This is most 

 easily done on vigorously growing susceptible plants. Plants 

 may be inoculated by dusting, brushing, or rubbing uredio- 

 spores onto leaflets, and small numbers of plants can be 

 inoculated with the cyclone-spore collector by reversing the 

 airflow. Diluting the spores with dry talcum powder enhances 

 dispersal of small quantities of inoculum (56). For inoculat- 

 ing larger numbers of plants, a larger duster can be fabri- 

 cated from a small jar, an atomizer bulb, a rubber stopper, 

 and glass and rubber tubing. 



A substantial supply of urediospores may be obtained by 

 inoculating flats of a susceptible cultivar and collecting ure- 

 diospores from the plants by shaking them over an enamel 

 tray. Slanting the tray slightly and tapping the bottom lightly 

 result in the gravitational movement of any soil, leaves, and 

 nonspore particles to a corner of the tray where they can be 

 removed. The urediospores are then tapped to the corner of 

 the tray and are scooped into a container for storage. 



Approximately 4,000 plants can be inoculated with 0.45 g of 

 urediospores. Seedlings for evaluation are grown in the 



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