**3. Results**

Ten studies were included. Calcineurin inhibitor therapy has been documented to be effective in various coronaviral diseases both in vitro as well as in vivo. So far, no data in immunocompetent patients on effects of CNI in human SARS-CoV-2 infections have been published. The results are shown in Table 1.

**Table 1.** Coronaviral serotypes and treatment with calcineurin inhibitors.

ALV = alisporivir, CNI = calcineurin inhibitor, CoV = coronavirus, CsA = cyclosporin A, CsAd = cyclosporine A derivatives, IFN-α = interferon alpha, MERS = Middle East respiratory syndrome, Tac = tacrolimus.

The available studies with in vivo data on SARS-CoV, MERS-CoV and SARS-CoV2 include studies performed on animal models of coronavirus-related diseases. SARS-CoV replication has been studied in mice, Syrian golden and Chinese hamsters, civet cats, and non-human primates [21], and MERS-CoV in mice, camelidae and non-human primates [21]. These animal studies investigated protease inhibitors, monoclonal and polyclonal antibodies, convalescent plasma, interferons, ribavirin, lopinavir/ritonavir in both SARS-CoV and MERS-CoV [21], but to our knowledge there are no animal data on SARS-CoV and SARS-CoV-2 addressing CNI therapy. Exceptions are the feline and turkey CoV, which do not have the possibility of spillover into human hosts [13,14]. The other exception is alisporivir, a nonimmunosuppressive cyclophillin inhibitor (CsA analog) in SARS-CoV, with strong in vitro dose-dependent antiviral properties against SARS-CoV-2 [16].

## **4. Discussion**

So far, the COVID-19 pandemic has led to more than 17 million SARS-CoV-2 infected patients and over 700000 deaths [7]. The number of cases is still, or again, on the rise in many countries and there currently is no geographic region where the pandemic seems totally under control.


**Author details**

Jungwhan Chon, Kidon Sung\* and Saeed Khan

\*Address all correspondence to<EMAIL_ADDRESS>
Division of Microbiology, National Center for Toxicological Research, US Food and Drug Administration, Jefferson, AR, USA


of water, minerals and various chemical compounds from the roots to the shoots. Drought stress increases the content of ABA in leaves, not only by promoting the biosynthesis of ABA but also by accelerating the ABA catabolic pathway [18–20]. In contrast, excessive water content in soil or the substrate causes an oxygen-poor environment (hypoxia), which becomes a major factor in inhibiting root respiration. This inhibits metabolic activities and ATP production in plants. These plant responses limit the supply of energy for the growth of the root and eventually result in poor plant growth [2,19]. In addition, flooding stress induces the accumulation of toxic compounds such as ethanol, lactic acid and acetaldehyde, and these compounds not only suppress plant growth but also cause adverse effects such as root dysfunction and low soil redox potential [21,22]. In this experiment, the inhibition of shoot and root growth under the 20% and 60% substrate water content conditions could be explained as being caused by drought and flooding stresses, respectively (Figure 2). These results imply that the substrate water content between these values, 45%, in which plants showed the most effective growth, may be adequate for the growth of *C. denticulatum*.

Stomatal closing due to drought stress as described above prevents water loss in the leaves, while it hinders transpiration and the allocation of photosynthetic assimilates, resulting in a reduction in photosynthetic rate [21,23]. Hypoxia conditions in soil caused by flooding restrict water absorption by roots and root hydraulic conductance, leading to stomatal closure [24,25]. In addition, Ahsan et al. [3] reported that soil flooding increases photorespiration and/or decreases the activities of RuBP and RuBP activase, which is one of the main reasons for the reduction in photosynthetic rate. In our study, substrate water content of 20% (drought) and 60% (flooding) decreased stomatal conductance and transpiration rate in the leaves of *C. denticulatum* (Figure 4B,C). The observed sap flow values also supported the results of stomatal conductance and transpiration rate. During the day, plants under the 20% and 60% substrate water content treatments had lower sap flow values than plants under the 45% treatment (Figure 5). In this experiment, the decrease in transpiration rate owing to soil drought and flooding seemed to have a direct effect on photosynthetic inhibition (Figure 4A). Mutava et al. [26] reported that drought-tolerant genotypes accumulate more ABA than drought-susceptible genotypes, leading to an increased stomatal closure in dry soil. In this experiment, the stomatal conductance of plants under the 20% treatment was significantly lower than that of plants under the 60% treatment (Figure 4B), suggesting that *C. denticulatum,* which naturally grows at the foot of a mountain in dry conditions, may be more sensitive to excessive soil water content than to dry soil. In this study, chlorophyll content was not significantly affected by substrate water content (Figure 4D). In general, constant or excessive water stress decreases chlorophyll content and ultimately accelerates leaf senescence [3]. However, it should be considered that the water level and duration conditions used in this study were not severe enough to affect a change in chlorophyll content. Plants have complex defensive mechanisms to survive in tough external environments, among which the antioxidant system plays an important role in overcoming environmental stresses. Soil-water-related stresses promote the generation of reactive oxygen species (ROS) owing to the limitation of photosynthetic processes, which can activate the antioxidant system [27–29]. The typical antioxidant bioactive compounds of *C. denticulatum* are types of HCAs including caftaric acid, chicoric acid, chlorogenic acid and 3,5-DCQA [5–7,14]. The results of this experiment showed that antioxidant capacity and total HCAs per dry weight of plants under the soil flooding treatment (60%) were higher than those of plants under other treatments (Figures 6B and 7A). This result can also be explained by the concentration effect because of growth inhibition. However, considering that shoot dry weights of 20% and 30% substrate water content treatments were similar to 60% treatment, not only the inhibition of photosynthesis by flooding stress but also hypoxia-produced ROS and toxic substances are thought to activate the biosynthesis pathway of secondary metabolites [21]. At 20% water content of the substrate, total phenolic content and HCAs content did not increase, probably owing to a lack of photosynthetic assimilates. However, although antioxidant capacity and total HCAs content per dry weight of plants grown under the 60% substrate water content treatment were significantly higher than in plants under the 45% treatment, their content per shoot were the highest under the 45% substrate water content treatment because of a remarkable

increase in the shoot dry biomass. Another study of the cultivation of *C. denticulatum* used the capillary wick culture system described in a previous study [14,30]. The results also showed that shoot fresh weight, photosynthetic rate, total phenolic content and antioxidant capacity per shoot of plants grown under 45% substrate water content were significantly the highest among treatments (Figure S1). These results showed that shoot biomass of *C. denticulatum* is directly related to the content of bioactive compounds, and that 45% water content of the substrate can increase not only the growth, but also the antioxidant capacity and phenolic content.

In conclusion, we confirmed that the growth and bioactive compounds of *C. denticulatum,* which is used as a plant-derived raw material for functional food, can be influenced by the water content of the substrate. The water content of 45% in the substrate increased the biomass of the shoot and root and increased phenolic content, antioxidant capacity and HCAs content per shoot. The possibility of using hydroponics for native plants was also verified by another experiment using the capillary wick culture system. The results of the current study are expected to be useful for the stable mass production of high-quality *C. denticulatum* in greenhouses or plant factories capable of controlling water content in the root zone.

**Supplementary Materials:** The following are available online at http://www.mdpi.com/2073-4395/10/3/388/s1, Figure S1: effects of capillary wick culture system.

**Author Contributions:** Methodology, investigation, resources, formal analysis, data curation, software, writing original draft preparation, visualization, S.-Y.P.; methodology, conceptualization, formal analysis, software, writing—original draft preparation, J.K.; supervision, funding acquisition, validation, writing—review and editing, M.-M.O. All authors have read and agreed to the published version of the manuscript.

**Funding:** This work was supported by Korea Institute of Planning and Evaluation for Technology in Food, Agriculture, Forestry and Fisheries (IPET) through the Agriculture, Food and Rural Affairs Research Center Support Program, funded by the Ministry of Agriculture, Food and Rural Affairs (MAFRA) (717001-07-02-HD240).

**Conflicts of Interest:** The authors declare no conflict of interest. The funders had no role in the design of the study; in the collection, analyses, or interpretation of data; in the writing of the manuscript, or in the decision to publish the results.

#### **References**

© 2020 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (http://creativecommons.org/licenses/by/4.0/).

## *Article* **Growth Response of Cassava to Deficit Irrigation and Potassium Fertigation during the Early Growth Phase**

## **Daniel O. Wasonga \* , Jouko Kleemola, Laura Alakukku and Pirjo S.A. Mäkelä**

Department of Agricultural Sciences, University of Helsinki, P.O. Box 27, FIN-00014 Helsinki, Finland<EMAIL_ADDRESS>(J.K<EMAIL_ADDRESS>(L.A<EMAIL_ADDRESS>(P.S.A.M.)

**\*** Correspondence<EMAIL_ADDRESS>Tel.: +358-(0)466-170-988

Received: 27 January 2020; Accepted: 24 February 2020; Published: 26 February 2020

**Abstract:** Cassava (*Manihot esculenta* Crantz) experiences intermittent water deficit and suffers from potassium (K) deficiency that seriously constrains its yield in the tropics. Currently, the interaction effect between deficit irrigation and K fertigation on growth and yield of cassava is unknown, especially during the early growth phase. Therefore, pot experiments were conducted under controlled greenhouse conditions using cassava cuttings. Treatments initiated at 30 days after planting included three irrigation doses (30%, 60%, 100% pot capacity) and five K (0.01, 1, 4, 16, and 32 mM) concentrations. The plants were harvested 90 days after planting. Decreasing irrigation dose to 30% together with 16 mM K lowered the leaf water potential by 69%, leaf osmotic potential by 41%, photosynthesis by 35%, stomatal conductance by 41%, water usage by 50%, leaf area by 17%, and whole-plant dry mass by 41%, compared with full-irrigated plants. Lowering the K concentration below 16 mM reduced the values further. Notably, growth and yield were decreased the least compared with optimal, when irrigation dose was decreased to 60% together with 16 mM K. The results demonstrate that deficit irrigation strategies could be utilized to develop management practices to improve cassava productivity by means of K fertigation under low moisture conditions.

**Keywords:** leaf area; *Manihot esculenta*; photosynthesis; tuber; water status

### **1. Introduction**

A global challenge for the agricultural sector is to produce more food with less water [1]. Developing new scientific strategies that allow crops to use water efficiently could be crucial in a world with a growing population [2]. Thus, water application strategies focused on increased agricultural water productivity, such as deficit irrigation coupled with potassium (K) fertigation to investigate multiple alternatives, have a pivotal role to play in sustainable crop production. Cassava (*Manihot esculenta* Crantz) is a major food crop for more than 800 million people in the tropics, providing more than 60% of daily calorific needs [3]. Cassava leaves are widely consumed, due to the high contents of protein, minerals, vitamins, lipids, and fiber, compared with roots that are mainly rich in carbohydrates [4,5]. Cassava is alternatively used as a processed food, animal feed, starch for pharmaceutical industries, and bioethanol for vehicles [6,7]. Even though cassava is considered drought-tolerant [8,9], the crop experiences intermittent water deficit [10] and suffers from K deficiency [11,12], which seriously constrains its yield in the tropics.

Water deficit restricts cassava growth and yield by decreasing the soil-water potential, which in turn limits stomatal conductance [13,14], resulting in reduced photosynthesis [15], number of leaves, and the individual leaf size [8,16]. It also leads to a reduction in shoot growth [17] and subsequent reduction in fresh and dry biomass [18,19]. Cassava shoot and root biomass can decrease by 70% under conditions of water deficit [20,21] but are more pronounced if the water deficit occurs during the first

1−5 months after planting [18,22] since plant leaf expansion and tuberous root development initiate during this period.

Cakmak and Engels [23] showed that water deficit increases the plant K requirement. K alleviates water deficit in plants by regulating the cell osmotic potential (Ψ*s*) to maintain the turgor pressure (Ψ*p*) required for cell functioning [24] and regulating the stomatal movement that aids in minimizing water losses during drought, leading to maintain the carbon dioxide (CO2) fixation [25]. K also plays a key role in partitioning photosynthates to storage roots [26,27] and activation of enzymes [28]. It also increases heat tolerance in plants [26]. Byju et al. [29] estimated that cassava requires a total uptake of 15.6 K kg ha−1 to produce a single metric ton of dry root yield. Thus, improving K nutrition in drought areas with low inherent soil fertility could increase cassava productivity. Lately, cassava yields have been improved through irrigation [30,31] and nutrient application [29,32]. Studies of K nutrition in cassava have also been performed [33,34] with granular K application. However, no studies have combined water deficit strategies and K fertigation in cassava to elaborate its effect on growth and yield. Moreover, understanding how young cassava plants respond to deficit irrigation and K fertigation during the early growth phase is essential for holistic agronomic management to ensure improvements in terms of growth and marketable yield under drought conditions. Therefore, the objective of this work is to assess the effects of interaction between deficit irrigation and K fertigation on growth and yield response of biofortified cassava during the early growth phase.

#### **2. Materials and Methods**

#### *2.1. Plant Material and Growth Conditions*

Four pot experiments were conducted with single-stem cuttings (25 cm) of yellow cassava "Mutura" cultivar (Kenya Agricultural and Livestock Research Organization (KALRO), Nairobi, Kenya). Cuttings were planted in 5-L pots containing 1.7 kg of pre-fertilized potting mix (pH 5.5, N–P–K: 17–4–25, organic matter: 25–40%, Kekkilä Karkea ruukutusseos, W R8014; Kekkilä Oy, Vantaa, Finland). The potting mix was saturated with water and allowed to drain overnight, and the maximum soil water holding capacity (1600 g pot−1 ) was calculated as the difference between water applied and water drained. The pots were placed in the greenhouse at the University of Helsinki, Finland, under controlled conditions with day/night temperatures of 28/20 ◦C and relative humidity of 55% ± 5%. High-pressure sodium lamps (Master son-t; Philips Lighting N.V., Eindhoven, The Netherlands) provided a 12-h photoperiod with photosynthetic photon flux density (PPFD) of 600 µmol photosynthetically active radiation (PAR) m −2 s −1 at the top of the canopy. The plants were watered every second day on the soil surface until drainage for 30 days, and the side shoots were trimmed to maintain single-stemmed plants. At 30 days after planting (DAP), treatments lasting 60 days were initiated. There were three irrigation doses (30%, 60%, 100% pot capacity) in all four experiments that were further split into a range of K (potassium chloride (KCl); Sigma-Aldrich Chemie GmbH, Munich, Germany) concentrations of 0.01 mM (EXP. I, III, IV), 1 mM (EXP. I, II, III, IV), 4 mM (EXP. II, IV), 16 mM (EXP. I, II, III), and 32 mM (EXP. III, IV) of irrigation water. Plants were watered every second day with full-strength Hoagland solution [35] in which the K concentration was modified. The experiments were arranged in a completely randomized design with four (EXP. I, in total 36 pots) to eight (EXP. II, III, IV, in total 72 pots each) replicates.

#### *2.2. Measurements*

Morpho-physiological traits were measured at 15-day intervals, beginning 30 DAP between 11:00 and 13:00 h from the three uppermost fully expanded leaves of each plant. The leaf temperature was measured, using an infrared thermometer (Fluke 574; Fluke Corporation, Everett, WA, USA). The chlorophyll content was measured with an Apogee MC-100 meter (Apogee Instruments, Logan, UT, USA). Net photosynthesis and stomatal conductance were measured with a portable photosynthesis meter (LI-6400; LI-COR, Lincoln, NE, USA). The plant height was measured from the soil level to the

tip of the plant. The leaf water potential (Ψ*w*) was measured according to McCutchan and Shackel [36] by first covering the leaflets with bags made of black plastic on the inside and aluminum foil on the outside to prevent leaf transpiration. After 1 h, the leaflets were detached and leaf Ψ*w* was measured, using a pressure chamber (Soilmoisture Equipment Corp; Goleta, CA, USA). The osmolality was analyzed from the leaves used for the Ψ*w* measurements, using a freezing-point depression osmometer (Micro-Osmometer 3300 M; Advanced Instruments, Norwood, MA, USA) as described by Mäkelä et al. [37]. The osmotic potential (Ψ*s*) was calculated from the osmolality values as π (osmotic pressure, MPa) = *cRT*, where *RT* (*R* is the gas constant (J mol–1 K–1), *T* is absolute temperature (K)) is 2.48 and *c* the osmolality (osmol kg–1). Leaf turgor (Ψ*p*) was estimated as Ψ*w*–Ψ*s* . The pots were weighed every second day before irrigation and K treatment application to monitor water usage.

The plants were harvested at 90 DAP by cutting the stems at the soil surface. Leaves were detached from the plants, and the roots were carefully washed with water to remove soil. The fresh weight of the leaves, stems, and roots was recorded. The green and senescent leaves were then separated, and the green leaf area was measured with a portable leaf area meter (LI-3000; LI-COR, Lincoln, NE, USA). The green leaves and tuberous roots were divided into two subsamples. One subsample of each was snap-frozen in liquid N2 and stored at −20 ◦C until further analysis. The other subsample was dried in a forced-air chamber at 70 ◦C for 72 h, weighed, ground to pass a 0.5-mm sieve using a centrifugal mill (ZM200; Retsch, Haan, Germany) and stored at room temperature until further analysis.

#### *2.3. Potassium Analysis*

The K content was analyzed from the ground leaf and root subsamples. Plant material (250 mg) was weighed into polytetrafluoroethylene (PTFE) Teflon tubes (CEM Corp; Matthews, NC, USA) and 6 mL of 15.2 M nitric acid (68% *w*/*v*; VWR International BVBA, Leuven, Belgium) and 1 mL of 9.8 M hydrogen peroxide (30% *w*/*v*; Merck KGaA, Darmstadt, Germany) were added for microwave digestion (MARS 240/50; MARSXpress, CEM). The digested samples were filtered through paper (Whatman grade no. 42, pore size 2.5 µm; GE Healthcare, Gloucester, Cheltenham, UK), diluted in purified water and stored at −20 ◦C. Elemental analysis was conducted with an inductively coupled plasma-optical emission spectrometer (iCAP 6200; Thermo Fisher Scientific, Cambridge, UK) with every 20th sample as standard.

#### *2.4. Statistical Analyses*

Data of the four experiments were combined and analyzed as one experiment, after subjecting to contrast analysis for experimental differences. To show the effects of irrigation doses, K concentrations, and their interactions as fixed effects on traits measured, a two-way ANOVA was carried out. Differences were considered significant when the *p*-values were <0.05, and means were compared using Tukey's multiple range test. In addition, a two-tailed Pearson correlation was calculated to measure the patterns of relationship among the traits measured. All statistical analyses were carried out using R program (version 3.5.1; R Development Core Team, Vienna, Austria) [38].

#### **3. Results**

#### *3.1. Physiological Parameters*

The irrigation and K doses showed significant interactive effects on leaf Ψ*w* and Ψs (Figure 1). When irrigation dose was decreased to 30% together with 0.01 mM K, leaf Ψ*w* and Ψ*s* were each lowered to −2.7 MPa by 90 DAP but increasing the K concentration to 32 mM increased leaf Ψ*w* to −1.6 MPa and leaf Ψ*s* to −1.8 MPa. Conversely, when irrigation dose was decreased to 60% together with 0.01 mM K, leaf Ψ*w* and Ψ*s* were each lowered to –2.3 MPa; however, increasing the K concentration to 32 mM increased leaf Ψ*w* to −0.8 MPa and leaf Ψ*s* to −1.3 MPa by 90 DAP. In general, leaf Ψ*w* and Ψ*s* values remained maximum in full irrigated plants together with 32 mM K.

The different irrigation and K doses significantly affected leaf Ψ*p* but showed no interactive effects (Table 1). Leaf Ψ*p* greatly reduced when irrigation dose was decreased to 30% rather than to 60%, compared with full-irrigated plants (100%). Notably, increasing the K concentration to either 16 or 32 mM resulted in increased Ψ*p* by 90 DAP, regardless of the irrigation doses. In addition, the Ψ*p* loss point or the critical potential was observed at 90 DAP, where leaf Ψ*p* was greatly reduced when irrigation dose was decreased to 30% and K concentration was 0.01 mM. *Agronomy* **2020**, *10*, 321 4 of 13 The different irrigation and K doses significantly affected leaf *Ψp* but showed no interactive effects (Table 1). Leaf *Ψp* greatly reduced when irrigation dose was decreased to 30% rather than to 60%, compared with full-irrigated plants (100%). Notably, increasing the K concentration to either 16 or 32 mM resulted in increased *Ψp* by 90 DAP, regardless of the irrigation doses. In addition, the *Ψp* loss point or the critical potential was observed at 90 DAP, where leaf *Ψp* was greatly reduced when

**Table 1.** Leaf turgor (Ψ*p*) of young cassava plants in response to deficit irrigation and K fertigation. The treatments were initiated 30 days after planting and lasted 60 days. The data from four separate experiments were combined and shown as the means ± standard error of 4–16 replicate plants. irrigation dose was decreased to 30% and K concentration was 0.01 mM. **Table 1.** Leaf turgor (*Ψp*) of young cassava plants in response to deficit irrigation and K fertigation. The treatments were initiated 30 days after planting and lasted 60 days. The data from four separate

Means followed by different letters in the same column are different (Tukey's test *p* < 0.05). DAP = days after planting; I = irrigation; K = potassium; S.E.M. = standard error of the mean; df = degrees of freedom. days after planting; I = irrigation; K = potassium; S.E.M. = standard error of the mean; df = degrees of freedom.

**Figure 1.** Leaf water potential *Ψw* (**A**), leaf osmotic potential *Ψs* (**B**), and water usage (**C**) of young cassava plants in response to deficit irrigation and K fertigation. The treatments were initiated 30 days after planting and lasted 60 days. The data from four separate experiments were combined and shown as the means ± standard error of 4 to 16 replicate plants. **Figure 1.** Leaf water potential Ψ*w* (**A**), leaf osmotic potential Ψ*s* (**B**), and water usage (**C**) of young cassava plants in response to deficit irrigation and K fertigation. The treatments were initiated 30 days after planting and lasted 60 days. The data from four separate experiments were combined and shown as the means ± standard error of 4 to 16 replicate plants.

Plant water usage followed a trend similar to that observed in leaf Ψ*w* (Figure 1)**.** Decreasing irrigation dose to 30% together with 0.01 mM K caused an 80% reduction in water usage between 30 and 90 DAP. However, when the K concentration was increased to 32 mM, water usage reduced by 45% compared with full-irrigated plants. Notably, decreasing irrigation dose to 60% together with 16 mM K only caused a 13% reduction in water usage compared with full-irrigated plants. Plant water usage followed a trend similar to that observed in leaf *Ψw* (Figure 1)**.** Decreasing irrigation dose to 30% together with 0.01 mM K caused an 80% reduction in water usage between 30 and 90 DAP. However, when the K concentration was increased to 32 mM, water usage reduced by 45% compared with full-irrigated plants. Notably, decreasing irrigation dose to 60% together with 16 mM K only caused a 13% reduction in water usage compared with full-irrigated plants. Moreover, the irrigation and K doses showed significant interactive effects on net

Moreover, the irrigation and K doses showed significant interactive effects on net photosynthesis, stomatal conductance, leaf temperature, and leaf chlorophyll (Figures 2 and 3). Decreasing irrigation dose to 30% together with 0.01 mM K reduced the net photosynthesis and stomatal conductance by 70% and leaf chlorophyll by 51% between 30 and 90 DAP. However, when the K concentration was increased to 16 mM, the net photosynthesis reduced by 35%, stomatal conductance by 41%, and leaf chlorophyll by 34% between 30 and 90 DAP, compared with full-irrigated plants. In contrast, decreasing irrigation dose to 60% together with 0.01 mM K reduced net photosynthesis by 56%, stomatal conductance by 42%, and leaf chlorophyll by 38% between 30 and 90 DAP. Moreover, increasing the K concentration to 16 mM reduced the net photosynthesis by 21%, stomatal conductance by 19%, and leaf chlorophyll by 18%. In all, decreasing irrigation dose to 60% together with 16 mM K resulted in the least reduction in net photosynthesis and stomatal conductance, although the least reduction in leaf chlorophyll was observed when irrigation dose was decreased to 60% together with 32 mM K. photosynthesis, stomatal conductance, leaf temperature, and leaf chlorophyll (Figures 2 and 3). Decreasing irrigation dose to 30% together with 0.01 mM K reduced the net photosynthesis and stomatal conductance by 70% and leaf chlorophyll by 51% between 30 and 90 DAP. However, when the K concentration was increased to 16 mM, the net photosynthesis reduced by 35%, stomatal conductance by 41%, and leaf chlorophyll by 34% between 30 and 90 DAP, compared with fullirrigated plants. In contrast, decreasing irrigation dose to 60% together with 0.01 mM K reduced net photosynthesis by 56%, stomatal conductance by 42%, and leaf chlorophyll by 38% between 30 and 90 DAP. Moreover, increasing the K concentration to 16 mM reduced the net photosynthesis by 21%, stomatal conductance by 19%, and leaf chlorophyll by 18%. In all, decreasing irrigation dose to 60% together with 16 mM K resulted in the least reduction in net photosynthesis and stomatal conductance, although the least reduction in leaf chlorophyll was observed when irrigation dose was decreased to 60% together with 32 mM K.

**Figure 2.** Net photosynthesis *PN* (**A**), stomatal conductance *gs* (**B**), and leaf temperature (**C**) of young cassava plants in response to deficit irrigation and K fertigation. The treatments were initiated 30 days after planting and lasted 60 days. The data from four separate experiments were combined and shown as the means ± standard error of 4 to 16 replicate plants. **Figure 2.** Net photosynthesis *PN* (**A**), stomatal conductance *gs* (**B**), and leaf temperature (**C**) of young cassava plants in response to deficit irrigation and K fertigation. The treatments were initiated 30 days after planting and lasted 60 days. The data from four separate experiments were combined and shown as the means ± standard error of 4 to 16 replicate plants.

The leaf temperature increased when the irrigation dose was decreased, but the leaf temperature declined when the K concentration was increased (Figure 2). At 90 DAP, the leaf temperature was highest (34 °C) when irrigation dose was decreased to 30% together with 0.01 mM K, but when the K was increased to 16 mM, the leaf temperature was low (26 °C). Likewise, when irrigation dose was The leaf temperature increased when the irrigation dose was decreased, but the leaf temperature declined when the K concentration was increased (Figure 2). At 90 DAP, the leaf temperature was highest (34 ◦C) when irrigation dose was decreased to 30% together with 0.01 mM K, but when the K was increased to 16 mM, the leaf temperature was low (26 ◦C). Likewise, when irrigation dose was

decreased to 60% together with 0.01 mM K, the leaf temperature was 28 °C, but when K was increased

decreased to 60% together with 0.01 mM K, the leaf temperature was 28 ◦C, but when K was increased to either 16 or 32 mM, the leaf temperature was 26 ◦C. In all, decreasing irrigation dose to 60% together with 32 mM K showed the lowest leaf temperature (25 ◦C) between 30 and 90 DAP compared with full-irrigated plants. *Agronomy* **2020**, *10*, 321 6 of 13 to either 16 or 32 mM, the leaf temperature was 26 °C. In all, decreasing irrigation dose to 60% together with 32 mM K showed the lowest leaf temperature (25 °C) between 30 and 90 DAP compared with full-irrigated plants. to either 16 or 32 mM, the leaf temperature was 26 °C. In all, decreasing irrigation dose to 60% together with 32 mM K showed the lowest leaf temperature (25 °C) between 30 and 90 DAP compared with full-irrigated plants.

*Agronomy* **2020**, *10*, 321 6 of 13

**Figure 3.** Leaf chlorophyll content of young cassava plants in response to deficit irrigation and K fertigation. Treatments were initiated 30 days after planting and lasted 60 days. The data from four separate experiments were combined and shown as the means ± standard error of 4 to 16 replicate **Figure 3.** Leaf chlorophyll content of young cassava plants in response to deficit irrigation and K fertigation. Treatments were initiated 30 days after planting and lasted 60 days. The data from four separate experiments were combined and shown as the means ± standard error of 4 to 16 replicate plants. fertigation. Treatments were initiated 30 days after planting and lasted 60 days. The data from four separate experiments were combined and shown as the means ± standard error of 4 to 16 replicate plants.

**Figure 3.** Leaf chlorophyll content of young cassava plants in response to deficit irrigation and K

#### plants. *3.2. Growth Parameters 3.2. Growth Parameters*

*3.2. Growth Parameters*  The irrigation and K doses showed interactive effects on plant height (Figure 4). The plants were 49% shorter by 90 DAP when irrigation dose was decreased to 30% but 27% shorter when irrigation dose was decreased to 60%, compared with full-irrigated plants. Notably, lowering the K concentration below 16 mM resulted in shorter plants, regardless of the irrigation doses. The plants were 72% shorter when irrigation dose was decreased to 30% together with 0.01 mM K but were 52% shorter when K was increased to 16 mM K by 90 DAP, compared with full-irrigated plants. The smallest difference in heights was obtained when irrigation dose was decreased to 60% together with The irrigation and K doses showed interactive effects on plant height (Figure 4). The plants were 49% shorter by 90 DAP when irrigation dose was decreased to 30% but 27% shorter when irrigation dose was decreased to 60%, compared with full-irrigated plants. Notably, lowering the K concentration below 16 mM resulted in shorter plants, regardless of the irrigation doses. The plants were 72% shorter when irrigation dose was decreased to 30% together with 0.01 mM K but were 52% shorter when K was increased to 16 mM K by 90 DAP, compared with full-irrigated plants. The smallest difference in heights was obtained when irrigation dose was decreased to 60% together with 32 mM K, compared with full-irrigated plants. The irrigation and K doses showed interactive effects on plant height (Figure 4). The plants were 49% shorter by 90 DAP when irrigation dose was decreased to 30% but 27% shorter when irrigation dose was decreased to 60%, compared with full-irrigated plants. Notably, lowering the K concentration below 16 mM resulted in shorter plants, regardless of the irrigation doses. The plants were 72% shorter when irrigation dose was decreased to 30% together with 0.01 mM K but were 52% shorter when K was increased to 16 mM K by 90 DAP, compared with full-irrigated plants. The smallest difference in heights was obtained when irrigation dose was decreased to 60% together with 32 mM K, compared with full-irrigated plants.

**Figure 4.** Plant height of young cassava plants in response to deficit irrigation and K fertigation. The treatments were initiated 30 days after planting and lasted 60 days. The data from four separate **Figure 4.** Plant height of young cassava plants in response to deficit irrigation and K fertigation. The treatments were initiated 30 days after planting and lasted 60 days. The data from four separate experiments were combined and shown as the means ± standard error of 4 to 16 replicate plants. **Figure 4.** Plant height of young cassava plants in response to deficit irrigation and K fertigation. The treatments were initiated 30 days after planting and lasted 60 days. The data from four separate experiments were combined and shown as the means ± standard error of 4 to 16 replicate plants.

experiments were combined and shown as the means ± standard error of 4 to 16 replicate plants. The plant leaf area, tuber number, leaf dry mass, stem dry mass, root dry mass, and whole-plant dry mass differed significantly with the irrigation and K doses and their interactions (Table 2). Decreasing irrigation doses substantially reduced these parameter values, while increasing the K increased their values. The plant leaf areas were 17% smaller and whole-plant dry mass was 41% lower when irrigation dose was decreased to 30% together with 16 mM K, compared with fullirrigated plants. In contrast, the plant leaf areas were 8% smaller and whole-plant dry mass was 13% lower when irrigation dose was decreased to 60% together with 16 mM K. This treatment The plant leaf area, tuber number, leaf dry mass, stem dry mass, root dry mass, and whole-plant dry mass differed significantly with the irrigation and K doses and their interactions (Table 2). Decreasing irrigation doses substantially reduced these parameter values, while increasing the K increased their values. The plant leaf areas were 17% smaller and whole-plant dry mass was 41% lower when irrigation dose was decreased to 30% together with 16 mM K, compared with fullirrigated plants. In contrast, the plant leaf areas were 8% smaller and whole-plant dry mass was 13% lower when irrigation dose was decreased to 60% together with 16 mM K. This treatment combination showed the least decrease compared with full-irrigated plants. Otherwise, the largest leaf areas were obtained in full-irrigated plants together with 16 or 32 mM K, which were similar, The plant leaf area, tuber number, leaf dry mass, stem dry mass, root dry mass, and whole-plant dry mass differed significantly with the irrigation and K doses and their interactions (Table 2). Decreasing irrigation doses substantially reduced these parameter values, while increasing the K increased their values. The plant leaf areas were 17% smaller and whole-plant dry mass was 41% lower when irrigation dose was decreased to 30% together with 16 mM K, compared with full-irrigated plants. In contrast, the plant leaf areas were 8% smaller and whole-plant dry mass was 13% lower when irrigation dose was decreased to 60% together with 16 mM K. This treatment combination showed the least decrease compared with full-irrigated plants. Otherwise, the largest leaf areas were obtained in full-irrigated

combination showed the least decrease compared with full-irrigated plants. Otherwise, the largest leaf areas were obtained in full-irrigated plants together with 16 or 32 mM K, which were similar, whereas the highest whole-plant dry mass was obtained in full-irrigated plants with 32 mM K.

whereas the highest whole-plant dry mass was obtained in full-irrigated plants with 32 mM K.

plants together with 16 or 32 mM K, which were similar, whereas the highest whole-plant dry mass was obtained in full-irrigated plants with 32 mM K.

**Table 2.** Plant leaf area, leaf dry mass, shoot dry mass, root dry mass, and whole-plant dry mass of 90-day-old cassava plants in four pot experiments. Deficit irrigation and K fertigation were initiated 30 days after planting and lasted 60 days. The data from separate experiments were combined and shown as the means of 4 to 16 replicate plants.

Means followed by different letters in the same column are different (Tukey's test *p* < 0.05). S.E.M. = standard error of the mean; df = degrees of freedom; I = irrigation; K = potassium.

The leaf, stem, and root dry mass followed trends similar to that observed with whole-plant dry mass. Decreasing irrigation dose to 30% together with 16 mM K resulted in fewer tubers. Conversely, the tubers were much fewer when the K concentration was lowered below 16 mM. Substantially high numbers of tubers were obtained when irrigation dose was decreased to 60% together with 16 or 32 mM K, which did not vary.

#### *3.3. Correlation of Physiological and Growth Traits*

Statistically significant relationships were observed among the traits measured (Table 3). Net photosynthesis was highly correlated (0.906 ≤ *r* ≤ 0.980; *p* < 0.05) with stomatal conductance, leaf Ψ*w*, leaf Ψ*s* , leaf chlorophyll, water usage, and whole-plant dry mass. Positive associations (0.719 ≤ *r* ≤ 0.897; *p* < 0.05) were also found between plant leaf area, plant height, tuber number, and water usage. However, the leaf temperature correlated negatively (−0.923 ≤ *r* ≤ −0.772; *p* < 0.05) with all traits measured.

*Agronomy* **2020**, *10*, 321

\*\*: differences between traits are significant at *p* < 0.01. LDM = leaf dry mass; RDM = root dry mass; WPD = whole-plant dry mass; TBN = tuber number; LA = leaf area; PLH = plant height; Chl = leaf chlorophyll; LT = leaf temperature; PN = net photosynthesis; gs = stomatal conductance; Ψ*w* = leaf water potential; Ψ*s* = leaf osmotic potential; Ψ*p* = leaf turgor; WUS = water usage.

#### *3.4. Potassium Content*

The irrigation and K doses significantly affected the K content but showed no interactive effects (Table 4). When irrigation dose was decreased to 30%, the K content in the roots was 14% lower, but when irrigation dose was decreased to 60%, the K content in the roots was 9% lower. Increasing the K concentration to 32 mM resulted in the highest K content in both the leaves and roots. In general, K contents were all higher in the leaves than in the roots.

**Table 4.** Potassium (K) content in the leaves and roots of 90-day-old cassava plants in four pot experiments. Deficit irrigation and K fertigation were initiated 30 days after planting and lasted 60 days. The data from separate experiments were combined and shown as the means of 4 to 16 replicate plants.

Means followed by different letters in the same column are different (Tukey's test *p* < 0.05). S.E.M. = standard error of the mean; df = degrees of freedom.

#### **4. Discussion**

Our findings show that water deficit and the K concentrations influence the water status of young cassava plants, thereby affecting the leaf gas exchange and causing a decline in growth and yield. Decreasing irrigation dose to 30% together with 0.01 mM K lowered leaf Ψ*w* and Ψ*s* greatly, while leaf Ψ*p* was five times lower at the end of our experimental period (90 DAP) compared with full-irrigated plants (100%). The decrease in Ψ*s* in response to the water stress imposed is an osmotic adjustment mechanism used by most plants to adjust to water-limited environments [39] and contributes to Ψ*p* maintenance at low Ψ*w* [40,41]. Osmotic adjustment enables leaf Ψ*p* maintenance for the same leaf Ψ*w*, thus supporting stomatal conductance [42], and improves root capacity for water uptake [43]. Moreover, our results showed that increasing the K concentration to 32 mM increased both leaf Ψ*w* and Ψ*s* . This effect could have been related to the high K+ solute concentration in the leaves, which seemed to increase the Ψ*s* , as evident from elemental K content analyzed in the leaves. Furthermore, K is one of the primary osmotic solutes that contribute to osmotic adjustment in plants by altering the Ψ*s* and enabling plant cells to retain water and maintain Ψ*p* [44,45].

Reduction in water usage parallelled the decrease in leaf Ψ*w*. Significant positive correlations (*r* = 0.923) observed between water usage and stomatal conductance suggest that the reduction in water usage was probably induced by stomatal closure. Cassava responds to initial water deficit by partial stomatal closure and nearly complete stomatal closure during extreme water deficit [8,17]. Duque and Setter [46] suggested that stomatal closure protects the leaf from severe water loss and protects photosynthetic systems and cellular structures from irreversible damage. Our results showed that effective water usage was greatest when irrigation dose was decreased to 60% together with 32 mM K, given the high amount of dry mass observed relative to full-irrigated plants. High dry mass production under water deficit is achieved when plants divert a large portion of available soil moisture towards stomatal conductance [47]. The water usage and stomatal conductance data further suggest

that partial stomatal closure occurred at about 60 DAP when the water level was decreased to 30% or 60% of pot capacity. The marked increase in water usage, regardless of the irrigation doses when the K concentration was increased to 32 mM, suggests that high levels of K increased the leaf Ψ*s* and consequently increased Ψ*w*, resulting in Ψ*p* maintenance and increased water usage.

The observed depletions in net photosynthesis was significantly associated with a significant reduction in stomatal conductance. Our results showed that the decline in net photosynthesis was more pronounced when irrigation dose was decreased to 30% together with 0.01 mM K, but less pronounced when irrigation dose was decreased to 60% of pot capacity. Even more, the strong positive correlation observed between net photosynthesis and stomatal conductance (*r* = 0.939) and leaf Ψ*w* (*r* = 0.941) suggests that photosynthesis was limited by stomatal closure in response to a limited water supply. The decline in leaf chlorophyll due to decreased irrigation doses additionally limited photosynthesis. This decline in leaf chlorophyll is considered a non-stomatal limiting factor [48] and is prevalent under water-deficit conditions, causing decreased photosynthetic activity [49]. Chlorophyll degradation also occurs in K-deficient plants [25], which further inhibits photosynthesis. Conversely, low-K nutrition diminishes Hill reaction activity [50] and the rate of production of adenosine triphosphate (ATP) and reduces nicotinamide adenine dinucleotide phosphate (NADP) in chloroplasts [51], resulting in reduced net photosynthesis. In contrast, increasing the K concentration to 16 mM increased net photosynthesis by increasing leaf Ψ*w* and leaf Ψ*p*, which in turn increased stomatal conductance, resulting in increased net photosynthesis. Moreover, Ψ*p* determines stomatal aperture and closure [52,53], and the extent of stomatal closure in cassava during water deficit levels corresponds to the decline in net photosynthesis [8,54]. The enhanced need for K by plants under water deficit appears to be related to the fact that K is required for the maintenance of photosynthetic CO2 fixation [25].

The leaf temperatures increased above the ambient greenhouse temperature (27 ◦C) when irrigation doses were decreased. Our results showed that increased leaf temperature corresponded to decreased net photosynthesis as indicated by the significant negative correlations between leaf temperature and net photosynthesis (*r* = −0.912), and between leaf temperature and leaf Ψ*w* (*r* = −0.898). This observation implies that the rise in leaf temperature was occasioned by stomata closure in response to a decline in Ψ*w*. High leaf temperature leads to heat stress, and photosynthesis is more sensitive to heat stress under water deficit [55]. Thus, C3–C4 plants such as cassava rely on evaporative cooling to lower leaf temperature [56]. Despite decreased irrigation doses, increasing the K concentration to 16 mM and above appeared to lower leaf temperature. This was attributed to the role of high K concentration, which increased stomatal conductance and, thus, prevented leaf temperature from reaching harmful levels [57].

Plant leaf area and plant height were greatly reduced when irrigation dose was decreased to 30% together with 0.01 mM K. These reductions could be linked to the decrease observed in leaf Ψ*w*, low Ψ*p*, decreased stomatal conductance, and declined net photosynthesis. Raza et al. [58] showed that water deficit initiates a series of biochemical and physiological processes that result in a reduction in crop growth and yield. The small leaf areas (0.37 m2 ) and short plants (56 cm) observed at the end of the experiment when irrigation dose was decreased to 30% together with 0.01 mM K were probably due to the effects of low leaf Ψ*p*. In comparison, plant leaf areas and plant height increased due to increased Ψ*p* when irrigation dose was decreased to 60% of pot capacity. These observations concur with Alves and Setter [9], who found decreased leaf area expansion in cassava 8 days after initiation of water deficit. Nesreen et al. [59] reported reduced leaf area, plant height, and stem diameter in cassava subjected to water deficit under greenhouse conditions. Moreover, optimal leaf area development is important to photosynthesis and dry mass yield [60].

Leaf dry mass, root dry mass, whole-plant dry mass, and tuber number were equally reduced when irrigation dose was decreased to 30% together with 0.01 mM K. These reductions can be largely attributed to the decline in net photosynthesis and reduced leaf areas that were influenced by the low Ψ*w*, Ψ*s* , and Ψ*p*. Duque and Setter [46] reported a 78% loss in total plant dry weight in cassava after 31-days of water deficit treatment. El-Shakwy and Cadavid [61] observed reductions in leaf

area index and shoot and root biomass when they initiated water stress in three cassava cultivars for 2 to 8 months after planting. Nevertheless, increasing the K concentration to 16 mM improved the growth and yield of young cassava plants, regardless of decreased irrigation doses. This could be ascribed to the high levels of K that reduced the negative effects on plant-water relationships and improved net photosynthesis. This observation agrees with the findings of Mengel and Arneke [44], who reported improved water status and high dry mass in cassava supplied with high levels of K (4 mM) in comparison to low-K (0.1 mM) treatments. There were no tubers when irrigation dose was decreased to 30% together with 0.01 mM K, whereas four tubers were observed when K was increased to 16 mM. High levels of K increase the translocation of photosynthates to the storage roots [62], and the photosynthates initiate cassava tuberous root growth during the early growth phase [8,21].

The critical nutrient contents of K observed in this investigation fall within the sufficient range as those obtained by Nguyeh et al. [63] in field-grown cassava at four months after planting. The decreased K contents observed when irrigation doses were decreased were probably due to the limited water supply, which possibly limited the K uptake from the rhizosphere. Plants experiencing both water and K deficiency tended to show decreases in K accumulation rates, while those grown in K-rich soil maintained high rates of K accumulation during most of the season [64]. Moreover, our findings show that increasing K from 16 to 32 mM did not result in additional growth and yield benefits, regardless of the irrigation doses. Thus, it appears that 32 mM K was excessive and could have limited the uptake of other cations, creating a nutrient imbalance [65].

#### **5. Conclusions**

Our investigation showed significant interactions between deficit irrigation and K, whereby decreasing irrigation dose to 60% together with 16 mM K resulted in the least reduction in growth and yield. Thus, it seems that deficit irrigation strategies could be used as a tool to develop management practices to improve cassava productivity by means of K fertigation under low moisture field conditions. The experiments allowed analytical investigation of the effects of the irrigation and K doses without interference from underlying abiotic and biotic factors. Nevertheless, the present investigation had certain limitations such as the use of pots, which could have constrained plant growth, and the use of a controlled greenhouse environment, which varies from field environments. Therefore, further tests with several cassava cultivars under field conditions are warranted to compare these findings.

**Author Contributions:** Conceived and designed the experiment D.O.W., P.S.A.M., and J.K.; contributed the reagents/materials P.S.A.M.; conducted the experiments D.O.W.; supervised the research P.S.A.M., J.K., and L.A.; performed the statistical analysis D.O.W., and P.S.A.M.; wrote the first draft of the manuscript D.O.W. All authors have read and agreed to the published version of the manuscript.

**Funding:** This work was supported by CIMO Finland (TM-17-10552) and the Ella and Georg Ehrnrooth Foundation. The open access publication fee was covered by the University of Helsinki.

**Acknowledgments:** We gratefully acknowledge Markku Tykkyläinen, Afrane Yaw, and Marjo Kilpinen for their technical assistance with the greenhouse and laboratory activities. We thank the Kenya Agricultural and Livestock Research Organization (KALRO) for providing the cassava cuttings used in this investigation.

**Conflicts of Interest:** The authors declare no conflict of interest. The funders played no role in the design of the study; in the collection, analyses, or interpretation of data; in the writing of the manuscript or in the decision to publish the results.

#### **References**

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*2.3. Knockout of GPR4 Decreases the Bax*/*Bcl-2 mRNA Ratio during Neurotoxin-Induced Apoptosis in SH-SY5Y Cells*

To determine the role of GPR4 in both MPP+- (1 mM) and H2O2- (125 μM) stimulated apoptotic cell death, we investigated the expression levels of the Bcl-2 family proteins (Bax and Bcl-2). Many studies suggest that the Bcl-2 family plays a critical role in the mitochondrial apoptotic pathway. Bax enhances the release of cytochrome C from the space of the mitochondrial intermembrane to the cytosol, resulting in apoptosis. In contrast, Bcl-2 prevents apoptosis through its prevention of cytochrome C release, thereby maintaining mitochondrial cellular integrity [29,30]. In this study, an RT-PCR was employed to assess the mRNA expression levels of GPR4, Bax, and Bcl-2 in 24 h serum-starved SH-SY5Y cells treated with either MPP+ (1 mM) or H2O2 (125 μM; Figure 3A).

**Figure 3.** The measurement of GPR4 mRNA expression and the Bax/Bcl-2 mRNA ratio in MPP+- and H2O2-treated SH-SY5Y cells that were stably GPR4-OE or GPR4-KO. (**A**) An RT-PCR illustrating the mRNA expression of pro-apoptotic Bax, anti-apoptotic Bcl-2, and GAPDH in SH-SY5Y, GPR4-OE, and GPR4-KO cells after stimulation with MPP+ (1 mM) and H2O2 (125 μM) in serum-free media for 24 h. (**B**) A semi-quantification of the GPR4 mRNA and Bax/Bcl-2 mRNA expressions relative to GAPDH. This semi-quantification of the respective mRNA expression levels was performed on ImageJ software; GAPDH was utilised as an internal control. Mean ± SEM (*n* = 3) was employed to express the data. Tukey's multiple comparison test was performed using a one-way ANOVA. Each \* *p* < 0.05 refers to the sample concentration compared with the same group of non-treated cells.

A semiquantitative analysis (Figure 3B) of the RT-PCR bands highlighted a more than 4-fold increase in the expression of GPR4 in the GPR4-OE cells without any treatment, compared with the non-treated SH-SY5Y cells. In comparison with the non-treated SH-SY5Y cells, neurotoxins increased the expression of GPR4 in the GPR4-OE cells by 3–4-fold (MPP+, 3.65 ± 0.03; H2O2, 3.31 ± 0.17), whereas no significant difference in the GPR4 expression of the GPR4-OE cells (MPP+, 0.53 ± 0.003; H2O2, 0.04 ± 0.003) was observed.

Interestingly, the ratio of Bax/Bcl-2 mRNA expression for non-treated GPR4-OE cells was slightly higher (1.40 ± 0.08) than that for the control SH-SY5Y cells, whereas the Bax/Bcl-2 mRNA ratio was slightly lower (0.73 ± 0.904) in the non-treated GPR4-KO group than that for the control SH-SY5Y cells. For the SHSY-5Y cells, treatment with MPP+ or H2O2 significantly increased the ratio of Bax/Bcl-2 mRNA expression (MPP+, 1.59 ± 0.02; H2O2, 1.55 ± 0.04). Yet, the ratio of Bax/Bcl-2 mRNA expression in MPP+- and H2O2-stimulated GPR4-OE cells was significantly higher (MPP+, 1.92 ± 0.03; H2O2, 1.88 ± 0.07) than the neurotoxin-treated SH-SY5Y and non-treated GPR4-OE cells. Meanwhile, the ratio of Bax/Bcl-2 mRNA expression in both MPP+- and H2O2-stimulated GPR4-KO cells was significantly lower (MPP+, 1.38 ± 0.02; H2O2, 1.25 ± 0.02) than both the neurotoxin-treated SH-SY5Y and GPR4-OE cells.


**1. Introduction**

Never has humanity experienced so palpably and unambiguously a feeling of collective fate. The common experience of facing danger and suffering disrupts our faith in life and evokes questions of meaning and existence.

Humanity faces the challenge of sustainability: global warming, climate change, new viruses, pandemic, and the new technology—Artificial Intelligence. From East to West, people find themselves in fear, exposed to the questions of their fate and real suffering. Nature demands that humankind join hands in the battle against the ecological problems. An awareness of the collective fate that has been forced upon us has the potential to mark a new kind of partnership for humanity as a whole.

Jewish tradition tried to teach human beings that their mission in life is to turn fate into destiny, to turn a passive existence into an active one—to move from being an object dictated to by powers greater than he or she, to a subject who determines his or

her own path and meaning in the world. The readiness of humanity to enter into the covenant of fate—the will of individuals to take responsibility for the community and join together in times of distress, out of a sense of obligation and responsibility—is worthy of honor and recognition.