BORON DEFICIENCY AND CHILLING INJURY INTERACTIONS IN TOBACCO TRANSPLANTS GROWN IN THE FLOAT SYSTEM
Abstract
Boron deficiency and chilling injury produce similar symptoms in young tobacco transplants. Mistaken identification of chilling injury as B deficiency has resulted in B toxicity when growers apply additional B to non-deficient plants. In order to distinguish between B deficiency and chilling injury, it is important to determine an approximate B deficiency threshold and to determine the effects of sub-optimal temperatures on growth and B uptake. In an experiment to determine tissue concentrations of B associated with B deficiency symptoms, transplants grown at a day/night temperature regime of 26/22°C exhibited B deficiency symptoms at 10–20 µg B g−1 dry matter. These tissue levels resulted from solution concentrations of 0.19–1.9 µM B (.002–.205 mg L−1 B). Root and shoot growth of flue-cured cultivars was near maximum at a 26/26°C day/night temperature regime. Burley cultivars were adapted to a wider temperature range for optimum growth compared to flue-cured cultivars. The B deficiency threshold decreased with decreasing temperatures and with increased day/night temperature differentials. Sub-optimal temperatures appeared to be the primary growth limiting factor for tobacco transplants in the float system with low B availability. Boron deficiency was a secondary factor in limiting growth.
INTRODUCTION
Quality of transplants is a critical factor in tobacco (Nicotiana tabacum) production. Good transplant quality can improve stand establishment, decrease stand variability, make harvesting more efficient, and improve the quality of the cured leaf product. Currently, the most prevalent system for production of tobacco transplants in the southeastern United States is the greenhouse float system 13.
Growers sometimes overlook management of boron (B) in float systems. Current recommendations 10 suggest that the fertilizer contain at least 0.02 percent B, a concentration not contained in all fertilizers utilized in float systems. In some instances, trace contamination levels of B in fertilizer and water sources may provide sufficient B for acceptable plant growth 1,9. Lack of attention to B fertilization creates the potential for B deficiencies in float systems. Conversely, supplemental applications of B can produce B toxicity if not carefully applied. Concentrations of B in the float solution or in plant tissue required to avoid deficiency in tobacco transplants is currently unknown.
Boron deficiency symptoms include interveinal puckering, leaf dimpling, discoloration, and eventually failure to maintain apical dominance and necrosis of the apical meristem. Boron is required for the production of stabile complexes with cell wall constituents necessary for structural integrity of tissues. Boron also appears to be necessary for growth of shoot apical meristems, root hairs, and pollen tubes. The high requirement of B for these tissues is probably the result of the need for B for the secretion of cell wall material 3. Lovatt 5 concluded that cessation of cell division in the apical meristem is the earliest and most prevalent result of B deficiency.
Chilling injury in young tobacco transplants is a common occurrence for plants grown in tobacco float greenhouses, especially in the burley tobacco production areas of North Carolina. The symptoms of chilling injury in young transplants, which include cupped leaf margins, puckering of the interveinal lamina tissue, lesions, and deformities of the leaf, are similar to those of B deficiency. Although not typically a major threat to the health of transplants, chilling injury can stunt growth and delay transplant date of tobacco seedlings. Symptoms are induced and/or exacerbated by a reduction in ambient air temperature followed by a return to warmer temperatures 6,8,11,12. Many tobacco float systems are susceptible to such fluctuating conditions due to inadequate ventilation systems. In the early morning hours when the sun is first rising, greenhouses can warm up rapidly. Temperature increases of seven to ten degrees C have been measured in greenhouses over the course of just 15 min in the early morning 2. This rapid increase in temperatures over a short period of time may be a key in symptom development in young tobacco transplants produced in the float system.
The similar symptoms induced by B deficiency and chilling injury may indicate interdependent effects on cell physiology and/or metabolism of these two stresses. Because little research has been conducted with tobacco float culture, an initial study was conducted to determine a B deficiency threshold for tobacco seedlings in the float system. A second study was conducted to examine the combined effects of temperature regime and B fertilization on transplant growth and B uptake by seedlings. The specific objectives of this study were: 1) to determine the B deficiency threshold of tobacco transplants grown in the float system; and 2) to determine effects of B deficiency and temperature regime on size, mass, and B concentration, and to examine interactions between B deficiency and chilling injury in tobacco transplants grown in the float system.
MATERIALS AND METHODS
General methods
Both studies were conducted in controlled-environment chambers within the NC State University Phytotron. Since individual tobacco cultivars can have distinctive uptake rates of B 7, four cultivars, two flue-cured and two burley, were used in this study. All seedlings were started on 0.14 M NO3 solution with no other nutrients in order to avoid salt injury to seedlings. At two weeks after seeding, nutrients were added to float solutions in both studies. With the exception of B, all studies had the same macronutrient and micronutrient concentrations. Macronutrient concentrations were: 0.6 M NO3, 0.8 M K, 0.25 M Ca, 0.16 M Mg, 0.02 M S, while micronutrient concentrations were: 10 µM Fe, 3.7 µM Mn, 0.3 µM Zn, 0.1 µM Cu, and 0.05 µM Mo. Trace B contamination was removed by running nutrient stock solutions through an Amberlite IRA-743 resin column (Sigma Corporation). Final B concentrations in nutrient stock solutions was 2 µg L−1 or less.
Nitrate and PO43− solution concentrations were measured weekly throughout the studies using an ion chromatograph (Dionex 2110i, AS4A 4 mm column). Replacement nutrients were added in the form of Mg(NO3)2 and KH2PO4 as needed to compensate for plant uptake.
Boron deficiency threshold
A seedling hydroponics system with four 15 L compartments with individual pumps to provide circulation and aeration of solution was used for this study. The hydroponics unit was located in a controlled-environment chamber set to a 26°/22°C ( ±1°C) day/night temperature regime with a 9-hour day period, reported to be near optimal for establishment of tobacco seedlings 4. The average photon flux density was 541 µmol m−2 sec−1, and relative humidity was maintained at greater than 50%. All nutrient solutions were maintained at a pH value between 5.5 and 6.5 by injecting 0.05 M H2SO4 with an automated pH control system. Each compartment contained a separate treatment: 0, 0.19, 1.9, or 19 µM B from B(OH)3. Boron concentrations were not renewed over the course of the study.
Tobacco seedlings were grown from uncoated seeds. Seeds were individually placed on agar plugs poured into holes (approximately 0.75 cm diameter) in the polyethylene lid covering each of the four 15 L compartments of the hydroponic unit. Burley varieties TN90 and KY14 and flue-cured varieties C371 and K326 were placed on the surface of plugs consisting of 0.8% agar and 0.14 M NO3−. Treatments were replicated in time and samples were treated as a completely randomized block. Sub samples were taken within each treatment to evaluate the effect of B on different tobacco varieties. Five tobacco seedlings per sub treatment were harvested 29 d after seeding to determine root and shoot fresh mass and dry mass (tissue was freeze-dried), leaf area, and tissue B concentrations.
Temperature effects on boron concentration
This study was conducted using a 4×2× 2 factorial treatment design in a split-split-plot arrangement. Temperature treatments (four levels) were applied to the whole plots, while B concentration (two levels) was applied to split plots and tobacco type (two levels) was applied to split-split plots. Controlled-environment chambers were programmed to provide four day/night temperature regimes: 26°/18°C, 32°/14°C, 21°/21°C, and 26°/26°C, with one chamber per regime. These four temperature regimes were selected to test the hypothesis that chilling injury is induced by a large day/night temperature differential instead of a low average temperature alone. Each regime either tested or served as a control for this hypothesis in the following manner: 26°/26°C – optimum temperature for maximum growth, control; 21°/21°C - sub-optimal temperature with no day/night differential, serves as a control to test the hypothesis that chilling injury is induced by a temperature differential; 26°/18°C - sub-optimal average temperature of 21°C and an 8°C day/night difference, tests hypothesis that chilling injury is induced by a temperature differential; 32°/14°C - sub-optimal average temperature of 21°C and an 18°C day/night difference, tests hypothesis that chilling injury is induced by an extreme temperature differential. Shoot and root dry mass were used as indicators of over-all plant health and thus for determining a B deficiency threshold and for determining plant response to B-level and temperature regime.
Photoperiod was 10 h with a 30-min night interruption in the middle of the dark period. Light intensity was greater than levels used in the initial B deficiency study, with an average photon flux density of 940 (±58) µmol m−2 s−1. Four 12 L plastic tanks were placed within each chamber to establish two replicates for each B treatments (19 µM and 0.19 µM B). Polystyrene seedling trays (288 cell, Speedling Corp., Sun City, FL) were cut down to fit the tanks (122 cells) and divided into quadrants. One cultivar was randomly assigned to each quadrant (same two cultivars of flue-cured and two cultivars of burley as used in B deficiency study). At each sampling date, four plants were randomly selected from each quadrant for measurement.
The trays were filled with a 1∶1 sand∶vermiculite medium that had been rinsed three times with 12 L of deionized water to remove B contamination. Sand was used in the mix in place of peat to facilitate control of B contamination. The trays were seeded by hand with uncoated tobacco seeds. Plants were thinned to two seedlings per cell after five days and then to one seedling per cell after 10 days. All seedlings were germinated and grown at a temperature of 26/18°C for the first two weeks after seeding. After this time, temperature and nutritional treatments were initiated.
Four tobacco seedlings per treatment combination were harvested on days 47 and 54 after seeding to determine root and shoot fresh mass, dry mass (tissue was freeze dried), and tissue B concentration. At the final harvest, plants were 15–20 cm tall with 8 to 10 leaves and considered to be of transplant size.
Tissue boron analysis
Shoot and root tissues were digested in concentrated high-purity HNO3 (Fisher, Optima grade) based on procedure two described by Zarcinas et al., 15. The procedure was modified to use 4 ml of nitric acid per sample due to the small mass of tissue in each sample. Each plant from the four plants per sample was divided into root and shoot tissues and individually analyzed. Samples were allowed to digest in HNO3 overnight and then were microwaved at five-percent power for fifteen minutes in a standard food-grade microwave oven (1000 watts). After digestion, samples were analyzed using inductively-coupled plasma atomic emissions spectroscopy with a Perkin Elmer Model 2000 DV emission spectrometer.
Statistical analysis
Statistical contrasts were utilized to make comparisons between treatments in the temperature study. The rationale for using this analysis compared to other more common pair-wise tests was the consequence of having the temperature treatment un-replicated. Due to time and space constraints, it was not feasible to replicate the temperature treatments and thus there was no available error term to assign to the temperature values. Therefore, we are only able to make statistical contrasts based on differences between the low and high B values for each temperature treatment. In the figures representing this data, the letters connoting significant difference are placed in the center of the two bars representing the low and high B values for each temperature treatment and the letters signify the statistical differences of the arithmetic differences for low and high B values.
There were no statistically significant differences in dry weight between the two flue-cured varieties so the eight plants were averaged for each tray before further statistical analysis was conducted. The same was true for the burley cultivars, and they, too, were combined for further analysis.
RESULTS AND DISCUSSION
Boron deficiency threshold
The B deficiency threshold for 29 day-old seedlings grown in a 26/22°C temperature regime occurred when shoot B levels were between 10 and 20 µg g−1. This threshold was set based on the observation that shoot B concentration, root dry matter, and shoot dry matter dramatically increased as solution B concentration increased from 0.19 µM (10 µg g−1 tissue B) to 1.9 µM (20 µg g−1 tissue B) (Figs. 1a, 1b, and 1c). Although no B deficiency threshold has been established for tobacco, our results are in agreement with a previous study that reported increased shoot growth between 0 and 46 µM B in float solution 9.
Citation: Tobacco Science 47, 47; 10.3381/1953.1
Temperature regime and B concentrations
Shoot B concentrations of flue-cured and burley types at different temperature and B treatments were determined at 47 and 54 days after seeding (Figs. 2 and 3). Plants in the 19 µM B treatment exhibited greater uptake of B than plants in the 0.19 µM B treatments for all temperature treatments and tobacco types. Plants in the low B treatments exhibited small differences in B tissue concentration regardless of temperature treatments or tobacco type at both sampling dates; therefore, we conclude that depletion of B at lower solution B concentrations was not strongly affected by the temperature treatments imposed in this study
Citation: Tobacco Science 47, 47; 10.3381/1953.1
Citation: Tobacco Science 47, 47; 10.3381/1953.1
At 47 days after seeding, flue-cured and burley plants grown in the high B treatments and the sub-optimal temperature treatments of 21/21°, 26/18°, and 32/14°C, had somewhat lower shoot B concentrations than plants grown in the 26/26°C treatment (Fig. 2). By 54 d after seeding, however, this trend was no longer evident (Fig. 3), possibly due to depletion of available solution B (solution B concentration was not renewed over the course of the study). Treatments generally had slightly greater B concentration values per unit mass at 54 days after seeding. The one exception to this trend was the flue-cured plants grown at the 26/26°C high B treatment, in which the tissue B concentration decreased from 35.9 µg g−1 to 17.7 µg g−1. This was most likely the result of a dilution effect due to the rapid increase in average shoot tissue mass, from 0.14 g plant−1 to 0.44 g plant−1, that occurred between the two sampling dates.
Shoot Mass
Similar to observations on tissue B concentration, shoot mass data reflected the trend of maximal biomass production in the 26/26°C temperature regime for the day 47 harvest (Fig. 4). Also, as in the tissue B concentration data, this trend was less evident on day 54 (Fig. 5). One obvious exception to this pattern was the previously mentioned flue-cured plants grown at 26/26°C and high B level, where plants displayed noticeably greater growth compared to all other treatments. These plants, however, did not have significantly greater B levels in the shoot tissue, which may indicate that flue-cured tobacco transplants are quite sensitive to sub-optimal temperature conditions regardless of whether it is the result of a lower average temperature or a large day/night temperature differential.
Citation: Tobacco Science 47, 47; 10.3381/1953.1
Citation: Tobacco Science 47, 47; 10.3381/1953.1
Another noteworthy observation on day 47, although also not statistically significant, is that the higher B concentration produces greater tissue mass for the 26/26°C treatments (Fig. 4). Interestingly, tissue mass production was favored by the lower B concentration at the more stressful temperature regimes of 26/18° and 32/14°C. This was more pronounced in the burley than the flue-cured types and may be an indication that less B is required for transplants grown with a day/night temperature differential.
Root mass
Root mass of 47 day-old flue-cured seedlings was consistently greater at the lower B values regardless of temperature regime (Fig. 6). For the flue-cured varieties, sub-optimal temperatures, but not low B levels, resulted in decreased root growth. When flue-cured and burley types were compared, it again appeared that at 47 d, flue-cured types grew best under a 26/26°C day/night regime and burley types were more tolerant of lower and fluctuating day/night temperatures. At 54 days after seeding, flue-cured transplants had the greatest root mass at 26/26°C. There were no obvious differences across temperatures for burley varieties, again suggesting that burley varieties are less sensitive to temperature than flue-cured varieties. All cultivars produced the greatest root mass in the low B treatments. Steinberg 14 reported that B deficiency increased root branching, which would be consistent with visual observations made during measurements.
Citation: Tobacco Science 47, 47; 10.3381/1953.1
Effects of temperature on the B deficiency threshold
At the largest day/night temperature differential of 32/14°C, B uptake was always below or within the B deficiency threshold of 10 to 20 µg g−1 dry matter (Figs. 2 and 3). Interestingly, the lower B treatment (0.19 uM) produced greater root and shoot biomass than the high B treatment at both sampling dates for this temperature regime. Plants in the low B treatment grew faster between days 47 and 54 than plants in the high B treatments. For example, the root systems of plants in the 32/14°C and low B treatment increased 165% (flue-cured) and 275% (burley) relative to the plants in the high B treatment.
The B deficiency threshold we established was determined for plants grown at optimal temperature conditions (26/22°C), and it is possible that other, more stressful, temperature regimes may alter the B deficiency threshold; it may be lower at lower temperatures. Possible mechanisms for such a shift in deficiency thresholds at sub-optimal temperatures include reduced transpiration and the related phenomena of reduced mass flow of nutrients, reduced nutrient uptake, and physiological chemical interactions. Shoot masses of flue-cured varieties at day 47 (Fig. 4) were greatest at low B levels in the 21/21° 26/18°, and 32/14°C treatments. A similar affect occurred in the 26/18° and 32/14°C treatments for burley. Tissue B concentration for all of the low B treatments mentioned above were well below the deficiency threshold previously determined, while the high B treatments for the respective samples would be classified as marginally- or non-deficient. (Fig 2). None of the above-mentioned samples displayed symptoms of B deficiency. This suggests that there was an interaction between temperature regime and B concentration in our study.
Our data indicate that a B deficiency threshold may be reduced under sub-optimal temperature conditions. Several hypotheses can be developed to explain this phenomenon. One is that there is a temperature and B deficiency interaction resulting in reduced shoot growth, and, thus, a reduced B requirement. If this is true, then the B toxicity threshold could also be lowered at sub-optimal temperatures and a marginal B toxicity could occur at lower temperatures even under B supply considered to be optimal at a warmer temperature.
Temperature Effects
It also appeared that flue-cured and burley varieties differ in their abilities to adjust to sub-optimal temperature regimes (Figs. 4–7). Flue-cured varieties produced maximal root and shoot biomass at the optimal temperature regime of 26/26°C. In contrast, burley varieties showed no discernable pattern in root and shoot biomass accumulation over the range of temperature regimes tested. Therefore, it may be concluded that burley is more tolerant of temperature extremes than flue-cured varieties. In considering B deficiency thresholds, we conclude that although the plant may be able to adjust its B deficiency threshold to a lower level during times of temperature stress, it is not able to attain its maximum shoot biomass-production potential during these times, particularly for flue-cured varieties.
Citation: Tobacco Science 47, 47; 10.3381/1953.1
SUMMARY AND CONCLUSIONS
The preliminary study indicated that the boron deficiency threshold for tobacco transplants growing at a 26/22°C day/night temperature regime is in the range of 10–20 µg B g−1 dry matter, which occurred at solution B concentration of 0.19 to 1.9 µM B. A relationship was found between tissue B concentration and day/night temperature regime. Even with adequate solution B concentrations (19 µM B), tissue B is lower at sub-optimal temperature regimes (21/21°C, 26/18°C, and 32/14°C) compared to an optimal temperature regime (26/26°C), especially for flue-cured varieties. Root and shoot growth of all varieties was good in the 26/26°C treatment, but the burley varieties also grew well in other temperature treatments.
This study suggests that symptoms commonly observed in commercial tobacco seedling production are those of cold injury. It is possible that sub-optimal temperature regimes are resulting in transient delays in B uptake. Even so, the value of supplemental B application during periods of cold injury is questionable since the plants should recover upon return to favorable temperatures. The danger of inducing B toxicity from over-application of B is greater than the potential growth benefit derived from the application.
Sub-optimal temperatures or stressful temperature differentials may decrease the functioning B deficiency threshold. It appears that environmental parameters, including day/night temperature regimes, affect the B deficiency threshold for a plant, in effect inducing a “sliding scale” for determining the B deficiency concentration. Finally, temperature is the immediate limiting factor in tobacco transplant growth in the float system, but under conditions of sub-optimal temperatures and low B concentration, B deficiency can be an additional limiting factor.
Influence of nutrient solution B concentration on shoot B concentration (A), root mass (B), and shoot mass (C) of four varieties of tobacco seedlings. Data are means of three replications.
B concentrations in 47 day-old shoot tissue at four day/night temperature regimes and two B concentrations in nutrient solution for A) Flue-cured and B) Burley tobacco types. Bars represent mean data of two replications. A previously established B deficiency threshold zone of 10 to 20 µg g−1 dry weight is shaded. Letters placed in the center of the two bars representing the low and high B values for each temperature treatment signify a statistical difference between low and high B values.
B concentrations in 54 day-old shoot tissue at four day/night temperature regimes and two B concentrations in nutrient solution for A) Flue-cured and B) Burley tobacco types. Bars represent mean data of two replications. A previously established B deficiency threshold zone of 10 to 20 µg g−1 dry weight is shaded. Letters placed in the center of the two bars representing the low and high B values for each temperature treatment signify a statistical difference between low and high B values.
Shoot masses of 47 day-old transplants at four day/night temperature regimes and two B concentrations in nutrient solution for A) Flue-cured and B) Burley tobacco types. Bars represent mean data of two replications. Letters placed in the center of the two bars representing the low and high B values for each temperature treatment signify a statistical difference between low and high B values.
Shoot masses of 54 day-old transplants at four day/night temperature regimes and two B concentrations in nutrient solution for A) Flue-cured and B) Burley tobacco types. Bars represent mean data of two replications. Letters placed in the center of the two bars representing the low and high B values for each temperature treatment signify a statistical difference between low and high B values.
Root masses of 47 day-old transplants at four day/night temperature regimes and two B concentrations in nutrient solution for A) Flue-cured and B) Burley tobacco types. Bars represent mean data of two replications. Letters placed in the center of the two bars representing the low and high B values for each temperature treatment signify a statistical difference between low and high B values.
Root masses of 54 day-old transplants at four day/night temperature regimes and two B concentrations in nutrient solution for A) Flue-cured and B) Burley tobacco types. Bars represent mean data of two replications. Letters placed in the center of the two bars representing the low and high B values for each temperature treatment signify a statistical difference between low and high B values.
Contributor Notes
The use of trade names in this publication is solely to ensure accurate description of experimental conditions. No endorsement or criticism of these products or similar products not mentioned is intended.