*Article* **Foliar Glycine Betaine or Hydrogen Peroxide Sprays Ameliorate Waterlogging Stress in Cape Gooseberry**

### **Nicolas E. Castro-Duque, Cristhian C. Chávez-Arias \* and Hermann Restrepo-Díaz**

Departamento de Agronomía, Facultad de Ciencias Agrarias, Universidad Nacional de Colombia, Bogotá 111321, Colombia; necastrod@unal.edu.co (N.E.C.-D.); hrestrepod@unal.edu.co (H.R.-D.)

**\*** Correspondence: ccchaveza@unal.edu.co; Tel.: +57-1-316-5000 (ext. 19018)

Received: 20 April 2020; Accepted: 13 May 2020; Published: 19 May 2020

**Abstract:** Exogenous glycine betaine (GB) or hydrogen peroxide (H2O2) application has not been explored to mitigate waterlogging stress in Andean fruit trees. The objective of this study was to evaluate foliar GB or H2O2 application on the physiological behavior of Cape gooseberry plants under waterlogging. Two separate experiments were carried out. In the first trial, the treatment groups were: (1) plants without waterlogging and with no foliar applications, (2) plants with waterlogging and without foliar applications, and (3) waterlogged plants with 25, 50, or 100 mM of H2O2 or GB, respectively. The treatments in the second trial were: (1) plants without waterlogging and with no foliar applications, (2) plants with waterlogging and without foliar applications, and (3) waterlogged plants with 100 mM of H2O2 or GB, respectively. In the first experiment, plants with waterlogging and with exogenous GB or H2O2 applications at a dose of 100 mM showed higher leaf water potential (−0.5 Mpa), dry weight (1.0 g), and stomatal conductance (95 mmol·m−2·s<sup>−</sup>1) values. In the second experiment, exogenously supplied GB or H2O2 also increased the relative growth rate, and leaf photosynthesis mitigating waterlogging stress. These results show that short-term GB or H2O2 supply can be a tool in managing waterlogging in Cape gooseberry.

**Keywords:** hypoxia; leaf gas exchange; waterlogging tolerance; organic compound; plant growth; *Physalis peruviana* L.

#### **1. Introduction**

Cape gooseberry (*Physalis peruviana* L.) is a plant that belongs to the *Solanaceae* family and its center of origin is located in the Andes, specifically in Peru, from where it expanded to various areas of the tropics and subtropics [1–3]. In Colombia, the production of this crop was 16,445 t, occupying 1312 ha during 2018 [4].

Climate change and variability alter the normal rainfall cycle causing floods of agricultural land and affecting crop production [5]. In Colombia, climate variability phenomena, such as the "La Niña" phenomenon, are characterized by an increase in rainfall that enhances the probability of floods [6,7]. In 2010, La Niña phenomenon produced an increase in rainfall, exceeding historical averages and causing a decrease in agricultural production from 7888 to 1515 t in Cundinamarca, one of the main producer departments of the country [4,7,8].

It has been reported that there is a high susceptibility of cultivated plants to waterlogging stress, affecting their growth, development, yield, and finally their survival [9,10]. One of the main effects of waterlogging is on plant growth. In this regard, several authors have observed that moderate or prolonged periods of O2 deficit in the soil cause a low leaf area [11], a reduction in plant height [12], and an alteration in stem diameter [13]. In Cape gooseberry, short periods of waterlogging stress (6 days) also cause a decrease in plant height, leaf area, and stem diameter [14,15].

A reduction of growth parameters due to waterlogging may be associated with an impairment of the leaf gas exchange properties (stomatal conductance), chlorophyll content, and efficiency of photosystem II (PSII) [11,16–18]. Plants susceptible to waterlogging have been reported to show stomatal closure 24 h after the exposure to stress [19]. On the other hand, the leaf chlorophyll content can drop due to imbalances in the nutrient uptake or increased ethylene synthesis, causing impairment or decrease in the efficiency of PSII [19–21]. A previous experiment has also shown low stomatal conductance, leaf chlorophyll content, and *Fv*/*Fm* ratio in Cape gooseberry plants under moderate waterlogging periods (4 days) [15].

Waterlogging alters the plant water status due to stomatal closure [22]. The negative effects of periods of oxygen deprivation on the leaf water potential have been reported in cacao [17], bean [16], and tomato [23]. Likewise, the relative water content (RWC) has been widely used to describe the plant water status and has been correlated with the level of soil moisture [24]. In this regard, the RWC is a reliable variable to measure the susceptibility of plants to waterlogging [17,18]. These variables have also been useful to evaluate the susceptibility or efficiency of management techniques to O2 deficit conditions in the soil in Andean fruit trees such as Lulo or Cape gooseberry [15,20,25].

Exogenous applications of compounds such as glycine betaine (GB) or hydrogen peroxide (H2O2) can help tolerate or lessen negative effects on plants under abiotic stress conditions by activating defense mechanisms or aiding plant growth, development, and productivity [26,27]. Some authors have reported that physiological parameters such as leaf gas exchange properties (photosynthesis), efficiency of PSII, water relations (water potential), growth, and antioxidant activity are favored by these compounds under waterlogging stress in different cultivated species [13,28]. Glycine betaine helps plants under abiotic stress conditions by acting as an osmolyte that protects cells [29], increases cell water retention [30], reduces levels of reactive oxygen species (ROS) and helps in the protection of the plasma membrane [31]. Regarding waterlogging stress, the exogenous application of this molecule has been little studied; however, Rasheed et al. [28] reported that GB applications caused an increase in plant biomass, leaf total chlorophyll, and K<sup>+</sup> concentration compared to fully waterlogged plants.

Hydrogen peroxide is a molecule that has also been studied to mitigate the effects of abiotic stresses in crops such as potato [32], tomato [33], bean [34,35], rice [36], maize [37] and soybean [13]. Different studies have concluded that exogenous H2O2 application helps leaf gas exchange properties (stomatal conductance and photosynthesis) [13], dry matter accumulation [35], leaf relative water content, and water potential [33,34], and plant height under different abiotic stresses [34,35]. Finally, the use of H2O2 has been little studied under waterlogging conditions. However, Andrade et al. [13] reported that pretreatments with H2O2 favored the increase in plant biomass, stomatal conductance, and net photosynthetic rate in soybean.

Increases in the intensity and frequency of rainfall in Colombia are estimated for the coming years [6,38]. For this reason, studies on the acclimatization response of Andean fruit trees to waterlogging scenarios have recently gained importance [14,20,21]. However, research on agronomic strategies to mitigate the negative impact of waterlogging with foliar GB and H2O2 sprays on Andean fruit trees has yet to be explored. Rasheed et al. [28] and Andrade et al. [13] mention the positive effect of these molecules on tolerance to waterlogging stress. For this reason, the objective of this study was to evaluate the exogenous application of different doses of GB or H2O2 on the physiological behavior of Cape gooseberry plants ecotype Colombia subjected to waterlogging, to determine the best molecule and dose to use to mitigate this stress.

#### **2. Results**

#### *2.1. First Experiment: Evaluation of Di*ff*erent Doses of Glycine Betaine (GB) or Hydrogen Peroxide (H2O2) under a Waterlogging Period*

Table 1 summarizes the effect of foliar GB and H2O2 sprays on the growth parameters of Cape gooseberry plants. Control plants without waterlogging (CWoW) (not exposed to waterlogging) generally showed the highest growth parameter values throughout the experiment compared to the other treatments. In this sense, foliar GB or H2O2 sprays mainly contributed to a greater stem length in Cape gooseberry plants under waterlogging conditions at 4 Days After Waterlogging (DAW), with

approximate stem length values of 21 cm, while plants with waterlogging and without any foliar compound sprays (control with waterlogging, CWW) had a height of 16.70 cm. At 4 DAW, it was also observed that the foliar applications of both compounds at their different doses favored the leaf area, stem diameter, and shoot dry weight of waterlogged plants. At 13 DAW, the obtained results of plant growth showed that foliar GB applications at a concentration of 100 mM caused an increase mainly on stem diameter (0.53 cm), leaf area (222.56 cm2), and shoot dry weight (1.06 g) in waterlogged plants compared to the CWW (0.42 cm, 116.14 cm2, and 0.39 g, respectively). Regarding foliar H2O2 applications, this compound directly affected the plant height (22.10 cm) of waterlogged plants, while the CWW showed values of 16.56 cm. Table 2 shows how foliar GB or H2O2 applications at their different doses influenced physiological variables such as leaf temperature, stomatal conductance (*gs*), efficiency of PSII (*Fv*/*Fm*), and water potential (Ψ*wf*) in Cape gooseberry leaves at 4 and 13 DAW, respectively. It is observed that waterlogging causes a higher leaf temperature (26.89 and 28.49 ◦C) and lower *gs* (157.10 and 42.76 mmol CO2·m−2·s<sup>−</sup>1) in plants at both sampling points. Foliar GB or H2O2 applications, mainly at a dose of 100 mM, caused a reduction in leaf temperature (22.27 and 24.71 ◦C, respectively) and an increase in *gs* (180.70 and 191.78 mmol CO2·m−2·s−1, respectively) at 4 DAW, with similar values to the ones recorded for plants without waterlogging (18.85 ◦C and 194.42 mmol CO2·m−2·s<sup>−</sup>1). Similar trends were also observed for the variables previously described at 13 DAW. On the other hand, the *Fv*/*Fm* ratio was also conditioned by the treatments at both points, with the lowest ratio being obtained in the CWW treatment (around 0.6). Furthermore, foliar applications of these compounds helped to increase this ratio (~0.77). Finally, the Ψ*wf* was higher in the control without waterlogging and in the GB treatment at a dose of 100 mM, compared to the other treatments in both samples. The Waterlogging Tolerance Coefficient (WTC) was obtained only at 13 DAW (Figure 1A), observing that the foliar GB application at 100 mM caused greater tolerance to waterlogging (0.52) compared to the rest of the treatments. Then, the correlation between leaf area and WTC (*r*<sup>2</sup> = 0.96) also confirmed that the foliar GB or H2O2 sprays at a concentration of 100 mM were the best at conferring tolerance to a waterlogging condition (Figure 1B).

**Figure 1.** (**A**) Waterlogging Tolerance Coefficient (WTC) at 13 Days After Waterlogging (DAW) and (**B**) correlation between the leaf area and the WTC of Cape gooseberry (*Physalis peruviana* L.) plants ecotype Colombia subjected to a waterlogging period with treatments of control with waterlogging (CWW), 25 mM of hydrogen peroxide (H2O2), 50 mM of hydrogen peroxide (H2O2), 100 mM of hydrogen peroxide (H2O2), 25 mM of glycine betaine (GB), 50 mM of glycine betaine (GB) and 100 mM of glycine betaine (GB). Evaluated 13 days after waterlogging (DAW). Data represent the average of ten plants ± standard error per treatment (*n* = 5). Bars followed by different letters indicate statistically significant differences according to the Tukey test (*p* ≤ 0.05).



#### *Plants* **2020**, *9*, 644


