Ultra-fine bubble irrigation promotes coffee (Coffea arabica) seedling growth under repeated drought stresses

ABSTRACT Coffee plants are likely to be highly susceptible to changes in climate, and several statistical studies based on projections of altered precipitation patterns have predicted negative effects on coffee growth including the loss of suitable cultivation areas in most coffee-producing countries. Increased drought occurrences owing to climate change are expected to be a new challenge for stable coffee production, because coffee production relies heavily on stable rainfall conditions. In recent years, ultra-fine bubble (UFB) water has been reported to be effective in alleviating environmental stresses for plant growth, especially drought stress. The objective of the present study was to determine the effect of UFB water irrigation on the growth of coffee seedlings to mitigate the stress of repeated droughts. To simulate an environment with frequent droughts, six pot experiments were conducted over 3 years by applying repeated drought stress to coffee plants in a greenhouse. The results showed that UFB water had a remarkable growth-promoting effect on coffee seedlings under drought conditions. In contrast, no significant effect on coffee growth was observed in the environment with sufficient nutrients, in which additional fertilizer was used. UFB water significantly increases the root length and surface area of coffee plants, which may promote water absorption and prevent leaf senescence (leaf cell collapse) under drought conditions. This leads to coffee plants adapting to drought conditions. Therefore, UFB water irrigation may be an effective measure to promote coffee growth in drought conditions. GRAPHICAL ABSTRACT


Introduction
Coffee is an important beverage crop and a valuable agricultural export commodity; it is the second largest traded commodity after petroleum and second most popular beverage after water (Atabani et al., 2019).Among the 90 species of the Coffea genus, Coffea arabica L. (Arabica coffee) is the most economically important species worldwide, with arabica coffee accounting for 75-80% of the coffee consumed globally (Bresciani et al., 2014).Coffee is mainly grown in tropical regions, such as Hawaii, Brazil, and Colombia, which are located between 30°N and 30°S (Vinícius de Melo Pereira et al., 2017).The optimal growing conditions for coffee include an average annual temperature of 18-21°C and annual rainfall of 1200-1800 mm (Da Matta, 2004).As a climate-sensitive perennial crop, coffee is likely to be highly susceptible to changes in climate (Pham et al., 2019).In recent years, guerrilla rainstorms have been increasing, followed by longterm dry periods, which causes drought stress on coffee growth owing to water shortages, even if there is no change in precipitation.Their apparent negative effects on coffee yield and quality have been shown.Moreover, pest and disease infestation are increasing, because coffee relies on abundant water sources (Jaramillo et al., 2011).
For these reasons, it is estimated that areas suitable for coffee cultivation could be significantly reduced in the near future.For example, in Ethiopia, coffee cultivation is currently good, but it has been reported that droughts caused by increasing temperatures and reduced precipitation have resulted in lower yields, decreasing the cultivation suitability of the country (Iscaro, 2014).Several statistical studies based on projections of altered precipitation patterns under current and ongoing climate change scenarios have predicted negative effects on the coffee industry, including the loss of suitable areas in most coffee-producing countries (Da Matta et al., 2019).Because the water environment significantly impacts coffee growth, quality, and yield, it is important for coffeeproducing regions (Worku & Astatkie, 2010).However, few studies have aimed to develop new cultivation management strategies to alleviate environmental stress.
Ultrafine bubble water (hereinafter referred to as 'UFB water') are defined as gas in a medium enclosed by an interface with a volume-equivalent diameter of less than 1 μm (ISO 20,480-1: 2017(ISO 20,480-1: (en), 2021)).In recent years, UFB water has been utilized for various industrial applications.Although micro-bubbles in water disappear after a few minutes, UFBs can persist for several months (Nishihara & Maeda, 2014).Some studies have reported the plant growth-promoting effects of UFB (e.g.Ebina et al., 2013;Liu et al., 2016;Purwanto et al., 2019), whereas others established negative or statistically similar growth to the control in some agronomic traits (e.g.Ahmed et al., 2018;Minamikawa et al., 2015;Mochizuki et al., 2019).Hydroponic cultivation with UFB water has been reported to promote the growth of young seedlings of Gramineae and legume crops under nutrientstress conditions (Iijima et al., 2020(Iijima et al., , 2022a)).Furthermore, it has been reported that the growth-promoting effect of UFB water on crops is much greater under osmotic stress conditions than that under normal conditions (Iijima et al., 2022b).No studies have examined the stress mitigating effects of UFB irrigation on perennial crops.We hypothesized that the use of UFB water may be effective in alleviating drought stress on coffee growth.
Therefore, the objective of this study was to evaluate the effect of UFB water on coffee growth and the mitigation of environmental stress under drought conditions due to climate change.In Experiments 1 and 2, the effects of UFB water on shoot and root growth were investigated.In Experiment 3, we examined the effects of UFB water in environments with sufficient nutrients using additional fertilizer.Finally, in Experiment 4, we examined the mechanism of the drought-mitigation effect of UFB water, as observed in Experiments 1 and 2.

Plant material
Coffee seeds (Coffea arabica 'Typica') were sown in plastic cell trays (23 × 23 × 43 mm) with a 6 mm-diameter hole at the bottom and filled with commercial soil (Green Plaza Yamacho original culture soil; Nara, Japan) on 11 October 2018, 6 December 2019, and 14 December 2020.The soil was composed of peat moss and ripe bark compost with coarse sand.The soil pH (H 2 O) and electrical conductivity were 5.93 and 0.98 mS cm −1 , respectively.The total NH 4+ -N, P 2 O 5 , and K 2 O (exchangeable K) contents were 9.5, 1.6, and 4.7 g kg −1 , respectively.After sowing, the seeds were grown in a plant growth room at 28°C and 23°C during day and night temperature, respectively, under a 14 h photoperiod and 318 ± 2 µmol m −2 s −1 of photosynthetically active radiation at the canopy top level (Iijima et al., 2017).After 4 months, the cotyledons of the seedlings were fully expanded, and the seedlings were transplanted into polypots (90 mm diameter, 80 mm deep) filled with the same soil.The coffee seedlings were grown in a glasshouse at Kindai University in Nara Prefecture (34°40′N, 135°43′E, 172 m a.s.l.).

Cultivation conditions
Six experiments were conducted in the greenhouse.Details about the experimental period, ambient temperature, and treatments in the six experiments are listed in Table 1.In experiment 1, approximately 10month-old coffee seedlings were used, with six replications for each treatment.In experiment 2, approximately 6-month-old coffee seedlings were used, with six replications for each treatment.In experiments 3 and 4, approximately 8-month-old coffee seedlings were used, with 20 replications in each treatment.In experiment 3, top dressing (N: P 2 O 5 : K 2 O = 0.2: 0.2: 0.2 g kg −1 dry soil) was applied during the middle of the treatment period to maintain the green leaf color of the coffee seedlings.This treatment was defined as sufficient fertilizer.The amount of water delivered through irrigation was set to be the same for both the non-UFB and UFB treatments.In all the experiments, insects and diseases were controlled by agrochemicals as required to avoid growth inhibition.

Treatment
Various combinations of the two water regimes (control and drought) and two types of water (tap water (non-UFB) and UFB) were tested using randomly arranged pot-grown coffee.UFB water was prepared by circulating deionized water 10 times through an ultra-fine bubble generator (EAT-SWHI, Eatech Co. Ltd., Kumamoto, Japan).The bubble concentration and size distribution were measured through nanoparticle tracking analysis using a NanoSight LM10 device (Malvern Panalytical, Tokyo, Japan).The concentration and average diameter of UFB were 0.7 × 10 8 mL −1 and 215 nm, respectively (Iijima et al., 2022b).Except for the drought period, the topsoil in both the control and drought conditions was wellwatered by applying approximately 3 mm of irrigation water every one or two days, depending on the soil moisture content of the topsoil.In the control condition (well-watered), surface irrigation was conducted continuously, whereas it was stopped under drought conditions during the drought period.The timing for the irrigation was determined based on the meteorological condition under which the plant was easily damaged by drought stress.Leaf rolling of the plants (leaf water potential was approximately −4.0 MPa) was observed when the drought treatment was stopped.One cycle consisted of stopping and restarting irrigation, and the coffee plants were subjected to repeated drought stress treatments during the experimental period.

Measurement
Plant samples were collected at the end of each experiment to calculate their leaf (except for Experiment 1), stem, and root dry weights, and plant height was measured simultaneously.Plant height was also measured at the beginning of the experiment to calculate plant height increase during the experimental period.The dry weight was measured after oven drying for 72 h at 80°C.In Experiment 4, the green leaf number, green leaf area, root surface area, and total root length were also measured.All green leaf blades were digitalized with a table scanner (35 cm × 25 cm; ScanSnap SV600, FUJITSU, Tokyo, Japan), and the public domain ImageJ (http://rsb.info.nih.gov/ij/)software was employed.In ImageJ, the images were contrasted to facilitate leaf area determination, and all measurements were performed.Root samples were gently washed with tap water and treated with 50% ethanol before scanning using WinRHIZO v. 2009b (Regent Instruments, Blain City, QC, Canada), according to Kato et al. (2010), to determine the total length and surface area of the root system.To measure electrolyte leakage which is often used as an indicator of drought damage in cell membrane, four plant samples in each treatment were selected by the plant height and leaf number and were represented by the average of all coffee plants in each treatment.The leaf disks (13 mm diameter) were punched from the fully expanded uppermost leaves of plants, according to Yamane et al. (2022).The leaf disks were incubated in 40 mL deionized water at 25°C for 24 h, and then, the initial conductance (Ci) of the solution was measured using a conductance meter (CT-27112B; DKK-TOA Corp., Tokyo, Japan).The solutions were autoclaved at 120°C for 20 min for electrolyte extraction.The conductance (C max ) of the autoclaved solution was determined after cooling to room temperature (approximately 25°C).The relative electrolyte leakage was calculated as (Ci/C max ) × 100.

Statistical analysis
A two-way analysis of variance (ANOVA) was performed to analyze the effect of water environment (E) and water treatment (T) in all the experiments.The statistical difference between non-UFB and UFB treatments within the same water regime was determined using Student's t-test.All statistical analyses were performed using Bell Curve for Excel (Social Survey Research Information Co., Ltd., Tokyo, Japan).

Experiment 1
All traits were significantly downregulated by drought treatment (Table 2).The UFB treatment significantly increased plant height (p < 0.05), shoot dry weight (p < 0.05) and root dry weight (p < 0.001) under drought conditions, but there was no significant effect under the control environment.Therefore, a strong interaction between environment and treatment was observed in root dry weight (p < 0.001).

Experiment 2
In Experiment 2-1 a weak effect of drought stress was observed only on the root biomass (Table 3).The effects of UFB (treatment) on plant height increase, shoot dry weight, leaf dry weight, and root dry weight were significant at the 5% level.The UFB treatment significantly enhanced plant height increase (p < 0.05) under drought environment, as well as shoot (p < 0.05) and leaf (p < 0.05) dry weights under the control environment.The interaction effect between UFB treatment and environment was not significant.In Experiment 2-2, the drought stress had a stronger impact affecting plant height increase and root dry weight.UFB treatment did not have any significant effect on any of the traits, and there was no interaction effect between UFB treatment and environment.

Experiment 3
In both Experiments 3-1 and 3-2, the drought stress strongly affected both the shoot and root biomass (Table 4).The UFB treatment did not have any significant effect on any of the traits both in Experiments 3-1 and 3-2, except for plant height increase in control environment in Experiment 3-1.

Experiment 4
Drought treatment significantly decreased all traits, except for the individual leaf area and specific root length, which significantly increased (Tables 5, 6).The effects of the UFB treatment on leaf and root dry weights were significant at the 1% and 5% levels, respectively.UFB treatment significantly increased shoot (p < 0.05), leaf (p < 0.01), and root (p < 0.01) dry weight under drought conditions, but not under control treatment.Similarly, the UFB treatment significantly enhanced leaf number (p < 0.001), total leaf area (p < 0.001), total root length (p < 0.001), and root surface area (p < 0.01) under drought conditions, but not under control treatment.UFB treatment did not affect the individual and specific leaf areas and specific root length under both control and drought conditions.Electrolyte leakage increased under the drought treatment, but UFB treatment significantly mitigated the damage of drought stress (Figure 1).

Discussion
The effect of UFB water irrigation on coffee plant growth was different among experiments.In Experiments 1 and 4, UFB water irrigation had a significant positive effect on shoot biomass under drought conditions.The results of the present study with young coffee seedlings (6-10  months old) were consistent with those of our previous studies, in which young annual crop seedlings of 1-2 weeks after seeding were used (Iijima et al., 2020(Iijima et al., , 2022a(Iijima et al., , 2022b)).In those previous studies, UFB water irrigation positively affected root elongation and activity; therefore, it is more efficient in the early stage, when the root system is not as well developed.The effect of UFB water on the total root length and root surface area was remarkable in drought environments in this study, probably because it was directly affected by physical contact with UFB water.The lateral root length of annual crops was also significantly affected by UFB water (Iijima et al., 2020(Iijima et al., , 2022a(Iijima et al., , 2022b)).Increased root surface area promotes water absorption and prevents leaf wilting (Amato & Ritchie, 2002).In addition, the fine root biomass of the southern highbush blueberry increased when UFB water irrigation was applied (Lai et al., 2022).Moreover, He et al. (2022) showed that UFB could improve crop water use efficiency even with a 20% reduction in water input.Thus, UFB water might alleviate drought stress with increasing total root length and root surface area under drought conditions.On the other hand, in Experiments 2-1 and 2-2, UFB effect was not clear under the drought environment except for plant height in Experiment 2-1.This might be because Experiment 2 is a short-term experiment (less than 2 months).Thus, a long-term treatment might be required for significant effect of UFB irrigation Table 6.Leaf number (LN; plant −1 ), individual leaf area (ILA; cm 2 ), total leaf area (TLA; cm 2 ), specific leaf area (SLA; cm 2 g −1 ), total root length (TRL; cm plant −1 ), root surface area (RSA; cm 2 plant −1 ), and specific root length (SRL; cm g −1 ) in experiment 4.  on coffee growth.Additionally, in Experiment 3, we found that there was no significant effect of UFB water on growth promotion under either control or drought conditions, when additional fertilizer was applied in an environment with sufficient nutrients.Iijima et al. (2020) reported that in the hydroponic cultivation of soybean, UFB water had a reduced growth-promoting effect under high nutrient conditions compared to that under non-nutrient environments.Furthermore, this trend was common to other Gramineae and legume crops (Iijima et al., 2022a).This is because the high colloidal stability and longevity of UFB in water promotes nutrient release or uptake in plant roots (Wang et al., 2021).Furthermore, UFB can improve soil microbial activity by increasing nutrient availability and oxygen content in soil (Zhou et al., 2020), which may lead to increased nutrient uptake by coffee seedlings.Lai et al. (2022) demonstrated that nitrogen absorption in blueberry seedlings was enhanced by UFB water irrigation.Therefore, the effect of UFB water was shown under low-nutrient conditions, similar to that observed in many annual Gramineae and legume crops, because UFB water irrigation may enhance nutrient absorption in coffee grown under low-nutrient conditions.The use of chemical fertilizers and pesticides in large-scale plantations in tropical regions has caused soil degradation and reduced food production (Ogura, 2018), and sustainable agriculture is a key focus in coffee cultivation.Hence, it is important for coffee farmers to use fertilizers more efficiently to reduce production costs and environmental impacts (Bruno et al., 2011).In addition, N deficiencies in the initial growth stages of coffee plants negatively affect productivity throughout the reproductive period (Parecido et al., 2022).Thus, the results of this experiment suggest that UFB water applications are effective for promoting growth under the conditions without additional fertilizers, implying that this could be a useful technology for sustainable coffee cultivation.Green leaf area is an important indicator of plant growth, light interception, and photosynthetic efficiency, which greatly affect growth and productivity (Blanco & Folegatti, 2005).The results of experiment 4 showed that the total leaf area was significantly increased under drought conditions with UFB water due to the increased leaf number, not by the individual and specific leaf areas.These results suggest that UFB water promotes biomass increase by enhancing physiological functions with increasing the leaf number and total root length.Yamane et al. (2022) reported that when coffee plants were subjected to hightemperature stress, leaf electrolyte leakage exceeded 20%, and the plant physiological function was damaged.After drought stress treatment, electrolyte leakage was 67.2% under non-UFB water irrigation, while it was 15.7% under UFB water irrigation.This indicated that the leaves of plants irrigated with non-UFB water were greatly damaged by drought treatment, whereas UFB water mitigated the drought stress in plants irrigated with it.Therefore, UFB water is considered to prevent the collapse of leaf cells and greatly reduce leaf senescence by increasing root surface area.
A relationship between environmental stress and excess reactive oxygen species (ROS) production has been demonstrated under various conditions (Choudhury et al., 2017), and excess ROS causes plant damage (Suzuki & Mittler, 2006).The acclimation of coffee plants to drought stress is strongly related to photosynthesis and respiration activity, ROS production, and expression of antioxidant enzymes (Menezes-Silva et al., 2017).Four types of ROS (O 2-, H 2 O 2 , .OH, and O 2 ) are produced by UFB water, and the stimulation of these exogenous ROS promotes seed germination (Liu et al., 2016).In contrast, UFB reduced ROS under osmotic stress conditions and contributed to stress mitigation (Iijima et al., 2022b).Therefore, further studies are necessary to examine the effects of UFB water on changes in ROS and plant morphology, especially root morphology, to elucidate the factors behind the growth-promoting and stress mitigation effects in detail.

Conclusion
UFB water exerted a growth-promoting effect on coffee seedlings, especially under drought conditions.UFB water increased the root length and surface area of coffee plants, which may promote water absorption and prevent leaf senescence (leaf cell collapse) under drought conditions.Therefore, UFB water irrigation may be an effective measure to promote coffee growth in a water scarce environment.In contrast, no significant effect on coffee growth was observed in an environment with sufficient nutrients.To elucidate the effect of UFB clearly, it should be further compared under different nutrient conditions.In addition, further studies should examine the effects of UFB water on changes in ROS and plant morphology to elucidate the mechanisms underlying the growth-promoting and stress mitigating effects in detail.

Figure 1 .
Figure 1.Electrolyte leakage of coffee leaf disks in experiment 4. Data are means of four plant samples in each treatment.Vertical bars indicate standard errors (n = 4).Note:** indicates significance at 1% probability, 'ns' indicates non-significance.Student's t-test was performed for the comparison between non-UFB and UFB.

Table 1 .
Details of treatments and environments in the six experiments.

Table 2 .
Plant height (cm) increase, and dry weight (DW; g plant −1 ) of both shoot and root in experiment 1.
* and *** indicate significance at 0.05 and 0.001 probability levels, respectively.'ns' indicates non-significance.Student's t-test was performed for the comparison between non-UFB and UFB.Two-way-ANOVA was performed using environment and treatment factors.

Table 3 .
Plant height (cm) increase, and dry weight (DW; g plant −1 ) of shoot, leaf, and root in experiment 2.
*, **, and *** indicate significance at 0.05, 0.01 and 0.001 probability levels, respectively.'ns' indicates non-significance.Student's t-test was performed for the comparison between non-UFB and UFB.Two-way-ANOVA was performed using environment and treatment factors.
*, **, and *** indicate significance at 0.05, 0.01, and 0.001 probability levels.'ns' indicates non-significance.Student's t-test was performed for the comparison between non-UFB and UFB.Two-way-ANOVA was performed using environment and treatment factors.

Table 5 .
Plant height (cm) increase, and dry weight (DW; g plant −1 ) of shoot, leaf, and root in experiment 4.