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Citation: Oliveira, I.A.d.; Reis,
L.d.N.A.d.; Fonseca, M.E.d.N.; Melo,
F.F.S.; Boiteux, L.S.; Pereira-Carvalho,
R.d.C. Geminiviridae and
Alphasatellitidae Diversity Revealed by
Metagenomic Analysis of Susceptible
and Tolerant Tomato Cultivars across
Distinct Brazilian Biomes. Viruses
2024,16, 899. https://doi.org/
10.3390/v16060899
Academic Editor: Grazia Licciardello
Received: 3 April 2024
Revised: 20 May 2024
Accepted: 24 May 2024
Published: 1 June 2024
Copyright: © 2024 by the authors.
Licensee MDPI, Basel, Switzerland.
This article is an open access article
distributed under the terms and
conditions of the Creative Commons
Attribution (CC BY) license (https://
creativecommons.org/licenses/by/
4.0/).
viruses
Article
Geminiviridae and Alphasatellitidae Diversity Revealed by
Metagenomic Analysis of Susceptible and Tolerant Tomato
Cultivars across Distinct Brazilian Biomes
Izaías Araújo de Oliveira 1, Luciane de NazaréAlmeida dos Reis 1, Maria Esther de Noronha Fonseca 2,
Felipe Fochat Silva Melo 1, Leonardo Silva Boiteux 1, 2, * and Rita de Cássia Pereira-Carvalho 1, *
1Department of Plant Pathology, University of Brasília (UnB), Brasília 70910-900, DF, Brazil;
eng.agro16@gmail.com (I.A.d.O.); lucianealmeidareis@outlook.com (L.d.N.A.d.R.);
ffochatsm@hotmail.com (F.F.S.M.)
2Embrapa Vegetable Crops (Hortaliças), National Center for Vegetable Crops Research (CNPH),Brasília
70275-970, DF, Brazil; maria.boiteux@embrapa.br
*Correspondence: leonardo.boiteux@embrapa.br (L.S.B.); rcpcarvalho@unb.br (R.d.C.P.-C.)
Abstract: The diversity of Geminiviridae and Alphasatellitidae species in tomatoes was assessed via
high-throughput sequencing of 154 symptomatic foliar samples collected from 2002 to 2017 across
seven Brazilian biomes. The first pool (BP1) comprised 73 samples from the North (13), Northeast
(36), and South (24) regions. Sixteen begomoviruses and one Topilevirus were detected in BP1. Four
begomovirus-like contigs were identified as putative novel species (NS). NS#1 was reported in
the semi-arid (Northeast) region and NS#2 and NS#4 in mild subtropical climates (South region),
whereas NS#3 was detected in the warm and humid (North) region. The second pool (BP2) comprised
81 samples from Southeast (39) and Central–West (42) regions. Fourteen viruses and subviral agents
were detected in BP2, including two topileviruses, a putative novel begomovirus (NS#5), and two
alphasatellites occurring in continental highland areas. The five putative novel begomoviruses dis-
played strict endemic distributions. Conversely, tomato mottle leaf curl virus (a monopartite species)
displayed the most widespread distribution occurring across the seven sampled biomes. The overall
diversity and frequency of mixed infections were higher in susceptible (16 viruses + alphasatellites) in
comparison to tolerant (carrying the Ty–1 or Ty–3 introgressions) samples, which displayed 9 viruses.
This complex panorama reinforces the notion that the tomato-associated Geminiviridae diversity is yet
underestimated in Neotropical regions.
Keywords: breeding; Solanum lycopersicum; high-throughput sequencing; single-stranded DNA
viruses; tolerance
1. Introduction
The Geminiviridae is the major family of plant-infecting, insect-transmitted, circular,
and single-stranded DNA (ssDNA) viruses [
1
]. This family comprises
≈
520 species as-
signed to 14 highly divergent genera, including Becurtovirus, Begomovirus,Capulavirus,
Curtovirus,Eragrovirus,Grablovirus, Mastrevirus,Topocuvirus,Turncurtovirus,Citlodavirus,
Maldovirus, Mulcrilevirus, Opunvirus, and Topilevirus [
1
]. Begomoviruses (genus Bego-
movirus) are transmitted under natural conditions by a complex of cryptic Bemisia tabaci
species (family Aleyrodidae, order Hemiptera). The Begomovirus is the largest genus
(with
≈
445 viruses), encompassing monopartite (with a single DNA–A segment) and
bipartite (with DNA–A and DNA–B segments) species [
2
]. Currently, all newly assigned
Begomovirus species must have a nucleotide identity of less than 91% to the entire DNA–A
segment in relation to isolates of other previously described species within the genus [3].
The DNA–A component of the New World begomoviruses comprises six open reading
frames (ORFs): one in the viral sense (AV1) and five in the complementary sense (AC1 to
Viruses 2024,16, 899. https://doi.org/10.3390/v16060899 https://www.mdpi.com/journal/viruses
Viruses 2024,16, 899 2 of 24
AC5). The AV1 gene codes for the coat protein (CP). The AV2 gene is present only in Old
World begomoviruses and codes for the movement protein (MP) [2]. The AC1 gene codes
for a protein involved in viral replication (REP), while the AC2 gene is responsible for
coding the transcription activating protein (TrAp). The AC3 gene encodes REN, a protein
that enhances viral replication [
4
] and the gene product of the AC4 gene is associated
with the expression of symptoms [
5
]. The AC5 gene codes for a protein related to viral
pathogenicity able to suppress the host post-transcriptional gene silencing [
6
]. Recently,
new ORFs were identified in the DNA–A component, including ORF AV3, which codes
for a 7.4 KDa protein without ascribed function [
7
]; ORF AC6, which codes for a protein
that plays a role in targeting the host mitochondria [
8
]; and ORF AC7, which codes for
a protein that interacts with AV2 and AC2 proteins, inhibiting RNA silencing and acting
as a pathogenicity factor [
9
]. On the other hand, the DNA–B component comprises the
ORF BV1 (=NSP) coding for the nuclear shuttle protein and ORF BC1 (=MP) coding for the
movement protein of New World begomoviruses [10].
Bipartite genomes share a common region (CR) of
≈
200 nucleotides with conserved
motifs (iterons) involved in viral replication [
11
]. Within the CR is located a conserved
nonanucleotide sequence (TAATATTAC) that corresponds to the site of origin of viral repli-
cation responsible for Rep binding [
11
]. Cognate iterons are invariable among DNA–A and
DNA–B components of the same virus [
12
]. Another conserved Rep domain interacts with
the plant retinoblastoma protein, being crucial for modulating the host gene expression [
13
].
Promoter regions (homologous to the ones of the CPs from New World begomoviruses)
display nearly palindromic DNA sequences with a conserved core (ACTT–N7–AAGT),
which is distinct from that of the Old World begomoviruses [
14
]. Some begomoviruses
also present associations with DNA satellites, which can either attenuate or intensify the
symptom expression depending on the relationship between the satellite and its helper
virus [15–17].
The first reports of tomato (Solanum lycopersicum L.) diseases induced by begomoviruses
in Brazil were carried out in the 1960s and 1970s, including the characterization of tomato
golden mosaic virus (TGMV), the first Neotropical species [
18
]. During this period, be-
gomoviruses occurred only sporadically with no major economic importance. However,
this scenario changed after the invasion of the whitefly B. tabaci MEAM 1 in the 1990s,
resulting in an explosion of regional outbreaks and a substantial emergence of novel bego-
moviruses [
19
]. Tomato is a major crop in Brazil, being cultivated across all major biomes,
including the warm and humid Amazon Forest; Caatinga (semi-arid scrubland); temperate
Southern fields; highland and lowland Cerrado (Savannah) areas; Atlantic Rain Forest;
Pantanal (floodplain area); the warm/lowland seashore zone; and the peculiar transition
zones of Amazon Forest–Caatinga,Cerrado–Caatinga, and Amazon Forest–Cerrado (Supple-
mentary Figure S1). However, little is yet known about the diversity of tomato-infecting
Geminiviridae and Alphasatellitidae across each of these biomes.
In the past decade, metagenomic approaches have facilitated the discovery and iden-
tification of many novel and highly divergent members of the Geminiviridae family [
1
].
Metagenomics has also been a fundamental tool to provide a more accurate panorama
about the diversity of tomato-infecting ssDNA viruses under Brazilian conditions [
20
],
allowing the detection of
≈
30 begomoviruses in association with this vegetable crop in the
country [20,21].
The employment of resistant cultivars is the most efficient strategy for the manage-
ment of New World and Old World begomoviruses in tomatoes [
22
–
26
]. Two distinct
introgression events [
27
] involving a segment of chromosome 6 of Solanum chilense (named
as Ty–1 and Ty–3 genes) allowed the development of cultivars with suitable levels of tol-
erance [
28
]. The Ty–1 gene (and its putative allele Ty–3) encodes for an RDRy-type RNA
polymerase [
27
], being effective against a wide array of begomoviruses [
27
]. For this reason,
the Ty–1 and Ty–3 introgressions are massively employed in tomato breeding programs
worldwide [
29
]. Interestingly, a putative ‘filtering effect’ of the tolerance factor Ty–1 on
Viruses 2024,16, 899 3 of 24
the diversity of begomoviruses in tomato crops has been observed in Central Brazil in
HTS-based surveys [20,21].
In the present work, a broader geographical (across seven Brazilian biomes) and
chronological (from 2002 to 2017) survey of the diversity of the tomato-associated Gemi-
niviridae species and their satellite DNAs was conducted via an HTS-based approach. The
present survey covered samples from the main tomato-producing areas, located in different
biomes across all five macro-regions of Brazil. From the breeding standpoint, the present
work represents a more extensive sampling of the diversity of these viral pathogens and in-
vestigates the potential impact of two tolerance factors (Ty–1 and Ty–3) on the composition
and dynamics of tomato-associated Geminiviridae populations.
2. Material and Methods
2.1. Leaf Samples from Tomato Plants with Begomovirus-Like Symptoms
One hundred and fifty-four (154) leaf samples from tomatoes exhibiting typical be-
gomovirus symptoms (mosaics, leaf deformation, and mottle) were obtained from field
surveys in tomato-producing areas in five macro-regions across seven different Brazilian
biomes from 2002 to 2017 (Supplementary Table S1). The five macro-regions were North
(13), Northeast (36), Central–West (42), Southeast (39), and South (24) (Supplementary
Table S1). These samples were selected to cover a broader geographical and chronological
snapshot.
2.2. DNA Extraction and Molecular Marker Confirmation of the Presence of the Ty–1 and
Ty–3 Introgressions
Foliar samples were stored in a freezer (–20
◦
C) and the total DNA was extracted
from them using a modified protocol 2X CTAB plus organic solvents as described [
30
].
The quantification of the DNA of the samples was carried out using a NanoDrop-1000
spectrophotometer (Thermo Fisher Scientific, Waltham, MA, USA), and the nucleic acid
integrity was assessed by electrophoresis (1% agarose gel). Total DNA from leaf samples
was used as a template (40 ng/
µ
L) in PCR reactions using specific primers for genomic
regions encompassing the codominant molecular markers linked to the Ty–1 [
31
] and Ty–
3 [
27
] introgressions. The PCR products were analyzed via electrophoresis (1% agarose
gel), stained with ethidium bromide, and visualized under UV light.
2.3. Enrichment of Circular DNAs by Rolling Circle Amplification—RCA and Confirmation of
Begomovirus Infection
DNA extracted from each individual sample (40 ng/
µ
L) was used as a template for
RCA—Rolling Circle Amplification [
32
]. The confirmation of begomovirus infection in
the individual samples (40 ng/
µ
L) was performed essentially as described [
33
], using the
two degenerated primer pairs (PAL1v1978/PAR1c496 and PBL1v2040/PRCc1), targeting
conserved regions of the DNA–A component and DNA–B components, respectively [33].
2.4. Preparation of Pools of Samples to High-Throughput Sequencing (HTS)
The RCAs of the samples were grouped into two pools (named as BP1 and BP2). Pool
BP1 encompassed samples from the North (13), Northeast (36), and South (24) regions,
while pool BP2 was composed of samples from the Southeast (39) and Central–West (42)
regions (Supplementary Table S1). After establishing the pools, the corresponding libraries
were sequenced on an Illumina NovaSeq-6000 (Agrega, Porto Alegre, RS, Brazil) and 150 bp
paired-end reads were generated.
2.5. Viral Sequence Analyses
The adapter sequences were removed from the HTS data, and the trimmed sequences
were subjected to de novo assembly using the CLC Genomics Workbench 23.0.1 program
(Qiagen, Hilden, Germany), with the default parameters. The contigs were subsequently
analyzed with the Geneious
®
11.1 program [
34
]. All contigs were compared with the
Viruses 2024,16, 899 4 of 24
viral RefSEq database available at NCBI (https://www.ncbi.nlm.nih.gov; accessed on 3
April 2024), using the BLASTn and BLASTx algorithms. The procedure was carried out
essentially as previously described [
20
,
35
]. The read files provided by HTS were mapped
to virus-like contigs to obtain the final sequence. The information of each individual
contig was extended with the help of the Geneious
®
11.1 program using the ‘Map to
reference’ tool (90 to 99% minimum overlap identity parameter). MUSCLE alignments
were performed in Geneious
®
11.1 and used for ORF annotation. After de novo assembly,
taxonomic prediction analyses were carried out with the contigs from both pools using
the Kaiju web server (http://Kaiju.binf.ku.dk/server, accessed on 3 April 2024) [
36
], with
the standard classification parameters. From these analyses, the sequences predicted to be
of viral origin were recognized. The largest sequences were selected and assembled. The
viral sequences were aligned with the reference genomes showing greater identities [
36
]
with the help of the Geneious
®
R11.1 program. This program was also used to assemble
the viral genome, annotation, and sequence alignments. For potential new viral/subviral
species, in addition to the ORF annotation, the intergenic region (present in monopartite
begomoviruses) and the common region (present in bipartite begomoviruses) were also
analyzed. In the common region, the nonanucleotide motifs and iterons were characterized
as well as the Rep Iteron-related domains (REP-IRDs), which allowed us to confirm that
pairs of DNA–A and DNA–B components were cognate [
14
,
15
]. For comparison across
isolates and viral species, the sequences were aligned using pairwise MUSCLE multiple
alignment with the help of the Sequence Demarcation Tool (SDT) program [37].
2.6. Detection of Viruses in Individual Samples by PCR with Virus-Specific Primers
Based on the sequences obtained by HTS sequencing, open and oppositely directed
primer pairs were developed using the primer design function of the Geneious
®
R11.1
program [
34
]. The primers were used to detect viruses in individual samples using a row
per column system. Sets of virus-specific primers were used to detect viral species in the
samples (Supplementary Table S2).
2.7. PCR Conditions Used to Detect ssDNA Viruses and Subviral Agents in Individual Samples
within Each Pool
PCR assays with species-specific primers were used to recover the viral genomes in
each individual DNA sample. Reactions were carried out in a total volume of 12.5
µ
L,
containing the following components: a Taq polymerase buffer (10
×
; 1.25
µ
L), 50 mM
MgCl
2
(40
µ
L), 2.5 mM dNTPs (0.25
µ
L), 10
µ
M forward and reverse primers (0.25
µ
L),
Milli-Q water (8.0
µ
L), and Taq polymerase 0.5 U (0.10
µ
L). The 35 amplification cycles were
divided into the following steps: initial denaturation (94
◦
C for 3 min), denaturation (94
◦
C
for 30 s), annealing (see temperatures in Supplementary Table S2) for 45 s, extension (72
◦
C
for 3 min), and final extension (72
◦
C for 10 min). The amplicons generated were visualized
in agarose gel (1%) stained in ethidium bromide and under UV light in a transilluminator
and photodocumented.
2.8. Validation via Sanger Dideoxy Termination Sequencing of the Amplicons Obtained with
Species-Specific PCR Primers
To validate the PCR primers used in virus-specific detection assays (Supplementary
Table S2), the amplicons generated by each primer pair were purified using a DNA purifi-
cation kit (Ludwig Biotech, Alvorada, RS, Brazil) and then subjected to Sanger dideoxy
termination sequencing at ACTGene Análises Moleculares (Alvorada, RS, Brazil). The
chromatograms were evaluated for their quality and further analyzed using the BLASTn
algorithm. The final sequences were compared to the ones available at the NCBI database
(https://www.ncbi.nlm.nih.gov, accessed on 3 April 2024) in order to confirm the ssDNA
viral and subviral species present in each sample.
Viruses 2024,16, 899 5 of 24
3. Results
3.1. Viral Diversity in the BP1 Pool (Composed of Samples from North, Northeast, and South
Brazilian Regions)
The HTS, conducted on the Illumina NovaSeq-6000 platform, provided DNA viral
genomic information of the BP1 pool (composed of tomato foliar samples collected in
the North, Northeast, and South regions) with the following raw reads: 7,230,366 reads
and 38,575 contigs with 137 of them corresponding to genomic segments of viruses as
indicated by the BLASTn analysis. HTS-derived genomic information and assembly of
contigs from the BP1 pool allowed the recovery of 15 begomovirus-like genomes, 4 of them
classified as putative new species—NS (Tables 1and 2). Eleven viruses were previously
reported as infecting tomatoes, including the monopartite tomato mottle leaf curl virus—
ToMoLCV [
38
,
39
], the bipartite tomato severe rugose virus—ToSRV [
38
,
40
], and Sida
micrantha mosaic virus—SimMV [
41
]. Among the 11 previously characterized Begomovirus
species, ToMoLCV displayed the highest read coverage (277,339), followed by ToSRV
(227,279), tomato golden leaf distortion virus—ToGLDV (170,461), tomato chlorotic mottle
Guyane virus—ToCMoGV (131,012), and Chino de tomate Amazonas virus—ChdTAV
(55,010). The additional viruses displayed lower read coverage numbers (Table 1). In
relation to the number of isolates, the ToMoLCV displayed the highest number (nine
isolates), followed by ToGLDV (three) and SimMV (two), and the other viruses were found
only in single isolates (Table 1). Sida yellow blotch virus—SiYBV (contig 6958) was the
only formerly described begomovirus that was not yet reported in association with tomato
crops in this pool.
As mentioned, four DNA–A segments exhibited identity levels lower than the thresh-
old of 91% nucleotide identity (Table 1), which is the current demarcation criterion for a
novel Begomovirus species [
3
]. Putative NS#1 (41,497 reads) shared 90.4% identity with
tomato interveinal chlorosis virus—ToICV (NC_038469.1), NS#2 (153,967 reads) shared
90.17% identity with ToMoLCV (MT215005.1), NS#3 (79.920 reads) shared 87.10% with
tomato bright yellow mosaic virus—ToBYMoV (NC_038468.1), and NS#4 (24.589 reads)
shared 82.80% with ToGLDV (HM357456.2). The HTS results from the BP1 pool (Table 2)
also allowed the recovery of complete DNA–B segments. Isolates of ToCMGV displayed
the highest coverage (129,354 reads) and the highest number of isolates (= four).
A partial genome related to tomato-associated geminivirus 1 (genus Topilevirus) was
also recovered (Tables 1and 2). A topilevirus-like virus (contig 8679) displayed low reads
(82) and only partial genomic sequence (2139 nucleotides) recovery (Table 1). The contig
associated with the Topilevirus-like partial genome shared a nucleotide identity of 90.27%
with tomato-associated geminivirus 1 (TAG1; MN527305.1) (Table 1).
3.2. Viral Diversity in the BP2 Pool (with Samples from Southeast and Central–West Regions)
The number of reads of the BP2 pool was in the order of 8,533,058, which generated
34,964 contigs, 145 of them corresponding to putative viral genome segments. HTS-derived
genomic information and assembly of contigs allowed the recovery of 14 viruses and/or
subviral agents (Tables 3and 4). Nine of them matched to previously reported tomato-infecting
begomoviruses viz.: Euphorbia yellow mosaic virus—EuYMV [
20
,
42
], tomato common mosaic
virus—ToCMoV [
43
], SimMV [
41
], ToSRV [
38
,
40
], tomato rugose mosaic virus—ToRMV [
20
,
24
,
38
], tomato yellow vein streak virus—ToYVSV [
44
,
45
], ToMoLCV [
38
,
39
], tomato golden vein
virus—TGVV [
20
,
45
], ToICV [
46
,
47
], and tomato yellow net virus—ToYNV [
21
]. ToRMV
displayed the highest number of reads (601,302), followed by ToSRV (590,532 reads), SimMV
(431,707), ToMoLCV (412,517), and putative NS#5 (369,619).
Regarding the number of isolates, the ToMoLCV displayed the highest number
(eleven), followed by TGVV (six), SimMV (five), and ToSRV, TRMV, and ToCMoV (three iso-
lates of each). The remaining viruses varied from one to two representative isolates (Table 3).
ToSRV displayed the highest number of reads (300,551 reads) and sequences (four) among
the recovered DNA–B segments (Table 4). The genome of a putative new begomovirus
(NS#5) was recovered, sharing 89.03% identity with a TGVV isolate (MN928612.1).
Viruses 2024,16, 899 6 of 24
Table 1. Code of the contigs, read coverage, assembled genome size, BLASTn coverage, sequence identity of the assembled virus, E-value, virus description, and
GeneBank accession number for the DNA–A segment of Geminiviridae viruses and subviral agents obtained by High-Throughput Sequencing (HTS) within pool BP1
(containing 73 foliar tomato samples from the North, Northeast, and South regions of Brazil). Contigs highlighted in gray and bold letters represent putative new
viral species.
Code of the
Contigs Read Coverage
Assembled
Genome Size
(nts)
BLASTn
Coverage (%)
Identity
(%) E-Value Virus Description * GeneBank
Accession Number
40 227,279 2593 100 99.96 0 Tomato severe rugose virus DNA–A 1MW573989.1
* 17,827 2660 100 99.85 0 Tomato yellow leaf deformation dwarf virus
DNA–A1NC_055586.1
38 131,012 2630 100 99.73 0 Tomato chlorotic mottle Guyane virus DNA–A 1MK878452.1
5 31,367 2694 99 99.52 0 Tomato rugose yellow leaf curl virus DNA–A 1KU682839.1
1098 9959 2661 100 98.99 0 Tomato yellow spot virus DNA–A 3KX348172.1
63 277,339 2634 100 98.97 0 Tomato mottle leaf curl virus DNA–A 4KX896408.1
66 55,010 2603 100 98.47 0 Chino del tomate Amazonas virus DNA–A 1NC_038443.1
7 170,461 2623 99 98.25 0 Tomato golden leaf distortion virus DNA–A 3HM357456.2
125 205,526 2631 100 97.00 0 Tomato mottle leaf curl virus DNA–A 6MT215005.1
144 156,309 2623 99 96.85 0 Tomato golden leaf distortion virus DNA–A 2HM357456.2
140 77,529 2627 100 96.66 0 Tomato mottle leaf curl virus DNA–A 5JF803247.1
44 194,632 2632 100 95.79 0 Tomato mottle leaf curl virus DNA–A 4KX896414.1
552 4407 2686 100 95.38 0 Sida micrantha mosaic virus DNA–A 2KC706535.1
105 152,241 2623 99 95.18 0 Tomato golden leaf distortion virus DNA–A 1HM357456.2
109 14,715 2661 100 95.11 0 Tomato yellow spot virus DNA–A 1KX348172.1
21 44,698 2622 99 94.82 0 Tomato bright yellow mottle virus DNA–A 1NC_038468.1
6958 2854 2643 99 94.54 0 Sida yellow blotch virus DNA–A 1MT103998.1
67 254,814 2631 100 92.49 0 Tomato mottle leaf curl virus DNA–A 1KX896414.1
68 129,337 2631 100 91.66 0 Tomato mottle leaf curl virus DNA–A 1JF803250.1
205 32,299 2685 100 91.01 0 Sida micrantha mosaic virus DNA–A 1EU908733.1
New species #1 41,497 2604 100 90.40 0 Tomato interveinal chlorosis virus DNA–A 2NC_038469.1
8679 82 2139 100 90.27 0 Tomato-associated geminivirus 1 1MN527305.1
New species #2 153,967 2631 100 90.17 0 Tomato mottle leaf curl virus DNA–A1MT215005.1
New species #3 7992 2657 99 87.10 0 Tomato bright yellow mottle virus DNA–A1NC_038468.1
New species #4 24,589 2612 97 82.80 0 Tomato golden leaf distortion virus DNA–A1HM357456.2
* Virus obtained from Kaiju online tool. Viruses with the same superscript number correspond to distinct isolates of the same species.
Viruses 2024,16, 899 7 of 24
Table 2. Code of the contigs, read coverage, assembled genome size, assembled genome size, BLASTn coverage, sequence identity of the assembled virus, E-value,
virus description, and GeneBank accession number for the DNA–B segment of begomoviruses obtained by High-Throughput Sequencing (HTS) within pool BP1
containing 73 foliar tomato samples from the North, Northeast, and South regions of Brazil.
Code of the
Contigs Read Coverage
Assembled
Genome Size
(nts)
BLASTn
Coverage (%)
Identity
(%) E-Value Virus Description * GeneBank
Accession Number
10 129,354 2593 100 99.92 0 Tomato chlorotic mottle Guyane virus DNA–B 2MK878451.1
98 3725 2609 100 99.00 0 Tomato yellow leaf deformation dwarf virus
DNA–B 2NC_060089.1
81 18,916 2535 100 97.09 0 Chino del tomate Amazonas virus DNA–B 2MG675220.1
2034 92,661 2662 83 96.76 0 Tomato chlorotic mottle Guyane virus DNA–B 1MK878451.1
1156 15,163 2634 100 93.70 0 Tomato yellow spot virus DNA–B 3KX348205.1
2913 82,057 2597 100 91.62 0 Tomato chlorotic mottle Guyane virus DNA–B 1MK878451.1
1778 1475 2583 100 88.31 0 Tomato crinkle leaf yellows virus DNA–B 3JN419011.1
299 2808 2619 99 83.98 0 Tomato yellow leaf deformation dwarf virus
DNA–B 3NC_060089.1
93 10,358 2565 85 82.05 0 Tomato interveinal chlorosis virus-2 DNA–B 2MK087039.1
8 14,354 2656 96 77.95 0 Tomato rugose yellow leaf curl virus DNA–B 1JN381822.1
* Virus obtained from Kaiju online tool. Viruses with the same superscript number correspond to distinct isolates of the same species.
Table 3. Code of the contigs, read coverage, assembled genome size, BLASTn coverage, sequence identity of the assembled virus, E-value, virus description, and
GeneBank accession number for the DNA–A segment of Geminiviridae viruses and subviral agents obtained by High-Throughput Sequencing (HTS) of pool BP2
containing 81 foliar tomato samples from the Southeast and Central–West regions. Contig highlighted in gray and bold letters corresponds to a putative new viral
species.
Code of the
Contigs Read Coverage Assembled
Genome Size (nts)
BLASTn Coverage
(%)
Identity
(%) E-Value Virus Description * GeneBank Accession
Number
38 46,535 1322 100 99.92 0 Alphasatellitidae sp.1MT214093.1
107 574,158 2591 100 99.88 0 Tomato severe rugose virus DNA–A 5MT733811.1
78 73,475 2631 100 99.81 0 Tomato mottle leaf curl virus DNA–A 3MT733813.1
255 1205 2628 100 99.51 0 Euphorbia yellow mosaic virus DNA–A 1MN782438.1
52 206,835 2622 100 99.50 0 Tomato chlorotic mottle virus DNA–A 2MT733804.1
79 57,409 2561 100 99.45 0 Tomato golden vein virus DNA–A 4KC706652.1
827 601,302 2698 99 99.32 0 Tomato rugose mosaic virus DNA–A 1MT215006.1
34 113,869 2561 100 98.83 0 Tomato golden vein virus DNA–A 1KC706646.1
Viruses 2024,16, 899 8 of 24
Table 3. Cont.
Code of the
Contigs Read Coverage Assembled
Genome Size (nts)
BLASTn Coverage
(%)
Identity
(%) E-Value Virus Description * GeneBank Accession
Number
14432 113 2636 100 98.79 0 Tomato yellow net virus DNA–A 2MT214096.1
47 12,561 2610 100 98.74 0 Euphorbia yellow mosaic virus DNA–A 2KY559437.1
211 245,699 2631 100 98.29 0 Tomato mottle leaf curl virus DNA–A 3MT733813.1
29 335,413 2606 100 98.05 0 Tomato rugose mosaic virus DNA–A 2MT215006.1
934 1694 2574 100 97.98 0 Tomato-associated geminivirus 2 1MN527305.1
37 154,963 2631 100 97.95 0 Tomato mottle leaf curl virus DNA–A 2MT214088.1
* 26,055 2560 100 97.89 0 Tomato yellow vein streak virus DNA–A 1KC136337.1
7 232,163 2622 100 97.83 0 Tomato chlorotic mottle virus DNA–A 3MT733804.1
268 4530 2671 99 97.78 0 Sida micrantha mosaic virus DNA–A 4JX415194.1
73 138,272 2561 100 97.77 0 Tomato golden vein virus DNA–A 4JF803259.1
* 4822 2667 100 97.76 0 Sida yellow mosaic virus DNA–A 1AY090558.1
101 264,219 2631 100 96.92 0 Tomato mottle leaf curl virus DNA–A 5MT215005.1
50 286,731 2637 95 96.83 0 Tomato rugose mosaic virus DNA–A 1MT215006.1
74 3857 2560 100 96.76 0 Tomato yellow vein streak virus DNA–A 1MN508216.1
40 229,047 2631 100 95.79 0 Tomato mottle leaf curl virus DNA–A 4MT733813.1
307 94,086 2676 100 95.70 0 Sida micrantha mosaic virus DNA–A 1KC706535.1
201 10,481 1365 100 94.59 0 Euphorbia yellow mosaic alphasatellite 1FN436008.1
49 203,042 2602 100 93.45 0 Tomato chlorotic mottle virus DNA–A 1MT733804.1
23 145,276 2631 100 94.19 0 Tomato mottle leaf curl virus DNA–A 1JF803247.1
* 431,707 2676 100 94.18 0 Sida micrantha mosaic virus DNA–A 1MT214092.1
* 204,035 2618 100 93.81 0 Tomato interveinal chlorosis virus DNA–A 1JF803253.1
56 412,517 2631 100 93.06 0 Tomato mottle leaf curl virus DNA–A 1MT733813.1
3065 123,209 2572 100 92.76 0 Tomato severe rugose virus DNA–A 1HQ606468.1
357 36,971 2675 100 91.76 0 Sida micrantha mosaic virus DNA–A 1MT214092.1
84 21,005 2556 100 91.68 0 Tomato golden vein virus DNA–A 1KC706653.1
New species #5 369,619 2561 100 89.03 0 Tomato golden vein virus DNA–A 1MN928612.1
45 18,143 2879 100 85.21 0 Tomato apical leaf curl virus 2MT135209.1
* Virus obtained from Kaiju online tool. Viruses and subviral agents with the same superscript number correspond to distinct isolates of the same species.
Viruses 2024,16, 899 9 of 24
Table 4. Code of the contigs, read coverage, assembled genome size, BLASTn coverage, sequence identity of the assembled virus, E-value, virus description, and
GeneBank accession number for the DNA–B segments of begomoviruses obtained by High-Throughput Sequencing (HTS) of pool BP2 containing 81 foliar tomato
samples from the Southeast and Central–West regions of Brazil.
Code of the
Contigs Read Coverage
Assembled
Genome Size
(nts)
BLASTn
Coverage (%)
Identity
(%) E-Value Virus Description * GeneBank
Accession Number
103 143,673 2571 100 99.73 0 Tomato severe rugose virus DNA–B 4MT215002.1
82 102,342 2597 100 99.58 0 Tomato chlorotic mottle virus DNA–B 6MT214087.1
16 137 2570 100 99.57 0 Tomato rugose mosaic virus DNA–B 7MT215007.1
14 47,649 2533 100 99.33 0 Tomato golden vein virus DNA–B 3MN928611.1
122 48,514 2551 100 98.55 0 Tomato golden vein virus DNA–B 9MT733807.1
132 142,262 2554 100 98.26 0 Tomato severe rugose virus DNA–B 2MT214085.1
121 147,477 2571 93 97.00 0 Tomato severe rugose virus DNA–B 1MT215002.1
* 45,881 2543 100 96.58 0 Tomato mild leaf curl virus DNA–B 1DQ336352.1
27613 4 318 100 96.54 0 Sida micrantha mosaic virus DNA–B 1AJ557452.1
3050 90,728 2597 85 90.55 0 Tomato rugose mosaic virus DNA–B 1MT214091.1
21 37,396 2527 97 95.50 0 Tomato golden vein virus DNA–B 1KC706660.1
499 2740 2556 100 94.97 0 Tomato yellow vein streak virus DNA–B 1MN508217.1
* Virus obtained from Kaiju online tool. Viruses with the same superscript number correspond to distinct isolates of the same species.
Viruses 2024,16, 899 10 of 24
Two members of the Topilevirus genus were also recovered: tomato-associated geminivirus
2—TAG 2 (contig 934) and ToALCV (contig 45). Contig 934 shared 97.98% identity with TAG 2
(Table 3). Contigs 185 and 45 shared 100% identity with each other and 85.21% identity with
ToALCV and 18,143 reads (Table 3). A minimum nucleotide identity of 78% is the demarcation
criterion for a new species in the genus Topilevirus [
48
,
49
]. It was also possible to recover two
species of alphasatellites associated with begomoviruses: a putative new Alphasatellitidae species
and Euphorbia yellow mosaic alphasatellite. Contig 38 corresponds to the satellite DNA of the
Alphasatellitidae family (with read coverage of 46,535), while the second satellite DNA recovered
was Euphorbia yellow mosaic alphasatellite (with 10,481 reads).
3.3. Comparative Viral Diversity: BP1 Pool (North, Northeast, and South Regions) Versus BP2
Pool (Southeast and Central–West Regions)
It was possible to notice in the HTS results a greater diversity in the BP1 pool encom-
passing samples from the North, Northeast, and South regions (16 viruses) in contrast with
the BP2 pool encompassing samples from Southeast and Central–West regions (14 viruses).
However, the BP2 virome displayed greater quantities of DNA–B segments (Table 4) in
addition to subviral agents (Table 3).
3.4. PCR Detection with Species-Specific Primers of Geminiviruses and Subviral Pathogens in
Individual Samples of the BP1 and BP2 Pools
3.4.1. Northern Region
In the Northern region of Brazil, PCR using species-specific primers allowed the
detection of begomoviruses previously reported in other geographical regions, including
ToMoLCV, SimMV, ToCMoGV, and ToYSV (Table 5). ToMoLCV was detected in the states of
Amazonas (sample AM–012), Roraima (RR–003), and Tocantins (TO–088). Likewise, SimMV
was detected in the states of Amazonas (AM–010), Roraima (RR–003 and RR–004), and
Tocantins (TO–045 and TO–046). ToCMoGV, a pathogen reported thus far only in French
Guiana [
50
], was found here in the state of Amazonas (sample AM–035). ToYSV infection
was observed in the states of Roraima (samples RR–003 and RR–004) and Tocantins (TO–
046). Endemic species were also detected, including ToBYMoV and NS#3, which present in
a mixed infection in a sample from the state of Tocantins (designated as TO–167).
3.4.2. Northeast Region
ToMoLCV, SimMV, and NS#1 were the begomoviruses detected in the Northeast
region (Table 5). Among these, ToMoLCV was the most prevalent, infecting 23 samples.
However, our report is the first confirmation of ToMoLCV infection in tomato plants from
the state of Ceará(samples CE–001; CE–011; and CE–012). SimMV was present in two
samples from the states of Bahia (BA–100) and Pernambuco (PE–011), while novel species
#1 was detected in samples from the states of Ceará(CE–001) and Pernambuco (PE–011
and PE–012) (Table 5). Therefore, the predominance of ToMoLCV isolates in this semi-arid
region must be highlighted.
3.4.3. South Region
ToSRV, ToMoLCV, SimMV, and two novel begomovirus species were detected in the
South region (Tables 5and 6). ToSRV was the most prevalent (sixteen samples), followed
by ToMoLCV (nine samples) and only one sample with SimMV. ToSRV and ToMoLCV
were present in samples from the states of Santa Catarina, Rio Grande do Sul, and Paraná.
SimMV was only present in the state of Paraná(PR–143). NS#2 and NS#4 were detected in
the state of Paraná. NS#2 was detected in two samples (PR–173 and PR–174) and NS#4 in a
single (PR–144) sample (Table 5).
Viruses 2024,16, 899 11 of 24
Table 5. Positive samples for viruses detected via PCR with species-specific primers in tomato cultivars without carrying either Ty–1 or Ty–3 or both introgression
events in samples collected across the five Brazilian regions.
Virus Acronyms *
(Number of Positive Samples)
Codes of the Isolates with Positive PCR Detection per Pathogen per Geographical Region
North Northeast South Southeast Central–West
ToSRV
(15 + 20 + 19 = 54) – –
PR–143; PR–173; PR–174; RS–033;
RS–012; RS–013; RS–014; RS–015;
SC–001; SC–002; SC–015; SC–030;
SC–032; SC–044; and SC–051
MG–013; MG–014; MG–108;
MG–109; MG–267; MG–292;
SP–003; SP–006; SP–008; SP–017;
SP–111; SP–205; SP–206; SP–154;
SP–173; SP–201; SP–239; SP–254;
SP–259; and SP–274
DF–663; DF–034; DF–209;
DF–487; GO–121; GO–033;
GO–034; GO–126; GO–127;
GO–204; GO–208; GO–211;
GO–212; GO–218; GO–589;
GO–604; GO–605; GO–617; and
GO–618
ToMoLCV
(2+23+9+14+10=58) AM–012 and RR–003
BA–034; BA–035; BA–050;
BA–100; BA–128; BA–134;
BA–143; BA–173; BA–174;
CE–001; CE–011; CE–012; PB–025;
PB–027; PE–027; PE–028; PE–011;
PE–012; PE–099; PE–100; PE–104;
PE–105; and PE–121
PR–144; PR–173; PR–174; RS–015;
RS–071; RS–095; SC–002; SC–015;
and SC–030
MG–013; MG–014; MG–109;
MG–292; MG–381; SP–003;
SP–056; SP–058; SP–111; SP–213;
SP–205; SP–154; SP–173; and
SP–201
DF–027; DF–024; DF–054;
DF–154; DF–487; GO–495;
GO–604; GO–605; GO–617; and
GO–618
ToCMoV
(16 + 11 = 27) – – –
MG–046; MG– 013; MG–014;
MG–084; MG–109; MG–267;
MG–292; MG–381; SP–004;
SP–006; SP–008; SP–017; SP–056;
SP–205; SP–239; and SP–254
DF–027; DF–024; DF–034;
DF–044; DF–054; DF–057;
DF–170; DF–209; GO–121;
GO–126; and GO–127
TGVV
(8 + 8 = 16) – – –
MG–046; MG–013; MG–014;
MG–108; MG–109; SP–003;
SP–017; and SP–206
DF–027; DF–024; DF–170;
DF–209; GO–121; GO–126;
GO–127; and GO–218
SimMV
(5+2+1+2+11=21)
AM–010; RR–003; RR–004;
TO–045; and TO–046 BA–100 and PE–011 PR–143 MG–267 and SP–173
DF–024; DF–034; DF–044;
DF–054; DF–170; DF–209;
GO–121; GO–033; GO–126;
GO–127; and GO–204
EuYMV
(1+3=4) – – – SP–003 DF–170; GO–204; and GO–208
ToCMoGV
(1) AM–035 – – – –
ToYSV
(3) RR–003; RR–004; and TO–046 – – – –
ToBYMoV
(1) TO–167 – – – –
Viruses 2024,16, 899 12 of 24
Table 5. Cont.
Virus Acronyms *
(Number of Positive Samples)
Codes of the Isolates with Positive PCR Detection per Pathogen per Geographical Region
North Northeast South Southeast Central–West
New species #1
(3) – CE–001; PE–011; and PE–012 – – –
New species #2
(2) – – PR–173 and PR–174 – –
New species #3
(1) TO–167 – – – –
New species #4
(1) – – PR–144 – –
New species #5
(5) – – – – DF–209, GO–121, GO–126,
GO–127, and GO–218
Alfasatellite
(3) – – – – DF–024, DF–027, and DF–057
ToALCV
(1) – – – SP–173 –
TAGV
(1) – – – – GO–495
* Viruses: Tomato severe rugose virus (ToSRV), tomato mottle leaf curl virus (ToMoLCV), tomato chlorotic mosaic virus (ToCMoV), tomato golden vein virus (TGVV), Sida micrantha
mosaic virus (SimMV), Euphorbia yellow mosaic virus (EuYMV), tomato yellow spot virus (ToYSV), tomato bright yellow mottle virus (ToBYMoV), tomato apical leaf curl virus
(ToALCV), and tomato-associated geminivirus (TAGV).
Viruses 2024,16, 899 13 of 24
Table 6. Positive samples for geminiviruses detected via PCR with species-specific primers exclusively in tomato cultivars carrying either Ty–1 or Ty–3 or both
introgression events in samples collected across the five Brazilian regions.
Virus Acronyms
(Total of Positive Samples)
Codes of the Isolates with Positive PCR Detection per Pathogen per Geographical Region
North Northeast South Southeast Central–West
ToSRV
(1+6+7=14) – – PR–112 MG–268; MG–291; SP–018;
SP–156; SP–240; and SP–252
DF–216; DF–235; DF–338;
DF–530; DF–546; DF–528; and
DF–541
ToMoLCV
(1+ 11 = 12) – – – SP–172
DF–155; DF–235; DF–530;
DF–546; DF–528; DF–541;
GO–124; GO–229; GO–342;
GO–499; and GO–526
ToCMoV
(3+3=6) – – – MG–268; SP–066; and SP–252
DF–216; DF–235; and GO–124
TGVV
(1+4=5) – – – SP–018 DF–216; DF–235; GO–124;
and GO–229
ToYNV
(1) – – – – GO–342
SimMV
(5) – – – – DF–216; DF–235; DF–338;
GO–005; and GO–124
EuYMV
(1) – – – SP–066 –
New species #5
(3) – – – –
DF–216, DF–235; and GO–124
ToALCV
ToALCV (1) – – – SP–172 –
Tomato severe rugose virus (ToSRV), tomato mottle leaf curl virus (ToMoLCV), tomato chlorotic mosaic virus (ToCMoV), tomato golden vein virus (TGVV), tomato yellow net virus
(ToYNV), Sida micrantha mosaic virus (SimMV), Euphorbia yellow mosaic virus (EuYMV), tomato yellow spot virus (ToYSV), and tomato apical leaf curl virus (ToALCV).
Viruses 2024,16, 899 14 of 24
3.4.4. Southeast and Central–West Regions
Seven begomoviruses, two topileviruses, and one alphasatellite were detected via PCR
with virus-specific primers in the pool of tomato samples of the Southeast and Central–
West regions (Tables 5and 6). ToSRV, ToMoLCV, ToCMoV, TGVV, SimMV, and EuYMV
were detected in both regions. In the Southeast region, ToSRV was the most prevalent
(26 samples), followed by ToCMoV (19 samples), ToMoLCV (15), TGVV (10), SimMV
(2), and EuYMV (2). ToCMoV and TGVV have not been reported as infecting tomatoes
in the São Paulo state, as well as the presence of isolates from the topilevirus ToALCV
(samples SP–172 and SP–173). For the Central–West region, the most prevalent virus was
also ToSRV (26 samples) followed by ToMoLCV (21), SimMV (16), ToCMoV (14), TGVV (12),
EuYMV (3), and ToYNV (1 sample). NS#5 was detected in eight samples. In this region, an
alphasatellite was also detected in the Federal District (DF–024, DF–027, and DF–057), in
addition to the topileviruses TAGV in the state of Goiás (GO–495), and ToALCV (SP–172
and SP–173) (Tables 5and 6).
3.5. Comparative Diversity of Samples with Versus without the Ty–1/Ty–3 Introgressions
The number of different geminiviruses and associated satellites detected as well as
the number of infected samples and the number of mixed infections (Table 5and Figures 1
and 2) were greater in samples without the Ty–1/Ty–3 introgressions (Figure 1). Altogether,
the number of viruses and subviral agents in susceptible plants was 16 viruses (plus one
alphasatellite) versus 9 viruses in plants with the Ty–1/Ty–3 introgressions (Table 5and
Figure 1).
Viruses 2024,16, 899 15 of 24
Viruses 2024, 16, x FOR PEER REVIEW 15 of 24
Figure 1. Begomoviruses, Topileviruses, and satellite DNAs detected (X axis) versus the number of tomato samples (Y axis) with the presence and
absence of resistance/tolerance factors (Ty–1/Ty–3). Viruses detected: tomato severe rugose virus (ToSRV), tomato mole leaf curl virus (ToMoLCV),
tomato chlorotic mole virus (ToCMoV), tomato yellow net virus (ToYNV), tomato golden vein virus (TGVV), Sida micrantha mosaic virus (SimMV),
Euphorbia yellow mosaic virus (EuYMV), tomato chlorotic mole Guyane virus (ToCMoGV), tomato yellow spot virus (ToYSV), tomato bright
yellow mole virus (ToBYMoV), tomato apical leaf curl virus (ToALCV), and tomato-associated geminivirus (TAGV).
0
10
20
30
40
50
60
Number of infected samples
Viruses detected
With Ty-1/Ty-3
Without Ty-1/Ty-3
Figure 1. Begomoviruses, Topileviruses, and satellite DNAs detected (X axis) versus the number of tomato samples (Y axis) with the presence and absence of
resistance/tolerance factors (Ty–1/Ty–3). Viruses detected: tomato severe rugose virus (ToSRV), tomato mottle leaf curl virus (ToMoLCV), tomato chlorotic mottle
virus (ToCMoV), tomato yellow net virus (ToYNV), tomato golden vein virus (TGVV), Sida micrantha mosaic virus (SimMV), Euphorbia yellow mosaic virus
(EuYMV), tomato chlorotic mottle Guyane virus (ToCMoGV), tomato yellow spot virus (ToYSV), tomato bright yellow mottle virus (ToBYMoV), tomato apical leaf
curl virus (ToALCV), and tomato-associated geminivirus (TAGV).
Viruses 2024,16, 899 16 of 24
Viruses 2024, 16, x FOR PEER REVIEW 16 of 24
Figure 2. Number of viruses found in mixed infections (X axis) in tomato samples with presence and absence of tolerance factors Ty–1/Ty–3 (Y axis).
0
5
10
15
20
25
30
2345
Number of infected samples
Number of viruses in mixed infections
With Ty-1/Ty-3
Without Ty-1/Ty-3
Figure 2. Number of viruses found in mixed infections (X axis) in tomato samples with presence and absence of tolerance factors Ty–1/Ty–3 (Y axis).
Viruses 2024,16, 899 17 of 24
4. Discussion
The most recent worldwide surveys revealed that more than 300 viral species are able
to infect the tomato crop [
51
–
54
]. The largest number of tomato-infecting viruses (221)
are classified as Begomovirus species (family Geminiviridae), comprising 66.97% of all viral
pathogens reported as infecting this vegetable crop thus far [
51
–
54
]. This scenario of exten-
sive begomovirus diversity is more likely to expand due to genetic plasticity of this group
of pathogens, which is generated via mutation, recombination, and pseudorecombination
events [55,56].
HTS platforms are allowing the discovery of new ssDNA viruses through virome
studies, thus making it possible to monitor the increase in viral diversity across different
biomes and over time [
20
]. We were able to recover genomes of Begomovirus,Topilevirus,
and subviral ssDNA species after a very extensive HTS-based virome of foliar tomato
samples was collected across seven Brazilian biomes: the warm and humid Amazon
Forest; Caatinga (semi-arid scrubland); highland and lowland Cerrado (Savannah) areas;
Atlantic Rain Forest; the warm/lowland seashore zone; and Cerrado–Caatinga and Amazon
Forest–Cerrado transition zones.
The situation of tomato-infecting begomoviruses in Brazil prior to our work indicated a
viral complex of more than 26 species [
21
]. Herein, we potentially added five more tomato-
infecting begomoviruses to this pathogenic complex, employing a very representative
temporal snapshot of samples (2002 to 2017). These novel begomoviruses will be further
characterized via biological and molecular assays. It is important to highlight that our data
on the dynamic changes in the relative prevalence across years/geographical areas as well
as the discovery of a new set of species gives support to the notion that recurrent surveys
must be conducted to provide updated panoramas of tomato-infecting begomoviruses.
Our survey also indicated that the diversity of ssDNA viral and subviral species is yet
largely underestimated in Neotropical areas. In addition, our results corroborate studies
showing the efficiency of HTS for assessment of ‘hidden’ viral richness across different
environments and hosts [20,21,57].
The five putative novel begomoviruses detected herein displayed endemic distribu-
tions. New Begomovirus species #1 was reported in the semi-arid Northeast region, whereas
begomoviruses #2 and #4 were collected in mild subtropical climates (South region). NS#3
was detected in the warm and humid (North) region, whereas putative NS#5 was occur-
ring in the continental highland areas (Central–West region). NS#1, detected in the states
of Ceará(CE–001) and Pernambuco (PE–011 and PE–012), displayed a DNA–A segment
(2604 nts) with 90.40% identity with isolated tomato interveinal chlorosis virus (NC_038469).
NS#2 (2631 nts) shared 90.17% identity with ToMoLCV (MT215005) and was detected in the
state of Paraná(PR–173 and PR–174). For NS#1 and NS#2, their cognate DNA–B segments
were not found, indicating that they are two putative monopartite species. However, more
extensive studies searching for these DNA–B cognate segments should be conducted in
order to verify their putative monopartite nature. NS#3 (2657 nts) displayed 87.1% with
tomato bright mottle virus (NC_038468.1), detected in the state of Tocantins (TO–167).
NS#4 displayed 80.2% identity with tomato golden leaf distortion virus (HM357456) and
was detected in a single sample in the state of Paraná(PR–144). It has a typical bipartite
begomovirus DNA–A segment of 2612 nucleotides (nts), with cognate DNA–B of 2565 nts.
Finally, NS#5, detected in the state of Goiás and the Federal District, presented a DNA–A
segment of 2561 nts and 89.3% identity with tomato golden vein virus (MN928612.1), its
DNA–B segment cognate with 2527 nts. All five new begomovirus species meet the species
demarcation criterion of less than 91% identity with other species in the genus [3].
PCR assays with species-specific primer pairs allowed us to verify the presence of novel
viruses as well as the geographical dispersion of previously described tomato-infecting
begomoviruses across distinct Brazilian regions. Thus far, only four begomoviruses as-
sociated with tomato plants were reported in the North region of Brazil [
58
]. Herein, a
new virus was detected in the state of Amazonas, which was previously considered as a
begomovirus-free area. We detected ToCMoGV in the AM–035 sample originating from
Viruses 2024,16, 899 18 of 24
Iranduba (AM) collected in 2016. This virus was already reported in French Guiana [
50
].
Also, in the North region, ToYSV (=Leonurus mosaic virus) was reported for the first time
in tomato plants in the states of Tocantins and Roraima. This ToYSV was detected in
the samples TO–046 (collected in 2008 in Gurupi District) and RR–003 and RR–004 (both
collected in Boa Vista City in 2013). Until now, reports of ToYSV infecting tomato plants
in Brazil were restricted to the Southeast region, in the state of Minas Gerais [
59
]. The
tomato infection by ToMoLCV and SimMV in the North of Brazil is also a novel report. It
is worth mentioning that the information on tomato-infecting begomoviruses occurring
in the North region (Amazon) is yet very limited, as is the knowledge of viral diversity in
this geographic area. Therefore, our study indicates that additional surveys may reveal a
peculiar novel set of endemic begomovirus species able to infect tomatoes and other crops.
Thus far, ToMoLCV is the prevalent tomato-infecting begomovirus in the warm and
semi-arid Northeast region of Brazil [
39
,
44
,
46
,
60
], corroborating the results of the present
study. However, before our results, there were no reports in the literature of infections in
tomato plants by SimMV in the Brazilian Northeast region. SimMV infection, reported here
for the first time, can be explained by the great transmission efficiency and the polyphagous
habit of the supervector B. tabaci [
61
] as well as by the frequent presence of weeds of the
genus Sida, which are often in association with commercial tomato cultivation [
62
,
63
]. This
observation reinforces the epidemiological importance of weeds as a repository and source
of inoculum for tomato-infecting viruses [63].
There is an overall lack of information about the panorama of begomovirus on toma-
toes in the South region of Brazil, which is composed of three states. Our work is the
first report of ToSRV in Rio Grande do Sul and SimMV in ParanáState in association with
tomato plants. ToSRV was previously registered in Santa Catarina in the year 2006 [
64
]
and also in Paranáin 2014 [
65
]. A recent survey in the state of Santa Catarina found that
ToSRV is limited to the metropolitan region of Florianópolis [
66
]. We also provide the first
confirmation of tomato plants infected by ToMoLCV across all states of the South regions
(Paraná, Rio Grande do Sul, and Santa Catarina). Our study conducted with a relatively
low number of samples (24) suggests that the viral diversity associated with tomatoes is
likely to be underestimated in this geographic region.
The number of begomoviruses in the Central–West region is very high, corroborating
previous studies in this geographic area [
20
,
24
,
29
,
43
,
44
,
46
]. This is the most important
geographical region for processing tomato production in the country. We observed a slight
prevalence of ToRMV over ToSRV (601,302 versus 590,532 reads) in the Central–West region.
However, our results from individual samples confirmed previous surveys that ToSRV is
the most prevalent begomovirus in tomato in this area [
20
,
39
,
40
], surpassing ToRMV, whose
prevalence was gradually decreasing under natural conditions. ToRMV and ToSRV belong
to a complex of bipartite tomato-infecting begomoviruses that share identical iterons. In
addition, these viruses have almost identical DNA–B sequences (98.2% identity). Previous
studies indicated that ToRMV and ToSRV are able to form pseudorecombinants in tomato
plants under experimental conditions in all possible combinations of single and mixed
infections [
67
]. However, there was a preferential detection of both genomic segments
from ToRMV over the DNA–A and DNA–B of ToSRV, and the accumulation of ToSRV in
mixed infections was reduced compared to that in single infection. In fact, ToSRV shows a
high adaptability, infecting a large number of hosts [
63
] and being present across different
regions of the country. These attributes of ToSRV may also explain its prevalence in the
Central–West region.
ToSRV, ToMoLCV, ToCMoV, and TGVV were found to be the most prevalent and with
wider geographical distribution across the temperate Southeast region. This region is the
most important tomato-producing area for the fresh-market consumption in the country
and outbreaks of begomoviruses are very often detected across all states [
20
]. The species
ToSRV and ToMoLCV are the most relevant from the tomato breeding standpoint since they
were often detected in association with tomato samples with and without the Ty–1/Ty–3
resistance factors, showing the high adaptive and dissemination capacity of the virus. The
Viruses 2024,16, 899 19 of 24
lower relative richness of novel begomoviruses outside the Southeast and Central–West
regions can be explained by the fact that these regions have been subjected, over the
past few decades, to a greater number of prospecting works and surveys of begomovirus
diversity via either conventional PCR strategies or via HTS [
20
,
24
]. The unequal number
of DNA–B segments observed across the pools may allow us to infer the significant use
of these segments in pseudorecombination events, allowing viruses to better adapt in the
absence of the cognate DNA–B segment and at the same time increase genetic variability.
Although endemic begomoviruses were detected in our survey, no Old World be-
gomoviruses were found in Brazil, suggesting, thus far, the exclusive invasion of non-
viruliferous populations of the exotic vector B. tabaci MEAM1. The greater number of novel
species in the BP1 pool can be explained by a large variation of the landscapes, encom-
passing distinct ecological niches and biomes. A second hypothesis of the higher number
of ssDNA viruses and subviral agents in BP1 could restrict employment of cultivars with
either Ty–1 or Ty–3 introgressions in the sampled regions.
Alphasatellite isolates were detected only in the Federal District, in two adjacent cities
of Gama (DF–024 and DF–027) and Ponte Alta (DF–057), revealing that, to date, this agent
is endemic to the central region of Brazil. Satellite DNAs are subviral agents that can
modulate viral pathogenesis depending on the interaction between the helper virus and
the host plant [
16
,
17
,
68
]. The presence of alphasatellites associated with tomato crops was
previously reported in the Central–West region of Brazil [
20
], corroborating the results
reported here. A closely related alphasatellite was formally reported in the weeds Euphorbia
heterophylla (KY559640.1), Sida spp. (KX348227.1), and Cleome affinis, with either EuYMV or
Cleome leaf crumple virus (ClLCrV) as helper viruses [69].
The TAGV (genus Topilevirus) was detected in a single sample in Central Brazil (GO–
495) collected in 2001 in the city of Planaltina de Goiás (GO). The first report of this
topilevirus in tomato plants was also carried out in Central Brazil [
70
]. However, we
detected the presence of the topilevirus ToALCV in Sao Paulo State (Southeast region) in
samples SP–172 and SP–173, both originating from Santo Antônio da Posse (SP) in 2015. As
far as we know, the presence of ToALCV infecting tomato plants was restricted to the central
region of Brazil [
71
]. The first reports of topileviruses associated with tomato crops were
carried out in Brazil [
70
] and in Argentina [
48
]. Currently, only two species are reported:
tomato-associated geminivirus [
70
] and tomato apical leaf curl virus [
48
]. Immediately
after the report, tomato apical leaf curl virus (ToALCV) was detected for the first time
infecting tomato plants in Central Brazil [
71
]. Since then, ToALCV has been reported to
be associated with tomatoes in other surveys across the Brazilian Central–West area [
71
].
Analyses based on the amino acids of the CP protein were used to propose that ToALCV
can be transmitted by the planthopper Micrutalis maleifera (family Membracidae). However,
transmission trials have not yet been carried out to confirm this hypothesis [
48
]. These
successive reports of these viruses show the rapid distribution capacity of topileviruses. In
fact, the result obtained here represents an expansion in the geographic distribution of the
genus since it is the first report outside Central Brazil.
Previous HTS-based surveys revealed that the Ty–1 factor might play a role as a “di-
versity filter”, reducing the number of ssDNA viruses and mixed infections in tomato plants
carrying this introgression [
20
]. Even with an unequal number of samples
(121 without and 33 with either Ty–1 or Ty–3 tolerance factors), the overall diversity ob-
served here was higher in susceptible samples (16 viruses + alphasatellites) in comparison
to tolerant samples (9 viruses). In addition, it is interesting to point out that four out
of five novel begomoviruses were detected in plants without either Ty–1 or Ty–3 genes.
Overall, these observations are also suggesting a ‘filtering effect’ of both tolerance factors
as previously observed [
20
]. In some cases, species-specific “filtering” was observed for
SimMV in the Central–West region, ToSRV in the South, ToMoLCV in the Northeast and
Southeast, as well as ToCMoV and TGVV in the Southeast region. However, additional
studies should be carried out employing controlled bioassays since local environmental
factors (e.g., high temperatures) might interfere with the mRNA and protein expression
Viruses 2024,16, 899 20 of 24
of these tolerant factors, misleading our conclusion about their full spectrum of efficiency.
We also could observe that the number of tomato plants carrying either Ty–1 or Ty–3 genes
displayed lower frequencies of both simple and mixed viral infections. In this regard,
our study is the first to assess the impact of the Ty–3 gene/allele on the dynamics of the
tomato/begomovirus pathosystem.
In addition, our results suggest that viruses that infect tomato plants with these toler-
ance genes may carry peculiar evolutionary/adaptive processes. For example, NS#5 and a
novel isolate of tomato apical leaf curl virus (SP–172) were detected only in tolerant plants
exhibiting severe begomovirus-like symptoms. Viruses able to replicate in plants with the
Ty–1 and Ty–3 resistance factors may be undergoing a differential evolutionary/adaptive
process, which could result in viral isolates with potentially superior capacity to overcome
resistance mediated by these genes. This selective force could be more intense especially
for begomoviruses with high adaptability and with greater dispersion and predominance
(e.g., ToSRV and ToMoLCV).
5. Conclusions
Herein, we uncovered a significant increase in the geographical amplitude of the
tomato-begomovirus pathosystem, encompassing different Brazilian biomes as well as
geographical regions. Similar to what was previously observed [
20
], ToSRV (a bipartite
species) and ToMoLCV (a monopartite species) were the prevalent begomoviruses in
the country, followed by TGVV and ToCMoV. Even though ToMoLCV is predominant
in the Northeast region, it is important to highlight that this begomovirus is the most
widely distributed, being present across seven biomes across all five macro-geographic
regions of Brazil. ToMoLCV is currently reaching areas where ToSRV was not yet able to
establish. In addition, when comparing ToSRV and ToMoLCV regarding their ability to
infect tomato plants with the presence of Ty–1/Ty–3 factors, both viruses displayed very
similar frequencies in these samples (14 versus 12 detections, respectively).
The adaptation to the Ty–1/Ty–3 tolerance factors may also be related to the diversity
of viruses with RNA genomes that are simultaneously infecting tomato plants with these
introgressions. It has already been found that the presence of the tomato chlorosis crinivirus
(ToCV) may reduce the efficiency of the Ty–1-mediated tolerance to tomato yellow leaf curl
virus—ToYLCV in Europe [
72
]. In this scenario, the use of gene pyramiding of multiple
resistance factors against criniviruses [
73
] and begomoviruses [
74
] would be a promising
strategy for generating phenotypic stable sources of resistance.
In conclusion, we demonstrated the efficiency of HTS-based platforms in combina-
tion with virus-specific PCR assays as tools for the large-scale study of the diversity of
Geminiviridae species across different regions over time. Novel species and novel tomato-
begomovirus interactions were detected. The present study also provided new insights
on the begomovirus distribution across Brazil and the confirmation of the ToSRV and
ToMoLCV as the most prevalent and the most disseminated pathogens as well as with the
best adaptation to the Ty–1/Ty–3 factors. The diversity detected in the susceptible samples
(16 viruses + alphasatellites) and the frequency of mixed infections was higher than the
ones with tolerance (9 viruses), suggesting that the Ty–1/Ty–3 genes may interfere with the
overall diversity.
This complex panorama reinforces the notion that the Geminiviridae diversity is yet
underestimated under Neotropical conditions. All these data will help to guide breeding
programs regarding the most effective control strategies and to update the status on
emergent and consolidated tomato-infecting begomoviruses in Brazil. Hence, we provide
a more accurate overview of the current situation of begomoviruses in tomato plants in
Brazil that could help the understanding of the population dynamics of these viruses and
their behavior in relation to the main tolerance genes used to control these viruses in the
country (Ty–1/Ty–3).
Viruses 2024,16, 899 21 of 24
Supplementary Materials: The following supporting information can be downloaded at: https://
www.mdpi.com/article/10.3390/v16060899/s1, Figure S1: Map illustrating the major Brazilian
biomes.; Table S1: Information on geographical regions, absence and/or presence of the molecular
markers associated with the resistance factors Ty–1 and Ty–3, year of collection and code of the
154 leaf samples of tomato cultivars (Solanum lycopersicum) used in the present study.; Table S2: List
of species-specific primers used to detect different viruses and satellite DNA in tomato samples and
details of information regarding the name of the primer, sequences, and annealing temperatures
(AT ºC).
Author Contributions: Conceptualization, I.A.d.O., M.E.d.N.F., L.S.B., and R.d.C.P.-C.; methodology,
I.A.d.O., L.d.N.A.d.R., F.F.S.M., L.S.B., M.E.d.N.F., and R.d.C.P.-C.; software, I.A.d.O., M.E.d.N.F.,
L.d.N.A.d.R., and R.d.C.P.-C.; validation, I.A.d.O., and L.d.N.A.d.R.; formal analysis, I.A.d.O. and
L.d.N.A.d.R.; investigation, I.A.d.O. and L.d.N.A.d.R.; resources, M.E.d.N.F., L.S.B., and R.d.C.P.-C.;
data curation, M.E.d.N.F. and L.S.B.; writing—original draft, I.A.d.O., L.d.N.A.d.R., L.S.B., and
R.d.C.P.-C.; preparation, I.A.d.O., L.d.N.A.d.R., L.S.B., and R.d.C.P.-C.; writing—review and editing,
I.A.d.O., M.E.d.N.F., L.S.B., and R.d.C.P.-C.; visualization, L.S.B. and R.d.C.P.-C.; supervision, L.S.B.
and R.d.C.P.-C.; project administration, M.E.d.N.F., L.S.B., and R.d.C.P.-C.; funding acquisition,
M.E.d.N.F., L.S.B., and R.d.C.P.-C. All authors have read and agreed to the published version of
the manuscript.
Funding: This research was supported by grants, scholarships, and post-doc fellowships from CNPq,
CAPES (Finance code 1), FAP–DF, and the Empresa Brasileira de Pesquisa Agropecuária (Embrapa),
Tomato Breeding project.
Informed Consent Statement: Not applicable.
Data Availability Statement: No new data were created.
Conflicts of Interest: The authors declare no conflicts of interest.
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