the surface crust of the central fruiting structure, or apothe- 

 cia, is fragmented to increase the inoculum. Approximately 

 1 ml of the suspension is distributed evenly over the surface 

 of the oatmeal agar in a petri dish. About 50 plates are 

 poured per liter of agar, and an increased ratio of about 1 :10 

 can be expected. Usually two increases are required to 

 obtain enough culture plates for an inoculation. Aseptic 

 conditions are maintained throughout these increases. Cul- 

 tures are incubated in the dark at 20°C for 21 days. At this 

 time a sample plate is inverted over a microscope slide for 1 

 to 2 h. This slide is examined at 100 x magnification to 

 ensure that the cultures are sporulating. 



Seedlings for inoculation are grown in metal flats filled with 

 a 1:1 mixture of soil and peat. Seeds are planted on about 

 4-cm centers and are inoculated when the seedlings have 7 

 to 10 fully expanded leaves. The plants take approximately 

 30 days to reach that stage in the greenhouse. 



Plants are moved to an inoculation-incubation chamber (54) 

 that is maintained at 20°C in the dark with saturated relative 

 humidity. Sporulating cultures are inverted and suspended 

 about 20 cm above the tops of the plants. The dishes are 

 spaced on about 15-cm centers and rearranged periodically 

 to ensure uniform spore coverage. The spores are small 

 enough for some lateral dispersion to occur. Cultures are 

 allowed to deposit spores for about 8 h before the plant sur- 

 faces are sprayed with a fine mist of distilled water, which is 

 terminated just before runoff. Cultures are left to deposit 

 spores on the plants for 48 h. Plants are retained in the 

 moist chamber an additional 24 h and are allowed to dry 

 slowly before returning them to the greenhouse. Because 

 the plants have been in the dark at a high humidity, they 

 should not be exposed immediately to full sunlight. Plants 

 should be moved into subdued light and then, during late 

 afternoon, should be moved into the greenhouse. 



At temperatures of 20° to 26°C, symptoms develop about 

 3 weeks after inoculation. Resistance is determined by the 

 size of lesions, the presence or absence of the apothecium, 

 and the leaf chlorosis and defoliation. We recommend a 1 to 

 5 scale for scoring disease severity. The classes are 1 = no 

 spots; 2 = very small black spots without apothecia; 

 3 = small black spots with small apothecia; 4 = large 

 black or brownish spots with large apothecia; and 5 = large 

 black or brownish spots with large apothecia accompanied 

 by chlorosis and defoliation. 



Plants rated 1 and 2 are considered resistant. When leaves 

 are heavily infected, spots may not develop normally. Thus, 

 leaves that are densely covered with class 2 spots may not 

 be truly resistant. Usually, heavily infected leaves of suscep- 

 tible plants will turn yellow and drop off. The chlorosis that 

 is typical of class 5 susceptibility on heavily infected sus- 

 ceptible leaves may be more striking in the field than in the 

 greenhouse. A susceptible check is useful in estimating the 



frequency of escapes. A resistant check is used to compare 

 results between tests. 



Field Methods 



F. I. Frosheiser 



University of Minnesota, St. Paul 



If plants are spaced about 30 cm apart in the row and about 

 60 cm between rows individual plants can be examined with 

 space to maneuver between rows. Wider spacing may result 

 in less infection. Alternate rows of a susceptible cultivar 

 may be useful in increasing disease potential but are usu- 

 ally unnecessary. Natural epiphytotics of common leaf spot 

 usually occur in Minnesota and adjacent States during late 

 summer or early fall. These epiphytotics are often relatively 

 pure and do not contain other diseases, especially in first- 

 year plantings. Plantings made in early May should be 

 clipped once in mid-July and then allowed to mature until an 

 epiphytotic develops. 



Each plant should be examined individually and rated on a 1 

 to 5 scale. The same scale recommended for laboratory 

 testing can be used in field evaluations. In 1971 Minnesota 

 field tests, the cultivars 'DuPuits', 'Cayuga', and 'Atlantic' 

 were scored for common leaf spot resistance along with 107 

 other cultivars. Percentages of resistant plants for the three 

 cultivars were 'DuPuits', 76; 'Cayuga', 40; and 'Atlantic', 14, 

 compared with 'DuPuits', 80; 'Cayuga', 32; and 'Atlantic', 

 20 percent as reported by Graham and others (28) in green- 

 house tests. Both laboratory and field tests appear suitable 

 to evaluate cultivars. 



Lepto Leaf Spot Resistance 



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



U.S. Regional Pasture Research Laboratory, University 



Park, Pa. 



Cultures of the fungus Leptosphaerulina briosiana (Poll.) 

 Graham & Luttrell are stored in soil under refrigeration. Cul- 

 tures for inoculum are started by depositing soil crumbs 

 containing the fungus onto plates of vegetable juice agar 

 (57). All cultures are grown at 21° ± 1°C under cool-white 

 fluorescent light, at about 500 lux, or preferably under long- 

 wave ultraviolet light (UV) (BLB40 lamps, 30 cm above the 

 plates). More ascospores are produced with the near-UV 

 lamps (53), but cool-white lamps will suffice. The resulting 

 fungus cultures are used as starter plates for increasing 

 inoculum. 



These cultures are flooded with sterile distilled water, and 

 the perithecia are manually scraped loose from the agar. 

 Approximately 1 ml of the fungal suspension is distributed 

 over the surface of the agar in each plate to be used as inoc- 

 ulum. About 50 plates are poured per liter of agar, and an 



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