Use of Ethephon and Calcium Acetate to Manipulate the Foliage Retention Rates of Camphor and Golden Shower Trees

Abstract

We evaluated the effect of water and ethephon (2-chloroethyl phosphonic acid) under different doses (500 mg·L⁻¹, 1000 mg·L⁻¹,2000 mg·L⁻¹, and 3000 mg·L⁻¹), with and without calcium acetate (CA) in two foliar applications on camphor and golden shower saplings. It was aimed for ethephon to replace pruning in reducing transpiration during transplantation. We adopted a completely randomized design as an experimental design. An adequate dose of the ethephon/CA solution must be able to defoliate more than 50% of the foliage and recover to more than 75% of the foliage between 11 May and 19 July. The result showed that defoliation started within one week of the first spray, reached the lowest foliage retention rates (LRRs) in one month, then re-foliated. The LRRs were correlated with the doses of ethephon in the means, but most of the treatments did not show statistical significance due to the large in-group variations among replicates. Adding CA raised the LRRs and alleviated the dieback, especially on camphor trees. Adding CA was necessary for camphor trees to re-foliate. The final foliage retention rate (FRR) was influenced by the ethephon dose, and different tree species showed different restoration abilities. The adequate dose for camphor and golden shower trees to have an LRR < 50% and an FRR > 75% was to spray 1000 mg·L⁻¹ of ethephon first, then spray another 2000 mg·L⁻¹ of ethephon and 8000 mg·L⁻¹ of CA three days later.

1. Introduction

The recovery of plant water balance is crucial for the survival of a transplanting tree. A loss of absorption roots upon transplant disrupts the plant water balance and results in water stress. Water stress can be mitigated by reducing transpiration and increasing the water uptake ability. A reduction in transpiration can be achieved by less foliage and stomata control [1,2]. The manual removal of leaves from the tree is an option, but it is time-consuming, and the labor cost is very expensive for large trees. It also causes skin irritation to workers due to leaf exudates.

Pruning is one of the most prevailing methods to remove leaves by cutting off branches and shoots to reduce transpiration and decrease the water demand. However, removing branches and shoots leads to losing the stored carbohydrates and photo-synthetic organs, which will stress the tree with reduced photosynthetic ability. The simultaneous growth of new shoots and new roots will compete for the limited carbohydrates left in the tree and retard the recovery of photosynthesis, respiration, and transpiration functions for survival [3,4]. The recovery of foliage must wait for the new branches and shoots to grow before new buds and sprouts can emerge from them, which is a lengthy process.

Defoliation using chemicals is popular in the pomology industry for harvest [5]. It is especially common for olive (Olea europaea L.), apple (Malus spp.), pear (Pyrus spp.), peach [Prunus persica (L.) Batsch], and citrus (Citrus spp.) [6,7,8]. Chemicals such as ethephon (2-chloroethyl phosphonic acid) and harvade (2,3 Dihydro-5, 6-dimethyl-l, 4-dithiin 1,1,4 tetroxide), and those in combination with Dupont-WK (D-WK) surfactant (principle functioning agent the dodecyl ether of polyethylene glycol), are usually used to defoliate nursery stocks [9,10]. Good defoliants are defined as removing at least 50% of foliage in 2–3 weeks, inexpensive, easy to apply, and less damaging to the plants [11]. However, leaf abscission and plant injury vary with tree species, weather, and the stage of tree growth. [12] The number of applications and the application rates also make a difference. Some defoliants, such as orange agents and 2,4-dichlorphenoxyacetic acid (2,4-d), are harmful to human beings and are prohibited by many governments [13,14].

Ethylene is a plant growth regulator which induces abscission and aging. Ethylene can degrade the substrate of the separation layer, thereby triggering the abscission of organs [15,16] and promoting the senescence of cells in the xylem, which causes xylem embolism and dries out the terminal shoots. One of the major reasons for dieback is xylem embolism due to the loss of hydraulic conductance [16,17,18]. However, ethylene does not harm living animals and humans, meaning it is safer than most defoliants. Ethephon releases ethylene upon degradation, which is commonly used to defoliate lemon, orange, olive, and sour cherry plants to save labor costs [19]. It is easy to apply and not expensive to use in large quantities in the field. However, a high dose of ethephon is phytotoxic and injures the plants through dieback. [13]. Sun et al. [20] reported that ethephon released abundant ethylene, causing damage in Ipomoea cairica. A dose higher than 4000 mg·L⁻¹ of ethephon can damage plant tissues, whereas a dose of 7200 mg·L⁻¹ can lead to plant death.

Larsen and Lowell [10] and Dozier et al. [11] reported that two applications of ethephon defoliated more leaves with less dieback than one single spray in their experiments. Plant injury appeared at the shoot terminals, and the gradient of injury was linearly correlated with dieback rates [11]. Xin et al. [21] reported that the efficacy of defoliation was affected by cumulated defoliant dosage. Two consecutive sprays allow for a lower concentration on each application than a single spray to achieve an effective threshold concentration [22,23]. The first spray induces the abscission process, and the second spray accumulates the concentration to reach the threshold level of ethylene.

Calcium can delay or prevent abscission through a deferral of pulvinar senescence to retard the ethylene effect [21]. It cements the cell wall by forming salt bridges between pectin components [24]. Martin et al. [25] reported that calcium spray mitigated the defoliation effect and plant injury by increasing free-form calcium in treated pecan leaves. Iwahori and Oohata [26] shared their experimental results on citrus defoliation that calcium acetate (CA) is effective in alleviating the plant injury of ethephon. Thus, it is worthwhile to investigate whether adding CA can substantially protect trees from excessive injury by ethephon.

There have been very few studies on defoliating large trees using defoliants before the transplant. Restoring foliage with less plant injury will enhance transplant survival and accelerate establishment.

The objective of this research was to identify the adequate ethephon dose with or without CA to defoliate more than 50% of the foliage in the process and recover to more than 75% of the foliage on the final date for camphor and golden shower trees.

2. Materials and Methods

The experiment was conducted between 11 May and 19 July 2020 at the nursery of Treegarden Corporation, which is located in Neihu district (latitude 25° N, longitude 121° E, 16 m above sea level), a suburb of Taipei City, Taiwan, within the subtropical climate zone. The 2-year-old camphor (Cinnamomum camphora (L.) J. Presl.) and golden shower (Cassia fistula L.) saplings were purchased from a nursery in Changhua County (30 m above sea level) in the middle of Taiwan on 10 February 2020. The camphor saplings were 80 cm high with an average of 50 leaves on the canopy. The golden shower saplings were 40 cm high with an average of 20 leaves on the canopy. They were transplanted from the 3″ pots to 5″ pots immediately and backfilled with peat moss and sand (50:50) for more soil volume and acclimation to the local climate (Figure 1A,B).

The experimental design included one water-sprayed control and eight ethephon-related treatments for each of the two species by using a completely randomized design. Each treatment had two sprays in 4 days. The water-sprayed control used water for both sprays. The eight ethephon-related treatments were all sprayed with 1000 mg·L⁻¹ of ethephon solution in the first spray on 11 May. The second spray was conducted three days later with four different doses of ethephon solution: 500 mg·L⁻¹, 1000 mg·L⁻¹, 2000 mg·L⁻¹, and 3000 mg·L⁻¹; and another four treatments involving the above four doses, each adding 8000 mg·L⁻¹ of CA (

Table 1. Nine treatments combining water and different doses of ethephon with and without calcium acetate (CA) solutions in two sprays in summer 2020. Two species, camphor and golden shower trees, were tested and compared. The first foliar spray was on 11 May, and the second spray was on 14 May. Each treatment had six replicates.

Treatment	First Spray	Second Spray
C	Water	Water
E1K+0.5K z	1000 mg·L−1 ethephon	500 mg·L−1 ethephon
E1K+1K	1000 mg·L−1 ethephon	1000 mg·L−1 ethephon
E1K+2K	1000 mg·L−1 ethephon	2000 mg·L−1 ethephon
E1K+3K	1000 mg·L−1 ethephon	3000 mg·L−1 ethephon
E1K+0.5K + CA y	1000 mg·L−1 ethephon	500 mg·L−1 ethephon + CA
E1K+1K + CA	1000 mg·L−1 ethephon	1000 mg·L−1 ethephon + CA
E1K+2K + CA	1000 mg·L−1 ethephon	2000 mg·L−1 ethephon + CA
E1K+3K + CA	1000 mg·L−1 ethephon	3000 mg·L−1 ethephon + CA

^z E_1K+0.5K treatment denotes 1000 mg·L⁻¹ of ethephon in the first spray and another 500 mg·L⁻¹ in the second spray, E_1K+1K treatment denotes 1000 mg·L⁻¹ of ethephon in the first spray and another 1000 mg·L⁻¹ in the second spray, …, etc. ^y + CA denotes the addition of 8000 mg·L⁻¹ of calcium acetate.

). Both sprays used a 1.5 L hand sprayer to apply the solution to both sides of the leaves until the liquid flowed down and avoided spraying the apical buds. No surfactant was used. Each treatment had six replicates. We set 50 days from the first spray as the experimental period, which was what most tree transplant projects used to evaluate the transplant survival.

We counted the number of leaves on each of the testing saplings on 11 May as the original leaf number and counted the leaf number on the saplings weekly to monitor the effect of ethephon/CA. The foliage retention rates of the saplings were calculated by the number of leaves remaining on the saplings divided by the original leave number. We categorized the foliage retention rates into five groups: 0%, 20%, 40%, 60%, 80%, and 100% by rounding the calculated percentages to the highest percentage of the group (Figure 2). For example, retention rates between 0–20% were recorded as 20%; retention rates between 20.1–40% were recorded as 40%, …, etc.

The treated saplings were placed in the open air without shading. Manual irrigation on the potting soil by pressurized handgun was provided every 2 days in the evening until the water flowed out of the pot while avoiding irrigation on the leaves. This was to simulate the transplantation practice in which we irrigated the trees regularly. No fertilization was provided during the experimental period.

An analysis of variance (ANOVA) was used to identify the significance of the effect among treatments in Costat (Version 6.400, Cohort Software, Ltd., Birmingham, UK). The least significant difference (LSD) was adopted to determine the significant variation at p < 0.05. SigmaPlot software (version 12.3; Systat Software, San Jose, CA, USA) was employed to draw line and scatter plot charts for ease of comprehension.

3. Results

Foliage retention rates under different treatments are shown in

Table 2. LRRs and FRRs of ethephon and/or calcium acetate treatments on camphor and golden shower trees using two-way completely randomized ANOVA. Lowercase letters indicate significant differences among treatments for a given trait (p < 0.05; LSD test). a, b, and c denote significant differences in performance among treatments. ns, *, **, ***: Non-significant or significant at p < 0.05, 0.01 or 0.001, respectively. ^Z: Please see Table 1 for treatment descriptions.

	Camphor		Golden Shower
	LRR	FRR	LRR	FRR
ethephon treatment Z
C (water)	77 a	77 a	63 a	63 bc
E1K+0.5K	42 a	70 a	65 a	95 a
E1K+1K	30 b	80 a	43 ab	47 c
E1K+2K	32 b	63 a	23 b	75 ab
E1K+3K	43 b	67 a	33 b	88 ab
without CA	30 b	55 b	37 b	74 a
with CA	59 a	87 a	54 a	73 a
Significance
Ethephon	***	ns	**	**
CA	***	***	*	ns
Ethephon × CA	**	***	*	ns

. The lowest retention rate (LRR) was the lowest weekly foliage retention percentage of a specific treatment in the experimental period. The final retention rate (FRR) was the foliage retention percentage on 19 July. The different lowercase letters represent the significance of the retention rates at p < 0.05 (Table 2). We set LRR < 50% and FRR > 75% as the criteria to choose the appropriate ethephon/CA solution at the transplantation.

The experimental design included two species and nine ethephon/CA treatments to generate 18 treatment combinations. Two-way completely randomized ANOVA was initially adopted to identify the significance of the effect on LRRs and FRRs from the ethephon and CA treatments (Table 2).

There were very significant interactions between ethephon and CA treatments for the LRRs and FRRs of camphor trees. The interaction between ethephon and CA treatments for the LRRs was also significant, although it was not significant for the FRRs on golden shower trees (Table 2). The significant interaction made the individual effect of ethephon and CA difficult to judge. We had to conduct one-way, completely random ANOVA to examine the effect of each of the ethephon/CA treatments of the two species individually (

Table 3. LRRs and FRRs of ethephon and/or calcium acetate treatments on camphor and golden shower trees using one-way completely randomized ANOVA. Lowercase letters indicate significant differences among treatments for a given trait (p < 0.05; LSD test). ns, **, ***: Non-significant or significant at p < 0.01, or 0.001, respectively. ^z Treatment description is listed in Table 1. Least square difference (LSD) at p < 0.05 significance.

	Camphor		Golden Shower
Treatments z	LRR	FRR	LRR	FRR
C (water)	77% a	77% a	63% ab	63% bc
E1K+0.5K	27% cd	47% bc	67% a	100% a
E1K+1K	20% de	70% ab	27% cd	33% c
E1K+2K	17% de	43% bc	13% d	73% ab
E1K+3K	10% e	40% c	17% d	100% a
E1K+0.5K + CA	57% ab	93% a	63% ab	90% ab
E1K+1K + CA	40% bcd	90% a	60% ab	60% bc
E1K+2K + CA	47% bc	83% a	33% bcd	77% ab
E1K+3K + CA	77% a	93% a	50% abc	77% ab
Significance	***	***	**	**

Different lowercase letters represent a significant difference in retention rates at p < 0.05.

).

3.1. Camphor

3.1.1. LRR

The ethephon treatment showed significant defoliation against the water-sprayed control. The higher doses of ethephon seemed to result in lower LRRs although only E_1K+0.5K showed significant variation against E_1K+3K. It was because of the large deviation among the replicates in the treatment group. E_1K+0.5K, E_1K+1K, E_1k+2K, and E_1K+3K each had 47%, 23%, 15%, and 13% of the LRRs.

Adding CA significantly raised the LRR levels. E_1K+0.5K + CA and E_1K+3K + CA had LRRs > 50% and disqualified themselves from being the adequate solution. E_1K+1K + CA and E_1K+2K + CA had 40% and 47% separately to meet the criteria.

E_1K+1K + CA and E_1K+2K + CA on the camphor trees met the LRR < 50% criteria.

3.1.2. FRR

The FRRs of most of the pure ethephon treatments abided by the dose-effect rule, except E_1K+1K. The control had the highest FRR of 77%, followed by E_1K+0.5K, E_1k+2K, and E_1K+3K at 47%, 43%, and 40%. E_1K+1K recorded 70% which was significantly higher than E_1K+3K. None of the ethephon treatments met the FRR > 75% criteria. It was not suggested to spray ethephon alone on camphor trees to defoliate at transplant.

The addition of CA raised the FRRs to meet and exceeded the criteria of FRR > 75%.

However, only E_1K+1K + CA and E_1k+2K + CA had an LRR < 50% to make them the adequate spray solution for camphor trees.

3.2. Golden Shower

3.2.1. LRR

The effect of ethephon was significant for most treatments except for E_1K+0.5K. Although not statistically significant, the higher doses of ethephon seemed to result in lower LRRs. E_1K+0.5K, E_1K+1K, E_1k+2K, and E_1K+3K each had 67%, 27%, 13%, and 17% of the LRRs.

Adding CA also raised the LRR levels of most treatments and exceeded the 50% LRR criteria except E_1K+2K + CA.

E_1K+1K, E_1K+2K, E_1K+3K, and E_1K+2K + CA met the LRR < 50% criteria.

3.2.2. FRR

Only E_1K+0.5K and E_1K+3K of the pure ethephon treatments meet the FRR > 75% criteria.

The addition of CA facilitated most of the treatments to achieve the FRR > 75% criteria, except E_1K+1K + CA.

Examining the LRR < 50% and FRR > 75% criteria, E_1K+3K and E_1K+2K + CA were the adequate doses for golden shower trees.

In summary, the proper dose to be used for transplanting camphor trees will need to add CA to have a reasonable re-foliation. Pure ethephon treatment was not advisable for camphor trees. As for golden shower trees, higher ethephon doses seemed to be more appropriate. E_1K+2K + CA was the adequate dose to be used for transplanting both camphor and golden shower trees.

3.3. Defoliation Pattern

The defoliation of the ethephon treatments occurred within 1 week after the first spray and continued over the next 2 weeks until the LRR was reached; it then remained stagnant for a few weeks, allowing the buds to grow into leaves. The control treatments showed different defoliation patterns due to heat stress. The movement curves below plotted the pathway of defoliation and recovery.

3.3.1. Camphor

The control plants retained full foliage until 15 June and then dropped to a 77% retention rate due to summer stress (Figure 3). The retention rates of various ethephon treatments took a dive within 1 week of the first spray on 11 May, reaching their LRRs between 1 June and 15 June, before bouncing back (Figure 3A). The variation within the treatment group showed large deviations. All ethephon treatments did not recover to FRR > 75%.

Adding CA delayed the timing of the LRRs by 2 weeks on 15 Jun and raised the LRR level of most treatments, leading to flatter curves. The deviation within the treatment group also became smaller, especially during the refoliation process (Figure 3B).

3.3.2. Golden Shower

The control plants retained full foliage until 22 June and then dropped to 63% retention rates due to summer stress (Figure 4). It was a week later than the camphor trees. The retention rates of various ethephon treatments dived within 1 week of the first spray on 11 May, reaching the LRR between 1 June and 15 June, before bouncing back (Figure 4A). The variation within the treatment group showed large deviations. Most treatments exhibited a good recovery, except for E_1K+1K, which only recovered to 60% as the FRR.

The addition of CA delayed the LRR timing for about 2 weeks and raised the LRR level for most treatments, leading to flatter curves. All the treatments bounced back to have FRRs higher than the control, except for E_1K+1K, which had a 3% lower retention rate than the control (Figure 4B). The variation within the treatment group did not contract like the camphor trees.

4. Discussion

4.1. Defoliation Pattern

The defoliation pattern of ethephon treatments was consistent with the research of Dozier et al. [11], who experimented with ethephon and harvade to defoliate apple nursery stock, reporting a similar defoliation pattern. Sterrett et al. [27] presented the experimental result of treating field tree branches of Norway maple (Acer platanoides L.), pin oak (Quercus palustris), and black locust (Robinia pseudoacacia L.) with different doses of endothall/ethephon. The maximum defoliation occurred in the third to fourth week after ethephon treatment, after which re-foliation commenced. Our ethephon treatments showed similar defoliation patterns.

The control treatments exhibited a loss of foliage after 15 June for the camphor trees and 22 June for the golden shower trees when the summer stress intensified. The foliage retention rates of the control did not subsequently recover on 19 July. This defoliation could have been induced by the heat and high vapor pressure deficit (VPD), which produced the water stress. Defoliation is a strategy for plants to counter stress. Irrigation every 2 days might not be appropriate for small potted plants in midsummer.

4.2. LRRs

The LRRs were influenced by the dose of ethephon but were not significantly different. The mean foliage retention rates under different treatments showed that the higher ethephon doses resulted in higher defoliation and lower retention rates. This is consistent with the research of Dozier et al. [11], where higher doses of ethephon resulted in higher defoliation with dieback. Thus, the dose of ethephon solution was negatively correlated with the retention rate. However, the deviation within the replicates was very large, which diminished the statistical significance.

CA increased the LRRs of most of the treatments for both species. It showed that CA could effectively reduce defoliation and alleviate higher-dose ethylene injuries. It is similar to the finding of Iwahori and Oohata’s [26] study on the defoliation and fruit drop of citrus. It is especially useful to raise the LRRs for ethylene-sensitive species such as camphor trees or slow-growing species requiring a long re-foliate lead. Severe injury to plants or dieback will impede the recovery of foliage. CA alleviates the ethylene damage to protect the cells.

4.3. FRRs

FRRs reflect the recovery of foliage, and the two species in our experiment exhibited very different recovery patterns. The tree species seemed to have different elasticities toward ethylene to influence their re-foliation pattern. The camphor trees could not recover sufficient leaves after the severe injury and loss of foliage due to the ethephon treatments. The golden shower trees seemed to possess a different sensitivity to ethylene. The golden shower trees had LRRs below 50% under ethephon treatments but recovered to 75% or more as the FRR. E_1K+0.5K did not defoliate much the golden shower tree leaves, implying that this concentration of ethephon might not be sufficient to reach the threshold concentration to defoliate. Their resistance to ethylene injury could be higher to tolerate higher doses. Smith and Whiting [28] reported a similar conclusion to Tyree and Ewers [29], that different species had different water potential and loss in hydraulic conductance, which influenced their plasticity towards water stress, plant injury, and dieback for the recovery of foliage.

CA raised the FRRs of camphor trees to a minimum of 83% foliage retention rates for all ethephon treatments in our study. The effect of their remedy benefit was obvious. Their effect on golden shower trees was also reasonable except for the E_1K+1K + CA. The golden shower trees seemed to be able to recover their foliage to a reasonable level even without CA.

4.4. Two Sprays versus One Spray

We used one dose spray of higher than 3000 mg·L⁻¹ ethephon doses in our earlier studies in 2019 to defoliate the camphor and ficus saplings and incurred serious diebacks. By adopting the two sprays of ethephon, which was similar to the experiments of Larsen and Lowell [9] and Dozier et al. [11], this experiment obtained reasonable results with less dieback. However, our experiment’s higher-dose treatments without CA still showed dieback on camphor trees under two sprays. The severe plant injury prevented the recovery of foliage from meeting the criteria.

We sprayed the mature leaves and avoided spraying apical buds and young leaves because they were more sensitive to ethylene [12]. However, most of the dieback in our experiment occurred on the terminal shoots and the minor branches. The young leaves and buds dried out and darkened, with the dieback stretching downward to the branch collar. This phenomenon deteriorated with time. Dieback usually results from xylem embolism induced by water stress [30]. Zimmermann [31] hypothesized that minor branches had a lower water potential during water stress, and their leaves were most susceptible to embolism due to hydraulic constriction. This caused some minor branches to lose their competitiveness in receiving water, thus worsening defoliation [29]. The terminal branches with very little foliage are more susceptible to embolism than branches with more foliage, which are stronger sinks that receive higher energy investment [32]. Dieback causes a loss of sink strength, which prevents defoliated terminal shoots from drawing water into the terminal shoot and dries out the apical shoots. That is the reason why the apical shoots dried out in our experiment, although they were not sprayed with ethephon. [11]. It is suggested that dieback shoots be pruned off to restore the source-sink relationship.

5. Conclusions

Since different tree species possess different plasticity toward ethylene, it is advisable to test the proper defoliation ethephon dose on the saplings of the specific species before applying it to large trees. CA increases the LRRs as a safety net to maintain retention rates at a minimum level, thus reducing plant injury, especially for high doses of ethephon. This study identified the adequate ethephon and ethephon + CA foliar spray solutions as E_1K+2K + CA to manipulate the retention rate to control transpiration. For species that are sensitive to ethylene or possess a slow-growing habit, antitranspirants may be an option to reduce transpiration by controlling the stomata, but their effect is short-term, and frequent applications may be needed [33,34].