Impact of water regimes on the physiological responses of dwarfing rootstocks in a young orchard of Tahiti acid lime

ABSTRACT

Citrus productivity is strongly influenced by water availability and rootstock selection. This study evaluated the physiological and productive responses of two commercial scion × rootstock combinations: Tahiti acid lime BRS EECB IAC Ponta Firme grafted onto the dwarfing trifoliate rootstock IAC 718 Flying Dragon (PF_FD), and IAC 10 grafted onto the semi-dwarfing citrandarin IAC 3152 Itajobi (10_152). Field experiments were conducted over three years (2022–2024) under three irrigation regimes—variable (VIS), fixed (FIS), and non-irrigated (NIS)—using a 3×2 factorial design with randomized blocks and drip irrigation. Physiological parameters (root length, leaf water potential, gas exchange, proline, and lipoperoxide content) and productive traits (fruit quality and cumulative yield) were assessed. The 10_152 combination showed superior root development, gas exchange performance, antioxidant regulation, and fruit yield, particularly under FIS and NIS, indicating enhanced drought tolerance. In contrast, PF_FD performed well only under VIS and was more sensitive to water deficit. Principal component analysis revealed that higher productivity and resilience were associated with greater root length, increased proline accumulation, higher CO₂ assimilation, and reduced oxidative stress. Furthermore, FIS irrigation was as effective as VIS in sustaining plant performance, while optimizing water use, suggesting a sustainable management strategy. Although Tahiti acid lime IAC 10 demonstrates vigorous early orchard growth, its susceptibility to wood pocket restricts its commercial use. Overall, citrandarin IAC 3152 Itajobi proved to be the most promising rootstock, suitable for both irrigated and non-irrigated orchards, whereas PF_FD is recommended only under optimal water conditions.

Key words
drought; gas exchange; irrigation; proline; tolerance

INTRODUCTION

Brazil is the second-largest producer of citrus fruits and the world’s leading producer and exporter of orange juice. In the 2023/24 harvest, the country produced 898.6 thousand tons, accounting for 50% of the global orange juice supply (IBGE 2024). Limes and lemons also play important roles in this export ranking, especially the Tahiti acid lime (Citrus latifolia Yu. Tanaka), with over 1.6 million tons produced in the 2022 harvest (IBGE 2023).

However, the citrus-growing area in the country has declined due to the biotic and abiotic challenges affecting the crop. These challenges require increased investment in technology, including the use of agricultural inputs and advanced equipment. As a result, many producers have opted to replace their orchards with less resource-intensive annual crops (Bassanezi et al. 2020). For this reason, years of research have enabled citrus plants to withstand adverse conditions throughout their long lifespan in the orchard. As perennial plants, they endure various challenges for at least 15 years, requiring continuous adaptation and resilience (Abobatta 2023).

It is essential not only to select individuals that are tolerant to adverse conditions, but also to choose those that produce smaller plant sizes, making orchard densification feasible (Bowman and Joubert 2020). This is because certain rootstocks can reduce the size of the scion variety by limiting vigor and promoting compact growth, thereby facilitating management, harvesting, and, importantly, irrigation (Donadio et al. 2019). The Flying Dragon trifoliate orange rootstock (Poncirus trifoliata var. monstrosa) is a variety recognized for its strong dwarfing effect on citrus scions, significantly reducing vegetative vigor and, consequently, the final tree size, which makes it extremely useful in high-density orchards (Pereira Costa et al. 2021). Flying Dragon also stands out for its resistance to soil-borne diseases and several common pathogens, such as the citrus tristeza virus, the fungus Phytophthora spp., and certain nematodes (Silva et al. 2025). However, while these rootstocks optimize water use, their smaller root systems make them more susceptible to water deficits, presenting a trade-off between efficiency and resilience (Borim de Souza et al. 2025).

That is why several technologies are used to enhance citrus productivity through water management in irrigation with uniformity and efficiency (Levidow et al. 2014). Even if drip irrigation ensures good uniformity of water distribution in the field, it is still necessary to have control over indicators of the water status required by the soil and plant to identify the appropriate irrigation, in addition to assuring the correct amount of frequency, to raise the efficiency of water use (Puig-Sirera et al. 2021).

However, the conscious use of water resources in agriculture is essential. Therefore, regardless of whether water supplementation is applied, it is advisable to select a rootstock variety capable of making more efficient use of this resource and translating it into higher productivity (Levidow et al. 2014). An example is the new IAC 3152 Itajobi citrandarin, a hybrid of Citrus sunki × Poncirus trifoliata cv. Rubidoux, which has shown advantages under drought conditions due to its ability to develop deeper root systems, that enhance water infiltration and absorption and consequently maintain higher leaf water potential. This contributes to greater photosynthetic efficiency, improved gas exchange, and the maintenance of active physiological responses even under water restriction (Borim de Souza et al. 2025). It is expected that, with adequate water supplementation, this rootstock will achieve even superior performance.

Therefore, determining the best management of water supplementation, associated with superior scion × roostock combinations that benefit the ability of a tree to tolerate adverse environmental factors, as well as maintaining optimum productivity, by responding promptly to the input provided, can guarantee economy of resources, security, and profit to the citrus market. Accordingly, the combination of the semi-dwarfing rootstock IAC 3152 Itajobi citrandarin with the Tahiti acid lime scion exhibits greater tolerance to water deficit, reflected in improved physiological performance (leaf water potential, gas exchange, proline accumulation, and lower lipid peroxidation) and higher productivity under fixed and non-irrigated regimes, compared with the dwarfing combination IAC 718 Flying Dragon trifoliate orange.

MATERIAL AND METHODS

Area and experimental design

The present experiment was installed in March 2020 at the Center of Agricultural Sciences of the Universidade Federal de São Carlos, Araras, São Paulo state, Brazil, located at geographic coordinates 22°18’49”S, 47°22’57”W, with an altitude of 695 m. The area comprises Dystrophic Red Latosol (Yoshida and Stolf 2024), characterized by a moderate A horizon and argillaceous texture. Nutritional analyses of the soil and leaves were carried out (Suppl. Mat.). The region’s weather is CWA, characterized by warm summers and dry winters (Köppen 1936). Throughout the experiment, the meteorological variables were monitored by a weather station located next to the experimental area (Suppl. Mat.).

Twelve-month-old nursery trees were planted in nine rows, using a spacing of 4 (inter-row) × 1.8 m (row, inter-plants), divided in a factorial design layout 3 × 2, with three different irrigation managements:

• Variable irrigation schedule (VIS);

• Fixed irrigation schedule (FIS) (2 mm.4 days^-1);

• Non-irrigation schedule (NIS);

The standard commercial combinations scion × rootstock were two: Tahiti acid lime BRS EECB IAC Ponta Firme grafted onto dwarfing variety, IAC 718 Flying Dragon trifoliate orange, (represented as PF_FD); and IAC 10 under semi dwarfing rootstock, IAC 3152 Itajobi citrandarin (represented as 10_152).

Irrigation in VIS treatment was done by indicating the water balance according to evapotranspiration and crop parameters, aiming to replace 100% of the evapotranspiration. Irrigation in FIS treatment was also done, aiming to replace the evapotranspiration blade totally every four days. For this, the system has two drippers per plant with an application intensity of 0.85 mm.h^-1, with dripper spacing of 0.5 m, pressure of 1.5 kgf.cm^-2, and flow rate of 2 L.h^-1. NIS treatment receives only rainfall water supply. The trial was arranged in a randomized block design with three replications, each plot containing four plants. Nutritional supplementation was carried out through weekly applications of 750 mL per 500 L of macro and micronutrients and phosphorus, interspersed, while NIS had the conventional fertilization, as indicated for the Tahiti acid lime at the correct stage of development.

Evaluations

Physicochemical quality of the Tahiti acid lime fruits

Ten fruits per plot were sampled in the summer (February 2024) for quality physicochemical measurements. The total mass of the fruit was obtained using an electronic scale with a maximum capacity of 15 g. The juice yield was determined after juicing fruits and calculated using the juice weight/fruit weight ratio and expressed as a percentage; the ratio was calculated using the soluble solids (SS)/titratable acidity (TA) ratio, which indicates the maturate stage in number; the ascorbic acid (vitamin C) contents were determined through pipetting, using 2 mL of juice with 50 mL of 1% oxalic acid solution. The solution was titrated using the DCPIP solution (2,6-dichlorophenolindophenol) until the pink color persisted for 15 seconds; the result was expressed in mm 100.g^-1 of juice, using Tillman’s solution.

Root development

Acrylic minirhizotron tubes were installed in June 2021, 40 cm from the plant trunk. Root images were collected in February 2024 (summer) using a CI-600 root scanner (Model CI-600-TA66). Images were taken from inside the access tube at depths ranging from 0 to 20 cm. Root length (mm) was measured from the images using RootSnap^® software (CID BioScience Inc., Camas, WA, United States of America).

Leaf water potential

During the winter (August 2021, 2022, and 2023), undamaged Tahiti acid lime leaves were collected from the different rootstocks/treatments in the middle third of the plant, in predawn. Three leaves were used per treatment. A Scholander-type pressure chamber from PMS Instrument (model 1000, Corvallis, United States of America) was used to assess the leaves’ water potential, and the values were expressed in MPa.

Gas exchange evaluation

The stomatal conductance to water vapor (gs), transpiration (E), photosynthesis (A), and intercellular CO₂ concentration (Ci) were measured in vivo for all plants. The analyses were performed in the youngest, fully expanded leaf using a portable infrared gas analyzer (LCpro, ADC, Hoddesdon, United Kingdom). Evaluations were performed in spring (October 2023), between 8 and 10 a.m., under ambient temperature, partial carbon dioxide pressure, and air-water vapor pressure. Photosynthetic active radiation (PAR) of 1,000 μmol.m^-2.s^-1 was supplied by a light unit mounted on the top of the leaf chamber.

Water deficit biomarkers

Lipoperoxides

Lipoperoxides were determined during summer (January 2024) and winter (August 2024). Undamaged Tahiti acid lime leaves were collected from the different rootstocks/treatments in the middle third of the plant, free of pests and disease symptoms, free of flowers, or free of fruits. Then, 0.3 g leaf tissue sample was homogenized with 0.5 mL of 0.1% trichloroacetic acid (TCA), which was then centrifuged for 10 min (15,000 × g, 4 °C). Subsequently, 1 mL of the supernatant was collected and mixed with 2 mL of 0.25% 2-thiobarbituric acid (TBA) diluted in 10% TCA in a test tube. The mixture was then incubated in a water bath at 95°C for 25 min, and the absorbance was finally measured at 532 and 600 nm in a spectrophotometer (Multiskan, Thermo Fisher Scientific, MA, United States of America). The data were calculated and expressed in mmol of lipoperoxides per gram of fresh tissue.

Proline

At the same time as lipoperoxide analysis, the proline content in Tahiti lime leaves was measured by homogenizing 0.5 g of fresh leaf tissue in 10 mL of 3% sulfosalicylic acid, followed by shaking the tubes every 15 min for 1 h. The samples were then centrifuged for 10 min (10,000 × g), and a 2 mL aliquot of the supernatant was collected for absorbance reading at 520 nm in a spectrophotometer (Multiskan, Thermo Fisher Scientific, MA, USA). The data were calculated and expressed in μmol of proline per gram of fresh tissue.

Fruit yield

From 2022 to 2024, monthly evaluations were conducted once ripe fruit appeared in the orchard. The harvest and weight of all fruit and the cumulative productivity of each year, expressed as yield (t.ha^-1), were determined. All evaluations were performed in two central trees per plot with four replications.

Statistical analysis

Data were subjected to analysis of variance (ANOVA), and any contrast between the means was evaluated using Tukey’s test at a 5% probability level, with the statistical package R (Foundation for Statistical Computing, Vienna, Austria). Normality tests were previously performed. A principal component analysis (PCA) was conducted using the FactoMineR package in RStudio (Foundation for Statistical Computing, Vienna, Austria), and results were visualized from the “factoextra” package.

The variables included in the PCA were root length, proline content, H₂O₂ concentration, net photosynthetic rate (A), leaf water potential (wp), and fruit yield. All variables were standardized (z-scores) before analysis to eliminate differences in scale. Additionally, the percentage of variance explained by the first two principal components was reported, and the direction and magnitude of the vectors (loadings) were used to interpret the relationships among variables.

RESULTS AND DISCUSSION

Physicochemical quality of the Tahiti acid lime fruits

The measurements obtained from the physical parameters of the Tahiti acid lime fruit showed significant differences between the combination (C) and water management (WM) factors (Table 1). The weight and juice yield were statistically higher in the fruit obtained from the 10_152 combination compared to PF_FD. The difference between the irrigation and dryland regimes also differed; the mass of the 10_152 fruit was higher when imposed on VIS, while the juice yield was higher in both managements with variable and fixed water availability, being significantly lower when imposed on NIS. A previous study on Citrange Carrizo under deficit irrigation (50% ETc) reported reduced productivity, primarily due to decreased fruit size and juice content resulting from the irrigation reduction (Primo-Capella et al. 2024).

Table 1
Physical parameters of Tahiti acid lime’s fruit (Araras, São Paulo state, Brazil).

Treatments		Mass (g)	Juice yield (%)
Combination (C)	10_152	67,08 A1	53,07 A
	PF_FD	61,78 B	50,26 B
Water management (WM)	VIS	72,13 a	53,28 a
	FIS	60,92 b	52,36 a
	NIS	60,25 b	49,35 b
F test	C	***	*
	WM	**	*
	C × WM	ns	ns
	CV	3,89	3,98

• ]
  VIS: variable irrigation schedule; FIS: fixed irrigation schedule; NIS: non-irrigation schedule; 10_152: IAC 10 with citrandarin IAC 3152 Itajobi; PF_FD: BRS EECB IAC Ponta Firme with trifoliata IAC 718 Flying Dragon;
• ^ns
  not significant;
• ^*
  significant at 5%,
• ^**
  significant at 1% according to the F test;
• ¹
  averages followed by the same letter, uppercase in the column for water management and lowercase in the column for combination scion/rootstock, do not differ (Tukey, 5%); CV: coefficient of variation.

The chemical parameters of Tahiti acid lime fruits showed a two-way factorial interaction (Fig. 1). The ratio of SS to acidity indicated that the 10_152 combination under NIS was statistically superior compared to other water management strategies and had a higher average than PF_FD under rainfed conditions. Meanwhile, the PF_FD combination exhibited its highest ratio under FIS, which was statistically greater than the other combination within the same treatment (Fig. 1a). Ascorbic acid levels were lower under the VIS management for the 10_152 combination, while the PF_FD combination also showed the lowest concentration under VIS compared to FIS. Under FIS and NIS water management, 10_152 had a higher vitamin C concentration than PF_FD (Fig. 1b). In a previous study on the 10_152 and PF_FD combinations, it was concluded that under more challenging conditions, plants with greater adaptability and resilience tend to accumulate more ascorbic acid. This accumulation is due to its antioxidant activity, which protects the plant from intensified free radicals caused by adverse environmental factors (Borim de Souza et al. 2025).

Figure 1
The physical quality of Tahiti acid lime’s fruits is subjected to irrigation management. (a) Ratio and (b) ascorbic acid (mm·g^-1 of juice), summer 2024.

Root development

Root length did not exhibit a significant two-way factorial interaction. However, within each factor, significant differences were observed among treatments. The combination 10_152 was statistically superior to PF_FD. Regarding the water management treatments, each showed distinct outcomes: NIS presented the greatest root length, followed by FIS, while VIS exhibited the least root development (Table 2).

Table 2
Root length of the two combinations of Tahiti acid lime and dwarfing rootstocks (Araras, São Paulo State, Brazil).

Treatments		Root length (mm)
Combination (C)	10_152	905,3 A1
	PF_FD	746,9 B
Water Management (WM)	VIS	505,9 c
	FIS	786,5 b
	NIS	1005,9 a
F test	C	***
	WM	*
	C × WM	ns
	CV	14,7

• VIS: variable irrigation schedule; FIS: fixed irrigation schedule; NIS: non-irrigation schedule; 10_152: IAC 10 with citrandarin IAC 3152 Itajobi; PF_FD: BRS EECB IAC Ponta Firme with trifoliata IAC 718 Flying Dragon;
• ^ns
  not significant;
• ^*
  significant at 5%; **significant at 1% according to the F test;
• ¹
  averages followed by the same letter, uppercase in the column for water management and lowercase in the column for combination scion/rootstock, do not differ (Tukey, 5%); CV: coefficient of variance.

In arboreal and perennial plants such as citrus, the root architecture developed is impacted by the rootstock used, and the management conditions imposed (Simpson et al. 2020). It is widely believed that dwarfing rootstocks have a poorly developed root system since the characteristic low vigor has little influence on large root extensions (Donadio et al. 2019). This may explain the lower means in combination with IAC 718 Flying Dragon, since it is an extremely dwarfing rootstock, while IAC 3152 Itajobi is a semi-dwarfing rootstock. Lastly, a previous comparison conducted by our research group demonstrated superior root development in combinations grafted onto 152, compared to FD, under both conventional soil management and dryland conditions (Borim de Souza et al. 2025).

However, as the roots are responsible for absorbing water and nutrients and are the first preceptors of water availability, it is thought that the performance of citrus under drought is linked to the allocation of carbon distributed in the roots, which varies between rootstocks. Those that are more tolerant to water deficit tend to accumulate a higher concentration of photoassimilates, allowing them to better search for water in the deeper layers of the soil (Silva et al. 2021). Greater root length allows for more efficient water uptake from deeper soil layers. For instance, in a previous study on apple trees (Malus domestica), a positive regulatory gene for root growth under drought conditions was identified, promoting root elongation and hydraulic conductivity (Zhang et al. 2024). This corroborates the explanation for better development, in both combinations, under FIS (partial hydric supplementation) and NIS management (no hydric supplementation). This is also justified by the fact that waterlogged soils tend to reduce the presence of oxygen, hindering better root development (Simpson et al. 2020).

Leaf water potential

The annual water potential evaluations conducted in August 2021, 2022, and 2023 revealed a two-way interaction (Fig. 2). The averages of the data collected in 2021 reached the most negative values, due to the juvenility of the plant and its lower capacity to tolerate climatic conditions (Syvertsen 1982). During the same year, there were no significant differences between the scion × rootstock combinations in the VIS and FIS managements, but in the management without water availability, NIS, 10_152 showed better water potential than PF_FD (Fig. 2a).

Figure 2
Water potential (MPa) of Tahiti acid lime’s leaves subjected to irrigation management. (a) 2022, (b) 2023, and (c) 2024.

In 2022, the effect of water management on the combinations shows that PF_FD had the least negative values only in VIS, while 10_152 maintained positive values in both VIS and FIS, with a significant reduction only in NIS. Statistically, the 10_152 combination maintained a better water potential than PF_FD in both FIS and NIS managements (Fig. 2b). Finally, the data for 2023 showed the least negative values, as both combinations developed and matured. However, PF_FD was once again the combination with the lowest capacity to retain water in its tissues when subjected to NIS management, in contrast to 10_152, which remained statistically unchanged in the three water management regimes (Fig. 2c).

Gas exchange evaluation

The measurements obtained in the winter of 2022 showed no significant differences, and in the following year, no significant interaction effect was observed among the values. However, within the water management factor, CO₂ assimilation (A) was lower under the NIS management compared to the others. Finally, in the winter of 2024, the values obtained across the four measurements showed no significant interaction between the factors: water management and scion × rootstock combinations. Nonetheless, significant differences were detected within each factor when evaluated independently (Table 3). The stomatal conductance (g_s) and transpiration (E) assessments within the scion-rootstock combination factor showed equivalent statistical differences, with the IAC 3152 Itajobi rootstock grafted with IAC 10 exhibiting the highest values in both measurements. Conversely, within the water management factor, the rainfed system (NIS) recorded the lowest values in both measurements.

Table 3
Gas exchange measured in vivo of Tahiti acid lime’s leaves, where gs (mol·m^-2s^-1), E (mmol·m^-2s^-1), and A (µmol.m^-2s^-1). (Araras, São Paulo state, Brazil).

Year	Measurements	Combination (C)		Water management (WM)			F test
		10_152	PF_FD	VIS	FIS	NIS	C	WM	C × WM	CV (%)
2022	gs	0,62	0,61	0,61	0,69	0,66	ns	ns	ns	3,33
	E	1,03	1,00	1,10	0,92	0,90	ns	ns	ns	4,65
	A	8,01	8,11	8,25	8,00	7,96	ns	ns	ns	4,02
2023	gs	0,72	0,71	0,75	0,7	0,69	ns	ns	ns	5,26
	E	0,59	0,63	0,66	0,61	0,60	ns	ns	ns	4,88
	A	6,33	6,29	6,45 a	6,5 a	6,06 b	ns	*	ns	3,33
2024	gs	0,75 A¹	0,70 B	0,93 a	0,91 a	0,34 b	*	**	ns	4,93
	E	0,47 A	0,35 B	0,44 a	0,48 a	0,32 b	*	*	ns	6,88
	A	7,71 A	6,98 B	7,56 a	8,01 a	6,68 b	**	**	ns	6,80

• VIS: variable irrigation schedule; FIS: fixed irrigation schedule; NIS: non-irrigation schedule; 10_152: IAC 10 with citrandarin IAC 3152 Itajobi; PF_FD: BRS EECB IAC Ponta Firme with trifoliata IAC 718 Flying Dragon;
• ^ns
  ns: not significant;
• ^*
  significant at 5%;
• ^**
  significant at 1% according to the F test;
• 1averages followed by the same letter, uppercase in the column for water management and lowercase in the column for combination scion/rootstock, do not differ (Tukey, 5%); CV: coefficient of variance.

Stomatal conductance (g_s) and leaf transpiration are closely related physiological processes that are strongly influenced by drought conditions. Under water stress, plants reduce stomatal aperture to minimize water loss through transpiration, a response mediated by hormones such as abscisic acid (ABA) (Borim de Souza et al. 2023).

Concurrently, stomatal closure to prevent water loss leads to a reduction in CO₂ uptake, which is essential for photosynthetic activity. Consequently, the recorded CO₂ assimilation values exhibited lower means in the PF_FD combination and the NIS management system, indicating lower drought tolerance and reduced water availability, respectively. For instance, a study conducted on Newhall navel orange (Citrus sinensis Osbeck cv. Newhall) and fertile orange (Citrus reticulata cv. Fertile Orange) observed that after 60 days without irrigation, Newhall navel orange exhibited the lowest values of stomatal conductance, transpiration, and CO₂ assimilation compared to fertile orange, which under the same conditions maintained gas exchange rates closer to those observed under normal environmental conditions (Jia et al. 2024).

In a previous study conducted with Flying Dragon and citrandarin IAC 3152 Itajobi, grown under rainfed conditions and conventional soil management (with tillage and no soil cover), gas exchange was reduced when compared to favorable environmental conditions. However, the citrandarin showed higher values of stomatal conductance, transpiration, and CO₂ assimilation than Flying Dragon, thus demonstrating greater tolerance to drier environments (Borim de Souza et al. 2025).

Water deficit biomarkers

No significant factorial interaction was observed for either lipoperoxide or proline levels, but statistically significant differences were found within each factor. Among the rootstock-scion combinations, the highest proline content was recorded in 10_152, whereas PF_FD exhibited the highest lipoperoxide accumulation in leaf tissue (Table 4). Similarly, irrigation regimes significantly influenced both parameters, with the NIS regime showing the highest mean values for proline and lipoperoxides, followed by FIS (Table 4).

Table 4
Water deficit biomarkers of Tahiti acid lime’s leaves (Araras, São Paulo state, Brazil).

Treatments		Proline (µmol·g-1)	Lipoperoxides (mmol·g-1)
Combination (C)	10_152	4,65 A1	0,0025 B
	PF_FD	3,59 B	0,0034 A
Water management (WM)	VIS	3,62 b	0,0028 b
	FIS	3,85 ab	0,0031 ab
	NIS	4,22 a	0,0036 a
F test	C	*	*
	WM	*	*
	C × WM	ns	ns
	CV	3,68%	12,32%

• VIS: variable irrigation schedule; FIS: fixed irrigation schedule; NIS: non-irrigation schedule; 10_152: IAC 10 with citrandarin IAC 3152 Itajobi; PF_FD: BRS EECB IAC Ponta Firme with trifoliata IAC 718 Flying Dragon;
• ^ns
  not significant;
• ^*
  significant at 5%;
• **significant at 1% according to the F test;
• ¹
  averages followed by the same letter, uppercase in the column for water management and lowercase in the column for combination scion/rootstock, do not differ (Tukey, 5%); CV: coefficient of variance.

Environmental stress-induced imbalances in plant metabolism lead to the overproduction of reactive oxygen species (ROS), such as lipoperoxides (H₂O₂). Excessive ROS generation impairs physiological functions and cellular homeostasis, ultimately causing oxidative damage and potential cell death (Shafqat et al. 2021). To mitigate oxidative stress, plants activate an integrated antioxidant defense system. One key component is proline, an osmoprotectant amino acid known for its role in ROS scavenging, redox regulation, and membrane stabilization, which collectively help maintain cellular integrity (Hayat et al. 2012).

In drought-tolerant genotypes, lower ROS accumulation and elevated proline levels are typically observed as stress severity increases. For instance, Citrus aurantium L. (sour orange) demonstrated reduced lipoperoxide content and increased proline accumulation under severe drought conditions when compared to 10 genetically diverse citrus rootstocks exposed to varying water deficits (Shafqat et al. 2021). A comparable response was previously reported for citrandarin IAC 3152 Itajobi, which exhibited enhanced antioxidant regulation through lower lipoperoxide concentrations and elevated proline levels, even under water-limited conditions (Borim de Souza et al. 2025).

Fruit yield

The yield data exhibited a significant two-way factorial interaction across all annual measurements (Fig. 3). In the 2022 season, the 10_152 combination showed the highest yields under the FIS treatment, followed by NIS and then VIS. Similarly, for the PF_FD combination, FIS also recorded the highest yields, followed by VIS and finally NIS. Furthermore, across all irrigation treatments, the 10_152 combination achieved higher productivity than PF_FD (Fig. 3a).

Figure 3
Yield (t·ha^-1) of Tahiti acid lime’s leaves subjected to irrigation management. (a) 2022, (b) 2023, and (c) 2024.

In the 2023 season, the 10_152 combination again recorded the highest mean yield under FIS. For PF_FD, the highest yield was also under FIS, followed by VIS and then NIS. When comparing the two combinations under different irrigation treatments, a significant difference was observed only under NIS—10_152 yielded significantly more than PF_FD (Fig. 3b).

Finally, in the 2024 season, no significant differences were observed between VIS and FIS treatments. Additionally, both combinations showed no significant differences in yield under these treatments. However, under the NIS regime, yields were lower compared to the other treatments. Notably, the 10_152 combination maintained higher productivity than PF_FD under NIS (Fig. 3c).

Productivity is closely associated with drought tolerance in citrus cultivation, particularly in regions characterized by low rainfall (Girardi et al. 2018). Previous studies have shown that during drought conditions, dwarfing rootstocks may exhibit up to 56% lower efficiency compared to vigorous rootstocks (Silva et al. 2023). This finding may explain the superior yield performance of the citrandarin IAC 3152 Itajobi in comparison to IAC 718 Flying Dragon, particularly under the NIS (non-irrigated) treatment. The advantage can be attributed to the fact that Itajobi is classified as a semi-dwarfing rootstock (Schinor et al. 2015), while Flying Dragon is a strongly dwarfing rootstock (Donadio et al. 2019).

Another critical factor influencing total fruit production is flowering, specifically the proportion of viable flowers that successfully develop into fruit. Floral induction in citrus is governed by exogenous stimuli. In tropical regions such as Brazil, drought conditions trigger flowering through the activation of hormonal pathways involving auxins, gibberellins, and cytokinins, which are key regulators of floral initiation in citrus (Gill et al. 2023). Therefore, as demonstrated by the yield and production efficiency data, VIS (full irrigation) and FIS (partial irrigation) are the most recommended irrigation strategies across citrus varieties, as they support optimal flower induction. In contrast, dry farming (NIS) is not a fully reliable approach, since water availability—either through rainfall or supplemental irrigation—is essential to ensure successful fruit set and complete fruit development (Agustí et al. 2022). Periods of prolonged drought or delays in the onset of spring rains may severely compromise citrus productivity (Abobatta 2023). Consequently, irrigation systems that align with citrus physiological needs can significantly enhance orchard productivity, potentially doubling fruit yields.

Unfortunately, we are monitoring wood pocket symptoms with greater severity in IAC 10 plants, a few symptoms in BRS IAC EECB Ponta Firme (PF), and no symptoms in IAC 5. Wood pocket is a physiological disorder or physiopathy caused by genetic and abiotic factors, such as high temperatures causing sectoral death in Tahiti, also called Persian or bear trees, leading to total extermination (Flores-de la Rosa et al. 2025). This information is essential for citrus breeding programs and may contribute to a better choice of varieties on the market.

Multivariate analysis

PCA was conducted to synthesize the variability of physiological and productive variables among treatments, combining different irrigation regimes and rootstocks. The first two principal components jointly explained 78.8% of the total variance (PC1: 54.6%; PC2: 24.2%), as illustrated in the biplot (Fig. 4). PC1 primarily separated the samples based on root length and proline content, which showed positive loadings, while H₂O₂ content and water potential (wp) were negatively loaded. This suggests that samples with greater root development and higher proline accumulation were associated with reduced oxidative stress. PC2, in turn, reflected secondary variation associated with carbon assimilation (A) and proline levels. The yield vector aligned in the same direction as A and wp, indicating a positive relationship among these variables, suggesting that higher photosynthetic rates and less water stress contributed to improved productivity.

Figure 4
Principal components analysis, where root: root length (mm); wp: water potential (MPa); A: CO₂ assimilation (µmol·m^-2s^-1); H₂O₂: Lipoperoxides (mmol·g^-1); proline (µmol·g^-1) and yields (T·ha^-1), which means are from the combinations of 10_152: IAC 10 with citrandarin IAC 3152 Itajobi, and PF_FD: BRS EECB IAC Ponta Firme with trifoliata IAC 718 Flying Dragon subjected to irrigation management.

In terms of sample distribution, the treatments FIS_10_152 and VIS_10_152 were positively associated with both PC1 and PC2, indicating superior physiological performance, as evidenced by higher values of yield, A, and wp. Conversely, the NIS_PF_FD and NIS_10_152 treatments were negatively aligned with PC1 and closely associated with higher H₂O₂ concentrations and lower productivity, highlighting the detrimental effects of rainfed conditions, especially in the more dwarfing rootstock (Flying Dragon).

The biplot reinforces the influence of both irrigation management and rootstock selection on plant physiological responses, highlighting that combinations involving citrandarin IAC 3152 Itajobi under partial irrigation (FIS) showed overall superior performance under moderate water stress conditions.

CONCLUSION

Based on the results, it can be inferred that the novel citrandarin IAC 3152 Itajobi presents significant physiological advantages for high-density citrus orchards. As a semi-dwarfing rootstock, it exhibits strong adaptability under suboptimal environmental conditions, maintaining efficient gas exchange, favorable water potential, enhanced root development, and consistent physicochemical fruit quality, even under water deficit conditions.

Under full irrigation (VIS), the Flying Dragon trifoliate rootstock also showed satisfactory performance in terms of gas exchange and fruit quality. However, its physiological and productive performance was notably limited under drought stress, reflecting its lower tolerance to water scarcity.

Overall, the data indicate that adequate water supply improves both physiological and productive responses across all rootstocks. The FIS irrigation regime (partial water availability) yielded responses comparable to those under VIS (full irrigation), offering the additional benefit of reduced water use, making it a viable and efficient alternative for managing Tahiti acid lime orchards with either rootstock. However, under adverse environmental conditions such as drought, only the IAC 3152 Itajobi rootstock is recommended, as it ensures higher final productivity.

ACKNOWLEDGMENTS

The authors thank the GD-Citros and GETI study groups, affiliated with the Center of Agricultural Sciences, for technical support, infrastructure, and assistance during the experimental and laboratory activities and Coordenação de Aperfeiçoamento de Pessoal de Nível Superior for the financial support.

• How to cite:
  Souza, A. J. B., Cristofani-Yaly, M., Bastianel, M., Devite, F. T., Gadanhoto, B. P., Arantes, A. C. C., Conceição, P. M., Bizari, D. R. and Azevedo, F. A. (2026). Impact of water regimes on the physiological responses of dwarfing rootstocks in a young orchard of Tahiti acid lime. Bragantia, 85, e20250169. https://doi.org/10.1590/1678-4499.20250169
• FUNDING
  Fundação de Amparo à Pesquisa do Estado de São Paulo
  Grants No.: 2021/10.123-9 and 2020/07045-3
  Coordenação de Aperfeiçoamento de Pessoal de Nível Superior
  Finance Code 001
• DECLARATION OF USE OF ARTIFICIAL INTELLIGENCE TOOLS
  Artificial intelligence tools were used solely for language editing and grammar improvement. The authors are fully responsible for the content, interpretation of the results, and conclusions presented in this manuscript.

DATA AVAILABILITY STATEMENT

All data analyzed during this study are included in this published article. The data supporting the findings of this study are available in the supplementary material of this article: https://doi.org/10.6084/m9.figshare.31622482

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Edited by

• Section Editor:
  Ivo Gonçalves https://orcid.org/0000-0002-8070-9292

Publication Dates

• Publication in this collection
  17 Apr 2026
• Date of issue
  2026

History

• Received
  26 Aug 2025
• Accepted
  20 Jan 2026