GROWTH OF BEGONIAS USING SALINE IRRIGATION WATER AND EXOGENOUS APPLICATIONS OF ABSCISIC ACID

By

CRISTINA MARTINEZ

A THESIS PRESENTED TO THE GRADUATE SCHOOL

OF THE UNIVERSITY OF FLORIDA IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF

MASTER OF SCIENCE

UNIVERSITY OF FLORIDA

2017

© 2017 Cristina Martinez

Dedicated to my mother, for being my first teacher, and to my sisters, Cecilia and Carolina,

who keep me grounded and continue to be my inspiration.

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ACKNOWLEDGMENTS

Firstly, I would like to express my sincerest gratitude to my advisor, Dr. Kimberly

Moore, for your continuous support and mentorship during my time at the University of

Florida. You welcomed me with open arms into your lab as an undergraduate student

and introduced me to the world of scientific research. Your door was always open for

me when I needed help or advice and for that I will be forever grateful.

I would like to acknowledge the other members of my graduate committee, Dr.

Brian Pearson and Dr. Travis Shaddox, for your insight and encouragement. My sincere

thanks also go to Luci Fisher, Dr. MunWye Chng, and my other labmates at both the

University of Florida and USDA Invasive Plant Research Laboratory for your invaluable

support, input, and friendship.

Most importantly, none of this could have happened without my family. To my

mother and sisters, Yvette, Cecilia, and Carolina, thank you for being my inspiration and

for always believing in me. To my fiancé, Patrick, thank you for pushing me to be the

best version of myself. Finally, I must express my profound gratitude to my late

grandmother and eternal cheerleader, Brunny, without whom I never would have

realized my love of horticulture. This thesis stands as a testament to your unwavering

love, patience, and encouragement.

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TABLE OF CONTENTS

page

ACKNOWLEDGMENTS .................................................................................................. 4

LIST OF TABLES ............................................................................................................ 7

LIST OF FIGURES .......................................................................................................... 9

LIST OF ABBREVIATIONS ........................................................................................... 10

ABSTRACT ................................................................................................................... 11

CHAPTER

1 RATIONALE ........................................................................................................... 13

Introduction ............................................................................................................. 13

Hypotheses ............................................................................................................. 15 Objective ................................................................................................................. 16

2 LITERATURE REVIEW .......................................................................................... 17

Water Usage by Florida’s Horticulture Industry ....................................................... 17 Saltwater Intrusion in South Florida ........................................................................ 18

Wastewater Reuse and Salinity .............................................................................. 19

Factors Affecting Salinity Levels in Containerized Plants ....................................... 21 The Physiology and Effects of Salt Stress .............................................................. 23 Abscisic Acid: a Plant Hormone Related to Stress Response ................................ 26

Use of Synthetic ABA in Horticulture ...................................................................... 27 Begonias ................................................................................................................. 29

3 GROWTH OF ‘LOOKING GLASS’ BEGONIAS USING SALINE IRRIGATION WATER FROM VARIOUS SOURCES .................................................................... 31

Introduction ............................................................................................................. 31 Materials and Methods............................................................................................ 32

Irrigation: Experiment 1 (Comparing Sources at Different Concentrations) ...... 32 Irrigation: Experiment 2 (Comparing Sources at Different Volumes) ................ 33

Results .................................................................................................................... 34 Experiment 1 (Comparing Sources at Different Concentrations) ...................... 34 Experiment 2 (Comparing Sources at Different Volumes) ................................ 35

Discussion .............................................................................................................. 35

4 EXOGENOUS APPLICATIONS OF ABSCISIC ACID IN THE PRODUCTION OF ‘ESCARGOT’ BEGONIAS EXPOSED TO SALINE IRRIGATION WATER ....... 43

6

Introduction ............................................................................................................. 43

Materials and Methods............................................................................................ 44

Results .................................................................................................................... 46 Discussion .............................................................................................................. 47

5 CONCLUSION ........................................................................................................ 55

LIST OF REFERENCES ............................................................................................... 58

BIOGRAPHICAL SKETCH ............................................................................................ 63

7

LIST OF TABLES

Table page 2-1 Recommended pour-through substrate value ranges transformed from SME

substrate values. Values adapted from Warnke and Krauskopf (1983) and Cavins et al. (2004). ........................................................................................... 30

3-1 Chemical analysis (pH, electrical conductivity EC, sodium Na, potassium K, nitrate NO3-N, and phosphate PO4-P) of stock solutions of wastewater, fertilizer solution, and salt water used to irrigate begonias. ................................ 38

3-2 Final shoot dry weight (SDW), root dry weight (RDW), and visual rating of begonia grown with seven solutions applied three times a week at 250 ml per irrigation for 6 weeks. All plants were fertilized with 5 g of 15-9-12 Osmocote prior to planting. Numbers followed by different letter are significantly different at p ≤ 0.05. ............................................................................................ 39

3-3 Leachate chemical analysis of pH, electrical conductivity (EC), nitrate (NO3-N), phosphate (PO4-P) sodium (Na) and potassium (K) from begonia plants irrigation with seven different solutions for 6 weeks. All plants were fertilized with 5 g of 15-9-12 Osmocote. Numbers followed by different letter are significantly different at p ≤ 0.05. ........................................................................ 39

3-4 Begonia shoot (SDW) and root (RDW) dry weight in containers watered with deionized water, waste water, fertilizer solution, or salt water applied at 100, 250, or 350 ml per irrigation (3x a week). All plants were fertilized with 5 g of 15-9-12 Osmocote prior to planting. Numbers followed by different letter are significantly different at p ≤ 0.05. Lower case letters signify analysis across all solutions and volumes, while upper case letters signify analysis across the averages by solution. .......................................................................................... 41

3-5 Final pour-through leachate analysis of pH, EC, NO3-N, PO4-P, Na, and K. There was no significant difference in leachate due to volume and no solution by volume interaction. All plants were fertilized with 5 g of 15-9-12 Osmocote prior to planting. Numbers followed by different letter are significantly different at p ≤ 0.05. ............................................................................................ 42

4-1 Chemical analysis (pH, electrical conductivity EC, sodium Na, potassium K, nitrate NO3-N, and phosphate PO4-P) of stock solutions of wastewater, fertilizer solution, and salt water. ........................................................................ 49

4-2 Stomatal conductances of begonia plants before and after an abscisic acid drench of either 0, 25, 50, or 100 ppm. All measurements are in measured in mmol/(m^2s). ...................................................................................................... 50

4-3 Final visual ratings and shoot dry weights (SDW) in grams of begonia plants 6 weeks after receiving an abscisic acid drench of 0, 25, 50, or 100 ppm and being irrigated with one of four saline solutions (liquid fertilizer, salt water, tap

8

water, and wastewater). Numbers followed by different letter are significantly different at p ≤ 0.05. ............................................................................................ 51

4-4 Final pour-through leachate analysis of pH, EC, NO3-N, PO4-P, K, Na, and Cl. All plants were fertilized with 5 g of 15-9-12 Osmocote prior to planting. ..... 54

9

LIST OF FIGURES

Figure page 3-1 Begonias grown with seven solutions applied three times a week at 250 ml

per irrigation for 6 weeks. ................................................................................... 38

3-2 Begonia plants irrigated with deionized water (top left), salt water (bottom left), wastewater (top right), and liquid fertilizer (bottom right) for 6 weeks.. ....... 40

4-1 Stomatal conductances of begonia plants 24 hours after an abscisic acid drench of either 0, 25, 50, or 100 ppm. No irrigation solution had been applied before these readings were taken. ......................................................... 49

4-2 Front and side views of begonias irrigated with tap water, wastewater, liquid fertilizer, and saltwater for 6 weeks. From left to right the abscisic acid drench rates are 0, 25, 50, and 100 ppm. ....................................................................... 52

4-3 Front and top views of begonias receiving an abscisic acid drench at 0, 25, 50, and 100 ppm. Plants were irrigated (from left to right) with tap water, wastewater, liquid fertilizer, and salt water for 6 weeks.. .................................... 53

10

LIST OF ABBREVIATIONS

ABA Abscisic acid

Cl Chloride

EC Electrical conductivity

ET Evapotranspiration

g/L Grams per liter

K Potassium

N Nitrogen

Na Sodium

NO3-N Nitrate nitrogen

pH Concentration of H ions

PO4-P Phosphate phosphorous

ppm Parts per million

RDW Root dry weight

SDW Shoot dry weight

SME Saturated media extract

11

Abstract of Thesis Presented to the Graduate School of the University of Florida in Partial Fulfillment of the Requirements for the Degree of Master of Science

GROWTH OF BEGONIAS USING SALINE IRRIGATION WATER AND EXOGENOUS

APPLICATIONS OF ABSCISIC ACID

By

Cristina Martinez

December 2017

Chair: Kimberly Moore Major: Horticultural Sciences

Salinity in irrigation water can be attributed to several factors, including saltwater

intrusion, wastewater reuse, or overfertilization. Containerized ornamental plants, such

as salt-sensitive begonias (Begonia sp.), are especially susceptible to the negative

effects of salinity stress. Our objective in the first 2 experiments was to compare the

growth and quality of salt-sensitive begonias (Begonia x ‘Looking Glass’) when irrigated

with saline water from various sources and at different volumes. Plants in the first

experiment were irrigated with solutions of either deionized water, liquid fertilizer, saline

water, or wastewater. In the second experiment, plants were irrigated with the same

solutions at 100, 250, or 350 mL Shoot dry weights were greatest in plants receiving the

half strength solution of liquid fertilizer and lowest in plants receiving the salt water.

Begonia plants that received 350 mL at each irrigation yielded the greatest shoot dry

weights. Electrical conductivities and levels of Na were generally inversely related to

plant growth. In our third study, the growth and quality of begonias (Begonia x

‘Escargot’) were compared after they were irrigated with different saline solutions,

including liquid fertilizer, salt water, and wastewater, and received exogenous drenches

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of abscisic acid (ABA) at either 0, 25, 50, or 100 ppm. Stomatal conductance decreased

24 hours after the ABA drench and returned to normal after 7 days. No significant

differences in biomass or visual quality were observed due to ABA application. These

results suggest that begonia plants can be grown to be saleable using saline irrigation

water, including wastewater.

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CHAPTER 1 RATIONALE

Introduction

Containerized plant production may be inclined to issues with high salinity in the

root zone due to the accumulation of salts in the growing media. In high enough

quantities, salts can cause osmotic imbalances in plants which mimic a physiological

drought stress. Too much sodium (Na) and chloride (Cl) will disrupt the metabolism of

cells, ultimately affecting plant growth and reducing overall yield. Nutritional imbalances

occur from poor ion exchange, with Na ions taking the place of other essential nutrients.

Excessive accumulation of Na and Cl in the foliage causes necrotic tissue to form on

the tips of leaves of many plants. Eventually, prolonged exposure to salts in the soil

solution can lead to plant death. Built up salts in the growing substrate can originate

from overfertilization or from salinity in the irrigation water.

Agriculture is the top utilizer of water in the state of Florida, with horticulture

being a large contributor (U.S. Geological Survey, 2015). Most nurseries in the state

rely on well water, rather than municipal water sources or surface water, to irrigate their

crops (U.S. Department of Agriculture, 2014). In the highly-populated areas of southeast

Florida, the Biscayne aquifer is the primary source of freshwater and is affected by

saltwater intrusion (Florida Oceans and Coastal Council, 2010). This shallow layer of

porous limestone supplies water to three of most productive counties for the output of

bedding and foliage plants in the state: Palm Beach, Broward, and Miami-Dade

counties.

The overutilization of groundwater, especially in coastal regions, is the number

one reason for saltwater intrusion (Langevin and Zygnerski, 2012). When wells are

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pumping up water faster than the aquifer can be replenished or recharged, then ocean

water moves further inland than naturally intended. The result is a brackish mixture of

freshwater from the aquifer and salty water from the ocean just offshore with an

interface ever-encroaching the inland well pumps. Another issue that contributes to the

higher levels of salinity in well water in coastal areas is the lowered water table

attributed to the draining of the Everglades (Barlow and Reichard, 2010). Back in the

early 20th century, this area was cleared for residential and agricultural development.

In addition, sea level rise related to climate change worsens the conditions conducive to

saltwater intrusion (Heimlich, et al., 2009).

When the usage of high quality irrigation water is restricted due to drought or as

an effect of saltwater intrusion, one possible solution from the standpoint of plant

growers is to use alternative water sources such as reclaimed wastewater. Wastewater

is both abundant and cost-effective. Currently, most of it is injected below the aquifer or

released into the ocean after treatment (South Florida Water Management District,

2014). Assuming the infrastructure exists, treated reclaimed wastewater costs a fraction

of traditional municipal water. It has been used for many years to irrigate landscapes,

turfgrass fields, and agricultural crops. One major benefit of using wastewater to irrigate

crops is that it may be utilized without restrictions during periods of extreme drought

(South Florida Water Management District, 2014). On the other hand, higher levels of

salts in wastewater may initially dissuade growers from using this resource.

There are a few methods that growers may resort to when dealing with salt build

up in containerized plant production. One option is to increase the volume of water

applied at each irrigation with the purpose of leaching out excessive salts. If the

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irrigation water itself is high in salinity, which may be the case when reclaimed

wastewater is the primary source, then a blend of the saline water from one source may

be created by mixing with high quality tap water. This will create a diluted mixture of

water with lower levels of salts.

Another avenue to explore is the application of plant growth regulators (PGR’s).

Exogenous applications of abscisic acid, a naturally occurring plant hormone, may be

the key to help maintain the quality of plants produced using low quality irrigation water.

Abscisic acid (ABA) is important in the plant’s response to environmental abiotic

stresses such as drought and salinity (Hartung, et al., 2002). For example, during

periods of drought when soil water potential is decreased plant roots will produce

abscisic acid to be moved up the stem and through the xylem to the leaves. ABA will

induce guard cells to close the stomata in the leaves to conserve water through a

reduced transpiration rate. ABA concentration in leaves and stomatal conductance are

linearly related (Steuer, et al., 1988). Typically, it is synthesized in the roots and then is

translocated through the xylem to the leaves. There, it sends a message to the guard

cells that causes them to lose their turgidity and close the stomata. The goal is to

conserve water loss by slowing or stopping transpiration. This is a survival mechanism

meant to help the plant endure short-term stresses. If applied to plugs or liners

immediately after transplant into the final container, abscisic acid may act as a primer to

prepare the young plant to better handle salinity stress.

Hypotheses

• Containerized begonias irrigated with the half strength solutions will grow to a

better quality and size because they will have a lower electrical conductivity than

the full strength solutions.

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• Using highly saline irrigation water, a moderate leaching fraction will cause plants

to grow larger because a smaller leaching fraction will accumulate too many salts

and a higher leaching fraction will leach out too many nutrients from the

controlled-release fertilizer.

• Exogenous application of abscisic acid in the form of a drench at time of

transplant will minimize the effects of salt stress because it will reduce

transpiration in the plants.

Objective

The purpose of this research was to measure and compare the size and quality

of begonias irrigated with water from various sources at different concentrations and at

different volumes. Begonia plants grown with saline water were examined after a one-

time exogenous application of ABA.

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CHAPTER 2 LITERATURE REVIEW

Water Usage by Florida’s Horticulture Industry

Florida’s floriculture industry is the second largest in the country in terms of crop

value, bringing in approximately $905 billion in 2012 (U.S. Department of Agriculture,

2015). Florida and California are among the top five states in annual bedding and

garden plant production. California leads in production of potted flowering plants for

indoor or patio use. Where Florida dominates is in foliage plant production with 67% of

the total crop value on a national scale. This accounts for approximately $547 million in

sales per year according to the U.S. Department of Agriculture’s Floriculture Crops 2014

Summary (2015).

Within the state of Florida there are over 4,100 irrigated horticultural operations,

mostly producing floriculture, bedding, and nursery crops (U.S. Department of

Agriculture, 2014). Of the areas being used for production in these operations, just short

of 8,500 acres are under protection and over 76,000 acres are out in the open. In total,

these horticulture operations use approximately 22.9 billion gallons of water per year.

For areas under protection, the most common method of irrigation is sprinklers.

Microirrigation and hand watering are the second and third most commonly used

methods, respectively. About 72% of the water used comes from ground well water.

Twenty-two percent of the water used is from on-farm surface water and only a small

percentage of irrigation water originates from off-farm water suppliers.

Agriculture has historically been the number one source of water withdrawal in

the state of Florida, accounting for 39% of total withdrawals (U.S. Geological Survey,

2016). In 2012, this equated to over 14 million gallons per day. Public water is currently

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the largest user of water, followed by agriculture. The supply of fresh water will become

more precious while municipalities and other agencies attempt to meet the general

population’s demand for fresh drinking water. Agriculture will continue to use between

2.75 and 3 billion gallons per day over the next 10 years. In order to sustain supplies of

freshwater, water use efficiency and conservation will need to be improved (Florida

Department of Environmental Protection, 2010).

Saltwater Intrusion in South Florida

The counties with the largest economic impact related to horticulture are Miami-

Dade, Orange, Palm Beach, Hillsborough, Broward, and Volusia counties, in order from

largest to smallest contributions (Hodges, et al., 2011). Three of these counties (Miami-

Dade, Palm Beach, and Broward) get their freshwater from the Biscayne aquifer. This

aquifer is already experiencing saltwater intrusion (Florida Oceans and Coastal Council,

2010).

Originally, south Florida’s hydrological system allowed for water to flow naturally

from Lake Okeechobee, through the Everglades, and eventually to the Florida Bay or

Gulf of Mexico. The Biscayne aquifer is recharged primarily via rainfall and from the

freshwater flowing through the Everglades that permeates the underground limestone

(Florida Oceans and Coastal Council, 2010). The Everglades and adjacent areas were

drained in the early twentieth century to allow for urbanization and agricultural

development. The large-scale drainage project was facilitated through the creation of a

system of drainage canals, which lowered the water table and has led to saltwater

contamination (Barlow and Reichard, 2010).

Higher Cl concentrations were documented in water samples from monitoring

and supply wells on the eastern coast of Florida as soon as the early of 1900’s, which

19

indicated an inward flow of saltwater into the Biscayne aquifer (Sonenshein, 1995). As

of August 2012, 37% of the 137 groundwater wells and stream gages tested by the U.S.

Geological Survey in South Florida had Cl concentrations greater than or equal to 1,000

mg/L, which is the threshold used to determine the extent of saltwater intrusion into the

Biscayne Aquifer. An additional 8% of the sites tested had Cl concentrations between

250 mg/L, the upper limit of the Florida Department of Environmental Protection’s

Secondary Drinking Water Standard for chloride, and 1,000 mg/L (U.S. Geological

Survey, 2016).

A study on how the aquifer in Broward County is affected by a Pompano Beach

well field showed that pumping large volumes of freshwater from the aquifer was the

largest factor contributing to saltwater intrusion (Langevin and Zygnerski, 2012).

Saltwater intrusion was exacerbated by sea level rise, which is expected to occur with

global climate change. The increased hydraulic backpressure on a coastal aquifer, such

as the Biscayne aquifer, reverses the flow of water from an outwards flow of freshwater

to the ocean to more of an inward flow of saltwater (Florida Oceans and Coastal

Council, 2010). This causes a serious threat of contamination to coastal water supply

wells that will worsen during periods of drought and during the dry winter and spring

season. A sea level rise of only about six inches would call for the implementation of

serious advanced water management strategies including conservation, storage,

wastewater reuse, and alternative supplies including desalination (Heimlich, et al.,

2009).

Wastewater Reuse and Salinity

Reuse of treated reclaimed wastewater reduces the reliance on traditional

freshwater sources. Reclaimed wastewater is in abundance, even during periods of

20

drought, and is already being used as an effective irrigation alternative in agriculture.

(da Fonseca, 2007) established landscape setting. According to the South Florida

Water Management District, approximately 266 million gallons of wastewater are reused

per day, which is only 31% of total created. In Broward and Miami-Dade counties, only

5% of treated wastewater is reused. The remainder is released into the ocean or

injected underground. Wastewater can lower production costs of growers as it can allow

for reduced application rates of fertilizer (Paranychianakis, et al., 2006). The cost of

connecting nurseries to wastewater treatment facilities may limit the usage of the water

resource (Fitzpatrick, 1985).

One of the major issues in using reclaimed water is that the salinity is usually

higher than other sources of water. Reclaimed wastewater is brackish with Na and Cl as

major ions and has total dissolved solids (TDS) between that of fresh water and ocean

water. It also contains nutrients essential to plant growth and developments, such as N,

P, and K (da Fonseca, 2007). Elevated levels of Na and Cl can be harmful or toxic to

some plans. For example, the effluent from the Southern Regional Wastewater

Treatment Plant (SRWTP) in Broward County Florida has higher conductivities and

salinities than what is recommended for reuse irrigation. The conductivities of the

effluent from the SRWTP ranged between 1 and 6 dS/m, which is dependent upon time

of year. The chloride, sodium, and total dissolved solid levels were an average of 384

mg/L, 844 mg/L, and 1906 mg/L, respectively. A study was done using blends of this

effluent with various electrical conductivities to irrigate turf and various typical landscape

shrubs and trees for a period of months. At the end of the study, it was concluded that

trees and shrubs treated with wastewater were similar in size to the control plants. The

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turfgrass benefited from the medium-strength blend, but still grew more in the full-

strength effluent than in the control (Karleskint, et al., 2011).

Salinity levels and the concentrations of various ions can vary depending upon

the source of the reclaimed water, the treatment of the wastewater , and the time of

year. Management practices vary when irrigating plants with reclaimed water due to the

levels of salts and nutrients in the reclaimed water. For example, fertilization may need

to be reduced. A significant amount of the nutrients in reclaimed water are readily

available to plants, including nitrogen and phosphorous. If application rates suggested

by the manufacturer are used in conjunction with reclaimed water, then there may be

elevated levels of nutrients in runoff or leachate (Martinez and Clark, 2009). When

irrigating with reclaimed wastewater, the soluble salts can be flushed out using an

increased volume of water beyond the volume that is used by the plant (Corwin and

Rhoades, 2007)

Factors Affecting Salinity Levels in Containerized Plants

Salt tolerance is typically evaluated by comparing the biomass produced by

plants in saline versus control conditions over a period of time. The salt tolerance of

plants is species-specific and can vary dramatically (Zapryanova and Atanassova,

2009). In some cases, even the selected cultivar can have varying salt tolerances

compared to other cultivars within the same species. Some cultivars absorb up to three

times the Cl as another cultivar of the same species (Sonneveld, 2000). Without taking

fertilization into consideration, the salinity of the growing substrate or of the soil in a

landscape setting depends on various factors involving the quality, volume, and

frequency of irrigation in addition to environmental conditions (Martinez and Clark,

2009).

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The salt content of the irrigation water depends on the source of the water (ex:

groundwater, municipal water, reuse water, etc.) and the quality of the water. Salinity

accumulates from irrigation water, especially in small root volumes or closed systems

(Sonneveld, 2000). These conditions are common in containerized plant

production. The volume of irrigation water being applied will alter the amount of water

leaching from the container. Generally, a greater leaching fraction will decrease the

accumulation of salts in the root zone. The electrical conductivity of a leachate of a

containerized ornamental is inversely related to the leaching fraction. In other words, as

the leaching fraction increases, the EC of the leachate decreases (Ku and Hershey,

1991). The frequency of irrigation will also affect the concentration of salts in the soil

solution. The more often the media can dry out, the higher the concentration of ions.

When the media is closer to saturation, the concentration will be less. If a high substrate

EC is caused by overfertilization, fertilization rates can be lowered or substrates can be

be flushed with clear water using a high leaching percentage. If irrigation water has a

high level of salts, the irrigation water can be supplemented with alternative water

sources to lower the EC (Whipker, et al., 2001).

If plants are grown in an open unprotected area, the amount of rainfall can also

impact salt levels in the growing media. Rainwater, which has a low salinity, will leach

ions from the soil solution in the container. The evapotranspiration (ET) rate also affects

concentration of salts in the media. The higher the ET rate, which can come from

increased temperatures and lower humidity, the less water there will be in the substrate.

It should be noted that plants under stress may close their stomata during periods of

23

stress, such as drought and in conditions of extreme salinity, which decreases

transpiration rates to conserve water.

The Physiology and Effects of Salt Stress

A plant’s response to salt stress depends upon the concentration of salts the

plant is exposed to, the length of time the plant is exposed to the salts, and the general

tolerance a species or cultivar has. Controlling water loss through the stomata, osmotic

and metabolic adjustments, and toxic ion homeostasis are all ways a plant can adapt to

salinity stress for it to survive and grow (Hasegawa, et al., 2000; Munns and Tester,

2008; Zhu, 2002).

Salinity in the soil environment can minimize the difference in water potential

between root cells and the soil. This potential can also become inverted and lead to

water loss (Boursiac, 2005). High salinity can lower a plant’s ability to take up water,

which can limit plant growth (Masrchner, 1995).

Plants exposed to high salinity are stressed due to osmotic pressure from higher

levels of ions. Osmotic adjustment within the cell via an uptake of inorganic ions or

organic solutes can be effective methods used by plants to tolerate salinity (Morgan,

1984). Osmoregulation is an active process by which a cell attempts to maintain the

concentration of salts within the vacuole through accumulation of ions and creation of

other solutes in the cytoplasm (Munns, 2002). These solutes often include sugars and

amino acids (Gorham, et al., 1985). Uptake of ions requires less energy than the

creation of organic solutes (Greenway and Munns, 1983). High levels of salts in the

cytoplasm are avoided by restricting salts to the vacuole or sorting them into different

tissues (Reddy, 1992).The compartmentalization of Na and Cl into the vacuole helps

24

supplement osmotic pressure and allows a greater influx of water into the cell

(Blumwald, 2000).

Toxic effects occur due to the high levels of ions that interfere with the uptake of

other nutrients. Plants either die or decrease in growth or yield (Küçükahmetler, 2002).

The salt tolerance of a plant is typically evaluated by the amount of biomass produced

in saline conditions compared to that produced under control conditions over a period of

time. Plants with lower salt tolerances will have greater reduction in biomass under

saline conditions than those with a higher salt tolerance (Munns, 2002). Mechanisms for

salt tolerance limit entry of salt or minimize the concentration of salt within the

cytoplasm of the plant’s cells. (Munns, 2002). Restriction of Na entry to shoot growth is

also linked with salt tolerance (Zurayk, et al., 1993).

Since salinity can limit cell division and expansion in all growing plant tissues,

effects of salt stress can be seen throughout the plant (Zidan, et al., 1990). A salt-

tolerant plant’s response to salinity is extremely similar to those due to stress from a

lack of water. An immediate response to salt stress is a reduction in leaf and root

elongation rate, followed by a partial recovery over the next few hours. As days

progress, leaf growth is more affected than the roots. Leaf emergence rates decline and

salt injury is visible in older leaves. More long-term effects include reduced leaf size,

difference in flowering time and seed production, and death of leaves and/or the entire

plant. Premature senescence of older leaves occurs first as increasing levels of salt

accumulate (Munns, 2002). Even without the death of the leaf, other symptoms can be

commonly seen, such as leaf burn, thickening, and abscission (Küçükahmetler, 2002).

In addition, root elongation and growth is limited by salinity stress (Neumann, 1995).

25

Containerized annuals subjected to solutions containing high levels of NaCl

exhibited multiple symptoms of salt stress. Salinity inhibited both plant height and

diameter in addition to the number of lateral branches. Bloom periods occurred earlier

and for a shorter period of time for plants under stress than for those in the control

group (Zapryanova and Atanassova, 2009). Increased NaCl concentrations decreased

both the weight and size of hydroponically-grown cut flowers, although there was no

effect on flower shape (Sonneveld, 2000). It has also been reported that salt can reduce

the total number and overall quality of flowers (Küçükahmetler, 2002).

Initial reduction in growth is due to the osmotic stress on the roots from excess

salts. It takes a period of days for internal injury due to toxic levels of salts accumulating

in the transpiring leaves. This reduces the amount of carbohydrates available for new

growth (Munns, 2002). Linear growth reductions have been seen in ornamental plants

exposed to salt once a certain threshold concentration of either Na or Cl has been

established in a substrate (Weinhold and Scharpf, 1997).

It has been demonstrated that solutions of the same conductivities but varying

levels of Na can have different effects on plant growth. If comparing two groups of

plants grown with solutions of the same EC but varying Na content, the one with higher

Na will have the greatest reduction in growth. Meanwhile, if you have two solutions with

the same Na content but varying ECs, the one with the higher EC will have the least

reduction in growth. In this scenario, cation competition restricts the uptake of Na. In

addition to overall growth, regrowth after the harvesting of cut flowers was also limited

more by NaCl content than by overall EC. New growth had higher content of both Na

and Cl than fully expanded leaves. Chloride was absorbed in higher quantities than Na

26

(Sonneveld, 2000). For most greenhouse plants, chloride concentrations of less than

108 ppm is usually of no concern for damaging foliar adsorption. Concentrations greater

than 144 ppm may cause toxicity by root assimilation (Peterson, 1996).

Abscisic Acid: a Plant Hormone Related to Stress Response

Abscisic acid, commonly known as ABA, can be found in all photosynthetic

organisms and is one of the five most important plant hormones. ABA acts as a

messenger that works aids in the growth and development of plants and in how plants

respond to the world around them. All cells in a plant have the ability to produce plant

hormones.

Abscisic acid was discovered in the early 1960s. It originally was called abscisin

II, after being found in relatively large concentrations in abscising cotton fruit

(Finkelstein, 2013). ABA is more involved with inhibitory actions rather than acting as a

stimulatory hormone. Physiological effects in plants related to ABA include inhibition of

fruit ripening, seed and bud dormancy, senescence, and flower induction or inhibition.

ABA is produced by plants during periods of stress. For example, during periods of

drought when soil water potential is decreased plant roots will produce abscisic acid to

be moved up the stem and through the xylem to the leaves (Hartung, et al., 2002). ABA

will induce guard cells to close the stomata in the leaves to conserve water through a

reduced transpiration rate. ABA concentration in leaves and stomatal conductance are

linearly related (Steuer, et al., 1988). Although roots are the primary source of ABA in

plants, leaves can also synthesize ABA that will be transported by the phloem to the

roots where it will be recirculated by the xylem. Other abiotic stresses that can induce

the increased production of abscisic acid include high salinity in the soil environment

and high temperatures (Hartung, et al., 2002). Most of the studies on the effects of ABA

27

in plants have been done through the use of genetic mutants that are either abscisic

acid deficient or insensitive. Other studies involved application of abscisic acid to plants

or measured through extraction of tissue (Cutler and Krochko, 1999).

Use of Synthetic ABA in Horticulture

Application of ABA to ornamental crops can help minimize water usage in

nurseries where plants are grown in small containers with limited water holding

capacity. Shipping from nurseries to garden centers and other retailers can potentially

be a very stressful time for plants, where they are exposed to high temperatures without

additional irrigation. ABA might be the key to ensuring the arrival of quality plants with

minimal stress.

After exogenous ABA applications, wilting in 6 ornamental species was delayed

by 1.7 to 4.3 days (Waterland, et al., 2010). In most plants, there was no difference in

wilt status and visual appearance between plants given a spray or a drench application

of ABA. Another trial found similar results, with wilting being delayed by 1.3 to 3.7 days

in 5 species of bedding plants after an ABA drench (Park, et al., 2016). Application rate

did not typically affect extension in shelf life in multiple species of flowering ornamentals

(Waterland, et al., 2010). However, others have found that higher concentrations of ABA

(1000 to 2000 ppm) reduce wilting by 1 extra day when compared to lower

concentrations (250 to 500 ppm) (Kim and van Iersel, 2008). It has been suggested

that similar responses in varying application rates may be due to the lower application

rate approaching the maximum amount of ABA that the plants can uptake and

metabolize (Blanchard, et al., 2007).

A rapid reduction in stomatal conductance has been documented within 4 hours

of an ABA drench. (Park, et al., 2016). ABA drenches in Salvia splendens reduced

28

stomatal conductance within 3 hours of application (Kim and van Iersel, 2008). A study

on perennial flowering Chrysanthemum x mortifolium found that plants sprayed with

ABA had longer periods of reduced stomatal conductance compared with plants that

had received a drench (Waterland, et al., 2010). Drenches may had been adsorbed and

metabolized more readily or been leached out with irrigation water. Variability in

extended drought tolerance were found among chrysanthemum cultivars, which could

be attributed to differences in sensitivity to ABA, growth habit, or water usage.

Plants rewatered after an extended period of drought that received an

exogenous application of ABA were often indistinguishable from plants that were

continuously irrigated during the same period (Waterland, et al., 2010). One issue that

may decrease the marketability of these plants was leaf chlorosis induced by the

application of ABA (Park, et al., 2016). Leaf abscisision was also strongly correlated

with ABA applications in salvia (Kim and van Iersel, 2008). Phytotoxicity has been

reported in viola (Viola ×wittrockiana) and lobelia (Lobelia erinusas) lower leaf necrosis

and leaf yellowing (Blanchard, et al., 2007).

ABA is a physiological antitranspirant. There are also physical antitranspirants,

such as resins, waxes, and polymers, although ABA has been shown to be more

consistent in enhancing water stress tolerance (Park, et al., 2016). Little research has

been done regarding the use of ABA to improve salt stress response in ornamentals.

Root drenches have been applied to multiple genotypes of potatoes with varying salt

stress resistance. Growth enhancement was found in all plants receiving ABA after

being irrigated with a complete nutrient solution with up to 180 mM NaCl. Plants had

increased shoot water content and reduced leaf necrosis when compared to untreated

29

potato plants (Etehadnia, et al., 2008). Endogenous levels of ABA were measured after

the application of an NaCl solution to the root zone of barley plants. Within 10 minutes,

the ABA concentrations in the leaves increased by up to 600% of original levels. A

decrease in transpiration and stomatal conductance immediately followed. ABA

concentrations returned to normal levels after 20 hours (Fricke, 2004).

Begonias

Begonias (Begonia sp.) are a large family of tropical plants grown as

ornamentals enjoyed for both their flowers and foliage. There are the following

horticultural classifications of begonias: cane-like, shrub, rhizomatous, semperflorens,

tuberous, rex, trailing-scandent, and thick-stemmed (American Begonia Society, 2014).

The ‘Escargot’ and ‘Looking Glass’ cultivars are both hybrids of rex begonias (B. rex)

crossed with other rhizomatous varieties and are notorious for their unique patterned

leaves. Rex begonias are considered to have light nutrient requirements and perform

best with a saturated media extract (SME) of 0.76 to 2.0 mS/cm or a pour through EC of

1.0 to 2.6 mS/cm (Whipker, et al., 2001). Recommended values for pour through and

SME substrate values in greenhouse crops are summarized in Table 2-1 (Cavins, et al.,

2004; Warncke and Krauskopf, 1983). In a study assessing salt tolerance of a multitude

of landscape plants, begonias were found to be sensitive to irrigation water with an EC

of 3 dS/m or less with 80% of the salts in the solution in the form of NaCl (Miyamoto, et

al., 2004). ABA drench applications increased the time before wilting by 2.3 days (from

8.7 days in the control group to 10 days in the treated group) in begonias (Begonia

semperflorens-cultorum) (Park, et al., 2016). A spray of a physical antitranspirant, β-

pinene polymer, caused floral damage to the begonia plants while an ABA drench did

not cause any damage.

30

Table 2-1. Recommended pour-through substrate value ranges transformed from SME substrate values. Values adapted from Warnke and Krauskopf (1983) and Cavins et al. (2004).

Nutritional Parameter Pour-through SME

pH 5.6-6.0 5.6-6.0

EC (mS/cm) 2.8-4.8 2.0-3.6

NO3-N (mg/L) 180-320 100-199

P (mg/L) 11-16 6-9

K (mg/L) 220-360 150-249

Ca (mg/L) 330+ 200+

Mg (mg/L) 100+ 70+

31

CHAPTER 3 GROWTH OF ‘LOOKING GLASS’ BEGONIAS USING SALINE IRRIGATION WATER

FROM VARIOUS SOURCES

Introduction

Saltwater intrusion to fresh groundwater reservoirs is attributed to sea level rise

and overutilization of groundwater and is an issue in some coastal regions. In August

2012, 37% of the 137 groundwater wells and stream gages tested by the U.S.

Geological Survey in South Florida had chloride (Cl) concentrations greater than or

equal to 1,000 mg/L, which is the threshold used to determine the extent of saltwater

intrusion (U.S. Geological Survey, 2016). Many nurseries that rely on well water

affected by saltwater intrusion will likely need to learn how to grow their crops using

lower quality water. Approximately three-quarters of irrigation water used by horticultural

operations is pumped from wells in the state of Florida (U.S. Department of Agriculture,

2015).

If the infrastructure exists, reclaimed wastewater is an alternative water source

(South Florida Water Management District, 2014). However, wastewater is known to

have low to moderate levels of nitrogen (N), potassium (K), sodium (Na), and chloride

(Cl), which may present a challenge to some growers. If suggested fertilizer rates are

applied to plants irrigated with wastewater, there may be elevated levels of salts in the

substrate (Martinez and Clark, 2009).

The salt tolerance of a plant can be evaluated by the biomass the plant produces

in saline conditions compared to control conditions, with salt sensitive plants having a

greater reduction in biomass as compared to tolerant plants (Munns, 2002). Salt-

sensitive begonias (Begonia sp.) are commonly grown for their foliage and flowers. In a

study assessing salt tolerance of a multitude of landscape plants, begonias were found

32

to be sensitive to irrigation water with an EC of 3 dS/m or less with 80% of the salts in

the solution in the form of NaCl (Miyamoto, et al., 2004). Increased irrigation volume

corresponds to a higher leaching fraction. Higher leaching fractions can be used to

combat salinity in irrigation water by flushing out excess salts and lowering the EC of

the soil solution (Ku and Hershey, 1991).

The objective of this study was to compare begonia growth when irrigated with

deionized (DI) water, wastewater, salt water or fertilizer solutions. A second objective

was to compare begonia growth with these solutions applied at low, medium, and high

volumes.

Materials and Methods

Liners of ‘Looking Glass’ begonias in 72 cell trays (AG 3, Inc., Eustice, FL) were

transplanted into 1 gallon pots filled with soilless Metro-Mix (Sun Gro Horticulture

Canada Ltd., Agawam, MA). A controlled release fertilizer (15𝑁 − 9𝑃2𝑂5 − 12𝐾2𝑂

Osmocote Plus 8 to 9 month, Scotts Co., Marysville, OH) was incorporated into the

growing substrate before planting at a rate of 5 g per pot. Pots were arranged in a

completely randomized design with 5 replicates per treatment. Plants were then grown

for 6 weeks beginning in May 2015 in an open sided greenhouse exposed to ambient

air temperatures at the University of Florida Fort Lauderdale Research and Education

center in Davie, FL. The average daily temperature and relative humidity were 25.8 °C

(78.5 °F) and 76%, respectively, for the duration of the trials. Environmental data was

collected by the Florida Automated Weather Network (FAWN) station located on-site.

Irrigation: Experiment 1 (Comparing Sources at Different Concentrations)

Plants were irrigated by hand 3 times a week with 250 mL of one of 7 solutions:

1) deionized water, 2) reclaimed wastewater, 3) a 1:1 (by volume) solution of

33

wastewater and deionized water, 4) liquid fertilizer (Jack’s Classic Blossom Booster

10𝑁 − 30𝑃2𝑂5 − 20𝐾2𝑂 with micronutrients, JR Peters Inc., Allentown, PA) at 6 grams

per liter (g/L), 5) liquid fertilizer at 3 g/L, 6) sea salt mixed with deionized water at 2 g/L,

and 7) sea salt mixed with deionized water at 1 g/L (Table 3-1). Wastewater was

collected from the Southern Regional Wastewater Treatment Plant in Hollywood, FL in

May 2015 and stored in sealed 55 gallon barrels with lids. Sea salt (Morton All-Purpose

Sea Salt, Morton Salt, Chicago, Il, USA) was mixed with deionized water to create

saline water. Control plants were irrigated with deionized water. Solutions used for

irrigation were full-strength at 6 dS/m or half-strength at 3 dS/m (Table 3-1). The 6 dS/m

solutions had an EC equal to that of the wastewater, while the 3 dS/m solutions were

diluted with deionized water so that the EC was equal to half that of the wastewater.

Irrigation: Experiment 2 (Comparing Sources at Different Volumes)

Plants were irrigated by hand 3 times per week with reclaimed wastewater, liquid

fertilizer, saline water, or deionized water from the same sources as in experiment 1. All

irrigation solutions, except for the deionized water, were mixed to have the same EC as

the wastewater (6 dS/m). Plants received either 100, 250, or 350 mL at each irrigation.

The 100, 250, and 350 mL applications were equivalent to leaching fractions of about

10%, 40%, and 60%, respectively.

For both experiments the visual quality of plants were rated using the following

scale: 5) excellent, 4) above average, 3) average, 2) below average, 1) poor, and 0)

dead. Above ground plant growth was harvested after 6 weeks and dried in an oven at

54.4°C (130°F) for a week to measure shoot dry weights. Leachate samples were

collected via the pour-thru method using deionized water. A Hanna Combo pH and EC

meter was used to measure the pH and EC (Hanna Instruments, Woonscket, RI).

34

Phosphate concentrations were measured using the ascorbic acid method with

absorbance at 440 nm using a Spectronic 200 Spectrophotometer (Thermo Electron

Corp, Waltham, MA). Concentrations of NO3-N, K, Na, and Cl were measured using

specific ion probes for an Accumet XL250 (Fisher Scientific, Waltham, MA).

Data from the two experiments were analyzed separately in SAS. For experiment

1, ANOVA was used to compare dry weights, electrical conductivities, visual ratings,

and values from nutritional analysis of the leachates across the different irrigation

solutions. A Duncan’s multiple range test was performed following a significant finding in

the ANOVA. For experiment 2, the same analysis was conducted to find potential

significant differences by the irrigation solutions and volumes.

Results

Experiment 1 (Comparing Sources at Different Concentrations)

In experiment 1, the greatest shoot dry weight (SDW) and visual rating were in

plants irrigated with half strength fertilizer (Table 3-2). The least SDW was in plants

irrigated with half- and full-strength salt water. The plants with the lowest visual rating

were the plants irrigated with the full-strength fertilizer and the full-strength salt

solutions.

There was no significant difference in leachate pH across the 7 irrigation

solutions (Table 3-3). The plants irrigated with the full-strength fertilizer and salt water

had higher leachate ECs. Leachate NO3-N, PO4-P, and K were also greatest for pots

irrigated with fertilizer solution. Growing media for the plants irrigated with the deionized

water had lower leachate ECs compared with other treatments. The Na levels were

highest in the full-strength liquid fertilizer and saline water solutions.

35

Experiment 2 (Comparing Sources at Different Volumes)

Plants grown with DI water and wastewater in the second experiment had a

larger SDW and RDW at harvest when compared to plants irrigated with saline water

and liquid fertilizer (Table 3-4). In general, for all 4 solutions plants that received 350 mL

at each irrigation had the largest SDW compared to plants that received 100 mL.

There was no significant difference in leachate pH, EC, NO3-N, PO4-P, K, and

Na due to volume and no solution by volume interactions (Table 3-5). Across the 4

solutions, there was no difference in leachate pH or PO4-P concentrations. Leachate

EC was greatest for pots irrigated with fertilizer or salt. Leachate NO3-N and K were

greatest for fertilizer while leachate Na was greatest in wastewater and fertilizer pots.

Plants irrigated with the deionized water had the lowest leachate EC, Na, and K

concentrations.

Discussion

Salinity can alter plant growth, development, and nutrient uptake, while

potentially causing toxicities and eventual plant death. Salts from the irrigation water

tend to accumulate in small root volumes such as those found in containerized plants

(Sonneveld, 2000). Plants exposed to high levels of salts in the soil solution resort to

physiological methods of tolerating stress, such as osmoregulation. This is the active

uptake of ions and other solutes to maintain a water potential high enough for growth in

spite of a lower environmental water potential (Morgan, 1984).

We observed reduced growth in both experiments where there were high ECs. In

experiment 1, the fertilizer and salt water treatments had final leachate ECs of 6.54 and

6.69, respectively. In experiment 2, the fertilizer and salt treatments had higher leachate

ECs. The quality, volume, and frequency of irrigation also affect the level of

36

accumulation of salts in the growing substrate (Martinez and Clark, 2009). In general, as

irrigation volume and leaching fractions increase, the electrical conductivity (EC) of the

soil solution decreases (Ku and Hershey, 1991).

In experiment 2, growth for plants irrigated with all of the solutions increased as

leachate volume increase. A drop in leachate EC was noted as irrigation volume

increased. In addition, it has been found that varying levels of Na in solutions with

comparable EC’s leads to differences in plant growth, with higher concentrations of Na

being attributed to the greater reductions in growth (Sonneveld, 2000).

The plants in these experiments irrigated with the salt solutions performed poorly

because of too much Na and not enough K. In a study involving containerized Jatropha

curcas plants, it was found that K and Na ions are competitive at the level of root uptake

and that elevated levels of K in the root zone were beneficial because it can restrict

uptake and transport of Na (Rodrigues, et al., 2013). This may be because the plants

had high affinity for K and an induced exclusion mechanism at the root level for Na.

Sodium and K compete for uptake due to high-affinity K transporters and nonselective

cations channels. It has been suggested that an increased concentration of K in saline

and sodic soils may improve the influx of K and reduce that of Na (Wakeel, 2013).

Overall, plants with high ECs and Na concentrations in the leachate had poor

growth. The wastewater and salt irrigation solutions had the lowest levels of K

compared with the liquid fertilizer. Wastewater at an EC equal to about 3 dS/m can be

used to grow begonias when a controlled release fertilizer is incorporated into the

growing substrate at a low rate of 5 g per 1 gallon pot. The ideal solution to irrigate

begonias with to maximize growth and quality is a liquid fertilizer such as the half

37

strength fertilizer solution used in experiment 1. Rex begonias are considered to have

light nutrient requirements and have been shown to perform best when pour-through

substrate ECs are between 1.0 and 2.6 mS/cm (Whipker, et al., 2001). For most

greenhouse crops, suggested pour-through substrate ECs fall between 2.8 and 4.8

mS/cm (Cavins, et al., 2004). The full strength fertilizer solutions used in both

experiments had nutrient concentrations, including Na, that were too high to yield

maximum growth.

38

Table 3-1. Chemical analysis (pH, electrical conductivity EC, sodium Na, potassium K, nitrate NO3-N, and phosphate PO4-P) of stock solutions of wastewater, fertilizer solution, and salt water used to irrigate begonias.

Solution pH EC

(dS/m)

NO3-N

(mg/g)

PO4-P

(mg/g)

Na

(mg/g)

K

(mg/g)

Half Wastewater 6.83 3.17 250 0.004 3.91 1.84

Wastewater 6.84 6.32 240 0.009 7.61 5.22

Half Fertilizer 6.42 2.53 870 7.2 2.24 94.80

Fertilizer 6.57 5.96 1600 14.8 6.03 183.95

Half Salt 6.82 2.53 110 0.003 2.79 0.03

Salt 6.84 5.28 170 0.008 6.54 0.05

Deionized water 7.00 0.00 0 0 0 0

Figure 3-1. Begonias grown with seven solutions applied three times a week at 250 ml per irrigation for 6 weeks. Photo taken by Cristina Martinez.

39

Table 3-2. Final shoot dry weight (SDW), root dry weight (RDW), and visual rating of begonia grown with seven solutions applied three times a week at 250 ml per irrigation for 6 weeks. All plants were fertilized with 5 g of 15-9-12 Osmocote prior to planting. Numbers followed by different letter are significantly different at p ≤ 0.05.

Solution SDW

(g)

RDW

(g)

Rating

Deionized water 10.79b 5.52a 3.0b

Half Wastewater 10.59bc 5.59a 3.5b

Wastewater 10.62b 5.54a 3.5b

Half Fertilizer 11.83a 5.50a 4.0a

Fertilizer 10.94b 5.14a 2.0c

Half salt 10.45c 5.36a 2.5c

Salt 10.22c 5.29a 2.0c

Table 3-3. Leachate chemical analysis of pH, electrical conductivity (EC), nitrate (NO3-

N), phosphate (PO4-P) sodium (Na) and potassium (K) from begonia plants irrigation with seven different solutions for 6 weeks. All plants were fertilized with 5 g of 15-9-12 Osmocote. Numbers followed by different letter are significantly different at p ≤ 0.05.

Solution pH EC

(dS/m)

NO3-N

(mg/g)

PO4-P

(mg/g)

Na

(mg/g)

K

(mg/g)

Deionized water 6.44a 2.63c 102d 0.03c 0.58c 8.82c

Half Wastewater 6.45a 3.49b 258c 0.02c 1.47c 8.93c

Wastewater 6.42a 3.95b 206c 0.02c 3.29b 13.16c

Half Fertilizer 5.67a 3.55b 1128b 0.93b 5.86b 99.13b

Fertilizer 5.60a 6.54a 1825a 1.89a 10.74a 200.9a

Half salt 6.47a 3.48b 199c 0.01c 4.58b 9.46c

Salt 6.30a 6.69a 253c 0.09c 10.55a 11.42c

40

Figure 3-2. Begonia plants irrigated with deionized water (top left), salt water (bottom

left), wastewater (top right), and liquid fertilizer (bottom right) for 6 weeks. Photo taken by Cristina Martinez.

41

Table 3-4. Begonia shoot (SDW) and root (RDW) dry weight in containers watered with deionized water, waste water, fertilizer solution, or salt water applied at 100, 250, or 350 ml per irrigation (3x a week). All plants were fertilized with 5 g of 15-9-12 Osmocote prior to planting. Numbers followed by different letter are significantly different at p ≤ 0.05. Lower case letters signify analysis across all solutions and volumes, while upper case letters signify analysis across the averages by solution.

Solution / mL SDW (g) RDW (g)

DI

100 10.46c 5.58c

250 11.15bc 6.04bc

350 11.27ab 6.31ab

Average 10.96A 5.97A

Wastewater

100 10.70c 5.56c

250 11.24bc 6.02bc

350 11.64ab 6.38ab

Average 11.19A 5.98A

Fertilizer

100 9.99c 4.88c

250 10.05bc 5.08bc

350 10.36ba 5.15ab

Average 10.13B 5.04B

Salt

100 9.59c 5.06c

250 10.08bc 5.17bc

350 10.46ab 5.41ba

Average 10.18B 5.25B

42

Table 3-5. Final pour-through leachate analysis of pH, EC, NO3-N, PO4-P, Na, and K. There was no significant difference in leachate due to volume and no solution by volume interaction. All plants were fertilized with 5 g of 15-9-12 Osmocote prior to planting. Numbers followed by different letter are significantly different at p ≤ 0.05.

Solution Volume pH

EC

(dS/m)

NO3-N

(mg/g)

PO4-P

(mg/g)

K

(mg/g)

Na

(mg/g)

Deionized

water

100 6.37 1.05 d 422.50 bc 0.19 b 27.65 b 1.36 bc

250 6.51 0.73 de 407.50 bc 0.18 b 14.17 b 0.67 c

350 6.52 0.45 e 435.00 bc 0.18 b 9.70 b 0.45 c

Fertilizer 100 5.70 5.01 ab 1625.00 a 0.23 a 192.62 a 4.543 ab

250 5.61 4.61 b 1507.50 ab 0.18 b 156.13 a 5.640 ab

350 5.56 4.18 bc 1287.5 b 0.21 ab 106.89 ab 3.778 ab

Salt water 100 6.39 5.84 a 169.00 c 0.11 b 20.19 b 6.590 a

250 6.36 4.25 bc 174.75 c 0.11 b 18.54 b 3.593 ab

350 6.37 2.84 c 160.75 c 0.07 c 19.79 b 1.878 b

Wastewater 100 6.43 1.37 d 207.50 c 0.15 b 18.91 b 2.590 b

250 6.25 1.82 cd 392.00 bc 0.18 b 45.18 b 6.998 a

350 6.45 1.47 cd 164.25 c 0.09 bc 14.94 b 2.748 b

43

CHAPTER 4 EXOGENOUS APPLICATIONS OF ABSCISIC ACID IN THE PRODUCTION OF

‘ESCARGOT’ BEGONIAS EXPOSED TO SALINE IRRIGATION WATER

Introduction

In coastal areas, elevated levels of salinity are attributed to saltwater intrusion

due to overutilization of freshwater wells and sea level rise (Langevin and Zygnerski,

2012; Heimlich, et al., 2009). Drought prone areas with limited water availability may

transition to the use of reclaimed wastewater to irrigate their crops, with variable levels

of salts being of concern to growers (Karleskint, et al., 2011). In cases that a salt

tolerant species or cultivar cannot be selected, the physical symptoms of salt stress can

cause crop loss and lower the marketability of crops. Accumulation of salts can lead to

leaf tip necrosis and overall plant stunting (Küçükahmetler, 2002).. ‘Escargot’ begonias

were selected as a salt-sensitive indicator crop to display potential effects of salt stress.

Abscisic acid (ABA) is a plant hormone involved in managing abiotic stresses, such as

drought and is available commercially in products labeled for use in production of

grapes (Hartung, et al., 2002). ABA works to conserve water in the short term by closing

the stomata in the leaves and reducing overall transpiration. Stomatal conductance (SC)

is used as a measure of transpiration. Elevated levels of salts in the soil solution lead to

osmotic imbalances and can sometimes simulate a drought stress due to reduced water

uptake (Boursiac, 2005). It is hypothesized that an ABA drench applied before plants

are irrigated with saline water may lower transpiration rates and improve plant tolerance

to salinity and potentially produce more marketable plants. The objective of this

research was to compare the growth and quality of ‘Escargot’ begonias irrigated with 3

saline solutions and treated with 4 ABA rates.

44

Materials and Methods

One gallon pots were filled with Atlas Mix (Atlas Peat & Soil, Boynton Beach, FL)

with a controlled release fertilizer (15𝑁 − 9𝑃2𝑂5 − 12𝐾2𝑂 Osmocote Plus 8 to 9 month,

Scotts Co., Marysville, OH) incorporated at a rate of 5 g per liter. ‘Escargot’ begonias

were transplanted into the pots from 72 cell liner trays (AG 3, Inc., Eustice, FL). Plants

were arranged on the benchtop in a completely random design with 5 replicates per

treatment. The begonias were grown from February to March 2017 in an open-sided

greenhouse exposed to ambient air temperatures at the University of Florida Fort

Lauderdale Research and Education Center in Davie, FL. During the 6 week trial period,

mean temperature and relative humidity were 21.6 °C (70.9 °F) and 74%, respectively.

Weather data was collected by the Florida Automated Weather Network (FAWN) station

located a few hundred yards away from the greenhouse.

Plants were irrigated by hand 3 times per week with 250 mL of one of the

following solutions (Table 4-1): 1) tap water, 2) reclaimed wastewater, 3) liquid fertilizer

(Jack’s Classic Blossom Booster 10𝑁 − 30𝑃2𝑂5 − 20𝐾2𝑂 with micronutrients, JR Peters

Inc., Allentown, PA) at 3 g/L or 300 ppm N, and 4) sea salt mixed with tap water at 2.5

g/L. Wastewater was collected from the Wastewater Treatment Plant in Davie, FL at the

beginning of the trial in February 2017 and stored in sealed 5 gallon buckets with lids.

Plants irrigated with liquid fertilizer were treated with the 300 ppm N fertilizer solution

once per week as a boost and received tap water twice per week. Sea salt (Morton All-

Purpose Sea Salt, Morton Salt, Chicago, Il, USA) was mixed with tap water to create

saline water. Control plants were irrigated with tap water.

In addition to the irrigation solution treatments, plants also received a one-time

100 mL drench of ProTone (20% ABA, Valent BioSciences, Libertyville, IL) immediately

45

after transplant. Protone was drenched at 0, 125, 250, or 500 mg/L (0, 25, 50, or 100

ppm ABA). This corresponds to 50%, 100%, and 200% of the manufacturer’s

recommended rate. Acceptable application rates for begonias were derived from

concentrations used to enhance water stress tolerance by Park et al. (2016).

Initial stomatal conductances on the largest leaf of 3 of the 5 replicates per

treatment were measured using a SC-1 Leaf Porometer (Decagon Device, Pullman,

WA). Stomatal conductance was measured again 4 and 24 hours after the exogenous

application of ABA. The first irrigation occurred immediately after the 24 hour stomatal

conductance readings were obtained. Weekly stomatal conductances were measured

until trial completion after 6 weeks.

Initial leachates were collected via the pour through method using deionized

water from 3 random plants before any irrigation solutions or ABA drenches were

applied. At the end of the 6 week period, final leachates were collected from all plants.

Leachates and treatment solutions were analyzed to determine pH, EC, and

concentrations of 𝑁𝑂3, 𝑃𝑂4, K, Na, and Cl. Leachate pH and EC were measured using

an Accumet Model 20 pH/Conductivity Meter (Fisher Scientific, Waltham, MA).

Phosphate concentrations were measured using the ascorbic acid method with

absorbance at 440 nm using a Spectronic 200 Spectrophotometer (Thermo Electron

Corp, Waltham, MA). Concentrations of NO3-N, K, Na, and Cl were measured using

specific ion probes for an Accumet XL250 (Fisher Scientific, Waltham, MA).

Visual quality of plants was noted after 6 weeks. Plants were rated using the

following scale: 5) excellent, 4) above average, 3) average, 2) below average, 1) poor,

46

and 0) dead. Shoot dry weights were measured using above ground plant growth that

was harvested and dried in an oven at 54.4°C (130°F) for one week.

All data collected were analyzed in R. ANOVA was used to compare stomatal

conductances, dry weights, electrical conductivities, visual ratings, and values from

nutritional analysis of the leachates across the different irrigation solutions. Tukey’s

HSD (honest significant difference) test was used for mean separation for significant

findings in ANOVA.

Results

Stomatal conductance at 24 hours post-drench was lower for plants receiving

ABA compared to control. (Figure 4-1). After 1 week, stomatal conductance was the

same for all ABA levels and remained so for 5 weeks. The one exception was the plants

irrigated with salt water, whose stomatal conductance began to drop half way through

the trial (Table 4-2Table 4-).

Shoot dry weight varied only by irrigation solution and was not affected by the ABA drench (Table 4-3). Plants irrigated with the salt water solution had the lowest shoot dry weights. Plants receiving the weekly liquid fertilizer boost at 300 ppm N had the highest shoot dry weight overall. Visual rating of plants also varied by irrigation solution (Table 4-3Table 4-). Mortality in the plants irrigated with the salt water containing 3 g/L sea salt reached 100% by 6 weeks after transplant. Begonias irrigated with tap water had the highest visual rating (Table 4-4

Figure 4-3. Front and top views of begonias receiving an abscisic acid drench at 0, 25, 50, and 100 ppm. Plants were irrigated (from left to right) with tap water, wastewater, liquid fertilizer, and salt water for 6 weeks. Photos taken by Cristina Martinez.

Table 4-). Plants irrigated with liquid fertilizer and wastewater were of slightly

below average quality.

Significant differences in electrical conductivities were found when comparing

leachates by irrigation solution at the end of the 6 week production period. Leachate

47

from plants irrigated with salt water had the highest EC’s, being consistently above 2

dS/m. Tap and wastewater had the lowest leachate EC’s and liquid fertilizer was

intermediate (Table 4-4). Leachates from plants irrigated with the liquid fertilizer had

significantly higher levels of Na compared with the other treatments. Levels of Cl were 7

to 8 times higher in the leachate of the plants irrigated with salt water compared to all

other treatments. Potassium levels were highest in plants receiving the weekly boost of

liquid fertilizer.

Discussion

A single exogenous ABA drench was short lived and did not have any significant

effect on plant growth and quality. There was a 24 hour delay between the ABA drench

and a measured decrease in stomatal conductance. This may be due to the time for the

uptake of ABA from roots to shoots. A foliar spray may have been active more quickly

and may potentially use less product. Reductions in stomatal conductance have been

measured within 3 or 4 hours of an ABA drench (Park, et al., 2016; Kim and van Iersel,

2008). Perhaps multiple sequential exogenous applications of ABA would be more

appropriate in ameliorating negative effects of salinity stress. Little research has been

conducted on the use of ABA too improve salt stress response in ornamental plants.

ABA drenches have been shown to enhance growth in potatoes irrigated with complete

nutrient solutions with up to 180 M NaCl (Etehadnia, et al., 2008). A lack of significance

in the decrease in stomatal conductance among the three ABA concentrations (25, 50,

and 100 ppm) suggests that lower application rates may be used for the same effect.

The suggested label rate for Protone drenches in grapes is 50 ppm ABA, so the 25 ppm

drench is half the label rate. ‘Escargot’ begonias may be more sensitive to lower rates of

ABA.

48

Plant growth was mostly influenced by the EC of the irrigation solution and not by

the rate at which the ABA was applied. The high mortality and low above ground

biomass in the plants irrigated with salt water may be attributed to higher ECs in the

irrigation solution. It has been documented that K and Na are competitive at the level of

root uptake and that elevated levels of K may restrict the uptake of Na (Rodrigues, et

al., 2013; Wakeel, 2013). The elevated K levels as seen in the liquid fertilizer may have

minimized uptake of Na by the plants and minimized accumulation in the tissue before

they became too toxic. It should be noted that K was present in all treatments due to the

presence of controlled release fertilizer incorporated into the potting media.

Overall, it appears that begonias are very sensitive to high ECs. Begonias have

been found to be show reduced growth when irrigated with solutions with an EC of 3

dS/m with 80% of the salts in the form of Na and Cl (Miyamoto, et al., 2004). Rex

begonias perform best when substrate pour-through ECs are between 1.0 and 26

mS/cm (Whipker, et al., 2001). Wastewater can successfully be used to irrigate

begonias. However, the source of the wastewater should be known and salinity levels

should be tested before use as they can fluctuate over time (Karleskint, et al., 2011).

Begonias may tolerate concentrations of up to 100 ppm ABA without any

symptoms of phytotoxicity, such as defoliation. Concentrations of 25 ppm were sufficient

in lowering stomatal conductance after 24 hours. More research must be done to

continue to test the efficacy of ABA drenches in improving salt tolerance of plants.

Weekly exogenous applications of ABA and lower liquid fertilizer concentrations may be

appropriate for begonias in future research. Effects of ABA on flower production of

begonias should also be studied.

49

Table 4-1. Chemical analysis (pH, electrical conductivity EC, sodium Na, potassium K, nitrate NO3-N, and phosphate PO4-P) of stock solutions of wastewater, fertilizer solution, and salt water.

Solutions pH EC

(dS/m)

NO3-N

(mg/g)

PO4-P

(mg/g)

K

(mg/g)

Na

(mg/g)

Cl

(mg/g)

Tap water 7.52 0.37 13.978 0.11 2.898 0.741 140.73

Wastewater 7.82 0.61 17.508 0.13 6.43 0.154 175.19

Liquid fertilizer 5.81 2.16 144.006 2.64 43.421 7.75 137.69

Saline water 7.47 2.81 44.83 0.07 2.786 136.2 1192.4

Figure 4-1. Stomatal conductances of begonia plants 24 hours after an abscisic acid

drench of either 0, 25, 50, or 100 ppm. No irrigation solution had been applied before these readings were taken.

y = -0.0001x3 + 0.0285x2 - 2.0207x + 119.18R² = 0.3951

0

20

40

60

80

100

120

140

160

180

200

0 25 50 75 100

Sto

mat

al C

on

du

ctan

ce (

mm

ol/

(m^2

s)

ABA Rate (ppm)

50

Table 4-2. Stomatal conductances of begonia plants before and after an abscisic acid drench of either 0, 25, 50, or 100 ppm. All measurements are in measured in mmol/(m^2s).

Solution ABA

Rate

(ppm)

Initial 4 Hours 24

Hours

1 Week 3 Weeks 6

Weeks

Tap water 0 161.6 a 127.9 a 143.8 a 156.5 a 129.9 b 127.4a

25 152.1 a 127.3 a 63.9 ab 131.8 a 124.6 b 141.3a

50 164.9 a 113.3 a 60.4 ab 147.3 a 99.0 b 151.7a

100 146.4 a 131.4 a 63.2 ab 96.7 a 228.4 a 192.8a

Liquid

fertilizer

0 144.6 a 141.3 a 121.2 ab 157.9 a 204.1 ab 147.2a

25 155.0 a 121.0 a 94.7 ab 142.1 a 125.3 b 196.1a

50 158.6 a 106.8 a 64.6 ab 115.8 a 165.4 b 120.8a

100 142.4 a 126.9 a 54.6 b 113.3 a 185.4 b 128.2a

Wastewater 0 155.8 a 114.4 a 103.6 ab 124.3 a 173.4 b 192.1a

25 169.1 a 151.7 a 91.9 ab 133.0 a 164.7 b 149.1a

50 160.5 a 90.3 a 68.1 ab 132.4 a 142.7 b 180.4a

100 143.8 a 111.1 a 78.2 ab 154.5 a 153.6 b 193.6a

Salt water 0 165.0 a 103.8 a 108.1 ab 104.7 a 93.0 bc NA

25 132.9 a 113.1 a 86.7 ab 130.4 a 54.3 c NA

50 171.3 a 116.6 a 95.0 ab 131.3 a 86.9 bc NA

100 141.9 a 84.4 a 58.3 b 114.2 a 76.6 bc NA

P>F

ABA Rate NS NS *** NS NS NS

Solution NS NS NS NS *** ***

Rate x

Solution

NS NS NS NS NS NS

Significance. codes: ‘***’ = 0.001, ‘**’ = 0.01, ‘*’ = 0.05, NS = not significant

51

Table 4-3. Final visual ratings and shoot dry weights (SDW) in grams of begonia plants 6 weeks after receiving an abscisic acid drench of 0, 25, 50, or 100 ppm and being irrigated with one of four saline solutions (liquid fertilizer, salt water, tap water, and wastewater). Numbers followed by different letter are significantly different at p ≤ 0.05.

Solution ABA Rate (ppm) Visual Rating SDW (g)

Tap water 0 3.4 a 1.256 a

25 3.0 a 1.020 a

50 3.0 a 0.940 a

100 3.6 a 1.326 a

Liquid fertilizer 0 2.6 ab 1.020 a

25 2.6 ab 1.192 a

50 3.2 a 1.382 a

100 2.8 a 1.524 a

Wastewater 0 2.6 ab 1.360 a

25 3.4 a 1.300 a

50 1.8 ab 0.788 ab

100 2.2 ab 1.068 a

Salt water 0 0.0 b 0.338 b

25 0.0 b 0.582 b

50 0.0 b 0.570 b

100 0.0 b 0.476 b

P>F

ABA Rate NS NS

Solution *** ***

Rate x Solution NS NS

Significance. codes: ‘***’ = 0.001, ‘**’ = 0.01, ‘*’ = 0.05, NS = not significant

52

Solution Front view Side view

Tap water

Wastewater

Liquid fertilizer

Salt water

Figure 4-2. Front and side views of begonias irrigated with tap water, wastewater, liquid fertilizer, and saltwater for 6 weeks. From left to right the abscisic acid drench rates are 0, 25, 50, and 100 ppm. Photos taken by Cristina Martinez.

53

ABA rate Front view Top view

0 ppm

25 ppm

50 ppm

100 ppm

Figure 4-3. Front and top views of begonias receiving an abscisic acid drench at 0, 25, 50, and 100 ppm. Plants were irrigated (from left to right) with tap water, wastewater, liquid fertilizer, and salt water for 6 weeks. Photos taken by Cristina Martinez.

54

Table 4-4. Final pour-through leachate analysis of pH, EC, NO3-N, PO4-P, K, Na, and Cl. All plants were fertilized with 5 g of 15-9-12 Osmocote prior to planting.

Solution ABA Rate

(ppm) pH

EC

(dS/m)

NO3-

N

(mg/g)

PO4-

P

(mg/g)

K

(mg/g)

Na

(mg/g)

Cl

(mg/g)

Tap water 0 5.75 0.68 63.72 0.51 39.11 1.14 79.30

25 6.21 0.62 47.98 0.32 25.95 1.07 91.18

50 6.06 0.70 64.29 0.42 30.89 1.20 81.05

100 6.07 0.68 51.15 0.41 29.90 1.09 104.16

Average 6.02 0.67 56.79 0.42 31.46 1.13 88.92

Liquid fertilizer 0 5.32 1.51 132.02 2.14 99.50 3.73 96.09

25 5.41 1.30 122.19 2.99 75.17 3.37 97.42

50 5.34 1.32 75.44 2.47 69.10 3.54 135.46

100 5.37 1.45 144.16 1.77 84.54 3.85 102.81

Average 5.36 1.40 118.45 2.34 82.08 3.62 107.95

Wastewater 0 5.99 0.79 71.27 0.61 33.31 1.31 143.14

25 5.82 1.11 54.32 0.65 46.49 1.92 135.64

50 6.08 0.74 69.04 0.41 34.90 1.22 122.39

100 6.36 0.70 45.98 0.42 26.92 0.95 121.67

Average 6.06 0.84 60.15 0.52 35.41 1.35 130.71

Salt water 0 6.24 2.45 99.86 0.47 24.68 2.22 857.18

25 6.16 2.41 93.37 0.65 32.24 2.39 863.36

50 6.00 2.36 66.45 0.56 34.01 2.73 813.08

100 6.29 2.23 82.53 0.44 29.89 3.13 760.99

Average 6.17 2.36 85.55 0.53 30.21 2.62 823.65

P>F

ABA Rate NS NS NS NS NS NS NS

Solution *** *** *** *** *** *** ***

Rate x Solution NS NS NS NS NS NS NS

Significance. codes: ‘***’ = 0.001, ‘**’ = 0.01, ‘*’ = 0.05, NS = not significant

55

CHAPTER 5 CONCLUSION

Access to high quality irrigation water is diminishing due to drought and saltwater

intrusion. Agriculture will need to learn how to manage salinity in irrigation water,

especially as water usage increases and facilities transition to the utilization of

alternative water sources. Producers of containerized ornamental plants especially have

a difficult challenge ahead of them, as they often grow many different species and

varieties of plants at once and containerized plants have a limited soil volume.

The objective of the first experiment conducted during this research was to

compare the growth and quality of rex begonias (Begonia x ‘Looking Glass’) watered

with different saline solutions, including wastewater, liquid fertilizer, and salt water.

These solutions were either at an EC equal to that of the wastewater (6 dS/m) or diluted

to half-strength (3 dS/m). The original hypothesis was that the plants irrigated with

solutions containing lower EC levels would grow to be larger than plants irrigated with

higher EC levels. Shoot dry weights were used as an indicator of plant size. Plants

irrigated with a half-strength solution of liquid fertilizer had the highest shoot dry

weights, while plants irrigated with the full-strength salt water had the lowest shoot dry

weights. Concentrations of soluble salts were highest in leachates from plants receiving

the full-strength fertilizer and salt solutions. Potassium was also high in the leachates

from the liquid fertilizer treatment. It was suggested that high EC and Na alone were not

sufficient to be detrimental to above ground biomass and that a greater presence of K in

the soil solution may help negate the effects of the Na.

The objective of the second experiment was to compare the effects of various

leaching fractions represented by different irrigation volumes (100, 250, or 350 mL) on

56

the same variety of begonias. It was hypothesized that the intermediate leaching

fraction would produce plants with the greatest shoot dry weight because too low of an

irrigation volume would accumulate high levels of salts and too high of an irrigation

volume would leach out too many nutrients from the controlled release fertilizer. It

resulted that the begonias receiving higher volumes at each irrigation (350 mL) had the

greatest shoot dry weights. Electrical conductivities in the leachate lowered as the

irrigation volume increased.

The objective of the third and final experiment was to determine whether an

exogenous application of ABA in the form of a drench immediately after transplant

would improve the growth and quality of begonias (Begonia x ‘Escargot) irrigated with

wastewater, liquid fertilizer, or salt water. In this experiment, the liquid fertilizer was

applied once weekly as a nutrient boost. It was originally thought that the ABA drench

would temporarily improve the plants’ tolerance to salt in the irrigation water by lowering

transpiration and creating more of an osmotic balance within the plant tissue. It was

found that a single ABA drench, regardless of concentration, was able to decrease

stomatal conductance after 24 hours. However, this did not last. Growth was mostly

affected by the irrigation solution. Plants irrigated with salt water had high mortality,

perhaps from the elevated EC and Na in the solution. High Na concentrations were

found in the leachates from plants receiving liquid fertilizer boosts on a weekly basis.

These leachates also had higher levels of K, which may have lowered the uptake of Na

by the plants

A salinity action plan should be developed to use when the irrigation water has a

high EC or elevated levels of Na or Cl. Lower fertilization rates should be used as high

57

nutrient levels can decrease plant size, especially if irrigation water is already high in

salts. Wastewater can be used to grow marketable begonia plants, but water quality

should be monitored as they are sensitive to high ECs. If growers are to consider

exogenous applications of ABA to help combat the negative effects of salinity stress or

other abiotic stresses, a concentration of 25 ppm in the form of a drench is sufficient to

see significant decreases in transpiration after 24 hours. Spray applications should be

considered in future experiments. The efficacy of multiple applications of ABA, perhaps

on a weekly basis, needs to be studied and may or may not be beneficial to plants

experiencing salinity stress.

58

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63

BIOGRAPHICAL SKETCH

Cristina Martinez is a native South Floridian and the oldest of 3 sisters. She

obtained her Associate in Arts from Broward College and transferred to the University of

Florida Fort Lauderdale Research and Education Center (FLREC) where she worked in

the ornamental horticulture lab under Dr. Kimberly Moore. She earned her Bachelor of

Science in plant science with an emphasis in environmental horticulture in December

2015 from the University of Florida. Before beginning her graduate coursework, she

completed an internship with Syngenta conducting greenhouse and field trials of

bedding plants. From there she accepted an internship through the Hispanic

Association of Colleges and Universities (HACU), which allowed her to work for the

USDA Invasive Plant Research Laboratory (IPRL) where she used moths and mites to

control an exotic climbing fern. While working for the USDA, she received her Master of

Science in horticultural sciences from the University of Florida in December 2017.