Baysal G

SOIL BORON DETERMINATION METHODS
AND
BORON UPTAKE IN TOMATOES
By
GÜRCAN DUYGU BAYSAL
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
2018
© 2018 Gürcan Duygu Baysal
To my mom, my dad, my sister, and Lila
ACKNOWLEDGMENTS
If a professor teaches ardently and encourages their students to learn, their students want
to learn more and develop themselves. I am very lucky that I have a helpful, altruist, and humane
advisor, Dr. Rao S. Mylavarapu. He always encourages me to learn, to improve my knowledge
and skills. Thanks to his advice and insight, I believe that I will use my ability of analytical
thinking and looking from different perspectives all my life. I would like to say thanks with my
whole heart for all of his support and help in my research and study.
I would like to thank my committee members Dr. George Hochmuth and Dr. John
Erickson for their precious time and advice.
I believe I am one of the luckier people in the world; since my childhood, my parents,
Dilek & Mehmet Baysal, and my grandmother (RIP) Naime Gur always gave their love and
encouraged me to be successful. They encouraged me to follow my dreams and I am realizing
my dream now. I am always proud of being their first love. Also, not only my sister but also my
first and best friend, Burcu Naime Baysal, who I thank for all her support and unforgettable
memories.
Additionally, I would like to thank my previous advisor Dr. Ibrahim Erdal for all his help
and trust. All of my academic study interests started with him and he convinced me to work hard
and be successful.
I also want to say thanks to my friends Ryan S. MacNeille and Lila. They are always
bringing me good luck and helping me build the confidence to finish my study. For not only their
technical assistance in research but also for their personal support, I would like to thank Laura
Jalpa, Rosemary Collins, Fernando Bortozolo, Sevda Yildirim, and Ahmet Durhan.
Finally, I would like to thank Mustafa Kemal Ataturk (RIP), his fellow soldiers, and the
Republic of Turkey which they founded.
4
TABLE OF CONTENTS
page
ACKNOWLEDGMENTS ...............................................................................................................4
LIST OF TABLES ...........................................................................................................................7
LIST OF FIGURES .........................................................................................................................9
LIST OF ABBREVIATIONS ........................................................................................................10
ABSTRACT ...................................................................................................................................13
CHAPTER
1 INTRODUCTION ..................................................................................................................15
Boron in Soil and Plants .........................................................................................................15

Boron Management ................................................................................................................16
Boron Uptake in Tomatoes .....................................................................................................17
2 LITERATURE REVIEW .......................................................................................................19
Study 1. Analytical Methods and Laboratory Protocol ..........................................................19

Study 2. Boron Uptake in Tomatoes.......................................................................................22
Boron Physiology in Plants .............................................................................................23
Boron Uptake in Plants ....................................................................................................25
Deficiency and Toxicity in Plants ...................................................................................27
Plant Availability .............................................................................................................29
Interactions with Other Nutrients ....................................................................................30
Species and Genotype......................................................................................................31
Boron Fertilizers ..............................................................................................................31
3 MATERIALS AND METHOD ..............................................................................................34
Study 1. Analytical Methods and Laboratory Protocol ..........................................................34

Sample Preparation ..........................................................................................................34
Laboratory Protocol Comparison ....................................................................................34
Determination Methods ...................................................................................................35
Hot-water extraction .................................................................................................35
Mehlich-3 extraction ................................................................................................36
Mehlich-1 extraction ................................................................................................36
Statistical Analyses ..........................................................................................................37
Site and Sample Preparation ............................................................................................38
Site and Sample Management .........................................................................................38
Plant Sampling.................................................................................................................39
Sample Digestion.............................................................................................................39
5
Verification ......................................................................................................................40
Statistical Analyses ..........................................................................................................40
4 RESULTS AND DISCUSSION .............................................................................................41
Study 1. Analytical Methods and Laboratory Protocol Study ................................................41

Laboratory Comparison ...................................................................................................41
Hot-water Extraction .......................................................................................................41
Instrument comparisons and boiling durations ........................................................41
Extraction delay comparison ....................................................................................43
Mehlich-3 Extraction .......................................................................................................44
Instrument comparison .............................................................................................44
Soil:extractant solution ratio comparison .................................................................44
Mehlich-1 Extraction .......................................................................................................46
Soil:extractant solution ratio adjustment comparison ..............................................46
Instrument comparison .............................................................................................47
Treatment to extraction delay comparison ...............................................................48
Replication with sandy loam soil .............................................................................49
Fruit Yield .......................................................................................................................50
Above-Ground Fresh Plant Weight .................................................................................50
Leaf B Content ................................................................................................................51
Petiole B Content .............................................................................................................52
Stem B Content................................................................................................................53
Root B Content ................................................................................................................53
Total B Content ...............................................................................................................54
Summary..........................................................................................................................54
5 CONCLUSION.......................................................................................................................64
Study 1. Analytical Methods and Laboratory Protocol Study ................................................64

LIST OF REFERENCES ...............................................................................................................66
BIOGRAPHICAL SKETCH .........................................................................................................78
6
LIST OF TABLES
Table page
4-1 Comparison of four different laboratories B measurement means ....................................55
4-2 Comparison of boiling duration measurement means from Hot-water extraction with
measurement from ICP-OES .............................................................................................55
4-3 Comparison of boiling duration recovery means from Hot-water extraction with
measurement means from ICP-OES ..................................................................................55
4-4 Comparison of boiling duration measurement means from Hot-water extraction with
measurement from spectrophotometer ...............................................................................56
4-5 Comparison of boiling duration recovery means from Hot-water extraction with
measurements from spectrophotometer .............................................................................56
4-6 Comparison of delays using Hot-water extraction with measurement means from
ICP-OES ............................................................................................................................56
4-7 Comparison of delays using Hot-water extraction with recovery means from ICP-
OES ....................................................................................................................................57
4-8 Comparison of instrument measurement means using Mehlich-3 extraction ....................57
4-9 Comparison of instrument recovery means using Mehlich-3 extraction ...........................57
4-10 Comparison of soil:extractant solution ratios using Mehlich-3 extraction with


recovery means from ICP-OES .........................................................................................58


recovery means from ICP-OES .........................................................................................58
4-14 Comparison of instrument measurement means using Mehlich-1 extraction with 1:1
soil:extractant solution ratio ...............................................................................................59
4-15 Comparison of instrument recovery means using Mehlich-1 extraction with 1:1
soil:extractant solution ratio ...............................................................................................59
4-16 Comparison of delays using Mehlich-1 extraction with measurement means from
ICP-OES using 1:1 soil:extractant solution ratio (premium sand) ....................................59
7
4-17 Comparison of delays using Mehlich-1 extraction with recovery means from ICP-
OES using 1:1 soil:extractant solution ratio (premium sand) ............................................59
4-18 Comparison of delays using Mehlich-1 extraction with measurement means from
ICP-OES using 1:1 soil:extractant solution ratio (sandy loam soil) ..................................60
4-19 Comparison of delays using Mehlich-1 extraction with recovery means from ICP-
OES using 1:1 soil:extractant solution ratio (sandy loam soil) .........................................60
4-20 Effect of B treatment on first season above-ground plant part weight ..............................60
4-21 Effect of B treatment on second season above-ground plant part weight..........................60
4-22 Effect of B treatment and method on first season leaf B content ......................................61
4-23 Effect of B treatment on second season leaf B content .....................................................61
4-24 Effect of B treatment and method on first season petiole B content .................................61
4-25 Effect of B treatment on second season petiole B content.................................................61
4-26 Effect of B treatment and method on first season stem B content .....................................61
4-27 Effect of B treatment on second season stem B content ....................................................62
4-28 Effect of method on first season root B content ................................................................62
4-29 Effect of B treatment and method on second season root B content .................................62
4-30 Effect of B treatment and method on first season total B content .....................................62
4-31 Effect of method on first season total B content ................................................................62
4-32 Effect of B treatment on second season total B content ....................................................63
8
LIST OF FIGURES
Figure page
1-1 Commercial B Production by Country ..............................................................................18
2-1 Process of B Uptake under Deficient Condition ................................................................33
9
LIST OF ABBREVIATIONS
AES Atomic Emission Spectrometry
Al Aluminum
ANOVA Analysis of Variance
ANSERV University of Florida, IFAS Analytical Services Laboratories
B Boron
B203 Boron Oxide
BOR1 Efflux-type boron transporter for xylem loading
Btl Recessive gene in Tomato plants responsible for ‘Brittle’ leaves and stems
Ca Calcium
CEC Cation Exchange Capacity
CO Cortex
CO2 Carbon Dioxide
CS Casparian Strip
Db Bulk Density
DDI Double-distilled Water
DI Distilled Water
DNA Deoxyribonucleic Acid
DTPA Diethylenetriaminepentaacetic Acid
EDTA Ethylenediaminetetraacetic Acid
EN Endodermis
EP Epidermis
Fe Iron
H3BO3 Boric Acid
10
HCI Hydrochloric Acid
IAA Indole-3-Acetic Acid
ICP Inductively Coupled Plasma
K Potassium
LSD Least Significant Difference
Mg Magnesium
MS Mass Spectrometry
N Nitrogen
Na Sodium
Na2B4O7.10H2O Borax
Na2B4O7.5H2O Etibor-48
Na2B8O13.H2O Etidot-67
Na2B8O7 Anhydrate Borax
NAPT North American Proficiency Testing Program
Ni Nickel
NIP5-1 Boric Acid Transporter
O2 Oxygen
OES Optical Emission Spectrometry
P Phosphorus
pH Potential Hydrogen
PHW Pressurized Hot-water
RG-II Rhamnogalacturonan II
RNA Ribonucleic Acid
Rutgers Boron-efficient Tomato Strain
S Sulfur
11
SAR Sodium Absorption Ratio
Si Silicon
T3238 Boron-inefficient Tomato Strain
ToMV Tomato Mosaic Virus
XY Xylem
Zn Zinc
12
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
SOIL BORON DETERMINATION METHODS

AND
BORON UPTAKE IN TOMATOES
By
Gurcan Duygu Baysal
August 2018
Chair: Rao Mylavarapu

Major: Soil and Water Sciences
Several methods have been used for determining Boron (B) concentrations in soils over
the years. However, soil properties such as pH, texture, organic matter, and mineralogy have
been found to directly influence determination of B. Also, B may be complexed in soils due to
transformations through one or more of its several oxidative states. Therefore, there is an express
need to identify a suitable and consistent method for routine laboratory analyses of soil B.
In this study, soil test methods such as Mehlich-1, Mehlich-3, and Hot-water extractions
were compared using standard and varying laboratory protocols for soil B determinations on
ICP-OES and spectrophotometer to help screen for an optimal method. Hot-water extraction
resulted in largely inconsistent measurements across all experiments but provided the most
consistent results with a boiling duration of 5 minutes. However, results from Hot-water
extraction varied as delay between B treatment and extraction increased. While Mehlich-3
extraction yielded more consistent results than Hot-water extraction when adjusting the
soil:extractant solution ratio from 1:10 to 1:1, Mehlich-1 yielded the most consistent results from
all tests when the soil:extractant solution ratio was adjusted to 1:1 from the standard 1:4.
13
A replicated greenhouse study was also conducted to determine uptake of B by tomato
plants grown in sandy soils. Tissue nutrient levels and plant growth parameters were measured in
plants that received granular or foliar treatments of B, with concentrations of 1, 2 and 3 mg kg-1,
and compared against a control. Due to severe insect and disease pressure, the yields in both
seasons were severely suppressed and therefore were not used to draw comparisons. The results
indicated that the effectiveness of application method may be influenced by pest-related factors
such as viruses, which may alter the uptake of B in different plant parts. In this study, we
observed that granular application improved B uptake in tomato plants more than foliar. This
information may be useful for further research in correlating the physiology of B on tomatoes
under different health conditions with varying B treatment methods.
14
CHAPTER 1
INTRODUCTION
Boron in Soil and Plants
Boron is an essential nutrient required for the development, growth, yield, and seed
improvement of higher plants. Although B is a micronutrient, requiring only a small quantity
to be effective, this essential element plays a significant physiological role in sustaining plant
growth and production. While B availability is crucial to commercial crop production, the
deficiency-toxicity range of B is narrow, a typical nature of essential micronutrients, making
the use of scientifically-reliable tools critical for optimal management and to ensure
commercial viability.
Boron is the 51st most common element in the Earth’s crust with an average concentration
below 10 mg kg-1 (Krauskopf, 1972) in borosilicate and borate forms, and is the only element
which is nonmetallic of the seven essential plant micronutrients. B was first discovered in 1808
by French chemists Joseph-Louis Gay-Lussac and Louis-Jaques Thénard (Onal and Burat, 2008)
and although it is not found as a standalone element in nature due to its natural reaction to
oxygen (Kilinc et al., 2001 and Onal and Burat, 2008), B has since been found in more than 150
mineral compositions and more than 250 types of rock.
It is estimated that the world’s B reserve is 1.176 billion tons, 72.2% of which (851
million tons), is found in Turkey (Onal and Burat, 2008). Countries contributing the majority of
commercial B production, in order of quantity, consist of Turkey, USA, Argentina, Bolivia,
Chile, China, Peru, Russia, and Kazakhstan (Kilinc et al., 2001). However, B production and
consumption rates in the USA are withheld for proprietary reasons (US Department of Interior,
US Geological Survey, 2011) (Figure 1-1).
15
Boron exists in soils in four primary forms: water-soluble, absorbed, organically bound,
and fixed in clay and mineral lattices. Of these forms, water-soluble B has the greatest
agricultural significance due its direct role in plant nutrition. Water-soluble reserves occur
naturally in soils predominantly as undissociated, electroneutral boric acid, B(OH)3 (Marschner,
1995), derived from sediments or plant material which originally formed from tourmaline, a
relatively insoluble borosilicate.
The total B content in normal surface soils ranges from 2 to 100 mg kg-1 (Swaine,
1955) with an average of about 30 mg kg-1. However, less than 5% of total soil B is plant-
available. The plant-availability of soil-applied B is dependent on a number of factors,
including the physical and chemical characteristics of the soil such as pH, texture, organic
matter, as well as the plant species and genotype, method of B treatment, irrigation practices,
environmental factors, and the interaction of B with other nutrients such as calcium (Ca) and
magnesium (Mg). The variability of plant-availability may be further complexed in soil due to
transformations through one or more of B’s several oxidative states.
Boron Management
Boron is one of the more widely applied micronutrients in agricultural fertilizers and is
available in a variety of forms and concentrations. Of the common B fertilizers, sodium
octaborate (Solubor) is the most concentrated B source that can be foliar applied as a liquid or
dust and is capable of dissolving more readily than other fertilizers. The more common B
treatment methods are broadcast, banded, and foliar spray or dust.
Management of B in agricultural soils requires employment of scientific tools such as
predictive and diagnostic soil testing as well as effective management of borate fertilizers to
achieve an appropriate balance between toxicity and deficiency. A suitable and consistent
16
method for routine laboratory analyses of soil B is therefore required. Several methods have
been used for determining B concentrations in soils over the years, including colorimetric,
fluorometric, and plasma-source methods, which employ plasma source OES and MS,
spectrophotometric, potentiometric, ionomeric methods, atomic spectrometric and nuclear
reaction analytical principles. In spite of several decades of research, the effectiveness of
these methods has been found to be generally inconsistent and often leading to conflicting
measurements, making the management of soil B sub-optimal.
Boron Uptake in Tomatoes
Although much research has been conducted on optimizing tomato production yield,
research on B partitioning in tomato plants is limited. Therefore, a laboratory study and
complementary greenhouse study were conducted with the objectives of (i) determining a
consistent and accurate analytical method and an appropriate laboratory protocol for
extractable B in soils, (ii) identifying a suitable complementary laboratory instrumentation for
reliable determination of extractable soil B, and (iii) determining B uptake and partitioning in
tomato plants grown on sandy soils with fertigation in the greenhouse.
17
Figure 1-1. Commercial B Production by Country
18
CHAPTER 2
LITERATURE REVIEW
Study 1. Analytical Methods and Laboratory Protocol
Over the years as technology has progressed, numerous methods have been tested for
determining accurate measurements of B in soil including colorimetric methods, fluorometric
method, plasma-source methods such as plasma-source OES and plasma source MS,
spectrophotometric methods, potentiometric methods, ionomeric methods, atomic spectrometric
methods, and nuclear reaction analytical methods (Sah and Brown, 1997).
Due to soil factors such as pH, texture, organic material, and minerology, the
recommended B determination method can vary according to soil properties. For acidic soils,
titrimetric procedures, spectroscopic methods, colorimetric methods and hot-water extraction
should be used for soil B analysis (Gupta, 1993j). On the other hand, alkaline soils are most
commonly tested with Hot-water extraction, saturated soil extracts, and spectroscopic methods
(Gupta, 1993l).
One of the earliest known tests was conducted in 1913 by Bertrand and Agulhon, who
used turmeric paper to determine small amounts of B in soil; however, it has since been
discovered that turmeric paper is not sensitive enough for small B variances (Berger and Truog,
1939). Before the introduction of Inductively Coupled Plasma-Atomic Emission Spectroscopy
(ICP-AES), it was discovered that extractable B can be determined with colorimetric methods by
using reagents such as carmine or Azomethine-H (Hatcher and Wilcox, 1950; Wolf, 1971; Sonon
et al., 2014). There were two common methods for plant and soil analysis using colorimetric
procedures (Dible et al., 1954). One of these methods, called quinalizarin method, utilized a
reaction between quinalizarin and boric acid in concentrated sulfuric acid which changed color
from pink to blue. The other procedure, called the curcumin method, relied on the production of
19
rose-colored rosocyanine which is produced when an acid borate solution containing curcumin is
evaporated. As technology continued to develop over time, so did the preferred analysis
methods. By 1996, ICP-AES was the most commonly used equipment for soil B analysis, even
with different determination methods (Keren, 1996; Sonon et al., 2014).
Berger and Troug (1939) developed the Hot-water extraction method by using a soil-to-
water ratio of 1:2 and boiling for 5 minutes. Gupta (1967) modified this method by increasing
boiling time from 5 to 10 minutes and later reported that ammonium acetate responds as a better
extractant compared to Hot-water (Gupta and Stewart, 1975). Odom (2008) later stated that the
most common boiling duration (5-minute) is not enough for extractable B in soil, and short
boiling durations produce more error than longer boiling durations; recommending a 10-minute
boiling duration for accurate B measurement.
Another of the more common soil tests is diethylenetriaminepentaacetic acid extraction
(DTPA), developed by Lindsay and Norvell in 1978. Although not included in the original
DTPA extraction method, plant-available B can be extracted with refluxing boiling water
(Berger and Truog, 1940; Goldberg and Suarez, 2014), which requires an additional extraction
step. When combined with sorbitol as a chelating agent, DTPA can provide simple multi-element
extraction capabilities using ICP-AES (Miller et al., 2000; Redd et al., 2008) but can require
additional monitoring of microbial contamination (Vaughan and Howe, 1994; Redd et al., 2008).
By 1994, Hot-water extraction became the most common procedure for determining
available B for plants in soils, but this procedure is tedious, time consuming, and needs some
additional considerations (De Abreu et al., 1994). More recent studies suggest highly favorable
results from a pressurized Hot-water (PHW) method in which B is extracted as boiling distilled
water is forced under pressure through soil.
20
In 1984, attempting to create a single chemical reagent that can extract all essential plant
nutrients, Mehlich improved the chemistry of his Mehlich-1 version of a “universal” soil
extractant and developed the Mehlich-3 extraction method (Mehlich, 1984). This chemical
improvement included salt, dilute acid, fluoride, and the addition of Ethylene-diamine-tetraacetic
acid (EDTA), which served to enhance the extraction of micronutrients. Mehlich-3 is one of the
more commonly used soil tests for simultaneous micronutrient determination and has since
shown promising results in estimating B levels in soils.
Zhang et al. (2014) stated that B analysis cannot be done with Mehlich-3 extraction using
a soil:extractant solution ratio of 1:10 due to Fluoride (F) which may release B from borosilicate
glass required for the test. Redd et al., (2008) also found that older ICP models may have
significant drift when measuring B and concluded that Mehlich-3 using a soil:extractant solution
ratio of 1:10 is not effective at predicting yield, suggesting that Mehlich-3 should not be
considered a universal extractant when including B. Allen et al., (2005) compared Hot-water,
pressurized Hot-water, DTPA-Sorbitol, and Mehlich-3 extraction results measured with ICP-
OES. According to results, Mehlich-3 consistently produced overestimated results compared to
the other extractants.
The Mehlich-1 extraction was developed by Dr. Adolf Mehlich in 1953 to determine the
bioavailability of phosphorus (P), potassium (K), calcium (Ca), and magnesium (Mg) in soils
(Mehlich, 1953) with a particular emphasis on P (Nelson et al., 1953). Mehlich-1 extractant is a
dilute double reagent, which is calibrated for macronutrient estimations in soils. One of the
primary limitations however is that Mehlich-1 extraction is only effective in the acidic range of
soil pH (Mylavarapu et al., 2014), leading to the need for the development of the more consistent
21
Mehlich-3 method for covering a wider range of soil pH and effectively extracting
micronutrients.
The North American Proficiency Testing program facilitates the soil testing processes
among 150 state and commercial laboratories and lists protocols for both Mehlich-1 and
Mehlich-3 as acceptable methods of B extraction, with six laboratories using Mehlich-1 and over
50 laboratories using Mehlich-3 (NAPT 2014).
With the understanding of potential variance in measurement accuracy, our objective was
to test and compare various determination methods and equipment, then adjust the analytical
parameters to identify an optimal extractable B measurement protocol.
Study 2. Boron Uptake in Tomatoes
Boron has been known to exist in many plants since 1910 (Agulhon) and was described
as being essential for plant development as early as 1915 (Maz´e). However, the essentiality of B
did not achieve widespread acceptance until Warington (1923) demonstrated that B was essential
to the healthy growth of Windsor broad beans. This discovery led to research by Brenchley and
Thornton (1925) in an attempt to determine the cause of B influence on the plant, suspecting that
there was a particular requirement of the Windsor broad bean plant which B is capable of
supplying. Subsequently, Sommer and Lipman (1926) published evidence for the establishment
of a general need for B by higher plants and outlined its effects on six non-leguminous dicots and
one graminaceous monocot (barley).
Upon repeated demonstrations that B is an essential element for higher plants, research
on determining its function began primarily by comparing the responses to B deficiency of
growing plant cells, resulting in many structural damage characteristics attributed to B
deficiency. However, many of these symptoms are often secondary effects of B deficiency
caused by changes in membrane permeability (Pilbeam and Kirkby, 1983). This fact,
22
compounded by the very small amount required for normal development, makes B probably the
least understood of all essential nutrients in plants (González-Fontes et al., 2008).
Boron Physiology in Plants
As these responses to B deficiency were documented, many scientists began describing
contribution of B to a variety of physiological processes in vascular plants, including synthesis
and lignification of cell walls and membranes, transport and metabolism of photosynthetic
products, nucleic acid metabolism, and phenolic acids and hormonal metabolism.
Synthesis and lignification of cell walls and membranes: Yamaguchi et al., (1986)
reported that B is distributed in the pectin of tomato leaf cell walls, and Matoh et al., (1992)
demonstrated that the majority of cell B is localized in the cell wall of tobacco plants.
Subsequently, Matoh et al. (1993) isolated a pectin-like cell wall component that bound most of
the cell B in radish roots.
An increasing number of publications support the idea that B plays a critical role in
plasma membrane integrity by binding membrane compounds containing cis-diol groups or
affecting activities of some enzymes related to membrane function or structure, or participating
phenolics metabolism (Blevins and Lukaszewski, 1998; Goldbach et al., 2001; Brown et al.,
2002,).
Transport and metabolism of photosynthetic products: Gauch and Dugger (1953) stated
that B treatment was shown to increase the export of sucrose from treated leaves by reacting with
sugar and forming a sugar-borate complex which moves through cellular membranes more
readily than non-borated sugar molecules. Subsequently, they described the secondary effects of
B-deficiency on O2 evolution, CO2 fixation and carbohydrate levels.
Nucleic acid metabolism: Birnbaum et al. (1974) described a systematic reduction in
growth of boron-deficient ovules in cotton plants which were affected by inhibitors of RNA and
23
DNA synthesis. Subsequently, Middleton et al. (1978) and Jarvis et al., (1983) described that B-
deficiency in roots of squash result in a complete failure of mitosis prior to cessation of DNA
synthesis and later described the increase of RNA synthesis in mung bean roots according to B
supply. In support of these findings, Sakcali et al. (2015) demonstrated a positive correlation
between DNA alterations and B supply in root growth of maize.
Phenolic acids and hormonal metabolism: Eaton (1940) stated that B is essential to the
formation of auxins in plants. Subsequently, Coke and Whittington (1968) described the
interrelationships between B and Indole-3-acetic acid (IAA). Later, Lewis (1980) described the
potential relationships between the metabolic role of B, synthesis of phenolic phytoalexins,
and the germination of pollen, speculating that the activity of enzymes concerned with the
metabolism of phenolic compounds are directly controlled by B. Subsequently, he outlined
the involvement of B and ethylene in the response to plant wounding. Ruiz et al. (1998a and
1998b) described the effects of B on the phenol content and metabolism in tobacco leaves.
Although these research studies offer extensive evidence that B plays an important role in
a variety of physiological processes, the requirements differ markedly within the plant kingdom
(Gupta, 1993i; Gupta 1993k) and the molecular basis for its roles is mostly unknown, making it
a difficult challenge to create a unified view of B role in plants. To date, the only established
molecular function of B in vascular plants is to cross-link two molecules of the cell wall pectic
polysaccharide referred to as rhamnogalacturonan II (RG-II) (Ishii and Matsunaga,
1996; Kobayashi et al., 1996; O’Neill et al., 1996, 2001; Blevins and Lukaszewski,
1998; Bolaños et al., 2004). It is believed that this cross-linking is required for the formation of a
pectin network within the cell wall. This is supported by studies in which plant cells grown
naturally in B-deficient conditions as well as mutants which lack the ability to synthesize normal
24
RG-II show severe developmental defects in cell wall organization and properties. However,
both the natural plants and mutant plants recover when grown in B-sufficient conditions,
indicating that the B-dependent stabilization of RG-II is essential for the structure of the cell wall
(Kobayashi et al., 1996; O’Neill et al., 2001; Reboul et al., 2011; Voxeur et al., 2011).
This supports historical research demonstrating that as much as 80-90% of B is located in
the plant cell wall (Loomis and Durst, 1991; Matoh et al., 1992; Hu and Brown, 1994; Hu et al.,
1996), undoubtedly indicating its primordial function of cell wall structure development
(Goldbach et al., 2007).
Boron Uptake in Plants
The uptake of B in plants was largely thought to be a passive transport process in which
boric acid entered the root apoplast along with other nutrients. However, Nuttall (2000),
Dordas et al., (2000) and Dordas and Brown (2000) have shown that boron absorption can also
occur by facilitated diffusion, through transmembrane channels. Much of the confusion
regarding B uptake and mobility was resolved with the identification of a borate exporter, BOR1,
(Takano et al., 2002) and subsequently, a boric acid channel, NIP5;1, (Takano et al., 2006).
A model has been developed based on the available data to describe the uptake of B from
soil and subsequent transportation to the shoots under B deficient conditions (Takano et al.,
2008; Miwa and Fujiwara, 2010; Miwa et al., 2010; Baxter and Dilkes, 2012). In this model, B
diffuses into the root epidermal apoplast from the soil and is then transported into the cytosol by
the influx protein NIP5;1, localized in the plasma membrane of the epidermis, cortex, and
endodermis. Boron then moves via the symplastic pathway through the cells of the stele until it
reaches the pericycle, at which point it is transported into the xylem by the efflux protein BOR1
(Figure 2-1).
25
Once in the xylem, B is transported to shoot tissue in the transpiration stream (Kohl and
Oertli, 1961) but little is known about the mechanism of B transport within the shoots. It has
been suspected that another influx protein may be involved in the transfer of B from the xylem to
phloem and export to sink tissues, Miwa et al. (2010) but evidence of its existence has not yet
been demonstrated.
A common theme has been found in which high B values are measured in leaves at older
stages of growth, compared to younger leaves. The severity of this theme varies across different
plants, peaking in corn leaves where the B concentration increases by a factor of eight as the
leaves age (Clark, 1975). Oertli and Richardson (1970) theorized that this is due to the mobility
of B in the plant system, in which B can easily translocate in the xylem but becomes immobile
once in the leaves.
This trend leads to an imbalance of B concentration along the height of the plant, where
the lower (older) leaves naturally have higher B concentration than the higher (younger) leaves.
This deficiency is easily controlled with B fertilization, but careful leaf samples should be tested
for B content wherever possible and compared against unfertilized control plots (Gupta, 1993b).
Typical B level in plants changes between 25 to 45 mg kg-1. The uptake and partitioning
of B by plants is dependent on the species with variances of sufficient levels. Gupta (1993c)
stated that of the variety of plants tested, sufficient B levels varied from 2.1 mg kg-1 in winter
wheat above-ground plant tissue to 432 mg kg-1 in celery leaflets. Boron sufficient levels of
broccoli leaf, whole carrot, cauliflower leaf, total above-ground corn material at vegetative stage,
cucumber leaf, potatoes leaf and whole radish are 70, 54, 36, 15-90, 40-120, 21-50 and 96-217
mg kg-1, respectively (Gupta, 1993c). Mature young leaves from the top of tomato plants have a
sufficient level of 30-75 mg kg-1, while whole tomato plants when 15 cm tall were found to have
26
B a sufficient level of 51-88 mg kg-1 (Gupta, 2008). Hochmuth (1991) stated that healthy tomato
leaves maintain B levels between 20 – 100 mg kg-1.
Deficiency and Toxicity in Plants
One of the primary driving factors for the enhanced research focus on B is related to the
widespread nature of its deficiency in a variety of plant species throughout the world, with 43
states in the USA reporting deficiencies (Gupta, 1993b). While only a small amount of B is
needed to promote the health and yields of plants, the range between adequate and toxic
concentrations of B is narrower than that of any other nutrient element. The B deficiency levels
of plants are also dependent on species and plant parts. Gupta (1993c) reported B deficiency
levels of a variety of plants as low as <0.3 mg kg-1 in winter wheat above-ground plant tissue and
as high as 12-40 mg kg-1 in recently matured sugar beet leaves. Boron deficiency levels of
broccoli leaf, carrot leaf, cauliflower leaf, total above-ground corn material, cucumber leaf,
potato leaf and whole radish are 2-9, 18, 23, <9, <20, <15 and <9 mg kg-1, respectively (Gupta,
1993c). Tomato crops have a moderate sensitivity to B deficiency and are capable of displaying
B deficiency symptoms throughout each stage of growth. Gupta (1993c) stated that mature
young leaves from the top of tomato plants have a deficiency level of <10 mg kg-1 while whole
tomato plants when 15 cm tall were found to have B deficiency related to < 12 mg kg-1 (Gupta,
2008).
Boron deficiency causes many anatomical, physiological, and biochemical changes, and
as stated above, many of these changes most likely represent secondary effects. Initial B
deficiency symptoms are observed on younger leaves due to B’s immobility in plants, causing
abnormal cell wall structure and subsequent plant-specific physical deficiency characteristics
(Fleischer et al., 1999; Ryden et al., 2003; Cakmak et al., 1995; Ruiz et al., 1998a; Cristobal et
al., 2002; Cristobal et al., 2008). Early B deficiency symptoms in tomato plants come in the form
27
of a blackened appearance on the growing point of the stems, stunted stems, and terminal shoots
that curl inward, yellow, and eventually die (Purvis and Carolus, 1964; Gupta, 1993b). Flower
damage also occurs early, leading to fruits which are poorly filled (Inden, 1975; Gupta, 1993b).
It is common for plants with B deficiency to set fruit with ridges, corky patches, and uneven
ripening, or even fail to set fruit entirely (Shorrocks, 1974; Gupta, 1993b). MacInnes and Albert
(1969) found that B deficiency developed more quickly at high light intensity on young tomato
plants.
Brown and Jones (1971) identified two strains of tomato which differ in their
susceptibility to B deficiency; T3238 (B‐inefficient) and Rutgers (B‐efficient). This was
achieved by controlling a single recessive factor (btl) which is responsible for the plant’s brittle-
stem condition in response to B deficiency (Wall and Andrus, 1962). When grown with B
concentration which limited the growth of the T3238 strain, the root B concentrations were
similar to that of the Rutgers strain, but the shoot B concentration was substantially less in the
shoots of the T3238 strain, suggesting that the roots control the transport of B.
Boron toxicity symptoms are very similar with B deficiency symptoms; under high B
levels, the plants can show marginal and tip chlorosis, and necrosis (Shorrocks, 1974; Gupta,
1993b). The two most frequent causes of B toxicity are the usage of irrigation water with high B
content and accidental B treatments such as composts which include B (Gupta et al., 1985;
Purves and MacKenzie, 1973; Gupta, 1993g). Toxicity in arid and semi-arid regions is
commonly attributed to the salinity of the soils and can be more negatively affected if the soils
have poor drainage (Gupta, 1993g). Similar to deficiencies, B toxicities are dependent on plant
species and plant parts with ranges observed from >10 mg kg-1 in winter wheat above-ground
plant parts to >800 in fully developed leaves without stem in sugar beets (Gupta, 1993c). Boron
28
toxicity levels of cabbage leaf, carrot leaf, total above-ground corn material, cucumber leaf and
potato leaf 132, 175-307, >100, >300 and >50 mg kg-1, respectively (Gupta, 1993c). Mature
young leaves from the top of tomato plants have a have a toxic level of >200 mg kg-1 (Gupta,
1993c) while whole tomato plants when 15 cm tall were found to have B toxicity level of > 172
mg kg-1 (Gupta, 2008). Hochmuch et al. (1991) stated that tomato leaves begin to show
symptoms of toxicity at levels above 150 mg kg-1.
Plant Availability
The plant availability of B in soil is highly variable, dependent on a variety of factors
including, but not limited to: soil texture, organic matter, salinity, pH, plant species and
genotype, interaction with other nutrients, irrigation practices, B treatment methods, and
numerous environmental factors. The effects of these variables are additionally compounded by
B’s multiple oxidative states.
Gupta (1993d) described the effects of soil texture by reporting that fine-textured soils
generally require more B than course-textured soils to produce similar B concentrations in plants
and that B recovery was higher in fine-textured soils than sandy clay loam soils. Soil texture can
also cause secondary effects on plant availability such as with heavy-textured soils which
typically emanate groundwaters lower in B than light-textured soils (Jain and Saxena, 1970). The
majority of surface water B concentrations vary between 0.1 to 0.3 mg kg-1 (Bingham, 1973).
Soil organic matter also increases the soil B concentration because it is affirmed that
organic material is one of the main B sources in acid soils (Gupta, 1993a; Gupta, 1993f). Soil
salinity also decreases the soil B concentration; Mehrotra et al. (1989) reported that there is an
antagonistic relationship between sodium adsorption ratio (SAR) of irrigation water and soil B
level. In general, B is less available in soils with a high pH level. Peterson and Newman (1976)
and Gupta and MacLeod (1977) determined that if the soil pH is higher than 6.3 to 6.5, the B
29
availability for plants will decrease. Wear and Patterson (1962), Barber (1971) and Gupta (1972)
have informed of limited B uptake with increased soil pH for alfalfa, soil beans, and barley.
Gupta and Cutcliffe (1972) found an important interaction between soil pH and brown heart in
rutabaga due to B deficiency; brown heart in rutabaga was found more at high pH than low pH.
Environmental factors also play a role in B availability. One such factor is the intensity of
light which accelerates plant growth and therefore develops more deficiency symptoms than
those grown under low light conditions (Gupta, 1993k). Temperature and moisture can also
affect plant availability of B. Generally, B deficiency can be seen in humid regions due to
increased leaching (Gupta, 1993d). While parent rock and derived soil are the primary sources of
soil B for plants, other environmental sources may contribute high B concentrations, such as
irrigation water, sewage sludge, or coal mine emissions (Gupta, 1993f).
Interactions with Other Nutrients
Plant availability and uptake of B can also be affected by naturally-occurring interactions
with other macronutrients in soil. Among these macronutrients, the greatest affect in B uptake by
plants is attributed to Nitrogen (N). Numerous scientists have noted that there is an inverse
relationship between N and B, in which high N depresses the level of B, which can sometimes be
beneficial in controlling the severity of B toxicity symptoms in some plants. Mack (1989) found
that in addition to increasing yields, higher rates of N were also beneficial in reducing B
deficiency in roots, subsequently reducing the required amount of B fertilizer. Gupta (1993d)
stated that B uptake increases by increasing K supply on alfalfa, and the existence of B
deficiency depends on both B and K concentration in soil. Scientists have also found a
synergistic relationship between B and P (Patel and Golakia, 1986; Singh and Singh, 1990;
Gezgin and Hamurcu, 2006; Uysal et al., 2017).
30
Additionally, high Calcium (Ca) entity reduces available B for plants. Tanaka (1967a,
1967b) explained that B uptake by radish (Raphanus sativus L.) reduced when the medium Ca
content increased. Drake et al. (1941) stated that the higher Ca/B ratios caused by B deficiency
are most likely due to the higher Ca concentration in tobacco leaf tissue. When studying on
pistachio trees, Kizilgoz et al., (2004) found that high Ca caused discrepancies in testing in
which tissue analyses showed B deficiencies from soils with sufficient B, showing significant
correlations between B uptake and Ca concentration in soil and water. Boron deficiency can also
be observed in soils with high lime content (Sahin et al., 2015).
Species and Genotype
Plant species and genotype are additional factor that affect B uptake. Baysal and Erdal
(2015) found that different varieties of apple plants measured different values of B uptake when
treated using the same granular fertilizer method. Erdal and Turkan (2016) applied foliar B
fertilizer to 5 different apple varieties and found that the different apple varieties were affected
differently. Taban and Erdal (1999) studied B granular treatment on different wheat varieties and
found that Triticum drum cultivars were affected more than Triticum aestivum cultivars.
Gorsline et al., (1968) found that particular hybrids of corn can exhibit genetic variability in their
B uptake as well as leaf concentration.
Boron Fertilizers
Boron fertilizers can promote quality and productivity of the plants (Gunes et al., 2003;
Gunes et al., 2011; Sahin et al., 2015). For this purpose, Borax, Borate, Anhydrous Borax,
Solubor, Boric Acid, Colemanite, Ulexite, and Boron Frits are used. Erdal and Erdoganyilmaz
(2017) applied different types of B fertilizers, which were Anhydrate borax (Na2B8O7), Boric
acid (H3BO3), Etibor-48 (Powder; Borax pentahydrate, Na2B4O7.5H2O), Boron oxide (B203),
Etidot-67 (Na2B8O13.H2O), Borax (Na2B4O7.10H2O), and Etibor-48 (Granule; Borax
31
pentahydrate, Na2B4O7.5H2O), to sunflower plants. According to this study, it was found that
although there were some differences between the seven sources, all sources increased the leaf B,
with the highest leaf B content being found from Etibor-48 treatment.
The required amount of B fertilizer may depend on plant species and application method.
Havlin et al. (2013) stated that Solubor treatment on broccoli requires 1-3 lb ac-1 in granular form
while foliar form requires 0.5-1 lb ac-1, whereas granular treatments on corn require 0.5 lb ac-1
and foliar treatments on soybeans require 0.1 lb ac-1. According to State Recommendations of
Boron for Various Crop - Report of 1988 Survey (Woodruff, 1988), the recommendation for
tomato at South Carolina, 2.24 kg/ha B, should be applied with broadcast or foliar spray
methods. Sultana et al. (2016) also recommended this same treatment method for tomato plants.
Haleema et al., (2018) stated that foliar B treatment increased tomato plant height, primary
number of branches, secondary number of branches, number of leaves, leaf area, and number of
fruits.
Gupta and Cutcliffe (1978) found that B results from band treatments are higher than
those from broadcast B treatments on rutabaga. On corn, band or foliar applied B results are
higher than broadcast B treatment (Touchton and Boswell, 1975; Peterson and MacGregor,
1966). Gupta and Cutcliffe (1978) stated that early foliar B fertilizing produces higher B
absorption than later foliar B fertilizing, and Mortvedt (1974) added because of high humidity
which provides open stomas and high photosynthesis rate, early B foliar treatment results in
increased absorption. Gupta (1993e and 1993h) indicated that granular B treatment resulted in
higher tissue B concentrations than foliar B treatment on barley. Erdal et al., (2016) conducted an
experiment comparing foliar and granular B treatments on apple and found that granular
32
treatments vastly affected leaf B concentrations while foliar treatments did not have a significant
impact.
The most reliable method of evaluating the deficiency and sufficiency of B in plants is
testing the plant tissue itself, as soil testing methods are not fully developed. Throughout
numerous tests of multiple plant types, it has been found that the available supply of B vastly
affects the distribution of B; in plants where B supply is adequate, the blades have higher B
content than the petioles, whereas the same plants have reversed results when there is a B
deficiency (Gupta, 1993c).
Figure 2-1. Process of B Uptake under Deficient Condition
33
CHAPTER 3
MATERIALS AND METHOD
Study 1. Analytical Methods and Laboratory Protocol
Sample Preparation
Correct sample preparation practices are critical to obtaining meaningful data,
particularly for B analyses. For all experiments on method determination and comparison,
premium dry play sand (Quikrete brand) was chosen as the source soil to ensure no B was
included. Solubor (4.878 g) was dissolved in 1-liter double-distilled water (DDI) water to
produce a B stock solution of 1000 mg kg-1 concentration. Premium dry play sand samples (20 g)
and 0, 10, 20, 30, 40, 50, 60, 70, 80, 90, and 100 µL B from stock solution were mixed uniformly
to prepare 0, 0.5, 1, 1.5, 2, 2.5, 3, 3.5, 4, 4.5, and 5 mg kg-1 treated soils, respectively. All
experiments other than laboratory protocol comparison used 5 replicates for each treatment. The
samples were filtered with Whatman 42 filter paper and tested with either ICP-OES or
spectrophotometer. Further soil sample preparations for different instruments are explained in
their respective sections below.
Laboratory Protocol Comparison
An experiment was conducted to compare the effectiveness and consistency of
extractable B measurements in soil. Soil samples were prepared with concentrations of 0, 1, 2,
3.5, and 4.5 mg kg-1 in two replicates each. Treated soil samples were sent to three different state
University laboratories for comparison of B analyses results. One set of samples was also
analyzed at the University of Florida, IFAS ANSERV Laboratories. The laboratories utilized the
following methods:
 Lab A: Hot-water extraction with Spectrophotometer and a 5-minute boiling duration
in distilled (DI) water with a soil-to-water ratio of 1:2.
34
 Lab B: Hot-water extraction with ICP-OES and a 12-15-minute boiling duration in a
CaC12 solution with a soil-to-solution ratio of 1:2.
 Lab C: Hot-water extraction with ICP-OES and a 5-minute boiling duration in DI
water with a soil-to-water ratio of 1:2.
 ANSERV Labs: Hot-water extraction with ICP-OES and a 5-minute boiling duration
in double-distilled (DDI) water with a soil-to-water ratio of 1:2.
Determination Methods
Separate experiments were conducted to identify an optimal analytical method and
protocol for extractable B measurement in soils using different standard laboratory procedures,
including Hot-water, Mehlich-3, and Mehlich-1 extractants. Additional experiments were
conducted to compare the effectiveness and consistency of the specific method’s procedures by
adjusting variables such as soil-to-solution ratios and delays between treatment and extraction,
and then analyzing the results compared to standard protocols.
Hot-water extraction
Soil samples were prepared with concentrations of 0, 0.5, 1, 1.5, 2, 2.5, 3, 3.5, 4, 4.5 and
5 mg kg-1. Treated soils placed in resealable plastic bags were boiled in DDI water for different
time durations. Boron content in the extractions were determined on both ICP-OES and
segmented-flow spectrophotometer.
An additional experiment was conducted to compare the results of varying boiling
durations, consisting of 5, 10 and 15 minutes. Soil samples were prepared with concentrations of
0, 0.5, 1, 1.5, 2, 2.5, 3, 3.5, 4, 4.5 and 5 mg kg-1, and tested with ICP-OES and segmented-flow
spectrophotometer.
A separate experiment was conducted to determine the effects of varying time periods
between soil B treatment and soil B measurement using Hot-water extraction method. Soil
35
samples were prepared with concentrations of 0, 1, 2, 3, 4 and 5 mg kg-1. The treated samples
were then stored for durations of 0, 6, 12, 24 and 48 hours prior to measurement and tested with
ICP-OES and segmented-flow spectrophotometer.
Mehlich-3 extraction
Soil samples were prepared with concentrations of 0, 0.5, 1, 1.5, 2, 2.5, 3, 3.5, 4, 4.5 and
5 mg kg-1. Mehlich-3 extracting solution consisting of Ammonium nitrate,
Ethylenediaminetetraacetic acid (EDTA), Ammonium Fluoride, Glacial acetic acid, and Nitric
acid (15.8M) was added to each sample in a 1:10 soil:extractant solution ratio using plastic cups
to eliminate concerns of B release from borosilicate glass, before shaken for 5 minutes. The
samples were then tested with ICP-OES and segmented-flow spectrophotometer.
An additional experiment was conducted to compare the results using an adjusted
soil:extractant solution ratio of 1:1. Soil samples were prepared with concentrations of 0, 1, 2, 3,
4 and 5 mg kg-1 and tested with ICP-OES.
Mehlich-1 extraction
Soil samples were prepared with concentrations of 0, 1, 2, 3, 4, and 5 mg kg-1. Mehlich-1
extracting solution consisting of Sulfuric Acid (0.0125M) and Hydrochloric Acid (0.05M) was
added to each sample in a 1:4 soil:extractant solution ratio using plastic cups to eliminate
concerns of B release from borosilicate glass and shaken for 5 minutes. The samples were then
tested with ICP-OES.
A replicated experiment was conducted to compare the results using an adjusted
soil:extractant solution ratio of 1:1. The samples were tested with ICP-OES and segmented-flow
spectrophotometer.
Measurements were compared in a separate procedure to observe the effects of varying
time periods between soil B treatment and soil B measurement using Mehlich-1 extraction
36
method. Soil samples were prepared with concentrations of 0, 1, 2, 3, 4 and 5 mg kg-1. The
treated samples were then stored for durations of 0, 6, 12, 24 and 48 hours prior to extraction and
tested with ICP-OES.
An additional experiment was conducted to test the 1:1 ratio using a sandy loam soil with
a pH of 6.5. Soil samples were collected from a virgin vegetation field on the University of
Florida campus, from a depth of 15 cm and treated with treatments of 0, 1, 2, 3, 4 and 5 mg kg-1
B. The treated samples were then stored for durations of 0, 6, 12, 24, and 48 hours prior to
extraction and tested with ICP-OES.
Statistical Analyses
Extraction data was tested by ANOVA with the means being separated using Tukey’s
multiple comparison tests by Least Significant Difference (LSD) at 0.05% level using JMP
Statistical Discovery (SAS Institute Inc. 2007).
Recovery performance; the percentage of B recovered from the applied concentration,
was analyzed for each extraction method by the formula:
Treatment
Recovery (%) = × 100 (3-1)
Result
A greenhouse study was conducted with tomatoes (Solanum lycopersicum) as a test plant
during two seasons at the UF/IFAS research facilities in Gainesville, FL. The first season
(summer) ran from 6/22/2017 – 11/17/2017 and the second season (fall) ran from 11/08/2017 –
04/03/2018. Pots filled with a premium dry play sand (Quikrete brand) were laid out in a
randomized block design with 0, 1, 2, and 3 mg kg -1 treatments in 6 replications.
37
Site and Sample Preparation
A Mehlich-3 extraction method was used to analyze the source soil, which showed a pH
of 9.5 and extractable nutrients consisting of 1 mg kg -1 P, 124 mg kg -1 Ca and 0 mg kg -1 Mg, 0
mg kg -1 Cu, 0 mg kg -1 Mn, 0 mg kg -1 B, and 0 mg kg -1 Zn. The sand was mixed with elemental
S to reduce the pH, yielding an average pH of 6.0. The sand was then transferred to 48 seven-
gallon plastic nursery pots and was packed to a bulk density 1.38 g cm-3.
For each of the two seasons, tomato (BNH 602) seeds were transplanted from nursery
seedling trays into the 48 pots and arranged in a completely randomized design in the
greenhouse-one plant per pot. In the summer season, seedlings were transplanted on 6/22/2017
and treatments were applied on 8/18/2017. In the fall season, seedlings were transplanted on
11/8/2017 and treatments were applied on 1/16/2018. Foliar treatments were applied uniformly
to each plant using 1, 2, and 3 mg kg-1 B concentrations (Solubor solution) in 20 ml plastic spray
bottles carefully and by covering the surface of the pots to avoid runoff from the leaf surfaces.
Granular treatments were applied using an automatic pipet directly to the soil in a circular pattern
at a distance of 3” from the base of the stem.
Site and Sample Management
An automatically scheduled irrigation system was installed in both greenhouses to apply
predefined irrigation to each individual plant. Irrigation water was tested for B and was
determined to be negligible (0.02 mg kg -1 B). All cultural, insect, and disease management were
conducted as per standard protocols described in IFAS Vegetable Production Guide (Freeman et
al., 2016).
During the fall season, a minor P deficiency was observed and a single foliar spray
containing 0.53% P was applied, resulting in a recovery of the plants. The fall season
experienced a severe infestation of white flies, requiring additional general insecticidal sprays.
38
The plants also exhibited symptoms of tomato mosaic virus, after which the symptomatic leaves
were cut from the plants and healthy leaves were cleaned on a routine basis to control the effects.
Plant Sampling
For both seasons, the fruit was inspected on a daily basis and removed from the plant
once it had reached the turning ripening stage. For the summer season, final harvest was
conducted 148 days after planting to collect the remaining plant samples from the pots. For the
fall season, final harvest was conducted 146 days after planting.
After collection, remaining plant samples were then oven-dried at 65°C for one week.
Once dried, the samples were then ground into a fine powder using a sample grinder (Laboratory
Model 3 Mill, Thomas Wiley, Swedesboro, NJ).
Sample Digestion
The digestion process was conducted in ANSERV Labs following standard procedure
(Mylavarapu, et al., 2017). Soil samples were transferred into 50-mL porcelain crucibles, placed
into a muffle furnace at 500°C, and allowed to ash for 4 hours. Porcelain crucibles were used as
sample receptacles due to their inability to leach chemicals, but were tested to ensure there was
no potential contamination for B. The results from this test yielded 0 mg kg -1 B (Figure 3-1).
Once each sample reached room temperature, 5 drops of DDI water were added to each
sample. 0.5 M Hydrochloric Acid (HCI) was then added to each sample and allowed to rest for
30 minutes before being thoroughly mixed with DDI water. The mixed solutions were then
filtered and transferred into plastic vials. The digested sample solutions were then analyzed using
ICP-OES for B, Ca, Mg, K, P, sulfur (S), aluminum (Al), iron (Fe), zinc (Zn), silicon (Si),
sodium (Na) and nickel (Ni).
39
Verification
Standard reference SRM 1573a tomato leaves from NIST Material Measurement Library
were tested for sample verification using the same method with 0, 1, 2, 3, 4 and 5 mg kg-1 B
treatments, each with 3 replications. These tests yielded a 92-95% recovery.
Statistical Analyses
Data was tested by ANOVA with the means being separated using Tukey’s multiple
comparison tests and Factorial Analysis by Least Significant Difference (LSD) at 0.05 level
using JMP Statistical Discovery (SAS Institute Inc. 2017).
40
CHAPTER 4
RESULTS AND DISCUSSION
Study 1. Analytical Methods and Laboratory Protocol Study
Laboratory Comparison
Hot-water extraction was the most common method, while the instrument and boiling
duration varied between laboratories. The results shown in Table 4-1 suggest that, although not
completely recovered, the consistency of applied B recovery using the ANSERV Labs procedure
was relatively the most reliable.
As mentioned, each laboratory had their own protocols (e.g. used different instruments
and boiling durations) thus resulting in highly varied soil B concentrations reported. Such
variations only further emphasized the expedited need for standardization of the procedures and
protocols in determination of soil B.
Hot-water Extraction
Since the 4 laboratories resulted in highly varied B measurements, in this study, we
decided to look at the accuracy of Hot-water extraction method in determining B measurements
by comparing results from 2 different instruments and 3 different boiling durations.
Instrument comparisons and boiling durations
For this experiment, measurement means and B recovery were compared between each
instrument (ICP-OES and spectrophotometer) for each boiling duration (5, 10, and 15-minutes)
using treatments 0, 0.5, 1, 1.5, 2, 2.5, 3, 3.5, 4, 4.5, and 5 mg kg-1 B. To simplify the
comparisons, analysis of variance (ANOVA) and two-factor multiple means comparisons using
Tukey-HSD were conducted for each instrument, using treatment and boiling duration as factors.
41
Analysis of variance showed significant interaction between boiling duration and
treatment for B measurements and recovery percentages made with ICP-OES and
spectrophotometer.
Measurements from ICP-OES were first analyzed. When B measurement means were
compared by Tukey-HSD (Table 4-2) between boiling duration and treatment, B measurements
were not significant with the exception of 5 mg kg-1 where, 5-minute boiling duration resulted in
significantly higher measurements compared to 10 and 15-minute durations. These results
indicate that boiling duration does not statistically affect measurements from ICP-OES,
suggesting that a 5-minute boiling duration is a more practical and reliable laboratory protocol
(Table 4-2).
Treatment
For the percent of B recovery, (Recovery (%) = × 100), from ICP-OES,
Result
results shown in Table 4-3 indicate that B recovery performance was not significantly affected
by boiling duration, further suggesting that a 5-minute duration is the optimal protocol for B
measurement because increasing the boiling duration beyond 5 minutes does not produce more
consistent results.
Results of measurement means from spectrophotometer shown in Table 4-4 showed no
statistically significant differences between boiling duration measurements.
For the percent of B recovery from spectrophotometer, results shown in Table 4-5
indicate that B recovery performance was not significantly affected by boiling duration with the
exception of 0.5 mg kg-1 which shows a statistical difference between 5-minute duration, and 1.5
mg kg-1, which shows a statistical difference between 15-minute duration.
42
Extraction delay comparison
An additional experiment was conducted to determine if delaying time of extraction (e.g.
extracting 24 hours after applying B treatment to soil) would have an effect on B measurements
when 5-minute boiling Hot-water extraction samples were analyzed by ICP-OES. Measurement
means from ICP-OES were compared from treatments of 0, 1, 2, 3, 4, and 5 mg kg-1 for delays of
0, 6, 12, 24, and 48 hours between treatment and extraction to test if the measurements change
according to time delayed.
Analysis of variance showed significant interaction between delayed extraction and
treatment for B measurements made with ICP-OES. With the exception of 0 and 1 mg kg-1, there
were significant differences between all measurement means from 0 hours and 48 hours,
indicating that a delay between soil B treatment and Hot-water extraction caused significant
variances in measurement (Table 4-6). This could be attributed possibly to reaction of B with the
soil and possible dissipation due to hydrolysis.
For the percent of B recovery, the results shown in Table 4-7 indicate that there were
statistical differences between delay periods, with a decrease in recovery as the delay increased.
Overall, Hot-water extraction with a 5-minute boiling duration and measurement with
ICP-OES resulted in more consistent results than the standard protocol as well as other adjusted
protocols but may be susceptible to variance caused by delays in time between soil B treatment
and extraction (Table 4-7). These variances may be the result of Hot-water extraction not
possessing the capacity to consistently adsorb B as it changes form over time. Therefore, we
decided to determine the consistency of Mehlich-3 and Mehlich-1 extraction methods on soil B
measurements.
43
Mehlich-3 Extraction
Instrument comparison
Results shown in Table 4-8 indicate that there were no significant differences between
instruments for treatments of 0, 0.5 and 1 mg kg-1. However, significant differences were
observed between instruments for all treatments between 1.5 and 4.5 mg kg-1, with a gradual
increase in measurement from spectrophotometer from 0.5 to 3 mg kg-1, and a gradual decrease
in measurement from 3 to 5 mg kg-1 (Table 4-8).
For the percent of B recovery, the results shown in Table 4-9 indicate that B recovery
from ICP-OES was mostly similar for each treatment with an average recovery of 109%, while
spectrophotometer showed a steady decrease in recovery between 1 and 5 mg kg-1 with an
average recovery of 207%. Although it may seem that recovery of B is higher from
spectrophotometer results than ICP-OES, this does not mean that it is recovering the correct
amount applied. A 207% average recovery means that spectrophotometer vastly overestimated
applied B treatments compared to ICP-OES, which more consistently recovered B amounts
correctly to what was applied (Table 4-9).
Soil:extractant solution ratio comparison
The standard Mehlich-3 soil:extractant solution ratio of 1:10 was adjusted to a
soil:extractant solution ratio of 1:1 to compare the consistency of measurements from ICP-OES
with both ratios using treatments of 0, 1, 2, 3, 4, and 5 mg kg-1. Analysis of variance (ANOVA)
showed significant interaction between ratio and treatment for B measurements and B recovery.
The results shown in Table 4-10 indicate that measurements from ICP-OES using the standard
Mehlich-3 extraction method resulted in significant differences between all
treatments. Measurements using Mehlich-3 with 1:10 ratio were consistently higher than the B
treatments. The measurement means had an average multiplication factor of 3.6. These results
44
indicated that when using the standard 1:10 ratio for Mehlich-3, ICP-OES resulted in
overestimation up to 3.6 times higher than the treatment levels (Table 4-10). These results align
with findings from Allen et al., (2005) who also reported significantly high measurements from
ICP-OES when using Mehlich-3 extraction. This may be caused by interactions with the glass
nebulizer and spray chamber in the ICP-OES machine, which come into contact with the solution
before entering the plasma.
Therefore, the ratio was changed from 1:10 to 1:1 to determine if it would optimize the
protocol and yield consistent results. The soil samples used for this experiment were prepared at
0 hours before extraction, which resulted in different measurements than those observed during
the instrument comparison, which used soil samples prepared 2 weeks prior to extraction. This
indicates that delays between soil B treatment and Mehlich-3 extraction may cause variances
similar to those seen from Hot-water extraction (Table 4-8 and Table 4-10).
The adjusted ratio of 1:1 also resulted in significant differences between all treatments.
These results indicated that the adjusted ratio resolved the multiplication factor observed from
the 1:10 ratio (Table 4-10).
The percentage of B recovery was compared between the standard ratio of 1:10 and the
adjusted ratio of 1:1. The results shown in Figure 4-2 showed that recovery percentages from
1:10 ratio decreased significantly from treatments of 1 to 3 mg kg-1 and resulted in no significant
differences between 3 and 5 mg kg-1. However, recovery percentages between the two ratios
were all significantly different. The standard 1:10 ratio resulted in an average recovery of 488%,
while the adjusted ratio of 1:1 resulted in an average recovery of 88.7% (Table 4-11).
Overall, these results suggest that for Mehlich-3, a ratio of 1:1 with measurement with
ICP-OES was found to recover close to 100 percent of the soil B treatments, suggesting that it
45
may be feasible for laboratories to re-evaluate the use of Mehlich-3 as an effective extractant of
soil B.
This indicates that an adjusted ratio (1:1) of Mehlich-3 may be a more consistent
extraction method than Hot-water extraction. Further research is required to determine if
increased ratio adjustments with more soil and less solution yield more consistent results.
Mehlich-1 Extraction
Soil:extractant solution ratio adjustment comparison
Mehlich-1 extraction method is another commonly used extractant for acid-mineral soils
(Mylavarapu et al., 2002). Although the soil samples in our study were not from acid-mineral
soils, we tested Mehlich-1 extraction to see if protocol adjustments could yield better results than
Hot-water and Mehlich-3 extractions. Therefore, an experiment was conducted to determine
whether adjusting the soil:extractant solution ratio of Mehlich-1 would impact B measurements
when treatments of 0, 1, 2, 3, 4 and 5 mg kg-1 B were analyzed with ICP-OES. The consistency
of the standard 1:4 ratio was tested and compared to a 1:1 ratio.
Analysis of variance (ANOVA) showed statistically significant interaction between ratio
and treatment for B measurements and B recovery. The results shown in Table 4-12 indicate that
measurements from ICP-OES using the standard Mehlich-1 extraction method resulted in
statistically significant differences between all treatments. Measurements using Mehlich-1 with
1:4 ratio were consistently higher than the B treatments (Table 4-12). The measurement means
had an average multiplication factor of 4.007 (Table 4-12). These results indicate, when using
the standard 1:4 ratio for Mehlich-1, ICP-OES consistently reported incorrect B measurements
four times higher than what was applied to the soil samples (Table 4-12). This may be caused by
interactions with the glass nebulizer and torch in the ICP-OES machine which come into contact
with the solution before entering the plasma.
46
Therefore, the ratio was changed from 1:4 to 1:1 to adjust an optimal protocol. The
adjusted ratio of 1:1 also resulted in significant differences between all treatments (Table 4-12).
The results indicate that the adjusted ratio (1:1) resolved the multiplication factor observed from
the 1:4 ratio (Table 4-12).
The percentage of B recovery was compared between the standard 1:4 ratio and the
adjusted ratio of 1:1. The results shown in Table 4-13 show that recovery percentages from 1:4
ratio have no significant differences. Additionally, the recovery percentages from 1:1 ratio also
have no significant differences (Table 4-13). However, recovery percentages between the two
ratios are all significantly different (Table 4-13). The standard 1:4 ratio resulted in an average
recovery of 400%, while the adjusted ratio of 1:1 resulted in an average recovery of 108% (Table
4-13). These results indicate that Mehlich-1 with adjusted ratio of 1:1 consistently recovered B
amounts correctly to what was applied (Table 4-13). Mehlich-1 at 4 times the volume of the soil
may have resulted in a false-positive signal in the ICP spectrum and therefore adjusting the ratio
to 1:1 could have optimized the emission to detect the B levels in the extracted solution to reflect
the treatments accurately. This observation may allow further options of refining the
soil:extractant ratios for the widely used Mehlich-3 reagent also.
Instrument comparison
An experiment was conducted to determine the consistency of Mehlich-1 extraction
method with a soil:extractant solution ratio of 1:1 on B measurement and recovery from two
instruments (ICP-OES and spectrophotometer) using treatments of 0, 1, 2, 3, 4, and 5 mg kg-1 B.
Analysis of variance (ANOVA) showed significant interaction between instrument and treatment
for B measurements and B recovery. Individual treatment measurements using Mehlich-1 with a
1:1 ratio had no significant differences between ICP-OES and spectrophotometer with the
exception of 5 mg kg-1 treatment (Table 4-14).
47
The percentage of soil B recovery was compared between ICP-OES and
spectrophotometer with the adjusted ratio of 1:1. As shown in Table 4-15, all recovery
percentages had no significant differences between ICP-OES and spectrophotometer with the
exception of 5 mg kg-1 treatment. ICP-OES resulted in an average recovery of 108% while
spectrophotometer resulted in an average recovery of 106% (Table 4-15). These results show that
soil B measurements using Mehlich-1 and a ratio of 1:1 can be taken from either ICP-OES or
spectrophotometer in concentrations up to 4 mg kg-1. However, spectrophotometer measurements
of 5 mg kg-1 are significantly lower than those from ICP-OES, indicating that ICP-OES is the
recommended instrument for Mehlich-1 extraction using a 1:1 ratio.
Treatment to extraction delay comparison
An experiment was conducted see if delaying time of extraction (e.g. extracting 24 hours
after applying B treatment to soil) would have an effect on B measurements when Mehlich-1
extraction samples with an adjusted ratio of 1:1 (soil:extractant) were analyzed by ICP-OES. Soil
B treatments of 0, 1, 2, 3, 4, and 5 mg kg-1 were compared using Mehlich-1 with a ratio of 1:1
and measurement with ICP-OES, with delays between treatment and extraction of 0, 6, 12, 24,
and 48 hours. Analysis of variance (ANOVA) showed significant interaction between delay
timing and treatment for B measurements made with ICP-OES. The results shown in Table 4-16
show that at 0 hours, B measurements from ICP-OES were higher than the treatments and were
different from each other. Additionally, as B concentrations increased in the soil samples, ICP-
OES reported increasingly high measurements at 0 hours but leveled to > 0.01 mg kg-1 by 48-
hours (Table 4-16).
For the percent of B recovery, the results shown in Table 4-17 indicate that, while 0 hours
yielded some statistical differences compared to longer delays, mostly there were no differences
between delay periods after 0 hours. This shows that extended delays between soil B treatment
48
and extraction do not significantly affect ICP-OES measurements using Mehlich-1 extraction
with a 1:1 ratio (Table 4-17).
Replication with sandy loam soil
A replicated experiment was conducted using Mehlich-1 with a soil:extractant solution
ratio of 1:1 and measurement with ICP-OES using sandy loam soil samples instead of premium
dry sand with treatments of 0, 1, 2, 3, 4 and 5 mg kg-1 B to evaluate the effect of the modified
laboratory protocol on B extraction and its consistency in determination of B concentration in
agricultural soils. The results shown in Table 4-18 indicate that ICP-OES extracted B
concentrations which are different from each other. With the exception of 3 and 5 mg kg-1
treatments, there were no significant differences between 0-hour and 48-hour measurements,
indicating that the amount of time between B treatment and Mehlich-1 extraction does not affect
measurements taken from ICP-OES (Table 4-18).
For the percent of B recovery, the results (Table 4-19) indicate that extended delays
between soil B treatment and extraction do not significantly affect ICP-OES measurements using
Mehlich-1 extraction with a 1:1 ratio (Table 4-19).
With Hot-water extraction, Gupta (1967) stated that increasing the boiling duration from
5 to 10 minutes positively affected the amount of extracted B on acidic podzol soils. Later,
Odom (1980) conducted a study on acidic soil and found that short boiling durations cause more
error than excessive boiling. On the contrary to these studies, we found that increasing boiling
time either did not affect the results or if it affected, 5-minute boiling resulted in more consistent
results on high pH soil.
Overall the Mehlich-1 results suggest that a ratio of 1:1 with measurement with ICP-OES
was found to recover close to 100 percent of the soil B treatments. These increases in recovery
from those seen from Mehlich-3 may be a result of the higher pH of Mehlich-1 solution
49
compared to Mehlich-3. The differences in B measurements observed between premium dry
sand and sandy loam soil may be the result of differences in adsorption. Therefore, further
research is required to test the consistency of this adjusted protocol on soil types with differing
chemical and physical soil characteristics.
This is a unique study that has not been researched before. Comparative analyses for
Mehlich-1 soil:extractant solution ratio adjustments are challenging to derive due to limited
research available on protocol adjustments for Mehlich-1 extraction. Mehlich-3 extraction
method is the most commonly used method and is cited in the literature as the most efficient
method. This study shows that there should be a consideration into adopting Mehlich-1
extraction method over Mehlich-3 and Hot-water extraction, especially when it comes to
measuring B. Additional findings will, therefore, necessitate further laboratory studies on the
effects of varied Mehlich-1 soil:extractant solution ratios with different soils and wider range of
B concentrations on plant available soil B determination.
Fruit Yield
Fruit yield data of both seasons was collected and analyzed, however, the data was
omitted due to a severe suppression in yields in both seasons as a result of insect and disease
pressure.
Above-Ground Fresh Plant Weight
The effect of treatment on above-ground fresh plant weight in first and second season
were found to be significant. However, method was not significant, indicating that B can be
applied either through granular or foliar form.
First and second season results as shown in Table 4-20 and Table 4-21. Results indicate
that control plants in each season (1868.2 and 66.63 kg ha-1, respectively) were significantly
50
lower than the other treatments, indicating that all treatments of B positively impacted above-
ground fresh plant weight.
Few articles are available that studied the effect of B application rates above 2 mg kg-1 on
tomato plants. While treatments of 0, 1, and 2 mg kg-1 are common for testing the response of
tomato plants to B application, treatments above 2 mg kg-1 are used to observe the effects of B
toxicity. For example, Gupta (1983) used 0, 1, 2 mg kg-1 on tomato to understand deficiency and
4 mg kg-1 to observe toxicity.
Our observations of treatment significance align with findings from Johnston and Dore
(1929) who found that B treatments (0.011 and 0.55 mg kg-1) positively impacted the green
weight of the tomato plants. Taban and Erdal (1999) conducted an experiment to observe B
effect on wheat types in greenhouse using granular B fertilizer in concentrations of 0, 1, 2 and 10
mg kg-1. In this study, the highest fresh above ground part weights came from 2 mg kg-1 B
application for all different wheat types. In contrast, 10 mg kg-1 B application caused a decrease
in the above ground part weights. This decreasing effect was similar to our observation at 3 mg
kg-1 treatment.
The weights of above plant parts were higher for first season compared to second season
which is likely the result of ToMV symptoms experienced in tomato plants during the second
season (Table 4-20 and Table 4-21). In addition to the effects of the virus, the lower weights of
the second season may be compounded by the removal of symptomatic leaves which was done to
decrease the spreading of the virus.
Leaf B Content
First and second season results shown in Table 4-22 and Table 4-23 indicate that
treatment had a significance on leaf B content. First season results also showed a statistical
significance for method and the interaction between method and treatment. The results in Table
51
4-22 show that granular method had a significant effect on leaf B content for the first season
while foliar method did not significantly affect leaf B content. For the second season, Table 4-23
shows that leaf B contents were similar regardless of B treatment applied.
The results agree with other studies in which soil applied B (Dursun et al., 2017; Davis et
al., 2003, Gupta, 1983) or foliar applied B (Davis et al., 2003) increased leaf B content in
tomatoes. Baysal and Erdal (2015) and Dunn et al. (2005) also stated that leaf B content
increases as a result of granular B treatment in apple and rice respectively.
When comparing first and second seasons (Table 4-22 and Table 4-23), the results
suggest that method only showed a positive effect during the first season. During the second
season, method did not show a positive affect which may be due to symptoms of ToMV which
may change the uptake of B under plant stress.
The decrease in leaf B content observed between the two seasons is likely due to
symptoms of ToMV observed during the second season in which the leaves curled, distorted, and
ultimately died.
Petiole B Content
The results showed a statistical significance for treatment for both seasons. Additionally,
there was a significance for method and the interaction between method and treatment for the
first season. The results in Table 4-24 show that control and treatments within foliar method
were the same for the first season, however, granular method results were different than foliar
and control group. The highest significant treatments which positively impacted petiole B
content came from granular treatment of 3 mg kg-1. Therefore, the data suggests that 3 mg kg-1 of
B is recommended in granular form as the optimal treatment in order to positively increase
petiole B content. However, for the second season, Table 4-25 shows that the highest B
concentration in petioles came from 1 and 3 mg kg-1 treatments.
52
Comparing first and second season, granular method’s effect on petiole B content was
higher than foliar method’s effect while second season petiole B content was not affected due to
method (Table 4-24 and Table 4-25). Literature could not be found which mentions B in petiole,
therefore, further research is needed to understand B function and transport in tomato petiole.
Stem B Content
The results showed a significance for treatment for both seasons as well as interaction
between the two main factors in the first season. Highest stem B content was recorded in
granular treatments of 2 and 3 mg kg-1 B (Table 4-26). For foliar method, all treatments resulted
in similar stem B content to the control group. For the second season, Table 4-27 shows that the
highest B concentration in petioles came from 1 and 3 mg kg-1 treatments.
Similar to what was observed for petioles, the first season stem B content resulted in
significance for treatment, method, as well as the interaction between treatment and method,
while the second season resulted in only a significance of treatment (Table 4-26 and Table 4-27).
The changes between seasons are likely due to the symptoms of ToMV observed during the
second season in which the stem size and weight decreased, likely causing a change in B uptake.
Literature search on B in plant stems have not yielded any results indicating that our study
maybe one of the first attempts to quantify B in plant stems.
Root B Content
The results showed a significance for method for both seasons. As Table 4-28 shows,
granular method produced higher root B content than foliar for the first season. The second
season, however, showed a statistical significance for method and the interaction between
method and treatment. The second season results shown in Table 4-29, indicate a difference
between foliar and granular treatments of 3 mg kg-1. This difference is likely related to symptoms
of ToMV observed during the second season in which the roots were stunted.
53
Reasons for these differences are unknown due to limited availability of similar research.
Therefore, further research is required to investigate potential causes.
Total B Content
The results showed a significance for treatment for both seasons. The first season results
in Table 4-30 show that granular method produced higher total B content than foliar method. For
the second season, Table 4-31 shows that the highest significant B content was observed in 2 mg
kg-1 B treatment.
The treatment and method were significant to the total B content for the first season while
only the treatment was significant for the second season. Lower uptake of total B during the
second season could be due to the ToMV where we removed leaves in order to prevent further
disease. Comparative research could not be found of total B, suggesting a need for additional
study in the area.
Summary
Overall, 2 mg kg-1 treatment produced the highest above-ground fresh plant weight and
total B as well as B content in leaves, petioles, and stems. Granular B fertilization method was
more effective during the first season while method of B application was not significant for the
second season. There were significant decreases between first and second seasons as a result of
ToMV. This data suggests that granular and foliar B fertilization positively impacted the growth
of tomato plants, but the effectiveness of treatment methods may be dependent on the overall
health of the plants. Healthy tomato plants in the first season showed that the plant uptake of B
was higher in 2 mg kg-1 treatments applied in granular form. However, granular method in the
second season was less effective on plants exhibiting symptoms of ToMV which resulted in
higher uptake of B from foliar B fertilizer, especially in the roots and stems, where the virus had
its most significant impact.
54
Table 4-1. Comparison of four different laboratories B measurement means
Laboratory 0 mg kg-1 1 mg kg-1 2 mg kg-1 3.5 mg kg-1 4.5 mg kg-1
ANSERV Labs 0.01 0.82 1.68 2.37 3.82
Lab A 0.02 0.09 0.28 0.77 1.44
Lab B 0.08 0.67 1.35 1.72 3.51
Lab C 0.90 0.67 0.40 0.15 0.00
Table 4-2. Comparison of boiling duration measurement means from Hot-water extraction with
measurement from ICP-OES
Treatment (mg kg-1) 5 min (mg kg-1) 10 min (mg kg-1) 15 min (mg kg-1)
0 0.01 o 0.03 o 0.01 o
0.5 0.42 mno 0.37 no 0.32 no
1 0.68 lmno 0.81 lmn 0.74 lmn
1.5 1.15 kl 1.10 klm 1.10 klm
2 1.61 jk 1.71 jk 1.25 kl
2.5 2.26 ghij 2.09 hij 2.06 ij
3 2.49 efghi 2.08 ij 2.44 efghi
3.5 3.28 bcd 2.79 defgh 2.42 fghi
4 3.1 cde 2.90 defg 3.11 cdef
4.5 3.78 bc 3.84 ab 3.77 bc
5 4.50 a 3.66 bc 3.24 bcd
Means followed by similar letter(s) in column do not differ significantly from one another.
Significant at p<0.05
Table 4-3. Comparison of boiling duration recovery means from Hot-water extraction with
measurement means from ICP-OES
Treatment (mg kg-1) 5 min (%) 10 min (%) 15 min (%)
0.5 84.4 ab 73.3 ab 64.4 b
1 67.5 ab 80.9 ab 74.2 ab
1.5 76.4 ab 73.0 ab 80.0 ab
2 80.4 ab 85.4 ab 62.5 b
2.5 90.5 ab 83.7 ab 82.3 ab
3 83.1 ab 69.4 ab 81.3 ab
3.5 93.6 a 79.6 ab 69.1 ab
4 78.1 ab 72.5 ab 77.7 ab
4.5 84.1 ab 85.2 ab 85.2 ab
5 90.1 ab 73.3 ab 64.9 ab
55
Table 4-4. Comparison of boiling duration measurement means from Hot-water extraction with
measurement from spectrophotometer
Treatment (mg kg-1) 5 min (mg kg-1) 10 min (mg kg-1) 15 min (mg kg-1)
0 0.55 lmn 0.18 mn 0.01 n
0.5 0.83 lm 0.63 lmn 0.32 mn
1 1.34 jkl 0.8 lmn 0.8 lmn
1.5 1.84 ljk 0.86 lm 1.20 kl
2 2.11 hij 1.9 ljk 1.25 kl
2.5 2.52 defghi 2.12 hij 2.06 hij
3 2.15 hi 2.48 defghi 2.44 efghi
3.5 2.27 ghi 2.72 cdefgh 2.42 fghi
4 2.97 bcdefg 3.11 abcdef 3.11 abcdef
4.5 3.18 abcdef 3.24 abcde 3.77 ab
5 3.78 a 3.52 abc 3.24 abcd
Table 4-5. Comparison of boiling duration recovery means from Hot-water extraction with
measurements from spectrophotometer
Treatment (mg kg-1) 5 min (%) 10 min (%) 15 min (%)
0.5 195.2 a 166.2 ab 126.7 cd
1 100.7 cdefgh 133.6 bc 80.1 hi
1.5 107.4 cdef 122.6 ab 57.6 defghi
2 102.6 cdefg 105.3 cdef 94.9 defghi
2.5 94.6 defghi 100.9 cdefgh 84.6 efghi
3 65.7 ghi 73.8 fghi 82.7 fghi
3.5 62.4 hi 64.8 ghi 77.8 fghi
4 84.0 efghi 70.3 fghi 77.7 fghi
4.5 86.9 efghi 70.7 fghi 72.9 fghi
5 76.5 fghi 75.6 fghi 70.4 fghi
Table 4-6. Comparison of delays using Hot-water extraction with measurement means from
ICP-OES
Treatment (mg kg-1) 0h (mg kg-1) 6h (mg kg-1) 12h (mg kg-1) 24h (mg kg-1) 48h (mg kg-1)
0 0.00 q 0.00 q 0.00 q 0.00 q 0.00 q
1 0.82 lmno 0.63 mnop 0.58 nop 0.33 pq 0.46 opq
2 1.67 ij 1.28 jkl 1.35 jk 0.94 klmn 1.1 klm
3 2.71 def 2.03 ghi 1.92 hi 1.59 ij 1.37 jk
4 3.27 bc 2.88 cde 2.43 efg 2.23 fgh 2.43 efg
5 4.32 a 3.57 b 2.92 cd 2.82 cde 3.27 bc
56
Table 4-7. Comparison of delays using Hot-water extraction with recovery means from ICP-
OES
Treatment (mg kg-1) 0h (%) 6h (%) 12h (%) 24h (%) 48h (%)
1 81.7 abcd 63.1 defg 57.5 efg 33.3 h 45.9 gh
2 83.5 abc 64.1 defg 67.4 bcde 47.2 fgh 54.9 efg
3 90.2 a 67.7 bcde 64.0 defg 53.1 efg 45.8 gh
4 81.7 abcd 72.0 abcde 60.8 efg 55.8 efg 60.7 efg
5 86.3 ab 71.4 abcde 58.4 efg 56.5 efg 65.5 cdefg
Table 4-8. Comparison of instrument measurement means using Mehlich-3 extraction

Treatment (mg kg-1) ICP-OES (mg kg-1) Spectrophotometer (mg kg-1)
0 2.6 def 2.7 def
0.5 0.9 fg 0.81 def
1 2.9 cde 3.82bcd
1.5 1.5 efg 5.11 ab
2 2.1 defg 5.67 ab
2.5 2.0 defg 5.99 a
3 2.53 def 6.72 a
3.5 2.46 def 6.42 a
4 2.95 cde 5.90 a
4.5 2.80 def 5.89 a
5 2.86 cde 4.79 abc
Table 4-9. Comparison of instrument recovery means using Mehlich-3 extraction

Treatment (mg kg-1) ICP-OES (%) Spectrophotometer (%)
0.5 171.6 cdefg 161.6 defgh
1 288.9 abc 382.3 a
1.5 98.7 fghi 340.6 ab
2 106.3 efghi 283.7. abc
2.5 78.1 fghi 239.4 bcd
3 84.2 fghi 244.1 bcde
3.5 70.2 fghi 183.4 cdef
4 73.8 fghi 147.5 defghi
4.5 62.3 ghi 65.8 hi
5 57.1 ghi 48.1 i
57
Table 4-10. Comparison of soil:extractant solution ratios using Mehlich-3 extraction with
Treatment (mg kg-1) 1:1 (mg kg-1) 1:10 (mg kg-1)
0 0.00 j 4.52 f
1 1.05 ij 5.87 e
2 2.02 hi 10.00 d
3 2.97 gh 13.81 c
4 4.10 fg 17.99 b
5 5.08 ef 22.24 a
recovery means from ICP-OES
Treatment (mg kg-1) 1:1 (%) 1:10 (%)
1 76.1 d 587.2 a
2 86.7 d 500.2 b
3 89.4 d 460.4 c
4 95.3 d 449.7 c
5 95.9 d 444.7 c
Treatment (mg kg-1) 1:1 (mg kg-1) 1:4 (mg kg-1)
0 0.01 j 0.00 j
1 1.09 i 3.96 f
2 2.07 h 8.25 d
3 3.20 g 11.89 c
4 4.47 f 16.03 b
5 5.54 e 19.90 a
recovery means from ICP-OES
Treatment (mg kg-1) 1:1 (%) 1:4 (%)
1 108.8 b 396.1 a
2 103.6 b 412.7 a
3 106.5 b 396.2 a
4 111.8 b 400.8 a
5 110.8 b 398.1 a
58
Table 4-14. Comparison of instrument measurement means using Mehlich-1 extraction with 1:1
soil:extractant solution ratio
Treatment (mg kg-1) ICP-OES (mg kg-1) Spectrophotometer (mg kg-1)
0 0.01 g 0.14 fg
1 1.09 efg 1.32 ef
2 2.07 de 2.23 cde
3 3.2 cd 3.4 bc
4 4.47 ab 4.69 a
5 5.54 a 2.79 cd
Table 4-15. Comparison of instrument recovery means using Mehlich-1 extraction with 1:1
soil:extractant solution ratio
Treatment (mg kg-1) ICP-OES (%) Spectrophotometer (%)
1 108.8 a 132.4 a
2 103.6 a 111.7 a
3 106.5 a 113.4 a
4 111.8 a 117.2 a
5 110.8 a 55.7 b
Table 4-16. Comparison of delays using Mehlich-1 extraction with measurement means from
ICP-OES using 1:1 soil:extractant solution ratio (premium sand)
Treatment (mg kg-1) 0 h (mg kg-1) 6 h (mg kg-1) 12 h (mg kg-1) 24 h (mg kg-1) 48 h (mg kg-1)
0 0.05 l 0.01 l 0.04 l 0.00 l 0.00 l
1 0.95 k 0.91 k 0.95 k 0.91 k 0.96 k
2 1.84 j 1.90 j 1.93 j 1.94 j 1.85 j
3 2.85 gh 2.93 g 2.86 gh 2.75 hi 2.71 i
4 3.91 d 3.83 de 3.78 ef 3.68 f 3.85 de
5 4.97 a 4.77 b 4.94 a 4.65 c 4.67 bc
Table 4-17. Comparison of delays using Mehlich-1 extraction with recovery means from ICP-
OES using 1:1 soil:extractant solution ratio (premium sand)
Treatment (mg kg-1) 0 h (%) 6 h (%) 12 h (%) 24 h (%) 48 h (%)
1 108.8 abc 102.1 def 103.2 def 108.8 abc 102.2 def
2 103.6 def 100.4 f 101.8 ef 103.0 def 100.1 f
3 106.5 bcd 100.0 f 100.7 f 101.9 ef 100.0 f
4 111.8 a 99.4 f 101.7 ef 105.3 cde 100.5 f
5 110.8 ab 100.2 f 101.1 ef 102.7 def 100.0 f
59
Table 4-18. Comparison of delays using Mehlich-1 extraction with measurement means from
ICP-OES using 1:1 soil:extractant solution ratio (sandy loam soil)
Treatment (mg kg-1) 0 h (mg kg-1) 6 h (mg kg-1) 12 h (mg kg-1) 24 h (mg kg-1) 48 h (mg kg-1)
0 0.05 l 0.01 l 0.04 l 0.00 l 0.00 l
1 0.95 k 0.91 k 0.95 k 0.91 k 0.96 k
2 1.84 j 1.90 j 1.93 j 1.94 j 1.85 j
3 2.85 gh 2.93 g 2.86 gh 2.75 hi 2.71 i
4 3.91 d 3.83 de 3.78 ef 3.68 f 3.85 de
5 4.97 a 4.77 b 4.94 a 4.65 c 4.67 bc
Table 4-19. Comparison of delays using Mehlich-1 extraction with recovery means from ICP-
OES using 1:1 soil:extractant solution ratio (sandy loam soil)
Treatment (mg kg-1) 0 h (%) 6 h (%) 12 h (%) 24 h (%) 48 h (%)
1 95.2 abcdefg 90.9 fg 94.7 abcdefg 91.2 efg 95.5 abcdefg
2 91.9 cdefg 94.8 abcdefg 96.5 abcdef 97.2 abcde 92.5 cdefg
3 94.9 abcdefg 97.7 abcd 95.4 abcdefg 91.6 defg 90.3 g
4 97.8 abc 95.7 abcdefg 94.6 abcdefg 92.1 cdefg 96.3 abcdefg
5 99.4 a 95.4 abcdefg 98.8 ab 92.9 bcdefg 93.4 abcdefg
Table 4-20. Effect of B treatment on first season above-ground plant part weight
Treatment (mg kg-1) Above-Ground Fresh Plant Weight (kg ha-1)
0 1868.2 b
1 4031.7 a
2 4569.0 a
3 3518.7 a
Table 4-21. Effect of B treatment on second season above-ground plant part weight
Treatment (mg kg-1) Above Ground Fresh Plant Weight (kg ha-1)
0 66.63 b
1 131.88 ab
2 203.94 a
3 180.73 a
60
Table 4-22. Effect of B treatment and method on first season leaf B content
Treatment (mg kg-1) Foliar (mg kg-1) Granular (mg kg-1)
0 22.64 bc 22.64 bc
1 15.91 c 31.82 ab
2 21.63 bc 45.84 a
3 20.68 bc 53.53 a
Table 4-23. Effect of B treatment on second season leaf B content

Treatment (mg kg-1) Leaf B (mg kg-1)
0 24.53 b
1 32.75 a
2 28.06 ab
3 32.26 a
Table 4-24. Effect of B treatment and method on first season petiole B content
0 19.45 cd 19.45 cd
1 16.72 d 22.60 bc
2 16.74 d 25.79 ab
3 19.58 cd 29.38 a
Table 4-25. Effect of B treatment on second season petiole B content

Treatment (mg kg-1) Petiole B (mg kg-1)
0 17.03 b
1 20.37 a
2 17.87 ab
3 20.43 a
Table 4-26. Effect of B treatment and method on first season stem B content
0 14.98 bc 14.98 bc
1 14.79 bc 13.66 bc
2 13.13 c 15.66 ab
3 13.64 bc 17.92 a
61
Table 4-27. Effect of B treatment on second season stem B content
Treatment (mg kg-1) Stem B (mg kg-1)
0 9.86 c
1 13.37 a
2 11.10 bc
3 12.87 ab
Table 4-28. Effect of method on first season root B content

Method Root B (mg kg-1)
Foliar 5.18 b
Granular 7.32 a
Table 4-29. Effect of B treatment and method on second season root B content
0 1.96 c 1.96 c
1 5.11 ab 4.60 ab
2 5.21 ab 4.51 ab
3 6.00 a 3.65 b
Table 4-30. Effect of B treatment and method on first season total B content
reatment (mg kg-1) Total B (g ha-1)
0 8.93 b
1 16.28 a
2 21.30 a
3 17.84 a
Table 4-31. Effect of method on first season total B content

Method Total B (g ha-1)
Foliar 12.61 b
Granular 19.57 a
62
Table 4-32. Effect of B treatment on second season total B content
Treatment (mg kg-1) Total B (g ha-1)
0 0.65 c
1 1.15 bc
2 1.69 a
3 1.61 ab
63
CHAPTER 5
CONCLUSION
Study 1. Analytical Methods and Laboratory Protocol Study
The data presented in this paper suggests that there is still a high level of variability in
soil B extraction and measurement protocols deployed by many laboratories, and thoughtful
protocol adjustments may be necessary to improve the consistency of results using these
methods. Additionally, extraction protocols should be carefully evaluated to determine if
potential variance can occur in relation to delays between B treatment and extraction.
In this study, evidence is presented to propose a protocol adjustment for Mehlich-1 extraction
from the standard soil:extractant solution ratio of 1:4 ratio to a modified soil:extractant solution
ratio of 1:1. Using this protocol adjustment, ICP-OES showed a consistent performance
improvement in soil B extraction over Hot-water and Mehlich-3 methods and the recovered
concentrations accurately reflected the B levels added. Delay between soil B treatment and
extraction times did not affect the accuracy and consistency of the results using Mehlich-1 (1:1)
and ICP-OES. Further work is required to evaluate the consistency of this adjusted protocols on
different soil extractants and soil types.
Boron application increased above-ground part weight and leaf, petiole, stem, root, and
total B content on tomatoes. Using the data from two seasons, the maximum return to B fertilizer
was obtained for tomato at 2 mg B kg-1 with granular form. We conclude that B addition of 2 mg
kg-1 (3 kg B ha-1) is sufficient to elevate tissue B levels. Similar studies with different soils are
needed to conclude if these tissue B level can be applied across other types of soils. Also, this
study suggests that while B treatment does significantly impact tomato plants, the effectiveness
64
of the treatment methods on plant part B content may be dependent on the overall health of the
plants.
65
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77
BIOGRAPHICAL SKETCH
Gürcan Duygu Baysal was born in Mersin, Turkey in 1988. In 2006, she graduated from
Antalya Aldemir Atilla Konuk Anatolian High School and started her undergraduate in Soil
Science and Plant Nutrition / Agricultural Engineering from Süleyman Demirel University in
2007 and completed in 2011. After her undergraduate education, she started her master’s in soil
science and plant nutrition and worked on the effects of soil boron treatment on mineral nutrition
of apple varieties, graduating in 2014. During her master’s education, she worked as a research
assistant and started to be a lecturer at Yenisarbademli Vocational College in Süleyman Demirel
University in 2012. In 2014, she received a scholarship from the Turkish Government to receive
a master’s degree in the USA and started her language education at University of Texas at
Austin, completing in October 2015. In January 2016, she started at the Soil and Water Sciences
Department at the University of Florida as a master’s student and completed in August 2018.
After completing her master’s degree, she will return to Turkey and work for the government as
an expert engineer in General Directorate of Combating Desertification and Erosion / Republic
of Turkey Minister of Forestry and Water Affairs.
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SOIL BORON DETERMINATION METHODS

GÜRCAN DUYGU BAYSAL

A THESIS PRESENTED TO THE GRADUATE SCHOOL

advisor, Dr. Rao S. Mylavarapu. He always encourages me to learn, to improve my knowledge

Erickson for their precious time and advice.

encouraged me to be successful. They encouraged me to follow my dreams and I am realizing

Republic of Turkey which they founded.

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

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

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

Boron in Soil and Plants .........................................................................................................15

2 LITERATURE REVIEW .......................................................................................................19

Study 1. Analytical Methods and Laboratory Protocol ..........................................................19

3 MATERIALS AND METHOD ..............................................................................................34

Study 1. Analytical Methods and Laboratory Protocol ..........................................................34

4 RESULTS AND DISCUSSION .............................................................................................41

Study 1. Analytical Methods and Laboratory Protocol Study ................................................41

Study 1. Analytical Methods and Laboratory Protocol Study ................................................64

LIST OF REFERENCES ...............................................................................................................66

BIOGRAPHICAL SKETCH .........................................................................................................78

4-1 Comparison of four different laboratories B measurement means ....................................55

4-8 Comparison of instrument measurement means using Mehlich-3 extraction ....................57

4-9 Comparison of instrument recovery means using Mehlich-3 extraction ...........................57

4-10 Comparison of soil:extractant solution ratios using Mehlich-3 extraction with

4-11 Comparison of soil:extractant solution ratios using Mehlich-3 extraction with

4-12 Comparison of soil:extractant solution ratios using Mehlich-1 extraction with

4-13 Comparison of soil:extractant solution ratios using Mehlich-1 extraction with

4-21 Effect of B treatment on second season above-ground plant part weight..........................60

4-23 Effect of B treatment on second season leaf B content .....................................................61

4-25 Effect of B treatment on second season petiole B content.................................................61

4-27 Effect of B treatment on second season stem B content ....................................................62

4-28 Effect of method on first season root B content ................................................................62

4-31 Effect of method on first season total B content ................................................................62

4-32 Effect of B treatment on second season total B content ....................................................63

1-1 Commercial B Production by Country ..............................................................................18

2-1 Process of B Uptake under Deficient Condition ................................................................33

AES Atomic Emission Spectrometry

ANOVA Analysis of Variance

ANSERV University of Florida, IFAS Analytical Services Laboratories

B203 Boron Oxide

BOR1 Efflux-type boron transporter for xylem loading

CEC Cation Exchange Capacity

CO2 Carbon Dioxide

DDI Double-distilled Water

DNA Deoxyribonucleic Acid

DTPA Diethylenetriaminepentaacetic Acid

EDTA Ethylenediaminetetraacetic Acid

H3BO3 Boric Acid

IAA Indole-3-Acetic Acid

ICP Inductively Coupled Plasma

LSD Least Significant Difference

Na2B8O7 Anhydrate Borax

NAPT North American Proficiency Testing Program

NIP5-1 Boric Acid Transporter

OES Optical Emission Spectrometry

PHW Pressurized Hot-water

RNA Ribonucleic Acid

Rutgers Boron-efficient Tomato Strain

T3238 Boron-inefficient Tomato Strain

ToMV Tomato Mosaic Virus

SOIL BORON DETERMINATION METHODS

Gurcan Duygu Baysal

Chair: Rao Mylavarapu