Drought Stress and Paper birch (Betula papyrifera) Seedlings: Effects of An Organic Biostimulant on Plant Health and Stress Tolerance, and Detection of Stress Effects with Instrument-Based, Noninvasive Methods

Effect of Biostimulant Application on Leaf Properties of Well-Watered Seedlings

There were no statistically significant differences between R (Roots) and NR (No Roots) treated seedlings (all P ≥ 0.10) for fresh leaf mass, dry leaf mass, leaf area, or LMA_Dry (Table 2). In spite of this, LMA_Fresh was marginally lower (P = 0.09) in leaves of R-treated seedlings compared to those in the NR treatment. R seedlings had significantly higher (P ≥ 0.01) foliar N concentrations (1.4%) than did NR seedlings (1.3%), but neither foliar C nor δ¹³C differed between the two biostimulant treatments.

Photosynthesis of the R-treated seedlings (7.0 μmol CO₂/m²/s) was somewhat higher than that of the NR seedlings (6.4 μmol CO₂/m²/s); the difference was just barely significant at the P ≤ 0.10 level (Table 2). F_v/F_m, Chl NDI, and PRI were also higher in the R-treated seedlings, but the difference between treatments was significant only for the reflectance indices (Table 2). The reflectance indices indicated that R-treated seedlings had higher chlorophyll contents, and higher chlorophyll:carotenoid ratios, than the NR seedlings.

Over the course of the experiment, R-treated seedlings grew 6% more than NR seedlings, but this difference in growth between biostimulant treated and control plants was not significant (P = 0.35).

Progression of the Drought Treatment

Over the course of the drought treatment, mean soil moisture content (SMC) of the well-watered plants remained similar (47 ± 6%) for both the R and NR treatments. For the well-watered plants, there was no apparent time trend in SMC (Figure 1; see p. 56). The droughted plants, on the other hand, showed a steady decline in SMC beginning after the final watering (day 5). The pattern of soil drying was similar for both the R and NR treatments (Figure 1). By day 20, SMC of the droughted plants was about 10% to 15%, and from day 30 through the end of the experiment, the figure was about 4 ± 3% for droughted seedlings in both R and NR treatments.

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Figure 1.

Time trend of volumetric soil moisture content over the course of the experiment. Plants in the “drought” treatment were watered for the last time on day 5 of the experiment. The soil drying curve depicts the progressive drought that resulted. Panel (A) shows data for pots without an organic biostimulant (Roots) treatment; (B) is for pots treated with the biostimulant. The soil drying curves are nearly identical in panels (A) and (B).

Detection of Drought Stress

The four physiological measures used as noninvasive methods to detect drought stress were plotted against SMC, with separate series plotted for the two biostimulant treatments (Figure 2; see p. 57). A three-parameter model with an exponential rise to maximum (specified as f(x) = y₀ + a × [1 – e^{−b × x}] + ε) was fit to these data. The parameters y₀ and a control the minimum and range of the data, respectively, while parameter b controls the curvature of the relationship, and ɛ indicates the stochastic regression residual. If b equals zero, then the relationship reduces to a flat line (y₀ + a).

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Figure 2.

Relationship between volumetric soil moisture content and four physiological variables over the course of a progressive drought. (A) net photosynthesis; (B) chlorophyll fluorescence F_v/F_m ratio; (C) chlorophyll normalized difference index (Chl NDI), an index of foliar chlorophyll content; and (D) photochemical reflectance index (PRI), an index based on xanthophyll cycle pigmentation. Data are plotted separately for the two biostimulant treatments (Roots, No Roots). Curves are based on a three-parameter model with an exponential rise to maximum (specified as f(x)=y₀ + a × [1 — e^b×v] + ε) fit to the data. No curve is shown for panel (C) because the coefficient b was not significantly different from 0. A dashed line is used to depict the curve for the No Roots plants, whereas a solid line is used for Roots-treated plants.

Across all four physiological variables, the R-treated seedlings had higher measured values across most of the entire range of SMC (0 to 50+%), which is in agreement with the above result (i.e., for the well-watered plants) that photosynthesis, F_v/F_m, Chl NDI, and PRI were all higher in the R-treated seedlings compared to the NR seedlings. This is particularly evident in Figure 2A, for example, from which it can be seen that there were far more R-treated seedlings with photosynthetic rates above 8 μmol/m²/s and far more NR-treated seedlings (at least at SMC > 10%) with photosynthetic rates below 6 μmol/m²/s. Thus, there was a clear tendency for the biostimulant treatment to improve the health of treated seedlings.

There was considerable scatter in the data, even at a given SMC, for each of the different physiological measurements. This scatter was especially pronounced for Chl NDI (Figure 2C), and in fact, for this variable, it was difficult to detect a clear relationship with SMC. For both the R- and NR-treated seedlings, the b coefficient was not significantly different from 0 (both P > 0.10), and R² values were very low (both models had R² ≤ 0.10, Table 3). On this basis, it is suggested that leaf chlorophyll content is not very sensitive to progressive drought stress.

In comparison, the other three physiological measures exhibited a far tighter relationship with SMC. For the photosynthesis, F_v/F_m, and PRI models, b coefficients were all significantly different from 0 at P ≤ 0.001. Model fits, as gauged by the coefficient of determination, R², were strongest for photosynthesis, intermediate for PRI, and weakest for F_v/F_m (Table 3). However, for all three measures, surprisingly low moisture contents were required for a functional response to be exhibited. For example, in all four cases, the modeled response f(x) did not fall more than 1 S.D. below the modeled maximum (y₀ + a) until SMC was below 15%. Photosynthesis of R-treated seedlings hit this threshold at 14.3%, whereas for NR seedlings the corresponding SMC was somewhat lower (11.9%). For PRI, SMCs of 9% to 10% were required for a similar magnitude of response, and for F_v/F_m the figures were even lower (4% to 6%). Although the R-treated seedlings had higher mean photosynthesis, F_v/F_m, and PRI, there was little or no evidence that the R treatment enabled seedlings to tolerate more severe drought before physiology was impaired (Figure 2).

Interpretation of Drought Effects on the Measured Physiological Variables

Results suggested that photosynthetic declines in the droughted seedlings were first triggered by stomatal closure, which led to reduced stomatal conductance. For both R-treated and NR seedlings in the drought treatment, conductances of below 0.05 mol H₂O/m²/s were associated with the lowest photosynthetic rates (≤ 4 μmol CO₂/m²/s). Stomatal effects on photosynthesis were probably larger than those of secondary physiological effects, and this may explain why F_v/F_m, PRI, and Chl NDI were all comparatively less sensitive indicators of drought stress.

Ögren (1990) found that drought-stressed and unstressed Salix leaves could be accurately differentiated on the basis of their fluorescence induction curves. Indeed, induction kinetics were of more use in this regard than measurements of photosynthetic capacity. However, results further demonstrated that the F_v/F_m ratio was of little use in identifying drought stress (see also Di Marco et al. 1988; Epron and Dreyer 1993; Lu and Zhang 1998; Tambussi et al. 2002), and Ögren (1990) concluded that drought stress does not affect photochemistry unless the stress becomes severe. Similarly, studying Quercus, Epron et al. (1992) found that photosynthesis of water-stressed trees was lower throughout the day than that of the control trees, but F_v/F_m differed by only a modest amount between treatments, and even then differences between treatments were only evident for several hours around midday. Thus, because the midday declines in F_v/F_m of drought-treated plants were found to be more or less fully reversible, Epron et al. (1992) suggested that this decline represented photoprotection and not photodamage to the PS II reaction center. In the present experiment, it was only at very low SMCs (i.e., extreme drought stress) that F_v/F_m began to show a nonreversible decline. On this basis, it is hypothesized that SMCs of 5% or less are required for drought-induced photodamage to occur in Betula papyrifera. This assumes the temperature and light regime of the present experiment—under higher irradiances (e.g., full sunlight, instead of the 20% full sun here), photodamage would likely occur under more mild drought stress (Lu and Zhang 1998). Furthermore, other experiments have demonstrated that even if PS II photochemistry does not show a drought response when fluorescence measurements are conducted on dark-adapted samples (i.e., the potential quantum yield, as given by F_v/F_m, does not change), there is still evidence of photosynthetic down-regulation in that the actual quantum yield of PS II (Φ_{ps II}) is decreased (Lu and Zhang 1998). This stress response can be detected by studying the fluorescence kinetics of light-adapted samples, for which pulse-modulated fluorescence techniques are required.

Tambussi et al. (2002) demonstrated that although chlorophyll content did not differ between control and severely water-stressed durum wheat plants (see also Epron and Dreyer 1993 for two Quercus spp.), a transmittancebased version of PRI was sensitive to even moderate drought. Results of that study confirmed that PRI is negatively correlated with nonphotochemical quenching (qN) of excess energy in the thylakoids through the xanthophyll cycle, and negatively correlated with the de-epoxidation state of the xanthophyll cycle pigment pool. It is likely that PRI performs better than F_v/F_m as a stress index precisely because it correlates with these photoprotective mechanisms, whereas nonreversible change in F_v/F_m requires that actual photodamage has occurred.

Effect of the Biostimulant Treatment

The biostimulant treatment appeared to have a modest effect on plant health (Table 2, Figure 2), but it did not result in greatly improved stress tolerance of treated plants (Figure 2), nor did it lead to a significantly enhanced growth rate. However, since carotenoid pigments are important for the de-excitation of harmful oxygen species (e.g., singlet oxygen radicals), and PRI indicated that the biostimulant-treated plants may have a higher chlorophyll:carotenoid ratio, it is suggested that the antioxidants in the Roots 3 formulation may help to reduce oxidative stress in the treated plants.

The effect of biostimulant treatment on plant growth and stress tolerance, as shown here, is weaker than that previously reported for a variety of different plant species [e.g., Coffea, Alnus, Pinus, and Populus, as well as several grasses (Berlyn and Sivaramakrishnan 1996)]. This may be due to the short duration of the present experiment and the comparatively large size of the birch seedlings relative to the dosage. Thus, experiments will be needed to determine the correct application rate for full-sized trees in a garden or landscape setting.

Detecting Stress with Noninvasive Physiological Measures

The photosynthetic rates we measured on well-watered plants (Table 2, for both R- and NR-treated seedlings) were somewhat lower than those previously measured on field-grown seedlings of the same species at a mid-elevation [550 m (1,800 ft) ASL] site in northern Vermont (≈ 10 μmol CO₂/m²/s) (Richardson and Berlyn 2002). Those same plants in Vermont also had higher mean Chl NDI (0.403) but lower PRI (−0.007) than the greenhouse-grown seedlings in the present experiment. The F_v/F_m of the well-watered plants in the present experiment was within the range considered “normal” (≈ 0.800) for dark-adapted, healthy, and unstressed leaves (Ball et al. 1994). F_v/F_m is the only one of the measures used here for which there is such a generally accepted standard against which measurements can be compared, but F_v/F_m was also less sensitive to drought stress than was photosynthesis or PRI. For photosynthesis and reflectance indices, site-specific differences in biotic and abiotic factors (e.g., microclimate, soil fertility, tree age) can have a significant effect on what is “normal” and hence what one considers to be “stressed.” Clearly, then, stress ultimately has to be a relative concept: It requires that we have some standard of “normal.” One-shot measurements of any physiological variable may tell the plant physiologist little about drought stress, since there is so much variation not only among species, but also within single species—i.e., across populations and even individuals (see, for example, the scatter apparent even for SMC > 30% in the different panels of Figure 2). Rather, monitoring plant health throughout the season, and detecting changes over time, may be the best way to implement an instrument-based stress monitoring program.

These results may be applicable to the detection of a wide range of stress agents or factors, since it has been previously noted that many stress factors produce similar stress responses (Larcher 1995). However, this also means that diagnosing, and prescribing treatment for, the causal stress factor can be exceptionally difficult, especially in the field. For example, Carter (1993) showed that a variety of stress factors all led to similar changes in the visible and near-infrared foliar reflectance spectra. Thus, particular stress agents do not appear to yield spectrally unique reflectance responses. A key may be distinguishing between the sometimes-unique primary effects of a stress factor (e.g., loss of turgor and resulting stomatal closure as a consequence of drought) from the resulting secondary effects (e.g., photoinhibition, chlorosis), which are more general responses to almost all stress factors. Unfortunately, at this stage, we don’t have field instruments capable of detecting the primary effects of most stressors, and, as demonstrated here, it was necessary for the stress to be well-developed before secondary effects could be detected by either reflectance or fluorescence methods.