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Identification of begomoviruses from different cryptic species of Bemisia tabaci in Bangladesh
Journal Pre-proof Identification of begomoviruses from different cryptic species of Bemisia tabaci in Bangladesh Mst Fatema Khatun, Hwal-Su Hwang, Jae-Kyoung Shim, Eui-Joon Kil, Sukchan Lee, Kyeong-Yeoll Lee PII:
S0882-4010(19)31417-2
DOI:
https://doi.org/10.1016/j.micpath.2020.104069
Reference:
YMPAT 104069
To appear in:
Microbial Pathogenesis
Received Date: 6 August 2019 Revised Date:
20 January 2020
Accepted Date: 12 February 2020
Please cite this article as: Khatun MF, Hwang H-S, Shim J-K, Kil E-J, Lee S, Lee K-Y, Identification of begomoviruses from different cryptic species of Bemisia tabaci in Bangladesh, Microbial Pathogenesis (2020), doi: https://doi.org/10.1016/j.micpath.2020.104069. This is a PDF file of an article that has undergone enhancements after acceptance, such as the addition of a cover page and metadata, and formatting for readability, but it is not yet the definitive version of record. This version will undergo additional copyediting, typesetting and review before it is published in its final form, but we are providing this version to give early visibility of the article. Please note that, during the production process, errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain. © 2020 Published by Elsevier Ltd.
1
Identification of begomoviruses from different cryptic species of Bemisia tabaci in
2
Bangladesh
3 4
Mst. Fatema Khatun1,2, Hwal-Su Hwang1,4, Jae-Kyoung Shim1,4, Eui-Joon Kil3, Sukchan Lee3,
5
Kyeong-Yeoll Lee1,4,5*
6 7
1
8
National University, Daegu, Republic of Korea
9
2
Division of Applied Biosciences, College of Agriculture and Life Sciences, Kyungpook
Department of Entomology, Bangabandhu Sheikh Mujibur Rahman Agricultural University,
10
Dhaka, Bangladesh
11
3
Department of Genetic Engineering, Sungkyunkwan University, Suwon, Republic of Korea
12
4
Institute of Plant Medicine, Kyungpook National University, Daegu, Republic of Korea
13
5
Institute of Agricultural Science and Technology, Kyungpook National University, Daegu,
14
Republic of Korea
15 16
*Corresponding author:
17
Dr. Kyeong-Yeoll Lee
18
Division of Applied Biosciences, College of Agriculture and Life Sciences, Kyungpook
19
National University, Daegu 41566, Republic of Korea
20
Tel.: 82-53-950-5759
21
E-mail: [email protected]
22
Abstract
23
Bemisia tabaci is a global species complex consisting of at least 40 cryptic species. It is also a
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vector for at least 100 species of begomovirus, many of which cause severe crop damage. The
25
relationship between begomoviruses and cryptic species of the B. tabaci species complex,
26
however, remains unclear. Our previous study [13] was identified four cryptic species (Asia I,
27
Asia II 1, Asia II 5, and Asia II 10) of B. tabaci from Bangladesh. Using those 110 whitefly
28
samples, vector-based PCR analysis identified 8 different begomovirus species: BYVMV,
29
BGYVV, OELCV, SLCCV, SLCV, TbCSV, ToLCBV, and ToLCNDV. The overall rate of
30
virus infection was 26.4%, and BYVMV and ToLCNDV were the most frequently detected in
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the B. tabaci vector. Virus infection rates for Asia I, Asia II 1, Asia II 5, and Asia II 10 were
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22.4% (15/67), 35% (7/20), 27.3% (6/22), and 100% (1/1), respectively. Each cryptic species
33
infected multiple virus species, but SLCCV, TbCSV, and BGYVV were each only detected in,
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Asia I, Asia II 1, and Asia II 5, respectively. This study demonstrates the geographic
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distribution of various begomoviruses in Bangladesh and their relationships with cryptic
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species of B. tabaci.
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Keywords: Begomovirus, Cryptic species, Genetic diversity, Vector, Whitefly
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Introduction
39
The genus Begomovirus is the largest group of the family Geminiviridae and consists
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of more than 320 species, each with a single-stranded circular DNA comprising between
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2,500 and 5,200 bases [1]. Many species of this genus cause serious damage to crops across
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the globe. Over the past three decades, the emergence of begomoviruses has resulted in crop
43
production reduction and economic losses in tropical and subtropical countries [2,3]. Tomato
44
yellow leaf curl virus (TYLCV) is highly virulent in tomatoes, and its worldwide spread has
45
resulted in significant losses for global tomato producers [3]. The spread of begomovirus,
46
however, has generally been associated with the global invasion of its vector, mainly
47
whiteflies [4].
48
Bemisia tabaci is the only known vector species of TYLCV [4]. It is considered a
49
species complex consisting of at least 40 cryptic species which are indistinguishable in body
50
structure but genetically different in regard to development, behavior, and physiological
51
characteristics [5-7]. Transmission of begomoviruses can be either general or specific to
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cryptic species of B. tabaci. However, the relationship of cryptic species to begomovirus
53
transmission remains poorly understood.
54
Transmission of begomovirus by B. tabaci is a circulative persistent manner [8].
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Viruses translocate to several important sites within the insect body after ingestion, such as
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midgut, filter chamber, hemolymph, and salivary glands [8,9]. The molecular interactions
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between virus coat proteins and receptors on the cellular membranes and the hemolymph of
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the vector is critical for effective translocation through the different tissues and organs therein
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[9]. This interaction is specific to each cryptic species and influence begomovirus
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transmission efficiency for these different vectors. For example, TYLCV is transmitted by
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two invasive B. tabaci species, Middle East Asia Minor 1 (MEAM1, formerly B biotype) and
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Mediterranean (MED, formerly Q biotype), at similar efficiencies, but Papaya leaf curl
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China virus (PaLCuCNV) is more efficiently transmitted by MEAM1 than MED [10]. A
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comparison of transmission efficiencies of Cotton leaf curl Multan virus (CLCuMuV) among
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four B. tabaci species revealed that CLCuMuV transmission by Asia II 1 was high, while that
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by MEAM1, MED, and Asia I was low [11], suggesting that transmission rates of certain
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begomoviruses are differentiated among different B. tabaci cryptic species.
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The genetic diversity of B. tabaci cryptic species has been studied in various countries
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and different geographic territories [5,12]. Our previous studies explored the genetic variation
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and geographic distribution of four cryptic species, including Asia I, Asia II 1, Asia II 5, and
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Asia II 10 as well as their endosymbiont profiles in Bangladesh [13,14]. We hypothesized
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that each cryptic species might possess differential transmission efficiencies of the different
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begomoviruses. Therefore, we analyzed the profiles of begomovirus infected in four cryptic
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species of B. tabaci identified in Bangladesh to understand the relationship between different
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species of B. tabaci and different begomoviruses. Generally, plant virus can be examined
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using plant samples which have pathogenic symptoms. However, vector-based survey
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technique has been conducted in several studies and provides several advantages [15,16].
78
This is easy to detect begomovirus by conducting PCR assay of whiteflies and to survey their
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geographic distribution. More importantly, this technique provide information of potential
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vectors that transmit specific species of plant viruses. This study enhances our understanding
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of the relationship of cryptic species of B. tabaci and begomovirus transmission.
82 83
Materials and Methods
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Whitefly collection
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We collected the whiteflies sample from 8 different crop plants in 35 different regions of
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Bangladesh in 2015 and 2018 (Table 1). At least 20 to 100 whiteflies were collected from
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each region. Upon collection, whiteflies were preserved in 70% alcohol and maintained at -
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20°C until further analysis. Identification of genetic diversity of B. tabaci and their
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endosymbiont profiles of the same samples were reported in previous studies [13,14].
90 91
DNA extraction
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Genomic DNA was extracted from individual adult whitefly using a PureLink Genomic DNA
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Mini Kit (Invitrogen, Carlsbad, CA) according to the manufacturer’s protocol. Briefly, the
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sample was placed in a 1.5 mL centrifuge tube containing 180 µL of digestion buffer and 20
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µL of proteinase K (50 µg/mL) and incubated at 55°C for 4 h. DNA samples were extracted
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and purified using genomic spin columns as described in the kit. DNA concentrations were
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evaluated using a NanoPhotometer™ (Implen, Schatzbogen, Germany).
98 99
Identification of begomoviruses in different cryptic species of Bemisia tabaci
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The presence of different begomoviruses in B. tabaci cryptic species collected from
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Bangladesh was assessed using polymerase chain reaction (PCR) to amplify a conserved
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region
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CCGTGCTGCTGCCCCCATTGTCCGCGTCAC-3’)
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CTGCCACAACCATGGATTCACGCACAGGG-3’). PCR amplification was carried out in a
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Gene PCR system as follows: 1 cycle of 2 min at 95°C, followed by 35 cycles of 40 sec at
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94°C, 1 min at 65°C, and 30 sec at 72°C, with a final extension for 7 min at 72°C. PCR was
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performed on the total reaction volume of 25 µL containing 13 µL Smart-Taq Pre-Mix
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(Solgent, Daejeon, Korea), 1 µL of each primer (10 pmol/µL), and 5 µL of template DNA
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solution (40 ng). The PCR products were separated via electrophoresis on a 1% agarose gel,
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stained with ethidium bromide solution, and visualized under ultraviolet (UV) light.
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Amplified PCR products were excised from the gel and purified using the Wizard® PCR
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Preps DNA Purification System (Promega, Madison, WI), cloned into the T-BluntTMeasy
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plasmid vector (Promega, Madison, WI), sequenced using a BigDye® Terminator Cycle
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Sequencing Kit (Applied Biosystems, Foster City, CA) and analyzed with a 3100 Capillary
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DNA Sequencer (Applied Biosystems, Foster City, CA) at the Solgent Sequencing Facility in
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Daejeon, Korea. Sequences were verified by comparison with those in the database using the
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BLAST algorithm at NCBI [17].
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of
the
begomovirus
sequence
using
the and
primers
BegomoF1
BegomoR1
(5’(5’-
119
Phylogenetic analyses and pairwise distance matrix of begomoviruses in Bangladesh
120
Phylogenetic trees were constructed using the maximum likelihood method based on the
121
HKY+G model, and evolutionary analysis was conducted in MEGA 6.0. We used 1000
122
bootstraps to the test the robustness of the phylogeny [18]. Pairwise comparisons were
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performed with the MUSCLE algorithm implemented by the Species Demarcation Tool
124
(SDT) version 1.2 [19,20].
125 126
Results
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Diversity and distribution of begomovirus in different species of the B. tabaci complex in
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Bangladesh
129
The total begomovirus infection rate was 26.4% (29/110) in B. tabaci collected from 35
130
locations across Bangladesh (Fig. 1; Table S1). Comparisons with begomovirus genetic
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sequences in the GenBank database resulted in the identification of eight different viruses:
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Bitter gourd yellow vein virus (BGYVV), Bhendi yellow vein mosaic virus (BYVMV), Okra
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enation leaf curl virus (OELCV), Squash leaf curl China virus (SLCCV) Indian strain,
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Synedrella leaf curl virus (SLCV), Tobacco curly shoot virus (TbCSV), Tomato leaf curl
135
Bangladesh virus (ToLCBV), and Tomato leaf curl New Delhi virus (ToLCNDV). Among
136
them, the highest infection rate was 9.1% for BYVMV, followed by 7.3% for ToLCNDV;
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infection rates for the other six viruses ranged from 0.9 to 2.7% (Fig. 3). TbCSV was found
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only in the Northern region, while other species were endemic mostly to the central and
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Southern regions of Bangladesh (Fig. 1). Seven of the eight species of begomovirus identified
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in this study showed at least a 99% sequence identity with related sequences in the GenBank
141
database (Table 1). However, SLCV was only a 96% match to the sequence KU933258
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identified in the Yunnan Province in China. Phylogenetic analysis of 8 begomoviruses
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showed that ToLCNDV, BGYVV and SLCCV were clustered together and closed to
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BYVMV but distant with another cluster of SLCV and OELCV (Fig. 2).
145
In Bangladesh, BYVMV and ToLCNDV were detected in all four cryptic B. tabaci
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species (Table 1, Fig. 4). Other viruses, however, were detected in only a single species;
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SLCCV in Asia I, TbCSV in Asia II 1, and BGYVV in Asia II 5.
148 149
Begomovirus identified in cryptic B. tabaci species collected from different host plants in
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Bangladesh
151
Whiteflies were collected from nine different host plants: bean, brinjal, cotton, dahlia,
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potato, sweet gourd, sweet potato, tobacco, and tomato (Table 2) to determine relationships
153
between begomoviruses and host plants. BYVMV was identified in whiteflies infesting seven
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different host plants, but SLCV and TbCSV were only found in whiteflies infesting brinjal; in
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fact, seven of the eight species of begomovirus identified were detected in whiteflies infesting
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brinjal (Table 2).
157 158
Relationships between begomovirus and endosymbionts identified from cryptic B. tabaci
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species in Bangladesh
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Our previous study showed that each B. tabaci species in Bangladesh presents with
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co-infection profiles containing the endosymbionts Arsenophonus, Cardinium, Rickettsia, or
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Wolbachia, but not Hamiltonella [14]. The relationship of acquired endosymbionts and
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begomoviruses was analyzed for each species (Table 1); however, we did not find any
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associated patterns between acquired viruses and endosymbionts therein.
165 166 167
Discussion This study identified the presence of eight species of begomovirus from four cryptic
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species of B. tabaci vector collected across Bangladesh. Viruses were detected in 26.4% of a
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total of 110 collected whiteflies. Asia I was found in six species, Asia II 1 in three species,
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Asia II 5 in five species, and Asia II 10 in one species. Infection rates ranged from 22 to 35%,
171
except for Asia II 10, which was only observed in a single individual. Infection rates for each
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virus varied across species. Both BYVMV and ToLCNDV were found in all four cryptic
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species at various rates. However, SLCCV, TbCSV, and BGYVV were only detected in Asia I,
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Asia II 1, and Asia II 5, respectively. These results suggest that each cryptic species is a
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potential vector for certain begomoviruses in a virus-specific manner.
176
Biodiversity among begomoviruses is rich in Southeast Asia and has been studied in
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several countries in this region [21-26]. Our analysis found that begomovirus diversity is also
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high in Bangladesh [15, 23-26]. Among them, BYVMV was the most frequently detected in
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all four cryptic species of Bangladeshi B. tabaci and was also found in a variety of host plants
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in the central region of Bangladesh. Our results suggest that BYVMV is widespread in this
181
country, infects many different crop plants, and is non-specifically transmitted by B. tabaci
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regardless of genetic variations among vectors. BYVMV has also been found in neighboring
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countries such as India, Pakistan, Thailand, and Africa [24-25]. A recent study demonstrated
184
that BYVMV could be transmitted by both invasive MEAM1 and indigenous Asia I, although
185
more efficiently by the former cryptic species [21]. Our study detected BYVMV more
186
frequently in Asia II 1 than in Asia I or Asia II 5, suggesting that transmission efficiency of
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BYVMV may rely on specific genetic features of B. tabaci only found in certain species.
188
ToLCV is common and destructive in tomato plants and can result in a total crop loss
189
during an upsurge in B. tabaci infestation [27]. ToLCV was first reported in Israel in 1930,
190
after which it gradually extended into Africa, Australia, Central America, and parts of Asia
191
including Bangladesh [27-30]. Six ToLCV species have been identified on the Indian
192
subcontinent [31]; they are ToLCBV, ToLCBDV, ToLCNDV, Tomato leaf curl Gujarat virus
193
(ToLCGV), Tomato leaf curl Karnataka virus (ToLCKV), and Tomato leaf curl Sri Lanka
194
virus (ToLCSV). In Bangladesh, all six different types of ToLCV have been reported [31],
195
and recently, 21 types of ToLCV were reported in India [32]. ToLCNDV has been found in
196
trans-replicate with both mono and bipartite begomoviruses, particularly in Europe, Asia, and
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Africa [33]. Our study detected ToLCNDV bipartite begomovirus in Asia I, Asia II 1, and
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Asia II 5 and ToLCBDV monopartite in Asia I and Asia II 5, all from central and Southern
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regions of Bangladesh. This suggests that ToLCV transmission may be common across
200
genetic groups of B. tabaci. Further study is required to determine the precise vector capacity
201
for the different virus strains of each cryptic species in this group.
202
SLCCV was first found in pumpkins from China in 1995 [34]. It has a restricted host
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range within the Cucurbitaceae family, infecting squash and pumpkin, but not melon or
204
cucumber [35]. It has been found in many Asian countries, including China, India, Pakistan,
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Malaysia, the Philippines, Thailand, and Vietnam [36,37]. SLCCV has also infected pumpkin
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in East Timor and determined the first complete genomic segment DNA-A sequence [38]. We
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detected this virus in Asia I collected from brinjal and tobacco, suggesting additional
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potential host plants for this virus. Further research is necessary, however, to determine
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infection parameters and virus symptoms in these plants. Two species of SLCCV, the China
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and India strains, are geographically distinct, and a third, similar to the Indian strain, was
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found in Pakistan [36]. In our study, SLCCV shared 96.3 to 95.7% genetic similarity with the
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Indian strain.
213
OELCV is a serious emerging disease in okra production in northern India [39-41].
214
OELCV occurrence has been reported in Pakistan, Saudi Arabia, Iran, Nigeria, and China
215
[42-46]. OELCV is associated with the alpha and beta satellites of cotton leaf curl virus
216
(CLCuV) infecting cotton in Pakistan [47]. Saleem et al. [48] demonstrated that transmission
217
of CLCuV was more efficient in Asia II 1 than MEAM1. Similarly, cotton leaf curl disease
218
has been associated with Asia II 1 in Pakistan [49]. Our study in Bangladesh found OELCV
219
in Asia I and Asia II 5, but not in Asia II 1; however, this discrepancy is not reliable because
220
our OELCV detection rate was only 1.8%. A more intensive study on a larger vector
221
population is required to clarify the relationship between OELCV transmission and different
222
genetic groups of B. tabaci.
223
SLCV is found in South India and China (NCBI database). This virus has a typical,
224
old-world, monopartite begomovirus genome that was evaluated by beta satellite. The present
225
study found SLCV in Asia I collected from brinjal in Southern Bangladesh, and the infection
226
rate was very low.
227
TbCSV was associated with tobacco leaf curl disease and tomato leaf curl disease in
228
the Yunnan province of China in 2001[50,51]. TbCSV has also been reported to infect pepper,
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common bean, sunflower, and tomato plants in India [52] and has been found in Punjab
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(Alipur) in tobacco cultivation areas [16]. This study only identified TbCSV in Asia II 1
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collected from northern Bangladesh, where tobacco cultivation is popular. Previous studies
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have shown that TbCSV transmission efficiency is low in MEAM1 and Asia II 3 [53]
233
although Asia 1 and Asia II 1 transmission have been reported, as well [50,53,54]. Of the four
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cryptic B. tabaci species examined in the present study, Asia II 1 was more frequently
235
infected with TbCSV begomovirus than the others [11].
236
This study evaluated virus profiles detected in four different cryptic B. tabaci species in
237
Bangladesh. Some viruses were detected in most or all of the four, while others were limited
238
to a single species. Our results suggest that relationships between begomovirus strains and
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cryptic species of B. tabaci are highly differentiated at the species level.
240
The PCR detection of begomoviruses in the vector’s body is not always associated with
241
the circular transmission of that particular virus because whiteflies ingested begomoviruses
242
from plants without restriction. Ingested viruses must translocate from the gut into the
243
hemolymph and salivary glands if they are to be transmitted into another plants [8,9].
244
Therefore, further studies are necessary to determine whether begomoviruses in vector
245
species are transmitted through host plants.
246
Declaration of interests: none.
247 248
Acknowledgments
249
We would like to thank the editors at Essay Review for critical English language revision of
250
the manuscript.
251 252
Author Contributions
253
M.F.K. and K.-Y.L. conceived and designed experiments, M.F.K., H. H., J.-K.S. Conducted
254
experiments, E.-J.K., S.L., K.-Y.L. analyzed the results, M.F.K., H. H., J.-K.S. wrote the
255
manuscript and prepared figures and tables, M.F.K., H. H., J.-K.S., E.-J.K., S.L., K.-Y.L.
256
revised and approved the manuscript. All authors reviewed the manuscript.
257 258
Disclosure statement
259
The authors report no conflicts of interest. The authors alone are responsible for all aspects of
260
this manuscript.
261 262
Funding
263
This work was supported by the Research Program for Exportation Support of Agricultural
264
Products, Animal and Plant Quarantine Agency, in the Republic of Korea under Grant #Z-
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1543086-2017-21-01.
266 267 268
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49. M.Z. Ahmed, P.J. De Barro, J.M. Greeff, S.X. Ren, M. Naveed, B.L. Qiu, Genetic identity
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(CLCuD) incidence in Pakistan. Pest Manag. Sci. 67 (2011) 307–17.
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397 398
51. Y. Xie, X. Zhou, Z. Li, Z. Zhang, G. Li, Identification of a novel DNA molecule associated with tobacco leaf curl virus. Chinese Sci. Bull. 47 (2002) 1273-1276.
399
52. S. Shilpi, A. Kumar, S. Biswas, A. Roy, B. Mandal, A recombinant tobacco curly shoot
400
virus causes leaf curl disease in tomato in a northeastern state of India and has potential
401
to trans-replicate a non- cognate betasatellite. Virus Gene 50 (2015) 87-96.
402
53. M. Jiu, X.P. Zhou, S.S. Liu, Acquisition and transmission of two begomoviruses by the
403
B and a non-B biotype of Bemisia tabaci from Zhejiang, China. J. Phytopathol. 154
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(2006) 587-591.
405
54. J. Hu, Z.L. Jiang, F. Nardi, Y.Y. Liu, X.R. Luo, et al., Members of Bemisia tabaci
406
(Hemiptera: Aleyrodidae) cryptic species and the status of two invasive alien species in
407
the Yunnan province (China). J. Insect Sci. 14 (2014) 281.
408 409
410
Figures and legends
411 412
Figure 1. Distribution of begomovirus by four cryptic species of Bemisia tabaci in
413
Bangladesh. Colored symbols represent the distributions of each begomovirus.
414 415
Figure 2. Phylogenetic analysis of begomovirus by maximum likelihood method based on
416
the HKY+G model. Evolutionary analyses were conducted in MEGA 6. Sequences identified
417
from Bangladesh in this study are shown in different colors and represented by sample name,
418
genetic group, begomovirus, and accession number. Bangladeshi sequences from GenBank
419
are shown in black.
420 421
Figure 3. Begomovirus detection rates from Bemisia tabaci collected in Bangladesh.
422
Numbers of virus sequences were counted from all Bemisia tabaci samples (n = 110).
423 424
Figure 4. Begomovirus detection rates from four cryptic species of Bemisia tabaci in
425
Bangladesh
1
Table 1. PCR detection of begomoviruses in Bemisia tabaci collected from various regions of Bangladesh Plant viruses ingested by B. tabaci Virus names
Accession numbers
A1+C2+W1 A1+C2+W1
SLCCV SLCCV
MK784259 MK784268
Identities (%) Genomic with reported organizations bemoviruses in GenBank Bipartite 99, DQ026296 Bipartite 99, DQ026296
Asia I
A1+W1
SLCCV
MK784265
Bipartite
99, DQ026296
Brinjal Brinjal
Asia I Asia I
A2+C2+R1 A2+C2
ToLCNDV MK784262 ToLCNDV MK784261
Bipartite Bipartite
99, EF043230 99, EF043230
Brinjal
Asia I
A1+R2+W1
ToLCNDV MK784251
Bipartite
99, EF043230
Bandarban
Dahlia
Asia I
A1+C2+W1
ToLCNDV MK784253
Bipartite
99, EF043230
BARI, Gazipur
Tomato
Asia I
A2+W1
ToLCNDV MK784258
Bipartite
99, KP641675
Rajshahi
Tomato
Asia I
C2+W1
BYVMV
MK784270
Monopartite
99, KJ462082
BARI, Gazipur
Sweet potato Sweet gourd Bean Brinjal Sweet potato Brinjal
Asia I
A1+W1
BYVMV
MK784254
Monopartite
99, KJ462082
Asia I
A1+C2+W1
BYVMV
MK784274
Monopartite
99, KJ462082
Asia I Asia I
A1+C2+W1 BYVMV MK784266 A1+C2+R2+W1 ToLCBDV MK784277
Monopartite Monopartite
99, KJ462082 99, KM383765
Asia I
A1+C2+W1
MK784256
Monopartite
99, KT394454
Asia I
A1+C2+R2+W1 SLCV
MK784250
Monopartite
95, KU933258
No
Sample names
Collection sites
Host plants
Genetic Co-infection of groups endosymbionts
1 2
Coxs-Br-7 Khul-Br-1
Brinjal Brinjal
Asia I Asia I
3
Digh-To-2
Tobacco
4 5
Coxs-Br-8 Coxs-Br-9 BARI-Br17 Band-Dah1 BARI-To21 Rajs-To342 BARI-Sp15
Coxsbazar Khulna Dighinala, Khagrachori Coxsbazar Coxsbazar BARI*, Gazipur
6 7 8 9 10 11
Satkh-Sg-3
Satkhira
12 13
Gopal-Be-4 Satkh-Br-6 BARI-Sp16 Band-Br-5
Gopalganj Satkhira BARI, Gazipur Bandarban
14 15
OELCV
1
16 17 18 19 20
Rang-Br-1 Dhak-To361 BARI-To19 BARI-Po20
Rangpur
Brinjal
Savar, Dhaka
Tomato
BARI, Gazipur BARI, Gazipur
Tomato Potato
Gopal-Br-1
Gopalganj
BARI-To13 BSMRAUTo-14 Rajs-To341
BARI, Gazipur BSMRAU, Gazipur Rajshahi
Tomato
24
Satkh-Be-1
Satkhira
Bean
25
Satkh-Be-2
Satkhira
Bean
26
Satkh-Sg-4
Satkhira
Sweet gourd
27
Satkh-Br-5
Satkhira
Brinjal
28
Gopal-Be-3 Gopalganj
Bean
29
BARI-Co18
Cotton
21 22 23
BARI, Gazipur
Brinjal Tomato Tomato
Asia II 1 Asia II 1 Asia II 1 Asia II 1 Asia II 1 Asia II 1 Asia II 1 Asia II 5 Asia II 5 Asia II 5 Asia II 5 Asia II 5 Asia II 5 Asia II 10
A2+C2+W1
TbCSV
MK784273
Monopartite
99, LC316184
A1+C2+W1
BYVMV
MK784263
Monopartite
100, KJ462082
A1+C2+W1
BYVMV
MK784257
Monopartite
99, KJ462082
A1+C2+W1
BYVMV
MK784249
Monopartite
99, KJ462082
A1+C2+W1
BYVMV
MK784267
Monopartite
99, KJ462082
A2+C2+R1
ToLCNDV MK784255
Bipartite
99, EF043230
A2+C2+R1
ToLCNDV MK784260
Bipartite
99, EF043230
A1+C2+W1
ToLCBDV MK784269
Monopartite
99, KM383765
A1+C2+W1
BYVMV
MK784272
Monopartite
99, KJ462082
A1+C2+W1
BGYVV
MK784271
Bipartite
99, KJ862841
A1+C2+W1
BGYVV
MK784276
Bipartite
99, KJ862841
A1+C2+W1
OELCV
MK784275
Monopartite
99, KT390454
A1+C2+W1
ToLCNDV MK784264
Bipartite
98, EF043230
A2+W1
BYVMV
Monopartite
99, KJ462082
2 2
MK784252
3
Table 2. Begomoviruses detected in four cryptic species of Bemisia tabaci collected from various host plants in Bangladesh Begomovirus
Cryptic species
Bhendi yellow vein mosaic virus (BYVMV)
Asia I, Asia II 1, Asia II 5, Asia Bean, brinjal, cotton, sweet gourd, sweet potato, potato, II 10 tomato
Tomato leaf curl New Delhi virus (ToLCNDV) Squash leaf curl China virus (SLCCV), Indian strain Tomato leaf curl Bangladesh virus (ToLCBDV) Okra enation leaf curl virus (OELCV) Bitter gourd yellow vein virus (BGYVV) Synedrella leaf curl virus (SLCV) Tobacco curly shoot virus (TbCSV)
Host plants
Asia I, Asia II 1, Asia II 5
Bean, brinjal, dahlia, tomato
Asia I
Brinjal, tobacco
Asia I, Asia II 5
Brinjal, tomato
Asia I, Asia II 1 Asia II 5 Asia I Asia II 1
Brinjal, sweet gourd Bean, sweet gourd Brinjal Brinjal
4 5
3
6
Table S1. Begomoviruses previously identified in Bangladesh by PCR detection from leaf and Bemisia tabaci samples Abbreviations Host plants
Sample types
Previousreports inBangladesh
Reported
ToLCJV-Mild
Tomato
leaf
Yes
2005
ToLCBDV
Tomato
leaf
Yes
2005
ToLCNDV
Tomato
leaf
Yes
2005
ToLCKV
Tomato
leaf
Yes
2005
ToLCGV
Tomato
leaf
Yes
2005
ToLCV
Tomato
leaf
Yes
2007
AYVM
Ageratum
leaf
Yes
2007
BGMV ChiLCuV
Bitter gourd Chilli
leaf leaf
Yes Yes
2007 2007
CYVMV
Croton
leaf
Yes
2007
BMV PLCV
Brinjal Papaya
leaf leaf
Yes Yes
2007 2007
PYVMV
Pumpkin
leaf
Yes
2007
SGYMV
Sponge gourd
leaf
Yes
2007
Squash leaf curl china virus
SLCCV
Brinjal, Tobacco
No
-
Bhendi yellow vein mosaic
BYVMV
Tomato, Sweet potato, Sweet gourd,
Yes
2007
Viruses Tomato leaf curl Joydebpur virus Tomato leaf curl Bangladesh virus Tomato leaf curl New Delhi virus Tomato leaf curl Karnataka virus Tomato leaf curl Gujarat virus Tomato leaf curl virus Ageratum yellow vein mosaic virus Bitter gourd mosaic virus Chilli leaf curl virus Croton yellow vein mosaic virus Brinjal mosaic virus Papaya leaf curl virus Pumpkin yellow vein mosaic virus Sponge gourd yellow mosaic virus
4
Bemisia tabaci Bemisia
virus
Bean, Okra
Okra enation leaf curl virus
OELCV
Sweet gourd
Tobacco curly shoot virus
TbCSV
Sweet gourd
Bitter gourd yellow vein virus
BGYVV
Sweet gourd, Bean
Synedrella leaf curl virus
SLCV
Brinjal
tabaci Bemisia tabaci Bemisia tabaci Bemisia tabaci Bemisia tabaci
7
5
No
-
No
-
Yes
2012
No
-
►Bemisia tabaci is a species complex of at least 40 cryptic species as well as a vector of begomoviruses. ► Four indigenous Asian cryptic species were identified in Bangladesh. ► Vector-based PCR analysis identified 8 different begomoviruses in Bangladesh. ► This study demonstrates the geographic distribution of various begomoviruses in Bangladesh and their relationships with cryptic species of B. tabaci.
S0882-4010(19)31417-2
DOI:
https://doi.org/10.1016/j.micpath.2020.104069
Reference:
YMPAT 104069
To appear in:
Microbial Pathogenesis
Received Date: 6 August 2019 Revised Date:
20 January 2020
Accepted Date: 12 February 2020
Please cite this article as: Khatun MF, Hwang H-S, Shim J-K, Kil E-J, Lee S, Lee K-Y, Identification of begomoviruses from different cryptic species of Bemisia tabaci in Bangladesh, Microbial Pathogenesis (2020), doi: https://doi.org/10.1016/j.micpath.2020.104069. This is a PDF file of an article that has undergone enhancements after acceptance, such as the addition of a cover page and metadata, and formatting for readability, but it is not yet the definitive version of record. This version will undergo additional copyediting, typesetting and review before it is published in its final form, but we are providing this version to give early visibility of the article. Please note that, during the production process, errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain. © 2020 Published by Elsevier Ltd.
1
Identification of begomoviruses from different cryptic species of Bemisia tabaci in
2
Bangladesh
3 4
Mst. Fatema Khatun1,2, Hwal-Su Hwang1,4, Jae-Kyoung Shim1,4, Eui-Joon Kil3, Sukchan Lee3,
5
Kyeong-Yeoll Lee1,4,5*
6 7
1
8
National University, Daegu, Republic of Korea
9
2
Division of Applied Biosciences, College of Agriculture and Life Sciences, Kyungpook
Department of Entomology, Bangabandhu Sheikh Mujibur Rahman Agricultural University,
10
Dhaka, Bangladesh
11
3
Department of Genetic Engineering, Sungkyunkwan University, Suwon, Republic of Korea
12
4
Institute of Plant Medicine, Kyungpook National University, Daegu, Republic of Korea
13
5
Institute of Agricultural Science and Technology, Kyungpook National University, Daegu,
14
Republic of Korea
15 16
*Corresponding author:
17
Dr. Kyeong-Yeoll Lee
18
Division of Applied Biosciences, College of Agriculture and Life Sciences, Kyungpook
19
National University, Daegu 41566, Republic of Korea
20
Tel.: 82-53-950-5759
21
E-mail: [email protected]
22
Abstract
23
Bemisia tabaci is a global species complex consisting of at least 40 cryptic species. It is also a
24
vector for at least 100 species of begomovirus, many of which cause severe crop damage. The
25
relationship between begomoviruses and cryptic species of the B. tabaci species complex,
26
however, remains unclear. Our previous study [13] was identified four cryptic species (Asia I,
27
Asia II 1, Asia II 5, and Asia II 10) of B. tabaci from Bangladesh. Using those 110 whitefly
28
samples, vector-based PCR analysis identified 8 different begomovirus species: BYVMV,
29
BGYVV, OELCV, SLCCV, SLCV, TbCSV, ToLCBV, and ToLCNDV. The overall rate of
30
virus infection was 26.4%, and BYVMV and ToLCNDV were the most frequently detected in
31
the B. tabaci vector. Virus infection rates for Asia I, Asia II 1, Asia II 5, and Asia II 10 were
32
22.4% (15/67), 35% (7/20), 27.3% (6/22), and 100% (1/1), respectively. Each cryptic species
33
infected multiple virus species, but SLCCV, TbCSV, and BGYVV were each only detected in,
34
Asia I, Asia II 1, and Asia II 5, respectively. This study demonstrates the geographic
35
distribution of various begomoviruses in Bangladesh and their relationships with cryptic
36
species of B. tabaci.
37
Keywords: Begomovirus, Cryptic species, Genetic diversity, Vector, Whitefly
38
Introduction
39
The genus Begomovirus is the largest group of the family Geminiviridae and consists
40
of more than 320 species, each with a single-stranded circular DNA comprising between
41
2,500 and 5,200 bases [1]. Many species of this genus cause serious damage to crops across
42
the globe. Over the past three decades, the emergence of begomoviruses has resulted in crop
43
production reduction and economic losses in tropical and subtropical countries [2,3]. Tomato
44
yellow leaf curl virus (TYLCV) is highly virulent in tomatoes, and its worldwide spread has
45
resulted in significant losses for global tomato producers [3]. The spread of begomovirus,
46
however, has generally been associated with the global invasion of its vector, mainly
47
whiteflies [4].
48
Bemisia tabaci is the only known vector species of TYLCV [4]. It is considered a
49
species complex consisting of at least 40 cryptic species which are indistinguishable in body
50
structure but genetically different in regard to development, behavior, and physiological
51
characteristics [5-7]. Transmission of begomoviruses can be either general or specific to
52
cryptic species of B. tabaci. However, the relationship of cryptic species to begomovirus
53
transmission remains poorly understood.
54
Transmission of begomovirus by B. tabaci is a circulative persistent manner [8].
55
Viruses translocate to several important sites within the insect body after ingestion, such as
56
midgut, filter chamber, hemolymph, and salivary glands [8,9]. The molecular interactions
57
between virus coat proteins and receptors on the cellular membranes and the hemolymph of
58
the vector is critical for effective translocation through the different tissues and organs therein
59
[9]. This interaction is specific to each cryptic species and influence begomovirus
60
transmission efficiency for these different vectors. For example, TYLCV is transmitted by
61
two invasive B. tabaci species, Middle East Asia Minor 1 (MEAM1, formerly B biotype) and
62
Mediterranean (MED, formerly Q biotype), at similar efficiencies, but Papaya leaf curl
63
China virus (PaLCuCNV) is more efficiently transmitted by MEAM1 than MED [10]. A
64
comparison of transmission efficiencies of Cotton leaf curl Multan virus (CLCuMuV) among
65
four B. tabaci species revealed that CLCuMuV transmission by Asia II 1 was high, while that
66
by MEAM1, MED, and Asia I was low [11], suggesting that transmission rates of certain
67
begomoviruses are differentiated among different B. tabaci cryptic species.
68
The genetic diversity of B. tabaci cryptic species has been studied in various countries
69
and different geographic territories [5,12]. Our previous studies explored the genetic variation
70
and geographic distribution of four cryptic species, including Asia I, Asia II 1, Asia II 5, and
71
Asia II 10 as well as their endosymbiont profiles in Bangladesh [13,14]. We hypothesized
72
that each cryptic species might possess differential transmission efficiencies of the different
73
begomoviruses. Therefore, we analyzed the profiles of begomovirus infected in four cryptic
74
species of B. tabaci identified in Bangladesh to understand the relationship between different
75
species of B. tabaci and different begomoviruses. Generally, plant virus can be examined
76
using plant samples which have pathogenic symptoms. However, vector-based survey
77
technique has been conducted in several studies and provides several advantages [15,16].
78
This is easy to detect begomovirus by conducting PCR assay of whiteflies and to survey their
79
geographic distribution. More importantly, this technique provide information of potential
80
vectors that transmit specific species of plant viruses. This study enhances our understanding
81
of the relationship of cryptic species of B. tabaci and begomovirus transmission.
82 83
Materials and Methods
84
Whitefly collection
85
We collected the whiteflies sample from 8 different crop plants in 35 different regions of
86
Bangladesh in 2015 and 2018 (Table 1). At least 20 to 100 whiteflies were collected from
87
each region. Upon collection, whiteflies were preserved in 70% alcohol and maintained at -
88
20°C until further analysis. Identification of genetic diversity of B. tabaci and their
89
endosymbiont profiles of the same samples were reported in previous studies [13,14].
90 91
DNA extraction
92
Genomic DNA was extracted from individual adult whitefly using a PureLink Genomic DNA
93
Mini Kit (Invitrogen, Carlsbad, CA) according to the manufacturer’s protocol. Briefly, the
94
sample was placed in a 1.5 mL centrifuge tube containing 180 µL of digestion buffer and 20
95
µL of proteinase K (50 µg/mL) and incubated at 55°C for 4 h. DNA samples were extracted
96
and purified using genomic spin columns as described in the kit. DNA concentrations were
97
evaluated using a NanoPhotometer™ (Implen, Schatzbogen, Germany).
98 99
Identification of begomoviruses in different cryptic species of Bemisia tabaci
100
The presence of different begomoviruses in B. tabaci cryptic species collected from
101
Bangladesh was assessed using polymerase chain reaction (PCR) to amplify a conserved
102
region
103
CCGTGCTGCTGCCCCCATTGTCCGCGTCAC-3’)
104
CTGCCACAACCATGGATTCACGCACAGGG-3’). PCR amplification was carried out in a
105
Gene PCR system as follows: 1 cycle of 2 min at 95°C, followed by 35 cycles of 40 sec at
106
94°C, 1 min at 65°C, and 30 sec at 72°C, with a final extension for 7 min at 72°C. PCR was
107
performed on the total reaction volume of 25 µL containing 13 µL Smart-Taq Pre-Mix
108
(Solgent, Daejeon, Korea), 1 µL of each primer (10 pmol/µL), and 5 µL of template DNA
109
solution (40 ng). The PCR products were separated via electrophoresis on a 1% agarose gel,
110
stained with ethidium bromide solution, and visualized under ultraviolet (UV) light.
111
Amplified PCR products were excised from the gel and purified using the Wizard® PCR
112
Preps DNA Purification System (Promega, Madison, WI), cloned into the T-BluntTMeasy
113
plasmid vector (Promega, Madison, WI), sequenced using a BigDye® Terminator Cycle
114
Sequencing Kit (Applied Biosystems, Foster City, CA) and analyzed with a 3100 Capillary
115
DNA Sequencer (Applied Biosystems, Foster City, CA) at the Solgent Sequencing Facility in
116
Daejeon, Korea. Sequences were verified by comparison with those in the database using the
117
BLAST algorithm at NCBI [17].
118
of
the
begomovirus
sequence
using
the and
primers
BegomoF1
BegomoR1
(5’(5’-
119
Phylogenetic analyses and pairwise distance matrix of begomoviruses in Bangladesh
120
Phylogenetic trees were constructed using the maximum likelihood method based on the
121
HKY+G model, and evolutionary analysis was conducted in MEGA 6.0. We used 1000
122
bootstraps to the test the robustness of the phylogeny [18]. Pairwise comparisons were
123
performed with the MUSCLE algorithm implemented by the Species Demarcation Tool
124
(SDT) version 1.2 [19,20].
125 126
Results
127
Diversity and distribution of begomovirus in different species of the B. tabaci complex in
128
Bangladesh
129
The total begomovirus infection rate was 26.4% (29/110) in B. tabaci collected from 35
130
locations across Bangladesh (Fig. 1; Table S1). Comparisons with begomovirus genetic
131
sequences in the GenBank database resulted in the identification of eight different viruses:
132
Bitter gourd yellow vein virus (BGYVV), Bhendi yellow vein mosaic virus (BYVMV), Okra
133
enation leaf curl virus (OELCV), Squash leaf curl China virus (SLCCV) Indian strain,
134
Synedrella leaf curl virus (SLCV), Tobacco curly shoot virus (TbCSV), Tomato leaf curl
135
Bangladesh virus (ToLCBV), and Tomato leaf curl New Delhi virus (ToLCNDV). Among
136
them, the highest infection rate was 9.1% for BYVMV, followed by 7.3% for ToLCNDV;
137
infection rates for the other six viruses ranged from 0.9 to 2.7% (Fig. 3). TbCSV was found
138
only in the Northern region, while other species were endemic mostly to the central and
139
Southern regions of Bangladesh (Fig. 1). Seven of the eight species of begomovirus identified
140
in this study showed at least a 99% sequence identity with related sequences in the GenBank
141
database (Table 1). However, SLCV was only a 96% match to the sequence KU933258
142
identified in the Yunnan Province in China. Phylogenetic analysis of 8 begomoviruses
143
showed that ToLCNDV, BGYVV and SLCCV were clustered together and closed to
144
BYVMV but distant with another cluster of SLCV and OELCV (Fig. 2).
145
In Bangladesh, BYVMV and ToLCNDV were detected in all four cryptic B. tabaci
146
species (Table 1, Fig. 4). Other viruses, however, were detected in only a single species;
147
SLCCV in Asia I, TbCSV in Asia II 1, and BGYVV in Asia II 5.
148 149
Begomovirus identified in cryptic B. tabaci species collected from different host plants in
150
Bangladesh
151
Whiteflies were collected from nine different host plants: bean, brinjal, cotton, dahlia,
152
potato, sweet gourd, sweet potato, tobacco, and tomato (Table 2) to determine relationships
153
between begomoviruses and host plants. BYVMV was identified in whiteflies infesting seven
154
different host plants, but SLCV and TbCSV were only found in whiteflies infesting brinjal; in
155
fact, seven of the eight species of begomovirus identified were detected in whiteflies infesting
156
brinjal (Table 2).
157 158
Relationships between begomovirus and endosymbionts identified from cryptic B. tabaci
159
species in Bangladesh
160
Our previous study showed that each B. tabaci species in Bangladesh presents with
161
co-infection profiles containing the endosymbionts Arsenophonus, Cardinium, Rickettsia, or
162
Wolbachia, but not Hamiltonella [14]. The relationship of acquired endosymbionts and
163
begomoviruses was analyzed for each species (Table 1); however, we did not find any
164
associated patterns between acquired viruses and endosymbionts therein.
165 166 167
Discussion This study identified the presence of eight species of begomovirus from four cryptic
168
species of B. tabaci vector collected across Bangladesh. Viruses were detected in 26.4% of a
169
total of 110 collected whiteflies. Asia I was found in six species, Asia II 1 in three species,
170
Asia II 5 in five species, and Asia II 10 in one species. Infection rates ranged from 22 to 35%,
171
except for Asia II 10, which was only observed in a single individual. Infection rates for each
172
virus varied across species. Both BYVMV and ToLCNDV were found in all four cryptic
173
species at various rates. However, SLCCV, TbCSV, and BGYVV were only detected in Asia I,
174
Asia II 1, and Asia II 5, respectively. These results suggest that each cryptic species is a
175
potential vector for certain begomoviruses in a virus-specific manner.
176
Biodiversity among begomoviruses is rich in Southeast Asia and has been studied in
177
several countries in this region [21-26]. Our analysis found that begomovirus diversity is also
178
high in Bangladesh [15, 23-26]. Among them, BYVMV was the most frequently detected in
179
all four cryptic species of Bangladeshi B. tabaci and was also found in a variety of host plants
180
in the central region of Bangladesh. Our results suggest that BYVMV is widespread in this
181
country, infects many different crop plants, and is non-specifically transmitted by B. tabaci
182
regardless of genetic variations among vectors. BYVMV has also been found in neighboring
183
countries such as India, Pakistan, Thailand, and Africa [24-25]. A recent study demonstrated
184
that BYVMV could be transmitted by both invasive MEAM1 and indigenous Asia I, although
185
more efficiently by the former cryptic species [21]. Our study detected BYVMV more
186
frequently in Asia II 1 than in Asia I or Asia II 5, suggesting that transmission efficiency of
187
BYVMV may rely on specific genetic features of B. tabaci only found in certain species.
188
ToLCV is common and destructive in tomato plants and can result in a total crop loss
189
during an upsurge in B. tabaci infestation [27]. ToLCV was first reported in Israel in 1930,
190
after which it gradually extended into Africa, Australia, Central America, and parts of Asia
191
including Bangladesh [27-30]. Six ToLCV species have been identified on the Indian
192
subcontinent [31]; they are ToLCBV, ToLCBDV, ToLCNDV, Tomato leaf curl Gujarat virus
193
(ToLCGV), Tomato leaf curl Karnataka virus (ToLCKV), and Tomato leaf curl Sri Lanka
194
virus (ToLCSV). In Bangladesh, all six different types of ToLCV have been reported [31],
195
and recently, 21 types of ToLCV were reported in India [32]. ToLCNDV has been found in
196
trans-replicate with both mono and bipartite begomoviruses, particularly in Europe, Asia, and
197
Africa [33]. Our study detected ToLCNDV bipartite begomovirus in Asia I, Asia II 1, and
198
Asia II 5 and ToLCBDV monopartite in Asia I and Asia II 5, all from central and Southern
199
regions of Bangladesh. This suggests that ToLCV transmission may be common across
200
genetic groups of B. tabaci. Further study is required to determine the precise vector capacity
201
for the different virus strains of each cryptic species in this group.
202
SLCCV was first found in pumpkins from China in 1995 [34]. It has a restricted host
203
range within the Cucurbitaceae family, infecting squash and pumpkin, but not melon or
204
cucumber [35]. It has been found in many Asian countries, including China, India, Pakistan,
205
Malaysia, the Philippines, Thailand, and Vietnam [36,37]. SLCCV has also infected pumpkin
206
in East Timor and determined the first complete genomic segment DNA-A sequence [38]. We
207
detected this virus in Asia I collected from brinjal and tobacco, suggesting additional
208
potential host plants for this virus. Further research is necessary, however, to determine
209
infection parameters and virus symptoms in these plants. Two species of SLCCV, the China
210
and India strains, are geographically distinct, and a third, similar to the Indian strain, was
211
found in Pakistan [36]. In our study, SLCCV shared 96.3 to 95.7% genetic similarity with the
212
Indian strain.
213
OELCV is a serious emerging disease in okra production in northern India [39-41].
214
OELCV occurrence has been reported in Pakistan, Saudi Arabia, Iran, Nigeria, and China
215
[42-46]. OELCV is associated with the alpha and beta satellites of cotton leaf curl virus
216
(CLCuV) infecting cotton in Pakistan [47]. Saleem et al. [48] demonstrated that transmission
217
of CLCuV was more efficient in Asia II 1 than MEAM1. Similarly, cotton leaf curl disease
218
has been associated with Asia II 1 in Pakistan [49]. Our study in Bangladesh found OELCV
219
in Asia I and Asia II 5, but not in Asia II 1; however, this discrepancy is not reliable because
220
our OELCV detection rate was only 1.8%. A more intensive study on a larger vector
221
population is required to clarify the relationship between OELCV transmission and different
222
genetic groups of B. tabaci.
223
SLCV is found in South India and China (NCBI database). This virus has a typical,
224
old-world, monopartite begomovirus genome that was evaluated by beta satellite. The present
225
study found SLCV in Asia I collected from brinjal in Southern Bangladesh, and the infection
226
rate was very low.
227
TbCSV was associated with tobacco leaf curl disease and tomato leaf curl disease in
228
the Yunnan province of China in 2001[50,51]. TbCSV has also been reported to infect pepper,
229
common bean, sunflower, and tomato plants in India [52] and has been found in Punjab
230
(Alipur) in tobacco cultivation areas [16]. This study only identified TbCSV in Asia II 1
231
collected from northern Bangladesh, where tobacco cultivation is popular. Previous studies
232
have shown that TbCSV transmission efficiency is low in MEAM1 and Asia II 3 [53]
233
although Asia 1 and Asia II 1 transmission have been reported, as well [50,53,54]. Of the four
234
cryptic B. tabaci species examined in the present study, Asia II 1 was more frequently
235
infected with TbCSV begomovirus than the others [11].
236
This study evaluated virus profiles detected in four different cryptic B. tabaci species in
237
Bangladesh. Some viruses were detected in most or all of the four, while others were limited
238
to a single species. Our results suggest that relationships between begomovirus strains and
239
cryptic species of B. tabaci are highly differentiated at the species level.
240
The PCR detection of begomoviruses in the vector’s body is not always associated with
241
the circular transmission of that particular virus because whiteflies ingested begomoviruses
242
from plants without restriction. Ingested viruses must translocate from the gut into the
243
hemolymph and salivary glands if they are to be transmitted into another plants [8,9].
244
Therefore, further studies are necessary to determine whether begomoviruses in vector
245
species are transmitted through host plants.
246
Declaration of interests: none.
247 248
Acknowledgments
249
We would like to thank the editors at Essay Review for critical English language revision of
250
the manuscript.
251 252
Author Contributions
253
M.F.K. and K.-Y.L. conceived and designed experiments, M.F.K., H. H., J.-K.S. Conducted
254
experiments, E.-J.K., S.L., K.-Y.L. analyzed the results, M.F.K., H. H., J.-K.S. wrote the
255
manuscript and prepared figures and tables, M.F.K., H. H., J.-K.S., E.-J.K., S.L., K.-Y.L.
256
revised and approved the manuscript. All authors reviewed the manuscript.
257 258
Disclosure statement
259
The authors report no conflicts of interest. The authors alone are responsible for all aspects of
260
this manuscript.
261 262
Funding
263
This work was supported by the Research Program for Exportation Support of Agricultural
264
Products, Animal and Plant Quarantine Agency, in the Republic of Korea under Grant #Z-
265
1543086-2017-21-01.
266 267 268
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410
Figures and legends
411 412
Figure 1. Distribution of begomovirus by four cryptic species of Bemisia tabaci in
413
Bangladesh. Colored symbols represent the distributions of each begomovirus.
414 415
Figure 2. Phylogenetic analysis of begomovirus by maximum likelihood method based on
416
the HKY+G model. Evolutionary analyses were conducted in MEGA 6. Sequences identified
417
from Bangladesh in this study are shown in different colors and represented by sample name,
418
genetic group, begomovirus, and accession number. Bangladeshi sequences from GenBank
419
are shown in black.
420 421
Figure 3. Begomovirus detection rates from Bemisia tabaci collected in Bangladesh.
422
Numbers of virus sequences were counted from all Bemisia tabaci samples (n = 110).
423 424
Figure 4. Begomovirus detection rates from four cryptic species of Bemisia tabaci in
425
Bangladesh
1
Table 1. PCR detection of begomoviruses in Bemisia tabaci collected from various regions of Bangladesh Plant viruses ingested by B. tabaci Virus names
Accession numbers
A1+C2+W1 A1+C2+W1
SLCCV SLCCV
MK784259 MK784268
Identities (%) Genomic with reported organizations bemoviruses in GenBank Bipartite 99, DQ026296 Bipartite 99, DQ026296
Asia I
A1+W1
SLCCV
MK784265
Bipartite
99, DQ026296
Brinjal Brinjal
Asia I Asia I
A2+C2+R1 A2+C2
ToLCNDV MK784262 ToLCNDV MK784261
Bipartite Bipartite
99, EF043230 99, EF043230
Brinjal
Asia I
A1+R2+W1
ToLCNDV MK784251
Bipartite
99, EF043230
Bandarban
Dahlia
Asia I
A1+C2+W1
ToLCNDV MK784253
Bipartite
99, EF043230
BARI, Gazipur
Tomato
Asia I
A2+W1
ToLCNDV MK784258
Bipartite
99, KP641675
Rajshahi
Tomato
Asia I
C2+W1
BYVMV
MK784270
Monopartite
99, KJ462082
BARI, Gazipur
Sweet potato Sweet gourd Bean Brinjal Sweet potato Brinjal
Asia I
A1+W1
BYVMV
MK784254
Monopartite
99, KJ462082
Asia I
A1+C2+W1
BYVMV
MK784274
Monopartite
99, KJ462082
Asia I Asia I
A1+C2+W1 BYVMV MK784266 A1+C2+R2+W1 ToLCBDV MK784277
Monopartite Monopartite
99, KJ462082 99, KM383765
Asia I
A1+C2+W1
MK784256
Monopartite
99, KT394454
Asia I
A1+C2+R2+W1 SLCV
MK784250
Monopartite
95, KU933258
No
Sample names
Collection sites
Host plants
Genetic Co-infection of groups endosymbionts
1 2
Coxs-Br-7 Khul-Br-1
Brinjal Brinjal
Asia I Asia I
3
Digh-To-2
Tobacco
4 5
Coxs-Br-8 Coxs-Br-9 BARI-Br17 Band-Dah1 BARI-To21 Rajs-To342 BARI-Sp15
Coxsbazar Khulna Dighinala, Khagrachori Coxsbazar Coxsbazar BARI*, Gazipur
6 7 8 9 10 11
Satkh-Sg-3
Satkhira
12 13
Gopal-Be-4 Satkh-Br-6 BARI-Sp16 Band-Br-5
Gopalganj Satkhira BARI, Gazipur Bandarban
14 15
OELCV
1
16 17 18 19 20
Rang-Br-1 Dhak-To361 BARI-To19 BARI-Po20
Rangpur
Brinjal
Savar, Dhaka
Tomato
BARI, Gazipur BARI, Gazipur
Tomato Potato
Gopal-Br-1
Gopalganj
BARI-To13 BSMRAUTo-14 Rajs-To341
BARI, Gazipur BSMRAU, Gazipur Rajshahi
Tomato
24
Satkh-Be-1
Satkhira
Bean
25
Satkh-Be-2
Satkhira
Bean
26
Satkh-Sg-4
Satkhira
Sweet gourd
27
Satkh-Br-5
Satkhira
Brinjal
28
Gopal-Be-3 Gopalganj
Bean
29
BARI-Co18
Cotton
21 22 23
BARI, Gazipur
Brinjal Tomato Tomato
Asia II 1 Asia II 1 Asia II 1 Asia II 1 Asia II 1 Asia II 1 Asia II 1 Asia II 5 Asia II 5 Asia II 5 Asia II 5 Asia II 5 Asia II 5 Asia II 10
A2+C2+W1
TbCSV
MK784273
Monopartite
99, LC316184
A1+C2+W1
BYVMV
MK784263
Monopartite
100, KJ462082
A1+C2+W1
BYVMV
MK784257
Monopartite
99, KJ462082
A1+C2+W1
BYVMV
MK784249
Monopartite
99, KJ462082
A1+C2+W1
BYVMV
MK784267
Monopartite
99, KJ462082
A2+C2+R1
ToLCNDV MK784255
Bipartite
99, EF043230
A2+C2+R1
ToLCNDV MK784260
Bipartite
99, EF043230
A1+C2+W1
ToLCBDV MK784269
Monopartite
99, KM383765
A1+C2+W1
BYVMV
MK784272
Monopartite
99, KJ462082
A1+C2+W1
BGYVV
MK784271
Bipartite
99, KJ862841
A1+C2+W1
BGYVV
MK784276
Bipartite
99, KJ862841
A1+C2+W1
OELCV
MK784275
Monopartite
99, KT390454
A1+C2+W1
ToLCNDV MK784264
Bipartite
98, EF043230
A2+W1
BYVMV
Monopartite
99, KJ462082
2 2
MK784252
3
Table 2. Begomoviruses detected in four cryptic species of Bemisia tabaci collected from various host plants in Bangladesh Begomovirus
Cryptic species
Bhendi yellow vein mosaic virus (BYVMV)
Asia I, Asia II 1, Asia II 5, Asia Bean, brinjal, cotton, sweet gourd, sweet potato, potato, II 10 tomato
Tomato leaf curl New Delhi virus (ToLCNDV) Squash leaf curl China virus (SLCCV), Indian strain Tomato leaf curl Bangladesh virus (ToLCBDV) Okra enation leaf curl virus (OELCV) Bitter gourd yellow vein virus (BGYVV) Synedrella leaf curl virus (SLCV) Tobacco curly shoot virus (TbCSV)
Host plants
Asia I, Asia II 1, Asia II 5
Bean, brinjal, dahlia, tomato
Asia I
Brinjal, tobacco
Asia I, Asia II 5
Brinjal, tomato
Asia I, Asia II 1 Asia II 5 Asia I Asia II 1
Brinjal, sweet gourd Bean, sweet gourd Brinjal Brinjal
4 5
3
6
Table S1. Begomoviruses previously identified in Bangladesh by PCR detection from leaf and Bemisia tabaci samples Abbreviations Host plants
Sample types
Previousreports inBangladesh
Reported
ToLCJV-Mild
Tomato
leaf
Yes
2005
ToLCBDV
Tomato
leaf
Yes
2005
ToLCNDV
Tomato
leaf
Yes
2005
ToLCKV
Tomato
leaf
Yes
2005
ToLCGV
Tomato
leaf
Yes
2005
ToLCV
Tomato
leaf
Yes
2007
AYVM
Ageratum
leaf
Yes
2007
BGMV ChiLCuV
Bitter gourd Chilli
leaf leaf
Yes Yes
2007 2007
CYVMV
Croton
leaf
Yes
2007
BMV PLCV
Brinjal Papaya
leaf leaf
Yes Yes
2007 2007
PYVMV
Pumpkin
leaf
Yes
2007
SGYMV
Sponge gourd
leaf
Yes
2007
Squash leaf curl china virus
SLCCV
Brinjal, Tobacco
No
-
Bhendi yellow vein mosaic
BYVMV
Tomato, Sweet potato, Sweet gourd,
Yes
2007
Viruses Tomato leaf curl Joydebpur virus Tomato leaf curl Bangladesh virus Tomato leaf curl New Delhi virus Tomato leaf curl Karnataka virus Tomato leaf curl Gujarat virus Tomato leaf curl virus Ageratum yellow vein mosaic virus Bitter gourd mosaic virus Chilli leaf curl virus Croton yellow vein mosaic virus Brinjal mosaic virus Papaya leaf curl virus Pumpkin yellow vein mosaic virus Sponge gourd yellow mosaic virus
4
Bemisia tabaci Bemisia
virus
Bean, Okra
Okra enation leaf curl virus
OELCV
Sweet gourd
Tobacco curly shoot virus
TbCSV
Sweet gourd
Bitter gourd yellow vein virus
BGYVV
Sweet gourd, Bean
Synedrella leaf curl virus
SLCV
Brinjal
tabaci Bemisia tabaci Bemisia tabaci Bemisia tabaci Bemisia tabaci
7
5
No
-
No
-
Yes
2012
No
-
►Bemisia tabaci is a species complex of at least 40 cryptic species as well as a vector of begomoviruses. ► Four indigenous Asian cryptic species were identified in Bangladesh. ► Vector-based PCR analysis identified 8 different begomoviruses in Bangladesh. ► This study demonstrates the geographic distribution of various begomoviruses in Bangladesh and their relationships with cryptic species of B. tabaci.