Ozone Symptom Identification Identifying Characteristics of Ozone Bioindicator Species

1. Ozone Symptom Identification Identifying Characteristics of Ozone Bioindicator Species Each slide is annotated to focus attention on certain diagnostic symptoms that may clarify what ozone injury does and does not look like in the field. Keep in mind that these are images of dried leaf samples. Injury on fresh, live plants may be slightly different in color and appearance. Reminder: If you are uncertain if a leaf injury symptom is caused by ozone exposure, please rate the plant as if it was ozone injury and send in a leaf sample for QA review. Use a plant press to prepare quality leaf vouchers. Leaves should be labeled and pressed on blotter paper with no overlap to preserve the injury symptoms. blackberry = 915 black cherry = 762; and pin cherry = 761 common and tall milkweed = 365 white ash = 541 (other ash species such as green ash are sometimes substituted) spreading dogbane = 366 (erect dogbane is sometimes substituted) big leaf aster = 364 sassafras = 931 yellow poplar = 621

2. BLACKBERRY 915 Identifying characteristics of Common Blackberry: (Rubus allegheniensis) Leaves alternate, palmately compound with 3 to 7 leaflets, averages 5, the middle leaflet tends to have a stalk and averages 31/4” to 5” in length. Upper leaf surface dark green, smooth to slightly pubescence, Lower surface green with velvety pubescence. First-year (non-flowering) canes are greenish to greenish-red, second-year (flowering) canes brown. Both first- and second-year canes are star-shaped and ridged, with stout prickles along the stem. Flowers borne in clusters, with small leaves at the base and gland-tipped hairs along the stalk, appearing from April through July. Fruit is dark violet to black, juicy, plump, and sweet, about ¾” long appearing June through August. leaflet Flower arching branches Stout prickles

3. Typical ozone injury symptoms on blackberry visible as red, interveinal pigmentation on the older foliage.

4. Classic purple-red ozone stipple occurring between the leaf veins and visible only on the upper-leaf surface. 915 Ozone stipple This is a single leaf of blackberry. A perfect leaf voucher consists of 3 leaves that have been pressed flat and that show obvious ozone injury symptoms, just like this one.

5. 915 Ozone stipple Classic ozone injury on blackberry. The photo image has caused some reddening of the leaf stipple. To the naked eye it is a darker purple-red than what is shown here. Note the line of green tissue along the leaf margins and the absence of injury symptoms where leaf overlap occurred.

6. This is not ozone injury. The underside of these leaves are covered with the dusty spores of the blackberry rust fungus. The upper-leaf surface discoloration is a uniform dark red that includes the smaller leaf veins and is not typical of ozone injury.

7. 915 Brown discoloration is not typical of ozone injury. This is not ozone injury. Bifacial, fungal-type lesions are abundant on the leaf surface.

8. This is not ozone injury This looks so much like ozone injury in the field that crews should collect this injury type and send it in for review by an expert. This is an example of a leaf with questionable ozone symptoms. With a hand lens, it is possible to see that the discoloration bleeds into all the smaller veins of the leaf. There is little classic ozone stipple. The injury appears concentrated at the leaf margins.

9. BLACK CHERRY 762 Identifying Characteristics of Black Cherry: (Prunus serotina) Leaves are 2” to 6” in length and 1 ¼” to 2” in width with finely serrated teeth on the margins, dark green and shiny on top with a paler green below. The midrib is prominently fringed beneath with white to brown hair. Fruit is a drupe that is arranged along a spreading or drooping axis at the end of a twig. The twigs have a bitter-almond smell and taste. Bark on younger trees is gray and smooth, reddish brown to nearly black, with conspicuous, narrow, horizontal lenticels. On older trees the bark is exfoliating in small, shiny, platy scales with upturned edges. Leaves of choke cherry, a similar species, have a hairless midrib beneath and are sharp toothed. Leaves of pin cherry are are longer and narrower with finely serrated margins. Brown fringe along midrib leaves

10. Classic ozone injury symptoms on a black cherry seedling. Note how the red-brown stipple is more pronounced on the mid-aged and older foliage. Ozone stipple

11. severe stippling 762 light stippling Ozone stipple no obvious stippling moderate stippling Classic distribution of ozone injury symptoms along a branch stem. Leaves toward the branch tip remain green; those toward the base of the branch show pronounced stippling.

12. 762 Classic interveinal ozone stipple. Note how fine the stippling is compared to the more blotchy, aggregated lesions that are visible on the previous slide. Some insect damage is also visible. Ozone stipple Ozone stipple

13. Leaves injured by ozone eventually lose much of their green color. Voucher leaf samples collected for QA review for ozone injury should be relatively green. Ozone stipple Good leaf samples Poor leaf samples: Ozone injured leaf is exhibiting premature senescence and has lost most of its chlorophyll.

14. Normal sized leaf sample Always collect normal sized leaves for the leaf vouchers. Avoid undersized leaves. Ozone-like stipple is visible on these undersized leaves of black cherry. There is disagreement amongst the experts as to whether ozone-like stipple on undersized leaves is attributable to ozone or is evidence of a non-specific stress response. This is not ozone injury. Undersized leaves are often found at the base of leaf branches in the inner crown. 762

15. Identifying characteristics of Common Milkweed: (Asclepiassyriaca) All parts of Common Milkweed exude latex when cut. Leaves opposite, or whorled. Twice as long (2” to 12”) as they are wide, with smooth margins and stems with latex. Upper leaf surface can be smooth to slightly hairy, lower densely hairy. Petiole is short and thick. Flowers borne in large clusters on stalks in the upper leaf axils. They appear rose or greenish-white, from June to August. Fruit is a large comma shaped pod. Monarch butterflies are a common sight on milkweed. Left photo: Common milkweed Right photo: Tall milkweed MILKWEED 365

16. Classic ozone injury symptoms on common milkweed. Note how the ozone stipple is more pronounced on the mid-aged and older foliage. Leaves at the top of the plant remain green. Towards the end of the growing season, the yellow, injured leaves at the base of the plant will drop off prematurely.

17. 365 Dark pigmented stipple on the upper leaf surface of milkweed. The veins and veinlets remain green as do the leaf margins. Ozone stipple

18. 365 Another good example of classic ozone stipple on common milkweed. The undersurface of this leaf is entirely green and free of any injury symptoms. Ozone stipple These tears in the leaf tissue are unrelated to ozone injury.

19. 365 This is a good example of a leaf voucher for milkweed. A voucher consists of 3 leaves that clearly show the ozone injury symptom. The stippling shown here is classic ozone stipple. A petiole label has been attached to the leaves to identify the ozone biosite where these leaf samples were collected. Ozone stipple State: 42 County: 13 HexNum: 4107814 PlotNum: 02 Date: 7/30/03 Species: 365 Labels may be attached to individual leaves or to a set of leaves as is shown in this slide.

20. 365 The stippled areas on the top leaf are surrounded by a yellow halo. The lesions show through to the underside of the leaf. The injury pattern is not typical of ozone stipple. This is not ozone injury. The discoloration on the bottom leaf may be attributable to hot, dry weather. No ozone stipple is apparent. This is not ozone injury.

21. On the top leaf, the stipple-like symptom clings to the leaf veins. Under magnification, many veins are discolored. The injury pattern is not typical of ozone injury. 365 This is not ozone injury. The gray-brown discoloration on the lower leaf may be due to improper voucher handling. If leaves are placed on top of each other in the plant press they will discolor in this way. Ozone injury could not be validated on this leaf sample.

22. 365 The injury pattern of the bleached and darkened tissues on this leaf sample are not typical of ozone injury. Under magnification, a stipple-like symptom is visible, but the lesions are not interveinal. The veins are discolored in the damaged areas. This is not ozone injury.

23. WHITE ASH 541 Identifying characteristics of White ash: (Fraxinus americana) Leaves are opposite and compound. Generally have 5-9 leaflets that are 2 ½” to 5” long. Twigs are round, shiny, mostly hairless. Buds are inset in the leaf scar. Bark is ashy gray, on older trees it becomes finely furrowed into close diamond-shaped areas separated by narrow interlacing ridges. Green ash = buds are set above the leaf scar. White ash = buds are inset in the leaf scar.

24. Classic purple-red ozone stipple on ash. The stippling occurs between the major veins on the upper-leaf surface. The undersurface of the leaf is green and entirely free of injury. Ozone stipple Dark brown lesion with a pale necrotic center - unrelated to ozone injury.

25. 541 Ozone stipple Severe ozone stippling on white ash. The lesions are dark purple-red in color and, under magnification, can be seen to be contained within the leaf veins. The undersurface of this leaf is clean and green.

26. This is not ozone injury. 541 Darkened lesions cover the leaf surface, often darkest along the midrib and other major leaf veins. This is not typical of ozone stipple. With a hand lens, it is possible to see white necrotic centers scattered throughout the discolored areas that may be attributable to insect feeding. There is honeydew and other evidence of insect activity on the undersurface. The leaf is generally messy and damaged by a variety of factors.. Ozone-like stipple visible along the edges of the heavily chewed tissues is assumed to be a response to a biotic stress agent, not ozone.

27. 541 The white to tan lesions visible on this leaf are characteristic of feeding activity by the ash plant bug. The undersurface of the leaf is covered with plant bug excrement. This is not ozone injury.

28. This ash leaf is infected with the ash ring spot virus. The reddened tissue is probably a result of virus infection and cannot be validated as ozone injury. Under magnification, the stippling appears less distinct than classic ozone stipple. 541 This is not ozone injury.

29. This is not ozone injury. 541 Large brown lesions have a tan necrotic center and are outlined by dark pigmented tissue. A fine stippling, similar to ozone injury, is visible in the surrounding tissue. This cannot be attributed to ozone due to the confounding presence of the more dominant stress agent. ozone-like stipple

30. 541 Leaf bronzing possibly due to exposure to hot, dry weather. The injury color and pattern are not typical of ozone injury. This is not ozone injury.

31. DOGBANE 366 Identifying characteristics of Spreading Dogbane: (Apocynum androsaemifolium) All parts exude a milky juice when broken. Leaves opposite, oval, oblong, or egg-shaped, with smooth margins and red stems. Dark green above, pale beneath, 2” to 3” long. Flowers small, pink, bell-shaped, with pink stripe in the center. Flowers appear from mid-June through August. Fruit a narrow, long pod with many seeds. Each seed has a long tuft of cottony hairs. Top and right photo: Spreading dogbane Far right photo: Erect dogbane

32. Classic upper-leaf surface ozone stipple. Note that the leaves are otherwise relatively green and healthy. The most severe stippling is towards the base of the plant stem. 366 Ozone stipple Light and scattered ozone stipple on the younger leaves Note the more pronounced stippling on the older leaves. Veins remain green.

33. Typical, dark brown to black, interveinal ozone stipple on dogbane. These leaves are so thin that the discoloration may be visible from the underside of the leaf. Ozone stipple 366

34. The brown color of these necrotic areas are not typical of ozone injury. 366 This is not ozone injury. The injury pattern (follows the leaf veins) and color of these lesions is not typical of ozone injury.

35. 366 This is not ozone injury. Clusters of necrotic lesions, especially along the leaf margin, are not typical of ozone injury.

36. This is not ozone injury. 366 The large brown lesions with a yellow margin visible on these 2 leaves are not symptomatic of ozone injury. On the smaller leaf it is possible to see ozone-like stipple on the leaf surface. However, because this stipple emanates from the larger fungal-type lesions, we can not consider this an ozone response. ozone-like stipple fungal-type lesions

37. BIG LEAF ASTER 364 Identifying characteristics of Big Leaf Aster: (Aster macrophyllum) Leaves are 4” to 8” in width, heart-shaped, with unevenly toothed margins. Leaf stem is purplish and nearly as long as the length of the leaf. Flowering stalk can reach 5’ in height. Flowers range in color from pale lavender to violet to light blue and are evident from August to September.

38. 364 Classic ozone stipple on big leaf aster. The stippling appears dark brown to black on the dried leaf samples. It is distinctly interveinal. There is no visible injury or discoloration on the undersurface of the leaf. Ozone stipple Larger, brown lesions are unrelated to ozone injury.

39. 364 Ozone stipple Dark pigmented ozone stipple. With a hand lens, it can be seen that the pin cushion like stipples are entirely contained by the leaf veins.

40. This is not ozone injury. 364 These large dark lesions of unknown origin are bifacial and appear to cross the smaller veins of the leaf.

41. 364 The tan stippling on this leaf sample obviously clings to the leaf veins as is characteristic of feeding injury caused by a phloem sucking insect. Insect excrement is visible on the undersurface of the leaf. This is not ozone injury.

42. SASSAFRAS 931 Identifying characteristics of Sassafras: (Sassafras albidum) Three leaf types: 1) entire and somewhat elliptical, 2) mitten-shaped, 3) three-lobed. All three leaf types can be found on the same tree. Twigs and leaves have a spicy odor and taste. Fruit is a blue drupe. Bark is red-brown, thick, furrowed and aromatic.

43. Classic ozone stipple on sassafras. Note how the red-brown stipple is more pronounced on the older foliage. Major leaf veins remain green.

44. Ozone injury on sassafras in the field is often described as leaf bronzing rather than leaf stippling. The injury pattern is clearly interveinal. The color on pressed leaf samples appears more brown or tan than purple-red. Ozone stipple leaf bronzing 931

45. Closer view of the bronze lesions characteristic of ozone injury on sassafras. 931 Ozone stipple Larger zones of necrotic tissue are unrelated to ozone injury.

46. Ozone stipple Photo Credit: ‘Diagnosing Injury to Eastern Forest Trees - Manual for Identifying Damage Caused by Air Pollution, Pathogens, Insects, and Abiotic Stresses’. Skelly, J.M. et al., 1987. Available from: Publications Distribution Center, Pennsylvania State University. Pigmented ozone stipple on sassafras at a field site used for biomonitoring.

47. Leaf sample with circular clusters of small pigmented spots that suggest infection by a biotic pathogen. This type of injury pattern is not characteristic of ozone injury. 931 This is not ozone injury.

48. SWEETGUM 611 Identifying characteristics of Sweetgum: (Liquidambar styraciflua) The leaves are star-shaped, deeply and palmately 5- to 7-lobed. Margins are finely serrated. Leaf surface is bright green above; pubescent in axils of the veins below; somewhat fragrant when crushed. Fruit is a 1” to 1 ¼” spiny ball, often hanging. Twigs are shiny, green to yellowish brown, somewhat fragrant when crushed. Bark is grayish brown; deeply furrowed into narrow, flaky ridges.

49. 611 Classic purple-red ozone stipple on sweetgum Ozone stipple Small pigmented stippling occurs between the large and small veins of the leaf. When stippling is severe, the color may be faintly visible on the undersurface of the leaf. Darker pigmented lesions with lighter centers tend to sit on the leaf veins. They are unrelated to ozone injury.

50. The leaf undersurface is free of ozone injury. Ozone stipple State: 3907365 County: 04 HexNum: 3008636 PlotNum: 1 Date: 8/19/03 Species: 611 A well prepared leaf voucher consists of 3 leaves that clearly show the upper-leaf surface pigmented stipple characteristic of ozone injury on sweetgum. All samples must be identified by state, county, hex number, and plot number, as well as sample date and species code.