Biochar-Improved Growth and Physiology of Ehretia asperula under Water-Deﬁcit Condition

Featured Application: Oak-wood biochar application at 15 tons ha − 1 could be recommended for ensuring the best physiological responses and highest growth of Ehretia asperula plants in water-deﬁcit conditions. Abstract: Ehretia asperula ’s physiological responses to growth performance following oak-wood biochar application under water stress conditions (WSC) and no water stress conditions (non-WSC) were investigated in a pot experiment. Biochar (WB) was incorporated into the soil at concentrations of 0, 5, 10, 15, and 20 tons ha − 1 before transplanting Ehretia asperula in the pots. One month after transplanting, Ehretia asperula plants were put under water stress by withholding water for ten days. Water stress signiﬁcantly decreased the growth and physiology of Ehretia asperula . Under WSC, the application of WB at the concentrations of 15 and 20 tons ha − 1 to the soil increased the plant height; number of leaves; fresh and dry weight of the roots, shoots, and leaves; F v /F m ; chlorophyll content; leaf relative water content; and soil moisture as well as decreased the relative ion leakage. The application of WB enhanced drought tolerance in Ehretia asperula plants by lowering the wilting point. The ﬁndings suggest that WB application at the concentration of 15 tons ha − 1 could be recommended for ensuring the best physiological responses and highest growth of Ehretia asperula plants.


Introduction
The genus Ehretiais is mainly distributed in the tropical areas of Asia, Africa, and Northern America and exhibits valuable pharmacological properties [1,2]. Ehretia asperula Zoll. et Mor, a species in the genus Ehretiais, was initially researched by Zollinger and Moritzi [3]. In Vietnam, Ehretia asperula has been used in traditional medicine for the treatment of gastritis and pimples, as an antitumor medication, and in antioxidation by some ethnic minorities for a long time. Moreover, the product of alcoholic extraction from the bark of Ehretia asperula can be used in the treatment of liver cancer, nose cancer, colon cancer, and HIV H-9 resistance [4,5].
Drought is considered one of the major environmental factors responsible for the reduction in plant growth and yield [6,7]. Drought stress adversely affects soil properties, thus restraining plant metabolism and growth [6][7][8][9]. On the other hand, drought stress can alter the physiological characteristics of plant leaves, such as lowering the leaf photosynthetic and transpiration rate, and stomatal conductance and, consequently, restraining crop The Ehretia asperula plants were propagated by stem cutting. The similar diameters of stem cuttings were inserted in a poly bag (70 mm in diameter × 100 mm in height) that had been filled with alluvial soil. Three months after rooting of stem cuttings, similar seedlings were transferred to the plastic pots (260 mm in diameter × 210 mm in height) which were filled with alluvial soil obtained from the experimental field at the Vietnam National University of Agriculture, Hanoi, Vietnam. Each pot contained 5 kg of dry alluvial soil. Biochar was incorporated into the plastic pots before transplanting the Ehretia asperula plants. The initial physical and chemical properties of the experimental soil were presented in Table 1. Oak-wood biochar (WB) was purchased from Gangwon Charmsoot Company, Hoengseong-gun, Gangwon Province, Korea. Biochar was produced at a temperature of 400 • C by pyrolysis. The proximate and ultimate analyses and physiochemical properties of oak-wood biochar were reported in Rajapaksha et al. [24] such as the pH: 10.17; EC: 2.15 dSm −1 , mobile matter: 31.42%; fixed matter: 56.04%; dissolved organic carbon (DOC): 14.6 mg L −1 ; ash content: 5.03%; C: 88.71%; H: 1.21%; N: 0.36%; O: 9.72%; molar H/C: 0.16; Molar O/C: 0.08; specific surface area: 270.76 m 2 g −1 ; pore volume: 0.12 cm 3 g −1 ; pore diameter: 1.10 mm.

Experimental Procedure
The pot experiment was carried out in net house at the Vietnam National University of Agriculture, Hanoi, Vietnam. A split-plot experimental design was adopted with three replications (one replication with 8 plastic pots for each treatment). The amount of biochar used was the main factor in this experiment, and five application rates were Appl. Sci. 2021, 11, 10685 3 of 14 employed (0, 26.5, 53.1, 79.6, and 106.2 g biochar/pot, which corresponded to 0, 5, 10, 15, and 20 tons biochar ha −1 , respectively. Drought stress conditions, considered a subfactor, included water-stress condition (WSC) and no water-stress condition (non-WSC). A brief design of this experiment is summarized in Table 2. The non-WSC comprised tap-water use during the growth of Ehretia asperula plants. One plant in each pot was watered daily with 200 mL of tap water. The WSC comprised tap-water use for one month following transplanting and subsequent induction of WSC by withholding irrigation for 10 days (from 11 May to 20 May); after 10 days of water stress, the plants were re-watered.

Growth Parameters
The growth rates of plant height were recorded from 4 May to 30 May (four times in pre-treating water stress period, five times in water stress period, six times in after re-watering period).
The growth rates of plant height (cm/checking time) = PH 2 − PH 1 PH 2 : Plant height this checking time; PH 1 : Plant height last checking time The plant height (cm), the number of leaves, and biomasses of Ehretia asperula plants were measured after 10 days of re-watering. The dry biomasses of roots, shoots, and leaves were determined, and their contents were measured by drying the fresh roots, shoots, and leaves samples at 80 • C until a steady weight in an oven (MOV-212F, Sanyo Electric Co., Ltd., Osaka, Japan).

Physiological Attributes
The methods used to determine the physiological attributes of the plants were from our previous work [25]. The quantum efficiency of photosystem II (F v /F m ) and chlorophyll content were also recorded from 4 May to 30 May (four times in pre-treating water stress period, six times in water stress period, and five times in after re-watering period). Chlorophyll contents were measured by using a chlorophyll meter (SPAD-502 Plus, Konica Minolta Sensing Inc., Osaka, Japan).
The chlorophyll fluorescences (F v /F m ) were measured by using a portable fluorometer (model OS-30p, Opti-Sciences Chlorophyll Fluorometer, Hudson, NY, USA). The initial fluorescence (F 0 ), maximum fluorescence (F m ), and potential quantum efficiency of photosystem II (F v /F m ) were measured. From these fluorescence data, the following parameters were calculated: variable fluorescence (F v = F m − F 0 ) and the effective absorbed energy-conversion efficiency of photosystem II (F v /F 0 ). Fluorescence determinations were performed between 08:00 h and 11:00 h on the same leaves. The leaves were submitted to a 30 min dark adaptation period using leaf-clip holders, so that all the reaction centers in the foliar region under analysis acquired the 'open' configuration, indicating the complete oxidation of the photosynthetic electron transport system.
The leaf relative water content was measured on the last day of withholding irrigation and 10 days after re-watering. To measure the leaf relative water content (RWC), 9 samples out of the 10 leaf discs, one per treatment, were obtained. Leaves were selected among the youngest fully expanded leaves. The leaf discs were immediately weighed (fresh weight; FW). The samples were bathed in distilled water (temperature range of 25-30 • C) inside a porous platform in order to obtain the turgid weight (TW). At the end of the imbibition period, the leaf samples were placed in a pre-heated oven at 80 • C for 48 h to obtain the dry weight (DW). Values of FW, TW, and DW were used to calculate RWC using the equation below.
RWC (%) = ((FW − DW)/(TW − DW)) × 100 The relative ion leakages in the leaves were measured on the last day of withholding irrigation. Relative ion leakage in the leaves was assessed by the leakage of electrolytes from the leaves of nine plants of similar size. Leakage of the electrolytes was determined with a conductivity meter (AG 8603, SevenEasy, Mettler Toledo, Switzerland). The leaf segments (disks of leaves with d = 1 cm 2 ) were washed, blotted dry, weighted, and placed in stopped vials filled with an exact volume of deionized water. The vials were then incubated for 2 h in darkness with continuous shaking before the conduction (C1) was measured. The vials were heated to 80 • C for 2 h, and the conduction (C2) was measured again. The electrolyte leakage was expressed as a percentage of relative ion leakage, which was calculated according to the following equation [26]: Relative electrolyte leakage (%) = C1/C2 × 100.
The soil moistures were measured by an Aquaterr-Model T300 Moisture Measurement Instrument (Aquaterr Model T300, Aquaterr, Costa Mesa, CA, USA). Measurements were taken at 15 cm soil depth on the last day of withholding irrigation.
The percentage of wilted plants under drought stress was recorded from the fifth day to the tenth day of withholding water. The plant was only considered wilting when 75% of its leaves were withered.

Data Analysis and Statistics
Growth and physiological parameters (growth rate of plant height, plant height, number of leaves, F v /F m , chlorophyll content, and soil moisture) were gathered from 15 randomly selected plants per treatment to be used for the statistical analysis. The measurement of the percentage of the wilted plant was obtained from 24 plants to be used for the analysis. Nine plants per treatment were randomly selected for the statistical analysis of the remaining growth and physiology parameters (the dry biomasses of roots, shoots, and leaves; relative ion leakage; and leaf relative water content).
The data recorded for the growth and photosynthetic efficiency of Ehretia asperula were statistically analyzed coefficient of variation and least significant difference using IRISTAT 5.0. Mean separations were estimated using Duncan's multiple range tests at p ≤ 0.05.

Effect of Biochar on Growth Rate of Plant Height
The growth performance of Ehretia asperula in different WB rates under WSC showed a reduction in the growth rate of plant height due to water stress compared with plants growing under non-WSC. The growth rate of the plant height of Ehretia asperula in all different WB rates under the non-WSC remained constant over time. In general, WSC reduced the growth rate of the plant height of Ehretia asperula while WB treatments increased the growth rate of plant height in both non-WSC and WSC. For instance, the highest value of growth rate of plant height was recorded with the 15 tons ha −1 WB treatment under both non-WSC and WSC ( Figure 1). creased the growth rate of plant height in both non-WSC and WSC. For instance, the highest value of growth rate of plant height was recorded with the 15 tons ha −1 WB treatment under both non-WSC and WSC ( Figure 1).   (Table 3).   (Table 3).    (Table 4).

Effect of Biochar on Chlorophyll Content
To examine the physiological performance of Ehretia asperula plants under WSC, we measured the change in chlorophyll content of Ehretia asperula plants in different WB rates between before (4 May) and after (30 May

Effect of Biochar on Photosynthetic Efficiency (F v /F m )
To examine the effect of water stress on physiological performance, we measured the changes in the quantum efficiency of photosystem II (F v /F m ) of Ehretia asperula plants in different WB rates from before (4 May) to after (

Effect of Biochar on Leaf Relative Water Content
WSC decreased the leaf relative water content of Ehretia asperula, whereas WB treatments increased the leaf relative water content significantly. The highest values of the leaf relative water content were recorded for the 15 tons ha −1 WB treatment under both non-WSC and WSC after 10 days withholding irrigation and after 10 days re-watering. The lowest values of the leaf relative water content were observed in plants growing in without WB treatment (0 tons ha −1 of WB treatment), followed by those grown with the 5 tons ha −1 WB treatment under the non-WSC and WSC. Although biochar noticeably increased the leaf relative water content, the difference in the leaf relative water content among the 15 and 20 tons ha −1 WB treatments was not significant under both the non-WSC and WSC after 10 days withholding irrigation and after 10 days re-watering ( Figure 4).

Effect of Biochar on Leaf Relative Water Content
WSC decreased the leaf relative water content of Ehretia asperula, whereas WB treatments increased the leaf relative water content significantly. The highest values of the leaf relative water content were recorded for the 15 tons ha −1 WB treatment under both non-WSC and WSC after 10 days withholding irrigation and after 10 days re-watering. The lowest values of the leaf relative water content were observed in plants growing in without WB treatment (0 tons ha −1 of WB treatment), followed by those grown with the 5 tons ha −1 WB treatment under the non-WSC and WSC. Although biochar noticeably increased the leaf relative water content, the difference in the leaf relative water content among the 15 and 20 tons ha −1 WB treatments was not significant under both the non-WSC and WSC after 10 days withholding irrigation and after 10 days re-watering ( Figure 4).

Effect of Biochar on Soil Moisture
The application of WB significantly increased the soil moisture under WSC. The soil moisture increased with increased WB rates under WSC. The soil enriched with the 20 tons ha −1 WB treatment gave the highest soil moisture value after 10 days of withholding irrigation. However, no significant difference in moisture of the soil enriched with the 15 and 20 tons ha −1 WB treatments after 10 days withholding irrigation in WSC was observed. The lowest values of the soil moisture were recorded with the without-WB treatment under WSC ( Figure 6).

Effect of Biochar on Soil Moisture
The application of WB significantly increased the soil moisture under WSC. The soil moisture increased with increased WB rates under WSC. The soil enriched with the 20 tons ha −1 WB treatment gave the highest soil moisture value after 10 days of withholding irrigation. However, no significant difference in moisture of the soil enriched with the 15 and 20 tons ha −1 WB treatments after 10 days withholding irrigation in WSC was observed.
The lowest values of the soil moisture were recorded with the without-WB treatment under WSC (Figure 6).

Effect of Biochar on Wilted Plant
The addition of Biochar to the soil enhanced the drought tolerance of Ehretia asperula plants by lowering the wilting point. In the without biochar treatment, the wilting point was reached on the fifth day withholding irrigation, but in the 15 and 20 tons ha −1 WB treatment groups, the wilting point was reached on the sixth day withholding irrigation. On the other hand, 100% of the plants had wilted by the eighth day withholding irrigation in the without biochar treatment, but in the 15 and 20 tons ha −1 WB treatment groups, only 80% of the plants had wilted by the tenth day withholding irrigation (Table 5).  Soil moisture (%)

Effect of Biochar on Wilted Plant
The addition of Biochar to the soil enhanced the drought tolerance of Ehretia asperula plants by lowering the wilting point. In the without biochar treatment, the wilting point was reached on the fifth day withholding irrigation, but in the 15 and 20 tons ha −1 WB treatment groups, the wilting point was reached on the sixth day withholding irrigation. On the other hand, 100% of the plants had wilted by the eighth day withholding irrigation in the without biochar treatment, but in the 15 and 20 tons ha −1 WB treatment groups, only 80% of the plants had wilted by the tenth day withholding irrigation (Table 5).

Discussion
Drought stress affects the growth, phenology, water and nutrient relations, photosynthesis, assimilate-partitioning, and respiration in plants, which results in the production of smaller organs [27]. These phenomena are controlled by physiological factors, such as plant hydraulic status, phytohormones, osmotic adjustment, and reactive oxygen species signaling [28,29]. In our study, water stress significantly decreased the growth and photosynthetic efficiency of Ehretia asperula, as demonstrated by the analysis of parameters such as plant height, number of leaves, dry biomasses of root, shoot, and leaves; F v /F m , chlorophyll content, and leaf relative water content.
Biochar not only improves the soil structure, fertility, and ion transfer ability but also increased the activity of microbes and nutrient holding and exchange capacity of the soil. Therefore, biochar addition is proposed as an effective management to improve crop performance [21], as supported by the results we obtained from the experiments on Ehretia asperula plants, for which all biochar treatments clearly improved the growth characteristics such as plant height; number of leaves; and dry biomasses of root, shoot and leaves. These results agree with previous data obtained by several researchers who reported that the application of biochar increased the plant height and leaf area of okra [30] and maize [31] under drought-stress conditions. In addition, Olmo et al. [32] reported that the application of biochar increased the biomass of field-grown wheat under semiarid Mediterranean conditions. Moreover, Vaccari et al. [33] also found that the application of biochar in silt clay soil increased the growth of tomato plants when compared with the control.
On the other hand, several studies have reported that biochar addition improved photosynthesis in plants under drought-stress conditions [18,34,35]. Biochar increased photosynthesis and water-use efficiency of okra under drought stress when compared with the control [30]. In addition, biochar increased the water-use efficiency of Chenopodium quinoa wild under drought stress [36] and increased the water-use efficiency of maize in sandy soil [37]. Therefore, in our study, WB treatments inversely increased the SPAD values, photosynthetic efficiency (F v /F m ), and leaf relative water content of Ehretia asperula significantly. These results agree with previous results obtained by Haider et al. [31], who reported that biochar application increased the leaf relative water content, transpiration rate, and osmotic potential of drought-stressed maize when compared with the control. In addition, Akhtar et al. [38] also showed that the application of biochar significantly improved physiological characteristics such as chlorophyll content, stomatal conductance, photosynthetic rate, water-use efficiency, and leaf relative water content as well as increased the stomatal density of tomato leaves under drought stress conditions. Biochar improves the physical and biological properties of soils and increases the water-holding capacity of soil under drought stress conditions [39]. In our study, the application of WB significantly increased soil moisture under WSC. On the other hand, WB application enhanced the drought tolerance of Ehretia asperula plants by lowering the wilting point. These results agree with previous results obtained by Akhtar et al. [38] and Chintala et al. [40], who found that biochar application greatly increased the water-holding capacity of soil and, consequently, enhanced plant physiological characteristics such as chlorophyll content, stomatal conductance, photosynthetic rate, and leaf relative water content. In addition, Mulcahy et al. [41] also demonstrated that biochar application in sandy soil significantly increased tomato seedling resistance to wilting.

Conclusions
Application of WB to the soil significantly increased the growth and physiology of Ehretia asperula plants under both non-WSC and WSC. On the other hand, WB addition enhanced the soil moisture and drought tolerance of Ehretia asperula plants under WSC by lowering the wilting point. The current study suggests that applying WB at a rate of 15 tons ha −1 could be recommended for ensuring the best growth and physiological responses of Ehretia asperula plants under WSC.