distribution and vertical transmission of...
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DISTRIBUTION AND VERTICAL TRANSMISSION OF SOUTHERN TOMATO VIRUS IN TOMATO
By
SEVGI COSKAN
A THESIS PRESENTED TO THE GRADUATE SCHOOL OF THE UNIVERSITY OF FLORIDA IN PARTIAL FULFILLMENT
OF THE REQUIREMENTS FOR THE DEGREE OF MASTER OF SCIENCE
UNIVERSITY OF FLORIDA
2017
© 2017 Sevgi Coskan
To my mom, dad and sister
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ACKNOWLEDGMENTS
My journey at Department of Plant Pathology in the University of Florida was a
significant contribution to both my personal life and professional career. I feel honor to
be a part of it. I consider myself as extremely fortunate, since I had the privilege
knowing so many amazing professionals who guided me during my master’s degree.
I would like to express my thanks and sincerest gratitude to my Academic
Advisor Distinguished Professor Jeffrey B. Jones for exemplary guidance, valuable
support, and generous help that kept me focused, and enabled me to pursue my
degree. I would also like to convey my appreciation to Professor Rosemary Loria, my
committee member and the chair of the Department of Plant Pathology, who
wholeheartedly and sincerely supported the completion of my thesis as well as the
challenges I encountered during this process. I express my deepest appreciation to
them, because the successful completion of my thesis could not have been possible
without their constant encouragement and belief in my success. I would like to extend
my gratitude to my committee member Dr. Svetlana Yuryevna Folimonova, for the
suggestions, support, and feedback through this process.
I would like to convey my deepest appreciation to my amazing parents and my
sister, whom always wish the best for me with an unconditional love, patience, and
support, and never let me to give up. I would also like to thank Ricardo Alcala-Briseno
for being such a great teacher, mentor, and friend who sacrificed his precious time for
showing and explaining the experiments in the laboratory for numbers of times. I need
to further thank Norsazilawati Saad for sharing her knowledge, giving me words of
encouragements and helping me under any circumstances with the best intentions. I
feel indebted to Debra Jones, Dr. Juliana Pereira-Martin, Dr. Sujan Timilsina, and
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Gerald V. Minsavage for their willingness to help and valuable contributions to my
thesis.
Very special thanks to Republic of Turkey Ministry of Food, Agriculture, and
Livestock for providing me the opportunity to have my master’s degree in the USA and
funding me as my sponsor. I started to study there as an inexperienced student,
therefore, I would also like to thank Polston Lab for the knowledge and skills that I have
gained through my research period and for truly challenging experiences which made
me stronger and determined person. Wishing to express my gratefulness to the all
department staff and officials, mainly Jessica Ulloa and Lauretta Rahmes for their
unfailing help and assistance. I am grateful to Patricia Soria, Kamaldeep Kaur, Warda
Boukari, and Salma Arous for being an excellent friend. Last but not the least, I am
thankful to my dearest friends Nesime Can, Meltem Mete, Pervin Ari Akin, and Gulen
Soyaslan, for their thoughts, good wishes, advices, and prayers. They listened to me
and made their presence left. Bulus Duman Ozkan desires the special
acknowledgement because without her, I might never have started this journey.
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TABLE OF CONTENTS
page
ACKNOWLEDGMENTS .................................................................................................. 4
LIST OF TABLES ............................................................................................................ 8
LIST OF FIGURES .......................................................................................................... 9
LIST OF ABBREVIATIONS ........................................................................................... 10
ABSTRACT ................................................................................................................... 13
CHAPTER
1 LITERATURE REVIEW .......................................................................................... 15
Introduction to Tomato ............................................................................................ 15 Tomato Production ........................................................................................... 15 Origin of Tomato ............................................................................................... 15
Introduction to Plant Viruses ................................................................................... 16 Seed Transmissible Viruses ................................................................................... 16
Economic Importance of Seed Transmitted Plant Viruses................................ 18 Seed Transmission of Tomato Viruses ............................................................. 20
Pollen Transmission ......................................................................................... 20 Mechanisms of Seed Transmission .................................................................. 21
Virus Genetic Variability .......................................................................................... 22 Plant Cryptic Viruses............................................................................................... 23 Amalgaviruses ........................................................................................................ 26
2 DISTRIBUTION OF STV ......................................................................................... 33
Introduction ............................................................................................................. 33 Materials and Methods............................................................................................ 36
Plant Sources ................................................................................................... 36 Nucleic Acid Extraction ..................................................................................... 37
Virus Detection ................................................................................................. 38 Cloning, Sequencing and Bioinformatic Analysis ............................................. 39
Results and Discussion........................................................................................... 39 Presence of STV among Cultivars ................................................................... 39 Distribution of STV at Different Leaf Ages in ‘Sweet Hearts’ Tomato Plants .... 40
Diversity of STV ................................................................................................ 41
3 VERTICAL TRANSMISSION OF STV .................................................................... 49
Introduction ............................................................................................................. 49
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Materials and Methods............................................................................................ 52
Cross Pollination .............................................................................................. 52
Seed Harvesting and Disinfection Treatments ................................................. 53 Seed Transmission ........................................................................................... 54 Total RNA extraction ........................................................................................ 54 Detection of STV .............................................................................................. 54
Results .................................................................................................................... 55
Discussion .............................................................................................................. 56
4 SUMMARY ............................................................................................................. 67
APPENDIX: CLUSTAL MULTIPLE SEQUENCE ALIGNMENT of 400 BP SEQUENCE AMPLIFIED WITH STV-SPECIFIC PRIMERS BY MUSCLE ............. 69
LIST OF REFERENCES ............................................................................................... 72
BIOGRAPHICAL SKETCH ............................................................................................ 80
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LIST OF TABLES
Table page 1-1 Seed transmitted viruses reported in tomato around the world .......................... 32
2-1 List of tomato cultivars tested for the presence of STV by RT-PCR ................... 45
2-2 Detection of STV in hypocotyl/cotyledon tissue and in developing leaves at different leaf ages of ‘Sweet Hearts’ tomato plants ............................................. 46
2-3 Detection of STV in leaves of five STV-infected plants when sampled simultaneously once the plants reached the 16th leaf stage (An STV-negative plant was used as a control for all leaves) .......................................................... 46
2-4 List of STV isolates used in this study ................................................................ 47
2-5 Pairwise comparison of percent identity nucleotide sequences of STV .............. 48
3-1 Seed transmission of STV following cross-pollination of STV-infected mother plants with pollen collected from STV-negative tomato plants ............................ 59
3-2 Seed transmission of STV following cross-pollination of STV-negative mother plants with pollen collected from STV-infected tomato plants ............................. 60
3-3 Seed transmission of STV following cross-pollination of STV-infected mother plants with pollen collected from STV-infected tomato plants ............................. 61
3-4 Seed transmission of STV following cross-pollination of STV-negative mother plants with pollen collected from STV-negative tomato plants ............................ 62
3-5 Virus infection in the progeny seedlings of crosses between STV-infected and STV-negative ‘Sweet Hearts’ parent plants ................................................. 62
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LIST OF FIGURES
Figure page 1-1 Full genome of STV ............................................................................................ 31
2-1 Hypocotyl plus cotyledon (1 to 2 weeks post germination) tested for the presence of STV ................................................................................................. 43
2-2 Leaf collected for testing for STV from a tomato plant with 12 leaves (7 weeks post planting) ........................................................................................... 43
2-3 Leaves collected from one plant for testing for STV. Age of leaf is shown from oldest (top left to youngest bottom right). ................................................... 44
3-1 Selected female flower ....................................................................................... 63
3-2 Female flower’s pistil from flower that had been emasculated (before cross-pollination) .......................................................................................................... 63
3-3 Selected male flowers as a source of pollen ...................................................... 64
3-4 Female flower following transfer of pollen to pistil .............................................. 64
3-5 Developing fruit, two-weeks post pollination ....................................................... 65
3-6 Cross-pollinated fruit ready for harvesting .......................................................... 65
3-7 Germination of seeds in plastic bag on moist paper towel .................................. 66
3-8 Germinated seedlings1 to 2 weeks after incubation in plastic bag on moist paper towel ......................................................................................................... 66
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LIST OF ABBREVIATIONS
ArMV Arabis mosaic virus
BBLV Blueberry latent virus
BBSV Broad bean stain virus
bp base pairs
BSMV Barley stripe mosaic virus
cDNA complementary DNA
CP coat protein
cm centimeter
CMV Cucumber mosaic virus
CSSV Cocoa swollen shoot virus
DNA deoxyribonucleic acid
dsRNA double-stranded RNA
FAOSTAT Food and Agriculture Organization Corporate Statistical Database
HCI Hydrochloric acid
H hybrid
kb kilobase
NCBI National Center for Biotechnology Information
OP open-pollinated
ORFs open reading frames
PCR polymerase chain reaction
PEMV Pea enation mosaic virus
PepMV Pepino mosaic virus
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PMV Peanut mottle virus
PZSV Pelargonium zonate spot virus
RCV Ryegrass cryptic virus
RdRp RNA-dependent-RNA-Polymerase
RhVA Rhododendron virus A
RMV Ryegrass mosaic virus
RNA ribonucleic acid
RT Reverse transcription
RT-PCR Reverse transcription polymerase chain reaction
RYMV Rice yellow mottle virus
SBMV Southern bean mosaic virus
ssDNA single-stranded DNA
ssRNA single-stranded RNA
STV Southern tomato virus
TAE Tris-acetate-EDTA
taq Thermus aquaticus
TBRV Tomato black ring virus
TBSV Tomato bushy stunt virus
TMV Tobacco mosaic virus
ToCV Tomato chlorosis virus
ToMV Tomato mosaic virus
TSP Tri-sodium phosphate
TSWV Tomato spotted wilt virus
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TSV Tobacco streak virus
TYLCV Tomato yellow leaf curl virus
TYMV Turnip yellow mosaic virus
USDA United States Department of Agriculture
VCVM Vicia cryptic virus M
VLPs virus-like particles
WHO World Health Organization
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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
DISTRIBUTION AND VERTICAL TRANSMISSION OF SOUTHERN TOMATO VIRUS
IN TOMATO By
Sevgi Coskan
August 2017
Chair: Jeffrey B. Jones Major: Plant Pathology
Southern tomato virus (STV) is a recently identified virus that is present in tomato
cultivars worldwide. STV is a monopartite virus with a double-stranded RNA genome
(3.5 kb) containing two overlapping open reading frames (ORFs) and is the type species
of the genus Amalgavirus (family Amalgaviridae). A reverse-transcription polymerase
chain reaction (RT-PCR) using virus-specific primers was used to detect STV in 24
tomato cultivars, which included hybrid and open pollinated cultivars. STV was detected
in plants produced by a number of seed companies, indicating that the virus is widely
distributed in the vegetable seed industry. STV was detected for the first time in the
following cultivars: ‘Agriset 761’, ‘Bonita’, ‘Florida Lanai’, ‘Mexico Midget’, and ‘Roma’.
Partial isolates of STV from this study and full genome sequences of STV from
GenBank were compared and found to have an average percentage diversity of less
than 1%. In order to look at the presence of STV in different developmental stages of
tomato plants, STV-infected and STV-negative ‘Sweet Hearts’ tomato plants were
grown and sampled for the presence of STV using RT-PCR at different developmental
stages. STV was detected in 75% of 1 to 2 week-old germinated seedlings. When
plants were grown and sampled at various leaf stages up to final sampling at 15th leaf
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stage, all positive plants were positive for STV and the negative plants remained
negative. The presence or absence of STV was consistent across all four
developmental stages. Seed transmission studies of progeny produced from crosses of
different combinations of parents that were STV-infected or -negative, revealed that
both ovule and pollen transmission of STV occurred at high rates.
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CHAPTER 1 LITERATURE REVIEW
Introduction to Tomato
Tomato Production
Tomato (Solanum lycopersicum, formerly Lycopersicon esculentum Mill.)
production worldwide, which in 2014 accounted for close to 171 million tons (FAOSTAT,
2017), is one of the most important and profitable vegetable crops, particularly in
Florida, and is widely distributed in the U.S.
The U.S. is the second largest country for tomato production and Florida ranked
first in fresh market tomato production in 2015 (USDA, 2017). In 2015, tomato
production in Florida was reported at approximately 33,000 acres and generated $453.1
million in gross sales (Freeman et al., 2016).
Origin of Tomato
Tomato originated in the Andean region, now part of Bolivia, Chile, Colombia,
Ecuador and Peru (Bai and Lindhout, 2007). The early events in domestication of
tomato remain unclear (Peralta and Spooner, 2007). Tomato was introduced to Europe
in the 16th century and reached an advanced level of domestication through Europe in
the 18th and 19th centuries (Sims, 1980; Bai and Lindhout, 2007). It is believed that
tomato was first introduced to Europe most probably from Mexico by Spanish
conquistadors (Blanca et al., 2012). By the 17th century, tomatoes were used just for
ornamental purposes since they were thought to be poisonous, like members of the
Solanaceae, Atropa belladonna and Mandragora sp. (Bergougnoux, 2014).
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Introduction to Plant Viruses
Viruses are obligate parasites that are not capable of replicating without a host
(Hull, 2014). Plant viruses are an important group of tomato pathogens that exist
worldwide (Hanssen et al., 2010). Diseases caused by viruses are a major problem
because of the difficulty in disease management (Jones et al., 2014). Plant damage
caused by viral diseases is difficult to predict due to various factors including plant
cultivar, geographic region, infection time, and virus strain (Jeong et al., 2014; Strange
and Scott, 2005).
In most situations, viral diseases are diagnosed based on the symptoms they
induce, which include leaf chlorosis, necrosis, malformation, mosaic, and stunting (Hull,
2009). However there are some cases in which viruses might not be visually diagnosed
since certain plant viruses do not cause any symptoms following infection (van der Want
and Dijkstra, 2006). In addition to that, sometimes virus-like symptoms are caused by
nutritional deficiencies, pest damage, and weather conditions (Jeong et al. 2014; Jones
et al., 2014). Hence compared to other pathogens, diagnosis of virus infection based on
symptoms is more complicated (Lievens et al., 2005).
Viral diseases usually produce a range of symptoms on plants with a scale of
severity such as mild to severe, or viruses can also cause no symptoms, or even plant
death (Jones et al., 2014; Hull, 2009). Variation in the virus symptoms can be influenced
by multiple factors such as the production systems, locations, and the type and age of
the cultivars.
Seed Transmissible Viruses
Seed transmission is defined as the process in which the virus is transferred from
seed to seedlings is referred to as seed transmission (Sastry, 2013). One of the most
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important and interesting aspects of plant-virus interactions is the plant`s ability during
seed development to protect their progeny against virus invasion coming from their
infected maternal plant tissues (Bennett, 1969). Viruses cannot infect plants directly
because they are not able to penetrate the host cell walls and they cannot get in the
plasma membrane, since there are no receptors in host cells for viruses (Atsumi et al.,
2015). Some viruses, however, are able to overcome those limitations and infect plants
by entering through wounds or via a vector. However, in some cases, viruses are also
capable of being transmitted to the next generation through infected seed originating
from infected parent plants (Bashir and Hampton, 1996; Bennett, 1969).
Quality seed plays a critical role in crop production and has a significant role in
yield (Gumus and Paylan, 2013). Therefore, seed health is one of the most important
factors to consider when attempting to produce quality seed. Worldwide, viruses are
generally transported to new regions on infected seed used for production (Mandahar,
1981). Germplasm exchange is another very common means for moving seed around
the world. As a result of this, viruses have been introduced to different geographical
areas (Bashir and Hampton, 1996).
Although seed may test positive for a particular virus, they do not always produce
infected plants. The frequency of seed infection resulting in seedling infection can vary
drastically. For instance, in one study, 62% of the tomato seed tested positive for
Tobacco streak virus (TSV), but only 11% of the seedlings were infected (Sdoodee and
Teakle, 1988). The location of the virus in seed also affected seed transmission to
seedlings. For instance, the presence of Turnip yellow mosaic virus (TYMV) in the seed
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coat of Arabidopsis thaliana did not result in seedling infection, and embryo invasion
was found to be necessary for seedling infection (de Assis Filho and Sherwood, 2000).
Seed transmission is important for dissemination of plant viruses (Mink, 1993;
Johansen et al., 1994; Sastry, 2013). For instance, seed-transmissible viruses
contributes to the ability of a virus to overwinter (Hull, 2014) and serve as a primary
inoculum source for vector transmission which may cause significant effects on crop
production (Akhtar and Kobayashi, 2010; Kil et al., 2016; Kim et al., 2015). Virus
transmission through seed has had a pronounced effect on virus survival from parents
to progeny and from one season to another (Akhtar and Kobayashi, 2010; Johansen et
al., 1994).
Approximately 20% of known plant viruses have been reported to be seed
transmissible (Johansen et al., 1994; Sastry, 2013). All seed-transmitted viruses are
vertically transmitted (i.e. the virus is transmitted from parents through ovule and/or
pollen to the progeny (Sastry, 2013). As reviewed by Sastry (2013) and by Card et al.
(2007) there are more than 231 viruses confirmed as being vertically transmitted, and
these include 17 genera of viruses that are reported as pollen transmitted by either
vertical and/or horizontal transmission.
Economic Importance of Seed Transmitted Plant Viruses
Seed transmission helps to evenly establish a virus in a crop at an early stage,
as a result of the presence of multiple foci (Hull, 2009). Hence, seed transmission might
have considerable economic significance in that the virus is introduced as primary
inoculum to a production field, and then once present in the field other transmission
mechanisms result in efficient secondary spread of the virus. Seed-transmissible
viruses can be devastating to crops (Sastry, 2013). As explained in the reviews by Mink
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(1993) and Johansen et al. (1994), when the host plant becomes infected with virus, the
infection mostly results in yield losses. According to Albrechtsen (2006), economic
losses caused by seed-transmitted viruses have both indirect impact such as chemical
management cost for vectors, and direct impact in terms of quality of crops and yield
reduction.
A significant example of the impact of a seed-transmitted virus is the high loss in
Barley stripe mosaic virus (BSMV) in Montana barley fields, which caused considerable
yield losses for many years (Carroll, 1980). The estimated total loss in currency was
more than $30 million between 1953-1970 (Carroll, 1983). In this case, to solve the
problem, a seed certification program was implemented which resulted in significantly
reduced yield losses.
Significant yield losses associated with seed infection can occur as a result of a
number of factors including the susceptibility of the host, the virus strain, and the host
stage when virus infection occurs (Albrechtsen, 2006; Hull, 2014; Lenardon et al.,
2001). For instance, Kuhn et al. (1978) reported that Peanut mottle virus (PMV) strain-
M2 had 31% yield loss in ‘Starr’ and 20% loss in both ‘PI-261945’ and PI-261946’;
however, strain-N in infected peanuts reported 47% yield loss in ‘PI-261945’, 68% in
‘Starr’, and no yield losses in ‘PI-261946’.
Viruses as well as their vectors can cause significant yield losses. For instance,
Hinz and Daebeler (1979) reported that when beans were infected with Pea enation
mosaic virus (PEMV) alone, the yield reduction in broad bean seeds was 6%. Yield
reduction was 50% with infestations of aphids in the absence of the virus. However,
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93% yield reduction was reported when the both the virus and the aphid vector were
present (Sastry, 2013).
Yield loss estimates are essential for establishing proper virus management
techniques in the field (Johansen et al., 1994; Mink, 1993). In other words, determining
the means of the virus transmission is important information in order to develop
strategies for controlling viral diseases (Atsumi et al., 2015); furthermore, knowing that a
virus is seed-transmitted is important for plant protection in that strategies are
developed to prevent or limit distribution of the virus (Kil et al., 2016). In summary,
based on the examples above, yield losses are affected by several factors.
Seed Transmission of Tomato Viruses
Seed transmitted viruses, including pollen transmitted viruses, create major
problems in tomato industries around the world (Lapidot et al., 2010). Although hybrid
tomatoes are cultivated and harvested in one region, they are traded all around the
world. Therefore, seed transmission can enable the distribution of viruses into non-
infected and new geographical areas. Currently, tomato is considered a host for 10
species of seed transmitted viruses from 8 known genera (Table 1-1) (Lister and
Murant, 1967; Sastry, 2013; Hanssen et al., 2010; Cicek and Yorganci, 1991; Hanada
and Harrison, 1977; Tomilnson and Faithfull, 1984; Hadas et al., 2004; Kil et al., 2016).
Pollen Transmission
Pollen can also be an important means in seed infection (Bailiss and Offei, 1990;
Card et al., 2007; Johansen, 1994; Sastry, 2013) and can result in long distance spread
in infected seed (Mink, 1993). When the viruses are pollen-transmissible, seed may be
infected which indicates vertical transmission, as the seedlings that develop contain the
virus (Roberts et al., 2003), or the healthy host might be infected via the fertilized flower,
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which represents horizontal transmission. However, it cannot be concluded that if a
virus is transmitted vertically by pollen, horizontal transmission will also occur and vice
versa (Card et al., 2007).
The mechanisms of pollen and seed transmission are closely related (Johansen
et al., 1994). Pollen-transmissible viruses are also commonly seed transmissible but not
necessarily the converse is true (Card et al., 2007). Broad bean stain virus (BBSV) is an
example of a virus that is reported to be seed transmissible but not pollen transmissible
(Brunt et al., 1996). Seventeen genera of plant viruses have been reported as being
transmitted through pollen vertically and/or horizontally (Atsumi et al., 2015).
Mechanisms of Seed Transmission
Seed transmission can be achieved in mainly two ways: 1) infection of the seed
coat or maternally derived seed parts, and 2) infection of the embryo. Tobacco mosaic
virus (TMV) infested tobacco seed is an example of seed-coat infection (de Assis Filho
and Sherwood, 2000), Southern bean mosaic virus (SBMV) on soybean is an example
of a maternally infected seed (Uyemoto and Grogan, 1977).
As reviewed by Mink (1993), Johansen et al. (1994) and Sastry (2013), during
seed infection, the embryo becomes infected by the virus during direct invasion or by
indirect invasion. Direct invasion is achieved when the virus invades the developing
embryo during embryogenesis. There is no clear explanation for how the virus crosses
the boundary between the female parent and resulting progeny generations in the ovule
(Wang and Maule, 1994).
Indirect invasion occurs when the virus invades the embryo before
embryogenesis by infection of reproductive tissues (e.g. pollen mother cell and
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megaspore mother cell) (Johansen et al., 1994). Interestingly, BSMV has been reported
to infect seed by direct and indirect invasion (Mandahar, 1981).
Virus Genetic Variability
According to Roossinck (2008), genetic variability is crucial for an organism’s
fitness and is common feature among viruses. When a mutation or recombination in the
virus genome results in a biological advantage, those new variants spread throughout
the viral population (Moya et al., 2004; Roossinck, 2008). Those viruses that recently
changed and succeed during the natural selection are described as emerging viruses
(Rojas and Gilbertson, 2008). Emerging viruses represent introduction of a new virus
species, an influx of genetic variability in an existing virus or the introduction of existing
viruses to new locations. According to the World Health Organization (WHO), emerging
viruses fall into two categories. They can represent those that have appeared in a
population for the first time, or those that have been present previously but are rapidly
increasing in incidence or geographic range. According to Rojas and Gilbertson (2008)
and Hanssen et al. (2010), emerging viruses can also be viruses that have been
changing or occupy and spread within new niches. Previously unknown viruses could
also be described as emerging viruses (Rojas and Gilbertson, 2008; Hanssen et al.,
2010).
Roossinck (2008) has categorized emerging viruses as individual viruses or
entire groups of viruses. Some of the emerging virus groups include criniviruses (family
Closteroviridae) (Tzanetakis et al., 2013) and whitefly-transmissible begomoviruses
(family Geminiviridae) (Leke et al., 2015). With regard to individual viruses, Rice yellow
mottle virus (RYMV) (Fargette et al., 2008) and Pepino mosaic virus (PepMV) in tomato
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(Gomez et al., 2012) are examples that have emerged in Africa and the Mediterranean
region, respectively.
Plant Cryptic Viruses
Over the past 120 years, numerous viruses have been reported that infect plants
(ICTV, 2015). Cryptic viruses were first described in plants in the late 1960s (Pullen
1968; 1969) and were described as viruses that are present at low titer, and are not
associated with disease symptoms.
Although cryptic viruses have been reported as symptomless (Lisa et al., 1981;
Roossinck, 2010; Sabanadzovic and Valverde, 2011), there are some reports of
symptoms related with those viruses (Antoniw et al., 1990). For instance, Natsuaki et al.
(1979) reported that most Japanese radish cultivars do not have symptoms; however,
some seedlings infected with cryptic viruses produce some symptoms. These
symptoms include leaf edges with mild yellowing and smaller lower leaves (Natsuaki et
al., 1979). Additionally, in the early 1970s, Plumb and colleagues (1973; 1974) found
that there are more severe symptoms when ryegrass is infected with both Ryegrass
mosaic virus (RMV) and Ryegrass cryptic virus (RCV) than RMV alone. Therefore these
findings revealed that between other viruses and cryptic viruses, there might be
synergistic interactions (Antoniw et al., 1990). Furthermore, Kassanis et al. (1978)
suggested that cryptic viruses might have an impact on the growth and development of
the host since they observed larger roots in healthy sugar beet plants compared to
those in beet cryptic virus-infected plants.
In general, most cryptic viruses do not produce visible symptoms in their hosts
and are transmitted only by seed and by pollen to the next generations (Boccardo et al.,
1987; Roossinck, 2010). According to Antoniw et al. (1990), their survival depends to a
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much larger extent than most plant viruses upon the successful growth and
reproduction of the host plant. Hence there are possibly strong selection pressures to
assure that cryptic viruses do not negatively affect the reproductive efficiency of their
host plants, and there might be selection for these viruses having positive effects in their
hosts (Antoniw et al., 1990).
Cryptic viruses also are not transmitted horizontally, which means they are not
transmitted mechanically or by any vector (Kassanis et al., 1977) or by grafting
(Boccardo et al., 1985). Additionally, cryptic viruses have been reported to only be
transmitted by seed and pollen (Roossinck, 2010; Sabanadzovic and Abou Ghanem-
Sabanadzovic, 2008; Sabanadzovic and Valverde, 2011). In other words, transmission
of the virus to the embryo is the only way to maintain the cryptic virus in the embryo
(Boccardo et al., 1987).
The first cryptic viruses were reported to be spherical viruses or virus-like
particles (VLPs) in sugar beet (Boccardo et al., 1987; Kassanis et al., 1977). In addition
to sugar beet, it was reported that cryptic viruses had wide host ranges and were found
in monocotyledonous and dicotyledonous plant species including alfalfa (Medicago
sativa) (Accotto et al., 1990), bean (Vicia faba) (Abou-Elnasr et al., 1985; Liu and Chen,
2009), beet (Beta vulgaris) (Szego et al., 2010), Brassica rapa (Li et al., 2016),
carnation (Dianthus caryophyllus) (Lisa et al., 1981), carrot (Daucus carota) (Willenborg
et al., 2009), hop trefoil (Medicago lupulina) (Accotto et al., 1990; Luisoni et al., 1987),
ryegrass (Lolium multiflorum) (Plumb, 1973; Plumb and Misari, 1974), meadow fescue
(Festuca pratensis) (Boccardo et al.,1983), pear (Pyrus pyrifolia) (Osaki et al.,1998),
pepper (Capsicum annuum) (Sabanadzovic and Valverde, 2011), pine (Pinus sylvestris)
25
(Veliceasa et al., 2006), radish (Raphanus sativus) (Chen et al., 2006), red clover
(Trifolium pratense) (Lesker et al., 2013), rose (Rosa) (James et al., 2015;
Sabanadzovic and Abou Ghanem-Sabanadzovic, 2008) , spinach (Spinacia oleracea)
(Park and Hahn, 2017), and white clover (Trifolium repens) (Boccardo and Candresse,
2005; Guy and Gerard, 2015).
All cryptic viruses found so far consist of small isometric particles with a dsRNA
genome and have been separated into two subunits: a particle about 29-32 nm in
diameter, and another particle about 37-38 nm in diameter (Boccardo et al., 1987;
Sabanadzovic and Valverde, 2011). Those cryptic viruses have two encoded proteins,
which consist of a coat protein and RNA-dependent-RNA-polymerase (RdRp)
(Boccardo and Candresse, 2005).
Plant cryptic viruses presently belong to the family Partitiviridae that currently
consists of five genera that infect plants, protozoa and fungi. Two of the genera,
Alphapartitivirus and Betapartitivirus, infect plants and fungi, while Cryspovirus infect
protozoa, Deltapartitivirus infect plants, and Gammapartitivirus only infect fungi (ICTV
2015). Recently, endornaviruses (Roossinck, 2011; Valverde and Gutierrez, 2007) have
been reported to have similar features as partitiviruses and as such both groups were
categorized as persistent viruses by Roossinck (2010). According to Roossinck (2010),
endornaviruses and partitiviruses have been described as having a persistent lifestyle in
their hosts because of the lack of symptoms in infected tissue, their lack of movement
proteins, their movement in plants only by cell division, and because they are
transmitted vertically but not horizontally. More recently amalgaviruses were
26
characterized and determined to be similar to the persistent viruses (Liu and Chen,
2009, Sabanadzovic et al., 2009, Sabanadzovic et al., 2010; Martin et al., 2011).
Amalgaviruses
The Amalgaviridae family consists of the dsRNA plant viruses, and contains only
the genus Amalgavirus that includes four species: Southern tomato virus (STV),
Blueberry latent virus (BBLV), Rhododendron virus A (RhVA), and Vicia cryptic virus M
(VCVM). Amalgaviruses are monopartite and contain approximately a 3.5kb dsRNA
genome with two partially overlapping coding regions (putative coat protein, RdRp)
(Martin et al., 2011). Little is known about Amalgaviridae replication. They are believed
to replicate in the cytoplasm (Liu and Chen, 2009). Only STV has been tested for
mechanical and graft transmission and was shown to be negative (Sabanadzovic et al.,
2009). Both STV (Sabanadzovic et al., 2009) and BBLV (Martin et al., 2011) have been
reported to be transmitted by seed.
The genomic organization of members of these viruses is similar to some of the
genus Totivirus that belong to the Totiviridae. Phylogenetic analysis shows this group of
viruses to be closer to the Partitiviridae family than the Totiviridae family (Martin et al.,
2011; Sabanadzovic et al., 2009). Nibert et al. (2014) proposed the new family,
Amalgaviridae, to indicate the similarities between the Totiviridae and the Partitiviridae.
As a result, this was approved as new virus family by ICTV (Adams et al., 2014).
STV, which is the type species of the Amalgavirus, is a virus with a monopartite
3.437 bp double-stranded RNA (dsRNA) genome that contains two overlapping coding
regions (putative CP, RdRp) (Sabanadzovic et al., 2009). The first ORF is p42 (42-kDa),
codes for a putative coat protein (CP), and the second ORF encodes p121 (121-kDA)
27
RNA-dependent RNA polymerase that has a + 1 ribosomal frameshift (Figure 1-1;
Sabanadzovic et al., 2009).
Like other amalgaviruses, there have been no observed or purified virus particles
associated with STV (Candresse et al., 2013; Isogai et al., 2011, Martin et al., 2011,
Sabanadzovic et al., 2009). Limited information is known about the replication of STV. It
is believed to replicate in the cytoplasm and to lack the ability to move systemically or
cell to cell (Liu and Chen, 2009; Sabanadzovic et al., 2009). STV, like amalgaviruses, is
not transmitted by mechanical inoculation or by graft transmission. It is known that STV
is transmitted through seed (Sabanadzovic et al., 2009; Martin et al., 2011), although it
is unknown whether seed transmission of STV is through pollen or ovules or both
(Coskan, unpublished data).
STV was first reported to be associated with tomato yellow stunt disease which is
characterized by reduction of fruit size, leaf chlorosis, and reduced crop yields in ‘UC
82’ and ‘Celebrity’ cultivars (Sabanadzovic et al., 2009, Candresse et al., 2013). Thus
the association between symptoms and the presence of STV is unclear. Since its
detection in tomato plants for the first time in the U.S. and Mexico (Sabanadzovic et al.,
2009), STV was also detected in France (Candresse et al., 2013), the Canary Islands in
Spain (Verbeek et al., 2015), China (Padmanabhan et al., 2015b), Bangladesh
(Padmanabhan et al., 2015a), and Italy (Iacono et al., 2015). Recently it was reported in
Florida (Alcala-Briseno et al., 2017) and in Korea (GenBank accession number:
LC270272.1.). Comparison of partial and complete sequences of STV isolates from
Italy, Mexico, Bangladesh, China, U.S. and France showed that the sequences had less
than 1% diversity (Iacono et al., 2015).
28
STV is often present in the field with other viruses as mixed infections. Verbeek
et al. (2015) detected STV along with PepMV, Tomato spotted wilt virus (TSWV), and
Tomato yellow leaf curl virus (TYLCV) in Spanish tomatoes. In China, Padmanabhan et
al. (2015b) found STV in a mixed infection with Cucumber mosaic virus (CMV), Tomato
chlorosis virus (ToCV), and TYLCV. STV was reported in France in a mixed infection
with Potato virus Y (PVY) and TMV (Candresse et al., 2013). In Bangladesh, STV was
found in a co-infection with PVY and CMV (Padmanabhan et al., 2015a). The recently
identified STV from Italy was in tomato plants also infected with PepMV and PVY
(Iacono et al., 2015).
Other members of Amalgaviridae, including VCVM (Liu and Chen, 2009) and
RhVA (Sabanadzovic et al., 2010), were identified in symptomless Vicia faba in
Hangzhou, Zhejiang Province, Eastern China, and symptomless rhododendron in Great
Smoky Mountains National Park, U.S. Little is known about these viruses. Like STV and
BBLV, they have an approximately 3.5 kb genome that contains two partially
overlapping putative open reading frames. As experimentally verified for STV
(Sabanadzovic et al., 2009) and BBLV (Martin et al., 2011), it is likely that VCVM and
RhVA are also seed transmissible.
The last identified Amalgavirus in family Amalgaviridae is BBLV. A new disorder
known as blueberry fruit drop disease was observed in the Pacific Northwest of North
America (British Columbia, Oregon, and Washington) in the early 2000s (Martin et al.,
2009; 2011). Even though it was later determined that there was no relationship
between BBLV and blueberry fruit drop disease, the virus was further characterized due
to its prevalence in the preliminary survey (Martin et al., 2011). This led to the isolation
29
of approximately a 3.5 kb dsRNA molecule, which was named BBLV (Martin et al.,
2011).
The genome organization of BBLV is similar to STV and possesses two partially
overlapping ORFs encoding for a replicase protein and an unknown protein (Martin et
al., 2011; Sabanadzovic et al., 2009). While the genome organization of BBLV is
analogous to the viruses in the family Totiviridae, its RdRp was found to be closely
associated with the members of the Partitiviridae family (Martin et al., 2011; Krupovic et
al., 2015). Thus, Nibert et al. (2014) placed BBLV in Amalgavirus in the family
Amalgaviridae along with STV (Adams et al., 2014).
Geographical distribution of BBLV is broad, as it has been detected in non- and
symptomatic plants in Arkansas, Michigan, New Jersey, and the Pacific Northwest, as
well as in blueberry germplasm from North America (Martin et al., 2011; 2012).
Comparison of partial and complete sequences of BBLV isolates from Japan and the
U.S. showed that BBLV has a very stable population structure, with less than 0.5%
diversity among isolates from the U.S. and Japan (Isogai et al., 2011; Martin et al.,
2011; 2012). BBLV is transmitted by seed in the absence of movement protein,
suggesting that it is replicating in the cytoplasm and moved by cell division (Martin et al.,
2011). Observation on some BBLV-infected high bush cultivars for almost a decade
indicated that, like other Amalgaviridae members, the presence of this virus in blueberry
is not a concern because infected plants remain symptomless (Martin et al., 2011). The
objectives of this research were 1) to determine the presence of STV in open pollinated
and hybrid tomato cultivars, 2) to determine how uniformly STV can be detected in
leaves at different plant growth stages, 3) to compare partial sequences of isolates of
30
STV in different tomato cultivars, and 4) to determine if STV is transmitted to the next
generation through ovules and/or through pollen.
31
Figure 1-1. Full genome of STV (photo courtesy of Sabanadzovic et al., 2009).
32
Table 1-1. Seed transmitted viruses reported in tomato around the world
Genome Family Genus Virus Abbreviation References
ssDNA Geminiviridae Begomovirus Tomato yellow leaf curl virus
TYLCV Kil et al., 2016.
ssRNA (+) Alphaflexiviridae Potexvirus Pepino mosaic virus PepMV Hanssen et al., 2010.
ssRNA (+) Bromoviridae Anulavirus Pelargonium zonate spot virus
PZSV Lapidot et al., 2010.
ssRNA (+) Bromoviridae Cucumovirus Cucumber mosaic virus
CMV Sastry, 2013.
ssRNA (+) Secoviridae Nepovirus Arabis mosaic virus ArMV Lister and Murant, 1967.
ssRNA (+) Secoviridae Nepovirus Tomato black ring virus
TBRV Hanada and Harrison, 1977.
ssRNA (+) Tombusviridae Tombusvirus Tomato bushy stunt virus
TBSV Tomlinson and Faithfull, 1984.
ssRNA (+) Virgaviridae Tobamovirus Tobacco mosaic virus
TMV Cicek and Yorganci, 1991.
ssRNA (+) Virgaviridae Tobamovirus Tomato mosaic virus ToMV Hadas et al., 2004.
dsRNA Amalgaviridae Amalgavirus Southern tomato virus
STV Sabanadzovic et al., 2009.
33
CHAPTER 2 DISTRIBUTION OF STV
Introduction
Many seed companies, farmers, distributors, and home gardeners rely on tomato
production profits every year. They face many challenges, but diseases are a major
impediment. Viral diseases have a critical economic impact on tomato production
(Hanssen et al., 2010). Management strategies for controlling viral diseases include
using cultural and chemical methods, which include avoiding the use of infected
transplants, weed management, chemical control of the vector, and, when available, the
use of resistant cultivars. A major concern is the spread of viruses to previously
unaffected places, which becomes more likely because of the global seed trade.
According to Hanssen et al. (2010), in recent years, many viral diseases have
emerged and are directly impacting fresh market tomato production worldwide. Some of
these viruses were known to affect crops other than tomato and have now been shown
to cause disease in tomato. Some are newly described virus species and others are
known tomato viruses that are becoming more prevalent.
Based on the criteria for emerging viral diseases outlined by Hanssen et al.
(2010), one might consider STV to be an emerging virus, even though it is not
associated with disease symptoms. STV was first detected in Mexico and Mississippi in
2005 (Sabanadzovic et al., 2009) and has been reported in several different locations
(Alcala-Briseno et al., 2017; Iacono et al., 2015; Padmanabhan et al., 2015a; 2015b;
Sabanadzovic et al., 2009). Although it has similarities with other viruses in Totiviridae
and Partitiviridae families, Sabanadzovic et al. (2009) was the first to propose that STV
is a new virus group and suggested it be placed in a new taxon. Hence, STV was
34
placed in a new genus, Amalgavirus, in the family Amalgaviridae with STV as the type
species (Adams et al., 2014).
Viruses in the Amalgaviridae, like those in the Endornaviridae and Partitiviridae,
are dsRNA viruses; unlike viruses in the Partitiviridae, members of the Amalgaviridae
and Endornaviridae do not have capsids. Partitiviruses are associated with plants and
fungi, whereas endornaviruses are associated with plants, fungi and oomycetes
(Fukuhara et al., 2006; Okada et al., 2011). Some viruses in all three families, which
infect plants, are efficiently transmitted vertically and infected plants remain
symptomless (Roossinck, 2010, Sabanadzovic et al., 2008, Valverde et al., 2007).
Therefore, these viruses could be considered cryptic viruses, although the term has
been specifically used for members of the Partitiviridae (Boccardo et al., 1987).
Endornaviruses and partitiviruses have been described as having a persistent lifestyle
in their hosts because of their lack of movement proteins, their movement in plants only
by cell division, the lack of symptoms in infected tissue, and their inability to transmit
horizontally (Roossinck, 2010). Amalgaviruses also behave in a persistent lifestyle.
Viruses in the Amalgaviridae (Sabanadzovic et al., 2009), Partitiviridae (Nibert et al.,
2014), and Endornaviridae (Valverde and Gutierrez, 2007) families are dispersed by
pollen and/or seeds. STV, like viruses in the Partitiviridae and Endornaviridae, is
disseminated by seeds and has been reported in hybrid tomato cultivars with 8 of 27
being positive; however, the contribution of pollen in STV transmission is unknown
(Sabanadzovic et al., 2009).
Information relating to STV and other amalgaviruses is limited. Because
persistent viruses seem to cause no economic impact, there has not been much
35
attention given to them. STV was detected in leaves, roots and fruit or seeds of tomato
plants (Gonzales et al., 2017); however, no systematic analysis of different ages of leaf
tissue was done to determine the overall distribution of STV.
Viruses possess potential for genetic diversity. Genetic variability is required
because viruses need to adapt constantly to a changing environment and shifting
selection pressure (Roossinck, 2008). According to Garcia-arenal et al. (2001), genetic
diversity in virus populations does not occur as a rule, because natural selection and
bottlenecks restrict diversity. Bottlenecks happen at several stages of the virus infection
cycle resulting in a reduction in the number of genomes that are present in a viral
population (Gutierrez et al., 2012). Comparison among isolates of STV from Italy,
Mexico, Bangladesh, China, USA, and France showed that the sequences had 99%
similarity (Iacono et al., 2015), so genetic variability among isolates of STV seems to be
low.
Although genetic diversity of STV appears to be low, the sequences that have
been published are strictly from hybrid tomato varieties and may have been from a
limited number of varieties. It is important to look at open-pollinated cultivars as well as
additional tomato hybrids to determine if greater genetic diversity exists. Breeding of
cultivars where breeding material may contain diverse STV populations may potentially
have an impact on the introduction of increased viral genetic variability. By determining
the presence of STV in diverse hybrid and open-pollinated cultivars followed by
sequence analysis of the isolates, we will gain insight into the potential diversity of the
virus in breeding programs.
36
The objectives of this study were 1) to determine the presence of STV in open
pollinated and hybrid tomato cultivars, 2) to determine how uniformly STV can be
detected in leaves at different plant growth stages and 3) to compare partial sequences
of isolates of STV in different tomato cultivars.
Materials and Methods
Plant Sources
Presence of STV in open pollinated and hybrid tomato cultivars. In a survey
of screening tomato cultivars for the presence of STV in hybrid (H) and open-pollinated
(OP) cultivars, 24 cultivars, including 13 hybrid tomato cultivars (2 cultivars were
obtained from two different sources) and 11 open-pollinated tomato cultivars were
obtained from different sources (Table 2-1). Cultivars were selected for diversity of
genetic background and commercial source. Two or three cultivars were selected from
the following tomato types: beefsteak, cherry, deep oblate, grape, plum, rootstock and
round (Table 2-1). Between 15 and 30 seeds were sowed and the plants were grown
under greenhouse conditions. Depending on the experiment, leaves from one to three
month old plants were sampled.
Presence of STV in leaves at different plant growth stages. To determine the
presence of STV in tomato plants at different stages of development, the hybrid cultivar
‘Sweet Hearts’ was chosen for testing because it was shown in this study to be infected
with STV at high rates (Table 2-1). For seedling testing, seeds were planted to test for
presence of STV at different stages of plant development. Twelve seeds were
germinated in a plastic bag on moist paper towels. The bags were then placed in an
incubator at 25-27°C. One to two weeks later at the post germination stage (hypocotyl
plus cotyledon) (Figure 2-1), the seedlings were separated from the seed coat and were
37
assayed for STV as described below. For testing tissue at different leaf stages, seed
were planted in seedling flats and grown in a greenhouse until the first true leaf stage
and then transplanted to 10-cm pots containing Sun Gro Metro Mix 830. The plants
were fertilized weekly with Peters Professional 20-20-20. The plants were grown in a
greenhouse and leaves were sampled for STV at the 1st, 4th, 7th, 12th and 15th leaf
stages. Ten plants were sampled at each time, with the seedling stage and 1st leaf
stage sampling requiring destruction of the entire plant. For samples taken (Figure 2-2)
at the 4th, 7th, 12th and 15th leaf stages, sampling was not destructive and the same
plants were used.
In a separate experiment, to address the second objective, the distribution of
STV within plants was determined when all leaf stages were sampled for STV at the
same time. Five ‘Sweet Hearts’ seeds were planted and grown under greenhouse
conditions until the 16th leaf stage. Once the plants reached the 16th leaf stage, leaves
were collected from the 3rd, 6th, 9th, 12th, and 15th leaves (Figure 2-3) of each of five
plants and assayed for STV.
Nucleic Acid Extraction
Total RNA was extracted from 60 to 80 mg of all samples except the seedlings
for objective 2, when only 18-30 mg of plant tissue was available for the initial sampling
at the 1-2 week post germination stage (hypocotyl plus cotyledon). Leaves were stored
at -80oC in case of the need to retest in the future.
An OmniBiotek Plant RNA mini kit (OmniBiotek Co. MA) was used according to
the manufacturer’s instructions to extract total RNAs. Extracted total RNAs were
visualized following electrophoresis through 1% agarose gel in 1X TAE (Tris-acetate-
EDTA) to check the RNA quality and then the RNA was quantified by
38
spectrophotometry using a Nanodrop (Thermo Scientific, Wilmington, ME). The total
RNA for each sample was adjusted to 100 ng/µl.
Virus Detection
Two primers, STV-F (5’-CGTTATCTTAGGCGTCAGCT-3’) and STV-R (5’-
GGAGTTTGATTGCATCAGCG- 3’), which amplify a 440 bp region that encompasses
the overlapping region between ORF 1 and ORF 2, were used for detection of STV
(Sabanadzovic et al., 2009). The cDNA was synthesized with ImProm-II™ Reverse
Transcriptase (Promega, Madison, Wl) in an RT step followed by DNA amplification in
PCR with Taq DNA Polymerase (New England Biolabs, Ipswich, MA), according to the
manufacturer’s instructions. Reverse transcription (RT) was carried out as follows: a
total volume of 10 µl containing 1 µM of virus-specific primers STV-F and STV-R and
RNA with an initial concentration of 100 ng was denaturated at 95°C for 5 min;
immediately after, 10 µl of mastermix containing a final concentration of 6 mM of MgCl2,
1 mM dNTPs, 20 U Rnasin and 1 U of reverse transcriptase was added and incubated
at 25oC for 5 minutes, 42oC for 1 hour and 70oC for 15 minutes. Subsequently the PCR
was done using 2 µl of cDNA in total of 20 µl of reaction mixture containing 10X
Standard Taq (Mg-free) Reaction Buffer, 2.5 mM MgCl2, 0.25 mM of dNTPs, 0.5 µM of
STV-F and STV-R primer, 0.25 mM of Spermidine and 0.625 U of Taq DNA
Polymerase. The thermocycler program was similar to Sabanadzovic et al. (2009) with
minor modifications. After the initial denaturation step at 95°C for 2 min, the cDNAs
were amplified for 35 cycles as follows: denaturation for 30s at 95°C, annealing for 30 s
at 61°C (annealing temperature modified based on preliminary results from gradient RT-
PCR), extension for 45 s at 72°C and a final extension for 10 min at 72°C. PCR
products were run by gel electrophoresis using 1X TAE buffer ethidium bromide-stained
39
1.5% agarose gel to determine whether the PCR amplicons had the correct size, and
consequently whether the plant was infected with STV. The PCR product using the
STV-specific primers has an expected size of 440 bp.
Cloning, Sequencing and Bioinformatic Analysis
The PCR products amplified by RT-PCR were purified using QIAquick PCR
Purification Kit (Qiagen, Valencia, CA) and cloned in pGEM-T Easy vector (Promega
Corp., Madison, WI). Escherichia coli JM109 cells (Promega, Madison, WI) were
transformed with the ligated products.
Plasmids were extracted with NucleoSpin® plasmid extraction kit (Macherey-
Nagel, Bethlehem, PA), and in order to confirm the presence of the amplification
product, plasmids were digested overnight with restriction endonuclease, EcoRI (New
England Biolabs, Ipswich, MA), and results were visualized in 1.5 % agarose gels to
determine if the plasmid contained the proper insert. One clone containing insert from
each cultivar was selected and sent to Eurofins Genomics (Huntsville, AL) for
sequencing using T7/SP6 primer pair to confirm the identity and to measure diversity.
Nucleotide BLAST was used to search for similar sequences in the GenBank
database. STV sequence alignment was conducted using ClustalW and MUSCLE.
Results and Discussion
Presence of STV among Cultivars
The data presented in Table 2-1 indicate that STV is distributed in both hybrid
and open pollinated tomato seeds. Twenty-five cultivars from companies and
distributors were tested for STV (Table 2-1). In total, 98 tomato seeds out of 571 tested
were shown to be virus-infected (17%). STV was found in the following hybrid cultivars:
‘Agriset 761’, ‘Bonita’, ‘Celebrity’ (Parkseed), and ‘Sweet Hearts.’ STV was detected in
40
both hybrid and open-pollinated cultivars. STV was detected for the first time in ‘Agriset
761’, ‘Bonita’, ‘Florida Lanai’, ‘Mexico Midget’ and ‘Roma’.
Although STV was reported to be a vertically transmitted virus (Sabanadzovic et
al., 2009) and was detected in the following hybrid cultivars including ‘Better Boy’,
‘Celebrity’, ‘Loica’, ‘Sweet Hearts’, ‘Trust’, and ‘UC-82’, in this study the virus was also
found in two additional hybrids, ‘Agriset 761’ and ‘Bonita’.
STV, which has been previously found in hybrid cultivars (Sabanadzovic et al.,
2009), had not previously been reported in open pollinated cultivars. We tested 11 open
pollinated cultivars and detected STV in three: ‘Florida Lanai’, ‘Mexico Midget’, and
‘Roma.’ Therefore, this is the first report of STV in open-pollinated cultivars.
The incidence of infection varied considerably from less than 6.7 to 90% (Table
2-1). In all hybrid cultivars tested, the highest incidence of seed transmission of STV
was found in cv. ‘Sweet Hearts’ (61.3%). In the open-pollinated cultivars, cv. ‘Florida
Lanai’ had the highest incidence of STV infection at 90%.
There appears to be wide variation in the presence of STV in at least one of the
hybrid varieties. In ‘Celebrity’, it was interesting to note that STV was found in 45% of
the ‘Celebrity’ seed from one distributor, but not detected in the other, while
Sabanadzovic et al. (2009) found it in 100% of the seeds sampled. However, they only
tested 5 seeds in that study and they did not report the exact seed source. On the other
hand, approximately 60% of ‘Sweet Hearts’ seed in this study and in that of
Sabanadzovic et al. (2009) were infected.
Distribution of STV at Different Leaf Ages in ‘Sweet Hearts’ Tomato Plants
In this experiment, leaf tissue was sampled at the various stages of growth from
‘Sweet Hearts’ tomato plants beginning at the hypocotyl/cotyledon stage and continuing
41
through leaf number 15. For the hypocotyl/cotyledon samples and leaf 1 samples, the
plants were destructively sampled; however, for leaves 4, 7, 12 and 15, the same plants
were used for each leaf age. STV was initially detected in 75% of the
hypocotyl/cotyledon samples (Table 2-2). STV was also detected in 50-60% of the
samples from leaves 1, 4, 7, 12, and 15. For leaf samples 4, 7, 12 and 15, the same
plants were always positive or always negative. This is the first report showing the
presence of STV in all tomato leaves.
In a separate experiment, to address objective 2, plants were grown to the 16th
leaf stage and then leaf tissue was sampled simultaneously from five known positive
‘Sweet Hearts’ plants at leaf ages 3, 6, 9, 12, and 15. STV was detected in all leaves
sampled from each of the 5 plants (Table 2-3). An STV negative plant was used as a
negative control and always tested negative. The virus was shown to be present in all
stages that were sampled. This distribution throughout the plant is similar to what has
previously been reported for other cryptic viruses such as in the Partitiviridae (Boccardo
et al., 1987).
Diversity of STV
Amplicons generated by RT-PCR were readily TA cloned and sequenced
directly. Partial sequences from representative isolates: ‘Agriset 761’, ‘Mexico Midget’,
‘Roma’ and ‘Sweet Hearts’ were submitted to GenBank and the accession numbers as
follows: KX949570 (STV-Agr761-11-17), KX949571 (STV-MM-2-6), KX949572 (STV-R-
11-6), and KX949573.1 (STV-SH-17m).
Amplicons of the 440 bp from four hybrid tomato cultivars, ‘Agriset 761’, ‘Bonita’,
‘Celebrity’, ‘Sweet Hearts’ and three open pollinated tomato cultivars ‘Florida Lanai’,
‘Mexico Midget’, and ‘Roma’, were compared with eight STV complete genome
42
sequences in GenBank, from Bangladesh (KT634055.1), Canary Islands and Spain
(KJ174690.1), China (KT438549.1), Florida (KX949574.1), Korea (LC270272.1),
Mississippi (EU413670.1), North Carolina (KT852573.1), and the reference sequence
from Mexico (NC_011591.1), were compared (Table 2-4). Comparison of the
sequences with those in GenBank showed that all the sequences analyzed and
described above displayed similarity greater than 98.75% (Table 2-5), corroborated with
previous studies that described very low genetic variability among STV isolates (Iacono
et al., 2015). A total of nine isolates from three partial sequences: ‘Agriset 761’, ‘Roma’,
‘Florida Lanai’, and six full genome STV sequences from Bangladesh, China, Florida,
Korea, Mexico, and Mississippi were 100% identical. The results revealed a total of four
nucleotide changes for ‘Celebrity’, one in ‘Bonita’, one in ‘Sweet Hearts’ and three in
STV-MM-2-6 (as reported by Alcala-Briseno et al., 2017) and two in China (as also
reported by Padmanabhan et al., 2015b) resulting in an overall similarity greater than
99% so diversity of STV among different isolates seems to be low.
43
Figure 2-1. Hypocotyl plus cotyledon (1 to 2 weeks post germination) tested for the presence of STV (photo courtesy of Sevgi Coskan).
Figure 2-2. Leaf collected for testing for STV from a tomato plant with 12 leaves (7
weeks post planting) (photo courtesy of Sevgi Coskan).
44
Figure 2-3. Leaves collected from one plant for testing for STV. Age of leaf is shown
from oldest (top left to youngest bottom right) (photo courtesy of Sevgi Coskan).
45
Table 2-1. List of tomato cultivars tested for the presence of STV by RT-PCR
Cultivars Type Shape Company
No. Plants Infection Rate (%)
Tested
(Infected/total)
A Grappoli D'inverno OP grape Baker Creek 0/20 0
Agriset 761 H round Petoseed 11/21 52.4
Better Boy H round Seminis 0/20 0
Better Boy H round ParkSeed 0/20 0
Bonita H round Rogers 10/20 50
Brandywine Red OP beefsteak Burpee 0/20 0
Celebrity H deep oblate Seminis 0/17 0
Celebrity H deep oblate Parkseed 9/20 45
Enpower H rootstock Nunhem 0/30 0
Florida 47 H round Seedway 0/20 0
Florida Lanai OP round UF Agricultural exp station 18/20 90
Heinz OP plum UC Davis germplasm collection
0/20 0
Large Red Cherry OP cherry Seed Savers Exchange 0/20 0
Mexico Midget OP cherry Seed Savers Exchange 1/15 6.7
Multifort H rootstock DeRuiter 0/30 0
Old Italiano OP beefsteak Baker Creek 0/20 0
Principe Borghese OP plum Seed Savers Exchange 0/20 0
Roma OP plum Burpee 10/20 50
Rutgers OP round Burpee 0/27 0
Super Beefsteak OP beefsteak Burpee 0/12 0
Sweet Chelsea H cherry Sakata 0/20 0
Sweet Elite H grape Sakata 0/22 0
Sweet Hearts H grape Sakata 38/62 61.3
46
Table 2-2. Detection of STV in hypocotyl/cotyledon tissue and in developing leaves at different leaf ages of ‘Sweet Hearts’ tomato plants
Plant stages
No. Plants STV-Infected*/Total
Tested Detection Rate (%)
Hypocotyl plus cotyledon
9/12 75
Leaf 1 5/10 50
Leaf 4 6/10 60
Leaf 7 6/10 60
Leaf 12 6/10 60
Leaf 15 6/10 60 *Positive for STV based RT-PCR. Table 2-3. Detection of STV in leaves of five STV-infected plants when sampled
simultaneously once the plants reached the 16th leaf stage (An STV-negative plant was used as a control for all leaves)
Leaf No. No. Plants Infected/Total
Tested
3 5/5
6 5/5
9 5/5
12 5/5
15 5/5
47
Table 2-4. List of STV isolates used in this study
Isolate Cultivar Location Sequence Accession Source
Agr761-11-17 Agriset 761 Florida Partial KX949570.1 In this study; Alcala-Briseno et al., 2017
Bo-239 Bonita Florida Partial N/A In this study
Ce-302 Celebrity Florida Partial N/A In this study
La-164 Florida Lanai Florida Partial N/A In this study
MM-2-6 Mexico Midget Florida Partial KX949571.1 In this study; Alcala-Briseno et al., 2017
R-11-6 Roma Florida Partial KX949572,1 In this study; Alcala-Briseno et al., 2017
SH-17m Sweet Hearts Florida Partial KX949573.1 In this study; Alcala-Briseno et al., 2017
Florida Sweet Hearts Florida Full genome KX949574.1 Alcala-Briseno et al., 2017
CN-12 not specified China Full genome KT438549.1 Padmanabhan et al., 2015b
GCN06 Mariana Canary I., Spain Full genome KJ174690.1 Verbeek et al., 2015
Gimcheon not specified Korea Full genome LC270272.1 NCBI GenBank Database
BD-13 not specified Bangladesh Full genome KT634055.1 Padmanabhan et al., 2015a
MT-1 not specified Mexico Full genome EF442780.1 Sabanadzovic et al., 2009
MS-7 not specified Mississippi Full genome EU413670.1 Sabanadzovic et al., 2009
NC12-03-08 not specified North Carolina Full genome KT852573.1 NCBI GenBank Database
48
Table 2-5. Pairwise comparison of percent identity nucleotide sequences of STV
*Sequences submitted to GenBank: Agriset 761 (KX949570), Mexico Midget (KX949571), and Roma (KX949572). **Sequences obtained from GenBank: Bangladesh (KT634055.1), Canary I and Spain (KJ174690.1), China (KT438549.1), Florida (KX949574.1), Korea (LC270272.1), Mississippi (EU413670.1), North Carolina (KT852573.1), and Mexico (EF442780.1). ***Sequences will be submitted to GenBank.
49
CHAPTER 3 VERTICAL TRANSMISSION OF STV
Introduction
Plant viruses cause significant problems in vegetable crops, being responsible
for reduction in seed germination when seed transmissible, causing significant crop
yield losses, and causing dramatic changes in shape and color of seeds (Gumus and
Paylan, 2013). Knowing how viruses are disseminated has a significant role in planning
proper disease management strategies. Generally, viruses are transmitted by two
common mechanisms: 1) when the virus is transmitted from parent to offspring (vertical
transmission), or 2) when the virus is transmitted among individuals of the same
generation (horizontal transmission) (Sastry, 2013; Simmons et al., 2011).
Approximately 20% of plant viruses are reported to be transmitted by seed
(Johansen et al., 1994, Sastry 2013). Viruses are obligate parasites that are incapable
of penetrating the cuticle and plant cell wall directly or directly entering the plasma
membrane (Hull, 2009). In the case of seed transmission, it has been reported that
developing embryos are largely, but not completely, protected from infection in
maternal-infected plant tissue (Bennett, 1969). Some viruses have developed strategies
to overcome the latter limitation by infecting embryos and therefore being transmitted to
the next generation through infected seeds (Bashir and Hampton, 1996).
Seed transmission plays a critical role in vertical transmission (Simmons et al.,
2011) and in viral dissemination (Yang et al., 1997). Pollen can also be an important
means of distributing plant viruses over great distances (Bailiss and Offei, 1990). When
the viruses are pollen-transmissible, the flower becomes infected during pollination
(horizontal transmission) and the seed may become infected and the resulting seedlings
50
that develop may be infected (vertical transmission). However, the virus when present in
the pollen may not be able to infect the embryo or may cause sterility in the ovule, and
thus not be transmitted vertically (Card et al., 2007). Even though it is known that there
is an intimate relationship between seed and pollen transmission, it cannot be assumed
if the pollen transmits the virus vertically, that there will always be horizontal
transmission or vice versa (Sastry, 2013). It has also been reported that pollen-
transmissible viruses are commonly seed-transmissible (Card et al., 2007).
Movement of seeds worldwide by commercial seed companies is a common
practice. Seeds are potential inoculum sources for plant viruses. For this reason the use
of clean seed is an important strategy to control diseases (Kuan, 1988). Virus-free
seeds are used as a prevention strategy for preventing introduction into areas where
specific diseases do not occur (Kuan, 1988).
Seed-transmitted viruses that are inefficiently transmitted may have significant
consequences when introduced into a new area if a vector is present that efficiently
transmits the virus resulting in devastation of the crop (Amari et al., 2009). Both local
and long distance movement of a seed transmitted virus rely on the rate of
transmission. Some viruses have low rates of seed transmission and without the vector
being present in production sites they are not considered as a concern for long distance
spread.
Knowing that a virus is transmitted via seed is crucial for developing plant
protection strategies and predicting virus distribution (Kil et al., 2016). If there is a low
incidence of seed infection, it is important to know what potential vectors are present in
51
the production fields where the seed are used such that proper management strategies
are implemented to control the vector and minimize the disease (Atsumi et al., 2015).
Virus transmission can occur in mainly two ways: infection of the embryo, and
infection of the seed coat or maternally derived seed parts (Sastry, 2013). Embryo
becomes infected either directly, when the virus present in the mother plant invades the
developing embryo during embryogenesis, or indirectly, when the virus infects
reproductive tissues before embryogenesis (Wang and Maule, 1997; Maule and Wang,
1996).
Sabanadzovic et al. (2009) determined STV is neither graft-transmitted nor
mechanically transmitted but is seed transmitted at high rates. Dissemination of STV
relies on the movement of seed and propagating material. Global seed trades and
propagative material distribution facilitate the spread of the virus. Seed transmission is a
typical characteristic of a persistent virus lifestyle as defined by Roossinck (2010). Plant
viruses that were classified by Roossinck (2010) as having a persistent lifestyle are
characterized by lacking movement proteins and therefore STV would be considered to
have a persistent lifestyle as it lacks a movement protein (Sabanadzovic et al., 2009).
Also characteristic of the persistent virus lifestyle described by Roossinck (2010) and
demonstrated by STV is vertical but not horizontal transmission (Sabanadzovic et al.,
2009).
A large number of viruses, including cryptic viruses (Partitiviridae), are reported
to be seed-transmissible, but information on the seed infection mechanism of these
viruses is limited and mostly inconclusive (Sastry, 2013). Cryptic viruses, currently
52
classified as part of the family Partitiviridae, are the most well studied group with shared
characteristics with amalgaviruses.
STV, the type species of the Amalgavirus (family Amalgaviridae), has a double
stranded RNA genome of approximately 3.5 kb and two partially ORFs. Like other
amalgaviruses, STV is not transmitted by mechanical inoculation and by grafting, but it,
like another member, BBLV, are the only members of the Amalgaviridae that have been
reported to be transmitted through seed (Martin et al., 2011; Sabanadzovic et al., 2009).
It is not clear how the virus moves from the parent to the embryonic tissues (Wang and
Maule, 1994). Although STV is a seed transmitted virus with a high rate of seed
infection (Sabanadzovic et al., 2009), it is not known what role pollen plays in vertical
transmission of STV from male to female plants.
Until now, like other members of Amalgaviridae, there have been no studies to
determine the role of pollen in seed transmission of STV. For a better understanding on
the seed transmission of STV, this study aimed to determine if both parents contribute
to seed transmission of STV to the next generation. We determined the role of pollen
from infected plants in seed infection when pollinating flowers of healthy and infected
female plants.
Materials and Methods
Cross Pollination
Viral transmission was evaluated using four combinations of STV-infected and
STV-negative female and male parents of the tomato cultivar ‘Sweet Hearts’. Plants
used in this experiment were grown from seed in the greenhouse. Before the pollination
process, plants were tested by RT-PCR to verify if they were infected with the virus or
were virus-free plants. In treatment 1, four female positive plants were pollinated using
53
pollen from three male plants negative for the virus. In treatment 2, four healthy female
plants were pollinated using pollen from three male plants infected with STV. In
treatment 3, four STV-infected female plants were pollinated using pollen from three
STV-infected male plants. In treatment 4, four STV-negative female plants were
pollinated using pollen from three STV-negative male plants. For each cross, the
flowers from female parents were emasculated when they were not open but just about
to show yellow (Figure 3-1) Using tweezers, the flower petals were removed (Figure 3-
2) without touching the stigma. Flowers were cross pollinated with fresh pollen by first
selecting flowers that were open as shown in Figure 3-3 then removing the flower from
the plant and gently tapping the flower to release the pollen onto fingertip. The pollen on
the fingertip was gently transferred to the stigma. Each crossed female flower was
labeled to identify them at harvest (Figure 3-4). When pollination was successful, fruit
will began to develop (Figure 3-5).
Seed Harvesting and Disinfection Treatments
When the fruit from cross-pollinated treatments had ripened (Figure 3-6), tissue
was collected as follows: each tomato fruit was cut in half and all the seeds in each fruit
(seeds from each fruit were kept separate) were transferred to a small beaker. To
disinfest, seeds were covered with 1M HCI (hydrochloric acid) and incubated for 30 min.
The HCI of the beaker was decanted and the seed were rinsed in tap water to remove
HCl solution and pulp was removed so that only seed and minimal pulp remained. Then
the seed were placed back into a beaker, immersed in TSP (tri-sodium phosphate, 400g
TSP in 3.78 L water) solution for 30 min and rinsed in tap water. Following the rinsing
step in tap water, the seeds were spread out on a several layers of paper towels and
54
allowed to dry at room temperature overnight. The seed from each fruit were then
placed in small envelopes and stored at room temperature.
Seed Transmission
Seeds from the treatments described above were germinated by placing at least
5 seed in a plastic bag on moist paper towels and then the bags (Figure 3-7) were
placed in an incubator at 25-27°C for 1 to 2 weeks until the weight of each seedling was
at least 13 mg (Figure 3-8). Hypocotyl and cotyledon tissue was collected (seed coat
was removed) and 60 seedlings from crosses for each treatment were tested
individually for STV by RT-PCR using STV specific primers (Sabanadzovic et al., 2009).
Total RNA extraction
Five seeds per cross (60 seeds per treatment) were germinated in the incubator
at 25-27°C for 1-2 weeks. Between 18-30 mg weight of germinated seedlings were
removed from the seed coat by tweezers then RNA was extracted using OmniBiotek
Plant RNA mini kit (OmniBiotek Co. MA) according to the manufacturer’s instructions.
Detection of STV
Two primers, STV-F (5’-CGTTATCTTAGGCGTCAGCT-3’) and STV-R (5’-
GGAGTTTGATTGCATCAGCG- 3’), which amplify a 440 bp region that encompasses
the overlapping region between ORF 1 and ORF 2 were used (Sabanadzovic et al.,
2009). RT step was carried out as follows: a total volume of 10 µl containing 1 µM of
virus-specific primers STV-F and STV-R and RNA with initial concentration of 100 ng
were denaturated at 95°C for 5 min, followed by reverse transcription in which 10 µl of
mastermix containing in a final concentration of 6 mM of MgCl2, 1 mM dNTPs, 20 U
Rnasin, and 1 U of reverse transcriptase ImProm-II™ Reverse Transcriptase. Two
microliters of cDNA were tested for the presence of STV by PCR (Sabanadzovic et al.,
55
2009). The PCR was done using 10X Standard Taq (Mg-free) Reaction Buffer, 2.5 mM
MgCl2, 0.25 mM of dNTPs, 0.5 µM of STV-F and STV-R primer, 0.25 mM spermidine
and 0.615 U of Taq Polymerase (New England Biolabs, Ipswich, MA). The thermocycler
program was similar to Sabanadzovic et al. (2009) with minor modifications. After an
initial denaturation step at 95°C for 2 min, the cDNAs were amplified as follows: 35
cycles included denaturation for 30 s at 95°C, annealing for 30 s at 61°C (annealing
temperature modified based on preliminary results from gradient RT-PCR), and
extension for 45 s at 72°C followed by a final extension for 10 min at 72°C. PCR
products were run by gel electrophoresis using 1X TAE buffer ethidium bromide-stained
1.5% agarose gel to determine if the PCR product had the correct size, and
consequently if the plant was infected with STV. The PCR product using the STV-
specific primers has an expected size of 440 bp.
Results
In order to determine the role of each parent in seed transmission of STV, a total
of 240 seeds (60 seed per treatment) representing the four treatments (Tables 3-1, 3-2,
3-3, and 3-4) were examined for the presence of STV. When the female parent was
STV-infected and the pollen was from a healthy parent, 70% of the progeny plants were
infected (Table 3-1). When the female parent was healthy and the pollen was from an
infected plant, 61.7% of progeny plants were infected, showing that STV can be
transmitted by pollen (Table 3-2). When both the female parent and male parent were
STV-infected, 100% of the progeny plants were infected (Table 3-3). As a negative
control where female parent and male parent were STV negative, 100% of the offspring
were STV-negative (Table 3-4).
56
Table 3-5 is the summary of the Table 3-1 through 3-4. The results clearly show
that STV was transmitted by both ovule and pollen to the progeny. It also appears that
the female parent had a higher contribution to virus transmission than the male parent.
Discussion
In this study, STV was shown to be transmitted via both the female and the male
parent. Although there are other reports addressing seed transmission in
Amalgaviridae, they have not reported the role of the male and female in transmission
(Sabanadzovic et al., 2009; Martin et al., 2011). Sabanadzovic et al. (2009) reported
high rates (70-90%) of vertical transmission for STV, but they did not report the parents’
contribution for the virus transmission since their source of seeds were tomato hybrid
seed and it was unknown which parent contributed STV. Similarly, Martin et al. (2011)
tested seedlings obtained from crosses in which both parents were infected with BBLV,
and therefore could not determine the parental contribution in seed transmission. In this
study, we demonstrated with infected female and male parents that seed transmission
for STV occurs at high rates, corroborating the previous report by Sabanadzovic et al.
(2009). We also showed that the female parent had a slightly greater contribution for the
STV transmission when compared to the male parent, although this may not be
statistically significant.
Clearly, based on these results, STV could be transmitted at high rates to the
next generation by either the ovule being infected in the female plant or being pollinated
by an infected male plant. In another study, STV was reported to be transmitted by seed
at high rates (70-90%), although they did not state transmission was from the female or
male parent (Sabanadzovic et al. 2009). However, this study for the first time
57
demonstrates pollen transmission (not horizontally) by a virus in the Amalgaviridae
family.
STV transmission through pollen was shown to occur at a high rate when the
female plant was free of the virus. Transmission of STV via pollen (61.7%) was more
efficient when compared to previous reports of cryptic virus transmission in two other
plant species (e.g. 50% in Vicia faba (Kenten et al., 1978), 43% in beet (Kassanis et al.,
1978), but less efficient compared to Endornavirus transmission (94%) (Moriyama et al.,
1996). Clearly pollen as well as ovule transmission seems to be a crucial means for
distribution of these persistent viruses.
Vertical transmission of persistent viruses when the female plant is infected is
quite common. As stated above, STV was more efficiently transmitted vertically when
the female parent was infected compared to the pollen parent. In the Partitiviridae,
Kassanis et al. (1978) reported a high rate of vertical transmission, 82%, when the
female BCV-infected parent and BCV-negative male parent were crossed, compared to
when the pollen parent was the source of the virus. For the Endornavirus (Moriyama et
al., 1996), 100% of rice seedlings were infected when the female parent was crossed
with pollen from a healthy plant. For persistent viruses, the probability for vertical
transmission is quite high when the female parent is infected.
One of the more interesting findings in this study was that the rate of
transmission of STV was considerably higher when both parents were infected. In that
case 100% of the progeny were infected. A similar situation occurred with BBLV in
which when both parents were infected, all progeny were positive for the virus (Martin et
58
al., 2011). Further investigations are necessary to identify the seed infection
mechanism.
59
Table 3-1. Seed transmission of STV following cross-pollination of STV-infected mother plants with pollen collected from STV-negative tomato plants
Treatment 1 Female STV- infected parent ID
Male STV-negative parent ID
No. of seeds Infected/Total
Infection Rate
42a 6-1b 3/5 60%
42 8-4 5/5 100%
42 9-3 2/5 40%
44 6-1 5/5 100%
44 8-4 2/5 40%
44 9-3 4/5 80%
47 6-1 3/5 60%
47 8-4 3/5 60%
47 9-3 4/5 80%
50 6-1 4/5 80%
50 8-4 5/5 100%
50 9-3 2/5 40%
Total - 42/60 -
Average - - 70% aPlants 42, 44, 47 and 50 were STV positive and were used for all crosses as STV positive female parents. bPlants 6-1, 8-4, and 9-3 were STV negative and were used for crosses as STV negative male parents.
60
Table 3-2. Seed transmission of STV following cross-pollination of STV-negative mother plants with pollen collected from STV-infected tomato plants
Treatment 2 Female STV- infected parent ID
Male STV-negative parent ID
No. of seeds Infected/Total
Infection Rate
6-1a 42b 2/5 40%
6-1 44 4/5 80%
6-1 48 5/5 100%
8-4 42 3/5 60%
8-4 44 2/5 40%
8-4 48 3/5 60%
9-1 42 3/5 60%
9-1 44 4/5 80%
9-1 48 5/5 100%
26-1 42 2/5 40%
26-1 44 1/5 20%
26-1 48 3/5 60%
Total - 37/60 -
Average - - 61.7% aPlants 6-1, 8-4, and 9-3 were STV negative and were used for crosses as STV negative female parents. bPlants 42, 44, 47 and 50 were STV positive and were used for all crosses as STV positive male parents.
61
Table 3-3. Seed transmission of STV following cross-pollination of STV-infected mother plants with pollen collected from STV-infected tomato plants
Treatment 3 Female STV-infected parent ID
Male STV-infected parent ID
No. of seeds Infected/Total
Infection Rate
42a 44b 5/5 100%
42 47 5/5 100%
42 50 5/5 100%
44 42 5/5 100%
44 47 5/5 100%
44 50 5/5 100%
47 42 5/5 100%
47 44 5/5 100%
47 50 5/5 100%
50 42 5/5 100%
50 44 5/5 100%
50 47 5/5 100%
Total - 60/60 -
Average - - 100% a bPlants 42, 44, 47 and 50 were STV positive and were used for all crosses as STV positive female parents and STV positive male parents.
62
Table 3-4. Seed transmission of STV following cross-pollination of STV-negative mother plants with pollen collected from STV-negative tomato plants
Treatment 4 Female STV- infected parent ID
Male STV-negative parent ID
No. of seeds infected/Total
Infection Rate
6-1a 8-4b 0/5 0%
6-1 9-1 0/5 0%
6-1 26-1 0/5 0%
8-1 6-1 0/5 0%
8-1 9-3 0/5 0%
8-1 26-1 0/5 0%
9-1 6-1 0/5 0%
9-1 8-4 0/5 0%
9-1 26-1 0/5 0%
26-1 6-1 0/5 0%
26-1 8-4 0/5 0%
26-1 9-1 0/5 0%
Total - 0/60 -
Average - - 0% abPlants 42, 44, 47 and 50 were STV-negative and were used for all crosses as STV positive female parents and STV-negative male parents. Table 3-5. Virus infection in the progeny seedlings of crosses between STV-infected
and STV-negative ‘Sweet Hearts’ parent plants
Parent Number of seedlings tested % of infected plants Female Male STV-infected STV-negative
STV-infected STV-negative 42 18 70
STV-negative STV-infected 37 23 61.7
STV-infected STV-infected 60 0 100
STV-negative STV-negative 0 60 0
63
Figure 3-1. Selected female flower (photo courtesy of Sevgi Coskan).
Figure 3-2. Female flower’s pistil from flower that had been emasculated (before cross-
pollination) (photo courtesy of Sevgi Coskan).
64
Figure 3-3. Selected male flowers as a source of pollen (photo courtesy of Sevgi Coskan).
Figure 3-4. Female flower following transfer of pollen to pistil (photo courtesy of Sevgi Coskan).
65
Figure 3-5. Developing fruit, two-weeks post pollination (photo courtesy of Sevgi
Coskan).
Figure 3-6. Cross-pollinated fruit ready for harvesting (photo courtesy of Sevgi Coskan).
66
Figure 3-7. Germination of seeds in plastic bag on moist paper towel (photo courtesy of
Sevgi Coskan).
Figure 3-8. Germinated seedlings1 to 2 weeks after incubation in plastic bag on moist
paper towel (photo courtesy of Sevgi Coskan).
67
CHAPTER 4 SUMMARY
STV is a recently described virus of tomato that is the type species of
Amalgavirus belonging to Amalgaviridae with a monopartite, small, approximately 3.5 kb
dsRNA genome that has two overlapping open reading frames (ORFs). The first ORF
encodes for putative coat protein (p42), and the second ORF codes for RdRp (p121)
that possesses a + 1 ribosomal frameshift. First described in tomato in U.S. and Mexico
in 2005, STV has more recently been reported in China, Bangladesh, France, Italy, the
Canary Islands and Spain and most recently in Florida and Korea. STV was first
reported to be associated with tomato yellow stunt disease, which is characterized by
leaf chlorosis, reduction of fruit size, and reduced crop yields; however, the virus is
frequently detected in mixed infections with other viruses, and has also been observed
to be asymptomatic in several cultivars. RT-PCR assay was performed for the detection
of STV in total RNA extracts from STV-infected tomato plants by using virus-specific
primers. PCR products were visualized by gel electrophoresis. STV was detected in
different tomato cultivars that were selected for diversity of genetic background and
commercial source. The virus was successfully detected in the following cultivars:
‘Agriset 761’, ‘Bonita’, ‘Celebrity’, ‘Florida Lanai’, ‘Mexico Midget’, ‘Roma’ and ‘Sweet
Hearts’ and similar frequency of virus was observed between open-pollinated and
hybrid cultivars.
In order to examine the diversity of partial STV isolates, RT-PCR amplification
products for each positive cultivar were evaluated following TA cloning. Amplicon
products of 440 bp cloned from four hybrid tomato cultivars, and three open pollinated
tomato cultivars, were compared with eight STV complete genome sequences in
68
GenBank using ClustalW and MUSCLE alignments. Isolates from ‘Agriset 761’, ‘Roma’,
‘Florida Lanai’ and six full genome STV sequences from Bangladesh, China, Florida,
Korea, Mexico, and Mississippi from GenBank were 100% identical. Four of the isolates
from this study had the slight variation that give the similarity ranged from 98.75 to
99.8% with the average percent similarity greater than 99%. So diversity of STV among
different isolates is low.
In order to determine distribution of STV in leaves at different plant growth
stages, plants were tested by RT-PCR at different stages of development: at the1‐2
week post germination stage and the youngest leaf from plants with 1st, and at 4th, 7th,
12th and 15th leaves. STV was detected in 75% of 1 to 2 week-old germinated seedlings.
Based on this study, STV can be detected as early as on the hypocotyl/cotyledon stage,
and the virus appears to be present at detectable levels in all leaves of a plant. This is
the first report showing the presence of STV in all tomato leaves on an individual plant.
Seed transmission studies revealed that STV could be transmitted to the next
generation by either ovule or pollen and at relatively high rates and these numbers
suggest that the female parent had a slightly greater effect on the transmission rate than
the male parent. Although suspected, this is the first demonstration of pollen
transmission by a virus in the Amalgaviridae.
69
APPENDIX CLUSTAL MULTIPLE SEQUENCE ALIGNMENT of 400 BP SEQUENCE AMPLIFIED
WITH STV-SPECIFIC PRIMERS BY MUSCLE (3.8) (FIRST SEVEN ISOLATES WERE FROM THIS STUDY)
70
71
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BIOGRAPHICAL SKETCH
Sevgi Coskan was born in Izmir, in western coast of Turkey. She is the eldest of
two children of her family. She received her bachelor’s degree in subprogram of plant
protection in the Agricultural Engineering Faculty of Agriculture - University of Adnan
Menderes, Aydin, Turkey in 2010. In 2011, she had an opportunity at Aegean Exporters
Associations to work on a pilot program that looked for agricultural practices,
knowledge, and technology. The program was a joint partnership between government,
private business, and Ege University. In 2012, the Republic of Turkey Ministry of Food,
Agriculture, and Livestock awarded her a full scholarship to study English, and also to
pursue a course of study at an institution leading to a master’s degree in plant pathology
with a minor in plant viruses in the United States of America. In 2013, she attended a
language program at the University of Pennsylvania. In 2015, she was accepted to the
Master of Science program in Plant Pathology Department at University of Florida. She
worked on distribution and vertical transmission of Southern tomato virus in tomato
under the supervision of Dr. Jeffrey B. Jones, Dr. Rosemary Loria, and Dr. Svetlana
Yuryevna Folimonova. Currently she is getting prepared to work in-service for General
Directorate of Agricultural Research and Policies of the Turkish Ministry of Food,
Agriculture, and Livestock as a plant virologist. She is also interested in pursuing a
doctoral degree that enables her to continue with her focus on plant virology. She
received her master’s from University of Florida in August 2017.