Short-term effect of fertilization and Captan on specific root respiration in hydroponically grown one-year-old loblolly pine

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CHAPTER 3 Short-term effect of fertilization and Captan on specific root respiration in hydroponically grown one-year-old loblolly pine (Pinus taeda L.) seedlings

Abstract.

Root respiration (RR) can make up 30-70% of the total soil CO2 efflux (EC). To properly model the effects of fertilization on net ecosystem productivity (NEP) we need to understand the direction and degree to which fertilization affects RR. Separating RR from respiring microorganisms living within the soil matrix has proven to be a major obstacle. Growing seedlings hydroponically is one of many methods for isolating RR while at the same time allowing measurements to be taken repeatedly with minimal disturbance to the seedling. We performed a pair of greenhouse studies to observe the effects of fertilization in the form of diammonium phosphate (DAP) on RR. Root respiration was measured using a Li-Cor 6200 infrared gas analyzer with a 0.25-L closed cuvette. The objectives were to determine how nutrient additions initially affect RR in one-year-old loblolly pine seedlings. Secondly, we wanted to determine if Captan [N-(trichloromethylthio) cyclohex-4-ene-1, 2-dicarboximide], a mild fungicide, could be used to reduce or eliminate ecto-mycorrhizae upon visual inspection and how this might influence the RR response to fertilizer. In experiment 1 we observed significant differences in the date x fertilizer (P=0.0003) and the date x Captan (P=0.0458) interactions. An analyses by date showed that, initially, at a high rate (100 ppm N, 49 ppm P) of fertilization RR was significantly (P< 0.10) increased relative to seedlings that did not receive fertilization. This increase was only temporary with rates returning to control levels by the end of the study. Experiment 2 also showed an initial increase in respiration rates with an eventual decline relative to control seedlings. No consistent trend was found between low (25 ppm N, 13 ppm P) and moderate (50 ppm N, 25 ppm P) rates of fertilization. Captan was shown to generally have no effect on RR. Captan and DAP both showed (visual inspection) a decrease in fine- roots and mycorrhizae, which could explain the reduction in respiration rates observed by the end of the studies in these treatments. We concluded that further research to quantify changes in mycorrhizal biomass need to be conducted to determine Captan’s effectiveness at reducing ecto-mycorrhizae without negatively affecting seedlings since we observed a 5% greater mortality in seedlings treated with Captan compared to seedlings that were not. In experiment 1, fertilized seedlings experienced high mortality rates. Water temperature (°C) was found to be a highly significant (P<0.0001) variable, but explained very little of the variance in RR leading us to conclude that something else, such as solar irradiance, was responsible for daily differences in respiration rates.

Introduction

Root respiration contributes from 30-70% of total soil CO2 efflux (EC) (Raich and Schlesinger 1992, Andrews et al. 1999, Maier and Kress 2000, Ekblad and Högberg 2001, Widén and Majdi 2001, Pangle and Seiler 2002, Ruess et al. 2003) making it a significant component of the global carbon cycle. Total root respiration (RR), as defined in this paper, is the sum of growth respiration (RG), which is CO2 emitted during the synthesis of new tissue, and CO2 emitted during the repair and replacement of plant tissues referred to as maintenance respiration (RM). While it is useful to partition RR into RG and RM respiration for purposes of understanding, there is no biochemical difference in the products of the two processes. Another component that is sometimes included in RR, because of its inherent difficulty in separating it, is rhizomicrobial respiration, which is respiration originating from organisms within the rhizosphere such as mycorrhizae and other organisms that are dependent on the plant for carbon and nutrients. Rhizomicrobial respiration can make up a significant portion of root respiration leading to large differences in reported respiration rates depending on whether included or separated during respiration measurements. Harley and Smith (1983) estimated that mycorrhizae associated with conifer roots accounts for 23-30% of respiration and Ek (1997) estimated mycorrhizae (Paxillus involutus) associated with European white birch (Betula pendula Roth) accounts for 11-25% of the root respiration. Biocides have been used to remove or significantly reduce mycorrhizae, which could allow for a better understanding of the effects of nutrient additions on root respiration. Captan® [preferred IUPAC name: N-(trichloromethylthio)cyclohex-4-ene-1,2-dicarboximide] is a mild fungicide, that at high concentrations, has been shown to reduce ectomycorrhizae (Pawuk et al. 1980, Ingham and Coleman 1984, Colinas et al. 1994) and vesicular-arbusculus mycorrhizal (VAM) development (Kough et al. 1987) without negatively affecting higher plants (Pawuk et al 1980, Marx and Rowan 1981, Wigand and Stevenson 1997).
Nitrogen is one of the most limiting elements to plant growth. The absorption and assimilation of nitrogen either as NH4+ or NO3- is energetically expensive for the plant, which can be observed by an increase in fine-root respiration (Bloom et al. 1992). Increases in root N concentrations have been shown to be positively correlated with RR (Ryan et al. 1996, Zogg et al. 1996, Widén and Majdi 2001, Burton et al.2002). It is hypothesized that increased nitrogen concentration increases RM. It requires both ATP generated from respiration and C-skeletons from stored carbohydrates or recent photosynthate to actively take up, reduce, and assimilate nitrogen ions into amino acids, nucleic acids, and other nitrogen containing compounds. In conifers, ammonium is the dominant source of N owing to its greater availability in acidic forest soils (Bedell et al. 1999). There is no extra energy costs involved in reducing ammonium as with nitrate, but unlike nitrates, ammonium must be metabolized immediately to avoid reaching toxic levels.
One problem with accurately estimating root respiration is separating it from CO2 that is being respired by soil organisms during decomposition of soil organic matter.
There are a number of methods for trying to separate RR from heterotrophic respiration (RH) each with their own strengths and weaknesses. Methods that employ root exclusion such as trenching (Haynes and Gower 1995, Boone et al. 1998, Lavigne et al. 2004) and tree girdling (Högberg et al. 2001, Singh et al. 2003) prevent disturbance of root-fungi associations and fine-roots, but deprive soil organisms of a much needed energy source (e.g. root exudates). Others have measured root respiration directly on excised roots in greenhouse (Gough and Seiler 2004) and field experiments (Zogg et al. 1996, Rakonczay et al. 1997b, Maier and Kress 2000, Widén and Majdi 2001, Ruess et al. 2003), or in situ by placing a sections of root in a cuvette and reburying it in the ground (Rakonczay et al.1997a), these methods allow measurements to be taken in the field, but preventing disturbance of fine-roots and fungal mycelium is unavoidable. Using carbon-13 (Andrews et al. 1999, Ekblad and Högberg 2001, Singh et al. 2003) or carbon-14 (Kuzyakov et al. 2002) labeling is a useful tool for separating root from RH, but is quite expensive. Growing seedlings hydroponically (Bloom et al. 1992, Cramer and Lewis 1993, BassiriRad et al. 1997, Lasa et al. 2002), or in sterilized soils (Lu et al. 1998) is a good method for gaining insight into how RR responds to a specific treatment, but as pointed out by Kuzyakov et al. (2002) is not necessarily representative of how a plant will react under natural conditions.
We have conducted a pair of greenhouse studies to explore the short-term effect of fertilization, in the form of diammonium phosphate (DAP), on specific fine-root respiration in one-year-old loblolly pine seedlings grown hydroponically. Specifically, we wanted to observe how RR responded to nutrient additions. We hypothesize that RR will increase as the rate of fertilization increases eventually decreasing as seedlings become nitrogen saturated. Secondly, we wanted to determine, upon visual inspection, if Captan, a mild fungicide, was effective in reducing or eliminating ectomycorrhizae and what effect this had on the RR response to fertilizer. These studies serve as pilot studies to determine to what direction and degree DAP will affect RR. We recognize no attempt was made to measure nitrogen or phosphorous uptake (root tissue N and P concentration), plant growth, or to quantify mycorrhizal biomass.

Materials and Methods

Experiment 1

The design was a completely randomized design with a 2×2 complete factorial with fertilization and fungicide. One-year-old improved loblolly pine clones, produced by somatic embryogenesis, were donated by Plum Creek Timber Co. on February 9, 2004 for use in this study. However, differences between clones were not investigated in this study. Twenty-eight seedlings were randomly assigned to each of 4 tanks and grown hydroponically in a greenhouse, under ambient light and temperature, for 10-weeks. On February 17, 2004 seedlings were washed and roots trimmed to approximately 15-cm length. Seedlings were refrigerated until February 27, 2004, at which point they were placed into tanks.
Each 38 L (10 gal) tank was wrapped in aluminum foil to keep out light, stabilize temperature, and minimize algae growth. Seedlings were floated using 5-cm thick high density construction foam, and oriented so that their tops were exposed to light and roots submerged in water. Foam also served to limit movement within the tanks and to prevent light from penetrating the surface (Figure 3.1). Tanks were filled with 28-L of tap water and aerated with a pump (1 per tank) split with a T-connector linking two air stones positioned at opposite ends of the tank. Each tank received a base rate of fertilizer weekly using water soluble 20-20-20 all purpose plant food with micronutrients (Peters Professional®, Spectrum Group Div. of United Industries Corp., St. Louis, MO) at a rate of 2 ppm N and 1 ppm P. Every week, the seedlings were re-randomized and relocated to an identical set of tanks allowing tanks to be sterilized, and seedlings to be re-randomized within their treatment group. By re-randomizing seedlings, between tanks and location within the greenhouse, we were able to treat this experiment as a completely randomized design as has been done in studies utilizing growth chambers (Teskey and Will 1999).
On March 19, 2004 tanks were randomly assigned one of four treatments: fertilizer (F), fertilizer and fungicide (FCAP), fungicide (CAP), and control (C). Treatments receiving fertilizer were brought to 100 ppm N and 49 ppm P in the form of diammonium phosphate (DAP) at the rate of 462 mg L-1, the equivalent of 98 ppm N and 48 ppm P, in addition to the base fertilization rate. From this point on fertilization rates will be referred as 100 and 0 ppm N. Seedlings receiving fungicide were dipped in a 1% solution of N-(trichloromethylthio) cyclohex-4-ene-1, 2-dicarboximide (Captan-50% a.i. WP, Bonide Products, Inc., Oriskany, NY) for 60-min. Captan treatments were applied on March 19, 2004 (10 days prior to pre-fertilization measurements) and each week (using the same solution) immediately following respiration measurements.
Pre-fertilization measurements were taken on March 29, 2004 to determine base respiration rates. Fertilizer treatments were applied on March 29, 2004 following pretreatment measurement. On April 1, 5, 12, and 23, 2004 the respiration measurements were taken on 80 seedlings using the Li-Cor 6200 infrared gas analyzer (IRGA) (Li-Cor Inc., Lincoln, Nebraska) with a 0.25-L cuvette chamber as a closed system, with a total system volume of 429-cm3. The instrument was zeroed before each sample date and recalibrated by running a known CO2 concentration through the system. RR was taken by removing a seedling from its tank, roots were patted dry with a paper towel, and approximately 10-cm2 section of root was immediately placed on a damp piece of paper towel in the cuvette (BassiriRad et al. 1997, Gough and Seiler 2004). After rates stabilized (usually within a couple minutes) CO2 evolution was measured over a 30-second period and respiration rate (µmol m-2root sec-1) calculated on a per unit root area using the following equation:
Where C = [CO2], t = time, P = atmospheric pressure, Vt = system volume, R = universal gas constant, and T = temperature.
Measured roots were marked with thin copper wire (so same section of root could be remeasured) and scanned with a USB-scanner at 300 dpi then immediately placed back in its tank. The scanned image was isolated using image software (Adobe Photoshop® 6.0, Adobe Systems Inc., San Jose, CA) and the root surface area (cm2) determined using WinRhizo 5.0A software (Regent Instruments Inc., Quebec, Canada). Water temperature was taken concurrently with respiration measurements using a Digi-sense temperature gauge (model no. 8528-20, Cole-Parmer Instrument Co., Niles, IL) to the nearest 0.1ºC.
A multivariate analysis of variance (MANOVA) with repeated measures was used to test the effects of fertilization and Captan on RR. Seedlings that died during the course of the study were completely removed from the dataset so that only seedlings repeatedly measured on every sampling date remained. The effects of temperature on RR were analyzed using simple linear regression. All analyses were performed using SAS version 9 (SAS Institute, Cary, NC) with an alpha level of 0.10.

Experiment 2

This study was designed as a completely randomized design with six replications of four levels of fertilization 2 ppm N and 1 ppm P (none), 25 ppm N and 13 ppm P (low), 50 ppm and 25 ppm P (mod), and 100 ppm N and 49 ppm P (High) applied to one-year-old loblolly pine seedlings grown hydroponically in a greenhouse at ambient temperature and light. From this point on fertilization rates will be referred to by their N concentrations. Open pollinated, improved seedlings were donated by Virginia Department of Forestry for use in this project. The design consisted of 38-L tanks (two per treatment) wrapped in aluminum foil filled with 28-L of tap water each containing 30 loblolly pine seedlings (as described above). On May 02, 2005 pre-fertilization measurements were taken to obtain a base root respiration rate and treatments applied immediately following. Fertilizer was applied in the form of DAP at a rate of 0, 23, 48, 98 ppm N, in addition to the 2 ppm N base solution, to bring tanks to their respective fertilization rates.
Root respiration rates were measured on six randomly selected seedlings (three per tank; 24 total per sampling period) using the Li-Cor 6200 with a 0.25-L closed cuvette chamber (as described above). Unlike the previous experiment, RR was measured using approximately 10-cm2 of excised root (<5-mm) and the seedling was discarded. A second change from the previous study involved determination of root surface area. Root area (cm2) was directly (e.g. roots were not pre-scanned) determined using the WinRhizo 5.0A software and root scanner (Regent Instruments Inc., Quebec, Canada). Measurements were taken 1, 2, 4, 8, and 23 days following fertilization. Every week seedlings were re-randomized within tanks, and tanks were randomly assigned a new position on the greenhouse bench allowing for tanks to be sterilized and the replacement of nutrient solutions. Differences between treatments for each measuring date were determined with an analysis of variance (ANOVA) using general linear model in SAS version 9 (SAS Institute, Cary, NC). An alpha level of 0.10 was considered significant. Analysis of repeated measures could not be utilized since seedlings were discarded after each measure.

Results

Experiment 1

The date by fertilizer interaction was found to be highly significant (P=0.0003). When data were analyzed by date, respiration rates were significantly (P<0.10) increased in fertilized relative to control seedlings by 33 and 35% on days 3 and 7 following fertilization, respectively. By the end of the experiment, rates were no longer statistically significant between fertilized and non-fertilized seedlings. Mean RR rates were found to decrease by 16 and 8% in fertilized seedlings relative to non-fertilized seedlings 14 and 25 days after fertilization, respectively (Table 3.1; figure 3.2A). When analyzed over the course of the experiment the fungicide x date interaction was found to be significant (P=0.0458). Seven days following fungicide treatment Captan treated seedlings had significantly (P=0.0402) lower respiration rates by 22% (Table 3.1, figure 3.2B). The date x Captan x fertilizer interaction was found to be slightly significant (P=0.0713) when analyzed over the study (Table 3.1). Seedlings treated with both fertilizer and Captan were slightly, but not statistically significantly, increased relative to non-fertilized seedlings and decreased relative to fertilizer only treated seedlings (Figure 3.3). Temperature was found to be a highly significant (P<0.0001) variable, but only accounted for 3% of the variation in RR.
Mortality was quite high is some of the treatments. The FCAP treatment had the highest mortality rate at 65% of seedlings that died during the course of the study. The second highest mortality was 55% in the F treatment. There was 35 and 30% seedling mortality in CAP and C treatments, respectively. When treatments that received fungicide were bulked together, there was 48% seedling mortality. Likewise, when treatments that received fertilizer were bulked together, seedling mortality was 58%.

Experiment 2

On two separate sampling dates (4 and 16 days after fertilization) there were significant (P<0.10) differences between treatments. Four days following fertilization seedlings that received a high rate of fertilization (100 ppm N) showed increased RR relative to control (2 ppm N) seedlings (Figure 3.4). Sixteen days following fertilization we found a reversal with seedlings receiving a high dose of fertilization showing a significant (P<0.10) decrease relative to control seedlings (Figure 3.4). We found no consistent trend or differences in RR in seedlings that received low and moderate rates of fertilization during the course of this study. Temperature only explained 19% of the variation in RR, but when date was included in the model both temperature and date explained 45% of the variation in RR.

Discussion

Our daily average RR ranged from 0.65 to 2.35 µmol CO2 m-2root sec-1 for the two greenhouse studies, which are consistent with values we found in the field (Chapter 2)
and in a greenhouse experiment (Gough and Seiler 2004) on excised loblolly pine roots.
Lipp and Andersen (2003) showed that excising roots of 3-year-old ponderosa pine (Pinus ponderosa Laws.) did not impact RR rates for up to six hours, although Rakonczay et al. (1997a) found that respiration rates dropped significantly (P<0.05) for the first 30 minutes after excision in eastern white pine (Pinus strobus L.), but then remained stable for 16 hours. Studies have shown that RR rates measured at ambient atmospheric CO2 concentrations were higher than when measured at ambient soil CO2 concentrations (Qi et al. 1994, Clinton and Vose 1999, McDowell et al. 1999). Since our main objective was to observe the response of RR to fertilization and not compare our rates with rates observed in the field no attempt was made to account for differences in CO2 concentration.
Temperature was a highly significant (P<0.0001) variable in both experiments 1 and 2, but explained only 3 and 19% of the variance, respectively. Due to the lack of explanatory power of temperature on RR, temperature was not used as a covariate. In contrast to our findings, others have shown that temperature exhibits a positive exponential relationship with RR (Ryan et al. 1996, Boone et al.1998, Atkin and Tjoelker 2003). The lack of correlation we observed between temperature and RR that we hypothesized was at least partly due to the small range in temperatures that the seedlings experience over the short duration of the experiment. Daily fluctuations in solar irradiance may have influenced differences in RR between sampling dates more so than temperature. Photosynthesis is necessary to provide plants and associated mycorrhizae with energy to carry out maintenance and growth, and has been shown to be closely linked with root respiration using carbon-13 labeling (Ekblad and Högberg 2001) and tree girdling (Högberg et al. 2001) experiments. As Lipp and Andersen (2003) have pointed out, the increased carbohydrate storage in woody plants may be enough to offset daily changes in solar irradiance.
Both greenhouse experiments showed an initial increase in RR between seedlings that received a high rate (100 ppm N) of fertilizer compared to seedlings that did not receive fertilization. This increase was found to be temporary with rates returning to, or decreasing below control levels within 30 days from the time of fertilization (Figures
3.2A and 3.4). Other researchers have also observed the transient nature of fertilization on RR (Maier and Kress 2000, Gough and Seiler 2004). Gough and Seiler (2004) studied RR in potted loblolly pine seedlings grown in a greenhouse over the course of one year, and found a significant (P<0.05) increase in RR on day 49 with rates returning to control levels by day 197 following fertilization in the form of DAP. Maier and Kress (2000) measured RR following three years of nutrient additions to an optimal foliar nutrition of 1.3% as urea in an 11-year-old loblolly pine stand located at the Southeast Tree Research and Education Site (SETRES) in North Carolina. The authors found no increase in RR due to fertilization except for the month of May when RR was significantly (P<0.05) higher in fertilized treatments relative to control treatments. Pangle and Seiler (2002) found no difference due to fertilization in RR one year after fertilization in two-year-old loblolly pine seedlings. Lu et al. (1998) grew 6-month-old Douglas-fir [Pseudotsuga menziesii (Mirb.) Franco] seedlings in root boxes under three levels of N fertilization (10, 50, and 200 mg l-1 N; in the form of NH4NO3). The authors found that RR in root boxes treated with the low level of N was significantly (P<0.05) less then seedlings grown at higher rates of fertilization.
Increases in RR could be due to increased maintenance costs associated with higher protein turnover (Bouma et al.1994, Ryan et al. 1996). Although, the short-term increase in RR that we observed is more likely a result of energy expended to absorb and assimilate nitrogen. The decrease in RR observed at the end of experiments 1 and 2 was unexpected. One explanation is after the initial increase we observed in RR following fertilization, seedlings became nitrogen saturated and were unable to metabolize the NH4+ fast enough resulting in ammonium toxicity since excess ammonium cannot be stored (Lasa et al. 2002). Support of this hypothesis comes from the high mortality rates observed in experiment 1 with seedlings that received fertilization. Interestingly, we did not see any mortality in the second experiment. We believe that by repeatedly measuring RR on the same plant, which we employed in experiment 1, may have stressed the seedlings, leaving them vulnerable to mortality. A second explanation for the observed decrease in respiration rates as the study progressed is that there were fewer fine-roots, and associated mycorrhizae, present (visual inspection) in seedlings that received high fertilization rates forcing us to use larger, more suberized roots sometimes referred to as brown roots, which are less physiologically active then white roots, resulting in lower respiration rates (Pregitzer et al. 1998, Widén and Majdi 2001, Lipp and Andersen 2003). Fertilization has been shown to decrease the proportion fine- to coarse-root biomass (Ryan et al. 1996, Albaugh et al. 1998, Maier and Kress 2000) and ectomycorrhizae (Wallander and Nylund 1992, Nilsson and Wallander 2003).
Upon visual inspection, seedlings that were treated with Captan showed less mycorrhizae and fine-roots then those which were not treated. Specific root respiration was significantly (P<0.10) decreased 7 days following a 1 hour Captan dip at 1% strength (Figure 3.2B). Although, this was the only day that significant differences were observed between treatments possibly caused by a dilution of the Captan with subsequent dips reducing its effectiveness. Reasons for the initial decrease can be attributed to a reduction in mycorrhizae or more likely a reduction of fine-roots. Captan has been shown to reduce ecto-mycorrhizae in longleaf pine (Pinus palustris Mill.) seedlings (Pawuk et al. 1980), but was not effective in reducing Pisolithus tinctorius or Thelephora terrestris (two common ecto-mycorrhizae used in nursery application) in loblolly pine (Marx and Rowan 1981). When applied as a soil drench, Captan has been shown to have no negative effects on seedling height or survival in pine (Pawuk et al. 1980, Marx and Rowan 1981). Conversely, we found our greatest mortality in seedlings that had been treated with both Captan and DAP, and found 5% greater mortality in Captan versus control treatments, indicating that Captan had a negative impact on seedling survival. Differences may have been attributed to the methods of application (drench versus dip), or the extra handling involved in Captan treated seedlings. Although we did not attempt to quantify ecto-mycorrhizal biomass, visually there was no difference between seedlings that were treated with Captan and seedlings that received fertilizer. Both reduced mycorrhizal formation and fine-roots. Interestingly, the significant (P=0.0713) date x Captan x fertilization interaction (Table 3.1) and differences between (F) and (FCAP) seedlings RR means, although not statistically significant, seven days following fertilization (Figure 3.3) suggests that mycorrhizae may be partially responsible for the increased respiration rates in response to fertilization. Specific root respiration in treatments which received fertilizer (presumably seedlings + mycorrhizae) increased to a greater extent than the seedlings that had received fertilizer and Captan on day seven. This decreased fertilizer response in Captan treated seedlings does not appear to be due to any negative effect of Captan since RR in Captan only treated seedlings did not differ from controls on day seven (Figure 3.3).
More work is needed to assess how Captan impacts mycorrhizal formation as well as any negative affects on the roots or root respiration rates. At this point we would conclude that use of Captan is not a good method for separating root and associated mycorrhizal respiration due to lack of knowledge of what effects it will have on respiration. Hydroponic studies are a good way to separate RR from RH as well as allow measurements to be repeated with minimal disturbance to the plant, but as with all modified environments care must be taken when trying to apply results to field situations.

Table of Contents
List of Tables
List of Figures
CHAPTER 1: Introduction
Background
Objectives
Literature Review
Literature Cited
CHAPTER 2: The effects of fertilization on heterotrophic, autotrophic, and total soil CO2 efflux in a two-year-old loblolly pine (Pinus taeda L.) clonal plantation located on the Virginia Piedmont
Abstract
Introduction
Material and Methods
Results
Discussion
Tables/Figures
Literature Cited
CHAPTHER 3: Short-term effect of fertilization and Captan on specific root respiration in hydroponically grown one-year-old loblolly pine (Pinus taeda L.) seedlings
Abstract
Introduction
Material and Methods
Results
Discussion
Tables/Figures
Literature Cited
CHAPTER 4: Conclusion
Findings and Implications
Future Research
Literature Cited
Comprehensive Literature Cited
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