Incidence of Intumescence Injury in Several Tomato Cultivars Under Different Calcium Conditions

Abstract

Intumescence injury is a physiological disorder characterized by abnormal outgrowth of epidermal and parenchymal cells on the adaxial and/or abaxial surfaces of leaves. The purpose of this study was to examine cultivar differences in the incidence of intumescence injury, the relation between different calcium (Ca) conditions and intumescence injury, and to investigate countermeasures against intumescence injury in tomatoes. We grew tomato plants under different Ca nutrient conditions and treated them under high relative humidity and low ultra-violet light conditions to investigate intumescence injury incidence. The degree of intumescence injury varied among different tomato cultivars under a normal Ca condition with 4.5 me·L⁻¹ of Ca. Intumescence occurred in cultivars that showed no injury incidence under the normal Ca condition when they were grown with a low Ca nutrient solution containing 0.5 or 2.5 me·L⁻¹ of Ca. Intumescence injury incidence decreased in cultivars that showed high incidence under the normal Ca condition when they were grown with a high Ca nutrient solution containing 9.5 or 24.5 me·L⁻¹ of Ca. The differences in the incidence of intumescence among cultivars was marked with different Ca nutrient solution concentrations. There was a negative correlation between the degree of intumescence injury and the Ca content in tomato shoots. Foliar spraying with Ca was found to reduce the incidence of intumescence injury. It was suggested that intumescence injury in tomatoes can be mitigated by using cultivars in which intumescence is less likely to occur, increasing the Ca concentration of shoots by providing more Ca nutrients to the plant, and spraying Ca solution directly on the leaves.

Introduction

Intumescence injury is a physiological disorder that is characterized by abnormal outgrowth and elongated epidermal or parenchymal cells on the adaxial surfaces of leaves or stems (Suzuki et al., 2020). It is also known as edema or oedema and commonly occurs in the Solanaceae family, which includes tomatoes (Lang and Tibbitts, 1983) and potatoes (Douglas, 1907). It has also occurred in sweet potatoes (Craver et al., 2014), Eucalyptus (Pinkard et al., 2006) and some ornamental plants such as ivy geraniums (Rangarajan and Tibbitts, 1994), Salvia, and Petunia (Wollaeger and Runkle, 2015). This physiological disorder has become a problem, particularly in greenhouse cultivation. In a controlled environment, intumescences occurred under conditions with a lack of ultraviolet (UV) irradiance (Lang and Tibbitts, 1983) and high relative humidity (RH) (Atkinson, 1893; Dale, 1901; Lang and Tibbitts, 1983).

Our previous studies found that tomatoes developed intumescence injury when the RH was high and UV radiation was insufficient, and the severity increased over time. The occurrence of intumescence injury was also found to be different depending on the tomato cultivar (Ozawa et al., 2018). Morphological studies found that intumescence injury occurred along with abnormalities in the cuticular layer of the adaxial surface of tomato leaves, and its severity could be detected by staining the leaves with toluidine blue O (Suzuki et al., 2020). In comparison to fully expanded leaves and young leaves, expanding leaves are more likely to suffer from intumescence injury.

In addition, intumescence injury observed in ‘Russet Burbank’ Potatoes was found to be associated with a calcium (Ca)-related disorder (Schabow and Palta, 2019). Ca is an important macronutrient that provides a structural role for cell walls and membranes, as well as serving as an intracellular messenger in cytosol (Marschner, 1995; White and Broadley, 2003). Ca is absorbed from the soil and transported in a cation form (Ca²⁺) from roots to shoots via xylem, and Ca apoplastic movement in the plants is dependent on the transpiration rate (González-Fontes et al., 2017; Jovanović et al., 2021). Cell wall pectin is gradually esterified by pectin methylesterase, cross-linked by Ca, and branches as the cells develop (de Freitas et al., 2012). When there is a temporary lack of sufficient Ca in growing plant tissues, it increases the membrane leakage as Ca functions as a cell wall stabilizer (de Freitas et al., 2012), thus making cell walls more susceptible to loosening. Hypertrophy with intumescence may be related to cell wall loosening.

Ca deficiency leads to some physiological disorders such as tip burn and brown heart in leafy vegetables, blossom end rot in peppers and tomatoes, and bitter pit in apples (White and Broadley, 2003), as well as internal browning in tomatoes (Norimitsu et al., 2017; Suzuki et al., 2019). According to a previous study (Mazumder et al., 2021), foliar Ca spray could increase the tomato growth yield and reduce the incidence of blossom end rot. It was also used to prevent various physiological disorders in fruits and vegetables such as apple, lettuce, and strawberry (Niu et al., 2021). This indicates that Ca deficiency may be mitigated by an adequate supply of Ca.

The present study was conducted to investigate differences in the incidence of intumescence injury among cultivars, and the relation between Ca conditions and intumescence injury. Several tomato cultivars were grown under different Ca nutrient conditions and then treated under high RH and low UV irradiance conditions to investigate their susceptibility to intumescence injury. Several tomato cultivars were grown and intumescence injury occurrences were observed. The degrees of intumescence injury were assessed by staining the tomato leaves with toluidine blue O. We also investigated the relationship between intumescence injury and Ca content in tomato shoots. An experiment utilizing a Ca foliar spray was also conducted to see if this method could prevent intumescence injury in tomato leaves.

Materials and Methods

Experiment 1: Investigation of the differences in susceptibility to intumescence injury among tomato cultivars

1.1.  Cultivar differences in susceptibility to intumescence injury at high RH and low UV under a normal Ca concentration nutrient condition

Twelve tomato cultivars (Solanum lycopersicum L.): ‘CF Momotaro York’, ‘Momotaro Eight’, ‘Momotaro Fight’, ‘Momotaro Natsumi’, ‘Momotaro Peace’, ‘Momotaro Sunny’, ‘Momotaro Select’, ‘Frutica’ (Takii Seed Co., Ltd., Kyoto, Japan), ‘CF Rinka 409’, ‘Reika’ (Sakata Seed Co., Ltd., Kanagawa, Japan), ‘Misora 64’ (Mikado Kyowa Seed Co., Ltd., Chiba, Japan), and ‘Hanami’ (Marutane Co., Ltd., Kyoto, Japan) were sown on June 22, 2018 in rockwool cube (75 × 75 × 75 mm; Grodan Delta, Grodan/Rockwool B.V., Roermond, the Netherlands), watered at the bottom with well water, and germinated for three days in an incubator (12 h light/12 h dark, 28°C, RH 80%; Nippon Medical & Chemical Instruments Co., Ltd, Osaka, Japan). Ten seedlings from each cultivar were transferred to a greenhouse under natural UV conditions at Shizuoka University in Japan and were supplied from the bottom with a nutrient solution up to three weeks after sowing. This nutrient solution was formulated based on the composition of Enshi formula nutrient solution at electrical conductivity (EC) 1.2 dS·m⁻¹ (Zhang et al., 2015) diluted in well water (EC 0.15 dS·m⁻¹), which contained a total of 4.5 me·L⁻¹ Ca, 4 me·L⁻¹ from chemical solution and 0.5 me·L⁻¹ from well water; this was used as the control solution under a normal Ca concentration condition in all experiments below.

Five tomato plants of each cultivar were then transferred into an incubator that was set to cause intumescence injury conditions (low UV, 12 h light/12 h dark photoperiod, 28 ± 1°C, RH: 85%) for three days. After growing in this incubator, the degree of intumescence injury to the 3rd and 4th leaves, on which intumescence mainly occurs, was measured using the same staining procedure as described by Suzuki et al. (2020). The staining was performed by immersing the samples in 0.1% Toluidine blue O aqueos solution for one minute and washing gently with distilled water. Digital images of the samples were obtained using a scanner (GT-F670; Seiko Epson Corporation, Nagano, Japan). The degree of intumescence was calculated by analyzing the digital images using ImageJ software (http://rsb.info.nih.gov/ij/). The degree of intumescence injury was measured in the same way in all experiments below.

1.2.  Cultivar differences in susceptibility to intumescence injury under different Ca concentrations

Twelve tomato cultivars were sown as in Experiment 1.1 on November 29, 2019. After four days, seedlings were transferred to a greenhouse and supplied with control nutrient solution from the bottom, as in Experiment 1.1. Based on these concentrations of Ca, 4.5 me·L⁻¹ (Ca 4.5 me·L⁻¹), four treatments of +5 me·L⁻¹, +20 me·L⁻¹, −2 me·L⁻¹, and −4 me·L⁻¹ were adjusted; therefore, the Ca concentrations of each nutrient solution were 9.5 me·L⁻¹, 24.5 me·L⁻¹, 2.5 me·L⁻¹, and 0.5 me·L⁻¹ (Ca 9.5 me·L⁻¹, Ca 24.5 me·L⁻¹, Ca 2.5 me·L⁻¹, and Ca 0.5 me·L⁻¹). The high Ca concentrations (9.5 me·L⁻¹ and 24.5 me·L⁻¹) were obtained by adding calcium nitrate (Ca(NO₃)₂) to the control nutrient solution. The 2.5 me·L⁻¹ concentration was made by removing (Ca(NO₃)₂) from the control nutrient solution, and the low Ca concentration (0.5 me·L⁻¹) was obtained by substituting sodium nitrate (NaNO₃) for calcium nitrate (Table S1). ‘CF Momotaro York’, ‘Momotaro Eight’, ‘Momotaro Fight’, and ‘Momotaro Select’ were grown using the low Ca concentration treatments (0.5 me·L⁻¹ and 2.5 me·L⁻¹). For the cultivation of ‘Frutica’, ‘Hanami’, ‘Misora 64’, ‘Momotaro Natsumi’, ‘Momotaro Peace’, ‘Momotaro Sunny’, ‘CF Rinka 409’, and ‘Reika’, high Ca concentration treatments (9.5 me·L⁻¹ and 24.5 me·L⁻¹) were applied.

The bottom watering method was used to grow all the tomatoes so that sufficient nutrient solution could be supplied. Four tomato plants of each cultivar under different Ca conditions were grown in a greenhouse until two weeks after sowing, then transferred into an incubator set to cause intumescence injury conditions the same as in Experiment 1.1. The average degree of intumescence injury to the 1st and 2nd leaves on which mainly intumescence occurred was measured using the same procedure used in Experiment 1.1. The degree of intumescence was set at 50% for wilted leaves in this experiment.

Experiment 2: Investigation of the correlation between the intumescence injury and Ca content in tomatoes shoots

‘Misora 64’ seeds were sown on July 24, 2020. Eight seedlings were grown under 0.5 me·L⁻¹, 4.5 me·L⁻¹ and 24.5 me·L⁻¹ Ca concentration nutrient solution until August 27. The bottom watering technique was applied twice daily to each treatment. Four plants were transferred to intumescence injury conditions the same as in Experiment 1.1. for four days and sampled on August 31. Four plants from the intumescence treatment were stained and an investigation of the degree of intumescence injury on all leaves from each treatment was performed as explained in Experiment 1.1. After scanning, all samples were dried in an oven for three days at a constant temperature of 80°C to obtain dry weights and then analyzed for Ca content.

Experiment 3: Correlation between the degree of intumescence and Ca content in various tomato cultivars

Tomato cultivars of ‘CF Momotaro York’, ‘Momotaro Select’, ‘Momotaro Natsumi’, ‘Misora 64’, ‘CF Rinka 409’, and ‘Reika’ were sown in a urethane medium (25 × 25 × 25 mm; LFS 023-4, Living Farm Ltd., Tokyo, Japan) containing well water on June 1, 2021, in an incubator as explained in Experiment 1.1. Seedlings were transferred on June 13 to 72-hole cell trays and grown hydroponically in containers in an artificial climate chamber (day/night: 12 h light/12 h dark, 25°C, RH: 50%; Hitachi, Ltd., Tokyo, Japan). They were supplied from the bottom with the control nutrient solution (same as Experiment 1.1).

After a week, the nutrient solution was changed to three different Ca concentrations: 0.5 me·L⁻¹, 4.5 me·L⁻¹ (control), and 24.5 me·L⁻¹ with eight plants supplied with each nutrient solution. On June 28, samples from each nutrient treatment were divided into two environmental treatments in the artificial climate chamber: A control condition (12 h light/12 h dark, 25°C, RH: 50%) and a low UV, high RH condition (low UV, 12 h light/12 h dark, 25°C, RH: 85%) for three days. The low UV condition was set by covering the plants using UV-cut film (Diastar UV Cut; Mitsubishi Chemical Agri Dream Co., Ltd, Tokyo, Japan). The degree of intumescence injury to all leaves and the Ca content in tomato shoots were assessed as explained for Experiment 1.1 and 2.

Experiment 4: Effect of Ca spray on the incidence of intumescence injury in tomato leaves

On March 11, 2022, seeds of the tomato cultivar ‘CF Rinka 409’ were sown and grown in the same manner as in Experiment 3. After one week, 60 seedlings were transferred and grown in a plant container (7.5 × 7.5 × 10 cm; VWR International, LLC, Radnor, PA, USA) with 110 mL of control nutrient solution and aeration flow. The plants were cultivated for 17 days in a greenhouse (as done in Experiment 1.1). On April 4, plants were moved into an artificial climate chamber under a low UV and high RH condition as explained in Experiment 3.

The Ca spray solution was made by diluting 2.5 mL calcium oxide 12% (Akuakaru, Nichieki Chemical Co., Ltd., Tokyo, Japan) into 1 L of distilled water. The Ca spray was applied to the adaxial and abaxial surfaces of tomato leaves once a day for 7 days. Five different treatments were used: no spray (Ctrl), distilled water spray (Distilled water spray), once a week (Ca-1x), twice a week (Ca-2x), and daily (Ca-7x). Each treatment was sprayed onto twelve tomato plants.

On April 11, a growth survey was carried out to measure the fresh weight, dry weight, number of leaves, and shoot and root lengths of the tomato plants. Assessment of the degree of intumescence injury was done as in Experiment 1.1. The dry weights for Ca content analysis were obtained as in Experiment 2.

Calcium content analysis

Dried samples of tomato shoots from Experiments 2, 3, and 4 were ground into fine powder in a pulverizer (Mini Speed Mill MS-05; Labonect, Ltd, Osaka, Japan) and sieved through a 212 μm sieve. Then, 20 mg samples were weighed into 15 mL polypropylene tubes (DigiTUBEs; GL Science Inc., Tokyo, Japan). Wet ashing was performed by adding 2 mL of concentrated nitric acid (60% HNO₃, guaranteed reagent, Fujifilm Wako Pure Chemical Corporation, Osaka, Japan) in a fume hood, covered loosely by a lid and samples were heated in a heat block set to 110°C for 2.5 h. After cooling, 2 mL of perchloric acid (HClO₄, guaranteed reagent, Fujifilm Wako Pure Chemical Corporation, Osaka, Japan) was added and the sample was heated again at 110°C for 2.5 h. The mixture was adjusted to 10 mL in polypropylene tubes (DigiTUBE, GL Sciences Inc.) and filtered using filter paper (5B; Advantec Toyo Kaisha, Ltd., Tokyo, Japan). The mixture was adjusted to 15 mL in a conical tube (Falcon^® conical tube; Corning Inc., Corning, NY, USA) and used as a stock solution.

The Ca content of tomato shoots in Experiment 2 was determined using an atomic absorption spectrometer (iCE3300, Thermo Fisher Scientific, Ltd., Waltham, MA, USA). Strontium chloride hexahydrate (99% SrCl₂·6H₂O, guaranteed reagent, Fujifilm Wako Pure Chemical Corporation) was used so that the final concentration of strontium (Sr) was 1,000 ppm. The Ca content of tomato shoots in Experiments 3 and 4 was measured using an Inductively Coupled Plasma (ICP) Spectrometer (iCAP 7000 Series, Thermo Fisher Science Co., Ltd.).

Statistical analysis

Statistical analysis was performed using JMP11 (SAS Institute Inc., Cary, NC, USA). A two-way ANOVA test was used to detect the effect of Ca concentration in the nutrient solution and to test tomato cultivars for degree of intumescence and Ca content. Tukey’s test was used to identify differences between Ca content in tomato leaves and shoots, as well as the degree of intumescence in different tomato cultivars, based on various Ca concentrations. Differences were considered significant when P < 0.05.

Results

Experiment 1: Differences in susceptibility to intumescence injury among tomato cultivars

1.1.  Cultivar differences in susceptibility to intumescence injury at high RH and low UV under a normal Ca concentration nutrient condition

Table 1 shows the differences in susceptibility to intumescence injury in 12 tomato cultivars under a high RH and low UV condition using a normal Ca 4.5 me·L⁻¹ concentration nutrient solution. On the third leaf, no intumescence injury was observed in ‘Momotaro Eight’, ‘Momotaro Select’, ‘Momotaro Fight’, or ‘CF Momotaro York’. The degree of intumescence injury was 0 to 10% in ‘Momotaro Natsumi’, ‘Momotaro Sunny’, ‘Hanami’, and ‘Misora 64’. It was more than 10% in ‘Frutica’, ‘CF Rinka 409’, and ‘Momotaro Peace’. ‘Reika’ showed the highest degree of intumescence injury among the twelve cultivars, at 32.6%. On the fourth leaf, no intumescence injury was observed in ‘Momotaro Eight’, ‘Momotaro Select’, ‘Momotaro Fight’, ‘CF Momotaro York’, ‘Momotaro Natsumi’, ‘Momotaro Sunny’, or ‘Frutica’. The degree of intumescence injury was 1.4% in ‘Hanami’ and 8.7% in ‘Misora 64’and it was more than 10% in ‘CF Rinka 409’, ‘Momotaro Peace’, and ‘Reika’. ‘Momotaro Eight’, ‘Momotaro Select’, ‘Momotaro Fight’, and ‘CF Momotaro York’ did not have any intumescence injuries.

Table 1

Cultivar differences in degree of intumescence injury (percentage data arcsine transformed) of tomato leaves at different leaf positions treated with normal Ca concentration (4.5 me.L⁻¹).

1.2.  Cultivar differences in susceptibility to intumescence injury under different Ca concentrations

Table 2 shows the degree of intumescence injury difference among tomato cultivars when treated with different Ca concentration solutions. The degree of intumescence injury was quantified on the first and second leaves of the tomato plants. Because we used younger seedlings than Experiment 1.1, the leaf position that showed intumescence injury was lower. The cultivars that showed no incidence under the normal Ca 4.5 me·L⁻¹ condition in Experiment 1.1. were treated with the low Ca concentration solution (Ca 0.5 me·L⁻¹ and Ca 2.5 me·L⁻¹). Two-way ANOVA showed that within cultivars, Ca concentration in the nutrient solution significantly affected the degree of intumescence injury in the leaves. In the 0.5 me·L⁻¹ Ca concentration-treated plants, intumescence injury was found to be highest in the ‘Momotaro Eight’ cultivar, and lowest in the ‘CF Momotaro York’ cultivar. Significant differences in the degree of intumescence injury among cultivars were no longer observed in the 2.5 me·L⁻¹ Ca concentration solution. The combined results from both treatment solutions indicated that ‘CF Momotaro York’ was a cultivar in which intumescence injury did not occur.

Table 2

Cultivar differences in susceptibility to intumescence injury of tomato leaves at different leaf positions in different Ca concentration solutions.

The cultivars that showed some intumescence injury under the normal Ca 4.5 me·L⁻¹ condition in Experiment 1.1 were examined under higher than normal concentrations of Ca treatment solution (Ca 9.5 me·L⁻¹ and Ca 24.5 me·L⁻¹). Two-way ANOVA tests showed that the cultivar differences, the Ca treatment solution, and the interaction between them resulted in significant differences in the degree of intumescence injury in the tomato leaves with the high-concentration Ca solutions. Intumescence injury incidence in the 4th leaf of ‘Misora 64’ was 8.7% under the normal 4.5 me·L⁻¹ Ca condition in Exp. 1.1, fell to 3.3% in the 1st leaf and 4% in the 2nd leaf under the 9.5 me·L⁻¹ Ca concentration condition and 2.5% in the 1st leaf and 1.8% in the 2nd leaf under the 24.5 me·L⁻¹ Ca concentration condition (Table 2). Among the cultivars, ‘CF Rinka 409’ showed the highest degree of intumescence injury when treated with the 9.5 me·L⁻¹ solution and injury was also observed on the first leaf treated with the 24.5 me·L⁻¹ solution. However, no significant differences in the degree of intumescence were observed among tomato cultivars on the second leaf when treated with the 24.5 me·L⁻¹ solution. This result suggested that ‘CF Rinka 409’ was a cultivar in which intumescence injury easily occurred, as it occurred even with a high Ca concentrated nutrient solution.

Experiment 2: Analysis of the correlation between intumescence injury and Ca content in tomato shoots

‘Misora 64’, which showed about 9% incidence under the normal Ca condition in Experiment 1.1 was examined under the high Ca concentration condition (Ca 24.5 me·L⁻¹), the low concentration condition (Ca 0.5 me·L⁻¹), and with the control solution (Ca 4.5 me·L⁻¹). The Ca content in the shoots and degree of intumescence were measured. The correlation between the intumescence injury and the Ca content in tomato shoots of cultivar ‘Misora 64’ is shown in Figure 1. The 0.5 me·L⁻¹ concentration Ca solution resulted in the highest degree of intumescence injury (about 40%) with the lowest Ca content being about 2.5 mg·g⁻¹DW in tomato shoots. The 4.5 me·L⁻¹ concentration Ca solution resulted in intumescence injury at about 15% with the Ca content being about 8.1 mg·g⁻¹DW in tomato shoots, and the 24.5 me·L⁻¹ concentration Ca solution resulted in intumescence injury of about 4% with the Ca content being about 12.8 mg·g⁻¹DW in tomato shoots. There was a negative correlation between the degree of intumescence injury and the Ca content in tomato shoots.

Fig. 1

Correlation between the degree of intumescence injury and the Ca content in tomato ‘Misora 64’ shoots. Ca 0.5 me·L⁻¹: Ca concentration of nutrient solution was 0.5 me·L⁻¹; Ca 4.5 me·L⁻¹: Ca concentration of nutrient solution was 4.5 me·L⁻¹; Ca 24.5 me·L⁻¹: Ca concentration of nutrient solution was 24.5 me·L⁻¹.

Experiment 3: Correlation between the degree of intumescence and Ca content in several tomato cultivars

The correlation between the degree of intumescence injury and Ca content was examined using ‘CF Momotaro York’, ‘Momotaro Select’, ‘Momotaro Natsumi’, ‘Misora 64’, ‘Reika’, and ‘CF Rinka 409’ which showed different degrees of intumescence in Experiment 1.1. ‘CF Rinka 409’ and ‘Reika’ developed a high degree of intumescence injury at about 11% and 6% when treated with the 0.5 me·L⁻¹ Ca concentration, while ‘Momotaro Natsumi’ and ‘Misora 64’ developed a lower degree of intumescence injury at about 0.3% and 2.4%. On the other hand, in ‘CF Momotaro York’ there was no incidence of intumescence injury, despite being under a low Ca concentration condition (Table 3). The degree of intumescence injury decreased in all cultivars along with higher concentrations of Ca in the nutrient solution. ‘CF Rinka 409’ showed a 10.8% degree of intumescence injury under the 0.5 me·L⁻¹ Ca condition, but this decreased to 0.1% under the 24.5 me·L⁻¹ Ca condition.

Table 3

Cultivar differences in susceptibility to intumescence injury and Ca content of tomato shoots at different Ca concentration supplies.

In this experiment, the Ca concentration and environmental conditions significantly affected the Ca content in tomato leaves, as well as the degree of intumescence injury, while the difference in tomato cultivars only affected the degree of intumescence injury in tomato leaves (Table 3). There were no significant difference in Ca content in the shoots among cultivars. The interaction between Ca concentration and environmental conditions significantly affected the Ca content and degree of intumescence in tomato leaves, while the interaction between Ca concentration and cultivars, as well as different cultivars and environmental conditions, only affected the degree of intumescence. Three-way ANOVA showed a significant interaction between Ca concentration, environment, and cultivar on the degree of intumescence injury of tomato leaves.

Experiment 4: Effect of Ca spraying on the incidence of intumescence injury in tomato leaves

The effect of Ca spraying on the intumescence injury in tomato leaves of cultivar ‘CF Rinka 409’ is shown in Table 4. Foliar Ca spraying did not significantly affect the fresh or dry weights of shoots. The shoot lengths were the with under the distilled water spraying, and the leaf number was highest when the foliar Ca spray was applied daily. Intumescence injury incidence was found to be highest when no foliar Ca spray was applied. Daily application of Ca spray significantly reduced the degree of intumescence injury. However, this treatment did not significantly affect the Ca content in the tomato leaves.

Table 4

Growth survey results, degree of intumescence and Ca content of tomato ‘CF Rinka 409’ shoots with foliar Ca spray applied.

Discussion

Susceptibility to intumescence injury was observed among 12 tomato cultivars in this study. Under the same Ca concentration conditions, the degree of intumescence injury varied among different cultivars. Intumescence injury occurred under 0.5 me·L⁻¹ or 2.5 me·L⁻¹ Ca concentration nutrient conditions in cultivars that showed no injury incidence under the normal 4.5 me·L⁻¹ Ca condition. The cultivars that showed high injury incidence under the 4.5 me·L⁻¹ Ca condition developed a high degree of intumescence injury under the 0.5 me·L⁻¹ Ca nutrient condition and this decreased under the 24.5 me·L⁻¹ Ca nutrient condition. The differences between cultivars in terms of intumescence injury incidence was marked when using different Ca concentration nutrient solutions. This could result could be applied to screening for cultivars in which intumescence injury is less likely to occur using a low Ca nutrient condition. Regarding cultivar differences, genetic factors may also contribute to the difference in the degree of intumescence injury between susceptible and tolerant cultivars, as suggested in potato leaves (Schabow and Palta, 2019). Prinzenberg et al. (2022) also suggested that the differences among intumescence injury severity in tomato are genetically dependent, with medium to high heritability. Miyama and Yasui (2021) conducted experiments to examine intumescence injury in several tomato cultivars, and the results showed that intumescence injury occurred in tomato cultivars with higher shoot/root ratios. Although only the effect of Ca was investigated in this study, other factors may be involved, and effects of other nutrients or the osmotic pressure of the nutrient solution on cultivar differences will be investigated in the future.

In this study, intumescence injury intensified under the 0.5 me·L⁻¹ Ca concentration nutrient condition. Intumescence injury occurs when cells become hypertrophic, and this phenomenon is likely caused by a decrease in the strength of cell walls. It was reported that insufficient Ca in a plant could result in cell wall and membrane breakdown (de Freitas et al., 2012; Jovanović et al., 2021). In our previous study, expanded cell walls were found to be wavy, which indicates a loss of cell integrity, under transmission electron microscopy and light microscopy observation (Suzuki et al., 2020). Cell wall rupture was also observed in intumescence injury in potato plants (Schabow and Palta, 2019). Moreover, Ca deficiency-loss of cell wall strength has also been observed with blossom-end rot in tomato plants (Suzuki et al., 2003).

In this study, the Ca contents in the shoots under low UV and high RH conditions were significantly lower than the control condition (Fig. 1; Table 3). Ca transport to the leaves is affected by the transpiration rate in plants. As an immobile macronutrient, Ca requires a higher transpiration rate to be transported from roots to leaves (Gilliham et al., 2011; Tanner and Beevers, 2001). When the transpiration is low, Ca transport to the leaves is reduced, resulting in a lower Ca content in the leaves. Transpiration rate and Ca uptake also decreased under a high RH condition (Adams and Holder, 1992), and high RH is known to be one of the factors that causes intumescence injury in plants (Atkinson, 1893; Dale, 1901; Lang and Tibbitts, 1983; Schabow and Palta, 2019). Therefore, it is suggested that an increase in intumescence injury occurrence under high RH could be related to a Ca deficit in tomato leaves.

In a potato study using ‘Russet Burbank’, intumescence injury occurred in 65% of plants when the Ca content averaged 6.63 mg·g⁻¹DW in the leaves and was 5% when the Ca content averaged 12.7 mg·g⁻¹DW (Schabow and Palta, 2019). In this study, using ‘Misora 64’, the degree of intumescence injury was 40% when the Ca content averaged 2.5 mg·g⁻¹DW in the shoots and was 15% when the Ca content averaged 8.1 mg·g⁻¹DW. The Ca content averaged 12.8 mg·g⁻¹DW in the shoots and the degree of intumescence was 4%. It is thought that when the Ca content in leaves or shoots is less than 10 mg·g⁻¹DW, the risk of developing intumescence injury increases.

Previous studies have shown that intumescence injuries are less likely to occur in young or fully expanded leaves (Suzuki et al., 2020). Expanding leaves require more Ca for the process of cell wall formation, as they are actively producing and expanding their cells (Chang et al., 2004; Collier and Tibbitts, 1982). Low availability of Ca in leaves resulted in easily ruptured and flexible cell walls (Hepler, 2005). It is thought that the Ca content in expanding leaves is important in relation to the occurrence of intumescence injury. It will be necessary to examine the relationship between the leaf position, stage, expansion rate, Ca content and occurrence of intumescence in the future.

In this experiment, intumescence was inhibited when a foliar Ca solution was sprayed on the leaves. Ca sprays have been suggested as a preventative technique for blossom-end rot, which is caused by Ca deficiency (Mazumder et al., 2021). Fruits are covered with a cuticle layer thicker than leaves and have no stomata, so the effect of spraying is weak. A foliar spray may be more effective than a fruit spray as it can be absorbed immediately by leaves (Gao et al., 2018; Niu et al., 2021). Ca spraying did not affect Ca contents in the shoots significantly, but it may have affected the surface cells in which intumescence injury occurred directly. Nutrients supplied via foliar spraying enter the leaves through stomata or penetrate the cuticle before being transferred to other organs (Geetha, 2019). Kumazaki et al. (2010) reported that foliar potassium (K) application helps to prevent leaf necrosis due to K deficiency. In the present study, intumescence injury significantly decreased with frequent Ca foliar spraying. This result agrees with that of a previous study which showed that 14 daily applications of calcium chloride foliar spray effectively reduced the risk of upper leaf necrosis in lily bulbs compared to twice a week application (Chang et al., 2004). Therefore, it is suggested that an increase in the frequency of foliar Ca spraying would be beneficial.

As a countermeasure against the occurrence of intumescence injury, creating environmental conditions that make it difficult for this to occur such as applying UV light and reducing RH are effective (Suzuki et al., 2020). It has also been reported that in addition to UV-B light, applying far-red, blue, or green light are also possible countermeasures against intumescence injury (Eguchi et al., 2016a, b; Hernández et al., 2016; Kubota et al., 2017; Morrow and Tibbitts, 1988; Wollaeger and Runkle, 2014). In addition, in this study, intumescence injury could be effectively suppressed by increasing the Ca concentration in the shoots. Growth in a nutrient solution with a high Ca concentration is another effective method for increasing the Ca concentration in shoots. However, when the Ca concentration increases, it may prevent K from being absorbed, resulting in K insufficiency, so caution is required (Paiva et al., 1998, Rhodes et al., 2018). Since there are differences in the incidence of intumescence injury among cultivars, using cultivars resistant to such injury is also important. Frequent foliar spraying of Ca solution on the expanding leaves can be used to prevent intumescence injury.

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