Short communication: morphophysiological response of kaffir lime (Citrus hystrix DC) subjected to defoliation and drought stress

ABSTRACT

Renowned for its leaf-spice attributes, kaffir lime has economic value that is worthy of intensifying amidst environmental threats, such as drought stress. This study aimed to analyze the morpho-physiological traits of kaffir lime in response to defoliation and drought stress. Kaffir lime was subjected to drought, defoliation, and a combination of both arranged in a randomised complete block design. Concerning morphology, defoliated lime produced a new flush that grows like a crown, while drought-treated plants displayed a bent pose with withered, edge-rolled, and drooping leaves. Higher leaf drop incidences were also noticed in drought-stressed plants. A combination of defoliation and drought stress produced severe flushing disruption by reducing 61% flush number, 77% flush fresh weight, and 74% flush dry weight, compared to undefoliated plants in normal irrigated conditions. Regardless of drought stress conditions, leaf production did not show significant differences among all defoliated plants. Defoliated plants maintained their photosynthetic rate and water use efficiency, leading to the absence of bent flush and rolling leaves as morphological markers of drought-treated plants. It is implied that defoliation can be a practical strategy for adapting kaffir lime in a drought environment.

KEYWORDS:

• Drought environment
• flushing
• morphological markers
• photosynthetic rate
• water use efficiency

Introduction

Global conditions are currently experiencing climate change phenomena that potentially endanger the agricultural sector (Sovacool et al. 2021; Bibi and Rahman 2023). Agricultural production should be performed in changing climatic variables, especially temperature, and rainfall intensity (Pope et al. 2022). Warmer global temperature has been noticed recently since there has been an increase of about 0.9°C from the nineteenth century (Eftekhari 2022). Moreover, the temperature in 2011–2022 was 1.1°C higher than that in the late nineteenth century, placing this finding as the warmest condition in the last 100,000 years (IPCC 2022). That change results in the incidence of extreme events such as floods, and drought (IPCC 2014; Bibi and Rahman 2023). Developing countries, especially in Asia and Sub-Saharan Africa, are the most vulnerable to being negatively impacted (IPCC 2014).

A developing country such as Indonesia is the place of origin of kaffir lime (Araujo et al. 2003), a unique citrus species with aromatic and bifoliate leaves (Budiarto et al. 2021a; Budiarto et al. 2023a) used for essential oils (Budiarto and Sholikin 2022) and food spice (Budiarto et al. 2019a) in numerous Asian cuisines (Setiyoningrum et al. 2018). Hence, the leaf became the main economic part (Efendi and Budiarto 2022) and is marketed in fresh, frozen, and dried forms (Wongpornchai 2012). The production and supply of kaffir lime leaf is also potentially hampered by climate change.

Climate change led to the incidence of prolonged hot and dry seasons, and ultimately resulted in drought stress problems (Ziogas et al. 2021). Drought stress is the most well-known limiting factor for citrus productivity and quality worldwide (Sato 2015). Previous studies have reported the effect of drought stress on citrus, i.e. alteration of gibberellin dan abscisic acid status (Mahouachi et al. 2005), antioxidants production for ROS scavenging (Hussain et al. 2018), photosynthetic rate reduction (Arjona-López et al. 2023), and eventually fruit yield drop (Zhang et al. 2018). Drought tolerance may differ among citrus species, as previously reported by Romero-Trigueros et al. (2021) that grapefruit (Citrus paradisi) has greater tolerance than mandarin (Citrus clementina). However, drought-related effects have not been widely studied in kaffir lime, compared to other Citrus species.

To overcome drought stress, an agronomic solution such as leaf removal/pruning is potentially applied. The leaf removal practice successfully improved the fig (Ficus carica) plant tolerance to drought stress (Jafari et al. 2017; Zare 2021). Severe pruning applied under drought conditions impedes leaf chlorosis incidence (Zare et al. 2021), and helps to maintain a greater stomatal conductance (Maurin and DesRochers 2013). Moreover, pruned grapes can balance their berry composition and development (Vitis vinifera) under drought conditions (Berhe 2022). The agronomic practice of defoliation has been adopted in kaffir lime to harvest its leaves yield (Budiarto et al. 2019b; Budiarto et al. 2023a) however, its effect to alleviate drought-negative problems has not been reported yet. Here, the present work tests the hypotheses that drought and defoliation would affect the morpho-physiological properties of kaffir lime and that defoliation can be alleviated the negative effect of drought stress. This study provides a valuable reference for the agronomic solution to address drought issues in kaffir lime plantations.

Materials and methods

Kaffir lime material

This study used kaffir lime seedlings that originated from grafting, combining kaffir lime var. Puri Agrihorti on the Citrus limonia rootstock. Selected seedlings possessed several characteristics, i.e. plant height (±100 cm), leaf number (±40 leaves), normal growth, bifoliate leaf characteristics, pest- and disease-free. Eight months after grafting, seedlings grew in the polybag with a diameter of 30 cm, containing growing media in the form of a well mixture of clay soil, goat manure, and rice husk. The seedling was raised under organic culture by monthly application of liquid fermented goat manure.

Seedlings were arranged in the Randomised Completely Block Design inside the greenhouse at the Bale Tatanen Padjadjaran (−6.9316851, 107.775863, 718 m above sea level), from July to August 2023. The use of greenhouse is aimed to eliminate the bias effect of rainfall so that the only water source is through irrigation. The experiment employed two factors, i.e. defoliation and drought stress. The first factor of defoliation was comprised of two levels, i.e. defoliated and undefoliated seedlings. Defoliation was performed manually by eliminating all leaves from the seedling, while undefoliated seedlings had 40 leaves attached to their canopy. The second factor of drought stress was also comprised of two levels, i.e. normal and drought stress. The stress was created by the regulation of watering once in two weeks, while normal seedlings were exposed by irrigation once in two days. All combination treatments were replicated five times. During the experimental period, seedlings were raised by manual weeding and pest inspection once a week.

Effect of defoliation and drought stress

Concerning the effect of defoliation and drought stress, all morpho-physiological observations and measurements were carried out at 4 weeks after treatment (WATs). The morphological appearance of treated kaffir lime was observed and documented in figures. Morphological responses in terms of leaf drop occurrence were measured only in the unpruned kaffir lime seedlings due to the absence of old leaves in all defoliated seedlings. The drop leaves were calculated by comparing the leaf number at pre- and post-drought stress. The old leaves were counted based on the number of retained leaves at post-drought stress. The weight of old leaves was measured on a fresh and dry basis. Flush production of kaffir lime seedlings was measured in the form of a flush number (flush per plant), flush dry weight (g), flush fresh weight (g), and flush length (cm). The proportion of leaves and branches in newly emerged flush was counted and displayed as a chart. Individual leaf length was measured manually with a ruler, while leaf weight and leaf area were calculated, following an earlier study by Budiarto et al. (2021b). Leaf production in response to defoliation and drought stress was also measured regarding leaf number (per plant), leaves dry weight (g), and leaves fresh weight (g). The leaves water content was the gap between fresh and dry weight and then was expressed in %.

Physiological traits of kaffir lime were measured by using the Li-6400XT instrument on a sunny day at 09.00 am. Intercellular CO₂ (μmol CO₂ mol⁻¹), leaf temperature (°C), transpiration rate (mmol H₂O m⁻² s⁻¹), photosynthetic rate (μmol CO₂ m⁻² s⁻¹) and stomatal conductance (mol H₂O m⁻² s⁻¹) were directly produced by that instrument, while intrinsic water use efficiency/WUE (μmol CO₂ mmol H₂O⁻¹) was counted by dividing the rate of photosynthesis by the rate of transpiration, following previous study (Roccuzzo et al. 2014; Budiarto et al. 2023b). All data were collected and subjected to analysis of variance and then continued to the Duncan multiple range test at α 0.05. All statistical data were processed in RStudio version 2023.06.2 + 561.

Results

Different morphological appearance due to defoliation and drought stress

The morphological appearance of kaffir lime plants differs due to defoliation and drought stress. Defoliation is prepared by removing 40 leaves on the initial days, leaving only a single main stem with some newly emerged flush at the apical parts, irrespective of drought treatment (Figure 1A, 1B). All defoliated seedlings display a similar flush appearance under drought-stressed and normal conditions (Figure 2A, 2B). Without defoliation (Figure 1C, 1D), drought-stressed kaffir lime plants exhibit a bent pose, while normal irrigated plants grow upright. In more detail, the bent flush of drought-stressed plants comprised withered, edge-rolled, and drooping leaves, while normal irrigated flush displays healthy and open-oriented leaves with towering growth patterns (Figure 2C, 2D).

Figure 1. Morphological appearance of kaffir lime at 4 WATs subjected to various treatments: defoliation and drought stress (A); only defoliation (B); only drought stress (C); control (D). Note: The yellow line in the figure is equal to 100 cm.

Figure 2. Morphological appearance of kaffir lime flush at 4 WATs subjected to various treatments: defoliation and drought stress (A); only defoliation (B); only drought stress (C); control (D). Note: The yellow line in the figure is equal to 30 cm.

Higher leaf drop incidences in drought-threatened plants

This research proved that drought-solely increased leaf drop incidence by about 50%, compared to normally irrigated plants, which only experienced 8% leaf drop (Figure 3A). 20 leaves still survive in the post-drought period, similar to the number of fallen leaves (Figure 3B). In normally irrigated plants, only 3 leaves fall off due to leaf senescence. Consequently, old leaves’ fresh- and dry-weight variables of drought-stressed seedlings were also reduced by about 52% and 48% compared to normally irrigated seedlings, respectively (Figure 3B).

Figure 3. The leaves drop occurrence leaving several old leaves on undefoliated kaffir lime seedlings during the post-drought stress period (4 WATs): the percentage of drop and old leaves (A) and the number and weight of old leaves (B). Note: Two compared seedlings experiencing leaf drop: drought stress (C); and control (D). Mean values above the bar inside the right chart followed by similar alphabet are not significantly different based on the Duncan Multiple Range Test at α = 0.05.

Flushing reduction by defoliation and drought condition

Flushing performance is an important component of leaf production, and it was significantly affected by defoliation and drought stress factors. Both factors significantly reduced flush number and flush weight; however, their effect was insignificant on flush length (Table 1). The highest significant reduction in flush number, flush fresh weight, and flush dry weight was found in the combination of defoliation and drought stress (treatment A) by about 61%, 77%, and 74% compared to control (treatment D), respectively. A significant reduction in flushing performance may be associated with a decline in vegetative growth (Table 1).

Table 1. Flushing characteristics of kaffir lime subjected to defoliation and drought condition.

Treatment	Flush number (flush per plant)	Flush length (cm)	The fresh weight of flush (g)	The dry weight of flush (g)
A	1.80 b	17.86 a	5.22 b	2.1 b
B	2.40 b	15.70 a	6.27 b	2.0 b
C	2.20 b	17.78 a	9.09 b	5.21 b
D	4.60 a	22.12 a	22.68 a	8.17 a

Note: Four compared treatments: defoliation and drought stress (A); only defoliation (B); only drought stress (C); control (D). Mean values followed by similar alphabet are not significantly different based on the Duncan Multiple Range Test at α = 0.05.

Effect of defoliation is more dominant than drought on flush proportion and leaf size

In the newly emerged flush, the leaf proportion was more dominant than the branches ones; however, there was a tendency for defoliation to reduce the leaf proportion (Figure 4). The higher leaf proportion in the newly emerged flush, as caused by the absence of defoliation, was supported by the individual leaf size data. Our finding in Table 2 highlighted the lower individual leaf size of newly emerged flush in defoliated plants than undefoliated ones. The reduction of individual leaf area, individual leaf length, and individual leaf fresh weight were caused by defoliation by about 54%, 35%, and 56%, respectively (Figure 4) (Table 2).

Figure 4. The proportion of leaf and branch in newly emerged flush at 4 WATS. Note: Four compared treatments: defoliation and drought stress (A); only defoliation (B); only drought stress (C); control (D).

Table 2. Individual leaf size of kaffir lime subjected to defoliation and drought condition.

Treatment	Individual leaf length (cm)	Individual leaf area (cm^2)	Individual leaf fresh weight (g)
A	8.84 b	19.65 b	0.58 b
B	8.48 b	18.24 b	0.54 b
C	11.76 a	32.99 a	1.00 a
D	13.02 a	39.80 a	1.22 a

Note: Four compared treatments: defoliation and drought stress (A); only defoliation (B); only drought stress (C); control (D). Mean values followed by similar alphabet are not significantly different based on the Duncan Multiple Range Test at α = 0.05.

Drought stress drop leaf production of undefoliated plants, not in defoliated ones

Leaf production performances, as the most important variable in kaffir lime agribusiness, displayed a significant decline as caused by defoliation and drought condition. Leaf number, leaf weight, and water content were not significantly different among all defoliated plants, with or without drought stress exposure (Table 3). In contrast, undefoliated kaffir lime plants experienced a significant drop in leaf production if drought stress was applied. That reduction was reported to be 45% in leaf number, 57% in leaf fresh weight, 44% in leaf dry weight, and 15% in leaf water content (Table 4).

Table 3. Leaf production of kaffir lime subjected to defoliation and drought condition.

Treatment	Leaf number (leaf per plant)	The fresh weight of leaves (g)	The dry weight of leaves (g)	The water content of leaves (%)
A	15.00 c	3.63 c	1.34 c	63 a
B	11.80 c	3.79 c	1.19 c	69 a
C	38.40 b	18.03 b	8.07 b	55 b
D	69.40 a	41.46 a	14.48 a	65 a

Note: Four compared treatments: defoliation and drought stress (A); only defoliation (B); only drought stress (C); control (D). Mean values followed by similar alphabet are not significantly different based on the Duncan Multiple Range Test at α = 0.05.

Table 4. Physiological properties of kaffir lime subjected to defoliation and drought condition.

Treatment	Pn	Sc	ICO	Tr	WUE	Lt
A	23.5 a	0.15 b	112.43 b	2.59 a	9.07 a	21.13 b
B	25.54 a	0.17 a	114.69 b	2.87 a	8.9 a	21.08 b
C	15.91 b	0.11 b	143.99 a	2.76 a	5.76 b	23.51 a
D	25.54 a	0.17 a	112.19 b	2.95 a	8.66 a	21.26 b

Note: Four compared treatments: defoliation and drought stress (A); only defoliation (B); only drought stress (C); control (D). Mean values followed by similar alphabet are not significantly different based on the Duncan Multiple Range Test at α = 0.05. Pn – Photosynthetic rate (μmol CO₂ m⁻² s⁻¹), Sc – Stomatal conductance (mol H₂O m⁻² s⁻¹), ICO – Intercellular CO₂ (μmol CO₂ mol⁻¹), Tr – Transpiration rate (mmol H₂O m⁻² s⁻¹), WUE – Water use efficiency (μmol CO₂ mmol H₂O⁻¹), Lt – Leaf temperature (°C)

Drought stress disrupt physiological properties of undefoliated plants, not in defoliated ones

Table 4 depicted a significant reduction effect of drought condition (treatment C) on intrinsic WUE, stomatal conductance, and photosynthetic rate compared to control plants (treatment D) for about 34%, 35%, and 38%, respectively. The photosynthetic rate is not significantly reduced by defoliation, in contrast to the stomatal conductance variable that displays a significant reduction in defoliated and undefoliated plants was 12% and 35% compared to the control, respectively. Lower stomatal conductance is usually accompanied by lower intercellular CO₂. However, opposite results were found in the present findings. Drought-subjected plants experienced a significantly higher intercellular CO₂ and leaf temperature than normal irrigated plants, for about 28% and 11%. Additionally, the present finding also revealed a significant reduction of WUE in the drought-stressed, undefoliated plants by about 34% compared to the control. In contrast, the defoliated plant subjected to drought stress seemed to have a similar WUE to control.

Discussion

Drought and defoliation affect the morpho-physiology of kaffir lime

Concerning morphological properties, drought-stressed kaffir limes displayed a bent flush with edge-rolled, and drooping leaves (Figure 1), similar to Hussain et al. (2023) who reported leaf rolling and wilting in stressed sour oranges (Citrus aurantium). Within the flush, the upper leaves depicted higher leaf rolling intensities, implying a higher drought sensitivity than the middle and lower leaves (Figure 2) in agreement with Zhou et al. (2021). Moreover, a high leaf drop incidence in drought-stressed plants (Figure 3A) implied the alteration of hormonal status, i.e. an increase in abscisic acid content (Mahouachi et al. 2005) that triggers the abscission of the leaf stalk, (Rodriguez et al. 2019). Ultimately, drought stress declined leaf growth (Abobatta 2021; Shafqat et al. 2021), which would eventually cause a decrease in canopy size (Costa et al. 2021). Apart from drought, defoliation also formed a smaller leaf size, implying the reduction of plant source capacity due to photosynthate limitations after leaf removal; a similar case with Budiarto et al. (2018) in mandarin and Yuan et al. (2005) in oranges.

Concerning physiological responses, drought stress disrupted the photosynthesis rate of undefoliated limes, in agreement with Pena-Rojas et al. (2004). Photosynthesis is suggested as a physiological marker of plant resistance to abiotic stress (Rahmat et al. 2023). The reduction of the photosynthesis rate could be caused by (i) a decline in stomatal conductance (Lawlor and Cornic 2002; Pena-Rojas et al. 2004), and (ii) leaf temperature-limiting factor, which was found only in undefoliated drought-stressed plants, as previously also reported by Lourkisti et al. (2022). Taken together, this study proposes the morpho-physiological alteration of kaffir lime under leaf removal and drought stress conditions.

Defoliation alleviates the negative effect of drought stress on kaffir lime

In agreement with our hypothesis, defoliated kaffir limes were suggested to be more tolerant to drought (Table 4) rather than undefoliated ones, as indicated by the absence of leaf rolling and wilting incidences. It was likely that defoliation helped to postpone the occurrence of drought symptoms (Namirembe et al. 2009). Pruned plants were supposed to have reduced water demand (Jackson et al. 2000; Lopez et al. 2012; Chen et al. 2016), as the consequence of the removal of certain canopy parts. The lowering water demand increases the WUE, which is proven in this study that the WUE of drought-exposed defoliated plants was as high as a control treatment with no drought conditions (Table 4). This work also showed no significant effect of defoliation on transpiration rate (p > 0.05) at the single leaf level; however, the declining leaf number in post-defoliated plants might suppress canopy area, and ultimately plant transpiration rate at the canopy level, as previously confirmed by numerous studies (Lopez et al. 2008; Jin et al. 2018; Devin et al. 2023). Pruned plant was reported to have a narrow xylem vessel, for lowering their susceptibility to xylem cavitation (Katul et al. 2003) and ultimately impede the rate of transpiration. In addition, pruned plants had greater stomatal control systems for optimising their net assimilation under stress conditions (Jin et al. 2018). This further supports the idea of defoliation as the feasible strategy to manage kaffir lime leaf production under drought-stress conditions.

Conclusion

The present study highlighted different morphological appearances of kaffir lime due to defoliation and drought stress. Defoliation removed a large portion of the existing canopy, leaving a new flush that grows like a crown. Normal irrigated plants grew upright, while drought-treated plants showed a bent pose comprised of withered, edge-rolled, and drooping leaves. Both defoliation and drought reduced flushing performance. However, the effect of defoliation is more dominant than drought on flush proportion and leaf size. Drought stress drop leaf production only in undefoliated plants, not in defoliated ones. Defoliated plants were able to maintain their photosynthetic rate and water use efficiency, leading to the absence of bent flush and rolling leaves as morphological markers of drought-treated plants. Pruning, especially defoliation, is strongly recommended, apart from harvesting activities, as a precautionary practice to plant kaffir lime in a drought environment.

Author contributions

RB, SM, and MAN were involved in the conception and design, MAR, and MHISJ in data analysis and interpretation; RB, and SA in the drafting of the paper, RB, DNS, AK, and SLS in revising it critically for intellectual content; RB in the final approval of the version to be published. All authors agree to be accountable for all aspects of the work.

Acknowledgments

The authors thank the team of Bale Tatanen Padjadjaran for the technical support during the fieldwork.

Disclosure statement

No potential conflict of interest was reported by the author(s).

Data availability statement

The data in the present finding are available from the corresponding author [RB] upon reasonable request.

Additional information

Funding

This work was supported by the Universitas Padjadjaran under the grant of Associate Professor Acceleration Research fiscal year 2023 [grant number: 1549/UN6.3.1/PT.00/2023]; fiscal year 2024 [grant number: 1579/UN6.3.1/PT.00/2024]; Universitas Padjadjaran for the article processing charge.