American Rose Annual, p. 125-133. (1961)
Comparable Susceptibilities of 50 Species and Hybrid Roses Inoculated With Black Spot Fungus from Plants Field-Grown in Maryland in 1959
John G. Palmer and Peter Semeniuk1

1 Pathologist and Horticulturist, respectively, Crops Research Division, Agricultural Research Service, United States Department of Agriculture, Beltsville, Maryland.
2 Complete scientific names for all cultivars of roses listed in Table 2.

MANY WORKERS have recorded the incidence of black spot on commercial varieties of roses (3, 8, 9) or incidentally in reports on other subjects (7). These reports indicate that immunity from black spot does not exist among the commercial cultivars. Most reports on species record only the presence or absence of black spot on the plants examined (1, 4, 10). Though Lyle (6) rated four species clones comparatively and reported the two clones of Rosa multiflora2 very resistant to if not immune from black spot, the reports for any one species probably describe different genetic clones with different resistances to black spot. In addition, subjectivity and variability in the rating procedures make the relation of any one rating to another uncertain.

Two immediate problems had to be solved in this study. First it was necessary to select 40 rose cultivars equally divided between commercial and species cultivars to form a permanent screen against which the virulence of inocula of the black spot fungus, Diplocarpon rosae Wolf, could be evaluated and with which the relative resistances of new cultivars, species, and seedling populations could be compared. In such tests, leaflets removed from bushes of the selected roses must not deteriorate during the 15 to 17 days necessary for development of black spot after inoculation. Second, an average infection value or index was necessary for each cultivar for ready comparison with other indices. A sequence of distinct, recognizable stages by which the infection would take place on leaflets of most cultivars and to which whole number values could be assigned was desirable. Therefore, a series of inoculations was planned to determine if these objectives were attainable and compatible.

METHODS

The work of Jenkins (5) here at Beltsville, which was supported by the American Rose Foundation, suggested solutions to these problems. He had inoculated with the black spot fungus rose leaflets removed from the plant and reported satisfactory comparisons with inoculations of leaflets attached to rose plants. Variations designed to improve the technique were studied during 1957 and 1958. By June, 1959, a satisfactory procedure had been worked out and was used during the frost-free season of that year. Experiments had indicated that the virulence of the black spot fungus declined in test-tube culture. Single-spore isolations required 8 to 12 weeks' growth with one sub-culturing before mature spores were produced in sufficient quantity to inoculate the 70 rose cultivars under study. The variation in times to formation of spores and the apparent loss of virulence in test-tube culture made use of cultures on artificial medium impractical as sources of inocula for reproducible tests of resistance to black spot.

In 1959, therefore, inoculations were made with spore suspensions prepared by scraping mature acervuli from leaflets of field-grown, unsprayed bushes of a hybrid tea, Red Radiance. The same plants always served as sources of leaves for inoculum in successive tests. The spores were suspended in rain water that had been collected in acid-cleaned glass containers and then sterilized by Seitz-filtering. The spore concentration was adjusted to approximately 2,250,000 spores per cc of inoculum. The inoculum was then distributed one drop per leaflet through a 26-gauge needle attached to a 5 cc hypodermic syringe. Drops of each inoculum were placed also in well-slides stored at 72° Fahrenheit in moist chambers, and germination of the first 100 spores seen was recorded after 24 and 48 hours.

TABLE 1.
Percent of positives in inoculations of 23 roses in which readings from each of 31 leaflets were obtained.

Inoculation date Positive rose cultivars
Number Percent
May 26 12 52
June 2 9 39
June 9 5 22
June 30 7 30
July 14 1 4a
August 10 16 69
October 7 19 83
October 14 20 87
October 20 15 65

 a lnoculum prepared on July 21 from infected leaves collected July 14

At least two plants of each rose cultivar or species clone being considered for the screen were propagated by budding or cutting from the original plant and were grown in the greenhouses. They were pruned so that four or five vegetative shoots developed consistently on each plant. On the day of inoculation three leaves were removed from three different shoots on plants of each clone to be tested. The first fully-expanded leaf below the shoot apex was selected, washed in running tap water, and blotted. One leaflet from each of the three leaves was rubbed lightly with a finger on the upper surface along the midrib and placed in a large, deep petri dish. This procedure insured inoculation of leaflets from three different sources and therefore leaflets with possible variations in physiology. Each dish had been fitted with thin discs of absorbent cotton, wetted, and then steam-sterilized. Each could be re-used two or three times after a sterilization if stains produced in deterioration of the leaflets had not been too great. For each of the 10 inoculations (Table 1) one person was responsible for each complete procedure, i.e. for selection of all leaves. After the inoculation was completed, the dishes were kept at 72° F. for 15 to 17 days. Observational data were recorded between the seventh and tenth and again between the fifteenth and seventeenth days. Each leaflet was examined at 20x magnification, and smears were made to ascertain the presence or absence of typical spores of D. rosae [Marssonina rosea (Lib.) Lind stage] if acervuli or acervulus-like structures were present.

TABLE 2.
Rose species and cultivars listed by increasing susceptibility to black spot inoculum obtained
from leaves of the rose cultivar Red Radiance field-grown at Reltsvile, Maryland during 1959.

Speciesa or cultivarb
A
Clone
B
Number
C
% positivec
D
Pointsd
E
Leaflets
F
Inoc. Index
G
1. Rosa multiflora Thunb. cv. Ginn 189 10 0 0 31 0
2. R. pisocarpa Gray 45 10 0 0 31 0
3. R. rugosa Thunb 184 8 0 0 24 0
4. R. x alba L. 121 8 0 4 26 0.15
5. R. x alba 179 8 0 4 25 0.16
6. R. woodsii Lindi. var. fendleri (Crép.) Rhbd. 73 10 10 7 31 0.23
7. R. sp. cv. Lasca (P.1. 236148)e 159 8 13 6 24 0.25
8. Rosa (F) cv. Goldilocks 624 10 20 8 29 0.28
9. R. soulieana Crép 101 10 10 9 31 0.29
10. R. x alba cv. Maxima 105 9 22 10 28 0.36
11. Rosa (F) cv. Chic 670 6 17 7 18 0.39
12. R. multiflora 165 8 37 10 24 0.42
13. R. helenae Rehd. & Wils 87 10 20 15 31 0.48
14. R. x alba var. suaveolens Dieck 11 9 33 14 28 0.50
15. R. setigera Michx 47 10 20 20 31 0.65
16. R. arvensis Huds 70 10 40 23 31 0.74
17. R. multiflora cv. Chennault (5828) 51 10 30 26 30 0.87
18. R. wichuraiana Crép 108 8 50 25 25 1.00
19. R. sp. cv. Pollmeg (P.I. 236151)e 169 7 43 32 28 1.16
20. R. sp. cv. Rubiginos (P.I. 236152)e 168 9 33 24 21 1.16
21. R. blanda Ait 68 10 70 35 29 1.21
22. R. spaldingii Crép 50 8 50 30 24 1.25
23. Rosa (F) cv. Goldilocks x (HT) cv. Kingcupg 187 8 40 33 25 1.32
24. R. wichuraiana 80 10 40 43 31 1.39
25. R. bracteata Wendi 54 10 50 46 31 1.48
26. Rosa (HT) cv. Georg Arends 256 10 60 49 31 1.58
27. Rosa (HP) cv. Black Prince 205 10 60 51 31 1.65
28. Rosa (HT) cv. Peace  616 10 80 53 29 1.83
29. Rosa (F) cv. Goldilocks x (HT) cv. Kingcupg 188 8 75 47 24 1.96
30. R. x alba 104 9 57 56 28 2.00
31. R. palustris Marsh 79 10 70 64 31 2.06
32. R. x noisettiana Thory f. manettii (Rivers) Rehd.h 58 10 70 64 31 2.06
33. Rosa (CH) cv. Old Blush 706 10 60 65 31 2.10
34. Rosa (HT) cv. Red Radiance 623 10 70 66 31 2.13
35. Rosa (LR) cv. Paul's Scarlet 702 10 80 67 31 2.16
36. Rosa (Cl) cv. Climbing Mermaid 708 10 80 67 31 2.16
37. Rosa (HP) cv. Mrs. John Laing 206 10 70 49 31 2.23
38. Rosa (HT) cv. Helen Traubel 620 10 60 71 31 2.29
39. Rosa (F) cv. Spartan 671 6 100 42 18 2.33
40. Rosa (F) cv. Garnette 621 10 80 75 31 2.58
41. Rosa (T) cv. Maman Cochet 626 10 90 80 31 2.58
42. Rosa (HT) cv. Nocturne 613 6 100 47 18 2.61
43. Rosa (HT) cv. Better Times 622 9 100 73 28 2.61
44. Rosa (F) cv. Ma Perkinsx g 56-2-7 6 87 49 18 2.72
45. Rosa (HT) cv. Confidence 602 8 75 69 25 2.76
46. Rosa (HT) cv. American Beauty 254 10 90 86 31 2.77
47. R. canina L 158 7 100 56 20 2.80
48. Rosa (HT) cv. Fred Howard 672 6 100 52 18 2.89
49. Rosa (F) cv. Ma Perkinsx g 56-5-2 7 100 66 21 3.14
50. R. laevigata Michx. 666 10 100 112 31 3.61
    1. With one exception (see footnote b) species names conform to Rehder, A. 1940. Manual of Cultivated Trees and Shrubs Hardy in North America. The MacMillan Co., New York. 996 pp.
    2. Variety names conform to McFarland, J. H. 1958. Modern Roses V. J. Horace McFarland Co., Harrisburg, Penn. 471 pp.
    3. See footnote a, Table 1. Each inoculation comprised 3-4 leaflets. If one or more leaflets were positive, the inoculation was positive.
    4. Cumulative points assigned as in Table 1.
    5. Roses in use or in testing as understock from Aalsmeer, Holland.
    6. Classification abbreviations according to McFarland, J. H. Modern Roses V. pp. vii-xi.
    7. Unnamed seedling clone.
    8. Authority cited for name of species (see footnote a) Rehder, A. 1949. Bibliography of Cultivated Trees and Shrubs. Arnold Arboretum. Jamaica Plain, Mass. 825 pp. (P. 315).

RESULTS AND DISCUSSION

Leaflets removed from many of the rose clones on hand, especially commercial cultivars, would not remain green and firm for the 15 to 17 days required. Leaflets of some of these cultivars such as the hybrid teas Frau Karl Druschki and Torch Song deteriorated within 10 days and adequate tests could not be obtained. None of these cultivars are listed in Table 2. Leaflets of a few cultivars such as the three Rosa x alba clones, the hybrid perpetual Black Prince and the hybrid tea Better Times deteriorated to some extent between the third and seventeenth days. However, readings could be obtained; the data are included in Table 2 if five readings were obtained from at least 15 leaflets. As the year progressed, new clones were added to replace those that had to be discarded because leaflets deteriorated. The prospective degree of contamination could be estimated during the spore-germination counts, and discolorations of the leaf that without magnification frequently resembled black spot resulted with some kinds of contamination. Removal of the inoculation drops with paper tissue after 48 hours eliminated most contamination.

Distinct stages in development of symptoms occur after invasion by the black spot fungus (Table 3) but generally are visible only on smooth-surfaced leaflets lacking hairs. As long as no evidences of infection were visible stage A was recorded. The first clear evidence that the fungus had invaded the leaflet was development of radiating strands of hyphae between the upper epidermis and cuticle (stage B). Such subcutinous strands might be followed immediately by blackening (stage C) with spore-bearing acervuli developing subsequently along the strands (stage E), or acervuli might develop along the strands before blackening occurs (stage D). Occasionally noticeable blackening did not develop although spore-bearing acervuli were present, i.e. R. woodsii var. fendleri frequently. Rarely acervulus-like structures lacked the typical two-celled spores of D. rosae. For these reasons examination of the spots and the contents of acervulus-like structures using magnification was necessary. Typical blackening occurred, but the fungus failed to sporulate on excised leaflets of R. bracteata in the eight day test of Jenkins (5). However, the 15 to 17 day test used in these inoculations generally resulted in sporulation. Following inoculations the more usual sequences were (a) stages A, B, either C or D, and finally E representing typical visible black spot and (b) stages A, B, and finally D. To arrange the rose cultivars in order of increasing susceptibility either the figures in column D or those in column G of Table 2 could be used. In each of the 10 inoculations, the dates for which are listed in Table 1, three or four leaflets were inoculated. If one acervulus from which typical spores of D. rosae could be obtained developed on one leaflet of the three or four, the single inoculation was positive. In Table 2, column D contains the percent of the total number of inoculations in column C in which one or more positive leaflets ultimately developed. Points (Table 3) were assigned on the basis of the condition of each leaflet on the 15th to 17th day after inoculation. The total points for all leaflets evaluated (Column E) divided by the total number of leaflets (column F) yielded an average (index of column G). The number of positive leaflets determined by the presence of typical spores and the stage to which an infection progressed are reflected in the index. If no leaflets showed evidence of invasion in any of the inoculations, the index was zero, and the rose cultivar was considered resistant to the inoculum used. The maximum possible index would be 4.00 and would occur only when all leaflet inoculated developed typical visual black spots that possessed spore-bearing acervuli.

TABLE 3.
Numerical values assigned stages of increasing severity of black spot infections on rose.a

Stage Description Points
A No evidence of infection 0
B Subcuticular hyphal strands 1
C Strands plus blackening; acervulus-like structures without spores 2
D Acervulus with spores but no blackening 3
E Acervulus with spores plus blackening 4

a Absence or presence of infection on a - or + scale would be determined by absence or presence of typical 2-celled spores of the black spot fungus. Therefore A, B and C would be negative for black spot infection and D and E positive.

In Table 2 species cultivars of roses are most resistant and commercial cultivars most susceptible to inocula obtained at Beltsville. Among the 12 most resistant cultivars (24 percent) only the two floribundas, Goldilocks and Chic, occur and among the 12 most susceptible cultivars only two species clones, Rosa canina and R. laevigata, occur. The latter rose is called Cherokee Rose in the trade. Though the two markedly resistant cultivars are floribundas, there is no evidence that any one of the three well-represented classes (floribundas, hybrid perpetuals, hybrid teas) is more susceptible than any other. The three clones of R. multiflora were resistant and the clone of R. noisettiana v. manetti was more susceptible to black spot. These results support the views of Lyle (6). R. multiflora cv. Ginn (Beltsville clone 189) had no leaflets that developed positives whereas R. multiflora cv. Chennault (5828) (Beltsville clone 51) had 22 percent and R. multiflora (Beltsville clone 165) had 29 percent of the inoculations develop positives. These results indicate that genetic variations governing black spot resistance can exist within a species. The two clones of R. wichuraiana differ, but procedural error could induce some variation. The wide variations within the five clones of R. x alba do not support this hypothesis since R. x alba is a species hybrid of unknown parentage.

Were the list of roses in Table 2 placed in sequence according to the percent of total inoculations that ultimately resulted in positives (column D) and then compared with the sequence in column G, discrepancies in the locations of some cultivars would occur. Only five cultivars would vary more than five places. If only one of three inoculated leaflets in 9 of 10 tests were to develop typical black spot, 90 percent of the tests would have been recorded positive but the index would have been low (0.90). The hybrid tea Mme. A. Meilland 'Peace' and the species clone R. blanda exemplify this type of response. If all three inoculated leaflets in 6 of 10 inoculations were positive and all three leaflets remained negative (stage A of Table 3) in the remaining four inoculations, only 60 percent of the tests could have been positive, but the index based on stages of infection would have been high (2.40). The hybrid teas Confidence and Helen Traubel exemplify this type of response. A sequence based upon column D in Table 2 would be useful mainly if immunity from black spot was the characteristic to be determined. The sequence presented, based upon column G, appears more nearly similar to the relative susceptibilities in the field.

This test does not and cannot measure the effect of black spot upon defoliation. The hybrid tea Red Radiance has been commonly used as a black spot resistant commercial cultivar (2, 7) and in field experiments at Beltsville has been paired with the hybrid tea Helen Traubel, which was thought to be much more susceptible. The comparative ratings in Table 2 clearly indicate that these two cultivars are almost equally susceptible. Obtaining an adequate supply of acervulus-bearing leaflets from the cultivar Helen Traubel grown in the field is difficult because leaves and leaflets abscise soon after invasion. Conversely, badly infected leaves are readily obtained from bushes of Red Radiance. Leaf retention after infection accounts for the use of the latter cultivar as the source of inoculum.

The analysis of data presented in Table 1 was based on a standard screen of 23 roses for which 31 leaflets were inoculated and indicates either that more cultivars are susceptible to black spot in late summer and early fall than during spring and midsummer or that black spot is more infective (virulent) then. Build-up of inoculum was not involved since the same volume of inoculum containing a standard concentration of spores was used. The period between June 27 and July 10, which just preceded the collection of leaves on July 14, was the most consistently hot period during 1959. Therefore daily, temperatures circa 90° F. may affect susceptibility to black spot in the hosts, virulence of D. rosae, or both.

LITERATURE CITED

  1. Frick, Louise. 1944. Untersuchugen über die Biologie and Pathogentkt von Diplocarpon rosae (Lib.) Wolf. Phytopath. Ztschr. 14:525-591.
  2. Geddes, Cleona H. 1952. Resistance of roses to Diplocarpon rosae—the cause of black spot. American Rose Annual 37:188-197.
  3. Green, D. E. 1932. Contribution from the Wisley Laboratory LX. Further observations on the black spot disease of roses (Diplocarpon rosae Wolf). Royal Horticulture Society Journal 57:58-62.
  4. Halsted, B. D. 1893. Fungous troubles of roses. New Jersey Agricultural Experiment Station Report 13:281 (1892).
  5. Jenkins, W. R. 1954. Variation in pathogenicity and physiology of Diplocarpon rosae (Lib.) Wolf, the rose black spot pathogen. Ph.D. Thesis. University of Maryland. 47 pp.
  6. Lyle, E. W. 1944. Understocks and black spot—Four rose understocks and their effect on the occurrence of black spot and growth of bushes. American Rose Annual 1944:160-162.
  7. McClellan, W. D., E. A. Taylor, and F. F. Smith. 1957. Relation of fungicide and miticide treatments to winter injury and spring black spot development on roses. Phytopath 47: 357-360.
  8. Piester, E. A. 1940. Relative susceptibility of rose varieties to black spot. Plant Disease Reporter 24:478-480.
  9. Rosen, H. R. 1944. Search for black spot resistance in roses. American Rose Annual 29:155-159.
  10. Seymour, A. B. 1929. Host index of the fungi of North America. Harvard University Press, Cambridge, Mass. 732 pp.