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Difference of Ca distribution before and after the onset of tipburn in lisianthus [Eustoma grandiflorum (Raf.) Shinn.] cultivars
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Difference of Ca distribution before and after the onset of tipburn in lisianthus [Eustoma grandiflorum (Raf.) Shinn.] cultivars Takanori Kuronuma, Nozomi Kinoshita, Masaya Ando, Hitoshi Watanabe* Center for Environment, Health and Field Sciences, Chiba University, Japan
A R T I C LE I N FO
A B S T R A C T
Keywords: Eustoma grandiflorum Ca deficiency Tipburn Systematic investigation Growth analysis Ca distributivity
Tipburn (leaf-tip necrosis) is severe problem for the production of lisianthus [Eustoma grandiflorum (Raf.) Shinn.] cultivars. A previous study has suggested that tipburn of 14 lisianthus cultivars are caused by the inability to translocate sufficient quantities of calcium (Ca) to the tips of the upper leaves. However, systematic studies are not available that identify the differences in Ca concentrations and distributivity before and after the onset of tipburn. To address this insufficiency in our knowledge, we used the lisianthus cultivars ‘Umihonoka’ (UH), ‘Voyage green’ (VG), and ‘Voyage pink’ (VP) and determined Ca concentrations and the dry weight of each organ at weekly intervals for 8 weeks. In addition, we evaluated the Ca distributivity from the ratios of the relative Ca increase rate (RGRCa) to the relative plant growth rate (RGR) by applying growth analysis methods. UH did not exhibit tipburn. In contrast, VG and VP exhibited tipburn after 4 and 5 weeks, respectively. At the same time, the concentrations and distributions of Ca in the roots increased and those of whole and new leaves decreased in each cultivar. Thus, it is suggested that the distribution of Ca in new leaves of the tipburn-sensitive cultivars VG and VP was below the threshold, which caused tipburn. Moreover, it was demonstrated that decreases in the distribution of Ca in whole and new leaves were mainly caused by not increase of the relative leaf growth rate (RGRleaf) but decrease of the relative leaf Ca increase rate (RGRleaf-Ca). We conclude that this approach more rigorously evaluates the influence of plant growth rate on the incidence of tipburn. Thefore, tipburn exhibited by lisianthus cultivars was caused by decreased distributions of Ca in new leaves, which was associated with an increase in the distribution of Ca in roots.
1. Introduction Lisianthus [Eustoma grandiflorum (Raf.) Shinn.] is an ornamental plant that is native to warm regions of the Southern United States and Northern Mexico. Its cultivars are mainly supplied as cut flowers. In Japan, the wholesale value in 2017 of lisianthus ranked fifth among cut flowers. However, tipburn [calcium (Ca) deficiency disorder] of certain lisianthus cultivars causes serious economic losses. Tipburn diminishes the quality of plants, thereby adversely affecting their marketability. The cause of tipburn has been intensively studied, including efforts to reduce damage to plants by controlling the environment [e.g., lettuce (Barta and Tibbitts, 1991; Choi and Lee, 2008; Misaghi and Grogan, 1978; Sago, 2016; Uno et al., 2016; Wissemeier and Zühlke, 2002), Chinese cabbage (Aloni et al., 1986; Kuo et al., 1981; Magnusson, 2002), strawberries (Bautista et al., 2009; Guttridge et al., 1981; Mason and Guttridge, 1975), and lilies (Chang and Miller, 2003, 2004; Chang and Miller, 2005)]. Research on lisianthus cultivars indicates that tipburn is mainly caused by the inability of the plant to translocate
⁎
adequate amounts of Ca to the tips of the upper leaves (Kuronuma et al., 2019). However, there is no systematic investigation, to our knowledge, which reports the differences in Ca concentrations and Ca distributivity before and after the onset of tipburn. Thus, the cause of cultivar-specific tipburn remains to be determined. It is well known that plant growth rate significantly influences tipburn severity and incidence (Collier and Huntington, 1983; Cox et al., 1976; Lee et al., 2013a; Saure, 1998). Because the Ca concentration of each organ is determined by the relationship between plant growth rate (dry weight) and Ca increase rate in each organ. Accordingly, Ca deficiency and its distributivity has a close relationship to plant growth rate and Ca increase rate. However, Ca deficiency and distributivity has been evaluated only through determination of the changes in Ca concentrations. Therefore, Ca distributivity (including Ca deficiency) must be determined using a more rigorous approach that measures Ca concentrations as well as the relationship between plant growth rate and Ca increase rate. For example, the Ca increase rate was quantified as Ca acquirement competence (Kuronuma et al., 2019) by applying growth
Corresponding author. E-mail addresses: [email protected] (T. Kuronuma), [email protected] (H. Watanabe).
https://doi.org/10.1016/j.scienta.2019.108911 Received 7 May 2019; Received in revised form 13 September 2019; Accepted 2 October 2019 0304-4238/ © 2019 Elsevier B.V. All rights reserved.
Please cite this article as: Takanori Kuronuma, et al., Scientia Horticulturae, https://doi.org/10.1016/j.scienta.2019.108911
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analysis methods (Garnier, 1991; Kuronuma and Watanabe, 2016a, b; Poorter and Remkes, 1990; Poorter et al., 1990). Here we aimed to determine the differences in Ca concentrations and distributivity before and after the onset of tipburn and to discuss the cause of tipburn in certain lisianthus cultivars. We further evaluated Ca distributivity (including Ca deficiency) and its associations with plant growth rates with Ca increase rates. 2. Material and methods 2.1. Plants The lisianthus cultivars were ‘Umi honoka’ (UH) (Sumika Agrotech Co., Ltd.), ‘Voyage pink’ (VP) (SAKATA SEED CORPORATION), and ‘Voyage green’ (VG) (SAKATA SEED CORPORATION), which were selected from a different group classified in a previous study (Kuronuma et al., 2019). UH has been classified tipburn–resistant cultivar’s group, and VG has been classified tipburn-sensitive cultivar’s group with low abilities to distribute Ca to the tips of their upper leaves. VP has been classified tipburn-sensitive cultivar’s group with low abilities to acquire Ca and distribute it to the tips of their upper leaves. In addition, Voyage series including the VG and VP is one of the most famous lisianthus cultivars in Japan. Thus, VG and VP were selected. Seeds were sown in plug flats (406 cells per tray) filled with seedling propagation medium (Metro Mix 350; Sun Gro Horticulture, Agawam, MA, USA). The seeded trays were maintained in a germination room at 24 °C under a 14-h light/10-h dark photoperiod. After two weeks, the trays were transferred to a controlled environmental system (phytotron), and the plants grown under the conditions as follows: 25 °C (light period) and 20 °C (dark period), 60 ± 5% humidity, 400 ppm CO2, 14h light (225 ± 25 μmol m−2 s-1) and 10-h dark. An irrigation system in the phytotron supplied a nutrient solution (62 ppm NO3-N, 4.6 ppm NH4-N, 10.2 ppm PO4-P, 104 ppm K, 16 ppm SO4-S, 40 ppm Ca, 12 ppm Mg, 2.8 ppm Fe, 1.6 ppm B, 0.8 ppm Mn, 0.08 ppm Zn, 0.04 ppm Cu, and 0.04 ppm Mo) by bottom watering for 10 min. Five weeks after transfer to the phytotron, plugs were transplanted into 0.25-L polyethylene pots filled with Metro Mix 350.
Fig. 1. Tipburn severity and incidence. Mean values and the SE (n = 12) (error bars) are shown.
2.4. Measurement of dry weight and Ca concentration Samples of plant organs were dried at 70 °C for 72 h, and weighed. Samples of two plants were combined, and Ca concentrations were determined using a Z-5300 polarized Zeeman atomic absorption spectrophotometer (Hitachi, Ltd., Tokyo, Japan). The Ca concentrations of the top one-fifth (leaf tip) and the remainder (leaf base) of each leaf were separately analyzed. To calculate total Ca concentrations, we quantified the whole-plant Ca content by adding the Ca content (Ca concentration × dry weight) of each organ. Total Ca concentrations were calculated by dividing the whole-plant Ca content by the mass of the whole plant. Ca concentrations of whole leaves were similarly determined. 2.5. Ca distributivity To evaluate the Ca distributivity of the lisianthus cultivars, we quantified Ca concentrations and their relationships between the plant growth rates and Ca increase rates (see Section 2.5.1).
2.2. Experimental design
2.5.1. Ca distributivity of leaves, stems, and roots Except for the early stages of the experiments, the dry weights and Ca contents of whole leaves, stems, and roots increased exponentially (Appendix 1). Thus, the rates of plant growth and increases in Ca content of each organ were quantified as the relative growth rate (RGR) and relative Ca increase rate (RGRCa), respectively, as follows:
To determine the differences in Ca concentration and distributivity before and after tipburn, cultivars were grown in the same phytotron under the environmental and irrigation conditions described above. First, four pots of each cultivar were randomly sampled in triplicate and then harvested at one week intervals for 8 weeks. Harvested plants were washed with distilled water and divided into roots, stems, leaves on the main stem, and other leaves. The leaves on the main stem were distinguished for each leaf position, which were numbered from the base to top leaves. Unfolded leaves were included with stems, because they were too small for analysis. 2.3. Tipburn severity and incidence Whole leaves were scored using arbitrary tipburn severity indices ranking from 0 to 1 (0, asymptomatic; 0.2, deformed leaf margins; 0.5, leaf-tip chlorosis; 1, leaf-tip necrosis). The severity of tipburn in each plant and at each leaf position was defined as follows: Tipburn severity in each plant = ∑ {(severity indices × leaf number) / whole leaves number per pot} × 100 (1)
RGR = (lnW2 – lnW1) / (T2 – T1)
(3)
RGRleaf = (lnWleaf2 – lnWleaf1) / (T2 – T1)
(4)
RGRstem = (lnWstem2 – lnWstem1) / (T2 – T1)
(5)
RGRroot = (lnWroot2 – lnWroot1) / (T2 – T1)
(6)
RGRCa = (lnWCa2 – lnWCa1) / (T2 – T1)
(7)
RGRleaf-Ca = (lnWleaf-Ca2 – lnWleaf-Ca1) / (T2 – T1)
(8)
RGRstem-Ca = (lnWstem-Ca2 – lnWstem-Ca1) / (T2 – T1)
(9)
RGRroot-Ca = (lnWroot-Ca2 – lnWroot-Ca1) / (T2 – T1)
(10)
where W1 and W2 are the whole-plant dry weights, Wleaf1 and Wleaf2 are the whole-leaf dry weights, Wstem1 and Wstem2 are the stem dry weights, Wroot1 and Wroot2 are the root dry weights, WCa1 and WCa2 are the whole-plant Ca contents, Wleaf-Ca1 and Wleaf-Ca2 are the whole-leaf Ca contents, Wstem-Ca1 and Wstem-Ca2 are the stem Ca contents, and WrootCa1, and Wroot-Ca2 are the root Ca contents at times T1 and T2,
Tipburn severity at each leaf position = ∑ {(severity indices × leaf number) / whole leaves number at each leaf position} × 100 (2) The incidence of tipburn is expressed as the percentage of plants in a cultivar exhibiting symptoms of tipburn. 2
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Fig. 2. Ca concentration in each leaf tip and base, and tipburn severity at each leaf position of UH. Mean values are shown, and error bars represent the SE (n > 3). *Significant differences between Ca concentrations in tips and those in the bases of individual leaves (Student t test, *P < 0.05, **P < 0.01).
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Fig. 3. Ca concentrations in each leaf tip and leaf base, and tipburn severity at each leaf position of VG. Mean values are shown, and error bars represent the SE (n > 3). * Significant differences between Ca concentrations in tips and those in the bases of individual leaves (Student t test, *P < 0.05, **P < 0.01).
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Fig. 4. Ca concentrations in each leaf tip and leaf base and tipburn severity at each leaf position of VP. Mean values are shown, and error bars represent the SE (n > 3). *Significant differences between Ca concentrations in tips and those in bases of individual leaves (Student t test, *P < 0.05, **P < 0.01).
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Table 1 Ca concentrations, relative Ca increase rate (RGRCa), relative growth rate (RGR), and RGRCa/RGR. Weeks
Total Ca concentrations (mg g
)
UH 0 1 2 3 4 5 6 7 8
3.1 2.9 2.8 2.7 3.1 2.9 3.0 3.1 3.4
RGRCa
−1
(mg-Ca mg-Ca VG
b ab ab a b ab b b b
RGR
3.1 3.3 3.1 3.1 3.1 2.8 3.0 3.4 3.9
VP B B B B B A AB BC C
2.3 2.8 3.0 2.7 2.8 2.9 3.0 3.0 3.6
a' a'b'c' b'c' a'b' a'b' b'c' c' c' d'
−1
week
−1
)
(g g
−1
RGRCa / RGR week
−1
(mg-Ca g-DW mg-Ca−1 g-DW−1)
)
UH
VG
VP
UH
VG
VP
UH
VG
VP
－ － 0.64 1.04 0.59 0.41 0.45 0.65 0.21
－ － 0.59 0.96 0.83 0.23 0.60 0.64 0.32
－ － 0.86 0.92 0.70 0.64 0.63 0.49 0.45
－ － 0.67 1.11 0.43 0.45 0.43 0.60 0.15
－ － 0.63 0.98 0.83 0.33 0.50 0.53 0.19
－ － 0.80 1.01 0.68 0.58 0.62 0.48 0.29
－ － 0.95 0.93 1.37 0.91 1.04 1.08 1.40
－ － 0.93 0.98 1.00 0.69 1.18 1.22 1.67
－ － 1.07 0.91 1.02 1.10 1.02 1.03 1.56
Mean values are shown, and significant differences among them are indicated by different letters (Tukey’s b test, P < 0.05). Bold type represents the values before and after the onset of tipburn.
3. Results and discussion
respectively. To evaluate Ca distributivity from the relationship between the relative rates of plant growth and increases in Ca concent, we calculated the ratios of the relative Ca increase rate to the relative growth rate (RGRCa/RGR). The weekly Ca accumulations in whole leaves, stems, and roots were calculated by subtracting the Ca content of each organ at “n” weeks from those at “n + 1” weeks. Weekly Ca distribution ratios in each organ were calculated by dividing the Ca accumulation in each organ per plant per week.
3.1. Tipburn severity and incidence 3.1.1. Tipburn severity and incidence UH did not exhibit detectable tipburn (Fig. 1), which is consistent with the results of a published study (Kuronuma et al., 2019). VG and VP exhibited tipburn 4 and 5 weeks, respectively, after the experiment commenced. The incidence of tipburn exhibited by VP and VG reached 100% the week after the first observation of tipburn. 3.1.2. Ca concentration and the tipburn severity at each leaf position The Ca concentration and the severity of tipburn at each leaf position are shown in Figs. 2–4, which show the results for each leaf position only when > 6 samples were collected, because the number of leaves of each plant differed. The first leaves (leaf position 1) dissolved and disappeared by 3 weeks. The Ca concentrations of the leaves of all cultivars decreased as a function of higher leaf position (Figs. 2–4). UH did not exhibit detectable tipburn at any leaf position (Fig. 2), and the Ca concentrations in the tips of the top leaves were > 2.0 mg g−1 until the next week. The Ca concentrations in the tips of the upper leaves were significantly higher compared with those at the bases, except at 8 weeks. In contrast, Ca concentrations in the tips of the top leaves of VG and VP scince 3 weeks were < 2.0 mg g-1 until the next week (Figs. 3 and 4). In addition, there are no significant differences between their Ca concentrations in tips of the upper leaves and those in bases. VG and VP exhibited tipburn in the upper leaves at 4 and 5 weeks, respectively. These results indicate that the tipburn-resistant cultivar UH translocated, at least, the threshold level of Ca to the upper (new) leaf tips. In contrast, although the tipburn-sensitive cultivars VG and VP translocated at least, the threshold level of Ca to upper (new) leaf tips during the early stages of plant growth, the Ca distributions of the upper (new) leaves gradually decreased. Consequently, tipburn would have occurred.
2.5.2. Ca distributivity in new leaves Quantification of Ca distributivity in new leaves was not used in the growth analysis methods, because the dry weights and Ca contents of new leaves did not exhibit an exponential increase. Thus, ⊿dry weights (increment of dry weight per week) and ⊿Ca content (increment of Ca content per week) of new leaves were calculated. The ⊿dry weights of new leaves from “n” to “n+1” weeks were calculated by the adding mean values of the dry weights of newly emerged leaves at “n+1” weeks to increment the mean values of the dry weights of leaves emerged at “n” weeks. Similarly, ⊿Ca content values were obtained, and the ratios of ⊿Ca content to ⊿dry weight were calculated.
2.6. Transpiration rates To investigate the relevance of Ca distributivity and water flow, six pots (2 pots × 3 replicates) per cultivar were randomly selected 4 weeks and 8 weeks after the experiment commenced. The transpiration rates of the representative upper, middle, and lower leaves were measured using an LI-6400 (LI-COR, Lincoln, NE) with an LED light source. Measurement conditions were as follows: 25 °C, 50% ± 10% humidity, 400 ppm CO2, and 250 μmol m−2 s-1 photosynthetic photon flux density. Transpiration rates of VP 4 weeks after start of the experiment were measured only in the upper and lower leaves, because the number of nodes was small.
3.2. Total Ca concentrations There was no significant difference in total Ca concentrations of VG and VP during tipburn (Table 1), and the ratios of RGRCa to RGR of VG and VP did not significantly differ before and after the onset of tipburn, because each value of each cultivar decreased at the same rate. In contrast, the total Ca concentrations and the RGRCa/RGR values of UH slightly decreased between 4 and 5 weeks. These results demonstrate that the Ca increase rate and plant growth rate per plant had little effect on the incidence of tipburn. Further, in tipburn-sensitive cultivars, Ca deficiency in the upper (new)
2.7. Statistical analysis Data were analyzed using SPSS v. 22.0 (IBM Corp. Japan, Tokyo, Japan). Differences in mean values were evaluated using the Student t test or Tukey’s b multiple comparisons test (One-way ANOVA analysis was conducted to assess the effects of the cultivars). The mean value and standard error (SE) of each value are presented.
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Fig. 5. Ca concentrations of each organ of UH, VG, and VP. Mean values are shown, and the error bars represent the SE (n = 6). Significant differences are indicated by different letters (Tukey’s b test, P < 0.05).
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Fig. 6. Weekly Ca accumulations in each organ of UH, VG, and VP.
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Fig. 7. Weekly Ca distribution ratios of each organ of UH, VG, and VP.
polyethylene pots. Hereafter, we discuss the Ca concentrations after 2 weeks. The Ca concentrations of the roots of UH at 4 weeks increased and those of whole leaves decreased. Similarly, the Ca concentrations of the roots of VG increased, whereas those of whole leaves decreased. The Ca concentrations of the roots of VP significantly increased and those of whole leaves significantly decreased between 4 and 5 weeks. Thus, the Ca distribution in the roots increased from 3 to 5 weeks, regardless of tipburn. This time interval was consistent with that before and after the onset of tipburn in VG and VP. The weekly Ca accumulation and distribution ratio of each organ (Figs. 6 and 7) also indicate that Ca accumulations in roots increased.
leaf tips may have been influenced by the relative leaf growth rate (RGRleaf), the relative leaf Ca increase rate (RGRleaf-Ca), or both. 3.3. Ca distributivity We investigated Ca distributivity by measuring the Ca concentration and the ratio of RGRCa to RGR of each organ. 3.3.1. Ca concentrations of leaves, stems, and roots The Ca concentrations of each organ of the three cultivars are shown in Fig. 5. Certain Ca concentrations of roots and leaves were significantly higher earlier during the experiment. These inexplicable results may be affected by transplanting the plug seedlings to 9
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Table 2 The ratios of the relative leaf Ca increase rate (RGRleaf-Ca) to relative leaf growth rate (RGRleaf), RGRleaf-Ca, and RGRleaf. Weeks
RGRleaf-Ca / RGRleaf
RGRleaf-Ca
(mg-Ca g-DW mg-Ca
0 1 2 3 4 5 6 7
→ → → → → → → →
1 2 3 4 5 6 7 8
−1
g-DW
−1
)
(mg-Ca mg-Ca
RGRleaf −1
−1
week
(g g−1 week−1)
)
UH
VG
VP
UH
VG
VP
UH
VG
VP
－ 1.03 0.86 1.59 0.53 0.72 0.85 3.18
－ 1.23 1.00 0.77 0.65 1.06 0.78 1.47
－ 1.13 0.81 1.00 0.67 0.89 1.04 1.94
－ 0.69 0.93 0.66 0.21 0.30 0.46 0.33
－ 0.75 0.96 0.62 0.20 0.47 0.33 0.27
－ 0.95 0.77 0.65 0.37 0.49 0.45 0.51
－ 0.67 1.08 0.42 0.39 0.41 0.54 0.10
－ 0.61 0.96 0.81 0.31 0.44 0.42 0.19
－ 0.84 0.95 0.65 0.55 0.55 0.43 0.26
Mean values are shown. Bold type represents the values from 2 to 5 weeks when the distribution of Ca in the roots of each cultivar increased. Table 3 The ratios of the relative stem Ca increase rate (RGRstem-Ca) to relative stem growth rate (RGRstem), RGRstem-Ca, and RGRstem. Weeks
0 1 2 3 4 5 6 7
→ → → → → → → →
1 2 3 4 5 6 7 8
RGRstem-Ca / RGRstem
RGRstem-Ca
RGRstem
(mg-Ca g-DW mg-Ca−1 g-DW−1)
(mg-Ca mg-Ca−1 week−1)
(g g−1 week−1)
UH
VG
VP
UH
VG
VP
UH
VG
VP
－ － 1.05 1.01 0.78 1.04 0.62 1.11
－ － 1.07 0.71 0.76 0.92 1.20 1.26
－ － 0.63 0.95 0.97 0.82 1.03 1.34
－ － 1.65 0.79 0.38 0.64 0.41 0.42
－ － 1.63 0.63 0.45 0.71 0.62 0.60
－ － 0.84 1.11 0.84 0.69 0.64 0.58
－ － 1.57 0.78 0.49 0.61 0.67 0.38
－ － 1.52 0.89 0.59 0.76 0.52 0.48
－ － 1.33 1.17 0.87 0.84 0.62 0.43
Mean values are shown. Bold type represents the values from 2 to 5 weeks when the distribution of Ca in roots of each cultivar increased. Table 4 Ratios of the relative root Ca increase rate (RGRroot-Ca) to the relative root growth rate (RGRroot), RGRroot-Ca, and RGRroot. Weeks
0 1 2 3 4 5 6 7
→ → → → → → → →
1 2 3 4 5 6 7 8
RGRroot-Ca / RGRroot
RGRroot-Ca
RGRroot
(mg-Ca g-DW mg-Ca−1 g-DW−1)
(mg-Ca mg-Ca−1 week−1)
(g g−1 week−1)
UH
VG
VP
UH
VG
VP
UH
VG
VP
－ 0.87 1.10 1.18 1.14 1.41 1.12 0.65
－ 0.70 1.01 1.14 0.80 1.20 1.14 2.22
－ 0.99 1.04 0.94 1.63 1.02 1.06 1.26
－ 0.52 1.17 0.47 0.81 0.60 0.81 0.10
－ 0.41 0.92 1.06 0.25 0.66 0.81 0.33
－ 0.68 1.18 0.68 1.00 0.74 0.50 0.41
－ 0.60 1.06 0.40 0.71 0.42 0.72 0.15
－ 0.59 0.91 0.92 0.31 0.55 0.71 0.15
－ 0.68 1.13 0.73 0.61 0.73 0.47 0.33
Mean values are shown. Bold type represents the mean values from 2 to 5 weeks when the Ca distribution in the roots of each cultivar increased. Table 5 Ratios of the Ca increase rate (⊿Ca content) to the plant growth rate (⊿dry weight) of new leaves, ⊿Ca content of new leaves, and ⊿dry weight of new leaves. Weeks
⊿Ca content /⊿dry weight of new leaves (mg-Ca g-DW
0 1 2 3 4 5 6 7
→ → → → → → → →
1 2 3 4 5 6 7 8
−1
)
⊿Ca content of new leaves
⊿dry weight of new leaves
(mg-Ca)
(g-DW)
UH
VG
VP
UH
VG
VP
UH
VG
VP
－ － 0.92 2.02 1.32 1.77 1.30 2.32
－ － 1.46 1.17 0.88 1.64 1.67 1.60
－ － 1.22 1.06 0.76 0.65 1.34 1.92
－ － 0.022 0.176 0.093 0.197 0.123 0.486
－ － 0.148 0.207 0.073 0.122 0.233 0.095
－ － 0.054 0.037 0.031 0.050 0.110 0.139
－ － 0.024 0.088 0.070 0.111 0.095 0.210
－ － 0.101 0.177 0.083 0.075 0.140 0.059
－ － 0.044 0.035 0.041 0.076 0.082 0.072
Mean values are shown. Bold type represents the values from 2 to 5 weeks when the Ca distribution in the roots of each cultivar increased.
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5 4
4W
Upper leaves
Transpiration rates (mmol m-2 s-1 )
Transpiration rates (mmol m-2 s-1)
T. Kuronuma, et al.
n.s.
Middle leaves Lower leaves
n.s.
3
b
2
a ab 1 0
7z
5
UH
3
5
3
VG
6
5
5 4
Middle leaves Lower leaves
3
b b'
a
13
9
6
10
4
VG
AB A
a' a'
1
UH
VP
B
ab
2
0
4
8W
Upper leaves
13
9
5
VP
Fig. 8. Transpiration rates at each leaf position in each cultivar 4–8 weeks after the experiment commenced. Mean values are shown, and the error bars represent the SE (n = 6). Significant differences among mean values are indicated by different letters (Tukey’s b test, P < 0.05). zleaf position measured.
at 5–6 weeks when the incidence of tipburn reached 100%. These results may be explained by (1) the decrease in ⊿Ca content, although the value of ⊿dry weight slightly increased (4–5 weeks), and (2) by the lower incremental value of the ⊿Ca content value compared with that of ⊿dry weight (5–6 weeks). These results demonstrate that the new leaf growth rate exerted little effect on the decrease in the distribution of Ca in new leaves and the incidence of tipburn. The increase in the distribution of Ca in roots and its decrease in leaves occurred at 2–5 weeks, which was consistent with that before and after the onset of tipburn in VG and VP. Thus, when a cultivar translocated, at least, the threshold level of Ca to new leaf tips, tipburn would not occur. In contrast, a cultivar that was unable to translocate the threshold level of Ca to new leaf tips would undergo tipburn, which must have been associated with the increase in the distribution of Ca in roots. Further, the Ca distributivity evaluated from the relationship between the rates of plant growth and increase in Ca content show that decreased Ca distribution in leaves was mainly caused by the decrease in the rate of Ca increase (RGRleaf-Ca and ⊿Ca content). Thus, this new approach more rigorously evaluates the influence of the rate of plant growth on the incidence of tipburn.
3.3.2. Relationship between plant growth rate (RGR) and Ca increase rate (RGRCa) of leaves, stems, and roots The ratios of the RGRCa to the RGR of each organ of the three cultivars are shown in Tables 2–4. Hereafter, to evaluate the differences in Ca distributivity of each organ before and after the onset of tipburn, we focus here on data acquired from 2 to 5 weeks. The decrease in the values of RGRleaf-Ca/RGRleaf (Table 2) were attributed to the higher decrement of RGRleaf-Ca compared with that of RGRleaf of each cultivar. Consequently, decreased Ca distribution (concentrations) of leaves mainly caused by a decrease of leaf Ca increase rate (RGRleaf-Ca), suggesting leaf growth rate has little effect on the incidence of tipburn in lisianthus. The changes in the values of RGRstem-Ca/RGRstem of UH and VG were similar to those of leaves (Table 3). There were no significant differences in the values of RGRstem-Ca/RGRstem of VP between before and after tipburn because their respective values decreased at the same rate. In contrast, the values of RGRroot-Ca/RGRroot of VG and VP increased, because the increment in the value of RGRroot-Ca was higher compared with that of RGRroot (Table 4). These results indicate that VG and VP accumulated Ca in their roots before and after the onset of tipburn. During this time, there was no significant change in the values of RGRroot-Ca/RGRroot of UH (Table 4), although the value of RGRCa/RGR per plant slightly decreased (Table 1). Further, the values of RGRroot-Ca and RGRroot of UH increased at the same rate. These results indicate that UH accumulated more Ca in roots. Thus, all cultivars accumulated Ca in roots before tipburn, which would be closely related to the decreased of distributions of Ca in leaves and the incidence of tipburn.
3.4. Transpiration rates The transpiration rates of the three cultivars at 4–8 weeks, during which the rates of transpiration of leaves became lower as a function of their upward positions (Fig. 8). This finding is consistent with the Ca concentration at each leaf position (Figs. 2–4). In contrast, although we assumed that transpiration rates of UH, which was highly efficient in distributing Ca to new leaf tips, were higher compared with those of the other cultivars. Further, the transpiration rates of the upper leaves of UH were slightly lower compared with those of VG and VP at 4 weeks. These findings suggest that the distributivity of Ca in new leaf tips of each cultivar cannot be evaluated solely by the varietal differences in values of transpiration rates in the upper leaves. To mitigate or eliminate tipburn, it will be necessary to identify the physiological factors that mediate the accumulation and distribution of Ca in lisianthus. For example, it has been suggested that the distributions of Ca in lisianthus cultivars are strongly influenced by Ca uptake caused by root pressure (Kuronuma et al., 2018). Further, Lee et al. (2013b) found that the expression of genes encoding major Ca2+ vacuole transporters in cabbage cultivars is associated with the accumulation of Ca2+ in the vacuoles of leaf cells. Flower bud formation may also have an influence on the increase in the distribution of Ca in roots. The extension of such studies will undoubtedly contribute to enhance the productivity and quality of lisianthus.
3.3.3. Ca distributivity of new leaves (2–5 weeks) The ratios of ⊿Ca content to ⊿dry weight of new leaves of UH decreased from 2.02 to 1.32 (Table 5). However, the values of ⊿Ca content/⊿dry weight were higher compared with those of the other cultivars, indicating that UH had a greater ability to distribute Ca to new leaf tips. Thus, tipburn would not have been exhibited by UH. Further, the changes in ⊿Ca content and ⊿dry weight of UH indicate that the rate of new leaf growth did not exert a significant effect on the decrease of Ca concentrations and Ca distributions in new leaves. The values of ⊿Ca content/⊿dry weight of the new leaves of VG decreased from 1.46 to 1.17 and reached a minimum (0.88) at 4–5 weeks when the incidence of tipburn reached 100%. These results may be explained as follows: (1) The incremental value of the ⊿Ca content was lower compared with that of the ⊿dry weight (3–4 weeks), and (2) the decrement of the ⊿Ca content during this time was higher compared with that of ⊿dry weight (4–5 weeks). The values of ⊿Ca content/⊿ dry weight of new leaves of VP decreased from 1.06 to 0.76. The values of VP reached a minimum (0.65) 11
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accumulation and distribution, and tipburn development in field-grown butterhead lettuce. Sci. Hortic. 21, 123–128. Cox, E.F., McKee, J.M.T., Dearman, A.S., 1976. The effect of growth rate on tipburn occurrence in lettuce. J. Hortic. Sci. 51, 297–309. Guttridge, C.G., Bradfield, E.G., Holder, R., 1981. Dependence of calcium transport into strawberry leaves on positive pressure in the xylem. Ann. Bot. 48, 473–480. Garnier, E., 1991. Resource capture, biomass allocation and growth in herbaceous plants. Trends Ecol. Evolution. 6, 126–131. Kuo, C.G., Tsay, J.S., Tsai, C.L., Chen, R.J., 1981. Tipburn of Chinese cabbage in relation to calcium nutrition and distribution. Sci. Hortic. 14, 131–138. Kuronuma, T., Watanabe, H., 2016a. Physiological and morphological traits and competence for carbon sequestration of several green roof plants under a controlled environmental system. J. Am. Soc. Hortic. Sci. 141, 583–590. Kuronuma, T., Watanabe, H., 2016b. Relevance of carbon sequestration to the physiological and morphological traits of several green roof plants during the first year after construction. Am. J. Plant Sci. 8, 14–27. Kuronuma, T., Watanabe, Y., Ando, M., Watanabe, H., 2018. Tipburn severity and calcium distribution in lisianthus (Eustoma Grandiflorum (Raf.) Shinn.) cultivars under different relative air humidity conditions. Agronomy 8 (10), 218. Kuronuma, T., Watanabe, Y., Ando, M., Watanabe, H., 2019. Relevance of tipburn incidence to the competence for Ca acquirement and Ca distributivity in lisianthus [Eustoma grandiflorum (Raf.) Shinn.] cultivars. Sci. Hortic. 246, 805–811. Lee, J.G., Choi, C.S., Jang, Y.A., Jang, S.W., Lee, S.G., Um, Y.C., 2013a. Effects of air temperature and air flow rate control on the tipburn occurrence of leaf lettuce in a closed-type plant factory system. Hortic. Environ. Biotech. 54, 303–310. Lee, J., Park, I., Lee, Z.W., Kim, S.W., Baek, N., Park, H.S., Park, S.U., Kwon, S.Y., Kim, H., 2013b. Regulation of the major vacuolar Ca2+ transporter genes, by intercellular Ca2+ concentration and abiotic stresses, in tip-burn resistant Brassica oleracea. Molecular boil. Reports 40, 177–188. Magnusson, M., 2002. Mineral fertilizers and green mulch in Chinese cabbage [Brassica pekinensis (Lour.) Rupr.]: Effect on nutrient uptake, yield and internal tipburn. Acta Agricul. Scandinavica. Section B-Plant Soil Sci 52, 25–35. Mason, G.F., Guttridge, C.G., 1975. The influence of relative humidity and nutrition on leaf tipburn of strawberry. Sci. Hortic. 3, 339–349. Misaghi, I.J., Grogan, R.G., 1978. Effect of temperature on tipburn development in head lettuce. Phytopathology 68, 1738–1743. Poorter, H., Remkes, C., 1990. Leaf area ratio and net assimilation rate of 24 wild species differing in relative growth rate. Oecologia 83, 553–559. Poorter, H., Remkes, C., Lambers, H., 1990. Carbon and nitrogen economy of 24 wild species differing in relative growth rate. Plant Physiol. 94, 621–627. Sago, Y., 2016. Effects of light intensity and growth rate on tipburn development and leaf calcium concentration in butterhead lettuce. HortScience 51, 1087–1091. Saure, M.C., 1998. Causes of the tipburn disorder in leaves of vegetables. Sci. Hortic. 76, 131–147. Uno, Y., Okubo, H., Itoh, H., Koyama, R., 2016. Reduction of leaf lettuce tipburn using an indicator cultivar. Sci. Hortic. 210, 14–18. Wissemeier, A.H., Zühlke, G., 2002. Relation between climatic variables, growth and the incidence of tipburn in field-grown lettuce as evaluated by simple, partial and multiple regression analysis. Sci. Hortic. 93, 193–204.
4. Conclusions The increase in the distribution of Ca in roots and its decrease in leaves occurred at 2–5 weeks, which was consistent with that before and after the onset of tipburn in VG and VP. The distributions of Ca in new leaves of the tipburn-sensitive cultivars VG and VP must fall below the threshold concentration of Ca during this time, in response to the increase in the distribution of Ca in roots. Moreover, evaluating the distributivity of Ca as a function of the relationship between the rates of plant growth and increase in Ca concent, demonstrates that the decrease in the distribution of Ca in leaves was mainly caused by the decrease in the rate of increase of Ca content in the leaves (RGRleaf-Ca and ⊿Ca content). Therefore, tipburn in these lisianthus cultivars was caused by the decrease in the distribution of Ca in new leaves, which was associated with the increase in the distribution of Ca in roots. Appendix A. Supplementary data Supplementary material related to this article can be found, in the online version, at doi:https://doi.org/10.1016/j.scienta.2019.108911. References Aloni, B., Pashkar, T., Libel, R., 1986. The possible involvement of gibberellins and calcium in tipburn of Chinese cabbage: study of intact plants and detached leaves. Plant Growth Regul. 4, 3–11. Barta, D.J., Tibbitts, T.W., 1991. Calcium localization in lettuce leaves with and without tipburn: comparison of controlled-environment and field-grown plants. J. Am. Soc. Hortic. Sci. 116, 870–875. Bautista, A.S., López-Galarza, S., Martínez, A., Pascual, B., Maroto, J.V., 2009. Influence of cation proportions of the nutrient solution on tipburn incidence in strawberry plants. J. plant nutrition. 32, 1527–1539. Chang, Y.C., Miller, W.B., 2003. Growth and calcium partitioning in Lilium ‘Star Gazer’ in relation to leaf calcium deficiency. J. Am. Soc. Hortic. Sci. 128, 788–796. Chang, Y.C., Miller, W.B., 2004. The relationship between leaf enclosure, transpiration, and upper leaf necrosis on Lilium ‘Star Gazer’. J. Am. Soc. Hortic. Sci. 129, 128–133. Chang, Y.C., Miller, W.B., 2005. The development of upper leaf necrosis in Lilium ‘Star Gazer’. J. Am. Soc. Hortic. Sci. 130, 759–766. Choi, K.Y., Lee, Y.B., 2008. Effects of relative humidity on the apparent variability in the incidence of tipburn symptom and distribution of mineral nutrients between morphologically different lettuce (Lactuca sativa L.) cultivars. Hortic. Environ. Biotechnol. 49, 20–24. Collier, G.F., Huntington, V.C., 1983. The relationship between leaf growth, calcium
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Difference of Ca distribution before and after the onset of tipburn in lisianthus [Eustoma grandiflorum (Raf.) Shinn.] cultivars Takanori Kuronuma, Nozomi Kinoshita, Masaya Ando, Hitoshi Watanabe* Center for Environment, Health and Field Sciences, Chiba University, Japan
A R T I C LE I N FO
A B S T R A C T
Keywords: Eustoma grandiflorum Ca deficiency Tipburn Systematic investigation Growth analysis Ca distributivity
Tipburn (leaf-tip necrosis) is severe problem for the production of lisianthus [Eustoma grandiflorum (Raf.) Shinn.] cultivars. A previous study has suggested that tipburn of 14 lisianthus cultivars are caused by the inability to translocate sufficient quantities of calcium (Ca) to the tips of the upper leaves. However, systematic studies are not available that identify the differences in Ca concentrations and distributivity before and after the onset of tipburn. To address this insufficiency in our knowledge, we used the lisianthus cultivars ‘Umihonoka’ (UH), ‘Voyage green’ (VG), and ‘Voyage pink’ (VP) and determined Ca concentrations and the dry weight of each organ at weekly intervals for 8 weeks. In addition, we evaluated the Ca distributivity from the ratios of the relative Ca increase rate (RGRCa) to the relative plant growth rate (RGR) by applying growth analysis methods. UH did not exhibit tipburn. In contrast, VG and VP exhibited tipburn after 4 and 5 weeks, respectively. At the same time, the concentrations and distributions of Ca in the roots increased and those of whole and new leaves decreased in each cultivar. Thus, it is suggested that the distribution of Ca in new leaves of the tipburn-sensitive cultivars VG and VP was below the threshold, which caused tipburn. Moreover, it was demonstrated that decreases in the distribution of Ca in whole and new leaves were mainly caused by not increase of the relative leaf growth rate (RGRleaf) but decrease of the relative leaf Ca increase rate (RGRleaf-Ca). We conclude that this approach more rigorously evaluates the influence of plant growth rate on the incidence of tipburn. Thefore, tipburn exhibited by lisianthus cultivars was caused by decreased distributions of Ca in new leaves, which was associated with an increase in the distribution of Ca in roots.
1. Introduction Lisianthus [Eustoma grandiflorum (Raf.) Shinn.] is an ornamental plant that is native to warm regions of the Southern United States and Northern Mexico. Its cultivars are mainly supplied as cut flowers. In Japan, the wholesale value in 2017 of lisianthus ranked fifth among cut flowers. However, tipburn [calcium (Ca) deficiency disorder] of certain lisianthus cultivars causes serious economic losses. Tipburn diminishes the quality of plants, thereby adversely affecting their marketability. The cause of tipburn has been intensively studied, including efforts to reduce damage to plants by controlling the environment [e.g., lettuce (Barta and Tibbitts, 1991; Choi and Lee, 2008; Misaghi and Grogan, 1978; Sago, 2016; Uno et al., 2016; Wissemeier and Zühlke, 2002), Chinese cabbage (Aloni et al., 1986; Kuo et al., 1981; Magnusson, 2002), strawberries (Bautista et al., 2009; Guttridge et al., 1981; Mason and Guttridge, 1975), and lilies (Chang and Miller, 2003, 2004; Chang and Miller, 2005)]. Research on lisianthus cultivars indicates that tipburn is mainly caused by the inability of the plant to translocate
⁎
adequate amounts of Ca to the tips of the upper leaves (Kuronuma et al., 2019). However, there is no systematic investigation, to our knowledge, which reports the differences in Ca concentrations and Ca distributivity before and after the onset of tipburn. Thus, the cause of cultivar-specific tipburn remains to be determined. It is well known that plant growth rate significantly influences tipburn severity and incidence (Collier and Huntington, 1983; Cox et al., 1976; Lee et al., 2013a; Saure, 1998). Because the Ca concentration of each organ is determined by the relationship between plant growth rate (dry weight) and Ca increase rate in each organ. Accordingly, Ca deficiency and its distributivity has a close relationship to plant growth rate and Ca increase rate. However, Ca deficiency and distributivity has been evaluated only through determination of the changes in Ca concentrations. Therefore, Ca distributivity (including Ca deficiency) must be determined using a more rigorous approach that measures Ca concentrations as well as the relationship between plant growth rate and Ca increase rate. For example, the Ca increase rate was quantified as Ca acquirement competence (Kuronuma et al., 2019) by applying growth
Corresponding author. E-mail addresses: [email protected] (T. Kuronuma), [email protected] (H. Watanabe).
https://doi.org/10.1016/j.scienta.2019.108911 Received 7 May 2019; Received in revised form 13 September 2019; Accepted 2 October 2019 0304-4238/ © 2019 Elsevier B.V. All rights reserved.
Please cite this article as: Takanori Kuronuma, et al., Scientia Horticulturae, https://doi.org/10.1016/j.scienta.2019.108911
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analysis methods (Garnier, 1991; Kuronuma and Watanabe, 2016a, b; Poorter and Remkes, 1990; Poorter et al., 1990). Here we aimed to determine the differences in Ca concentrations and distributivity before and after the onset of tipburn and to discuss the cause of tipburn in certain lisianthus cultivars. We further evaluated Ca distributivity (including Ca deficiency) and its associations with plant growth rates with Ca increase rates. 2. Material and methods 2.1. Plants The lisianthus cultivars were ‘Umi honoka’ (UH) (Sumika Agrotech Co., Ltd.), ‘Voyage pink’ (VP) (SAKATA SEED CORPORATION), and ‘Voyage green’ (VG) (SAKATA SEED CORPORATION), which were selected from a different group classified in a previous study (Kuronuma et al., 2019). UH has been classified tipburn–resistant cultivar’s group, and VG has been classified tipburn-sensitive cultivar’s group with low abilities to distribute Ca to the tips of their upper leaves. VP has been classified tipburn-sensitive cultivar’s group with low abilities to acquire Ca and distribute it to the tips of their upper leaves. In addition, Voyage series including the VG and VP is one of the most famous lisianthus cultivars in Japan. Thus, VG and VP were selected. Seeds were sown in plug flats (406 cells per tray) filled with seedling propagation medium (Metro Mix 350; Sun Gro Horticulture, Agawam, MA, USA). The seeded trays were maintained in a germination room at 24 °C under a 14-h light/10-h dark photoperiod. After two weeks, the trays were transferred to a controlled environmental system (phytotron), and the plants grown under the conditions as follows: 25 °C (light period) and 20 °C (dark period), 60 ± 5% humidity, 400 ppm CO2, 14h light (225 ± 25 μmol m−2 s-1) and 10-h dark. An irrigation system in the phytotron supplied a nutrient solution (62 ppm NO3-N, 4.6 ppm NH4-N, 10.2 ppm PO4-P, 104 ppm K, 16 ppm SO4-S, 40 ppm Ca, 12 ppm Mg, 2.8 ppm Fe, 1.6 ppm B, 0.8 ppm Mn, 0.08 ppm Zn, 0.04 ppm Cu, and 0.04 ppm Mo) by bottom watering for 10 min. Five weeks after transfer to the phytotron, plugs were transplanted into 0.25-L polyethylene pots filled with Metro Mix 350.
Fig. 1. Tipburn severity and incidence. Mean values and the SE (n = 12) (error bars) are shown.
2.4. Measurement of dry weight and Ca concentration Samples of plant organs were dried at 70 °C for 72 h, and weighed. Samples of two plants were combined, and Ca concentrations were determined using a Z-5300 polarized Zeeman atomic absorption spectrophotometer (Hitachi, Ltd., Tokyo, Japan). The Ca concentrations of the top one-fifth (leaf tip) and the remainder (leaf base) of each leaf were separately analyzed. To calculate total Ca concentrations, we quantified the whole-plant Ca content by adding the Ca content (Ca concentration × dry weight) of each organ. Total Ca concentrations were calculated by dividing the whole-plant Ca content by the mass of the whole plant. Ca concentrations of whole leaves were similarly determined. 2.5. Ca distributivity To evaluate the Ca distributivity of the lisianthus cultivars, we quantified Ca concentrations and their relationships between the plant growth rates and Ca increase rates (see Section 2.5.1).
2.2. Experimental design
2.5.1. Ca distributivity of leaves, stems, and roots Except for the early stages of the experiments, the dry weights and Ca contents of whole leaves, stems, and roots increased exponentially (Appendix 1). Thus, the rates of plant growth and increases in Ca content of each organ were quantified as the relative growth rate (RGR) and relative Ca increase rate (RGRCa), respectively, as follows:
To determine the differences in Ca concentration and distributivity before and after tipburn, cultivars were grown in the same phytotron under the environmental and irrigation conditions described above. First, four pots of each cultivar were randomly sampled in triplicate and then harvested at one week intervals for 8 weeks. Harvested plants were washed with distilled water and divided into roots, stems, leaves on the main stem, and other leaves. The leaves on the main stem were distinguished for each leaf position, which were numbered from the base to top leaves. Unfolded leaves were included with stems, because they were too small for analysis. 2.3. Tipburn severity and incidence Whole leaves were scored using arbitrary tipburn severity indices ranking from 0 to 1 (0, asymptomatic; 0.2, deformed leaf margins; 0.5, leaf-tip chlorosis; 1, leaf-tip necrosis). The severity of tipburn in each plant and at each leaf position was defined as follows: Tipburn severity in each plant = ∑ {(severity indices × leaf number) / whole leaves number per pot} × 100 (1)
RGR = (lnW2 – lnW1) / (T2 – T1)
(3)
RGRleaf = (lnWleaf2 – lnWleaf1) / (T2 – T1)
(4)
RGRstem = (lnWstem2 – lnWstem1) / (T2 – T1)
(5)
RGRroot = (lnWroot2 – lnWroot1) / (T2 – T1)
(6)
RGRCa = (lnWCa2 – lnWCa1) / (T2 – T1)
(7)
RGRleaf-Ca = (lnWleaf-Ca2 – lnWleaf-Ca1) / (T2 – T1)
(8)
RGRstem-Ca = (lnWstem-Ca2 – lnWstem-Ca1) / (T2 – T1)
(9)
RGRroot-Ca = (lnWroot-Ca2 – lnWroot-Ca1) / (T2 – T1)
(10)
where W1 and W2 are the whole-plant dry weights, Wleaf1 and Wleaf2 are the whole-leaf dry weights, Wstem1 and Wstem2 are the stem dry weights, Wroot1 and Wroot2 are the root dry weights, WCa1 and WCa2 are the whole-plant Ca contents, Wleaf-Ca1 and Wleaf-Ca2 are the whole-leaf Ca contents, Wstem-Ca1 and Wstem-Ca2 are the stem Ca contents, and WrootCa1, and Wroot-Ca2 are the root Ca contents at times T1 and T2,
Tipburn severity at each leaf position = ∑ {(severity indices × leaf number) / whole leaves number at each leaf position} × 100 (2) The incidence of tipburn is expressed as the percentage of plants in a cultivar exhibiting symptoms of tipburn. 2
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Fig. 2. Ca concentration in each leaf tip and base, and tipburn severity at each leaf position of UH. Mean values are shown, and error bars represent the SE (n > 3). *Significant differences between Ca concentrations in tips and those in the bases of individual leaves (Student t test, *P < 0.05, **P < 0.01).
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Fig. 3. Ca concentrations in each leaf tip and leaf base, and tipburn severity at each leaf position of VG. Mean values are shown, and error bars represent the SE (n > 3). * Significant differences between Ca concentrations in tips and those in the bases of individual leaves (Student t test, *P < 0.05, **P < 0.01).
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Fig. 4. Ca concentrations in each leaf tip and leaf base and tipburn severity at each leaf position of VP. Mean values are shown, and error bars represent the SE (n > 3). *Significant differences between Ca concentrations in tips and those in bases of individual leaves (Student t test, *P < 0.05, **P < 0.01).
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Table 1 Ca concentrations, relative Ca increase rate (RGRCa), relative growth rate (RGR), and RGRCa/RGR. Weeks
Total Ca concentrations (mg g
)
UH 0 1 2 3 4 5 6 7 8
3.1 2.9 2.8 2.7 3.1 2.9 3.0 3.1 3.4
RGRCa
−1
(mg-Ca mg-Ca VG
b ab ab a b ab b b b
RGR
3.1 3.3 3.1 3.1 3.1 2.8 3.0 3.4 3.9
VP B B B B B A AB BC C
2.3 2.8 3.0 2.7 2.8 2.9 3.0 3.0 3.6
a' a'b'c' b'c' a'b' a'b' b'c' c' c' d'
−1
week
−1
)
(g g
−1
RGRCa / RGR week
−1
(mg-Ca g-DW mg-Ca−1 g-DW−1)
)
UH
VG
VP
UH
VG
VP
UH
VG
VP
－ － 0.64 1.04 0.59 0.41 0.45 0.65 0.21
－ － 0.59 0.96 0.83 0.23 0.60 0.64 0.32
－ － 0.86 0.92 0.70 0.64 0.63 0.49 0.45
－ － 0.67 1.11 0.43 0.45 0.43 0.60 0.15
－ － 0.63 0.98 0.83 0.33 0.50 0.53 0.19
－ － 0.80 1.01 0.68 0.58 0.62 0.48 0.29
－ － 0.95 0.93 1.37 0.91 1.04 1.08 1.40
－ － 0.93 0.98 1.00 0.69 1.18 1.22 1.67
－ － 1.07 0.91 1.02 1.10 1.02 1.03 1.56
Mean values are shown, and significant differences among them are indicated by different letters (Tukey’s b test, P < 0.05). Bold type represents the values before and after the onset of tipburn.
3. Results and discussion
respectively. To evaluate Ca distributivity from the relationship between the relative rates of plant growth and increases in Ca concent, we calculated the ratios of the relative Ca increase rate to the relative growth rate (RGRCa/RGR). The weekly Ca accumulations in whole leaves, stems, and roots were calculated by subtracting the Ca content of each organ at “n” weeks from those at “n + 1” weeks. Weekly Ca distribution ratios in each organ were calculated by dividing the Ca accumulation in each organ per plant per week.
3.1. Tipburn severity and incidence 3.1.1. Tipburn severity and incidence UH did not exhibit detectable tipburn (Fig. 1), which is consistent with the results of a published study (Kuronuma et al., 2019). VG and VP exhibited tipburn 4 and 5 weeks, respectively, after the experiment commenced. The incidence of tipburn exhibited by VP and VG reached 100% the week after the first observation of tipburn. 3.1.2. Ca concentration and the tipburn severity at each leaf position The Ca concentration and the severity of tipburn at each leaf position are shown in Figs. 2–4, which show the results for each leaf position only when > 6 samples were collected, because the number of leaves of each plant differed. The first leaves (leaf position 1) dissolved and disappeared by 3 weeks. The Ca concentrations of the leaves of all cultivars decreased as a function of higher leaf position (Figs. 2–4). UH did not exhibit detectable tipburn at any leaf position (Fig. 2), and the Ca concentrations in the tips of the top leaves were > 2.0 mg g−1 until the next week. The Ca concentrations in the tips of the upper leaves were significantly higher compared with those at the bases, except at 8 weeks. In contrast, Ca concentrations in the tips of the top leaves of VG and VP scince 3 weeks were < 2.0 mg g-1 until the next week (Figs. 3 and 4). In addition, there are no significant differences between their Ca concentrations in tips of the upper leaves and those in bases. VG and VP exhibited tipburn in the upper leaves at 4 and 5 weeks, respectively. These results indicate that the tipburn-resistant cultivar UH translocated, at least, the threshold level of Ca to the upper (new) leaf tips. In contrast, although the tipburn-sensitive cultivars VG and VP translocated at least, the threshold level of Ca to upper (new) leaf tips during the early stages of plant growth, the Ca distributions of the upper (new) leaves gradually decreased. Consequently, tipburn would have occurred.
2.5.2. Ca distributivity in new leaves Quantification of Ca distributivity in new leaves was not used in the growth analysis methods, because the dry weights and Ca contents of new leaves did not exhibit an exponential increase. Thus, ⊿dry weights (increment of dry weight per week) and ⊿Ca content (increment of Ca content per week) of new leaves were calculated. The ⊿dry weights of new leaves from “n” to “n+1” weeks were calculated by the adding mean values of the dry weights of newly emerged leaves at “n+1” weeks to increment the mean values of the dry weights of leaves emerged at “n” weeks. Similarly, ⊿Ca content values were obtained, and the ratios of ⊿Ca content to ⊿dry weight were calculated.
2.6. Transpiration rates To investigate the relevance of Ca distributivity and water flow, six pots (2 pots × 3 replicates) per cultivar were randomly selected 4 weeks and 8 weeks after the experiment commenced. The transpiration rates of the representative upper, middle, and lower leaves were measured using an LI-6400 (LI-COR, Lincoln, NE) with an LED light source. Measurement conditions were as follows: 25 °C, 50% ± 10% humidity, 400 ppm CO2, and 250 μmol m−2 s-1 photosynthetic photon flux density. Transpiration rates of VP 4 weeks after start of the experiment were measured only in the upper and lower leaves, because the number of nodes was small.
3.2. Total Ca concentrations There was no significant difference in total Ca concentrations of VG and VP during tipburn (Table 1), and the ratios of RGRCa to RGR of VG and VP did not significantly differ before and after the onset of tipburn, because each value of each cultivar decreased at the same rate. In contrast, the total Ca concentrations and the RGRCa/RGR values of UH slightly decreased between 4 and 5 weeks. These results demonstrate that the Ca increase rate and plant growth rate per plant had little effect on the incidence of tipburn. Further, in tipburn-sensitive cultivars, Ca deficiency in the upper (new)
2.7. Statistical analysis Data were analyzed using SPSS v. 22.0 (IBM Corp. Japan, Tokyo, Japan). Differences in mean values were evaluated using the Student t test or Tukey’s b multiple comparisons test (One-way ANOVA analysis was conducted to assess the effects of the cultivars). The mean value and standard error (SE) of each value are presented.
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Fig. 5. Ca concentrations of each organ of UH, VG, and VP. Mean values are shown, and the error bars represent the SE (n = 6). Significant differences are indicated by different letters (Tukey’s b test, P < 0.05).
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Fig. 6. Weekly Ca accumulations in each organ of UH, VG, and VP.
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Fig. 7. Weekly Ca distribution ratios of each organ of UH, VG, and VP.
polyethylene pots. Hereafter, we discuss the Ca concentrations after 2 weeks. The Ca concentrations of the roots of UH at 4 weeks increased and those of whole leaves decreased. Similarly, the Ca concentrations of the roots of VG increased, whereas those of whole leaves decreased. The Ca concentrations of the roots of VP significantly increased and those of whole leaves significantly decreased between 4 and 5 weeks. Thus, the Ca distribution in the roots increased from 3 to 5 weeks, regardless of tipburn. This time interval was consistent with that before and after the onset of tipburn in VG and VP. The weekly Ca accumulation and distribution ratio of each organ (Figs. 6 and 7) also indicate that Ca accumulations in roots increased.
leaf tips may have been influenced by the relative leaf growth rate (RGRleaf), the relative leaf Ca increase rate (RGRleaf-Ca), or both. 3.3. Ca distributivity We investigated Ca distributivity by measuring the Ca concentration and the ratio of RGRCa to RGR of each organ. 3.3.1. Ca concentrations of leaves, stems, and roots The Ca concentrations of each organ of the three cultivars are shown in Fig. 5. Certain Ca concentrations of roots and leaves were significantly higher earlier during the experiment. These inexplicable results may be affected by transplanting the plug seedlings to 9
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Table 2 The ratios of the relative leaf Ca increase rate (RGRleaf-Ca) to relative leaf growth rate (RGRleaf), RGRleaf-Ca, and RGRleaf. Weeks
RGRleaf-Ca / RGRleaf
RGRleaf-Ca
(mg-Ca g-DW mg-Ca
0 1 2 3 4 5 6 7
→ → → → → → → →
1 2 3 4 5 6 7 8
−1
g-DW
−1
)
(mg-Ca mg-Ca
RGRleaf −1
−1
week
(g g−1 week−1)
)
UH
VG
VP
UH
VG
VP
UH
VG
VP
－ 1.03 0.86 1.59 0.53 0.72 0.85 3.18
－ 1.23 1.00 0.77 0.65 1.06 0.78 1.47
－ 1.13 0.81 1.00 0.67 0.89 1.04 1.94
－ 0.69 0.93 0.66 0.21 0.30 0.46 0.33
－ 0.75 0.96 0.62 0.20 0.47 0.33 0.27
－ 0.95 0.77 0.65 0.37 0.49 0.45 0.51
－ 0.67 1.08 0.42 0.39 0.41 0.54 0.10
－ 0.61 0.96 0.81 0.31 0.44 0.42 0.19
－ 0.84 0.95 0.65 0.55 0.55 0.43 0.26
Mean values are shown. Bold type represents the values from 2 to 5 weeks when the distribution of Ca in the roots of each cultivar increased. Table 3 The ratios of the relative stem Ca increase rate (RGRstem-Ca) to relative stem growth rate (RGRstem), RGRstem-Ca, and RGRstem. Weeks
0 1 2 3 4 5 6 7
→ → → → → → → →
1 2 3 4 5 6 7 8
RGRstem-Ca / RGRstem
RGRstem-Ca
RGRstem
(mg-Ca g-DW mg-Ca−1 g-DW−1)
(mg-Ca mg-Ca−1 week−1)
(g g−1 week−1)
UH
VG
VP
UH
VG
VP
UH
VG
VP
－ － 1.05 1.01 0.78 1.04 0.62 1.11
－ － 1.07 0.71 0.76 0.92 1.20 1.26
－ － 0.63 0.95 0.97 0.82 1.03 1.34
－ － 1.65 0.79 0.38 0.64 0.41 0.42
－ － 1.63 0.63 0.45 0.71 0.62 0.60
－ － 0.84 1.11 0.84 0.69 0.64 0.58
－ － 1.57 0.78 0.49 0.61 0.67 0.38
－ － 1.52 0.89 0.59 0.76 0.52 0.48
－ － 1.33 1.17 0.87 0.84 0.62 0.43
Mean values are shown. Bold type represents the values from 2 to 5 weeks when the distribution of Ca in roots of each cultivar increased. Table 4 Ratios of the relative root Ca increase rate (RGRroot-Ca) to the relative root growth rate (RGRroot), RGRroot-Ca, and RGRroot. Weeks
0 1 2 3 4 5 6 7
→ → → → → → → →
1 2 3 4 5 6 7 8
RGRroot-Ca / RGRroot
RGRroot-Ca
RGRroot
(mg-Ca g-DW mg-Ca−1 g-DW−1)
(mg-Ca mg-Ca−1 week−1)
(g g−1 week−1)
UH
VG
VP
UH
VG
VP
UH
VG
VP
－ 0.87 1.10 1.18 1.14 1.41 1.12 0.65
－ 0.70 1.01 1.14 0.80 1.20 1.14 2.22
－ 0.99 1.04 0.94 1.63 1.02 1.06 1.26
－ 0.52 1.17 0.47 0.81 0.60 0.81 0.10
－ 0.41 0.92 1.06 0.25 0.66 0.81 0.33
－ 0.68 1.18 0.68 1.00 0.74 0.50 0.41
－ 0.60 1.06 0.40 0.71 0.42 0.72 0.15
－ 0.59 0.91 0.92 0.31 0.55 0.71 0.15
－ 0.68 1.13 0.73 0.61 0.73 0.47 0.33
Mean values are shown. Bold type represents the mean values from 2 to 5 weeks when the Ca distribution in the roots of each cultivar increased. Table 5 Ratios of the Ca increase rate (⊿Ca content) to the plant growth rate (⊿dry weight) of new leaves, ⊿Ca content of new leaves, and ⊿dry weight of new leaves. Weeks
⊿Ca content /⊿dry weight of new leaves (mg-Ca g-DW
0 1 2 3 4 5 6 7
→ → → → → → → →
1 2 3 4 5 6 7 8
−1
)
⊿Ca content of new leaves
⊿dry weight of new leaves
(mg-Ca)
(g-DW)
UH
VG
VP
UH
VG
VP
UH
VG
VP
－ － 0.92 2.02 1.32 1.77 1.30 2.32
－ － 1.46 1.17 0.88 1.64 1.67 1.60
－ － 1.22 1.06 0.76 0.65 1.34 1.92
－ － 0.022 0.176 0.093 0.197 0.123 0.486
－ － 0.148 0.207 0.073 0.122 0.233 0.095
－ － 0.054 0.037 0.031 0.050 0.110 0.139
－ － 0.024 0.088 0.070 0.111 0.095 0.210
－ － 0.101 0.177 0.083 0.075 0.140 0.059
－ － 0.044 0.035 0.041 0.076 0.082 0.072
Mean values are shown. Bold type represents the values from 2 to 5 weeks when the Ca distribution in the roots of each cultivar increased.
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5 4
4W
Upper leaves
Transpiration rates (mmol m-2 s-1 )
Transpiration rates (mmol m-2 s-1)
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n.s.
Middle leaves Lower leaves
n.s.
3
b
2
a ab 1 0
7z
5
UH
3
5
3
VG
6
5
5 4
Middle leaves Lower leaves
3
b b'
a
13
9
6
10
4
VG
AB A
a' a'
1
UH
VP
B
ab
2
0
4
8W
Upper leaves
13
9
5
VP
Fig. 8. Transpiration rates at each leaf position in each cultivar 4–8 weeks after the experiment commenced. Mean values are shown, and the error bars represent the SE (n = 6). Significant differences among mean values are indicated by different letters (Tukey’s b test, P < 0.05). zleaf position measured.
at 5–6 weeks when the incidence of tipburn reached 100%. These results may be explained by (1) the decrease in ⊿Ca content, although the value of ⊿dry weight slightly increased (4–5 weeks), and (2) by the lower incremental value of the ⊿Ca content value compared with that of ⊿dry weight (5–6 weeks). These results demonstrate that the new leaf growth rate exerted little effect on the decrease in the distribution of Ca in new leaves and the incidence of tipburn. The increase in the distribution of Ca in roots and its decrease in leaves occurred at 2–5 weeks, which was consistent with that before and after the onset of tipburn in VG and VP. Thus, when a cultivar translocated, at least, the threshold level of Ca to new leaf tips, tipburn would not occur. In contrast, a cultivar that was unable to translocate the threshold level of Ca to new leaf tips would undergo tipburn, which must have been associated with the increase in the distribution of Ca in roots. Further, the Ca distributivity evaluated from the relationship between the rates of plant growth and increase in Ca content show that decreased Ca distribution in leaves was mainly caused by the decrease in the rate of Ca increase (RGRleaf-Ca and ⊿Ca content). Thus, this new approach more rigorously evaluates the influence of the rate of plant growth on the incidence of tipburn.
3.3.2. Relationship between plant growth rate (RGR) and Ca increase rate (RGRCa) of leaves, stems, and roots The ratios of the RGRCa to the RGR of each organ of the three cultivars are shown in Tables 2–4. Hereafter, to evaluate the differences in Ca distributivity of each organ before and after the onset of tipburn, we focus here on data acquired from 2 to 5 weeks. The decrease in the values of RGRleaf-Ca/RGRleaf (Table 2) were attributed to the higher decrement of RGRleaf-Ca compared with that of RGRleaf of each cultivar. Consequently, decreased Ca distribution (concentrations) of leaves mainly caused by a decrease of leaf Ca increase rate (RGRleaf-Ca), suggesting leaf growth rate has little effect on the incidence of tipburn in lisianthus. The changes in the values of RGRstem-Ca/RGRstem of UH and VG were similar to those of leaves (Table 3). There were no significant differences in the values of RGRstem-Ca/RGRstem of VP between before and after tipburn because their respective values decreased at the same rate. In contrast, the values of RGRroot-Ca/RGRroot of VG and VP increased, because the increment in the value of RGRroot-Ca was higher compared with that of RGRroot (Table 4). These results indicate that VG and VP accumulated Ca in their roots before and after the onset of tipburn. During this time, there was no significant change in the values of RGRroot-Ca/RGRroot of UH (Table 4), although the value of RGRCa/RGR per plant slightly decreased (Table 1). Further, the values of RGRroot-Ca and RGRroot of UH increased at the same rate. These results indicate that UH accumulated more Ca in roots. Thus, all cultivars accumulated Ca in roots before tipburn, which would be closely related to the decreased of distributions of Ca in leaves and the incidence of tipburn.
3.4. Transpiration rates The transpiration rates of the three cultivars at 4–8 weeks, during which the rates of transpiration of leaves became lower as a function of their upward positions (Fig. 8). This finding is consistent with the Ca concentration at each leaf position (Figs. 2–4). In contrast, although we assumed that transpiration rates of UH, which was highly efficient in distributing Ca to new leaf tips, were higher compared with those of the other cultivars. Further, the transpiration rates of the upper leaves of UH were slightly lower compared with those of VG and VP at 4 weeks. These findings suggest that the distributivity of Ca in new leaf tips of each cultivar cannot be evaluated solely by the varietal differences in values of transpiration rates in the upper leaves. To mitigate or eliminate tipburn, it will be necessary to identify the physiological factors that mediate the accumulation and distribution of Ca in lisianthus. For example, it has been suggested that the distributions of Ca in lisianthus cultivars are strongly influenced by Ca uptake caused by root pressure (Kuronuma et al., 2018). Further, Lee et al. (2013b) found that the expression of genes encoding major Ca2+ vacuole transporters in cabbage cultivars is associated with the accumulation of Ca2+ in the vacuoles of leaf cells. Flower bud formation may also have an influence on the increase in the distribution of Ca in roots. The extension of such studies will undoubtedly contribute to enhance the productivity and quality of lisianthus.
3.3.3. Ca distributivity of new leaves (2–5 weeks) The ratios of ⊿Ca content to ⊿dry weight of new leaves of UH decreased from 2.02 to 1.32 (Table 5). However, the values of ⊿Ca content/⊿dry weight were higher compared with those of the other cultivars, indicating that UH had a greater ability to distribute Ca to new leaf tips. Thus, tipburn would not have been exhibited by UH. Further, the changes in ⊿Ca content and ⊿dry weight of UH indicate that the rate of new leaf growth did not exert a significant effect on the decrease of Ca concentrations and Ca distributions in new leaves. The values of ⊿Ca content/⊿dry weight of the new leaves of VG decreased from 1.46 to 1.17 and reached a minimum (0.88) at 4–5 weeks when the incidence of tipburn reached 100%. These results may be explained as follows: (1) The incremental value of the ⊿Ca content was lower compared with that of the ⊿dry weight (3–4 weeks), and (2) the decrement of the ⊿Ca content during this time was higher compared with that of ⊿dry weight (4–5 weeks). The values of ⊿Ca content/⊿ dry weight of new leaves of VP decreased from 1.06 to 0.76. The values of VP reached a minimum (0.65) 11
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accumulation and distribution, and tipburn development in field-grown butterhead lettuce. Sci. Hortic. 21, 123–128. Cox, E.F., McKee, J.M.T., Dearman, A.S., 1976. The effect of growth rate on tipburn occurrence in lettuce. J. Hortic. Sci. 51, 297–309. Guttridge, C.G., Bradfield, E.G., Holder, R., 1981. Dependence of calcium transport into strawberry leaves on positive pressure in the xylem. Ann. Bot. 48, 473–480. Garnier, E., 1991. Resource capture, biomass allocation and growth in herbaceous plants. Trends Ecol. Evolution. 6, 126–131. Kuo, C.G., Tsay, J.S., Tsai, C.L., Chen, R.J., 1981. Tipburn of Chinese cabbage in relation to calcium nutrition and distribution. Sci. Hortic. 14, 131–138. Kuronuma, T., Watanabe, H., 2016a. Physiological and morphological traits and competence for carbon sequestration of several green roof plants under a controlled environmental system. J. Am. Soc. Hortic. Sci. 141, 583–590. Kuronuma, T., Watanabe, H., 2016b. Relevance of carbon sequestration to the physiological and morphological traits of several green roof plants during the first year after construction. Am. J. Plant Sci. 8, 14–27. Kuronuma, T., Watanabe, Y., Ando, M., Watanabe, H., 2018. Tipburn severity and calcium distribution in lisianthus (Eustoma Grandiflorum (Raf.) Shinn.) cultivars under different relative air humidity conditions. Agronomy 8 (10), 218. Kuronuma, T., Watanabe, Y., Ando, M., Watanabe, H., 2019. Relevance of tipburn incidence to the competence for Ca acquirement and Ca distributivity in lisianthus [Eustoma grandiflorum (Raf.) Shinn.] cultivars. Sci. Hortic. 246, 805–811. Lee, J.G., Choi, C.S., Jang, Y.A., Jang, S.W., Lee, S.G., Um, Y.C., 2013a. Effects of air temperature and air flow rate control on the tipburn occurrence of leaf lettuce in a closed-type plant factory system. Hortic. Environ. Biotech. 54, 303–310. Lee, J., Park, I., Lee, Z.W., Kim, S.W., Baek, N., Park, H.S., Park, S.U., Kwon, S.Y., Kim, H., 2013b. Regulation of the major vacuolar Ca2+ transporter genes, by intercellular Ca2+ concentration and abiotic stresses, in tip-burn resistant Brassica oleracea. Molecular boil. Reports 40, 177–188. Magnusson, M., 2002. Mineral fertilizers and green mulch in Chinese cabbage [Brassica pekinensis (Lour.) Rupr.]: Effect on nutrient uptake, yield and internal tipburn. Acta Agricul. Scandinavica. Section B-Plant Soil Sci 52, 25–35. Mason, G.F., Guttridge, C.G., 1975. The influence of relative humidity and nutrition on leaf tipburn of strawberry. Sci. Hortic. 3, 339–349. Misaghi, I.J., Grogan, R.G., 1978. Effect of temperature on tipburn development in head lettuce. Phytopathology 68, 1738–1743. Poorter, H., Remkes, C., 1990. Leaf area ratio and net assimilation rate of 24 wild species differing in relative growth rate. Oecologia 83, 553–559. Poorter, H., Remkes, C., Lambers, H., 1990. Carbon and nitrogen economy of 24 wild species differing in relative growth rate. Plant Physiol. 94, 621–627. Sago, Y., 2016. Effects of light intensity and growth rate on tipburn development and leaf calcium concentration in butterhead lettuce. HortScience 51, 1087–1091. Saure, M.C., 1998. Causes of the tipburn disorder in leaves of vegetables. Sci. Hortic. 76, 131–147. Uno, Y., Okubo, H., Itoh, H., Koyama, R., 2016. Reduction of leaf lettuce tipburn using an indicator cultivar. Sci. Hortic. 210, 14–18. Wissemeier, A.H., Zühlke, G., 2002. Relation between climatic variables, growth and the incidence of tipburn in field-grown lettuce as evaluated by simple, partial and multiple regression analysis. Sci. Hortic. 93, 193–204.
4. Conclusions The increase in the distribution of Ca in roots and its decrease in leaves occurred at 2–5 weeks, which was consistent with that before and after the onset of tipburn in VG and VP. The distributions of Ca in new leaves of the tipburn-sensitive cultivars VG and VP must fall below the threshold concentration of Ca during this time, in response to the increase in the distribution of Ca in roots. Moreover, evaluating the distributivity of Ca as a function of the relationship between the rates of plant growth and increase in Ca concent, demonstrates that the decrease in the distribution of Ca in leaves was mainly caused by the decrease in the rate of increase of Ca content in the leaves (RGRleaf-Ca and ⊿Ca content). Therefore, tipburn in these lisianthus cultivars was caused by the decrease in the distribution of Ca in new leaves, which was associated with the increase in the distribution of Ca in roots. Appendix A. Supplementary data Supplementary material related to this article can be found, in the online version, at doi:https://doi.org/10.1016/j.scienta.2019.108911. References Aloni, B., Pashkar, T., Libel, R., 1986. The possible involvement of gibberellins and calcium in tipburn of Chinese cabbage: study of intact plants and detached leaves. Plant Growth Regul. 4, 3–11. Barta, D.J., Tibbitts, T.W., 1991. Calcium localization in lettuce leaves with and without tipburn: comparison of controlled-environment and field-grown plants. J. Am. Soc. Hortic. Sci. 116, 870–875. Bautista, A.S., López-Galarza, S., Martínez, A., Pascual, B., Maroto, J.V., 2009. Influence of cation proportions of the nutrient solution on tipburn incidence in strawberry plants. J. plant nutrition. 32, 1527–1539. Chang, Y.C., Miller, W.B., 2003. Growth and calcium partitioning in Lilium ‘Star Gazer’ in relation to leaf calcium deficiency. J. Am. Soc. Hortic. Sci. 128, 788–796. Chang, Y.C., Miller, W.B., 2004. The relationship between leaf enclosure, transpiration, and upper leaf necrosis on Lilium ‘Star Gazer’. J. Am. Soc. Hortic. Sci. 129, 128–133. Chang, Y.C., Miller, W.B., 2005. The development of upper leaf necrosis in Lilium ‘Star Gazer’. J. Am. Soc. Hortic. Sci. 130, 759–766. Choi, K.Y., Lee, Y.B., 2008. Effects of relative humidity on the apparent variability in the incidence of tipburn symptom and distribution of mineral nutrients between morphologically different lettuce (Lactuca sativa L.) cultivars. Hortic. Environ. Biotechnol. 49, 20–24. Collier, G.F., Huntington, V.C., 1983. The relationship between leaf growth, calcium
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