CHARACTERIZATION OF TWO BEGOMOVIRUSES ISOLATED FROM Sida
santaremensis Monteiro AND Sida acuta Burm. f
By
HAMED ADNAN AL-AQEEL
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
2003
Copyright 2003
by
Hamed Adnan Al-Aqeel
This dedicated to my family my father Dr. Adnan, my mother Fareda and my wife Hanin.
TABLE OF CONTENTS page LIST OF TABLES............................................................................................................. vi
LIST OF FIGURES .......................................................................................................... vii
ABSTRACT....................................................................................................................... ix
CHAPTER 1 HISTORY AND LITERATURE REVIEW .................................................................1
Geminivirus History .....................................................................................................1 Taxonomy and Nucleotide Functions...........................................................................3 Begomoviruses .............................................................................................................5 The Genus Sida.............................................................................................................6 Viruses Infecting Sida spp. ...........................................................................................7 Begomoviruses Infecting Sida spp. in Florida............................................................10
2 CHARACTERIZATION OF A NEW BEGOMOVIRUS ISOLATED FROM Sida
santaremensis Monteiro in Florida.............................................................................12
Materials and Methods ...............................................................................................13 Virus Source ........................................................................................................13 Begomovirus Detection .......................................................................................13 Cloning and Sequencing......................................................................................14 Molecular Characterization of the Virus .............................................................15 Biological characterization..................................................................................15
Biolistic inoculation .....................................................................................16 Whitefly inoculation.....................................................................................16
Detection of SiGMoV in Test Plants...................................................................17 Results.........................................................................................................................18
Phylogenetic Analysis .........................................................................................18 Nucleotide and Amino Acid Sequence Analysis.................................................19
Biological Characterization ........................................................................................19 Discussion...................................................................................................................27
iv
3 AN EPIDEMIC IN TOMATO CAUSED BY VARIANTS OF Sida golden mosaic virus ............................................................................................................................29
Materials and Methods ...............................................................................................29 Sample Source .....................................................................................................29 PCR Analysis and Restriction Analysis ..............................................................29 Cloning ................................................................................................................30 Gap and Blast Analysis .......................................................................................30 Phylogenetic Analysis .........................................................................................30
Results.........................................................................................................................31 Partial Sequence Analysis from Tomato and S. acuta ........................................31 Phylogenetic Analysis .........................................................................................33
Discussion...................................................................................................................44 LIST OF REFERENCES...................................................................................................46
BIOGRAPHICAL SKETCH .............................................................................................51
v
LIST OF TABLES
Table page 2-1 Comparison of the nucleotide sequence identity of the DNA-A of Sida golden
mottle virus ...............................................................................................................21
2-2 Comparison of the nucleotide sequence identity of the DNA-B of Sida golden mottle virus ...............................................................................................................21
2-3 Comparison of the open reading frame nucleotide and common region sequences identity of the DNA-A of Sida golden mottle virus .................................................22
2-4 Comparison of the open reading frame and common region nucleotide sequences identity of the DNA-B of Sida golden mottle virus .................................................22
2-5 Comparison of the open reading frame amino acid sequences similirity of the DNA-A of Sida golden mottle virus.........................................................................23
2-6 Comparison of the open reading frame amino acid sequences similirity of the DNA-B of Sida golden mottle virus .........................................................................23
2-7 Host range study of SiGMoV...................................................................................24
3-1 The nucleotides identity of partial sequences of SiGMV DNA-A...........................38
3-2 The nucleotides identity of partial sequences of SiGMV DNA-B...........................38
3-3 The Common region nucleotides identity of SiGMV DNA-A sequences isolated from tomato and S. acuta .........................................................................................39
3-4 The Common region nucleotides identity of SiGMV sequences isolated from tomato and S. acuta ..................................................................................................39
3-6 The nucleotide identity of partial sequences DNA-B sequences isolated from tomato and S. acuta ..................................................................................................41
vi
LIST OF FIGURES
Figure page 2-1 Sida santaremensis infected with Sida golden mottle virus showing typical ........20
2-2 Phylogenic tree of complete nucleotide of a component of selected begomoviruses with SiGMoV................................................................................25
2-3 Phylogenic tree of complete nucleotide of B component of selected begomoviruses with SiGMoV................................................................................26
3-1 Partial sequence of DNA-A (S3-C7A) amplified from Sida acuta collected from Citra Field, Florida. .......................................................................................33
3-2 Partial sequence of DNA-A (T3-C8A) amplified from tomato plant collected from Citra Field, Florida ........................................................................................34
3-3 Partial sequence of DNA-A (T5-C2A) amplified from tomato plant collected from Citra Field, Florida ........................................................................................34
3-4 Partial sequence of DNA-A (T10-C8A) amplified from tomato plant collected from Citra Field, Florida ........................................................................................35
3-5 Partial sequence of DNA-A (T10-C10A) amplified from tomato plant collected from Citra Field, Florida ........................................................................................35
3-6 Partial sequence of DNA-A (T12-C6A) amplified from tomato plant collected from Citra Field, Florida ........................................................................................36
3-7 Partial sequence of DNA-B (S3-C4B) amplified from Sida acuta collected from Citra Field, Florida.................................................................................................36
3-8 Partial sequence of DNA-B (T12-C3B) amplified from tomato plant collected from Citra Field, Florida ........................................................................................37
3-9 Partial sequence of DNA-B (T12-C5B) amplified from tomato plant collected from Citra Field, Florida ........................................................................................37
3-10 Partial sequence of DNA-B (T12-C7B) amplified from tomato plant collected from Citra Field, Florida ........................................................................................37
vii
3-11 Partial sequence of DNA-B (T12-C9B) amplified from tomato plant collected from Citra Field, Florida ........................................................................................38
3-12 Phylogenic tree of partial nucleotide sequence of DNA-A of selected begomoviruses with the SiGMV and the SiGMV sequences isolated from tomato and S. acuta................................................................................................42
3-13 Phylogenic tree of partial nucleotide sequences of DNA-B of selected begomoviruses with the SiGMV and the SiGMV sequences isolated from tomato and S. acuta................................................................................................43
viii
Abstract of Thesis Presented to the Graduate School
of the University of Florida in Partial Fulfillment of the Requirements for the Master of Science
Characterization of Two Begomoviruses Isolated from Sida santaremensis Monteiro and Sida acuta Burm. f.
By
Hamed Adnan Al-Aqeel
December 2003
Chair: Jane E. Polston Major Department: Plant Pathology
A new bipartite begomovirus was isolated and characterized from Sida
santaremensis. The proposed name of this new begomovirus is Sida golden mottle virus
(SiGMoV). The SiGMoV DNA-A is not similar to any characterized DNA-A
begomovirus obtained by Blast analysis. However, the SiGMoV DNA-B shows some
similarity with Tomato mottle virus and Abutilon mosaic virus. SiGMoV was able to
infect Lycopersicon esculentum Mill. (FL Lanai), Phaseolus vulgaris L. (Topcrop),
Gossypium hirsutum L. (elta Pine 70), Nicotiana benthamiana (Domin), and N.
tabacum L. (V20) based on biolistic inoculation.
In fall of 2002, an epidemic was observed in a tomato field in Citra, FL. The plants
in this field were 100% infected and showed symptoms of small upwardly-curled leaves
with chlorotic margins, and stunting of the plants, that were nearly identical to those
described for Tomato yellow leaf curl virus. The amplification of 1254-1295 nt fragment
with degenerate primers PAR1c496 /PAL1v1978 and the amplification of 616-639 nt
ix
fragment with degenerate primers, PBL1240/PCRc154, suggests the presence of a
bipartite begomovirus. Analysis of these partial sequences showed that the epidemic was
caused by a strain of Sida golden mosaic virus. Gap and phylogenetic analyses showed
the presence of two diverse DNA-B sequences.
x
CHAPTER 1 HISTORY AND LITERATURE REVIEW
Geminivirus History
Long before geminiviruses were identified to cause plant diseases, the symptoms
caused by these viruses were noted. A poem written by the Empress Koken in Japan in
752 AD, which described the beauty of yellow veins of Eupatorium chinense L. leaves,
may be the earliest record of a geminivirus [28]. For many years, plants with
geminivirus-incited symptoms of yellow leaf veins and bright golden mosaics were
selected and cultured long before the cause of these symptoms was known. There is a
record in 1809 of the collection and movement (from the West Indies to Europe) of
Abutilon sellovianum var. marmorata plants with mosaic symptoms now known to be
caused by Abutilon mosaic virus [61].
Economic losses caused by geminiviruses were not described until the end of the
1800s, when several disease outbreaks that we now know to be caused by geminiviruses
were reported from various locations around the globe. In 1894, cassava mosaic disease
was reported in cassava in East Africa [63]. The cause of this disease is now known to be
the geminivirus, African cassava mosaic virus (ACMV). Five years later, epidemics of
beet curly top disease in sugar beet were reported from California [44, 61]. The cause of
this disease was later identified as the geminivirus, Beet curly top virus (BCTV). A
disease of maize known as streak disease was reported in South Africa in 1901 [22]. The
cause of this disease is the geminivirus, Maize streak virus (MSV).
1
2
The viral nature of geminiviruses was suggested in some of the earliest studies of
viruses. In 1899, Beijerinck compared the mosaic symptoms of tobacco and mosaic
symptoms of A. sariatum Dicks. ex Lindl. and concluded that they were related [14, 30].
Seven years later, Zimmermann suggested that the mosaic disease of cassava was caused
by a virus [63]. By 1925, Story studied the symptoms and ability of leafhoppers to
transmit streak disease of maize and concluded that streak disease of maize was caused
by a virus which was transmitted by a leafhopper [62]. By 1931, Kirkpatrick reported the
whitefly as a vector of leaf curl of cotton [39]. In approximately 1932, Ghesuiere
suspected that whiteflies were the vector of the causal agent of cassava mosaic disease
[63]. This suspicion was later confirmed by Storey in 1934 and Golding in 1936 [63].
The unique characteristics of geminiviruses were not clear until the 1970s, at which
time geminate virus particles were observed by electron microscopy and the nature of the
viral nucleic acid was determined. Bennett in 1971[48], observed small spherical bodies
in filtered phloem sap of sugar beet infected with BCTV; his observation was confirmed
by Mumford [48] in 1974. In 1972, Plasvic and Maramorisch observed isometric particles
in thin sections of maize infected with MSV. This observation was confirmed in 1974 by
Bock et al. [4]. Three years later in 1977, the nucleic acid of geminivirus was identified
as a single-strand of DNA [27]. One year later, geminiviruses were recognized as a new
virus group [44].
In 1978, plant viruses were classified into families. The Geminiviridae family
consists of plant viruses with a single-stranded DNA (ssDNA) genome that is
encapsidated into a unique geminate capsid structure [44]. Geminivirus genomes are
either bipartite or monopartite. Bipartite genomes are divided into two components: A
3
component (DNA-A) and B component (DNA-B). DNA-A contains the gene required for
encapsidation of progeny and viral DNA replication. DNA-B contains the genes required
for viral movement (for movement of viral DNA from host cytoplasm to host nucleus;
and for cell-to-cell movement in infected host plants) [45, 49]. In monopartite
geminiviruses, all of the genes are found in one component [45]. An intergenic region
(IR) contains the common region (CR) found in all monopartite and bipartite
geminiviruses. The IR is believed to play a role in the initiation of DNA replication. In
bipartite genome geminivirus, the CR is highly conserved between DNA-A and DNA-B
[38].
Although geminiviruses have a small genome (about 5000 nt for bipartite and about
2800 nt for monopartite viruses) and few genes, they have an efficient means of
replication. The strategy of replication of the ssDNA genome begins by converting
ssDNA into double-stranded DNA (dsDNA) starting at the stem loop. This dsDNA is
used as a template to amplify viral dsDNA and to produce mature ssDNA in a process
known as a rolling-circle replication mechanism [24]. Recently, Jeske reported
recombination-dependent replication as another method of geminivirus replication [36].
Taxonomy and Nucleotide Functions
Geminiviruses are currently divided into four genera (based on genome
organization and structure, host range, and insect vector) [19]. The genera are Curtovirus,
Topocuvirus, Mastrevirus, and Begomovirus.
The Curtoviruses, type species BCTV, have a monopartite genome, are transmitted
by leafhoppers, and infect dicot plants. Seven proteins are encoded by the Curtovirus
genome. Three proteins are encoded on the viral-sense (v-sense) strand: the movement
protein (MP) which is responsible for cell-to-cell movement; the capsid protein (CP)
4
which is responsible for forming the viral capsid; and the V2 protein that converts
double-stranded DNA to single-stranded DNA. Four proteins are encoded on the
complementary sense (c-sense) strand: the Replication initiation protein (Rep) by which
viral replication starts; the replication enhancer protein (REn); and C4 protein (which
determines symptom expression). An extra open reading frame is also recognized (known
as the C2) whose function is unknown [6, 24].
Topocuvirus, type species Tomato pseudo-curly top virus (TPCTV), has only one
member virus that has a monopartite genome; is transmitted by the treehopper; and
infects dicot plants. Six proteins are encoded by the TPCTV genome. On the v-sense
strand, two proteins are encoded: the V2 and the CP. On the c-sense strand four proteins
are encoded: Rep, C2, REn, and C4 [5, 6].
Mastrevirus, type species MSV is a genus that consists of viruses with a
monopartite, genome that are transmitted by leafhoppers; and infect both monocots and
dicots. The genome consists of two intergenic regions: one large (LIR) and one small
(SIR) located on opposite sides on the viral genome. Two features are unique to this
genus: the first is the presence of an ~80 nt-long DNA sequence annealed to a region
within the SIR, which is present inside the viral particle. The second feature is the
presence of a splicing event on the c-sense transcript. Four proteins are encoded by the
genome; two on the c-sense strand (the MP and CP); and two on the v-sense strand (the
Rep A protein and the Rep protein) [25, 38, 51].
Begomovirus, type species Bean golden mosaic virus (BGMV), is the largest genus
in the family. The viruses in this genus are transmitted by the whitefly (Bemisia tabici)
and they infect primarily dicot plants. Most begomovirus species consist of a bipartite
5
genome and few are monopartite. DNA-A encodes five proteins which are the CP, on the
v-sense strand, and the Rep, TrAP (a transcriptional activator), REn, and C4 on the c-
sense strand. DNA-B encodes two proteins: the nuclear shuttle protein (NSP) and the MP
on the c-sense and v-sense strands, respectively. [5, 18, 24, 61].
Recently small circular single stranded satellite DNAs (DNA ß) have been found to
be associated with some Old World monopartite begomoviruses. The DNA ß is about
1330 nucleotides and several have been isolated and sequenced [7, 16]. It is believed that
the DNA ß play an important roll in the severity of the symptoms, begomovirus
pathogenicity, and host range of the associated begomovirus [46].
Begomoviruses
Begomoviruses can be one of the biggest threats to tomato production. In the early
1990s, 95% of tomato fields were destroyed in the Dominican Republic due to
begomoviruses, primarily Tomato yellow leaf curl virus (TYLCV) [47]. In the 1991-1992
production season, the begomovirus Tomato mottle virus (ToMoV) cost the tomato
growers in Florida about $140 million [47].
The whitefly Bemisia tabaci is the vector of begomoviruses. When adults feed on
infected plants; virus is usually transferred with food material through the salivary canal
to the mid-gut and from the mid-gut it passes into the hemolymph. The virus is then
circulated with normal hemolymph. It then passes into the salivary glands. As the
whitefly feeds in healthy plants, the virus is transmitted with the saliva to the plant via the
salivary canal [13, 35]. The coat protein of begomoviruses has been shown to play an
important role in the circulation of the virus in the vector [33].
6
The Genus Sida
Sida is the Greek word for a water plant, but the allusion to this genus is still
unclear. According the USDA data base (www.itis.usda.gov), there are about 27 species
of Sida worldwide. Sida is usually found in roadsides, gardens, waste places, barn yards,
canal banks, and fallow and cultivated fields. The way to grow Sida is either by asexual
propagation using cuttings of young green stems or by cultivation of seed under direct
sun light and dry conditions. Seed of Sida spp. are covered with a thick layer of an
unknown chemical that blocks water from penetrating, leaving the seed in a dormant state
[55].
In 1975 Ghosal and his group were able to analyze S. cordifolia L. chemically. The
chemical analyses showed S. cordifolia contains three types of chemicals: β-
phenethylamines (viz. β-phenethylamines, ephedrine, and pseudoephedrine),
carboxylated tryptamines (S-(+)-N-methyltryptophane methyl easter and hypaphorine),
and quinazoline alkaloids (vasicinone, vasicinol, and vasicine). Moreover, different parts
of the plant contain the same chemicals but in different concentrations. Ghosal reported
the concentrations of those chemicals changed with plant age [23].
Genomic analysis of a selected Sida spp. done by Hazra showed that they have
chromosome numbers that range from 2n=14 to 2n=32. In details, S. rhombifolia var. C,
S. rhombifolia var. D, and S. rhombifolia var. E are 2n=14. S. acuta, S. rhombifolia var.
A, and S. rhombifolia var. B are 2n=28. S. cordifolia, S. glutinosa Comm. ex Cav., and
S. veronicaefolia Lam. are 2n=32 [29].
Sida plants are good source of fiber; and some Sida species are used in traditional
medicine. S. rhomboidea L. and S. cordifolia are used for their anti-inflammatory activity
[20, 64]. S. cordifolia contains a high amount of ephedrine and pseudoepherdrine
7
components which have medical uses. In nature, Sida plants play an important roll in
reducing erosion of nitrogen, organic carbon, calcium, potassium, and sodium from soil
[40, 41].
Viruses Infecting Sida spp.
Viruses that infect Sida spp were considered as a part of Infection Chlorosis of
Malaveace virus group for many years. In the nineteenth century, the major tools used by
botanists to classify and identify the causal agent of plant diseases were symptom
expression, ability to see the pathogen with a microscope, and the method of
transmission. Based on the presence of mosaic symptoms, inability to visualize any
pathogen in infected cells [42], and transmission by grafting, a group of plant viruses was
classified as one group, known as the Infectious Chlorosis of Malaveace. The written
record begins with the movement of A. striatum with a mosaic symptoms to Europe from
the Caribbean in 1868 [37]. One year later, Lemoine was able to transfer the Infectious
Chlorosis of Malaveace to another species of Abutilon by grafting. In the same year,
Masters reported the graft transmission of Infectious Chlorosis of Malaveace from A.
pictum `Thompsonii` to other Malaveace species including S. napaea Cav. [30, 37]. In
1899, Beijerinck suggested the viral nature of Infectious Chlorosis of Malaveace after
comparing symptom expression of A. striatum and tobacco infected with Tobacco mosaic
virus (TMV) [30]. Between 1904 and 1908, Baur studied the transmission of Infectious
Chlorosis of Malaveace from A. indicum L. by sap and seed. He reported the inability to
transmit the symptoms by sap or seed. Interestingly, he concluded that the lack of seed or
sap transmission was because the too low virus titer in the seed to produce a disease in
new seedlings [37]. Today we know begomoviruses are not seed transmitted and hard to
be sap transmitted. In 1926, Hein proved the Infectious Chlorosis of Malaveace cause the
8
degradation of plastids and the disease move from cell-to-cell [31]. In 1927, Hertzsch
was the first person to recognize variation within Infectious Chlorosis of Malaveace. He
recognized the existence of two types of viruses within the Malvaceae. He call them Type
A and Type B; each had a unique host range and produced different symptoms in the
same hosts [37] . By 1931, Cook reported from the West Indies that seeds of A. hirtum
Lam. produced only healthy green seedlings, and concluded that the Infectious Chlorosis
is not seed transmissible [37].
High temperature, hot water or sulphuric acid treatments, and physical disruption of
seed coat are the major methods used to break the dormancy of the seed [17].
Although in 1899, Beijerinck suggested the viral nature of Infectious Chlorosis of
Malaveace, the nature of this disease was not clear for many botanists. Several
hypotheses were raised by scientists until the 1940s to explain Infectious Chlorosis of
Malaveace. One was that the nature of Infectious Chlorosis of Malaveace was
spontaneous and due to genetic crossing between white and green genes [60]. Another
popular hypothesis referred to metabolic and enzymatic activity of plant cells as a reason
for mosaic symptoms [59]. A third one suggested the presence of an ultramicroscopic
pathogen [42] which ultimately replaced all other hypotheses by the 1940s. In 1943,
Silberschmidt studied Infectious Chlorosis of Malaveace using three species of Sida
showing mosaic symptoms (S. acuta, S. rhombifolia, and S. cordifolia). In his study he
was able to observe the limitations of moving the symptoms from one species to another
[58]. He explained these results by concluding that some species of Sida have immunity
against the Infectious Chlorosis of other species. Today we know that different Sida
species can be infected with different begomoviruses or different viruses. In 1945, just
9
two years after Silberschmidt’s work, the whitefly was reported to be the vector of
Infectious Chlorosis of Malaveace [11]. In 1946 Orlando and Silberschmidt published a
paper proving the whitefly was the vector of Infectious Chlorosis of Malaveace using S.
rhombifolia [50]. Those two papers are considered to be one of the earliest reports
demonstrating the ability of the whitefly to vector a begomovirus in Western
Hemisphere.
The begomoviruses that infect Sida species were also considered to be strains of
Abutilon mosaic virus for a time. This was because of the similar symptoms and the
ability of some Sida begomoviruses to infect species of both Sida and Abutilon [11, 50].
In 1953, Costa and Bennett suggested again that the whitefly was the vector of a virus
they called AbMV after studying whiteflies population on Sida sp [10]. In 1955, Costa
published a study on AbMV that naturally infecting Sida (this at indication that Costa
mixed between the begomoviruses infecting Sida with AbMV). He reported the ability to
transmit a begomovirus infecting S. micrantha ST. Hill. and S. rhombifolia to other plants
by means of whiteflies [11]. In 1960, Costa published study on the mechanical
transmission of a begomovirus from Sida (which was referred to as AbMV) to selected
plant hosts. He also reported on the difficulty in transmission of geminiviruses by
mechanical means [12].
Species of Sida with mosaic symptoms have been reported from many locations in
Latin America [3]. In Puerto Rico, S. carpinifolia L.f. with other species of Sida shows
mosaic virus symptoms have been reported from different places in the island. These
mosaic symptoms believed to be transmissible via whitefly [3]. In El Salvador, mosaic
10
symptoms were observed in Sida spp and were shown to be transmissible to healthy Sida
spp and cotton [3].
Viruses other than begomoviruses have been reported to infect species of Sida. S.
alba in Zimbabwe was demonstrated to be a host of Turnip mosaic virus which belongs
to the Potyviridae family [8]. In Nigeria S. acuta and S. rhombifolia were able to be
inoculated with Okra mosaic virus which belongs to the Tymoviridae family [1].
Begomoviruses that infect species of Sida were not characterized until the 1990s,
by which time sequencing was the primary method used to characterize and compare
different begomovirus species. In 1997, Hofer et al. reported a new bipartite begomovirus
which was isolated from S. rhombifolia in Costa Rica and know as Sida golden mosaic
Costa Rica virus (SiGMCRV) (GenBank Accession No. X99550 and X99551) [33]. In
the same year, Frischmuth et al. reported two bipartite begomoviruses with one extra
DNA-B isolated from S. rhombifolia in Honduras. The first one is called Sida golden
mosaic Honduras virus (SiGMHV) (GenBank Accession No. Y11097 and Y11098), the
second is the Sida yellow vein virus (SiYVV) (Accession No. Y11099 and Y11100) [21],
and the DNA-B has the Genbank Accession No. AJ250731 [34]. Recently, two DNA-A
have been reported from Brazil from Sida spp. (Genbank Acc. No. AY090555 and
AY090558) [19].
Begomoviruses Infecting Sida spp. in Florida
Probably one of the earliest study on Sida begomoviruses in Florida was published
in 1930 when Kunkel reported the ability of a mosaic disease to infect S. rhombifolia and
other Sida spp. by budding or grafting but not by mechanical methods [43]. He also
showed the this mosaic disease was not transmitted through seeds and pointed out that it
resembled AbMV based on symptoms and method of transmission [43]. In 1953, Costa
11
and Bennett reported that Sida spp. in Orlando, Florida, were probably infected with
AbMV and hypothesis that this virus may transmissible by the whitefly (Bemisia tabaci)
[10]. By 1990s, scientists in three labs at the University of Florida begin studying
begomoviruses of Sida. In 1993, the laboratory of E. Hiebert in Gainesville was able to
characterize a begomovirus that infects S. acuta known Sida golden mosaic virus
(SiGMV) (GenBank Accession No. AF049336 and AF039841) [32]. In Homestead,
partial sequences of two DNA-As from S. acuta were reported (GenBank Accession No.
U77963, U77964) [19]. In Bradenton, begomovirus-like symptoms were observed in S.
santaremensis.
CHAPTER 2 CHARACTERIZATION OF A NEW BEGOMOVIRUS ISOLATED FROM Sida
santaremensis Monteiro in Florida
The genus Sida is a group of wild plants that is distributed throughout both the New
and Old World [3, 9, 20, 41]. Several species of Sida have been reported as hosts of
whiteflies, specifically Bemisia tabaci Genn. biotype B, as well as begomoviruses [10].
Begomoviruses, a genus of plant viruses that belong to the family Geminiviridae, are
plant viruses with a single-stranded circular DNA genome. The whitefly, B. tabaci, is the
only known insect vector of begomoviruses [35]. The relationship between Sida spp.,
begomoviruses, and whiteflies has been recognized since the 1950s [10-12].
Recently, several begomoviruses have been characterized from different species of
Sida in the New World [21, 33]. In Costa Rica a bipartite begomovirus known as Sida
golden mosaic Costa Rica virus (SiGMCRV) has been isolated and characterized from S.
rhombifolia L. [33]. In Honduras two bipartite begomoviruses, known as Sida golden
Honduras mosaic virus (SiGMHV) and Sida yellow vein virus (SiYVV), and an extra B
component (DNA-B) have been isolated and characterized from S. rhombifolia [21]. In
Brazil, two A components (DNA-A) have been sequenced and characterized from Sida
spp. [19]. In Jamaica, a partial clone of a begomovirus was obtained from S. urens L. and
partial sequences of other begomoviruses have been found in an unreported species of
Sida.
There are ten species of Sida found in Florida (http://www.plantatlas.usf.edu) and
bright golden mosaic symptoms, typical of those caused by begomovirus, have been
12
13
observed in several species. Several begomoviruses have been reported from S. acuta
Burm. f. found in several counties. In S. acuta from Dade Co., two partial sequences of
begomovirus DNA-A were obtained (Genbank Acc. No. U77963, U77964; data not
published). Sida golden mosaic virus (SiGMV) was found in S. acuta in Alachua Co.
(Genbank Acc. No. AF049336 and AF039841) (data not published).
This study reports on the identification and characterization of a new begomovirus
isolated from Sida santaremensis Monteiro in Manatee Co. FL.
Materials and Methods
Virus Source
The virus was isolated from a plant of S. santaremensis showing bright golden
mosaic symptoms (Fig. 2-2), which was originally collected from behind greenhouses
located at the University of Florida, Gulf Coast Research and Education Center,
Bradenton, FL. in January 1997. Plants were identified to species by curators at the
Florida Museum of Natural History, University of Florida, Gainesville, FL. A culture of
the virus was maintained in the greenhouse by periodically reproducing infected plants
through cuttings made from young stems with symptomatic leaves.
Begomovirus Detection
DNA was extracted from leaves of S. santaremensis which displayed golden
mosaic symptoms using a modification of a protocol reported by Doyle and Doyle [15].
The plant tissue was ground in CTAB buffer in the absence of liquid nitrogen, and DNA
was precipitated in isopropanol for one hour at -20°C. Degenerate primer pairs
(PAR1c496/PAL1v1978, and PBL1240/PCRc154) were selected to detect begomovirus
DNA [56]. PAR1c496/PAL1v1978 amplify an ~1100 bp from the begomovirus A
component (DNA-A) of most bipartite begomoviruses and a ~1300 bp fragment from
14
most monopartite begomoviruses. This fragment includes the 3´ end of the putative Coat
Protein gene (CP), the entire common region (CR), and a part of the putative Replication
Association Protein gene (Rep) [56]. PBL1240/PCRc154 amplify an ~600 bp fragment
from the B component (DNA-B) of most bipartite begomoviruses. This fragment includes
the 3´ end of the putative Nuclear Shuttle Protein gene (NSP) [65] and part of the CR
sequence [56]. The PCR reaction contained 2.5 mM Mg , 50 pM of each primer, 12.5
pM of of dNTPS, 12.5 mM Spermidine, and 1U Taq polymerase. The PCR condition was
started with a DNA denaturation step of 94°C for 5 min. followed by 35 cycles of 60 sec.
of denaturing, 60 sec. of annealing at 55°C, and 60 sec. of extension at 72°C. The
reaction was terminated with a final extension at 72°C for 5 min. The PCR reaction was
carried out using gene amp PCR system 9700 or 2700 (Applied Biosystems, The Perkin
Elmer Corp. Norwalk, CT).
+2
Cloning and Sequencing
The amplicons obtained with the above mentioned primers were cloned and
sequenced. Sequences of the fragments were used to design primers using Wisconsin
package (GCG) which would amplify the DNA from the remainder of the genome. After
obtaining the complete sequences of DNA-A and DNA-B, a restriction map was
constructed for both. In order to obtain an infectious clone, a single restriction site (ApaI)
at the 5´ end of the Rep gene was identified for DNA-A and a single restriction site
(NcoI) in the Movement Protein gene (MP) was identified for DNA-B. The DNA
extracted from leaves of S. santaremensis was, digested with the respective enzyme and a
DNA fragment of ≈2600 bp was obtained. This DNA was gel purified using a gel
purification kit (Qiagen Sciences, Germantown, MD) and cloned into plasmids. The
15
linear full length DNA-A was cloned into pBluescript® KS (-) [Stratagene, La Jolla, CA]
and the DNA-B into pLitmus 28 (New England Biolabs, Beverly, MA).
Molecular Characterization of the Virus
After obtaining the complete sequences of DNA-A and DNA-B, open reading
frames were determined using Vector NTI software (Infomax, Frederick, MD). Sequence
comparisons were made by NCBI BLAST using the NCBI taxonomy database
(http://www.ncbi.nlm.nih.gov/). Based on this analysis the 13 begomoviruses with the
highest nucleotide sequence identity to SiGMoV were selected for further comparisons.
The nucleotide sequences of whole genomes as well as individual genes were compared.
The same begomoviruses where used in the phylogenetic analysis at which the
alignment of full length nucleotide sequences would begin at the ATAATT sequences of
the stem loop [2]. The comparison were based on maximum parsimony using the
PAUP*s heuristic method with the bisection-reconnecting branch swapping. The
Bootstrap value was set to be based on 500 replicates. Display tree was with no rooting
using the midpoint rooting option.
Biological characterization
A host range study was conducted using two methods of inoculation, biolistic
inoculation with the infectious clones and whitefly inoculation. SiGMoV from S.
santaremensis biolisticly which had been inoculated with the infectious clones and give a
positive result for SiGMoV using PCR and dot spot hybridization. The host plants were
grown from seed in a greenhouse. The host plants tested in this study were: common bean
(P. vulgaris), cotton (G. hirsutum), N. benthamiana, tobacco (N. tabacum), pepper
(Capsicum annuum L. ‘Calwonder‘), S. santaremensis, and tomato (L. esculentum).
16
Biolistic inoculation
The infectious clones were grown overnight in 400 ml of 2XYT media with 1%
Ampicillin and the plasmid DNA was extracted using QIAGEN Plasmid Maxi Kit
(Qiagen Sciences, Germantown, MD). Approximately 5.8 µg/µl of the DNA-A plasmid
and 2.4 µg/µl of DNA-B plasmid DNA were obtained. The viral insert of the DNA-A
was released from the plasmid by an overnight digestion with ApaI which cut at the
insertion site. NcoI was used in overnight reaction to release the DNA-B from the
plasmid. The restriction reaction was stopped by precipitating the DNA using 0.1 vol.
sodium acetate and 3 vol. isopropanol. The DNA was then dissolved in 50 µl of water
and the concentration of DNA was determined using a spectrophotometer. Both DNA-A
and DNA-B were mixed together to make a total of 25.0 ng, which was bound to sterile
1.0 µm in diameter spherical gold particles (Biorad, Hercules, CA). This mixture was
then treated with 2.5 M calcium chloride and 0.1 M spermidine and allowed to sit for 15
minutes at room temperature. Then, it was washed with 70% isopropanol followed by
100% isopropanol. Finally the mixture was re suspended on 60µl of 100% isopropanol.
About 10 µl of gold and DNA mixture were biolisticly inoculated into each host plant
using a gene gun [57].
Whitefly inoculation
A virus-free whitefly colony was established by allowing the virus-free whiteflies
to feed on cotton. After 21 days, a new generation of adults was collected and used in
transmission experiments.
Virus-free adult whiteflies were given an acquisition access period of 3 days on
SiGMoV-infected S. santaremensis. These S. santaremensis plants were three-week old
plants propagated as cuttings from S. santaremensis plants that had been biolisticly
17
inoculated with SiGMoV. The S. santaremensis plants used as acquisition hosts showed
strong mosaic symptoms and were positive by PCR analysis for SiGMoV. The selected
host plants were introduced to whiteflies that feed on S. santaremensis, and the S.
santaremensis plants were shaken so that the whitefly adults could be removed. The S.
santaremensis, was then isolated in a different cage. Whiteflies were given a 3 day
inoculation access period which was terminated by the addition of a drench of
imidacloprid, a systemic insecticide (Bayer Corp., Kansas City, Missouri). Inoculated
plants were kept in an isolated cage in greenhouse.
Detection of SiGMoV in Test Plants
The presence of SiGMoV in test plants was determined by visual assessment of
symptoms beginning two weeks after inoculation and continuing for two months in
summer months. In winter months the symptoms were recorded every three weeks
starting at three weeks after inoculation. Each time symptoms were recorded a leaf
sample was collected from each plant and analyzed by PCR and by dot spot
hybridization.
Samples were tested for virus using dot spot hybridization. The full length DNA-B
of the virus was used as a probe under conditions of high stringency [52].
The presence of SiGMoV was confirmed in plants testing positive by dot spot
hybridization using PCR. Plant samples collected from N. benthamiana, N. tabacum,
common bean, and tomato were extracted as described above. Plant samples of S.
santaremensis, cotton and pepper were extracted using the protocol described by
Porebski [54]. Degenerate primer pairs PAR1c496/PAL1v1978 for the DNA-A and
PBL1240 and PCRc154 for DNA-B were used [56]. The homology analysis using Vector
18
NTI software of those primers and SiGMoV shows that primers PAR1c496/PAL1v1978
have a homology of 91.2% and 88.8% at binding sites, and primers PBL1240/PCRc154
have the homology 87.5% and 69.7%. The positive results were further analyzed using a
set of primers to specifically bind to SiGMoV. They were JAP85
3´GCTCTCTCGCTCAAAAGTCTAG5´ which binds in the CR of SiGMoV and the
degenerate primer AC1048 [65] which binds in the 5´ end of the CP and has a homology
of 87.7% with SiGMoV.
Results
S. santaremensis (common name: moth fanpetals) is a species of Sida that was
reported from Hillsborough and Pinellas counties in Florida
(http://www.plantatlas.usf.edu). However, according to the USDA plant database S.
santaremensis is not native to the U.S.A (http://plants.usda.gov/topics.html). This is the
first report of this species of S. santaremensis in Manatee Co.
Full length sequences of both DNA-A and DNA-B were obtained from
symptomatic plants of S. santaremensis. The sequences were numbered beginning at the
first nucleotide of the CR sequence shared by DNA-A and DNA-B. The DNA-A was
found to have five open reading frame and the DNA-B was found to have two open
reading frames which is an arrangement typical of many bipartite begomoviruses
[19].The sequence identity of the CR (125 nt) between DNA-A and DNA-B was 95.2%.
Phylogenetic Analysis
The phylogenic analysis of SiGMoV DNA-A indicates that the DNA-A does not
cluster with any characterized begomovirus (Fig. 2-2). However, the DNA-B clustered
within the AbMV group (Fig. 2-3).
19
Nucleotide and Amino Acid Sequence Analysis
A comparison of SiGMoV DNA-A and DNA-B nucleotide sequences with ten
other characterized begomoviruses confirmed the results obtained by the phylogenetic
analysis (Tables 2-1 and 2-2). The comparison shows that DNA-A of SiGMoV
nucleotide sequences identities ranged from 78.6 to 83.0% (Table 2-1.) Sida golden
mosaic Honduras virus and Sida golden yellow vein virus had the greatest nucleotide
sequences identity with SiGMoV. A comparison of the nucleotide sequence identities of
DNA-B of SiGMoV showed a range of 66.5 to 78.3%, the most similar virus being
AbMV (Table 2-2).
A comparison of selected regions and open reading frames did not reveal any close
relationships with other begomoviruses. The CR of DNA-A of SiGMV was somewhat
similar to that of PYMV-VE (87.1%) but the CR of the DNA-B showed less identify with
PYMV-VE (60.8%) than with PYMV (80.7%) (Tables 2-3 and 2-4). The comparison of
open reading frames on the DNA-A with those of SiGMoV showed no significant
identities (Table 2-3). Similar results were obtained using the amino acid sequence
similarities of the open reading frames on DNA-A (Table 2-5). However, on DNA-B the
nucleotide and amino acid sequence of the putative MP gene of SiGMoV was fairly
homologous (>90%) to the MP of several characterized begomoviruses (Tables 2-4 and
2-6).
Biological Characterization
The biological characterization was carried out using two methods of transmission,
biolistic inoculation and whitefly transmission, on selected host plants. The detection of
SiGMoV was carried out using: symptom expression, PCR analysis, and dot spot
hybridization. N. benthamiana, N. tabacum, S. santaremensis, bean, tomato and cotton
20
were all susceptible to infection with SiGMoV by biolistic inoculation (Table 7). Viral
DNA was detected by PCR and dot spot hybridization in these plants two weeks and four
weeks after inoculation. However, symptoms were only observed in species, N.
benthamiana, P. vulgaris, and S. santaremensis . In N. benthaniana a mild mosaic was
observed two weeks after inoculation. Four weeks after inoculation the symptoms
observed in N. benthaniana were mosaic, leaf cupping, and shorting. In beans the
symptoms appearred three weeks after inoculation and these were a mild mosaic and
stunting of the plant. In whitefly transmission, only two plants were inoculated from
SiGMoV-infected S. santaremensis plants. Two plants of N. tabacum were determined to
be infected based on PCR and dot spot hyridization. No symptoms were produced in this
plant. Figure 2-1: Sida santaremensis infected with Sida golden mottle virus showing
typical mosaic symptoms.
Figure 2-1. Sida santaremensis infected with Sida golden mottle virus showing typical
21
Table 2-1. Comparison of the nucleotide sequence identity of the DNA-A of Sida golden mottle virus with the 13 most closely related begomoviruses identified by BLAST analysis
Begomovirus ACC. NO. % Sequence Identity
Sida golden mosaic virus AF049336 82.1 Sida golden mosaic Honduras virus Y11097 83.0 Chino del tomato virus-[IC] AF101476 82.1 Sida golden yellow vein virus Y11099 83.0 Potato yellow mosaic virus-Venezuela D00940 81.6 Chino del tomato virus- [H6] AF226665 81.9 Tomato mottle Taino virus AF012300 79.7 Abutilon mosaic virus X15983 81.5 Abutilon mosaic virus-HW U51137 81.5 Bean dwarf mosaic virus M88179 81.5 Potato yellow mosaic Trinidad virus AF039031 78.6 Sida golden mosaic Costa Rica virus X99550 79.6 Tomato mottle virus-[Florida] L14460 79.6 ACC. No. : GenBank Accession number 1 Begomovirus sequences were selected from the first 13 sequences obtained by a Blast analysis. Table 2-2. Comparison of the nucleotide sequence identity of the DNA-B of Sida golden
mottle virus with the 13 most closely related begomoviruses identified by BLAST analysis
Begomovirus ACC. NO. % Sequence Identity
Abutilon mosaic virus X15984 78.3 Tomato mottle virus-[Florida] L14461 77.7 Abutilon mosaic virus-HW U51138 77.0 Tomato mottle Taino virus AF012301 76.3 Sida golden mosaic virus AF049341 75.0 Sida yellow vein virus Y11100 73.4 Sida golden mosaic virus* (Honduras) AJ250731 72.7 Sida golden mosaic Honduras virus Y11098 72.6 Bean dwarf mosaic virus M88180 72.4 Sida golden mosaic Honduras virus- yellow vein Y11101 72.1 Sida golden mosaic Costa Rica virus X99551 72.0 Chino del tomato virus-[IC] AF101478 70.2 Potato yellow mosaic virus-Venezuela D00941 67.8 Potato yellow mosaic Trinidad virus AF039032 66.5 Chino del tomato virus-[B52] AF226666 70.9 ACC. No. : GenBank Accession number
22
Table 2-3. Comparison of the open reading frame nucleotide and common region sequences identity of the DNA-A of Sida golden mottle virus with the 13 most closely related begomoviruses identified by BLAST analysis
% Sequence Identity Begomovirus CR CP Rep TrA
P REn AC4
Potato yellow mosaic virus-Venezuela
87.1 82.1 83.4 78.3 80.3 67.4
Tomato mottle Taino virus 78.4 83.0 80.9 78.3 79.6 61.6 Sida golden mosaic Honduras virus 65.1 86.3 82.4 83.7 85.6 69.8 Sida golden mosaic Costa Rica virus 61.3 82.4 80.5 82.2 81.7 65.1 Sida yellow vein virus 61.6 87.5 81.7 83.0 83.3 76.7 Potato yellow mosaic Trinidad virus 61.3 82.8 78.4 79.1 81.1 64.0 Sida golden mosaic virus 60.0 87.7 81.3 81.4 82.6 67.4 Bean dwarf mosaic virus 56.5 85.3 80.4 82.0 81.1 66.3 Abutilon mosaic virus 55.7 85.5 81.3 78.9 81.1 67.4 Abutilon mosaic virus-HW 54.8 85.2 80.5 82.2 81.8 69.1 Chino del tomato virus-[H6] 54.8 86.7 80.9 83.0 83.3 64.0 Chino del tomato virus-[IC] 54.0 87.0 81.3 83.0 83.3 69.8 Tomato mottle virus-[Florida] 60.0 86.0 79.0 84.2 83.3 81.6
Table 2-4. Comparison of the open reading frame and common region nucleotide sequences identity of the DNA-B of Sida golden mottle virus with the 13 most closely related begomoviruses identified by BLAST analysis
% Sequence Identity Begomovirus CR NSP MP
Tomato mottle Taino virus 76.8 79.0 93.2 Sida golden mosaic virus*(Honduras) 62.4 76.3 93.5 Sida yellow vein virus 62.4 75.9 93.2 Sida golden mosaic Honduras virus 62.1 75.9 94.2 Sida golden mosaic Honduras virus- yellow vein 62.1 75.9 93.5 Bean dwarf mosaic virus 58.9 77.4 92.9 Sida golden mosaic virus 58.4 82.1 94.2 Abutilon mosaic virus 57.6 75.1 93.2 Tomato mottle virus-[Florida] 57.6 80.1 93.9 Abutilon mosaic virus-HW 55.2 73.9 89.8 Chino del tomato virus-[IC] 52.8 75.5 90.5 Sida golden mosaic Costa Rica virus 52.4 74.7 91.8 Potato yellow mosaic Trinidad virus 60.8 79.5 69.1 Potato yellow mosaic virus 80.7 79.4 68.5 Chino del tomato virus-[B52] 53.6 82.9 74.8
23
Table 2-5. Comparison of the open reading frame amino acid sequences similirity of the DNA-A of Sida golden mottle virus with the 13 most closely related begomoviruses identified by BLAST analysis
Begomovirus CP Rep TrAP REn AC4 Abutilon mosaic virus 92.1 87.1 85.9 84.9 73.3 Abutilon mosaic virus-HW 90.4 83.9 89.9 86.4 70.9 Bean dwarf mosaic virus 93.6 86.7 85.9 87.1 72.1 Chino del tomato virus-[H6] 90.6 86.7 86.1 88.6 68.6 Potato yellow mosaic virus- Venezuela 92.8 91.2 85.3 84.9 72.1 Potato yellow mosaic Trinidad virus 92.4 84.1 84.5 85.6 68.6 Sida golden mosaic virus (Honduras) 93.6 87.4 84.5 91.7 75.6 Sida golden mosaic Costa Rica virus 91.3 86.9 84.4 89.2 68.6 Sida golden mosaic virus 92.8 86.0 86.8 87.1 73.3 Sida golden mosaic Honduras virus 93.2 88.5 86.8 90.9 76.7 Sida yellow vein virus 93.6 85.4 86.8 89.4 81.4 Chino del tomato virus-[IC] 93.6 87.0 86.1 88.6 74.4 Tomato mottle Taino virus 91.6 86.2 83.7 86.4 95.1 Tomato mottle virus-Florida 92.3 85.8 85.7 86.4 65.9
Table 2-6. Comparison of the open reading frame amino acid sequences similirity of the DNA-B of Sida golden mottle virus with the 13 most closely related begomoviruses identified by BLAST analysis
Begomovirus NSP MP Sida golden mosaic virus 87.2 96.3 Tomato mottle virus-Florida 85.2 96.3 Tomato mottle Taino virus 84.8 95.9 Bean dwarf mosaic virus 84.1 95.6 Sida golden mosaic virus* (Honduras) 81.7 95.2 Sida golden mosaic Honduras virus 81.7 95.9 Abutilon mosaic virus 81.3 95.9 Sida yellow vein virus 81.3 95.2 Sida golden mosaic Honduras virus-yellow vein 81.3 94.9 Chino del tomato virus-[IC] 80.9 93.9 Abutilon mosaic virus-HW 80.6 93.2 Sida golden mosaic Costa Rica virus 80.2 95.2 Potato yellow mosaic Trinidad virus 73.6 91.8 Potato yellow mosaic virus- Venezuela 73.4 92.2 Chino del tomato virus-[B52] 94.2 81.0
24
Table 2-7. Host range study of SiGMoV using selected plants at which number of positive SiGMoV to the total number of plant
Plant Biolistic inoculation infectivity1
(infected/inoculated)
Whitefly inoculation infectivity 2
(infected/inoculated) Nicotiana benthamiana 8/24 0/6 N. tabacum L. (V20) 9/15 2/6 Phaseolus vulgaris L. (Topcrop) 8/24 0/6 Gossypium hirsutum L. (Delta Pine 70) 20/25 0/6 Sida santaremensis Monteiro 11/12 0/0 Lycopersicon esculentum Mill. (FL Lanai)
9/25 0/6
1 25 plants were used in each biolistic inoculation and 5 were used as negative controls. 2 6 plants were used in each whiteflies transmission and 1 was used as a negative control.
25
Figure 2-2. Phylogenic tree of complete nucleotide of a component of selected
begomoviruses with SiGMoV. SiYVV: sida yellow vein virus, SiGMHV: Sida golden mosaic Honduras virus, SiGMCRV: Sida golden mosaic Costa Rica virus, BDMV: Bean dwarf mosaic virus, CdTV-[H6]: Chino del tomato virus-[H6], CdTV-[IC]: Chino del tomato virus-[IC], AbMV-HW: Abutilon mosaic virus-HW, AbMV: Abutilon mosaic virus, SiGMV: Sida golden mosaic virus, ToMoV-[FL]: Tomato mottle virus-Florida, SiGMoV: Sida golden mottle virus, PYMTV-TT: Potato yellow mosaic Trinidad virus, PYMV-VE: Potato yellow mosaic virus- Venezuela, ToMoTV: Tomato mottle Taino virus.
26
Figure 2-3. Phylogenic tree of complete nucleotide of B component of selected
begomoviruses with SiGMoV. CdTV-[B52]: Chino del tomato virus-[B52], CdTV-[IC]: Chino del tomato virus-[IC], , PYMTV-TT: Potato yellow mosaic Trinidad virus,, PYMV-VE: Potato yellow mosaic virus- Venezuela, SiGMCRV: Sida golden mosaic Costa Rica virus, SiGMHV: Sida golden mosaic Honduras virus, SiGMHV*: B Strain of Sida golden mosaic Honduras virus, SiYVV: Sida yellow vein virus, SiGMHV-YV: Sida golden mosaic Honduras virus-yellow vein, BDMV: Bean dwarf mosaic virus, SiGMV: Sida golden mosaic virus, SiGMoV: Sida golden mottle virus, , ToMoV-[FL]: Tomato mottle virus-Florida, ToMoTV: Tomato mottle Taino virus, AbMV-HW: Abutilon mosaic virus-HW, AbMV: Abutilon mosaic virus. * Virus is in gene bank but there is no acronym for it.
27
Discussion
A new begomovirus, Sida golden mottle virus (SiGMoV) was isolated and
characterized from S. santaremensis showing golden mosaic symptoms in the leaves. The
nucleotide sequence identity of the CR of SiGMoV between DNA-A and DNA-B is
95.2%. The CR identity between DNA-A and DNA-B have been reported as low as
80.0% [26], also SiGMoV was able to infect and cause golden mosaic symptoms in S.
santaremensis by biolistic inoculation. This suggests that the DNA-A and DNA-B are
those of the same virus.
Based on the results of phylogenetic analysis, nucleotide sequence and amino acid
sequence comparisons, the SiGMoV DNA-A sequence was unique and was not clustered
with any characterized begomovirus DNA-A sequence. Using the same analyses, the
SiGMoV DNA-B sequence clustered with and shared a theoretical common ancestor with
viruses in the AbMV group.
The data obtained from host range study showed different results depending on
method used. By biolistic inoculation, SiGMoV was able to infect L. esculentum, P.
vulgaris, G. hirsutum, N. benthamiana, and N. tabacum. However, by whitefly
inoculation using SiGMoV-infected S. santaremensis, SiGMoV was able to infect N.
tabacum and at a lower rate of transmission than by biolistic inoculation. The method
used to inoculate may influence the apparent host range. In biolistic inoculation a
concentrated reproductive form of the geminivirus, double-stranded linear DNA, is bound
to gold particles which are delivered directly into a variety of host plant cells using high
velocity. The efficiency of biolistic inoculation is dependent upon the purity and the
concentration of the DNA, and other parameters which are under the control of the
researcher. However in whitefly transmission, a virion, containing a single-stranded
28
circular DNA, is delivered into phloem parenchyma cells by the whitefly stylet. The
efficiency of whitefly transmission is dependent upon the virus titer of the inoculum
source plant, the distribution of virus within the source plant, and the feeding preference
of the whiteflies, all of which are difficult to control by the researcher. These differences
may explain the discrepancy between the results obtained using the two inoculation
methods. These results indicate that SiGMoV can replicate in six plant species.
However, it is not clear whether the whiteflies are able to transmit SiGMoV from S.
santaremensis to these hosts.
There is as yet no reported economic significance of SiGMoV. Even though
SiGMoV is able to replicate in bean, tomato, cotton, and tobacco, no epidemics in these
crops have been reported. This could be because whiteflies are not able to acquire and
transmit SiGMoV from S. santaremensis to these crops. This could also be due to a
limited geographic distribution of SiGMoV. The geographic distribution of SiGMoV has
not been determined, but may be limited as S. santaremensis, the only known natural host
of SiGMoV, has only been found in two counties in Florida. However, since SiGMoV
was able to replicate in several hosts, there is a potential for SiGMoV to become a
pathogen. The ability of whiteflies to acquire and transmit SiGMoV, a more extensive
host range, and the geographic distribution of SiGMoV need to be established in order to
better assess the potential of SiGMoV to cause crop losses.
CHAPTER 3 AN EPIDEMIC IN TOMATO CAUSED BY VARIANTS OF Sida golden mosaic virus
A tomato field near Citra, FL was 100% infected with a virus that produced
symptoms identical to those caused by Tomato yellow leaf curl virus (TYLCV), a
begomovirus found throughout Florida [53]. However, there was no identifiable source of
TYLCV. This study was undertaken to identify the virus causing the symptoms in tomato
and identify the source of the virus.
Materials and Methods
Sample Source
Samples were collected from symptomatic plants of tomato and S. acuta growing in
and around the tomato field. S. acuta was identified to species by curators at the Florida
Museum of Natural History, University of Florida, Gainesville, FL.
PCR Analysis and Restriction Analysis
DNA was extracted from symptomatic plants of S. acuta [54]. The DNA was then
used as a template for polymerase chain reaction (PCR). Degenerate primers, PAR1c496
/PAL1v1978, which amplify an ~1100 bp fragment of the DNA-A of most bipartite
begomovirus and an ~1300 bp fragment from most monopartite begomovirus [56] were
used to amplified the DNA-A. This fragment includes the 3´ end of the putative Coat
Protein gene (CP), the common region (CR), and part of the putative Replication
Association Protein gene (Rep) [56]. Degenerate primers PBL1240/PCRc154 which
amplifies an ~600 bp fragment of the begomovirus DNA-B which includes the 3´ end of
29
30
the putative Nuclear Shuttle Protein gene (NSP) and almost the entire CR [56, 65] were
also used amplified the DNA-B.
The amplicons obtained with the described primers were restricted using AluI,
EcoRI, BglI, BglII, ApaI, and NcoI restriction enzymes and compared with a predicted
restriction map of SiGMV generated by Vector NTI software (Infomax, Frederick, MD).
Cloning
One partial SiGMV variant was obtained from S. acuta. Six partial sequences of
DNA-A and four partial sequences of DNA-B were obtained from tomato. The partial
sequences were cloned using pGEM®-T easy vector system (Promega, Madison, WI,
USA 53711) and sequenced.
Gap and Blast Analysis
The partial begomovirus sequences obtained from S. acuta and tomato were
compared using Gap method in Wisconsin package program (GCG). Sequence
comparisons were made by NCBI BLAST using the NCBI taxonomy database (
http://www.ncbi.nlm.nih.gov/ ). The CR of the partial DNA-A and DNA-B sequences
were determined and compared. In partial DNA-B the CR were missing at least two
nucleotides.
Phylogenetic Analysis
The partial DNA-A and DNA-B sequences were used in phylogenetic analysis. The
first 12-14 begomoviruses generated by Blast were also compared with these sequences
and a phylogenic tree was constructed for DNA-A, DNA-B. [2]. The comparison were
based on maximum parsimony using the PAUP*s heuristic method with the bisection-
reconnecting branch swapping The Bootstrap value was set to be based on 500 replicates.
Display tree was with no rooting using the midpoint rooting option.
31
Results
Even though the symptoms in tomato closely resembled those of TYLCV, the
presence of 1254 bp to 1295 bp fragments amplified by the degenerate primers
PAR1c496 /PAL1v1978 and 616 bp to 639 bp fragments amplified by the degenerate
primers PBL1240/PCRc154 suggested the presence of a bipartite begomovirus. After
obtaining the partial begomovirus sequences isolated from tomato and S. acuta, they were
compared with each other, with SiGMV, and with known begomoviruses.
The presence of S. acuta with SiGMV-like symptoms and high population of
whitefly vector in and around the field suggested a possible role of SiGMV in the
epidemic. A comparison of restriction enzyme patterns of DNA-A and DNA-B fragments
amplified from S. acuta and tomato with the predicted restriction sites of SiGMV,
indicated that the fragments amplified from S. acuta and tomato were very similar to
those of SiGMV. There were 6 restriction enzyme sites predicted from SiGMV DNA-A
and 5 to 7 of these sites were found in DNA-A fragments amplified from S. acuta and
tomato. There were 4 restriction enzyme sites predicted from SiGMV DNA-B and 3 to 3
of these sites were found in DNA-B fragments amplified from S. acuta and tomato.
Partial Sequence Analysis from Tomato and S. acuta
The partial nucleotide sequences amplified from S. acuta are shown for DNA-A
(Fig. 3-1) and DNA-B (Fig.3-7). The five DNA-A partial sequences amplified from
tomato are presented in (Fig 3-2 – 3-6) and the four DNA-B partial sequences are
presented in (Fig. 3-8 and 3-11).
There were no significant differences among the five DNA-A sequences amplified
from tomato that ranged from 97.9% to 98.7% (Table 3-1). There were no significant
32
differences between the DNA-A sequences amplified from tomato and that amplified
from S. acuta or SiGMV that ranged from 94.6% to 98.4% (Table 3-1).
However, the nucleotide sequence identities among the four DNA-B sequences
amplified from tomato and the one sequence from S. acuta were more variable than those
of the DNA-A sequences, and ranged from 67.7% to 99.2% (Table 3-2).
The analysis of the CR sequences of the partial sequences amplified from tomato
and S. acuta showed some differences among the DNA-A sequences that ranged from
93.2% to 100% (Table 3-3) and among the DNA-B sequences that ranged from 94.5%-
99.3% (Table 3-4). There were also no significant differences found between DNA-A and
DNA-B CR sequences that ranged from 93.8% to 98.6% (Table 3-4). A comparison of
the CR of the partial sequences (DNA-A and DNA-B) amplified from tomato and S.
acuta and that of SiGMV DNA-A showed some differences that ranged from 91.7% to
95.9% (Table 3-4). The same was observed with SiGMV DNA-B CR that range from
94.5% to 96.6% (Table 3-4).
The sequence analysis of DNA-A partial sequences amplified from tomato and S.
acuta and characterized begomoviruses shows some similarity between partial sequences
amplified from tomato and S. acuta and Tomato mottle virus—[Florida] that ranged from
86.4%-87% (Table 3-5).
The sequence analysis of DNA-B partial sequences amplified from tomato and S.
acuta and characterized begomoviruses showed a variable similarity among partial
sequences amplified from tomato and S. acuta with characterized begomovirus which can
be divided into two groups. The first group shared some similarity with Tomato mottle
virus- Florida with similarity of 76.1% (Table 3-6). The second group shared some
33
similarity with Sida golden mosaic Costa Rica virus that ranged from 76.4%-77.9%
(Table 3-6).
Phylogenetic Analysis
SiGMV strains DNA-A and SiGMV DNA-A cluster with Ablution mosaic virus
group (Fig. 3-12).
In the DNA-B Phylogenetic analysis, the SiGMV sequences are divided into two
groups: the first group includes T12-C3B and T12-C9B that cluster with ToMoV-{FL]
and Tomato mottle Taino virus group (Fig. 3-13).
The other group includes T12-C5B and T12-C7B, SiGMV, and S3-C4B that
clusters with SiGMVRV and BDMV group (Fig. 3-13).
1 TCTTGAATCA CCTTCTACTA TGAGACTTAA TGGTCTGTCT GGCCGCGCAG 51 CGGAACCTGT TCCAAAAAAT TTATCCGCCC ACTCTTGCAT CTCGTCGGGA 101 ACGTTAGTGA AAGAGGAGAG TTGAAATGGA GGAACCCACG GTTCCGGAAC 151 CTTAGCGAAT ATCCTCTCTA AGTTGGAGCG GATGTTATGA TTCTGCAAGA 201 CAAAATCCTT TGGCTGTTCT TCCCTTAAAA CCGCTAAGGC AGATTGAACA 251 GAATCTGCAT TTAACGCCTT GGGCATATGA ATCATTAGCA GTCTGCGGGC 301 CTCCTCTAGC TGATCTGCCG TCGATCTGGA ATTCTCCCCA TTCCAGTGTA 351 TCACCGTCCT TGTCGATGTA GGACTTGTCC GTCGGAGCTG GATTTAGCTC 401 CCTGNTATGT TTGGATGGAA ATGTGCTGAC CTGGTTGGGG AGACCAGATC 451 GAAGAATCTG TTATTCTTGC ACTGATATTT CCCTTCGAAC TGTATGAGCA 501 CATGGAGATG AGGCTCCCCA TTCTCGTGAA GCTCTCTGCA GATTTTGATG 551 AACTTCTTGT TCACTGGGGT ATTTAGGCTT TGTATTGGGA AAGTGCTTCT 601 TCTTTAGTCA GAGAGCACTG GGGATATGTG AGGAAATAGT TTTTGGACTG 651 AACTCGAAAT TTCTTTGGCG GGGGCATTTT TGTAATAAGA AGTGGGACTC 701 CAGTTGAGGT ACTCTAATTG AGCCCTCTCA AACTTGCTCA TTCAATTGGA 751 GTATTAGAGT CTCATATATA GTAGAACCCT CTATAGAACT CTCAATCTGG 801 TTCACACACG TGGCGGCCAT CCGGATATAG TATTACCGGA TGGCCGCGCG 851 CCCCCCCTGG TGCCGTACAC TCTCGCGCGA TCTTTAATTT CAATTAAAGA 901 TGGTCCCAGA CGCTCTCGTC CAATCAGGTC GCGTCTGACG AGTCTAGATA 951 TTTGCAACAA CTTGGGCCCT AAGTTGTTGG GTGTCTGCTA TAAATGAAAG 1001 AGACTTTGGC CCACTGCTTT TAACTCAAAA TGCCTAAGCG CGATTTGCCA 1051 TGGCGCTCTA TGGCGGGAAC CTCAAAGGTT AGCCGCAACG CTAACTATTC 1101 TCCTCGTGGA GGTAGTGGGC ACAAGAGTTA ACAAGGCCTC TGAATGGGTG 1151 AACAGG Figure 3-1. Partial sequence of DNA-A (S3-C7A) amplified from Sida acuta collected
from Citra Field, Florida.
34
1 GCCCACATTG TCTTTCCAGT GTCTTCCCCA TGTACAGAAA GCCATGCAGT 51 ATTATCTTCC CCGTTGCATC TGCAGGCCCA CATTGTCTTT CCTGTTCTTG 101 AATCACCTTC TACTATGAGA CTTAATGGTC TGTCTGGCCG CGCAGCGGAA 151 CCTGTTCCAA AAAATTCATC CGCCCACTCT TGCATCTCGN TCGGGAACGT 201 TAGTGAAAGA GGAGAGTTGA AATGGAGGAA CCCACGGNTT CCGGAACCTT 251 AGCGAATATC CTCTCTAAGT TGGAGCGGAT GTTATGATTC TGCAAGACAA 301 AATCCTTTGG CTGTTCTTCC CTTAAAACCG CTAAGGCAGA TTGAACAGAA 351 TCTGCATTTA ACGCCTTGGG CATATGAATC ATTAGCAGTC TGCGGGCCTC 401 CTCTCGCTGA TCTGCCGTCG ATCTGGAATT CTCCCCATTC CAGTGTATCA 451 CCGTCCTTGT TCGATGTAGG ACTTGACGTC GGAGCTGGAT TNTAGCTCCC 501 TGTTATGTTT GGATGGAAAT GTGCTGACCT GGTTGGGGAG ACCAGATCGA 551 AGAATCTGTT ATTCTTGCAC TGATATTTCC CTTCGAACTG TATGAGCACA 601 TGGAGATGAG GCTCCCCATT CTCGTGAAGC TCTCTGCAGA TTTTGATGAA 651 CTTCTTGTTC ACTGGGGTAT TAAGGCTTTG TAATNGGGAA AAGTGCTTCT 701 TCTTTAGTCA GAGAGCACTG GGGATATGTG AGGAAATAGT TTTTGGACTG 751 AACTCGAAAT TTCNTTTGCG GTGGCATTTT TGTAATAATG AGTGGGACTC 801 CAGTTGAGGT ACTCCAATTG AGCCCTCTCA AACTTGCTCA TTCAATTGGA 851 GTATTAGAGT CTCATATATA GTAGAACCCT CTATAGAACT CTCAATCTGG 901 TTCNCACACG TGGCGGCCAT CCGCTATAAT ATTACCGGAT GGCCGCGCGC 951 CCCCCCTGGT GCCGTACACT CTCGCGCGAT CTTTAATTTC AATTAAAGAT 1001 GGTCCCAGAC GCTCTCGTCC AATCAGGTCG CGTCTGACGA GTCTAGATAT 1051 TTGCAACAAC TTGGGCCCTA AGTTGTTGGG TGTCTGCTAT AAATGAAAGA 1101 TACTTTGGCC CACTGCTTTT AACTCACAAT GCCTAAGCGC GATTTGCCAT 1151 GGCGCTCTAT GGCGGGAACC TCAAAGGTTA GCCGCAACGC TAACTATTCT 1201 CCTCGTGGAG GTAGTGGGCC AAGAGTTAAC AAGGCCTCTG AATGGGTGAA 1251 CAGG Figure 3-2. Partial sequence of DNA-A (T3-C8A) amplified from tomato plant collected
from Citra Field, Florida
1 TCTTGAATCA CCTTCTACTA TGAGACTTAA TGGTCTGTCT GGCCGCGCAG 51 CGGAACCTGT TCCAAAAAAT TCATCCGCCC ACTCTTGCAT CTCGTCGGGA 101 ACGTTAGTGA AAGAGGAGAG TTGAAATGGA GGAACCCACG GTTCCGGAAC 151 CTTAGCGAAT ATCCTCTCTA AGTTGGAGCG GATGTTATGA TTCTGCAAGA 201 CAAAATCCTT TGGCTGTTCT TCCCTTAAAA CCGCTAAGGC AGATTGAACA 251 GAATCTGCAT TTAACGCCTT GGCATATGAA TCATTAGCAG TCTGCGGGCC 301 TCCTCTCGCT GATCTGCCGT CGATCTGGAA TTCTCCCCAT TCCAGTGTAT 351 CACCGTCCTT GTCGATGTAG GACTTGACGT CGGAGCTGGA TTTAGCTCCC 401 TGTTATGTTT GGATGGTAAT GTGCTGACCT GGTTGGGGAG ACCAGATCGA 451 AGAATCTGTT ATTCTTGCAC TGATATTTCC CTTCGACTGT ATGAGCACAT 501 GGAGATGAGG CTCCCCATTC TCGTGAAGCT CTCTGCAGAT TTTGATGAAC 551 TTCTTGTTCA CTGGGGGTAT TTAGGCTTTG TAAATTGGGA AAGTGCTTCT 601 TCTTTAGTCA GAGAGCACTG GGGATATGTG AAGGAAATAG TTTTTGGACT 651 GAACTCCAAA ATTNCTTTGG CGGGGGCATT TTTGTAATAA TGAGTGGGAC 701 TCCAGTTGAG GTACTCCAAT TGAGCCCTCT CAAACTTGCT CATTCAATTG 751 GAGTATTAGA GTCTCATATA TAGTAGAACC CTCTATAGAA CTCTCAATCT 801 GGTTCACACA CGTGGCGGCC ATCCGCTATA ATATTACCGG ATGGCCGCGC 851 GCCCCCCCTG GTGCCGTACA CTCTCGCGCG ATCTTTAATT TCAATTAAAG 901 ATGGTCCCAG GCGCTCTCGT CCAATCAGGT CGCGTCTGAC GAGTCTAGAT 951 ATTTGCAACA ACTTAGGGCC CAAGTTGTTT GGTGTCTGCT ATAAATGAAA 1001 GAGACTTTGG CCCACTGCTT TTAACTCAAA ATGCCTAAGC GCGATTTGCC 1051 ATGGCGCTCT ATGGCGGGAA CCTCAAAGGT TAGCCGCAAC GCTAACTATT 1101 CTCCTCGTGG AGGTAGTGGG CACAAGAGTT AACAAGGCCT CTGAATGGGT 1151 GAACAGG Figure 3-3. Partial sequence of DNA-A (T5-C2A) amplified from tomato plant collected
from Citra Field, Florida
35
1 CTTGAATCAC CTTCTACTAT GAGACTTAAT GGTCTGTCTG GCCGCGCAGC 51 GGAACCTGTT CCAAAAAATT CATCCGCCCA CTCTTGCATC TCGTCGGGAA 101 CGTTTGTGAA AGAGGAGAGG TGAAATGGAG GAACCCACGG TTCCGGAACC 151 TTAGCGAATA TCCTCTCTAA GTTGGAGCGG ATGTTATGAT TCTGCAAGAC 201 ATAATCTTTT GGCTGTTCTT CCCTTAAAAC CGCTAAGGCA GATTGAACAG 251 AATCTGCATT TAACGCCTTG GCATATGAAT CATTAGCAGT CTGCGGGCCT 301 CCTCTAGCTG ATCTGCCGTC GATCTGGAAT TCTCCCCATT CCAGTGTATC 351 ACCGTCCTTG TCGATGTAAG ACTTGACGTC GGAGCTGGAT TTAGCTCCCT 401 GTATGTTTGG ATGGAAATGT GCTGACCTGG TTGGGGAGAC CAGATCGAAG 451 AATCTGTTAT TCTTGCACTG ATATTTCCCT TCGAACTGTA TGAGCACATG 501 GAGATGAGGC TCCCCATTCT CGTGAAGCTC TCTGCAGATT TTGATGAACT 551 TCTTGTTCAC TGGGGTATTT AGGCTCTGTA ATTGGGAAAG TGCTTCTTCT 601 TTAGTCAGAG AGCACTGAGG ATATGTTAGG AAATAGTTTT TGGACTGAAC 651 TCGAAGTTTC TTCGGCGGTG GCATTTTTGT AATAAGAAGT GGTACTCCAG 701 TTGAGGTACT CCAATTGATC CCTCTCAAAC TTGCTCATTC AATTGGAGTC 751 TAGAGTCTCA TATATAGTAG AACCCTCTAT AGAACTCTCA ATCTGGTTCA 801 CACACGTGGC GGCCATCCGC TATAATATTA CCGGATGGCC GCGCGCCCCC 851 CCTGGTGCCG TACACTCTCG CGCGATCTTT AATTTCAATT AAAGATGGTC 901 CCAGACGCTC TCGTCCAATC AGGTCGCGTC TGACGAGTCT AGATATTTGC 951 AACAACTTGG GCCCTAAGTT GTTGGGTGTC TGCTATAAAT GAAAGAGACT 1001 TTGGCCCACT GCTTTTAACT CAAAATGCCT AAGCGCGATT TGCCATGGCG 1051 CTCTATGGCG GGAACCTCAA AGGTTAGCCG CAACGCTAAC TATTCTCCTC 1101 GTGGAGGTAG TGGGCCAAGA GTTAACAAGG CCTCTGAATG GGTGAACAGG 1151 CCCATGTACA GAAAGCCCTG CAGTATTAAT CACTAGTGAA TTCGC Figure 3-4. Partial sequence of DNA-A (T10-C8A) amplified from tomato plant collected
from Citra Field, Florida
1 TCTTGAATCA CCTTCTACTA TGAGACTTAA TGGTCTGTCT GGCCGCGCAG 51 CGGAACCTGT TCCAAAAAAT TCATCCGCCC ACTCTTGCAT CTCGTCGGGA 101 ACGTTAGTGA AAGAGGAGAG GTGAAATGGA GGAACCCACG GTTCCGGAAC 151 CTTAGCGAAT ATCCTCTCTA AGTTGGAGCG GATGTTATGA TTCTGCAAGA 201 CAAAATCTTT TGGCTGTTCT TCCCTTAAAA CCGCTAAGGC AGATTGAACA 251 GAATCTGCAT TTAACGCCTT GGCATATGAA TCATTAGCAG TCTGCGGGCC 301 TCCTCCAGCT GATCTGCCGT CGATCTGGAA TTCTCCCCAT TCCAATGTAT 351 CACCGTCCTT GTCGATGTAG GACTTGACGT CGGAGCTGGA TTTAGCTCCC 401 TGTATGTTTG GATGGAAATG TGCTGACCTG GTTGGGGAGA CCAGATCGAA 451 GAATCTGTTA TTCTTGCACT GATATTTCCC TTCGAACTGT ATGAGCACAT 501 GGAGATGAGG CTCCCCATTC TCGTGAAGCT CTCTGCAGAT TTTGATGAAC 551 TTCTTGTTCA CTGGGGTATT TAGGCTTTGT AATTGGGAAA GTGCTTCTTC 601 TTTAGTCAGA GAGCACTGGG GATATGTGAG GAAATAGTTT TTGGACTGAA 651 CTCGAAATTT CTTTGGCGGT GGCATTTTTG TAATAATGAG TGGGACTCCA 701 GTTGAGGCAC TCCAATTGAG CCCTCTCAAA ACTTGCTCAT TCAATTGGAG 751 TCTGGAGTCC CATATATACT AGAACCCTCT ATAGAACTCT CAATCTGGTT 801 CGCACACGTG GCGGCCATCC GCTATAATAT TACCGGATGG CCGCGCGCCC 851 CCCCTGGTGC CGTACACTCT CGCGCGATCT TTAATTTCAA TTAAAGATGG 901 TCCCAGACGC TCTCGTCCAA TCAGGTCGCG TCTGACGAGT CTAGATATTT 951 GCAACAACTT GGGCCCTAAG TTGTTGGGTG TCTGCTATAA ATGAAAGAGA 1001 CTTTGGCCCA CTGCTTTTAA CTCAAAATGC CTAAGCGCGA TTTGCCATGG 1051 CGCTCTATGG CGGGAACCTC AAAGGTTAGC CGCAACGCTA ACTATTCTCC 1101 TCGTGGAGGT AGTGGGCCAA GAGTTATCAA GGCCTCTGAA TGGGTGAACA 1151 GG Figure 3-5. Partial sequence of DNA-A (T10-C10A) amplified from tomato plant
collected from Citra Field, Florida
36
1 TCTTGAATCA CCTTCTACTA TGAGACTTAA TGGTCTGTCT GGCCGCGCAG 51 CGGAACCTGT TCCAAAAAAT TCATCCGCCC ACTCTTGCAT CTCGTCGGGA 101 ACGTTAGTGA AAGAGGAGAG TTGAAATGGA GGAACCCACG GTTCCGGAAC 151 CTTAGCGAAT ATCCTCTCTA AGTTGGAGCG GATGTTATGA TTCTGCAAGA 201 CGAAATCCTT TGGCTGTTCT TCCCTTAAAA CCGCTAAGGC AGATTGAACA 251 GAATCTGCAT TTAACGCCTT GGCATATGAA TCATTAGCAG TCTGCGGGCC 301 TCCTCTCGCT GATCTGCCGT CGATCTGGAA TTCTCCCCAT TCCAGTGTAT 351 CACCGTCCTT GTCGATGTAG GACTTGACGT CGGAGCTGGA TTTAGCTCCC 401 TGTATGTTTG GATGGAAATG TGCTGACCTG GTTGGGGAGA CCAGATCGAA 451 GAATCTGTTA TTCTTGCACT GATATTTCCC TTCGAACTGT ATGAGTACAT 501 GGAGATGAGG CTCCCCATTC TCGTGAAGCT CTCTGCGGAT TTTGATGAAT 551 TTCTTGTTCA CTGGGGTATT TAGGCTTTGT AATTGGGAAA GTGCTTCTTC 601 TTTAGTCAGA GAGCACTGGG GATATGTGAG GAAATAGTTT TTGGACTGAA 651 CTCGAAATTT CTTAGGCGGT GGCATTTTTG TAATAAGAAG TGGTACTCCA 701 GTTGAGGTAC TCCAATTGAG CCCTCTCAAA CTTGCTCATT CAATTGGAGT 751 CTGGAGTCTC ATATATAGTA GAACCCTCTA TAGAACTCTC AATCTGGTTC 801 ACACACGTGG CGGCCATCCG CTATAATATT ACCGGATGGC CGCGCGCCCC 851 CCTTGGTGCC GTACACTCTC GCGCGATCTT TAATTTCAAT TAAAGATGGT 901 CCCAGACGCT CTCGTCCAAT CAGGTCGCGT CTGACGAGTC TAGATATTTG 951 CAACAACTTG GGCCCTAAGT TGTTGGGTGT CTGCTATAAA TGAAAGAGAG 1001 TTTGGCCCAC TGCTTTTAAC TCAAAATGCC TAAGCGCGAT TTGCCATGGC 1051 GCTCTATGGC GGGAACCTCA AAGGTTAGCC GCAACGCTAA CTATTCTCCT 1101 CGTGGAGGTA GTGGGCCAAG AGTAAACAAG GCCTCTGAAT GGGTGAACAG 1151 G Figure 3-6. Partial sequence of DNA-A (T12-C6A) amplified from tomato plant collected
from Citra Field, Florida
1 GCTACGACTC AGTCTAGCTG TCAACTGCGA CGCCGTCGAC GGGAATTGCA 51 GAATTATCTC AGTTAGGTCA TGGGAAAGTT GATACTCGTC CCGGTGCGAC 101 TCTATGTAGT TGAAGGCACT CGGAGGATTT ACTAACTGAG ATTCCATTTG 151 AAGAAGAAAG GCCGCGCAGC GGAACCGATT GCTGAAGTTG AATCGGAAAA 201 AAGATGTCAG GAATTCTCGT GAAGAACAGT ATTTGAACCC TTGTTGAAGA 251 TGAACACTTT TTCTGGGAAA CCCAGAAAGT TGGTGAAGAA GTTGAGGAAC 301 ACTCGTCTAA CCTCTTATGA AAGTGGGTGG GTTGTTGAGA AAGAGGAGAA 351 ATCTGGTGAT GAAAGTTTAG GATGATAGTG AGTTAGATCT GGTAGTGTCT 401 ATAAATAGAC CCAGATTTTA TGTTGTTGGT AAAGAACGTC TATGAGAAGT 451 TTTTACTTCT GTTTAATGGC ATTTTTGTAA TAATGAGTGG GACTCCAGTT 501 GAGGTACTCC AATTGAGCCC TCTCAAACTT GCTCATTCAA TTGGAGTATT 551 AGAGTCTCAT ATATAGTAGA ACCCTCTATA GAACTCTCAA TCTGGTTCAC 601 ACACGTGGCG GCCATCCGA Figure 3-7. Partial sequence of DNA-B (S3-C4B) amplified from Sida acuta collected
from Citra Field, Florida
37
1 GCTACGACTG AGCCTCGCCG TCAACTGCGA CGCCGTGGAA GGAAATTGCA 51 GTATTATCTC AGTTAGGTCA TGTGAAAGCT GATATTCGTC CCGGTGAGAT 101 TCTATGTAAT TGAAAGCGTT CGGAGGATTA ACTAACTGAG AATCCATATG 151 AGGAAGAAAG GCCGCGCAGC GGAACCGATT GCTGAAGTTG AATCGGGAAG 201 AAGATGAACA ACTGATGAAC AGGACGAACA GTGTTCGATG GCTGAGTTTA 251 GATCTCGAAG AAGGTAAAGG CGTAACTTTG TTTCTGTGTT TGAGAGTGTC 301 GGATCTTTCT GACAGTTACT GTTTAGAAGA TTTAAGAACG AAAATTTGTT 351 TAACCCTTGA TGTTTATGAG AAAGAAAGGA GTGTTGATGA ATAATTTGGG 401 AGAATTCTGG AAATGAAGTA GTTTGTGTAT GAACCCAGAA CTTCTGGGTT 451 GACGGGTATT TAAAATGGGA AAGGGTTCAT CAACCGGTGG CATTCTTGTA 501 ATAATGAGTG GGACTCCAGT TGAGGTACTC CAATTGATCC CTCTCAAACT 551 TGCTCATTCA ATTGGAGTCT AGAGTCTCAT ATATAGTAGA ACCCTCTATA 601 GAACTCTCAA TCTGGTTCAC ACACGTGGCG GCCATCCGA Figure 3-8. Partial sequence of DNA-B (T12-C3B) amplified from tomato plant collected
from Citra Field, Florida
1 GCTACGACTC AGCCTCGCCG TCAACTGCGA CGCCGTCGAC GGAAATTGCA 51 GAATTATCTC AGTTAGGTCA TGGGAAAGTT GATACTCGTC CCGGTGAGAC 101 TCTATGTAGT TGAAGGCGCT CGGAGGATTT ACTAACTGAG ATTCCATTTG 151 AAGAAGAAAG GCCGCGCAGC GGAACCGATT GCTGAAGTTG AATCGGGAAA 201 AGATGTCAAG AATTCTCGTG AAGAACAGTA TATGAACCCC CCTTGAAGAT 251 GAACACTTTT TCTGGGAAAC CCAGAAAGTT GGTGAAGAAG TTGAGGAACA 301 CTTGTCTAAC CTCTCTTGAA AGTGGGTGTG TTGTTGAGAA AGAGGAGAAA 351 TCTGGTGATG AAAATGAGGA TGATAGTGAG TTAGATCTGG TAGTGTCTAT 401 AAATAGACCC AGATATTATG TTGTTGGTAA AGAACGTCTA TGAGAAGTTT 451 TTACTTCTGT TCAATGGCAT TTTTGTAATA AGAAGTGGTA CTCCAGTTGA 501 GGTACTCCAA TTGAGCCCTC TCAAACTTGC TCATTCAATT GGAGTCTGGA 551 GTCTCATATA TAGTAGAACC CTCTATAGAA CCCTCAATCT GGTTCACACA 601 CGTGGCGGCC ATCCGA Figure 3-9. Partial sequence of DNA-B (T12-C5B) amplified from tomato plant collected
from Citra Field, Florida
1 GCTACGACTG AGCCTCGCCG TCAACTGCGA CGCCGTCGAC GGAAATTGCA 51 GAATTATCTC AGTTAGGTCA TGGGAAAGTT GATACTCGTC CCGGTGAGAC 101 TCTATGTAGT TGAAGGCGCT CGGAGGATTT ACTAACTGAG ATTCCATTTG 151 AAGAAGAAAG GCCGCGCAGC GGAACCGATT GCTGAAGTTG AATCGGGAAA 201 AGATGTCAAG AATTCTCGTG AAGAACAGTA TATGAACCCC CCTTGAAGAT 251 GAACACTTTT TCTGGGAAAC CCAGAAAGTT GGTGAAGAAG TTGAGGAACA 301 CTTGTCTAAC CTCTCTTGAA AGTGGGTGTG TTGTTGAGAA AGAGGAGAAG 351 TCTGGTGATG AAAATGAGGA TGATAGTGAG TTAGATCTGG TAGTGTCTAT 401 AAATAGACCC AGATATTATG TTGTTGGTAA AGAACGTCTA TGAGAAGTTT 451 TTACTTCTGT TCAATGGCAT TTTTGTAATA AGAAGTGGTA CTCCAGTTGA 501 GGTACTCCAA TTGAGCCCTC TCAAACTTGC TCATTCAGTT GGAGTCTGGA 551 GTCTCATATA TAGTAGAACC CTCTATAGAA CTCTCAATCT GGTTCACACA 601 CGTGGCGGCC ATCCGT Figure 3-10. Partial sequence of DNA-B (T12-C7B) amplified from tomato plant
collected from Citra Field, Florida
38
1 GCTACGACTG AGCCTCGCCG TCAACTGCGA CGCCGTGGAA GGAAATTGCA 51 GTATTATCTC AGTTAGGTCA TGTGAAAGCT GATATTCGTC CCGGTGAGAT 101 TCTATGTAAT TGAAAGCGTT CGGAGGATTA ACTAACTGAG AATCCATATG 151 AGGAAGAAAG GCCGCGCAGC GGAACCGATT GCTGAAGTTG AATCGGGAAG 201 AAGATGAACA ACTGATGAAC AGGACGAACA GCGTTCGATG GCTGAGTTTA 251 GATCTCGAAG AAGGTAAAGG TATAACTTTG TTTCTGTGTT TGAGAGTGTC 301 GGATCTTTCT GACAGTTACT GTTTAGAAGA TTTAAGAACG AAAATTTGTT 351 CAACCCTTGA TGTTTATGAG AAAGAAAGGA GTGTTGATGA ATAATTTGGG 401 AGAATTCTGG AAATGAAGTA GTTTGTGTAT GAACCCAGAA CTTCTGGGTT 451 GACGGGTATT TAAAATGGGA AAGGGTTCAT CAACCGGTGG CATTCTTGTA 501 ATAATGAGTG GGACTCCAGT TGAGGTACTC CAATTGATCC CTCTCAAACT 551 TGCTCATTCA ATTGGAGTCT AGAGTCTCAT ATATAGTAGA ACCCTCTATA 601 GAACTCTCAA TCTGGTTCAC ACACGTGGCG GCCATCCGT Figure 3-11. Partial sequence of DNA-B (T12-C9B) amplified from tomato plant
collected from Citra Field, Florida
Table 3-1. The nucleotides identity of partial sequences of SiGMV DNA-A isolated from tomato and Sida collected from Citra Field
T3-C8A
T5-C2A
T10-C8A
T10-C10A
T12-C6A
S3-C7A
SiGMV-A
T3-C8A 100% 97.9% 98.3% 98.7% 98.5% 97.8% 96.0% T5-C2A 100% 97.9% 98.3% 98.2% 98.5% 95.9% T10-C8A 100% 98.3% 98.3% 98.4% 94.6% T10-C10A
100% 98.2% 98.4% 95.9%
T12-C6A 100% 98.4% 95.9% S3- C7A 100% 96.2% SiGMV-A 100% T3-C8A: SiGMV DNA-A sequence from tomato 3 clone 8, T5-C2A: SiGMV DNA-A sequence from tomato 5 clone 2, T10-C8A: SiGMV DNA-A sequence from tomato 10 clone 8, T10-C10A: SiGMV DNA-A sequence from tomato 10 clone 10, T12-C6A: SiGMV DNA-A sequence from tomato 12 clone 6, S3-C7A: SiGMV DNA-A sequence from Sida 3 clone 7, SiGMV-A: Sida golden mosaic virus DNA-A Table 3-2. The nucleotides identity of partial sequences of SiGMV DNA-B isolated from
tomato and Sida collected from Citra Field: - S3-C4B T12-C3B T12-C5B T12-C7B T12-C9B SiGMV-B S3-C4B 100 67.9% 95.8% 95.3% 67.7% 96.1% T12-C3B 100 68.9% 68.9% 99.2% 68.0% T12-C5B 100 99.2% 68.7% 95.3% T12-C7B 100 69.0% 95.3% T12-C9B 100.0% 68.0% SiGMV-B 100.0% S3-C4B: SiGMV DNA-B sequence isolated from sida 3 clone 4, T12-C3B: SiGMV DNA-B sequence isolated from tomato 12 clone 3, T12-C5B: SiGMV DNA-B sequence isolated from tomato 12 clone 4, T12-C7B: SiGMV seqence of DNA-B isolated from tomato 12 clone 7, T12-C9B: SiGMV DNA-B sequence isolated from tomato 12 clone 9, SiGMV-B: Sida golden mosaic virus DNA-B
39
Table 3-3. The Common region nucleotides identity of SiGMV DNA-A sequences isolated from tomato and S. acuta
T3-C8A T5-C2A T10-C8A T10-C10A T12-C6A S3-C7A T3-C8A 100% 95.9% 95.9% 96.6% 97.3% T5-C2A 95.9% 95.9% 96.6% 97.3% T10-C8A 94.5% 99.3% 95.9% T10-C10A 95.2% 93.2% T12-C6A 96.6% T3-C8: SiGMV DNA-A sequence from tomato 3 clone 8, T5-C2: SiGMV DNA-A sequence from tomato 5 clone 2, T10-C8: SiGMV DNA-A sequence from tomato 10 clone 8, T10-C10: SiGMV DNA-A sequence from tomato 10 clone 10, T12-C6: SiGMV DNA-A sequence from tomato 12 clone 6, S3-C7: SiGMV DNA-A sequence from Sida 3 clone 7, SiGMV-A: Sida golden mosaic virus DNA-A. . SiGMV-B: Sida golden mosaic virus DNA-B
Table 3-4. The Common region nucleotides identity of SiGMV sequences isolated from tomato and S. acuta: -
S3-4B* T12-3B* T12-5B* T12-7B* T12-9B* SiGMV-A
SiGMV-B
T3-C8A 98.6% 97.3% 94.5% 94.5% 96.6% 95.2% 96.6% T5-C2A 98.6% 97.3% 94.5% 94.5% 96.6% 95.2% 96.6% T10-C8A 95.9% 96.6% 97.3% 97.3% 96.6% 95.2% 95.2% T10-C10A 95.2% 94.5% 93.8% 93.8% 94.5% 91.7% 93.2% T12-C6A 95.9% 95.2% 98.6% 98.6% 95.2% 95.9% 95.9% S3-C7A 97.3% 95.2% 95.9% 95.9% 95.2% 95.2% 96.6% S3-4B* 98.0% 96.0% 95.0% 97.3% 93.9% 95.9% T12-3B* 95.2% 94.5% 99.3% 91.8% 95.2% T12-5B* 98.0% 94.5% 93.8% 94.5% T12-7B* 95.2% 93.8% 94.5% T12-9B* 91.8% 95.2% SiGMV-A 93.9% * the CR miss at least two necleotides.T3-C8: SiGMV DNA-A sequence from tomato 3 clone 8, T5-C2: SiGMV DNA-A sequence from tomato 5 clone 2, T10-C8: SiGMV DNA-A sequence from tomato 10 clone 8, T10-C10: SiGMV DNA-A sequence from tomato 10 clone 10, T12-C6: SiGMV DNA-A sequence from tomato 12 clone 6, S3-C7: SiGMV DNA-A sequence from Sida 3 clone 7, S3-C4B: SiGMV DNA-B sequence isolated from sida 3 clone 4, T12-C3B: SiGMV DNA-B sequence isolated from tomato 12 clone 3, T12-C5B: SiGMV DNA-B sequence isolated from tomato 12 clone 4, T12-C7B: SiGMV seqence of DNA-B isolated from tomato 12 clone 7, T12-C9B: SiGMV DNA-B sequence isolated from tomato 12 clone 9, SiGMV-A: Sida golden mosaic virus DNA-A, and SiGMV-B: Sida golden mosaic virus DNA-B
40
Table 3-5. The nucleotide identity of partial sequences DNA-A sequences isolated from tomato and S. acuta at Citra, FL with begomoviruses generated by Blast Begomovirus ACC. No. T3-
C8A T5-C2A
T10-C8A
T10-C10A
T12-C6A
S3-C7A
SiGMV-A
ChTV-[IC] AF101476 82.8% 82.9% 83.1% 83.4% 83.2% 82.8% 82.2% AbMV X15983 83.9% 85.9% 84.6% 84.7% 84.2% 83.9% 84.5% ToMoV-[FL] L14460 86.6% 86.5% 86.5% 86.4% 86.7% 86.6% 87.6% ChTV - [H8] AF226664 81.5% 81.8% 81.8% 82.1% 82.7% 81.5% 82.0% ChTV - [H6] AF226665 81.6% 81.7% 82.0% 82.0% 82.6% 81.4% 81.9% AbMV -HW U51137 82.9% 82.4% 83.1% 83.0% 82.8% 82.7% 83.0% SiYVV Y11099 83.1% 82.8% 83.1% 83.1% 83.5 83.0% 83.2% ToMoTV AF012300 76.8% 76.7% 77.3% 77.3% 77.0% 76.5% 77.3% SiGMV-YV AJ577395 76.2% 76.3% 76.5% 76.7% 76.7% 76.0% 76.6% SiGMHV Y11097 79.8% 79.6% 79.3% 80.0% 79.9% 79.4% 79.3% SiGMCVR X99550 77.2% 76.9% 77.0% 79.0% 77.4% 76.8% 77.3% BDMV M88179 77.8% 77.6% 78.0% 81.0% 78.3% 77.6% 78.3% PYMTV-TT AF039031 78.3% 78.3% 77.8% 77.9% 78.1% 78.0% 78.1% ACC. NO. Accession number, T3-C8: SiGMV DNA-A sequence from tomato 3 clone 8, T5-C2: SiGMV DNA-A sequence from tomato 5 clone 2, T10-C8: SiGMV DNA-A sequence from tomato 10 clone 8, T10-C10: SiGMV DNA-A sequence from tomato 10 clone 10, T12-C6: SiGMV DNA-A sequence from tomato 12 clone 6, S3-C7: SiGMV DNA-A sequence from Sida 3 clone 7, SiGMV-A: Sida golden mosaic virus DNA-A, ChTV-[IC]: Chino del tomato virus-[IC], AbMV: Abutilon mosaic virus, ToMoV-[FL]: Tomato mottle virus-Florida, ChTV-[H6]: Chino del tomato virus-[H6], ChTV-[H8]: Chino del tomato virus-[H8], AbMV-HW: Abutilon mosaic virus-HW, SiYVV: Sida yellow vein virus, ToMoTV: Tomato mottle Taino virus, SiGMHV: Sida golden mosaic Honduras virus, SiGMCRV: Sida golden mosaic Costa Rica virus, BDMV: Bean dwarf mosaic virus, and PYMTV-TT: Potato yellow mosaic Trinidad virus- Trinidad and Tobago
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Table 3-6. The nucleotide identity of partial sequences DNA-B sequences isolated from tomato and S. acuta at Citra, FL with begomoviruses generated by Blast
Begomovirus ACC. No. T12-C3B
T12-C5B
T12-C7B
T12-C9B
S10-C4B
SiGMV-B
ChTV-[IC] AF101478 64.6% 68.9% 68.7% 64.3% 68.6% 67.8% AbMV X15984 76.2% 63.4% 63.4% 75.9% 62.4% 61.5% ToMoV-[FL] L14461 76.1% 66.6% 66.7% 76.1% 66.6% 66.9% SiGMHV-YV
AJ250731 62.2% 66.2% 65.7% 62.6% 65.1% 62.5%
SiGMV-YV Y11101 62.2% 66.1% 65.6% 62.7% 65.0% 62.3% AbMV-HW U51138 75.1% 61.6% 61.6% 75.0% 61.3% 60.3% SiYVV Y11100 62.1% 66.1% 65.7% 62.4% 65.0% 62.3% ToMoTV AF012301 68.6% 59.2% 59.3% 68.8% 59.4% 58.5% SiGMHV Y11098 62.8% 63.0% 62.7% 75.0% 65.6% 63.0% SiGMCRV X99551 60.2% 77.9% 77.9% 60.4% 76.4% 75.8% BDMV M88180 60.3% 75.1% 74.8% 60.3% 75.2% 74.3% PYMTV-TT AF039032 63.3% 64.9% 64.9% 64.4% 65.1% 63.7% S3-C4B: SiGMV DNA-B sequence isolated from sida 3 clone 4, T12-C3B: SiGMV DNA-B sequence isolated from tomato 12 clone 3, T12-C5B: SiGMV DNA-B sequence isolated from tomato 12 clone 4, T12-C7B: SiGMV seqence of DNA-B isolated from tomato 12 clone 7, T12-C9B: SiGMV DNA-B sequence isolated from tomato 12 clone 9, SiGMV-B: Sida golden mosaic virus DNA-B, ChTV-[IC]: Chino del tomato virus-[IC], AbMV: Abutilon mosaic virus, ToMoV-[FL]: Tomato mottle virus-Florida, SiGMHV-YV: Sida golden mosaic Honduras virus- yellow vein, SiGMV-YV: Sida golden mosaic- yellow vein , SiYVV: Sida yellow vein virus, ToMoTV: Tomato mottle Taino virus, SiGMHV: Sida golden mosaic Honduras virus, SiGMCRV: Sida golden mosaic Costa Rica virus, BDMV: Bean dwarf mosaic virus, and PYMTV-TT: Potato yellow mosaic Trinidad virus- Trinidad and Tobago
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Figure 3-12. Phylogenic tree of partial nucleotide sequence of DNA-A of selected
begomoviruses with the SiGMV and the SiGMV sequences isolated from tomato and S. acuta. PYMTV-TT: Potato yellow mosaic Trinidad virus- Trinidad and Tobago, SiGMHV: Sida golden mosaic Honduras virus, SiGMCRV: Sida golden mosaic Costa Rica virus, BDMV: Bean dwarf mosaic virus, SiYVV: sida yellow vein virus, ToMoTV: Tomato mottle Taino virus, SiYVHV: Sida yellow vein Honduras virus, ChTV-[IC]: Chino del tomato virus-[IC], ChTV-[H6]: Chino del tomato virus-[H6], ChTV-[H8]: Chino del tomato virus-[H8], AbMV-HW: Abutilon mosaic virus-HW, AbMV: Abutilon mosaic virus, ToMoV-[FL]: Tomato mottle virus-Florida, SiGMV: Sida golden mosaic virus, T3-C8: SiGMV DNA-A sequence from tomato 3 clone 8, T5-C2: SiGMV DNA-A sequence from tomato 5 clone 2, T10-C8: SiGMV DNA-A sequence from tomato 10 clone 8, T10-C10: SiGMV DNA-A sequence from tomato 10 clone 10, T12-C6: SiGMV DNA-A sequence from tomato 12 clone 6, S3-C7: SiGMV DNA-A sequence from Sida 3 clone 7
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Figure 3-13. Phylogenic tree of partial nucleotide sequences of DNA-B of selected begomoviruses with the SiGMV and the SiGMV sequences isolated from tomato and S. acuta. ChTV-[IC]: Chino del tomato virus-[IC], PYMTV-TT: Potato yellow mosaic Trinidad virus- Trinidad and Tobago, S3-C4B: SiGMV DNA-B sequence isolated from sida 3 clone 4, T12-C3B: SiGMV DNA-B sequence isolated from tomato 12 clone 3, T12-C5B: SiGMV DNA-B sequence isolated from tomato 12 clone 4, T12-C7B: SiGMV seqence of DNA-B isolated from tomato 12 clone 7, T12-C9B: SiGMV DNA-B sequence isolated from tomato 12 clone 9, ToMoV-[FL]: Tomato mottle virus-Florida, ToMoTV: Tomato mottle Taino virus, AbMV: Abutilon mosaic virus, AbMV-HW: Abutilon mosaic virus-HW, SiGMHV: Sida golden mosaic Honduras virus, SiGMHV-YV: Sida golden mosaic Honduras virus- yellow vein, SiGMHV*: Strain of Sida golden mosaic Honduras virus, SiYVHV: Sida yellow vein Honduras virus, SiGMCRV: Sida golden mosaic Costa Rica virus, BDMV: Bean dwarf mosaic virus, SiGMV-B: Sida golden mosaic virus DNA-B
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Discussion
The Samples were collected from putatively SiGMV infected tomatoes and S.
acuta from experimental field near Citra, FL., Begomovirus DNA was extracted, isolated
and characterized. Partial DNA-A and DNA-B fragments were cloned, sequenced and
subjected to Gap sequencing and phylogenetic analysis. The partial DNA-A sequences
comparisons revealed no significant differences between samples acquired from tomato
and Sida. Furthermore the partial DNA-A sequence analysis suggested theses variants
were related to ToMoV-[FL].
However, the partial DNA-B sequences showed greater diversity and were divided
into two groups, the first group was related to ToMoV-[FL] and the second was related
to a group of viruses that included SiGMV.
The high level of homology in the nucleotide sequence of the CR between DNA-A
and DNA-B confirmed that these components do support each other. The diversity
observed in the sequences of DNA-B may be due to recombination events. It is possible
that this recombination took place at some time in the past or could be relatively current
and ongoing series of events These results suggest that S. acuta was the inoculation
source for the epidemic of SiGMV in tomato. This is the first report of S. acuta acting as
a virus source for tomato and possible recombination host source for Begomoviruses.
The suggested recombination of the DNA-B in S. acuta could have an impact on
the host range and virulence of Begomoviruses capable of using S. acuta as a host. The
possibility of Begomoviruses using S. acuta as a recombination host could have a
dramatic impact on cultural practice and crop selection where S. acuta occurs, which may
lead to elimanite the S. acuta or change the crops in the farming area specially in South
45
East United State. However, the scientific aspect of naturally recombination occurrences
in S. acuta may lead to more attention to S. acuta.
A complete nucleotide sequence of the partial DNA-A and DNA-B sequences from
infected plants of tomato and S. acuta would help to understand the relationship between
SiGMV, these variants, and recombination. More study on S. acuta begomovirus and the
S. acuta the weed host must be achieved to understand the recombination events that can
be due to the lack of stringency of replication or because of begomovirus movement to S.
acuta. In addition, biolistic inoculation of infectious clones of SiGMV and SiGMV
variants to tomato is required to determine if the SiGMV sequence variants that caused
the epidemic in tomato should be classified as a strain of SiGMV. Also, whitefly feeding
preference and virus aquision from sida speacies must be study to determine the
efficiency of whiteflies to acquire and transmission.
Finally, the occurrence of recombination and the whitefly preference and feeding to
and from Sida species will play an importance role in introducing new begomoviruses.
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BIOGRAPHICAL SKETCH
Hamed Sayed Adnan Al-Aqeel was born on August 25, 1975, in Kuwait City, State
of Kuwait. He received his Bachelor’s degree in Microbiology in 1998. In 1999 he
received a scholarship from Kuwait University to continue his graduate studies toward
Master and Doctor of Philosophy degrees in plant viruses. In the same year he married
Hanin Altarkeet. In summer 2000 he joined the University of Florida as a graduate
student and since then he has been working under the supervision and guidance of Dr
Jane Polston and her lab group and under the support of the committe members, family,
and friends. On February 17, 2001 he becomes a father to Ali Hamed Sayed Adnan Al-
Aqeel. Upon completion of his M.S degree, Hamed is looking forward to completing to
his PhD degree under the same supervisor at the same lab.
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