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Grulke NE 2003 Physiological basis of ozone injury assessment in Sierra Nevadan conifers. In: Bytnerowicz 

A, Arbaugh M, Alonso R. Assessment of ozone distribution and its effects on Sierra Nevada 

ecosystems. Elsevier Publishers, The Hague, Netherlands (in press). 

The physiological basis of O3 injury assessment attributes 

in Sierran conifers 

N.E. Grulke 

Pacific Southwest Research Station, USDA Forest Service 

4955 Canyon Crest Drive, Riverside, CA 92507 

Abstract 

The generalized effects of ozone (O3) exposure on plants are well known, but translating 

responses known under controlled conditions to effective tools for assessments under 

field conditions can be complex. There are two methods generally used for assessing 

yellow pine (ponderosa and Jeffrey) response to oxidant exposure in the Sierra Nevada: 

the Ozone Injury Index (OII) and the Forest Pest Management (FPM) assessment. For 

both methods, chlorotic mottle and needle retention form the basis of the assessment. The 

response of these two key attributes to cumulative O3 exposure and uptake, nitrogen 

deposition (either associated with O3 deposition as a correlative environmental factor or 

as an experimental manipulation), and drought stress are described using field examples 

from both ponderosa and Jeffrey pine. Specific suggestions are offered for future, 

repeated field assessments of O3 injury for consistency, and to help interpret historical 

data sets. 

Key words: O3 injury assessments, chlorotic mottle, needle retention, ponderosa pine, 

Jeffrey pine, drought stress 

Introduction 

The generalized effects of O3 exposure on plants are well known, and have been 

reviewed from several viewpoints over the last decade (De Kok and Tausz, 2001; Heath 

and Taylor, 1997; Pell et al., 1997; Schrauder et al., 1997; Matyssek et al., 1995; 

Bytnerowicz and Grulke, 1992). In general, oxidant exposure results in lower 

photosynthetic capacity, higher respiration, lower stomatal conductance, lower nutritional 

content of tissues, as well as elemental imbalances. Oxidant exposure alters within plant 

priorities for resources: less carbon is allocated to roots, and less foliar biomass is 

retained. 

The specific effects of O3 exposure on conifers are well described (Darrall, 1989; Reich, 

1987). Most of the research on O3 effects has been conducted on herbaceous species (i.e., 

crops). Several important differences between herbaceous and coniferous species affect 

the total foliar uptake of the pollutant, the timing of the uptake, and the effect of multiple 

stressors. However, the underlying mechanisms of O3 injury response in conifers and 

herbaceous species are assumed to be similar. 

One of the most important differences between herbaceous and conifer species relevant to 

oxidant exposure is lower stomatal conductance. Stomatal conductance in conifers is 
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roughly half that of herbaceous species, so that at the same O3 exposure level, O3 uptake 

in conifers is proportionately reduced. However, conifer foliage is generally longer-lived, 

and active over a greater portion of the year. A longer period of physiological activity 

increases the total O3 uptake over the course of the year, and greater foliar longevity 

increases total O3 exposure over the lifetime of the leaf. Conifers are physiologically 

active in early spring and late fall, during times of lower oxidant concentrations. These 

periods can contribute significantly to a net positive carbon balance over the course of the 

year. Patterson and Rundel (1989) reported that Jeffrey pine had significant stomatal 

opening (one third that of a summer day) in mid winter with 2 m of snow on the ground. 

At least pole-sized and larger trees can mitigate reductions in carbon acquisition due to 

oxidant exposure in the summer with carbon assimilation on favorable days in the winter. 

Conifers have thicker cuticles relative to herbaceous species. Although cuticular uptake 

of O3 is believed to be negligible (Kerstiens and Lendzian, 1989), other associated 

pollutants (e.g., HNO3) in the southern Sierra Nevada and in the Transverse Range may 

compromise cuticular integrity (Bytnerowicz et al., 1999) and contribute to the total 

stress experienced. Needle longevity may be an advantage from the point of view of 

nutrient storage important in reparation responses to pollutants. However, cuticular 

lesions may be more common in older needles and trans-cuticular loss of water is 

unlinked to plant carbon gain. The potential for “leaky” cuticles to contribute to whole 

canopy pollutant uptake in mature trees (not to be confused with pollutant deposition to 

foliar surfaces) has yet to be quantified under field conditions. 

The longevity of the whole tree also holds advantages and disadvantages for response to 

pollutants. Greater longevity of conifers translates into greater environmental volume 

from which to scavenge resources, a greater proportion of resources carried over from 

year to year and the potential to buffer the individual from environmental stressors and 

allocate more resources to plant defense (antioxidants). Conversely, longevity can also 

work against the tree in the form of multiple, cumulative stressors. A series of drought 

years can reduce O3 uptake, but can also reduce carbon and nutrient acquisition, reducing 

resource allocation to defenses (antioxidants, resins against insect infestation), rendering 

the tree more susceptible to O3 injury in subsequent year of greater precipitation, greater 

stomatal conductance, greater O3 uptake, and injury. These differences in resource 

acquisition and allocation all contribute to altering carbon and nitrogen dynamics within 

the tree. 

Nitrogen deposition modifies the effects of oxidant exposure. Nitrogen deposition, in wet 

or dry particulate form, ultimately increases site fertility. This can occur to excess (“N 

saturation”) as has been shown by Fenn et al. (1996). Increased nitrogen availability 

decreases carbon allocation to roots, further exacerbating the effects of oxidant exposure 

on roots. Increased nitrogen availability also exacerbates O3 exposure effects on foliage 

turnover: fewer needle age classes are retained (Gower et al., 1993). Nitrogen 

amendments counteract the effect of oxidant exposure on photosynthesis by increasing 

nitrogen available for photosynthetic pigments and enzymes, increasing stomatal 

conductance, but deleteriously resulting in increased O3 uptake. On the other hand, 

nitrogen amendments may mitigate the degree of foliar injury from oxidant pollution via 

higher nitrogen available for reparation of photosynthetic pigments. Nitrogen 

amendments also modify the antioxidant defense system in complex ways (Polle, 1998). 
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In additional to these effects, estimated stomatal uptake of gaseous nitrogen oxides may 

be significant (Bytnerowicz et al., 1996) and phytotoxic (Tausz, 2001). 

Over much of the Sierra Nevada, nitrogen deposition is not believed to be significant in 

the mid elevation forests. In the south central Sierra Nevada, estimates of deposition are 

in the range of 2-4 kg ha-1yr

-1 (Bytnerowicz et al., 2002; Chorover et al., 1994). However, 

during prescribed or wildfire events, local NOx concentrations can become quite high 

(Bytnerowicz et al., 2002). With increasing population, fossil fuel use, continued 

excessive fertilizer use in the San Francisco Bay area and San Joaquin Valley, and 

increasing anthropogenically-caused fires, we can expect nitrogen deposition to Sierra 

Nevada ecosystems to become a chronic problem. In areas experiencing moderately high 

O3 and above (e.g., > 280 ppm h based on 24 h exposure over the 6 month growing 

season) such as the southern Sierra Nevada, nitrogen deposition may also be a significant 

co-stressor. In Sequoia National Forest near Mountain Home, high nitrogen deposition is 

suspected to cause forest tree responses similar to those found in the Transverse Range in 

southern California (Fenn et al., this volume). 

The focus of this chapter is western conifer response to oxidant exposure. Most of the 

examples are based on ponderosa and Jeffrey pine (Pinus ponderosa Dougl. Ex Laws. 

and Pinus jeffreyi Grev. & Balf.) responses because these two species are present on 

nearly all of the forest plots surveyed for O3 injury in the Sierra Nevada. Ozone exposure 

in the Sierra Nevada is greatest at lower elevations, and decreases with increasing 

elevation and distance from source (Bytnerowicz et al., 2002). Consequently, coniferous 

species that form lower treeline, such as Coulter (Pinus coulteri D. Don) and ponderosa 

pine, will be exposed and potentially have much greater injury than species at upper 

treeline, such as lodgepole pine (Pinus contorta Dougl. ex. Loud.), red fir (Abies 

magnifica A. Murr.), and Jeffrey pine. The mid-elevation, Sierran mixed conifer zone is 

dominated by white fir (Abies concolor (Gord.& Glend.) Lindl. ex Hildebr.), ponderosa 

and Jeffrey pine, incense cedar (Libocedrus decurrens Torr.), and relatively small 

amounts of sugar pine (Pinus lambertiana Dougl.). Unique to the Sierra Nevada are also 

stands of giant sequoia (Sequoiadendron giganteum (Lindl.) Buchholz) locally dominant 

in “groves.” Of these species, ponderosa and Jeffrey pine are the most sensitive conifers 

to O3 exposure (Miller et al., 1983), and as such, are the “bell weathers” for forest health 

in the Sierran mixed conifer zone. 

The interaction of environmental factors, plant phenology (the timing of growth events 

and birth and mortality of plant parts), physiological status (nutritional or moisture status, 

and dormant or actively growing within the year), and tree age (interannual differences in 

resource acquisition and requirements for allocation) all contribute to the complexity of 

conifer response to O3 exposure. Of relevance to this book, however, is the translation of 

the whole tree physiological response to O3 exposure, in the collection of relatively few 

morphological (visible) attributes that are used to assess O3 injury in the field. 

The objective of this chapter was to describe seasonal variation in attributes used to 

assess O3 injury, under a range of O3 exposures and nitrogen deposition fluxes typical for 

the Sierra Nevada. The relationship between O3 exposure and O3 uptake, and their 

influence on the biological response attributes used to assess injury in the field will be 

discussed. Responses in different microsites (mesic vs. xeric) and in years of significantly 

different water availability (average or above and 20% below average) will be used to 
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elucidate the effect of drought as an additional environmental stressor. Perhaps the most 

useful application of this information is to help determine in what month, and in which 

year to make consistent O3 injury assessments based on what we know about the effect of 

environmental factors and temporal variation in attributes. 

Attributes used in O3 injury assessments of conifers 

The attributes used in O3 injury assessments include foliar chlorotic mottle and or 

necrosis, both branch (number of needle age classes retained) and within-whorl needle 

retention, needle length, and proportion of the live crown remaining. Trees used in 

assessment have already been selected for dominance or co-dominance, and to minimize 

the effect of pathogens (bark beetle, mistletoe, bud worm). 

There are two commonly used assessment methods used in Sierra Nevadan forests: O3

injury index (OII; Miller et al., 1996a) and Forest Pest Management (FPM, Pronos et al., 

1978). In the OII assessment, five attributes are utilized for dominant or co-dominant 

yellow pine: chlorotic mottle, number of whorls retained, needle retention within whorls, 

needle length, and percent live crown. The higher the OII score, the more oxidant injury. 

In the FPM assessment, the first needle age class that exhibits chlorotic mottle for more 

than one third of that foliage age class is recorded. In this assessment, the lower the score, 

the more oxidant injury. Arbaugh et al. (198) has recently described these methods in 

more detail, as well as their development. 

Additional attributes correlated with oxidant exposure have also been reviewed and 

statistically assessed for significant correlation with level of chlorotic mottle in Grulke 

and Lee (1997). In that paper, eight attributes were highly correlated with level of 

chlorotic mottle: foliar nitrogen or chlorophyll content (interchangeable in the 

multivariate analysis), number of whorls retained, a quantitative measure of within whorl 

retention, a measure of wood production (basal area increment or branch diameter), 

spring root carbohydrate content, and distance to the nearest conspecific neighbor as a 

measure of inferred competition. Foliar antioxidant concentrations and activities have 

also been clearly correlated with oxidant exposure levels and degree of foliar chlorotic 

mottle (Tausz et al., 2001). Tissue chemistry, and biochemical attributes (carbohydrates, 

pigments, antioxidants) are currently not a component of any field assessment of oxidant 

injury: they are costly, and time consuming to assess. However, these as well as other 

attributes may be chosen for future assessments. Biochemical attributes may be 

particularly important in assessing response of species that are asymptomatic. 

What is chlorotic mottle? 

Of all the attributes used in O3 assessment on many different species in the Sierra 

Nevada, visible injury to the leaf or needle is the first step of O3 injury assessment. In 

yellow pine, the visible injury is chlorotic mottle (Fig. 1; Miller et al., 1996a; 1963). The 

two most common assessments, FPM and OII survey yellow pine. The FPM method of 

O3 assessment is wholly based on the first needle age class with significant chlorotic 

mottling, functionally greater than 10% on more than a third of the needles of that whorl. 

Forty percent of the OII score is based on chlorotic mottle of all needle age classes 

retained (up to 6 years). 

Although no quantitative assessment techniques similar to that used for yellow pine have 

been developed for other species, foliar symptoms may also develop in response to O3
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exposure. In white fir, chlorotic mottling on the needle margins or at the tip or necrosis is 

apparent with O3 exposure (Taylor et al., 1980). Seedlings of giant sequoia up to three 

years old also have chlorotic mottling of needles under current ambient pollutant levels 

(Miller et al., 1996b). Older giant sequoia trees at ambient or experimentally elevated O3

levels do not have visible symptoms on foliage (Grulke et al., 1989; 1996). Incense cedar 

is also asymptomatic at ambient O3 levels. Other pines, such as lodgepole and sugar 

appear to be more tolerant to O3 exposure than the yellow pines (Miller et al., 1983), but 

have similar symptoms: chlorotic mottle that begins near the tip. Most of the pines 

exhibit some foliar injury at current ambient O3 levels (approximately 260 ppm h, 

cumulative O3 exposure over the 6 month growing season; National Park Service ARD 

data base) in Sequoia National Park, the most polluted National Park in the country. 

Because ponderosa pine is the most sensitive conifer to oxidant exposure, some 

individuals exhibit visible symptoms even at relatively clean sites in the southern 

Cascade Mountains in California (Grulke, 1999). There, O3 concentrations are just 

slightly elevated over global background levels (38 to 42 ppb per hour averaged over the 

six month growing season; Grulke, 1999). At this O3 exposure level (180 ppm h), 1% of 

the trees exhibited foliar injury symptoms. 

Because O3 concentrations are commonly moderate and above during the growing season 

in the western United States (Lee and Hogsett, 2001), and because of its high 

phytotoxicity, O3 probably has the most negative effect of any air pollutant at ambient 

levels on tree vigor and growth (Reich 1987). Once in the substomatal cavity, O3 reacts 

with water and or cell membranes or walls to form reactive oxygen species (Polle, 1998). 

It is not clear if reactive oxygen species are caused by reaction of O3 and water (Byvoet 

et al., 1995; Grimes et al., 1983), or O3 and constituents of the cell wall and membrane 

(Mehlhorn et al., 1990). Highly reactive oxygen species disrupt membrane integrity, 

pigment structure, and oxidize proteins (Mehlhorn et al., 1986) contributing to chlorotic 

mottle development and or cell death. Reactive oxygen species may also act as a signal to 

initiate or coordinate other processes such as ethylene production, which induces 

senescence (Pell et al., 1997; Schraudner, et al., 1997). Reactive oxygen species are 

detoxified by antioxidant systems in the apoplasm, cytoplasm, and chloroplast (Alscher et 

al., 1997; Heath and Taylor, 1997; De Kok and Tausz, 2001). 

Photosynthesis is inhibited by O3 exposure (Weber et al., 1993): both pigment structure 

and RUBP carboxylase activity are altered (Schweizer and Arndt, 1990). When the 

photosynthetic pigments have been damaged, the pigment must be fully broken down 

(and or new N and Mg must be taken up and transported to the leaf) for pigment to be 

regenerated (Bjorkman and Demig-Adams, 1995). The relationship between chlorotic 

mottle estimates and chlorophyll content or leaf nitrogen content is not precise (Fig. 2). 

Chlorotic mottle is by definition patchy and on a whole needle basis, nitrogen content on 

a per leaf basis may not differ between symptomatic and asymptomatic needles. Needles 

exposed to both O3 exposure and drought stress had higher nitrogen content than that of 

controls because significant foliar biomass was lost, and nitrogen may have been 

translocated out of senescing into remaining needles (Temple and Riechers, 1995). This 

explains in part for the within-growing season changes in chlorotic mottle estimates: 

older needles (generally more symptomatic) within the whorl may be lost through the 

growing season, and thus the chlorotic mottle estimate decreases for the whole whorl. 
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Higher chlorotic mottle is significantly correlated to lower net assimilation in Jeffrey pine 

(Patterson and Rundel, 1995). 

Factors influencing the expression of chlorotic mottle 

Perhaps the simplest demonstration of the effect of environmental stressors on the 

expression of chlorotic mottle can be made by plotting the relationship between chlorotic 

mottle and cumulative O3 exposure or uptake for three sites differing in pollutant 

exposure and between years with average or below-average total annual precipitation 

(Fig. 3). These sites have been described previously (O3 exposure, Grulke, 1999; O3

uptake, Grulke et al., 2002), and range from roughly 60 (Barton Flats) to 70 (Strawberry 

Peak), to 80 ppb (Crestline) average O3 concentration per hour (24 h per day, April 15 

through October 15), and low (6-9 kg ha-1

yr

-1) to high nitrogen deposition (30-40 kg ha1

yr

-1; Kiefer and Fenn, 1997). 

In an average precipitation year, percent chlorotic mottle increased regularly with 

cumulative O3 exposure, but the same level of cumulative O3 exposure did not elicit the 

same level of chlorotic mottle between sites in that year. In the 20% below average 

precipitation year, chlorotic mottle developed more quickly at the moderate and 

moderately high pollution site: the peak expression occurred at the end of June. At both 

of these sites, there was a significant reduction in chlorotic mottle in the last month of the 

growing season at the two most polluted sites, and in the last two months of the growing 

season at the moderate pollution site. Drought stress decreased chlorotic mottle of the 

whole whorl because the older, more damaged needles within the whorl were lost (Miller 

et al., 1996b). When cumulative O3 uptake (calculated by a physiologically based model, 

or empirically) is used instead of exposure in the relationship, there was no improvement 

in the relationship with percent chlorotic mottle. These results strongly suggest that 

interannual comparisons of ozone injury should not be made between years that 

significantly differ in soil moisture availability (e.g., average vs. below-average 

precipitation years). 

The most polluted site based on cumulative O3 exposure was also the site of the greatest 

O3 uptake by the trees, whether it was a year of average or below-average precipitation. 

The converse was true for the moderate pollution site, Barton Flats (Grulke et al., 2002a). 

The maximum chlorotic mottle within the growing season roughly followed the rank 

order of cumulative O3 exposure at the three sites (Fig. 4). For example, at the most 

polluted site, maximum chlorotic mottle was 35% at the most polluted site, 20% at the 

moderately high pollution site, and 8% at the moderate pollution site. In the drought year, 

the seasonal maximum occurred at much lower cumulative O3 exposure. If the seasonal 

minimum and maximum percent foliar chlorotic mottle from all sites is plotted with 

respect to cumulative O3 exposure, there appears to be a relationship (increase in 

chlorotic mottle with increase in O3 exposure), but the relationship is not statistically 

significant (Adj. r2

 = 0.31; P = 0.06). A slightly better fit was obtained with cumulative 

O3 uptake (Adj. r2

 = 0.35; P = 0.04). However, the cumulative O3 exposure at the time of 

maximum seasonal expression was not significantly correlated to the maximum percent 

chlorotic mottle. Cumulative O3 uptake as calculated by a physiological model, 

TREGRO, had the same predictive capability as that of statistically calculated O3 uptake 

(Adj. r2

 = 0.34; P = 0.04; CC Fan and WA Retzlaff, unpubl. simulation for ponderosa 

pine using the same data set). Site O3 exposure alone does not wholly explain the 

physiological response and may interact with environmental factors in unexpected ways. 
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However, a significant correlation coefficient was obtained between a chlorotic mottle 

score and several O3 exposure indices (7 and 24 h SUM00, and 7 and 24 h SUM06; 

Salardino and Carroll, 1998) with a greater number of sites distributed along the Sierra 

Nevada. 

Foliar oxidant injury usually accelerates only after the upper soil horizons have dried out 

(Grulke and Balduman, 1999). Although foliar N and chlorophyll at high nitrogen 

deposition and O3 exposure sites is high early in the summer, foliar N and chlorophyll 

content decreases with increasing O3 exposure after mid summer (Grulke 1999). 

Cumulative O3 exposure, high radiation, and drying of the upper soil horizons 

(insufficient uptake of N to replenish damaged pigments) perhaps all contribute to the 

expression of chlorotic mottle. Cumulative O3 exposure early in the summer is significant 

(Grulke et al., 2002a), but upper soil horizons are rich in nitrogen and fine root mass is 

high until they dry out, even in a year of average precipitation, in mid to late July. When 

fine roots (and associated mycorrhizae and microbial communities) die off in nearsurface soil horizons, trees rely on deep roots for water (Hubbert et al., 2001). Even at 

sites where the ground water is also rich in nitrogen (Fenn and Poth, 1999), chlorotic 

mottle still develops. Either deep roots (and associated mycorrhizae) are not as effective 

at nitrogen uptake, or oxidant damage is too great for reparation activities to keep up with 

N uptake from the pedosphere. In the autumn with favorable soil temperatures and 

moisture permitting, fine roots and mycorrhizae grow in the near surface soil horizons, 

and chlorotic mottle can significantly decrease (Fig. 5). 

Because OII integrates foliar response of up to six needle age classes, theoretically it 

should not be correlated to cumulative O3 exposure in any one year of assessment unless 

site cumulative O3 exposure is fairly consistent from year to year. In order to generate a 

meaningful value for site O3 exposure, O3 exposure should be accumulated over the 

average number of years that needles are retained on the branches, as described in 

Arbaugh et al. (1998) and tested in Salardino and Carroll (1998). The OII scoring system 

works well to rank significant differences in site to site injury level. The FPM is also a 

robust scoring system that works well to coarsely rank stand injury under a wide range of 

environmental co-stressors. Integrating cumulative O3 exposure for this score is even 

more problematic than for OII because chlorotic mottle is not integrated over all needle 

age classes, but is based on the needle age where chlorotic mottle is first signficantly 

expressed. In both scoring systems, chlorotic mottling is modified by the degree of 

overwinter reparation of damaged pigments (see Fig. 5). 

Perhaps the most effective use of the FPM and OII score would be to conduct canopy 

assessments in average or above-average precipitation years, preceded by at least one 

average or above precipitation year to best rank injury in widely ranging geographic sites. 

Assessment in a below-average precipitation year, or of stands with a disproportionate 

number of trees in xeric microsites could significantly decrease the OII score, or increase 

the FPM score (Fig. 6) if assessments were made on the same day of year. Both scores 

reflect a microsite difference in O3 uptake, based on both leaf-level and canopy-level 

measurements (Grulke et al., subm.). Trees in mesic sites had 20% greater seasonal O3

uptake than Jeffrey pine trees in xeric sites. Choosing trees in consistently mesic 

microsites should increase the chance of a relationship between site O3 exposure and 

foliar injury development. Assessing canopy injury at the same phenological stage (such 

as near or at the end of needle elongation, correlated with upper soil horizons drying to 
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<4% soil moisture; Grulke et al., 2001; 1998) would significantly aid in interannual 

comparisons of foliar injury. 

The level of chlorotic mottle could differ between sites with the same cumulative O3

exposures because of associated nitrogen deposition at these sites. Greater nitrogen 

availability could aid repair of damaged pigments, but it could also increase stomatal 

conductance (via stimulated photosynthesis), and thus O3 uptake. From a biochemical 

point of view, nitrogen-limited trees had greater antioxidant concentrations than that of 

controls (Polle, 1998). Conversely, excess nitrogen in fertilizer manipulations increased 

secondary metabolites with antioxidizing capacity (Pell et al., 1995). 

Experimental applications of nitrogen to trees in consistently mesic or xeric microsites on 

the same slope may help determine the direction of change expected with concurrent 

environmental stressors (O3 exposure, drought) and enhancers (nitrogen deposition). 

After three years of experimental amendments of nitrogen availability (+50 kg ha-1yr

-1), 

mid canopy chlorotic mottle decreased, foliar nitrogen increased slightly, foliar carbon 

decreased, and foliar C:N decreased relative to control trees in mesic microsites for 

mature Jeffrey pine in Sequoia National Park (Fig. 7). The opposite trends occurred in 

midcanopy foliage of trees in xeric microsites. In a similar experiment, N-fertilized (+50 

kg ha-1yr

-1) mature yellow pine at a dry site had lower bole growth than that of controls 

(Fenn, unpubl. data). The two experiments had similar background O3 exposures, 

approximately 260 ppm h accumulated over the growing season. At a high pollution site 

(approximately 320 ppm h seasonal O3 exposure, 30-40 kg ha-1yr

-1 background N 

deposition) with more moisture availability, additional N amendments (+50 kg ha-1yr

-1) 

increased bole growth. In dry sites, perhaps N amendments decrease fine root mass and 

pedosphere N uptake. In mesic sites, fine root (+ mycorrhizae) may be able to take up and 

transfer more N to the foliage. Perhaps foliar carbon content of fertilized trees is lower in 

mesic sites because of within-tree allocation to bole growth (or other tissue), Nstimulated foliar growth reduced specific leaf weight, or increased respiration due to 

greater O3 exposure in this site. 

The level of chlorotic mottle could differ between sites with the same cumulative O3

exposures because of differences in drought stress. Jeffrey pine in xeric microsites had 

20% lower O3 uptake than mesic microsite trees, but chlorotic mottle was greater at all 

canopy levels in current and previous year needles, and in four year old needles in the 

upper canopy (Fig. 8; Grulke et al., subm.). This may be a field example of free radical 

production within the chloroplast due to drought stress, lower stomatal conductance, 

lower Ci, and high radiation loads (Tausz et al., 2001; Foyer et al., 1994). These trees had 

greater antioxidant concentrations relative to mesic microsites in June, prior to the onset 

of seasonal drought stress (Tausz et al., 2002). In late summer, drought stress and O3

uptake combined to decrease carbon acquisition in ponderosa pine (Grulke et al, 2002b). 

In simulations with white fir, a 25% reduction in annual precipitation at moderate O3

levels was protective against injury as assessed by tree biomass (Retzlaff et al., 2000). 

Drought stress imposed at greater levels in combination with moderate O3 exposure was 

deleterious to carbon acquisition in white fir. 

Factors influencing needle retention (branch-level; whorl-level) 

There are two types of needle retention measurements of interest in O3 injury 

assessments: the number of live needle age classes on the branch, and the within needle 
 9

age class (whorl) retention. The former is represented with a simple count, and the latter 

is either estimated by thirds (Miller et al., 1996a) or measured ([length of the branch with 

live needles] / [branch elongation growth]) in a given year; Grulke and Balduman, 1999). 

In OII, the branch-level needle retention (the number of needle age classes or whorls 

retained on the branch), and the within-whorl needle retention is 40% of the O3 injury 

score. Long-lived foliage acts as a storage location for nitrogen, but with a carbon cost 

for maintenance (Chapin 1980). Ozone exposure accelerates needle senescence (Heath 

and Taylor 1997; Pell and Dann, 1991), with the net result that N can be translocated out 

of senescing tissue to replace damaged photosynthetic pigments. Ozone exposure can 

also increase foliar respiration due to reparation: it may become too costly to maintain 

older needles with lower assimilation rates. 

Nitrogen deposition, positively correlated with O3 exposure, also accelerates needle loss 

(Gower et al., 1993), but via a different mechanism. Nitrogen amendments increase foliar 

nitrogen contents, and older leaves may have redundant nitrogen with too high a carbon 

cost for maintenance. Trees with nitrogen amendments in Sequoia National Park had 

higher foliar nitrogen content, and fewer needle age classes relative to control trees in 

mesic microsites (Fig. 7; Grulke, unpubl. data). 

The FPM score is described as the youngest needle age class with significant chlorotic 

mottle development. However, the whorl number and the level of chlorotic mottle may 

co-vary, as suggested by the ratio of chlorotic mottle and foliar retention as a new index 

(“CM/FR,” Salardino and Carroll, 1998). Ponderosa pine trees with known allelic and 

genotypic diversity at a site with both high O3 and nitrogen deposition illustrate the 

interaction between chlorotic mottle and needle retention (Staszak et al., subm.; PrusGlowacki et al., 1999). Ponderosa pine with a genetic signature indicating sensitivity to 

O3 had two strategies: (1) many needle age classes retained, but high chlorotic mottle 

across all needle age classes (e.g., FPM score, 0); and (2) few needle age classes retained, 

but little chlorotic mottle (e.g., FPM score, 3). Also, ponderosa pine with a tolerant O3

genetic signature less than 2 m from a conspecific tree had morphological and tissue 

chemistry attributes similar to a sensitive tree (lower foliar nitrogen, higher chlorotic 

mottle, fewer needle age classes) than typical for a “tolerant” tree. The genetic, 

morphological, and tissue chemistry attributes for 60 trees were identified using cluster 

analysis (Staszak et al., subm.). 

Drought stress also reduces both the number of needle age classes retained, and retention 

within a needle age class. The response of both types of needle retention is illustrated in 

Fig. 9 for four sites differing in pollutant exposure. The number of needle age classes 

retained significantly declined with increasing pollutant exposure, as has been described 

previously by both Pronos et al. (1978) and Miller et al. (1996a). At the relatively clean 

atmosphere site, there was little difference in within-whorl needle retention between an 

average and below-average precipitation year. In the southern Californian sites within 

increasing pollutant exposure, there was significant loss of both whorls and within-whorl 

needle retention in a drought year. Drought stress acts synergistically with O3 exposure to 

promote premature senescence of foliage. Both nitrogen (from reduced root mass and 

uptake) and carbon (increasingly lower net carbon balance with reduced stomatal 

aperature, lower assimilation, and constant respiratory losses) may be limiting in a 

drought year to facilitate this response. 
 10

The quantitative measure of within-whorl retention is a better measure of needle retention 

than the estimated needle retention score of Miller et al. (1996a) for ponderosa pine. 

Ponderosa pine loses needles regularly from the lower or oldest needles to the upper or 

younger needles within a whorl. However, Miller et al.’s (1996a) needle retention score is 

a better measure for Jeffrey pine because needles are lost erratically within the whorl. 

Male cone scars can sometimes be mistaken for needle loss within the whorl: their scars 

would reduce estimated needle retention and increase the OII score (implying greater 

injury). Each male cone develops at the expense of fascicle production (for lodgepole 

pine, McDick et al., 1990), thus needle mass will necessarily be lower in whorls that bear 

them. Female cones develop from terminal buds, thus form at the expense of branch 

growth (for Monterey pine, Pinus radiata D. Don, Cremer 1992). Both male and female 

cone production accounted for 5% of total aboveground production in this species, but 

this would not be detected as a reduction in crown mass in a field assessment. 

Within-canopy variability in O3 injury expression 

Both OII and FPM assessments are conducted in lower canopy due to ease of access for 

extensive surveys. To illustrate the difference in expression in O3 injury within the 

canopy, morphological and tissue chemistry attributes in three canopy positions of 30 

trees each in mesic and xeric microsites were measured. The following attributes were 

used in the multivariate analysis: chlorotic mottle, % foliar nitrogen and carbon, foliar 

C:N, average number of needle age classes retained, proportion of the branch foliated, 

and branchlet diameter. These attributes were chosen based on the statistical analysis of 

ponderosa pine described in Grulke and Lee (1997), and subsequent application to Jeffrey 

pine (Grulke et al., subm.). Table 1 provides summary statistics for these attributes for 

upper, mid, and lower canopy of mesic and xeric trees (prior to multivariate grouping). 

In contrast to that reported by Muir and Armentano (1988) for ponderosa pine, needle 

retention (number of whorls retained on the branch and within-whorl foliar retention) in 

the lower canopy was significantly lower than that in the upper canopy of Jeffrey pine, 

regardless of microsite. Less foliage in mid-canopy in mesic microsites may be an 

indication of greater O3 uptake in this microsite. The greatest portion of the canopy foliar 

mass is in the middle third of the tree, followed by the upper canopy, then the lower 

canopy in ponderosa pine (from whole trees harvested in Grulke and Balduman, 1999). 

Foliar loss in mid canopy due to oxidant injury, or oxidant and drought stress, is perhaps 

more critical than loss in lower canopy. Except for the number of whorls retained on the 

branch and % foliar N, all other attributes significantly differed between microsites. 

Except for % chlorotic mottle, needle length, and % foliar carbon, all other attributes 

measured significantly differed with canopy position. 

A tree model (Fig. 10) was used to illustrate which attributes, at what values 

differentiated the two groups of trees (1.0 = all mesic trees; 2.0 = all xeric trees; values 

between 1 and 2 indicate the relative proportion of trees in the two microsites) at each 

canopy level. For example, the average number of whorls retained was not used to 

differentiate groups in the mid canopy, but was significant in the upper canopy. Whorl 

number was marginally useful in lower canopy in differentiating groups. Some attributes 

(the proportion of the branch foliated; foliar N and C content) significantly assisted in 

differentiating between groups of trees at all canopy levels. 
 11

Divisive clustering (S-Plus 2000; figures not presented) was applied to sort trees into 

different groups according to the value of the same attributes (used in the tree model 

above) in each canopy position. This multivariate technique yielded the best separation of 

microsites, with the highest cluster coefficient. However, the groups were robust: other 

multivariate analyses yielded the same end groups. Many different combinations of 

attributes were used to optimize the resulting cluster coefficient. Although some of the 

attributes could have been excluded for one or more of the canopy positions, a consistent 

approach was used across all three canopy levels. 

Clustering analysis applied to the three canopy layers differed in the number of groups 

identified (three for lower and upper canopy, and two for mid canopy measurements). 

The first group largely consisted of mesic site trees at all canopy levels, and the second 

group largely consisted of xeric site trees at all canopy levels suggesting whole tree 

differences in the expression of these attributes in the two microsites. The third group 

consisted of a relatively few number of trees (2 out of 3 trees were mesic in the third 

group of the upper canopy; 5 out of 6 trees were xeric in the third group of the lower 

canopy). In Group 1, these attributes misclassified 50% of the trees when measured in 

upper canopy, 15% of the trees when attributes were measured in mid canopy, and 20% 

of the trees when measured in the lower canopy. If the upper canopy acts as a sink for 

resources, attributes measured there would be less effective at indicating microsite 

location. In Group 2, these attributes misclassified 40% of the trees when measured in the 

upper canopy, 65% of the trees when measured in mid canopy, and 60% of the trees 

when measured in the lower canopy. Trees in mesic microsites were more consistent in 

expression of attributes correlated with O3 injury in yellow pine. 

Conclusions 

The O3 injury assessments that have been developed for yellow pine are robust and were 

designed for a coarse assignment of forest health. They do well at this and scores reflect 

O3 exposure regimes averaged over many years. Of the many known physiological 

responses to O3 exposure in conifers, only a few attributes are used to integrate the whole 

tree response to the stressor. The intent of this chapter was to review responses of foliar 

chlorotic mottle and needle retention, the key attributes relevant to field assessments of 

O3 injury. Correlative and manipulative examples of foliar chlorotic mottle and needle 

retention response to (1) O3 exposure and uptake, (2) nitrogen deposition frequently 

associated with O3, and (3) drought stress in years of significantly below-average annual 

totals help to describe the range of field responses expected. Understanding sources of 

variation in the key O3 injury assessment attributes may improve between-observer, and 

between-year assessments, and may help interpreting historical data sets. 

Several recommendations are offered for future field assessments of O3 injury. Because 

Jeffrey pine has lower stomatal conductance, greater antioxidant defense, and differing 

patterns of loss within a needle age class, scores should be reported separately for the two 

species of yellow pine at any one site. Interannual differences in the development (and 

loss) of chlorotic mottle are significant: the peak foliar injury may appear two months 

earlier in a drought year relative to a year of average precipitation, although the 

maximum level of foliar injury developed in a given site appears to roughly correlate 

with interannual, average site O3 exposure. The differences reported in leaf- and canopylevel gas exchange and thus O3 uptake for trees in mesic and xeric microsites, the 

manifestation of O3 uptake in chlorotic mottle, tissue chemistry, and needle retention 
 12

strongly suggests strongly for choosing trees in consistently mesic microsites for 

consecutive, interannual comparisons, or for restricting repeated assessments to years of 

+ 10% average precipitation years. Due to reduced foliar production within whorls with 

male cones, these branches should be avoided in field assessments. Although lower 

canopy is more easily accessible for large surveys, it is the least representative of the 

canopy. Future field surveys should be based in the mid canopy, allowing the capability 

of remote sensing to be linked to ground based measures of O3 injury. 

O3 injury assessments have been carefully developed for yellow pine, and it is possible 

that this approach can be applied to both sugar and lodgepole pine because despite their 

relative tolerance to O3, both exhibit chlorotic mottling on their needles. Ozone injury 

assessments will probably never be developed for giant sequoia, white fir, or incense 

cedar because visible foliar symptoms do not develop to any appreciable extent. 

Although subtle physiological responses to O3 exposure can be demonstrated in white fir 

(Retzlaff et al., 2000) and giant sequoia (Grulke et al., 1989; 1996), the physiological 

response is not translated to a consistent set of morphological responses unique to O3

exposure. Without the definitive chlorotic mottle that PR Miller (1963) correlated to O3

exposure, the changes in growth and needle retention observed cannot be conclusively 

assigned to the stressor in a field survey. Oxidative stress can, however, be supported on 

asymptomatic species using biochemical techniques. 

Acknowledgments 

This chapter largely summarizes research supported over the last decade by the 

Terrestrial Ozone Program, U.S. Environmental Protection Agency, PRIMENET (a joint 

NPS/EPA program), and the Air Quality Division of the National Park Service. The 

views presented here have not been reviewed or specifically condoned by either agency. 

Special thanks to Paul Miller, for introducing me to O3 injury effects, and Annie 

Esperanza, who has provided logistical support and field knowledge for much of this 

work. 

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 17

Figure captions 

Fig. 1 Example of chlorotic mottle development on ponderosa pine on the left. The oldest 

foliage within a needle age class senesces first, as seen in the previous year whorl in 

photo on the right. This photo was taken at the end of July in a drought year (1994) at 

Strawberry Peak, in the San Bernardino Mountains. 

Fig. 2 Relationship between chlorotic mottle index and leaf nitrogen content (% dry 

weight). Samples were taken in an above average precipitation year (1992), and current 

year foliage was collated for five branches per tree. The chlorotic mottle index 

corresponds to the following score: 0: no chlorotic mottle; 1: 1-6%; 2: 7-25%; 3: 26-50%; 

4: 51-75%; 5: 76-100% chlorotic mottle. 

0.6

0.8

1.0

1.2

1.4

1.6

1.8

2.0

0.0 0.5 1.0 1.5 2.0

Current year needles, ozone injury score

Current year needles, %N
 18

Fig. 3 Relationship between chlorotic mottle and cumulative O3 exposure at three sites in 

the San Bernardino Mountains (CR – highly polluted; SP – moderately high pollution; 

BF – moderate pollution) in a year of average (1993) and below-average total annual 

precipitation (1994). Chlorotic mottle of the previous year only has been plotted because 

this needle age class accounts for most of the branch carbon gain in ponderosa pine. 

Chlorotic mottle was monitored monthly on five branches per tree on twelve trees per 

site. 

Fig. 4 The seasonal maximum (from Fig. 3) is plotted for three sites across a pollution 

gradient with respect to O3 exposure (lower graph), O3 uptake (middle graph, as 

estimated from an empirical model, Grulke et al., 2002a; and upper graph, as estimated 

from a physiological model, TREGRO). Data in a year of average precipitation (1993) 

and a drought year (1994) is presented based on a 120 yr record maintained by the San 

Bernardino County Water District. The percent chlorotic mottle is not well correlated to 

cumulative O3 exposure. Cumulative O3 uptake, whether empirically or physiologically 

modeled, was a better predictor of response. 

Crestline

0 50 100 150 200 250 300

0

5

10

15

20

25

30

35

40

45

93

94

Barton Flats

Cumulative O3 exposure, ppm h

0 50 100 150 200 250 300

Chlortic mottle (percent)

0

5

10

15

Strawberry Peak

0 50 100 150 200 250 300

0

5

10

15

20

25
 19

Fig. 5 Typical phenology of chlorotic mottle in each needle age class, given for one 

branch, over the course of three years, in the San Bernardino Mountains (Grulke, unpubl. 

data). 

STRAWBERRY PEAK 

TREE #1290, BRANCH #4

0

5

10

15

20

25

30

35

40

45

50

111133182204243146179205235271163192239272

SAMPLE DATE, DAY OF YEAR (1993, 1994, 1995)

PERCENT CHLOROSIS

95 

94 

93 

92 

91 

90 
 20

Fig. 6 OII and FPM scoring for Jeffrey pine trees in mesic and xeric microsites, at lower 

and mid canopy. The OII score is much less sensitive to canopy position than FPM. 

Mesic trees had poorer health as indicated from both the OII (higher score, more injury) 

and the FPM (lower score, more injury) assessment techniques. Leaf-level and canopylevel measures of gas exchange indicate that trees in mesic sites had 20% greater seasonal 

O3 uptake than trees in xeric sites (Grulke et al., subm.) given the same site O3 exposure. 

Fig. 7 Within canopy variability in carbon, nitrogen, and C:N ratio in Jeffrey pine, and 

effect of 3 years of nitrogen amendments on mid canopy tissue chemistry. Each bar 

represents the average and + 1 S.D. of foliage from 32 trees. Within canopy foliar 

chemistry differed between trees in mesic and xeric microsites. Nitrogen amendments 

increased foliar nitrogen, decreased carbon, and decreased C:N of mid canopy foliage on 

mesic trees. It had the opposite effect on tissue chemistry of xeric trees. Trees were 

assigned to different microsites based on topographic position, proximity to perennial 

surface water, and ability of mesic site trees to increase bole growth in average and above 

precipitation years (Grulke et al., subm.). 

OZONE INJURY INDEX

0

5

10

15

20

25

30

35

40

45

LOW MID

CANOPY POSITION

SCORE

MESIC

XERIC

FOREST PEST MANAGEMENT INDEX

0.0

0.5

1.0

1.5

2.0

2.5

3.0

3.5

LOW MID

CANOPY POSITION

SCORE

MESIC

XERIC
 21

Fig. 8 Within-canopy variability in foliar chlorotic mottle in Jeffrey pine, Sequoia 

National Park. Each bar at each canopy position (U = upper; M = middle; L = lower) 

represents chlorotic mottle of current (dark grey), previous year (light grey), and four 

year old needles (black). Bar length represents the average and + 1 S.D. of 32 trees in 

each microsite (same trees as presented in Fig. 7). 

Fig. 9 Example of both branch-level and whorl-level needle retention in a year of aboveaverage precipitation (1993) and below average precipitation (1994) (from Grulke and 

Balduman, 1999). Within-whorl retention is a quantitative measure constructed from the 

[length of the branchlet with green needles retained] / [total branchlet length]. In this 

case, the foliated proportion of the branch is calculated for each branch age class, not the 

entire branch as in Miller et al. (1996). Reprinted courtesy of Kluwer Academic 

Publishers B.V., Water, Air, and Soil Pollution, Volume 116, Figure 1, p 242. 

MESIC

WHORLS 1,2,4

CHL MOTT

0 5 10 15 20 25 30

CANOPY POSITION

L

M

U

XERIC

WHORLS 1,2,4

CHL MOTT

0 5 10 15 20 25 30

CANOPY POSITION

L

M

U
 22

Fig. 10. Tree models of morphological and tissue chemistry attributes that significantly 

contribute to distinguishing between trees in mesic and xeric microsites in the upper, mid, 

and lower third of the canopy. Numbers at the base of the dendritic line indicate the 

proportion of trees in mesic (1.0) or xeric (2.0) microsites that satisfy the series of criteria 

in higher level branches. Foliage or branch tissue of the previous year was measured in 

thirty Jeffrey pine trees in each microsite, in Sequoia National Park in mid August, 2000. 

BR LEN = branch length; CM = chlorotic mottle; C.N = C:N ratio in needles; DIAM = 

branch diameter; FOL = proportion of branchlet with needles retained (see Grulke and 

Balduman 1999); N LEN = needle length; WHL = number of live needle age classes 

retained; 

UPPER CANOPY 

MID CANOPY 

|

BR.LEN<71.75

BR.LEN<35.75

WHL<4.5 CM<1.75

N.LEN<15 WHL<4.25

WHL<4.5

1.000 1.500

1.800 1.167 1.714 2.000

1.200 1.000

|

BR.LEN<43.75

C.N<45.825

DIAM<10.2775

CM<3.25

N.LEN<15.625

N.LEN<14

C.N<49.71

CM<3.25

FOL<73.3227

1.429

2.000 1.400

2.000

1.667

1.200 1.000

1.000 1.200

1.600
 23

LOWER CANOPY 

|

FOL<67.5187

CM<5.5

BR.LEN<29

WHL<4.75

DIAM<10.8425

BR.LEN<40

C.N<49.1

1.167 1.400

1.000

1.714 1.333

2.000 1.600

1.400