UTILITY AND LIMITATIONS OF CHLOROPHYLL FLUORESCENCE FOR THE DETERMINATION OF GROWTH LIMITATIONS IN TREES

J. A. Peterson, J. W. Groninger, J. R. Seiler, P. Mou

Research Specialist, Research Associate, Professor, Assistant Professor, respectively,

Virginia Polytechnic Institute and State University, Blacksburg, Virginia

Abstract--To aid in silvicultural decision making, a fast, accurate and inexpensive means to determine the effects of environmental stresses, especially tree nutrition, on tree vigor is needed. In the past several years, chlorophyll fluorescence technologies have been developed that are both inexpensive and easily used under field conditions. To explore the applicability of fluorescence measures to southern industrial forests, a series of pilot studies were established. It was determined that photochemical efficiency varies with the nutritional status of loblolly pine (Pinus taeda) and sweetgum (Liquidambar styraciflua) seedlings, with the water status of loblolly pine but not sweetgum seedlings, and with crown position in loblolly pine plantations. With further refinement and field testing, chlorophyll fluorescence may be a useful tool in tree nutrition management.

INTRODUCTION

Chlorophyll fluorescence is a relatively new technology that in recent years has become much easier to utilize as a field measurement. It is a physiologically based measurement that measures the efficiency of the light harvesting mechanism associated with photosystem II. As such, fluorescence has been shown to be sensitive to water (Lenham, 1994, Oogren 1990), mineral nutrition, chilling (reviewed in Mohammed et al., 1995) and light intensity (Groninger and others, 1996). Fluorescence can be measured rapidly, usually taking only 30 seconds (following a 10-30 minute dark acclimation period) and is nondestructive, allowing for repeat measures on one leaf. Further, chlorophyll fluorescence can be very consistent, with coefficients of variation of 3 percent not uncommon. Finally, the instrumentation described within this paper was relatively affordable, with a purchase price of under $10,000.

While the sensitivity of fluorescence parameters to environmental stresses is well known, the role of chlorophyll fluorescence in silvicultural decision making is largely unexplored. To apply fluorescence-based measures to silvicultural problems, a large body of data, under a variety of experimental conditions are needed. The pilot studies described in this paper were intended to provide information on potential uses and shortcomings of chlorophyll fluorescence as a silvicultural tool. Specifically, four studies are described in this paper. Study 1 examined the influence of varying fertilizer levels on chlorophyll fluorescence over time for sweetgum seedling sprouts. Study 2 examined the influence of drought on both loblolly pine and sweetgum seedling fluorescence. Study 3 examined fertilizer levels and shade on both loblolly pine and sweetgum seedling fluorescence. Study 4 examined fluorescence characteristics in the upper and lower crowns of thinned and unthinned loblolly pine stands.

The overall objectives of these studies were to: Determine the sensitivity of loblolly pine and sweetgum chlorophyll fluorescence to several stresses; To determine whether fluorescence can be used alone or in combination with other measures to make diagnoses in multiple stress situations; Finally, to determine the feasibility of developing a fluorescence-based diagnostic tool to identify growth limitations in southern forests.

METHODS

Four separate studies are described in this paper. All fluorescence measurements were taken with a P.K. Morgan CF-1000 Chlorophyll Fluorescence Measurement System (P.K. Morgan Instruments, Inc., Andover, MA) after at least a 15 minute dark acclimation period. Three greenhouse studies described below were undertaken in the Virginia Polytechnic Institute and State University climate-controlled greenhouse in Blacksburg, Virginia. The one field study was conducted at the Reynolds Homestead Forest Resource Research Station in Critz, VA.

Study 1 - Sweetgum Nutrition

On June 28, 1996 15 sweetgum seedlings that had been greenhouse-grown in peat moss and sand in 1 liter pots for approximately one year were transplanted into pure sand in 8 inch pots. All seedlings received an initial dose of Peters Soluble Trace Element Mix (Peters Fertilizer Products, Fogelsville, PA). After transplanting, the seedlings were placed in five blocks, and fertility treatments were randomly assigned to the three seedlings within each block. Fertility treatments included no, low, and high fertilization rates applied weekly as 20-20-20 NPK Peter’s Professional Water Soluble Fertilizer (Peters Fertilizer Products, Fogelsville, PA). This equaled 0.0442 grams N, 0.0190 P and 0.0367 K per week for the high treatment and 0.0133, 0.0057, and 0.011 grams per week N,P,and K, respectively, for the low fertilizer treatment. The seedlings were top-clipped on July 12, leaving an 8 centimeter "stump". After the seedlings resprouted and leaves appeared to be fully expanded (59 days after top-clipping), fluorescence measurements commenced. Measurements were taken at irregular intervals, including 59, 105, 130, 203, and 221 days after top-clipping. For the first measurement, the uppermost fully expanded leaves were measured. All measurements taken on or after the 105th day after top clipping were taken on leaves that were at 75 percent of the total height of the seedling. Fluorescence measurements were taken over 30 seconds, with an incident light level of 800 micromoles per meter squared per second. Statistical analysis consisted of a one-way ANOVA with five blocks.

Study 2 - Influence of Drought on Loblolly Pine and Sweetgum

Twelve seedlings each of loblolly pine and sweetgum seedlings were grown from seed in 1 liter pots in a 2:1:1 volume:volume:volume mixture of peatmoss, perlite and vermiculite. Treatment consisted of induction of drought by witholding water, followed by daily measurements until the individual seedling exhibited net negative carbon exchange rates (Lenham, 1994). The loblolly pine and the sweetgum seedlings were 391 and 174 days old, respectively when droughting began. Fluorescence measurements were taken over 15 seconds, with an incident light level of 1000 micromoles per meter squared per second. Seedling Fv/Fm was regressed against days of drought.

Study 3 - Influence of Shade and Nutrition on Loblolly Pine and Sweetgum

On March 16, 1996, 40 loblolly pine and 40 sweetgum germinals were transplanted into 5 gallon pots containing pure sand. One half of the seedlings of each species were then placed in either full greenhouse sun or under 75 percent shade cloth. One half of each of these sets of seedlings received high amounts of fertilizer, one half received low fertilizer. The high fertilizer treatment received 14.73 grams 6-month time release Sierra 15-10-10 time release fertilizer plus micros (Grace Sierra, Milpitas, CA), which equaled 0.0752 grams N, 0.0432 P and 0.0567 K per week. The low fertilizer treatment received one half of this amount. Fluorescence measures were taken prior to harvest, on September 9, 1996. Fluorescence measurements were taken over 30 seconds, with an incident light level of 800 micromoles per meter squared per second. Statistical analysis consisted of a two-way ANOVA with two fertilizer levels and two light levels.

Study 4 - Influence of Thinning and Crown Position on Loblolly Pine

In March of 1980, one-half of three replicate eight-year-old loblolly pine stands were mechanically thinned by 50 percent from an initial 10 x 10 foot spacing. Scaffolding was erected to permit access to both the upper and lower crown foliage (Ginn and others., 1991; Peterson and others, in press). In May of 1992 (four years after thinning), fluorescence measurements were taken in both the upper and lower crown of two sample trees within each treatment x block combination. Fluorescence measurements were taken over 10 seconds, with an incident light level of 1000 micromoles per meter squared per second.

RESULTS AND DISCUSSION

A variety of fluorescence measurements describing a Kautsky fluorescence induction curve can be obtained from the P.K. Morgan CF-1000 (Greaves and others, 1991). The photochemical efficiency of photosystem II is an ideal measurement for measuring environmental stresses on the health of the photosynthetic mechanism. The photochemical efficiency of photosystem II is estimated by Fv/Fm, which is the ratio of variable fluorescence (Fv) to maximum fluorescence (Fm). Most forest trees usually exhibit Fv/Fm values of 0.6 to 0.8. Further, Fv/Fm provided the most consistent results with regards to fertility and other environmental stresses. For these reasons, Fv/Fm will be the primary fluorescence parameter discussed in this paper.

Study 1 - Sweetgum Nutrition

Greaves (1991) reported that saturating light is necessary to estimate Fv/Fm, but maintained that Fv/Fm should not vary over a wide range of measurement light intensities. Mohammed and others (1995) indicated that the measurement light intensity should be tested with the plant material under study. In our measurements of sweetgum seedlings it was determined that Fv/Fm was reduced when incident light was increased from 800 to 1000 micromoles per meter squared per second across all three fertilizer treatments. To maintain light levels close to saturation and to reduce the drop in efficiency associated with very high light levels, measurements were taken at 800 micromoles per meter squared per second.

Across all fertilizer levels there was little difference in Fv/Fm between lower and upper crown leaves when measured 105 days after top clipping. However, it was determined that leaves that originated at 75% of total shoot height expressed less variation and greater differences between treatments than very young leaves.

Differing levels of fertilizer had a large effect on seedling height and diameter growth. Seedling height and diameter were significantly greater with high fertilization (Table 1).

Table 1--Height (from sprout attachment) and diameter (measured 2.5 centimeters above attachment) of greenhouse-grown sweetgum sprouts provided with three levels of fertilizer.

Examining photochemical efficiency over a greenhouse "growing season", several trends become apparent (Figure 1). Fv/Fm values for these seedlings ranged from 0.6 to 0.73. The highest fertilization level consistently had the highest Fv/Fm values of the three fertilizer levels, and the low fertilizer treatment was consistently greater than the non-fertilized treatment. Further, the difference in Fv/Fm between levels was significant (p = 0.10) on all but the last sampling date. Under controlled environmental conditions, the nutritional status of sweetgum sprouts influences photochemical efficiency.

Figure 1--Change in Fv/Fm over time for greenhouse-grown sweetgum sprouts grown under high, medium and low fertilizer levels.

Overall, a general increase followed by a decrease in Fv/Fm was observed over time across all three treatments, with the high fertilizer treatment expressing the highest values later in the season (Figure 1). The largest differences in Fv/Fm between the three treatments were observed during the greenhouse equivalent of "mid- to late-summer". A possible explanation for the late season decrease in Fv/Fm values may be leaf senescence. The leaves of unfertilized trees were changing color and abscising by the end of the study. Interestingly, when fluorescence was measured on the new, late season flushes of two well-fertilized trees, relatively high Fv/Fm values were observed (Figure 1).

Study 2 - Influence of Drought on Loblolly Pine and Sweetgum

Sweetgum photochemical efficiency apparently did not change significantly in response to drought (p = 0.117), even though the seedlings had net negative carbon exchange rates after several days (Figure 2). In contrast, loblolly pine photochemical efficiencies declined as the seedlings became increasingly stressed (p = 0.005). This has two implications. First, if fluorescence is to be used to determine the nutritional status of loblolly pine, drought stress may result in an artificially low Fv/Fm value. Second, the decrease in Fv/Fm over time during a drought indicates that chlorophyll fluorescence may be a useful tool in measuring the water status of loblolly pine.

Figure 2--Change in Fv/Fm with drought for greenhouse-grown loblolly pine and sweetgum seedlings.

The usefulness of chlorophyll fluorescence as an indicator of water stress apparently varies by species. Oogren (1990) found that Fv/Fm did not provide any useful drought stress information, and that photochemistry in Salix leaves is unaffected except by severe drought stress. Conroy and others (1986) did not directly report Fv/Fm, but found that a variety of other fluorescence parameters in Pinus radiata were affected by drought stress. Further, these parameters often interacted with P deficiency.

Study 3 - Influence of Shade and Nutrition on Loblolly Pine and Sweetgum

Shade did not significantly change seedling photochemical efficiency in either sweetgum or loblolly pine (Table 2). Groninger et. al (1996) found that the Fv/Fm change with shading apparently varies by species. Fv/Fm values of loblolly pine and Liriodendron tulipifera did not change with shade, but Pinus strobus and Acer rubrum Fv/Fm values were reduced by shade.

Table 2--Fv/Fm and total seedling mass (g) of green-house grown sweetgum and loblolly pine seedlings grown under two light levels and two fertilizer levels.

The fertilizer levels used in this study were overall quite high. Consequently, there was no difference in sweetgum mass between fertilizer treatments (Table 2). Pine mass was greater with the high fertilizer treatment, but no difference in Fv/Fm was detected between the two fertilizer treatments (Table 2). However, under full sun conditions (Table 3) seedling biomass was significantly greater with high amounts of fertilizer and Fv/Fm was significantly greater for the high fertilizer treatment. This indicates that loblolly pine chlorophyll fluorescence is sensitive to tree nutrition, even under very fertile conditions.

Table 3--Fv/Fm and total seedling mass (g) of green-house grown loblolly pine seedlings grown under full sun and two fertilizer levels.

Apparently the actual amount of nutrition applied to the seedlings is closely related to observed Fv/Fm. The highest Fv/Fm values observed in Study 1 (Figure 1) ranged from 0.711 to 0.723. Sweetgum in this study had mean Fv/Fm values of 0.723 for the low fertilizer treatment and 0.726 for the high fertilizer treatment. The actual amount of fertilizer applied weekly was very similar to the amount of fertilizer applied to the high fertilizer treatment in study one. The low fertilizer treatment had only 15 percent less N, 14 percent more P, and 22 percent less K than in study one.

Study 4 - Influence of Thinning and Crown Position on Loblolly Pine

When averaged over crown position, thinning did not change photosynthetic efficiency, with an overall mean Fv/Fm value of 0.788. If the greenhouse results of study 3 are scalable to large trees in the field, an Fv/Fm value of 0.788 is expected to be reflective of adequate nutrient availability. Indeed, the site index at base age 25 years was 80 feet (with weed control), and foliar analysis of these trees indicated that the foliar N content was greater than 1.2 percent (Ginn and others, 1991), which is considered to be above the critical level for loblolly pine (Wells and Allen, 1985).

Upper crowns were found to have significantly greater Fv/Fm values than lower crowns (0.8 vs. 0.775; p = 0.05). As indicated by study 3, the decreased Fv/Fm values in the lower crown are probably not solely induced by shading. Lower needles were likely in overall poorer health, which is reflected in lower photochemical efficiency.

A significant interaction between crown position and thinning was observed (p = 0.033). In control stands the upper crown had very high mean Fv/Fm values (0.806) and the lower crown had relatively low mean values (0.772). Thinning tended to decrease the difference between the upper and lower crowns. In thinned stands the upper crown had a mean value of 0.795 and the lower crowns averaged 0.778. This suggests that thinning may influence fluorescence results if samples are taken from only one crown position.

CONCLUSIONS

Chlorophyll fluorescence is a fast, accurate and inexpensive measurement that has developing applications in commercial forestry (Hawkins and Binder, 1990). The fluorescence parameter Fv/Fm, which estimates photochemical efficiency appears to vary with sweetgum and loblolly pine nutrition status, leaf age, loblolly pine water status, and crown position. Bjorkman and Demmig (1987) suggest a theoretical maximum Fv/Fm of 0.832 for a wide variety of C3 species under specific measurement conditions. If the theoretical maximum of loblolly pine and sweetgum can be determined for a standardized set of field measurement conditions, and if Fv/Fm values of field trees consistently vary with nutrition as do the greenhouse seedlings utilized in this experiment, fluorescence may prove to be a useful tool that can be used in conjunction with leaf area, and soil and foliar nutritional analyses in both loblolly pine and sweetgum nutrition management. Especially encouraging is the variation of sweetgum fluorescence with soil nutrition coupled with a lack of sensitivity to water stress.

Before the effectiveness of fluorescence as a management tool can be evaluated, several important issues remain to be addressed. It is not known if field nutritional deficiencies can be detected, which portion of the crown of larger trees offer the most consistent or early results, how much fluorescence varies between individuals, stands, or on a regional basis, or to what extent the influence of nutritional deficiencies is masked by drought, heat or other field stresses.

LITERATURE CITED

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