CHARACTERIZATION OF TWO BEGOMOVIRUSES ISOLATED FROM Sida …ufdcimages.uflib.ufl.edu/UF/E0/00/28/38/00001/alaqeel_h.pdf · Characterization of Two Begomoviruses Isolated from Sida

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

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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

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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





3-7 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



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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

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http://www.itis.usda.gov/servlet/SingleRpt/RefRpt?search_type=author&search_id=author_id&search_id_value=41302

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).

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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

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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

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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).

http://www.ncbi.nlm.nih.gov/

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).

http://www.plantatlas.usf.edu/

http://plants.usda.gov/topics.html

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

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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.

http://www.itis.usda.gov/servlet/SingleRpt/RefRpt?search_type=author&search_id=author_id&search_id_value=41302

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.


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).


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


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


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


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


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


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


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


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


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

41

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

42

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.

51

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