Carica papaya L. - Semantic Scholar

Scientia Horticulturae 202 (2016) 173–183

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Chemical oxygen fertilization reduces stress and increases recovery and survival of flooded papaya (Carica papaya L.) plants Qasim A. Thani a , Bruce Schaffer a,∗ , Guodong Liu b , Ana I. Vargas a , Jonathan H. Crane a a b

University of Florida, Tropical Research and Education Center, 18905 S.W. 280 Street, Homestead, FL 33031, USA Horticultural Sciences Department, University of Florida, P.O. Box 110690, Gainesville, FL 32611, USA

a r t i c l e

i n f o

Article history: Received 19 January 2016 Received in revised form 29 February 2016 Accepted 2 March 2016 Available online 12 March 2016 Keywords: Flooding Alcohol dehydrogenase Oxygen enrichment Net CO2 assimilation Stomatal conductance

a b s t r a c t In many parts of the world papaya (Carica papaya L.) is prone to hypoxic stress due to soil flooding as a result of severe storms or hurricanes. Two experiments were conducted to test the effects of root zone hypoxia on physiology, recovery and survival of papaya and to determine if negative impacts of root hypoxia can be reduced by chemically enriching the root zone with oxygen. In Experiment 1, seedlings in soil were divided into three flooding treatments: (1) 100% of roots submerged, (2) ∼75% of roots submerged, or (3) non-flooded; and three oxygen fertilization treatments: (1) 0 g CaO2 , (2) 2.28 g CaO2 g, or (3) 4.57 g CaO2 applied to the soil prior to flooding. In soil, CaO2 is broken down to H2 O2 which then releases oxygen to the rhizosphere. Therefore, in Experiment 2, plants in a hydroponic solution were divided into six treatments: (1) aeration of the hydroponic solution and no H2 O2 added to the solution, (2) no aeration and no H2 O2 added; (3) no aeration and 200 ␮l of 3% H2 O2 l−1 added daily, (4) no aeration and 500 ␮l of 3% H2 O2 l−1 added daily, (5) no aeration and 1000 ␮l of 3% H2 O2 l−1 added daily, or (6) no aeration and 2000 ␮l of 3% H2 O2 l−1 added daily. In soil, flooding of ∼75% or 100% of roots for two days decreased net CO2 assimilation (A), stomatal conductance (gs ), the leaf chlorophyll index, and the ratio of variable to maximum chlorophyll fluorescence (Fv/Fm). After plants were unflooded, these variables recovered to levels similar to those of the non-flooded treatment for plants with ∼75% of the roots submerged but did not recover in plants with 100% of the roots submerged if no CaO2 was applied to the soil. If 2.28 or 4.57 g of CaO2 was applied to the soil, A, gs , leaf chlorophyll index, and Fv/Fm recovered to values similar to those of non-flooded plants. Addition of CaO2 to the soil also minimized reductions in leaf, stem, root and plant dry weights and increased survival of plants with 100% of roots submerged. For plants in the hydroponic solution, A and gs were generally lower in the non-aerated treatments than in the aerated treatment. If 500 or 1000 ␮l H2 O2 l−1 was added to the solution, A of plants in the non-aerated solution tended to recover to levels similar to those of plants in the aerated solution. Root ADH activity tended to be greater in the non-aerated treatment with no H2 O2 added to the solution than in any of the treatments with H2 O2 added. This study demonstrated that chemical oxygen enrichment of the root zone reduces flooding stress and increases recovery and post-flooding survival of papaya. © 2016 Elsevier B.V. All rights reserved.

. 1. Introduction In several areas throughout the world, plants are subjected to low soil oxygen concentration in the root zone as a result of flooding due to severe storms or hurricanes (Schaffer, 1998; Schaffer et al., 1992). Flooding events are expected to increase globally as a result of climate change (IPCC, 2014). Papaya (Carica papaya L.) is con-

∗ Corresponding author. E-mail address: bas56@ufl.edu (B. Schaffer). http://dx.doi.org/10.1016/j.scienta.2016.03.004 0304-4238/© 2016 Elsevier B.V. All rights reserved.

sidered sensitive to low soil oxygen content (Balerdi et al., 2005). In a preliminary study, net CO2 assimilation (A) and stomatal conductance of H2 O (gs ) of papaya decreased after one day of flooding and continued to decline until plants were unflooded (Rodríguez et al., 2014). When the entire root system of papaya plants was completely submerged in water for more than two days, 11 days after they were unflooded plants permanently wilted (Rodríguez et al., 2014). Oxygen deficiency in the root zone (hypoxia) occurs at soil concentrations lower than 2 mg O2 l−1 H2 O, although the O2 concentration at which plants become hypoxic differs among plant species (Gibbs and Greenway, 2003). For example, roots of avocado (Persea americana Mill.) withstood oxygen levels of 1 mg O2

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Q.A. Thani et al. / Scientia Horticulturae 202 (2016) 173–183

Table 1 Dissolved oxygen content in Krome very gravelly soil solution for plants with ∼75% or 100% of roots submerged in H2 O, one and two days after flooding treatments were initiated. ∼75% of roots submerged

100% of roots submerged

Dissolved O2 concentration (mg l−1 )

Dissolved O2 concentration (mg l−1 )

Treatment

Day 1

Day 2

Day 1

Day 2

0 g CaO2 2.28 g CaO2 4.57 g CaO2

5.10 ± 0.41ca 5.55 ± 0.26b 6.28 ± 0.28a

5.52 ± 0.24b 3.56 ± 0.40ba 6.06 ± 0.37a

3.63 ± 0.92b 7.00 ± 0.76a 8.03 ± 1.09a

4.38 ± 0.89b 5.38 ± 1.70ba 7.19 ± 1.50a

a Data represent means ± 1 standard deviation of 5 replications per treatment. Different letters indicate significant differences (P ≤ 0.05) among CaO2 treatments according to a Waller-Duncan K-ratio Test.

l−1 H2 O in a hydroponic solution for 10 days with no root damage, whereas concentrations below 1 mg O2 l−1 H2 O or complete lack of O2 in the solution (anoxia) resulted in root damage (Curtis, 1949). There has been a considerable amount of research on physiological responses of plants to root hypoxia or anoxia. Among the earliest plant responses to low root zone oxygen content as a result of flooding are reductions in A and gs (Schaffer et al., 1992). These reductions in A and gs have been observed before visible stress symptoms occur. Thus, leaf gas exchange measurements have been useful for quantifying stress in response to soil hypoxia or anoxia caused by flooding of the root zone. Flooding has also been observed to negatively affect leaf chlorophyll content (Mielke and Schaffer, 2010) and the ratio of variable to maximum chlorophyll fluorescence (Fv/Fm) (Else et al., 2009; Mielke and Schaffer, 2010), an indication of damage to Photosystem 2 (Krause and Weis, 1991). Lack of oxygen in the rhizosphere can inhibit biochemical and physiological functions in plants. Some plant species can tolerate hypoxia in the root zone for several days whereas others tolerate root zone hypoxia for less than a few hours (Vartapetian and Jackson, 1997). The difference in tolerance to low root zone oxygen concentrations is due to variations in anatomical, morphological or metabolic responses among plant species (Vartapetian and Jackson, 1997). When oxygen content of the rhizosphere is low, a shift occurs from aerobic to anaerobic respiration (Chan and Ronald, 1992). There are two types of anaerobic respiration, fermentation and lactic acid respiration. As a result of root zone hypoxia or anoxia, lactic acid respiration can lead to a build-up of lactic acid, thereby reducing the pH of the cytoplasm (Davies et al., 1974). The resulting cytoplasmic acidosis often leads to cell death. Plants avoid cytoplasmic acidosis by shifting from lactic acid respiration to alcohol respiration with ethanol as the end product of the fermentation pathway (Roberts et al., 1982). During fermentation, acetaldehyde (the biochemical precursor to ethanol) is much more toxic to plant cells than ethanol and may result in cell death during anaerobic metabolism (Drew, 1997; Vartapetian and Jackson, 1997). The alcohol dehydrogenase (ADH) enzyme catalyzes the reduction of acetaldehyde to ethanol. When oxygen concentration in the root zone is low, ADH activity can increase. For example, flooded Trifolium subterraneum plants had 30-fold greater ADH activity than non-flooded plants (Francis et al., 1974). Increased ADH activity can improve a plant’s tolerance to hypoxia or anoxia (Chung and Ferl, 1999; Gibbs et al., 2000; KatoNoguchi, 2000; Morimoto and Yamasue, 2007; Preiszner et al., 2001). For example, Trifolium repens plants with high ADH activity under flooding stress showed greater flooding tolerance than plants with low ADH activity (Chan and Ronald, 1992). Thus ADH activity in flooded roots can be used as an indicator of potential flood tolerance. Chemical oxygen fertilization of the root zone, with slow release (solid) formulations such as calcium peroxide (CaO2 ) or magnesium peroxide (MgO2 ), or fast release (liquid) formulations such as hydrogen peroxide (H2 O2 ) or carbamide peroxide (CH4 N2 O·H2 O2 ), is a potential method of alleviating root hypoxia

(Liu and Porterfield, 2014; Liu et al., 2012, 2013). Injecting H2 O2 into the irrigation water increased oxygen in the soil (Gil et al., 2009a) as well as water use efficiency and biomass of avocado (Gil et al., 2009a). Hydrogen peroxide decomposes in the soil, releasing O2 which is needed for aerobic metabolism in the roots (Gil et al., 2009a,b). When H2 O2 comes in contact with water, it reacts to give off 0.5 mol of O2 per mole H2 O2 as shown in the equation H2 O2 + H2 O → 0.5O2 + 2H2 O (Gil et al., 2009a). In soil, solid oxygen compounds (i.e., CaO2 , MgO2 ) breakdown to H2 O2 which then provides oxygen to the rhizosphere (Liu and Porterfield, 2014). Application of MgO2 to the soil prior to flooding increased flooding tolerance of chrysanthemum by increasing A, lowering the intercellular CO2 concentration in the leaves, and increasing root dry weight (Wang and Yeh, 2015). Thus, amending soil with slow release solid oxygen compounds such as MgO2 has the potential to reduce stress caused by low oxygen concentration in the root zone. The objectives of this study were to test the hypotheses that: (1) plant stress and damage caused by flooding a portion of, or the entire root system of papaya can be reduced or alleviated by amending soil with CaO2 , a slow release solid formulation; and (2) adding H2 O2 (the breakdown product of CaO2 in soil) to a hydroponic solution can reduce stress of papaya plants caused by low oxygen in the root zone. 2. Materials and methods 2.1. Study site and plant material Two experiments were conducted in a greenhouse at the University of Florida, Tropical Research and Education Center in Homestead, Florida (25.5◦ N latitude and 85.5◦ W longitude). In the first experiment (Experiment 1) plants in soil in containers were used to test physiological plant responses to flooding and soil applications of CaO2 . The second experiment (Experiment 2) was conducted in a hydroponic solution to test: (1) physiological plant responses to different concentrations of H2 O2 (the breakdown product of CaO2 in the soil) and (2) the effects of H2 O2 on ADH activity of papaya. Growing plants in a hydroponic solution allowed for sampling of root tips free of soil damage because intact, non-damaged root-tips are necessary for accurate determination of root ADH activity. At the top of the canopy, plants in the greenhouse received 80% of outside photosynthetic photon flux (PPF) as determined with a quantum sensor and LI-1000 light meter (LiCor Instruments, Lincoln, NE, USA). In each experiment, air temperature in the greenhouse was monitored and recorded with a Hobo Pro v2 logger (Onset Computer, Bourne, Massachusetts, USA). During the first experiment, daily air temperatures ranged from 23.1 ◦ C to 36.7 ◦ C with a mean of 23.7 ◦ C. During the second experiment, air temperature ranged from 24.4 ◦ C to 32.1 ◦ C with a daily mean of 27.1 ◦ C. In each experiment, papaya (C. papaya L. cv. Red Lady) seeds were soaked in tap water for 24 h and then sown in flats containing Promix® potting medium (Premier Tech, Quebec, Canada).


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2.2. Experimental design

Fig. 1. Effect of flooding and soil application of CaO2 on net CO2 assimilation (A) of papaya (Carica papaya L.) in Krome very gravelly loam soil. Symbols represent means and error bars represent ±1 std. dev. Different letters indicate significant differences according to a repeated measures ANOVA (P ≤ 0.05).

In Experiment 1, three days after germination, each seedling was transplanted into 0.4-L plastic pots containing Krome very gravelly loam soil, classified as a loamy-skeletal, carbonatic hyperthermia lithic rendoll (Noble et al., 1996). This is the soil type in the major tropical fruit production area of southern Florida. In Experiment 2, three days after seedling emergence each seedling was transplanted (bare root) into a 5.1-cm diameter plastic mesh basket. Baskets were placed in an aeroponic system to allow for rapid root development. The aeroponic system consisted of a tank half-filled with deionized water, a misting pump, tubing and sprinklers that continuously misted the roots with 10% Hoagland solution (Hoagland and Arnon, 1950). After two weeks, plants that had at least four leaves were transferred from the aeroponic system to a hydroponic system where roots were submerged in 1000 ml of 10% Hoagland solution.

In Experiment 1, three-week-old seedlings in Krome very gravelly loam soil were divided into three flooding treatments and three CaO2 application treatments in a 3 × 3 factorial design for a total of nine treatments. Plants were flooded by submerging each plant pot into a 2.5-l plastic bucket filled with tap water to: (1) about 2 cm above the soil surface (100% of the roots submerged), (2) filling the bucket with water to cover the lower 75% of the plant pot (∼75% of the roots submerged), or (3) 0% of the roots submerged (non-flooded control). Non-flooded plants were manually irrigated daily until water started to drain from the bottom of each pot which was sufficient to keep adequate soil moisture based on previous experience. The oxygen enrichment treatments, based on the amount of CaO2 added to the soil were: (1) 0 g, (2) 2.28 g, or (3) 4.57 g CaO2 . These CaO2 concentrations were selected to provide a range of concentrations of O2 released in the soil solution based on previous experiments with six-month-old papaya plants in larger pots (Thani, 2016). Calculations were then made to account for the smaller pot size in the present experiment. CaO2 was applied evenly to the soil surface a few minutes prior to beginning the flooding treatments. Treatments were arranged in a randomized complete block design with five single-plant replications per treatment. This experimental design accounted for possible temperature variations in the greenhouse based on distance from the cooling pads. Plants were unflooded after a statistically significant difference (P ≤ 0.05) in either A or gs was observed between the non-flooded control treatment and any root submergence treatment, which occurred after 2 days of root submergence. After plants were unflooded, measurements of plant variables continued for 16 days. In Experiment 2, plants were divided into the following five treatments: (1) control, aeration of the hydroponic solution and no H2 O2 added to the solution. Tygon® tubing connected to an aquarium pump was placed into a plastic container with the hydroponic solution and roots of each plant (replication) and air was constantly bubbled into the Hoagland solution through a stone diffuser attached to the end of the tubing; (2) no aeration and no H2 O2 added to the hydroponic solution; (3) no aeration and 200 ␮l of 3% H2 O2 l−1 added to the hydroponic solution daily, (4) no aeration and 500 ␮l of 3% H2 O2 l−1 added to the hydroponic solution daily, (5) no aeration and 1000 ␮l of 3% H2 O2 l−1 added to the hydroponic solution daily, and (6) no aeration and 2000 ␮l of 3% H2 O2 l−1 added to the hydroponic solution daily. The H2 O2 concentrations chosen for this experiment were based on preliminary studies (Thani, 2016) in order to provide a wide range of H2 O2 concentrations among treatments but not cause phytotoxicity. There were five single-plant replications (blocks) per treatment for non-destructive plant measurements (A, gs leaf chlorophyll index, Fv/Fm) and an additional five single-plant replications per treatment for sampling a portion of the root tips for ADH activity. The experiment was arranged as a randomized complete block design. The treatment period was terminated when a statistically significant difference (P ≤ 0.05) in A or gs was observed between the aerated treatment and any of the non-aerated treatments. 2.3. Dissolved oxygen, temperature, pH and redox potential in the root zone The dissolved oxygen content in the soil solution (Experiment 1) or hydroponic solution (Experiment 2) for each plant was determined daily with a dissolved oxygen meter (Pro2030 dissolved oxygen/conductivity meter, YSI Inc., Yellow Springs, OH, USA). The pH of the soil or hydroponic solution was measured in all treatments with a pH probe attached to a pH/voltmeter (Accumet model 13-620-115, Fisher Scientific, Pittsburgh, PA, USA). The temperature of the hydroponic solution was measured daily in each

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Fig. 2. Effect of flooding and soil application of CaO2 on stomatal conductance (gs ) of papaya (Carica papaya L.) in Krome very gravelly loam soil. Symbols represent means and error bars represent ±1 std. dev. Different letters indicate significant differences according to a repeated measures ANOVA (P ≤ 0.05).

treatment with a temperature probe attached to a pH/voltmeter (Accumet model 13-620-115, Fisher Scientific, Pittsburgh, PA, USA). 2.4. Leaf gas exchange, chlorophyll index and chlorophyll fluorescence In each experiment, A and gs were measured daily on one newly matured, fully-expanded leaf per plant of five singleplant replications per treatment with a CIRAS-3 portable gas exchange system (PP Systems, Inc., Amesbury, MA, USA). Measurements were made at a light saturated photosynthetic photon flux (PPF) of 1000 ␮mol m−2 s−1 , a reference CO2 concentration of 390 ␮mol mol−1 and an air flow rate into the leaf cuvette of 200 ml min−1 . Leaf chlorophyll index was measured daily with a SPAD 502 meter (Konica Minolta Sensing, Osaka, Japan) on a newly matured leaf of 5 plants (replications) per treatment during the flooding period and twice weekly after plants were unflooded.

Fig. 3. Effect of flooding and soil application of CaO2 on the leaf chlorophyll index of papaya (Carica papaya L.) in Krome very gravelly loam soil. Symbols represent means and error bars represent ±1 std. dev. Different letters indicate significant differences according to a repeated measures ANOVA (P ≤ 0.05).

Fv/Fm was determined with an OS-30 hand-held chlorophyll fluorimeter (Opti-Sciences, Inc., Hudson, NH, USA) daily during the flooding period and twice weekly after plants were unflooded. 2.5. Root ADH activity In Experiment 2, during the flooding period, approximately one gram of roots was harvested daily from five plants (replications) per treatment and placed in a plastic zip-lock bag. Samples were then stored at −80 ◦ C for two to five days until ADH extraction. At the end of the experimental period final root samples were collected using the same procedures. Root ADH activity was determined according to a modification of the method described by Xie and Wu (1989). One gram


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coefficient of NADH (Liu et al., 2012; Schomburg et al., 2012). Protein content of the extract was determined based on the Bradford assay using a protein assay kit (Bio-Rad Laboratories, Hercules, CA, USA). ADH activity of the extract was expressed as nmol NADH min−1 mg protein−1 . 2.6. Plant survival, leaf area and tissue dry weight At the end of Experiment 1, the number of surviving plants and the total leaf area of each plant were determined for plants in each treatment. Plant mortality was determined by the total loss of leaves, browning of the vascular system, and the lack of any new fresh tissue for several weeks after these symptoms were observed. Leaf area was determined with a LI-1000 leaf area meter (LiCor Instruments, Lincoln, NE, USA). All plants were then harvested and roots from each plant were washed in tap water to remove the soil. All plant tissues were oven-dried at 70 ◦ C to a constant weight and then leaf, petiole, stem, root and total plant dry weights were determined. 2.7. Data analyses Statistical interactions between flooding and CaO2 treatments were determined by two-way analysis of variance (ANOVA) for all dependent variables. Net CO2 assimilation, gs , leaf chlorophyll index and Fv/Fm were analyzed by repeated measures ANOVA. ADH activity and plant dry weights were analyzed by a one-way ANOVA and a Waller-Duncan K-ratio test to determine significant differences among treatment means. All data analyses were done using the SAS 9.4 statistical software package (SAS Institute, Cary, NC, USA). 3. Results 3.1. Effects of flooding and CaO2 on plants in soil (Experiment 1)

Fig. 4. Effect of flooding and soil application of CaO2 on variable to maximum chlorophyll fluorescence ratio (Fv/Fm) of papaya (Carica papaya L.) in Krome very gravelly loam soil. Symbols represent means and error bars represent ±1 std. dev. Different letters indicate significant differences according to a repeated measures ANOVA (P ≤ 0.05).

of fresh root tips was weighed and homogenized in an extraction buffer solution composed of 50 mM Tris-HCL (pH 8.0), 1 mM EDTA, 0.5 mg ml−1 DTT, and 12 ␮M ␤-mercaptoethanol. The extraction solution was centrifuged at 4 ± 0.1 ◦ C at 10,000 rpm for 5 min and 50 ␮l of enzyme solution was added to 450 ␮l of a reaction solution composed of 50 mM Tris-HCL (pH 9.0), 1 mM EDTA and 1 mM NAD. The mixture was incubated in 1.5 ml micro centrifuge tubes in a water bath at 30 ◦ C for 3 min. Then 50 ␮l of 15% ethanol and 450 ␮l of enzyme and reaction solution were added to a cuvette. After a reaction time of 1 min, NADH concentration in the cuvette was determined with a Beckman DU-640 Spectrophotometer (Beckman Instruments, Fullerton, California, USA) at an absorbance of 340 nm. ADH activity was calculated as an average from 5 single-plant replicates using a value of 6.22 mM−1 cm−1 as the molar extinction

For plants with ∼75% of the roots submerged one day after flooding treatments began, dissolved oxygen content of the soil solution increased as CaO2 application rate increased. Two days after flooding treatments began, plants with either 2.28 or 4.57 g CaO2 applied had higher dissolved oxygen concentrations in the soil solution than plants with no CaO2 applied (Table 1). For plants in the 100% root submersion treatment, one and two days after flooding treatments were started, application of 2.28 or 4.57 g CaO2 resulted in higher dissolved oxygen contents than in the treatment with no CaO2 applied (Table 1). Addition of CaO2 to the soil raised the pH of the soil solution for plants with ∼75% or 100% of the roots submerged, but there was no significant difference in the pH of the soil solution between the 2.28 and 4.57 g CaO2 treatments (data not shown). In the ∼75% root submergence treatment, addition of either 2.28 or 4.57 g CaO2 raised the mean pH of the soil solution from 8.8 (no CaO2 applied) to 10.4 one day after flooding treatments began and from 8.7 (no CaO2 applied) to 11.0 two days after flooding treatments began (data not shown). For the 100% root submergence treatment, addition of 2.28 or 4.57 g CaO2 raised the mean pH of the soil solution from 8.7 (no CaO2 applied) to 11.0 one day after flooding treatments began and to 12.0 two days after flooding treatments began (data not shown). Regardless of the amount of CaO2 applied to the soil, A of plants with ∼75% or 100% of roots submerged was lower than that of the non-flooded treatment 48 h after flooding treatments began (Fig. 1). When no CaO2 was applied to the soil, A and gs were significantly lower in the 100% root submergence treatment than in the non-flooded treatment, even 16 days after plants were unflooded (Fig. 1). However, when 2.28 or 4.57 g CaO2 was added to the soil

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Table 2 Survival of papaya (Carica papaya L.) seedlings in Krome very gravelly loam soil with 0%, ∼75%, or 100% of roots submerged in H2 O with different concentrations of CaO2 added to the soil (Experiment 1). CaO2 application rate (g)

0 2.28 4.57

Amount of roots submerged (%) 0 Plant survival (%)

∼75

100

100 100 100

60 80 100

40 80 100

prior to flooding, A of plants in the ∼75% and 100% root submergence treatments returned to values similar to those of plants in the non-flooded treatment by 15 days after plants were unflooded (Fig. 1) and gs of plants with ∼75% or 100% of roots submerged also returned to values similar to those of plants in the non-flooded treatments by 16 days after plants were unflooded (Fig. 2). The leaf chlorophyll index was significantly lower in the ∼75% and 100% root submergence treatments than in the non-flooded treatment six days after flooding began for plants receiving 2.28 g CaO2 , and nine days after flooding began (three days after plants were unflooded) for plants in the 4.57 g CaO2 treatment (Fig. 3). When 0 or 2.28 g CaO2 was applied to the soil, the leaf chlorophyll index of the ∼75% and 100% root submergence treatments never recovered to levels similar to those of the non-flooded treatment. However, when 4.57 g CaO2 was applied to the soil, the leaf chlorophyll index returned to levels similar to those of plants in the non-flooded treatment 18 days after flooding commenced (16 days after plants were unflooded) (Fig. 3). The Fv/Fm was lower in plants in the ∼75% and 100% root submergence treatments than in the non-flooded treatment three to six days after flooding treatments began for plants in all CaO2 treatments (Fig. 4). When no CaO2 was applied to the soil, Fv/Fm of plants in the ∼75% and 100% root submergence treatments did not return the levels similar to those of the non-flooded treatment. However, 18 days after flooding treatments began (16 days after plants were unflooded), Fv/Fm of flooded plants (∼75% or 100% root submergence) with 2.28 or 4.57 g CaO2 applied was not significantly different from Fv/Fm of plants in the non-flooded treatment (Fig. 4). The total leaf area of plants with no CaO2 applied was significantly lower (P ≤ 0.05) in the 100% (9.8 cm2 ) and ∼75% (75.2 cm2 ) root submergence treatments than in the control treatment (173.9 cm2 ). When 2.28 g CaO2 was applied to the soil, the total leaf area was significantly lower (P ≤ 0.05) in the 100% root submergence treatment (38.8 cm2 ) than in the non-flooded control treatment (153.5 cm2 ). When 4.57 g CaO2 was added to the soil, there were no significant differences (P > 0.05) in total leaf area among flooding treatments. When 4.57 g CaO2 was applied to the soil, leaf areas were 166.6, 162.2 and 77.3 cm2 for the 100%, ∼75%, and 0% root submergence treatments, respectively. When no CaO2 was applied to the soil, leaf, petiole, root and total plant dry weights were lower for plants with 100% of the roots submerged than non-flooded plants (Fig. 5). Also, when no CaO2 was applied, stem dry weight was lower for plants with 100% of the roots submerged than non-flooded plants. However, when 4.57 g CaO2 was applied to the soil, there were no significant differences in leaf, petiole, stem, root, or total plant dry weight among the three flooding treatments (Fig. 5). At the end of the experiment, all non-flooded plants had survived. When no CaO2 was applied to the soil 60% of the plants survived ∼75% root submergence and 40% of plants survived 100% root submergence (Table 2). When 2.28 g CaO2 was applied to the soil, 80% of the plants survived when either ∼75% or 100% of roots were submerged. However, when 4.57 g CaO2 was applied to the

soil, 100% of the plants survived submergence of ∼75% or 100% of roots (Table 2).

Fig. 5. Effect of flooding and soil application of CaO2 on leaf, stem, root, and total plant dry weights of papaya (Carica papaya L.) in Krome very gravelly loam soil. Bars represent means and error bars represent ±1 std. dev. Different letters indicate significant differences according to a repeated measures ANOVA (P ≤ 0.05).


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Fig. 6. Effects of aeration and adding 0, 200, 500, 1000, or 2000 ␮l of 3% H2 O2 l−1 to a non-aerated hydroponic solution on dissolved oxygen content in the solution. Symbols represent means and error bars represent ± 1 std. dev. Asterisks indicate a significant difference between the non-aerated and aerated treatment at each H2 O2 concentration according to a repeated measures ANOVA (P ≤ 0.05).

3.2. Effects of H2 O2 on plants in a hydroponic solution (Experiment 2) Temperatures of the culture solution ranged from 24.5 ◦ C to 26.5 ◦ C (data not shown). There were no significant differences in pH of the hydroponic solution among treatments (P > 0.05). The pH of the solution for all treatments combined ranged from 7.6 to 7.9 on Day 1, from 8.4 to 8.9 on Day 2, from 8.7 to 8.8 on Day 3 and from 8.7 to 8.9 on Day 4 (data not shown). The dissolved oxygen content of the hydroponic solution was significantly lower in the non-aerated treatments with 0, 200, 500, or 1000 ␮l H2 O2 l−1 applied than in the aerated treatment starting

on Day 1. The dissolved oxygen content of the non-aerated 2000 ␮l H2 O2 l−1 treatment was lower than that of the aerated treatment starting on Day 6 (Fig. 6). Net CO2 assimilation tended to be lower for plants in the non-aerated treatments at each H2 O2 application rate than in the aerated treatment throughout the experiment, although these differences were not statistically significant on several measurement dates (Fig. 7). By the end of the experiment, A of plants in the nonaerated treatment with 2000 ␮l H2 O2 l−1 added to the solution was very similar to that of the aerated plants, whereas A of plants in the non-aerated treatment treatments with 0, 200, 500, or 1000 ␮l H2 O2 l−1 added to the solution was lower than that of plants in the

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Fig. 7. Effects of aeration or adding 0, 200, 500, 1000, or 2000 ␮l of 3% H2 O2 l−1 to a non-aerated hydroponic solution on net CO2 assimilation (A) of papaya (Carica papaya L.). Symbols represent means and error bars represent ±1 std. dev. Asterisks indicate a significant difference between the non-aerated and aerated treatment at each H2 O2 concentration according to a repeated measures ANOVA (P ≤ 0.05).

aerated treatment, although these differences were only statistically significant for the 0 and 200 ␮l H2 O2 l−1 treatments (Fig. 7). By the end of the experiment, gs was lower for plants in the non-aerated treatments with either 0, 200, 500, or 1000 ␮l H2 O2 l−1 applied to the solution than in the aerated treatment (Fig. 8). However, when 2000 ␮l H2 O2 l−1 was applied to a non-aerated solution, gs of plants in the non-aerated treatment was similar to that of the aerated treatment (Fig. 8). There were no significant differences in leaf chlorophyll index (data not shown) or Fv/Fm (data not shown) between the aerated treatment the non-aerated treatments at each H2 O2 application rate.

In all treatments, there was an increase in ADH activity one day after treatments began (Fig. 9). By the end of the experiment (Day 14), ADH activity in all treatments returned to levels close to those prior to beginning the treatments (Day 0). There was a significant difference in ADH activity among treatments only on the second day after treatments began (Fig. 9). On Day 2, ADH activity was significantly higher in plants in the non-aerated treatment with no H2 O2 added to the solution than in plants in the aerated treatment. There was no significant difference in ADH activity among any of the other treatments.


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Fig. 8. Effects of aeration or adding 0, 200, 500, 1000 or 2000 ␮l of 3% H2 O2 l−1 to a non-aerated hydroponic solution on stomatal conductance (gs ) of papaya (Carica papaya L.). Symbols represent means and error bars represent ±1 std. dev. Asterisks indicate a significant difference between the non-aerated and aerated treatment at each H2 O2 concentration according to a repeated measures ANOVA (P ≤ 0.05).

4. Discussion This study demonstrated that oxygen enrichment of the soil by addition of CaO2 reduces flooding stress and increases recovery and survival of papaya when 100% of the root system is submerged for at least two days. Soil application of CaO2 prior to flooding resulted in increased oxygen bioavailability in the soil solution. This increase in soil bioavailable oxygen content reduced flooding stress and increased plant recovery and survival, thereby avoiding the negative consequences of anaerobic metabolism on physiology and growth of papaya. Flooding of ∼75% or 100% of roots of papaya in Krome very gravelly loam soil for two days decreased several physiological variables

related to photosynthesis (A, gs , leaf chlorophyll index, Fv/Fm). In plants with ∼75% of roots submerged, levels of these physiological variables recovered to values similar to those of non-flooded plants within 13 days after plants were unflooded. When 100% of roots were submerged, A and gs declined and did not recover to levels similar to those of non-flooded plants even after plants were unflooded for 13 days. Root and whole plant dry weights and plant survival were also negatively impacted when ∼75% or 100% of roots were submerged compared to non-flooded plants. Flooding of papaya also inhibited leaf expansion and resulted in leaf chlorosis and abscission and thus a reduction in total leaf area. Leaf abscission in flooded plants may have been due to an increase in ethylene. Although ethylene was not measured in this study, flooding has

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Fig. 9. Effects of aeration or adding 0, 200, 500, 1000 or 2000 ␮l of 3% H2 O2 l−1 to a non-aerated hydroponic solution on alcohol dehydrogenase (ADH) enzyme activity in roots of papaya (Carica papaya L.). Bars represent means and error bars represent ±1 std. dev. Different letters indicate significant difference among treatments on each day according to a Waller-Duncan K-ratio test (P ≤ 0.05).

been shown to result in increased ethylene concentration leading to leaf abscission (Schaffer et al., 1992). For example in avocado trees, leaf abscission in flooded plants corresponded with increased ethylene accumulation in leaves (Gil et al., 2009b). Several researchers have observed that prolonged flooding causes a cessation of root and shoot growth, wilting, decreased nutrient uptake, and often plant death (Schaffer et al., 1992). Rodríguez et al. (2014) tested the effects of flooding duration (one to six days in daily increments) on physiology of 10-month-old papaya plants in Promix® potting medium. They found that A, gs, Fv/Fm, and leaf chlorophyll index decreased in plants flooded for two or more days and continued to decline after plants were unflooded. In that study, nearly all plants with 100% of roots submerged for two or more days permanently wilted by day 11. In a subsequent study, Rodriquez et al. (2014) also observed that when 100%, 75%, 50%, or 0% of the root system of papaya was inundated for 3 days, only plants with the 100% of the root system submerged did not recover from flooding stress after plants were unflooded. Those observations and observations from the present study indicate that papaya can tolerate of up ∼75% of the root system continuously submerged for at least 3 days, but is very sensitive to flooding of the entire root system. Chemically enriching flooded Krome very gravely loam soil with oxygen by adding 2.28 or 4.57 g of CaO2 increased the dissolved oxygen concentration in the soil but also significantly increased pH of the soil solution from 8.8 to up to 11.0. Oxygen enrichment of the soil helped papaya plants with 100% of the roots submerged recover from flooding stress after they were unflooded as evidenced by A and gs recovering to levels that were near those of the nonflooded control plants, greater plant tissue dry weights, and an increased percentage of plant survival compared to flooded plants with no CaO2 applied. Soil pH has little effect on physiology and growth of papaya, which is able to tolerate a wide range of soil pH (Campostrini and Glenn, 2007). The increased soil pH as a result of CaO2 application observed in the present study had no apparent inhibitory effect on plant recovery from flooding stress. Similar to our observations with papaya, Wang and Yeh (2015) observed that application of MgO2 to the soil prior to flooding chrysanthemum [Dendranthema × grandiflorum (Ramat.) Kitam.] increased flood tolerance by increasing A, lowering the intercellular CO2 concentration in the leaves, and increasing root dry weight. In the present study, addition of CaO2 to the soil prior to flooding 100% of the root system of papaya resulted in greater leaf area and leaf,

stem, root, and total plant dry weights compared to non-flooded plants. Liu et al. (2013) observed that application of slow-release [Ca(OH)2 ] or fast-release (CH4 N2 O·H2 O2 ) oxygen compounds to the root zone increased leaf chlorophyll content in Italian basil (Ocimum basilicum) by 58% and 53%, respectively and increased plant dry weight by up to 15%. In the hydroponic solution (Experiment 2), A and gs of papaya were generally lower for plants in non-aerated treatments than in the treatment with the aerated solution. If 500–1000 ␮l H2 O2 l−1 was added to the solution, A of plants in the non-aerated solution tended to recover to levels similar to those of aerated plants by the end of the experiment. This was interesting because addition of H2 O2 to the solution did not result in a measurable increase in dissolved oxygen content in the solution. It is possible that any additional oxygen resulting from the application of H2 O2 to the solution was metabolized by the plant, thus resulting in no detectable oxygen increase in the solution. In a study with corn (Zea mays L.) Liu and Porterfield (2014) found that the addition of H2 O2 to a hydroponic solution increased the concentration of bioavailable O2 , but that the O2 concentration of the solution decreased rapidly and that the additional O2 was mostly consumed within 2.5 h after addition of H2 O2 . Gil et al. (2009a) observed that when 1 mg kg−1 of 50% H2 O2 was injected into irrigation water applied to potted avocado plants, water use efficiency and tree biomass increased significantly. Similarly, adding H2 O2 to the soil increased growth of Z. mays (Melsted et al., 1949) and flooded tomato (Lycopersicon esculentum Mill.) (Bryce et al., 1982). In the present experiment with papaya in a hydroponic solution, biomass was not determined because the duration of the experiment was deemed too short for any biomass differences among treatments to be detected. However, the alleviation of the negative impact of hypoxia on A when H2 O2 was added to the medium suggests that oxygen enrichment of the root zone would have eventually reduced the negative impact of hypoxia on plant biomass over a longer time period. Previous studies of maize in hydroponic solution showed that the addition of H2 O2 to Hoagland solution increased the O2 concentration in the solution because the root surfaces had a sufficient quantity of catalase enzyme to catalyze the release of O2 from H2 O2 (D. Liu, unpublished data). Catalase is ubiquitous in aerobic organisms and therefore papaya root surfaces should have a sufficient amount of the enzyme to catalyze the release of O2 from H2 O2 in a hydroponic solution. Additionally, in the present study, the containers and nutrient solutions were not sterilized, and thus bacteria and other living microorganisms were presumably available to assist in catalyzing the breakdown of H2 O2 to release oxygen to the root zone. In the present study, in all non-aerated treatments, root ADH activity increased to a maximum two days after treatments began and by 14 days after treatments were initiated, ADH activity in each of the non-aerated treatments was similar to that observed before the treatment period. Similarly, Tanksley and Jones (1981) observed that ADH activity in tomato continued to increase for 50 h after plants were subjected to low root zone O2 conditions. Increased ADH activity was also observed in young corn seedlings as a result of low root zone O2 concentrations (Andrews et al., 1994). Trifolium repens plants with high ADH activity under flooding stress exhibited greater flooding tolerance than non-flooded plants with lower ADH activity (Chan and Ronald, 1992). Root ADH activity is often correlated with ethanol fermentation indicating that flood tolerance is related to the shift from aerobic to anaerobic respiration in the roots leading to ethanol fermentation (Chan and Ronald, 1992). The production of ethanol from pyruvate in the fermentation process to maintain NAD+ regeneration is catalyzed by ADH (Chung and Ferl, 1999). Thus, the increase in ADH activity in papaya plants under low root zone O2 conditions may play an important role in


short-term survival of papaya plants when root respiration shifts from aerobic to nonaerobic pathways. In the present study, there was no statistically significant effect of adding H2 O2 to the non-aerated hydroponic solution on ADH activity, probably due to the large between-plant (replication) variability within treatments. Although not statistically significant, ADH tended to be greater in the non-aerated treatment with no H2 O2 added to the solution than in any of the non-aerated treatments with H2 O2 added. Similarly, root ADH activity decreased in roots of flooded Z. mays when MgO2 was added to the potting medium compared to plants with no MgO2 added to the medium (Liu and Porterfield, 2014). This suggests that chemical oxygen enrichment of the root zone by application of fast-release (H2 O2 ) liquid or slow-release solid oxygen compounds can mitigate the effects of hypoxia on plant stress, possibly by providing sufficient bioavailable oxygen to the roots to prevent a shift from aerobic to anaerobic respiration. The results of this study indicate that plant stress and damage caused by flooding a portion of, or the entire root system of young papaya plants can be reduced or alleviated by amending the soil with CaO2 , a slow release solid oxygen compound. Also, adding H2 O2 (the break down product of CaO2 in soil) to a hydroponic solution can reduce plant stress due to low oxygen in the root zone. Thus, the potential exists to use chemical oxygen fertilization with compounds such as CaO2 to reduce flooding stress and increase plant survival of papaya in flood-prone areas. However, flooding effects on fruit crops often differ based on soil type and physical and chemical properties of the soil (Schaffer et al., 1992, 2006). Therefore, further studies are needed to compare the effects of amending soil with CaO2 as well as other solid oxygen compounds, such as MgO2 , among several different types of flooded soil. Also, the present study was conducted only with young seedling papaya plants. Further studies are warranted to assess the effects of solid oxygen fertilizers on reducing flooding stress and increasing recovery and survival of mature papaya plants from short-term flooding. References Andrews, D.L., Drew, M.C., Johnson, J.R., Cobb, B.G., 1994. The response of maize seedlings of different ages to hypoxic and anoxic stress. Plant Physiol. 105, 53–60. Balerdi, C.F., Crane, J.H., Schaffer, B., 2005. Managing Your Tropical Fruit Grove Under Changing Water Table Levels. Florida Cooperative Extension Service. Institute of Food and Agricultural Sciences, University of Florida, Publication HS957 https://edis.ifas.ufl.edu/hs202. Bryce, J.H., Focht, D.D., Stolzy, L.H., 1982. Soil aeration and plant growth response to urea peroxide fertilization. Soil Sci. 134, 111–116. Campostrini, E., Glenn, D.M., 2007. Ecophysiology of papaya: a review. Braz. J. Plant Physiol. 19, 413–424. Chan, J.W.Y., Ronald, S.B., 1992. Variation in alcohol dehydrogenase activity and flood tolerance in white clover, Trifolium repens. Evolution 46 (3), 721–734. Chung, H.J., Ferl, R.J., 1999. Arabidopsis alcohol dehydrogenase expression in both shoots and roots is conditioned by root growth environment. Plant Physiol. 121, 429–436. Curtis, D.S., 1949. Further investigations on avocado decline: effect of oxygen supply in nutrient solution on avocado and citrus seedlings studied in greenhouse tests. Calif. Agric. 3 (12), 8–9. Davies, D.D., Grego, S., Kenworth, P., 1974. The control of the production of lactate and ethanol by higher plants. Planta 118, 297–310. Drew, M.C., 1997. Oxygen deficiency and root metabolism: injury and acclimation under hypoxia and anoxia. Annu. Rev. Plant Physiol. Plant Mol. Biol. 48, 223–250. Else, M.A., Janowiak, F., Atkinson, C.J., Jackson, M.B., 2009. Root signals and stomatal closure in relation to photosynthesis: chlorophyll a fluorescence and adventitious rooting of flooded tomato plants. Ann. Bot. 103, 313–323.

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