![Assessing F1 plants derived from selected Solanum pimpinellifolium lines for their resistance to ToBRFV as evaluated for their absorbance values (virus titers) using an ELISA test. F1 plants from four crosses: 1-F1: 327-1 [PI 390712 (S)] x 326 [PI 390711 (R)]; 2-F1: 328-1 [PI 390713 (R)] x 326-1 [PI390711 (R)]; 3-F1: 333-1 [PI 390718 (S)] x 329-1 [PI 390714 (R)]; and 4-F1: 333-1 [PI 390718 (S)] x 332-1 [PI 390717 (R)] were evaluated for their resistance to ToBRFV, in comparison to the mock inoculation control and a positive control (ToBRFV+).](https://www.researchgate.net/publication/377309075/figure/fig3/AS:11431281216900470@1704975267884/Assessing-F1-plants-derived-from-selected-Solanum-pimpinellifolium-lines-for-their_Q320.jpg)
Available via license: CC BY
Content may be subject to copyright.
Article Not peer-reviewed version
Evaluation of Tomato Germplasm
against Tomato Brown Rugose Fruit
Virus and Identification of Resistance in
Solanum pimpinellifolium
Namrata Jaiswal , Bidisha Chanda , Andrea Gilliard , Ainong Shi , Kai-Shu Ling *
Posted Date: 10 January 2024
doi: 10.20944/preprints202401.0785.v1
Keywords: Tobamoviruses; tomato brown rugose fruit virus (ToBRFV); genetic resistance; tomato; Solanum
lycopersicum
Preprints.org is a free multidiscipline platform providing preprint service that
is dedicated to making early versions of research outputs permanently
available and citable. Preprints posted at Preprints.org appear in Web of
Science, Crossref, Google Scholar, Scilit, Europe PMC.
Copyright: This is an open access article distributed under the Creative Commons
Attribution License which permits unrestricted use, distribution, and reproduction in any
medium, provided the original work is properly cited.
Article
Evaluation of Tomato Germplasm against Tomato
brown rugose fruit virus and Identification of
Resistance in Solanum pimpinellifolium
Namrata Jaiswal 1,†,‡, Bidisha Chanda 1,†,§, Andrea Gilliard 1, Ainong Shi 2 and Kai-Shu Ling 1,*
1 United States Department of Agriculture - Agricultural Research Service, U.S. Vegetable Laboratory,
Charleston, SC 29414, USA; Namrata.jaiswal@usda.gov (N.J.); bidisha.chanda@gmail.com (B.D.);
Andrea.gilliard@usda.gov (A.G.); kai.ling@usa.gov (K.S.L.)
2 Department of Horticulture, University of Arkansas, Fayetteville, AR 72701, USA; ashi@uark.edu (A.S.)
* Correspondence: kai.ling@usda.gov; Tel.: +1 843-402-5313
† These authors contributed equally to this work.
‡ Current Address: United States Department of Agriculture - Agricultural Research Service, Crop
Production and Pest Control Research Unit, West Lafayette, IN 47907, USA.
§ Current Address: Sakata Seed America, Salinas, CA 93906, USA.
Abstract: Tomato is one of the most important vegetable crops grown worldwide. Tomato brown
rugose fruit virus (ToBRFV), a seed-borne tobamovirus, poses a serious threat to tomato
productions due to its ability to break the resistant genes (Tm-1, Tm-2, Tm-22) in tomato. The
objective of this work was to identify new resistance source(s) of tomato germplasm against
ToBRFV. To achieve this aim, a total of 476 accessions from 12 Solanum species were tested with the
ToBRFV US isolate for their resistance and susceptibility. As a result, a total of 44 asymptomatic
accessions were identified as resistance/tolerance, including 31 accessions of S. pimpinellifolium, one
accession of S. corneliomulleri, four accessions of S. habrochaites, three accessions of S. peruvianum and
five accessions of S. subsection lycopersicon hybrid. Further analysis using serological tests identified
four highly resistant S. pimpinellifolium lines, PI 390713, PI 390714, PI 390716 and PI 390717. The
inheritance of resistance in the selected lines was verified in next generation and confirmed by RT-
qPCR. To our knowledge, this is a first report of high resistance to ToBRFV in S. pimpinellifolium.
These new genetic resources will expand the genetic pool available for breeders to develop new
resistant cultivars of tomato against ToBRFV.
Keywords: tobamoviruses; tomato brown rugose fruit virus (ToBRFV); genetic resistance; tomato;
Solanum lycopersicum
1. Introduction
Tomato (Solanum lycopersicum L.) is one of the most important vegetable crops worldwide. In
the last few years, the tomato industry has faced a serious threat by the emerging tomato brown
rugose fruit virus (ToBRFV), a seed-borne tobamovirus causing disease outbreaks to tomato
productions in many countries around the world [1,2]. This emerging tobamovirus was first
discovered to infect tomatoes in Jordan and Israel in 2014-2015 [3,4]. Since then, disease outbreaks
caused by ToBRFV have been reported in at least 25 countries in five continents, including Asia [5–
10], Africa [11], Europe [12–22], North America [23–27] and South America [28]. A handful of other
countries also reported outbreaks through the European and Mediterranean Plant Protection
Organization [29]. Thus, ToBRFV has been considered as a global pandemic on tomato and pepper
[1,2]. The rapid spread of ToBRFV outbreaks around the globe is likely caused by several factors:
including seed-borne, mechanical transmission and resistance breaking to the popular Tm-22 gene in
tomato, as well as increasing off-shore commercial seed production and global trade activities of seed
and produce. The potential dire consequences of ToBRFV on tomato and pepper has prompted many
Disclaimer/Publisher’s Note: The statements, opinions, and data contained in all publications are solely those of the individual author(s) and
contributor(s) and not of MDPI and/or the editor(s). MDPI and/or the editor(s) disclaim responsibility for any injury to people or property resulting
from any ideas, methods, instructions, or products referred to in the content.
Preprints (www.preprints.org) | NOT PEER-REVIEWED | Posted: 10 January 2024 doi:10.20944/preprints202401.0785.v1
© 2024 by the author(s). Distributed under a Creative Commons CC BY license.
2
countries to impose quarantine status to ToBRFV [2]. In the United States, the USDA-APHIS issued
a Federal Order in 2019 to inspect imported tomato and pepper seeds and produce from selected
countries with ToBRFV.
With few options available for viral disease management, planting a disease-resistance cultivar
would be the most economic and eco-friendly measure for ToBRFV disease management. Several
resistance genes (Tm-1, Tm-2 and Tm-22) have been used for tomato breeding to control
tobamoviruses on tomato [30–32]. The Tm-1 gene was derived from S. habrochaites PI 126445, the Tm-
2 from S. peruvianum PI 126926 and the Tm-22 gene from S. peruvianum PI 128650 [1]. Although Tm-2
and Tm-22 are allelic, the Tm-22 gene is the most effective and durable against many strains of tobacco
mosaic virus (TMV) and tomato mosaic virus (ToMV) [33,34]. However, the emerging ToBRFV breaks
the popular Tm-22 gene in tomato [4,35,36] that has been used in tomato breeding for tobamovirus
control in the past 60 years [37]. This resistance breaking ability renders all commercial tomato
cultivars vulnerable to ToBRFV infection, necessitating the urgent need to screen tomato germplasm
collections for new sources of ToBRFV resistance.
Genetic resistance is one of the most effective approaches to combat the emerging disease caused
by ToBRFV. The term ‘tolerance’ is defined as a plant showing no symptoms in spite of being infected
by the virus [38]. The term ‘resistance’ is an infected plant showing no symptoms and also in reduced
virus titer from a systemic infection in comparison to a closely related control plant [39,40]. The term
‘immunity’ is an inoculated plant showing no symptoms and tested negative for the virus [41].
Several efforts have been made in the search for new sources of genetic resistance to ToBRFV [42–45].
There is certainly remarkable complexity in the genetic resistance to ToBRFV, with more Solanum
species in tolerance and few in resistance. These tolerant lines include S. chilense [43,45], S.
lycopersicum [42], S. lycopersicum var. cerasiforme [43], S. ochranthum [43,44], S. penellii [45], S.
peruvianum [43,44], S. pimpinellifolium [42,43,45], and S. habrochaites [43,44]. There is little public
information available on genetic inheritance, quantitative trait loci (QTLs) analysis and molecular
marker development [42].
To characterize the genetic complexity of resistance to ToBRFV in tomato, the objective of the
present study was to conduct a large-scale screening of two core collections of tomato germplasm
maintained at the Tomato Genetics Resource Center (TGRC) in the University of California Davis and
the USDA Plant Genetic Resource Unit in Geneva, NY for their resistance against the ToBRFV US
isolate. The outcomes of the present study would supply novel genetic materials for genetic study
and genomic analysis for molecular markers development to accelerate tomato breeding for
resistance to ToBRFV.
2. Results
2.1. Primary screening of USDA and TGRC tomato core germplasm collections for resistance to ToBRFV
The present project was initiated in November 2019 to screen the tomato core collections from
USDA and TGRC for their resistance to ToBRFV. A total of 476 tomato accessions, including 86
accessions from TGRC and 390 accessions from USDA. The first experiment was conducted using
TGRC materials. Among 86 lines, three did not germinate, all others yielded 1-12 seedlings (average
7), which were used for virus inoculation. To minimize any potential escape, all test seedlings were
inoculated twice (one week apart). The first symptom reading was conducted at 5 weeks after the
first inoculation. The second symptom reading was conducted in 8 weeks after inoculation. In
addition to visual reading of symptom expression on the test plants, leaf tissues from systemic leaves
were collected for lab testing using a TMV enzyme-linked immunosorbent assay (ELISA) kit (Agdia,
USA) that is known to cross react serologically with other tobamoviruses infecting tomato, including
ToBRFV. A combination of the symptom expression (0: No symptoms; 1: Mild mosaic; 2: Mosaic; 3:
Mosaic, leaf deformed; 4: Severe mosaic, leaf deformed, mottling; and 5: Severe mosaic, leaf
deformed, mottling and string leaves) and the absorbance readings in ELISA was used to determine
resistance (asymptomatic with low to nondetectable ELISA absorbent readings), tolerance
(asymptomatic with high ELISA absorbance readings), and susceptibility (mild mosaic to severe
Preprints (www.preprints.org) | NOT PEER-REVIEWED | Posted: 10 January 2024 doi:10.20944/preprints202401.0785.v1
3
shoe-string leaves and higher ELISA readings) (Figure 1; Supplementary Table S1). Based on these
criteria, there was no resistant line identified in the TGRC materials, but one accession of S.
habrochaites (LA2107) was considered tolerance to ToBRFV (Table 1). The remaining 82 accessions
were susceptible to ToBRFV (Supplementary Table S1).
Figure 1. Symptom classes of ToBRFV infection on tomato germplasm used to calculate a disease
severity index (DSI). 0: No symptoms; 1: Mild mosaic; 2: Mosaic; 3: Mosaic, leaf deformed; 4: Severe
mosaic, leaf deformed, mottling; and 5: Severe mosaic, leaf deformed, mottling and string leaves.
The second experiment was conducted in March 2020 using 390 Plant Introductions (PIs)
collected from the USDA Plant Genetic Resources Unit in Geneva, NY. Among them, 14 lines did not
germinate. For the 376 germinated lines, 1-13 seedlings (average 8) per line were tested for their
resistance to ToBRFV. Due to the large number of plants tested in this experiment, we rated disease
symptoms carefully from 0 to 5 (Figure 1) to generate a disease severity index (DSI) for each line. The
DSI value that was lower than <20% was considered tolerance to ToBRFV (Table 2). Those lines with
DSI values that were >20% were considered susceptible to ToBRFV (Supplementary Table 2). Based
on these criteria, 43 PIs from five Solanum species, including one accession of S. corneliomulleri (PI
129144), three accessions of S. habrochaites (PI 126445, PI 126445, and PI 247087), three accessions of S.
peruvianum (PI 306811, PI 390667, PI 390671), and 31 accessions of S. pimpinellifolium (PI 127805, PI
143524, PI 143527, PI 211838, PI 230327, PI 344102, PI 344103, PI 346340, PI 390692, PI 390693, PI
390694, PI 390695, PI 390698, PI 390699, PI 390700, PI 390702, PI 390710, PI 390712, PI 390713, PI
390714, PI 390716, PI 390717, PI 390720, PI 390722, PI 390723, PI 390724, PI 390725, PI 390726, PI
390727, PI 390750 and PI 432362) and five accessions of S. subsection lycopersicon hybrid (PI 127799,
PI 129143, PI 143522, PI 233930 and PI 237640), were identified as tolerance to ToBRFV (Table 2,
Supplementary Table S2). Besides those 43 PIs with tolerance, 333 accessions tested were susceptible
to ToBRFV (Supplementary Table S2).
These two preliminary screenings resulted in the identification of 44 accessions with tolerance
to ToBRFV, with 43 USDA and one TGRC tomato accessions (Table 2, supplementary Tables S1 and
S2). From those resistant accessions, one S. habrochaites, three S. lycopersicon hybrid, one S. peruvianum,
and eight S. pimpinellifolium accessions were still segregating (those lines with 18%>DSI > 3.4% in
Table 2).
Preprints (www.preprints.org) | NOT PEER-REVIEWED | Posted: 10 January 2024 doi:10.20944/preprints202401.0785.v1
4
Table 1. Summary of tomato core germplasm in USDA and the Tomato Genetic Resource Center
screened for resistance to tomato brown rugose fruit virus.
Species
USDA
PIs
TGRC
Accession
s
Total
number of
accessions
screened
Number of
accessions in
resistance/tolerance
Solanum arcanum 1 9 10 0
Solanum chilense 0 17 17 0
Solanum corneliomulleri 6 11 17 1
Solanum habrochaites 17 33 50 4
S. huaylasense 0 3 3 0
Solanum lycopersicum 10 0 10 0
Solanum lycopersicum var.
cerasiforme 1 0 1 0
Solanum neorickii 1 0 1 0
Solanum pennellii 1 0 1 0
Solanum peruvianum 64 9 73 3
Solanum pimpinellifolium 136 4 140 31
Solanum subsect. lycopersicon hybrid 153 0 153 5
Total 390 86 476 44
Table 2. Tomato germplasm with resistance/tolerance to tomato brown rugose fruit virus.
Plant ID Taxonomy Disease severity index (%)
PI 129144 Solanum corneliomulleri 0
PI 126445 Solanum habrochaites 17.6
PI 209978 Solanum habrochaites 0
Preprints (www.preprints.org) | NOT PEER-REVIEWED | Posted: 10 January 2024 doi:10.20944/preprints202401.0785.v1
5
PI 247087 Solanum habrochaites 0
LA 2107 Solanum habrochaites 0
PI 306811 Solanum peruvianum 16
PI 390667 Solanum peruvianum 0
PI 390671 Solanum peruvianum 0
PI 127805 Solanum pimpinellifolium 14.2
PI 143524 Solanum pimpinellifolium 14.2
PI 143527 Solanum pimpinellifolium 0
PI 211838 Solanum pimpinellifolium 0
PI 230327 Solanum pimpinellifolium 0
PI 344102 Solanum pimpinellifolium 0
PI 344103 Solanum pimpinellifolium 0
PI 346340 Solanum pimpinellifolium 0
PI 390692 Solanum pimpinellifolium 0
PI 390693 Solanum pimpinellifolium 0
PI 390694 Solanum pimpinellifolium 0
PI 390695 Solanum pimpinellifolium 0
PI 390698 Solanum pimpinellifolium 0
PI 390699 Solanum pimpinellifolium 18
PI 390700 Solanum pimpinellifolium 0
PI 390702 Solanum pimpinellifolium 11.4
PI 390710 Solanum pimpinellifolium 0
Preprints (www.preprints.org) | NOT PEER-REVIEWED | Posted: 10 January 2024 doi:10.20944/preprints202401.0785.v1
6
PI 390712 Solanum pimpinellifolium 0
PI 390713 Solanum pimpinellifolium 0
PI 390714 Solanum pimpinellifolium 0
PI 390716 Solanum pimpinellifolium 0
PI 390717 Solanum pimpinellifolium 0
PI 390720 Solanum pimpinellifolium 8.4
PI 390722 Solanum pimpinellifolium 0
PI 390723 Solanum pimpinellifolium 16
PI 390724 Solanum pimpinellifolium 0
PI 390725 Solanum pimpinellifolium 3.4
PI 390726 Solanum pimpinellifolium 0
PI 390727 Solanum pimpinellifolium 0
PI 390750 Solanum pimpinellifolium 0
PI 432362 Solanum pimpinellifolium 8.8
PI 127799 Solanum subsect. lycopersicon hybrid 17.8
PI 129143 Solanum subsect. lycopersicon hybrid 0
PI 143522 Solanum subsect. lycopersicon hybrid 18
PI 233930 Solanum subsect. lycopersicon hybrid 0
PI 237640 Solanum subsect. lycopersicon hybrid 3.4
2.2. Rescreening of selected lines to verify their resistant properties to ToBRFV
Through self-pollination, single plants from those lines with resistance/tolerance to ToBRFV
were advanced to a new generation (S1). To assess whether the identified resistance was inheritable
to a new generation, we germinated S1 seeds from five selected S. pimpinellifolium lines with putative
resistance/tolerance (line 327: PI 390712; line 328: PI 390713; line 329: PI 390714; line 331: PI 390716;
line 332: PI 390717) or one susceptible (line 333: PI 390718) (Supplementary Table S2) and tested S1
Preprints (www.preprints.org) | NOT PEER-REVIEWED | Posted: 10 January 2024 doi:10.20944/preprints202401.0785.v1
7
seedlings for their responses to ToBRFV infection. Interestingly, our re-test results verified the
resistance properties for line 328: PI 390713, with three other lines (line 329: PI 390714, line 331: PI
390716 and line 332: PI 390717) still segregating for their resistance to ToBRFV as assessed by the
ELISA absorbance values with a threshold absorbance level (OD405nm = 0.31) for resistance on
individual plants (Figure 2). When ELISA readings from individual plants were combined, the mean
absorbance readings for four resistant lines (line 328: PI 390713, line 329: PI 390714, line 331: PI 390716,
and line 332: PI 390717) remained below the threshold (0.31), whereas the two susceptible lines had
higher absorbance readings, 0.956 for the line 327: PI 390712 and 1.36 for the line 333: PI 390718,
relative to 1.239 for the positive control (ToBRFV+) on tomato ‘Moneymaker’ (Figure 3). As expected,
there were genetic impurities observed among individual plants in some germplasm materials. For
example, the line 326: PI 390711 which showed a segregation for resistance to ToBRFV with a disease
severity index at 48% in the preliminary screening (Supplementary Table S2) was still segregating
among individual S1 plants (Figure 2A). This would need additional self-pollination and further
selection of resistant individuals in advance generations to stabilize the genetic property of resistance.
In addition to using the serological test by ELISA to assess the relative virus titers on tested
tomato plants, we also employed a ToBRFV-specific RT-qPCR technology [36] to evaluate the virus
titers on ToBRFV-inoculated tomato plants. While a Ct value for the ToBRFV-inoculated tomato
‘Moneymaker’ plant was as low as 10.86 (in high virus titer), a similar low Ct value (12.16) was also
observed on a ToBRFV-susceptible S. pimpinellifolium Line 333 (PI 390718). As expected, a tomato
plant containing Tm-1 and Tm-22 (LA2830) showed the same low Ct value (12.68) due to the resistance
breaking by ToBRFV. In contrary, three ToBRFV-inoculated plants from the ToBRFV-resistant S.
pimpinellifolium Line 332 (PI 390717) had high Ct values (26.19, 23.31 and 24.65), which were in the
same level of a high Ct value (low virus titer) as to the background reading (25.93) generated from a
mock-inoculated tomato plant (Figure 4).
Figure 2. Re-evaluating of individual plants from selected Solanum pimpinellifolium lines confirmed
their resistance to ToBRFV but also revealed a segregating population. Absorbance values at OD405nm
for two individual plants derived from seeds generated from self-pollination (S1) of four putative
ToBRFV-resistant lines, A. PI 390711 (line 326); B. PI 390714 (line 329); C. PI 390716 (line 331); and PI
390717 (line 332) along with buffer, healthy tomato and ToBRFV-infected tomato as controls for
comparisons. The dashed line (OD405nm = 0.31) is the threshold level of resistance.
Preprints (www.preprints.org) | NOT PEER-REVIEWED | Posted: 10 January 2024 doi:10.20944/preprints202401.0785.v1
8
Figure 3. Re-evaluation of selected Solanum pimpinellifolium lines for their resistance to ToBRFV using
an ELISA test to assess virus titers on leaf tissue samples collected from systemic leaves. Based on the
threshold level (OD405nm as 0.31), plants from four lines were considered resistance (PI 390713, PI
390714, PI 390716 and PI 390717), whereas plants from two other lines (PI 390712 and PI 390718) were
susceptible to ToBRFV. Buffer, healthy tomato and ToBRFV-infected tomato were included in the
same ELISA test as controls for comparison.
Figure 4. Comparative evaluation on selected tomato lines for their resistance to ToBRFV using RT-
qPCR to assess the virus titers on leaf tissue samples collected from systemic leaves. The Ct values
from 3 plants of the ToBRFV-resistant line (332-1, 332-2, and 332-3: PI 390717), in comparison to that
of the ToBRFV-susceptible line (333: PI 390718), LA2830 (Tm-1 and Tm-22), as well as ToBRFV-infected
‘Moneymaker’ and its mock inoculation control.
Preprints (www.preprints.org) | NOT PEER-REVIEWED | Posted: 10 January 2024 doi:10.20944/preprints202401.0785.v1
9
2.3. Testing F1 progenies for their resistance to ToBRFV
In addition to evaluating of the self-pollinated seedlings in the S1 generation for their resistance
to ToBRFV, we also evaluated four F1 hybrids generated from crosses between 327-1: PI 390712 (S) x
326: PI 390711 (R), 328-1: PI 390713 (R) x 326-1: PI 390711 (R), 333-1: PI 390718 (S) x 329-1: PI 390714
(R), and 333-1: PI 390718 (S) x 332-1: PI 390717 (R). F1 plants derived from all these four crosses were
susceptible to ToBRFV, suggesting that the resistance to ToBRFV in S. pimpinellifolium is recessive.
Although all four F1 plants had lower absorbance values (Figure 5) than that of the positive control
(1.00), the ELISA readings were still higher than those readings obtained from the S1 lines (Figure 3).
Since these F1 plants were tested in the same ELISA plate as those from the S1 plants, their absorbance
values were relatively comparable.
Figure 5. Assessing F1 plants derived from selected Solanum pimpinellifolium lines for their resistance
to ToBRFV as evaluated for their absorbance values (virus titers) using an ELISA test. F1 plants from
four crosses: 1-F1: 327-1 [PI 390712 (S)] x 326 [PI 390711 (R)]; 2-F1: 328-1 [PI 390713 (R)] x 326-1
[PI390711 (R)]; 3-F1: 333-1 [PI 390718 (S)] x 329-1 [PI 390714 (R)]; and 4-F1: 333-1 [PI 390718 (S)] x 332-
1 [PI 390717 (R)] were evaluated for their resistance to ToBRFV, in comparison to the mock inoculation
control and a positive control (ToBRFV+).
3. Discussion
In the present study, by screening a total of 476 tomato core accessions from the USDA and
TGRC tomato germplasm collections (Table 1, Supplementary Tables S1 and S2), we identified 44
accessions with tolerance to ToBRFV US isolate (Table 2). A large proportion (31 of 44 or 70%) of these
tolerant lines belong to S. pimpinellifolium. In addition, a number of tolerant lines were also identified
from four other species, including S. corneliomulleri (1), S. habrochaites (4), S. peruvianum (3), and S.
subsection lycopersicon hybrid (5) (Table 2). The high genetic diversity of tolerance/resistance to
ToBRFV is in general agreement with the results obtained from earlier studies by other groups using
different isolates of ToBRFV [42–45]. Due to various sources of tomato germplasm collections used
for evaluation in the present study, these 44 accessions of tomato germplasm with
resistance/tolerance properties to ToBRFV are not overlapping with previous studies (Tables 2 and
3). The reason for these new additions is likely that we focused our efforts mainly on the USDA
tomato germplasm collections not previously extensively examined. The smaller number of 86
tomato germplasm accessions from TGRC also had little overlapping to those used in previous
studies [42–45].
Preprints (www.preprints.org) | NOT PEER-REVIEWED | Posted: 10 January 2024 doi:10.20944/preprints202401.0785.v1
10
There is a diversity of genetic sources of tolerance to ToBRFV in tomato germplasm. Several
tobamoviruses are known as harmful pathogens of tomato crop. Among them, the emerging resistant
breaking ToBRFV has posed a serious threat to the profitable tomato productions around the world
[1]. However, ToBRFV, a recently emerged plant virus [3], has been shown to infect all known
genotypes of tomato, including those carrying Tm-1, Tm-2, and Tm-22 resistance genes [4,36,46]. With
no available commercial tomato cultivars with ToBRFV resistance at the moment, growers adopt
preventative measures to protect their tomato crops from virus spread in the production greenhouse
facilities. Several effective disinfectants have been selected and recommended to growers for virus
control [47–54]. However, breeding for disease resistance is still the most powerful and economic
way to control viral diseases [55]. Thus, genetic resistance would be the most effective strategy to
combat the emerging ToBRFV. Several authors recently reported tolerance/resistance to ToBRFV in
genotypes of S. lycopersicum, S. pimpinellifolium, S. habrochaites and S. ochrantum [42–45]. Although a
high number of genetic resources identified with resistance/tolerance to ToBRFV, the majority of
them are considered as tolerance (asymptomatic) with some levels of virus infection (Table 3).
Table 3. Diversity of germplasm resources with resistance/tolerance to ToBRFV.
Zinger et al.,
2021 [38]
Kabas et a.,
2022 [41]
Jewehan et al.,
2022a [39]
Jewehan et
al., 2022b
[40]
This study
Total
lines
160 44 636 173 476
Tolerant
lines
S.
pimpinellifolium
(9); S.
Lycopersicum
(8)
S.
pimpinnelifolium
(1); S. penellii
(1); and S.
chilense (2)
S.
pimpinelifolium
(26); S. chilense
(1); S.
lycopersicum
var.
cerasiforme (4)
S.
corneliomulleri
(1); S.
habrochaites (8);
S. peruvianum
(3); S.
pimpinellifolium
(27); and S.
subsect.
lycopersicon
hybrid (5)
Resistant
lines
S. lycopersicum
(1)
S. ochrantum
(5)
S.
habrochaites
(9);
S.
peruvianum
(1)
S.
pimpinellifolium
(4)
Because we directly used the seeds that were provided by the germplasm repository for our
primary screening, individual plants in certain accessions from the germplasm materials might
develop various levels of symptom expression. We considered plants with a disease severity class in
less than 1 (or a disease severity index < 20%) as tolerance. In this case at least one of their plants in
an accession should be asymptomatic. For those accessions to be considered as resistance to ToBRFV,
in addition to their low disease severity index, some of their plants should also contain a reduced
level of virus titer as assessed by ELISA absorbance values, lower than 0.31 (a threshold for resistance)
or by RT-qPCR. Through single plant selection in advance generations, it is very possible to generate
a resistant plant with stable inheritance of genetic resistance to ToBRFV. For the S1 generation, two
plants per line were tested individually with an ELISA test using leaf tissue samples collected from
Preprints (www.preprints.org) | NOT PEER-REVIEWED | Posted: 10 January 2024 doi:10.20944/preprints202401.0785.v1
11
upper and lower portions of the plant. As shown in Figure 2, the two S1 plants tested demonstrated
some levels of genetic segregation for resistance in several resistance lines. Therefore, advance
generation through single plant self-pollination is underway to obtain lines with stable inheritance
of resistance to ToBRFV.
Based on disease severity class, if every plant (average 7-8 plants per accession) developed a
mild mosaic symptom in class 1 (DSI 20%), they are considered as susceptible to ToBRFV. If one or
more plants in an accession was rated as asymptomatic in class 0, then the DSI will be less than 20%,
such as those accessions in DSI numbers (3.4% and 18.0%, Supplementary Table S2). Those accessions
would be considered as tolerance to ToBRFV because at least one plant was asymptomatic which
could be advanced by single plant selection through self-pollination. For those accessions to be
considered as resistance, some or all of the test plants in an accession would need to produce a
significant low level of the virus titer as defined with a threshold at 0.31 in the absorbance value from
an ELISA test. Through advance generation, those resistant plants with a low absorbance value will
be selected for developing a resistant line through self-pollination. Although the line 327 (PI 390712)
was rated as asymptomatic in the primary screening (Table 2, Supplementary Table S2), in the S1
generation, two plants had higher ELISA readings, which can only be considered as tolerance, but
not resistance to ToBRFV (Figure 3). On the other hand, the line 326 (PI 390711) which was rated as
susceptible in the preliminary screening (Supplementary Table S2) was segregating for resistance to
ToBRFV in S1 plants (Tables 2) where some resistant individuals could be identified through single
plant selection in advance generations.
ELISA has been extensively used for virus detection and its OD405nm absorbance values are used
for relative quantification of virus titers in infected plant tissue samples when evaluated along with
proper controls (i.e., blank, health tissue and positive virus-infected controls). In fact, the same ELISA
method is used by Zinger et al. 2021 [42] for their evaluation of tomato germplasm materials with
resistance to ToBRFV. In the present study, we conducted an extensive screening with a large number
of tomato germplasm (476 accessions), each with average 7-8 plants, phenotyping with careful
symptom observation with disease severity classes were used to make initial assessment to identify
resistance/tolerance plants. ELISA test with absorbance values was used only as a secondary to assess
relative virus titers. We did not claim immunity for our selected materials, only disease resistance
with reduced virus titers in comparison to those readings from the susceptible controls. The low virus
titers observed from those resistant lines were confirmed through the use of RT-qPCR (Figure 4).
Nevertheless, several lines of Solanum species have been considered as resistance (with no
detectable level of the virus based on appropriate laboratory tests, by either ELISA [42] or PCR [43,44].
To decide a true resistance, it is necessary to conduct lab tests, using either serological tests [42]) or
molecular tests/bioassay [43,44] to determine the presence and concentration of the virus in the
systemic tissues. These rigorous tests identified only one source of S. lycopersicum [42] and several
accessions of S. habrochaites and S. peruvianum as ToBRFV resistance [44]. The sexual incompatibility
between S. ochranthum and S. lycopersicum limits its utility for tomato breeding [43]. Therefore, the
resistant S. pimpinellifolium lines identified in this study would offer additional choices of genetic
resources likely to be useful for tomato breeding against ToBRFV. Even for an experienced breeder,
it is still a challenge to use S. habrochaites or S. peruvianum to cross with tomato (S. lycopersicum). S.
pimpinellifolium is a close relative to S. lycopersicum, and the intercross between them is readily
compatible in tomato breeding. Therefore, those S. pimpinellifolium lines with ToBRFV resistance
identified in the present study would offer better genetic materials for breeders to choose in making
crosses with their elite tomato lines. Although the resistance to ToBRFV in selected S. pimpinellifolium
is not an immunity, to our knowledge, this is the first report in finding a true resistance with
significant lower virus titers in several S. pimpinelllifolium accessions (PI 390713, PI 390714, PI 390716
and PI 390717). This resistance is verified in a separate study (Ling’s lab, unpublished data), where
we had used one of the identified S. pimpinellifolium lines (PI 390717) to generate F2 populations and
applied genome re-sequencing technology and quantitative trait locus (QTL) analysis to identify
single nucleotide polymorphisms (SNPs) that are associated with the ToBRFV resistance in S.
Preprints (www.preprints.org) | NOT PEER-REVIEWED | Posted: 10 January 2024 doi:10.20944/preprints202401.0785.v1
12
pimpinellifolium. Molecular marker technology (i.e., Kompetitive Amplified Specific PCR) will be
developed to easing tomato breeding process using marker-assisted selection for ToBRFV resistance.
4. Materials and Methods
4.1. Plant germplasm materials
A total of 476 plant germplasm accessions representing the core collections of Solanum species,
including 390 Plant Introductions (PIs) from the United States Department of Agriculture (USDA)
National Plant Germplasm System (NGPS) and 86 accessions from the Tomato Genetic Resources
Center (TGRC) at University of California, Davis was evaluated for their resistance/tolerance to
ToBRFV through mechanical inoculation and symptom expression on tomato seedlings. The 11
Solanum species and number of accessions used in this study were S. arcanum (10); S. chilense (17); S.
corneliomulleri (17); S. habrochaites (50); S. huaylasense (3); S. lycopersicum (11); S. neorickii (1); S. pennellii
(1); S. peruvianum (73); S. pimpinellifolium (140); and S. subset. lycopersicon hybrid (153) (Table 1). Two
experiments were conducted, the first was with 86 accessions of tomato materials from TGRC and
the second was with 390 PIs supplied by USDA NPGS. For each accession, 12 seeds were planted
individually in a 36 seed-starter tray that was filled with soilless growth medium, Metro-Mix 360
(Sun Gro Horticulture, Agawam, MA, USA) for germination in a greenhouse. Most of seeds from the
germplasm collections were able to germinate, with an average of 7-8 seedings per accession
germinated and used for resistance screening.
4.2. Virus culture and mechanical inoculation
The ToBRFV US isolate CA18-01 (GenBank Accession No. MT002973; [56]) was collected on a
tomato plant from a greenhouse in California [25] and isolated through serial passages on a local
lesion host of Nicotiana tabacum var. Samsun to obtain a pure culture [36], which was used for this
evaluation. We maintained the pure virus culture of ToBRFV on ‘Moneymaker’ tomato plant in an
insect proof BugDorm (BioQuip Products, Compton, CA) in a containment greenhouse with
temperature at 25oC with 12-14 hours natural sunlight. The virus inoculum was prepared by grinding
the symptomatic leaves (1:5 w/v) in a plastic tissue extraction bag containing 1× phosphate-buffered
saline solution, pH 7.0 (140 mM NaCl, 8 mM Na2HPO4, 1.5 mM KH2PO4, 2.7 mM KCl, and 0.8 mM
Na2SO3) using a Homex-6 tissue homogenizer (Bioreba AG, Switzerland). The freshly prepared virus
inoculum was kept on ice until used. Ten days old seedlings (in 2-3 leaf stage) were used for
mechanical inoculation. Seedlings were lightly dusted with carborundum (320 grit, ThermoFisher
Scientific, USA) followed by rub-inoculation as determined in our previous study [36]. The inoculated
seedlings were placed under shade for several hours to minimize potential injury from direct
sunlight, then moved and maintained in a containment greenhouse for 4-8 weeks. Symptom
expression on the inoculated plants was observed weekly. Both positive and negative controls were
included in the screening experiments. The buffer-treated healthy plants were used as a negative
control (mock inoculation). Tomato ‘Moneymaker’ plants inoculated with the same ToBRFV culture
were used as a positive control. Test plants were visually scored for the presence of symptoms,
including mosaic, mottling, necrotic spots, leaf deformation, shoestring leaves, and plant stunting
(Figure 1). To confirm the presence or absence of ToBRFV on the test plants, after the final reading
on symptoms, a young systemic leaf was collected in a plastic bag and processed for serological test
(enzyme-linked immunosorbent assay, ELISA). To confirm virus infection, a bulk sample consisting
of one small leaf from each plant in one line was collected and tested to assess the virus titer
(Supplementary Table S1). For those PIs from USDA, only those lines with asymptomatic plants were
collected in a bulk per accession and used for an ELISA test. Those test plants from other lines that
expressed typical disease symptoms were infected by ToBRFV and therefore not tested.
Preprints (www.preprints.org) | NOT PEER-REVIEWED | Posted: 10 January 2024 doi:10.20944/preprints202401.0785.v1
13
4.3. Virus detection through a serological test using Enzyme-linked immunosorbent assay
Although a number of tomato germplasm tested were asymptomatic based on visual
observation, we were unsure which ones had true resistance to ToBRFV (low or no detectable
absorbance readings in ELISA) or just tolerance (high absorbance readings that were similar to those
of symptomatic plants). ELISA was conducted to quantify the virus titer on the inoculated plants in
each genotype. We used a commercial ELISA kit for tobacco mosaic virus (TMV, a tobamovirus with
serological cross reaction to ToBRFV) to detect ToBRFV following the manufacturer’s instructions
(Agdia, Elkhart, USA). Approximately 200 mg leaf tissue from each sample was collected in an
individual plastic bag and homogenized with a Homex6 tissue homogenizer (Bioreba AG,
Switzerland) in 4.0 ml of 1x ELISA general extraction buffer (GEB) (Bioreba AG, Switzerland).
Absorbance readings at OD405nm were quantified using a SpectraMax ELISA microplate reader
(Molecular Devices, San Jose, USA). An absorbance value that showed at least twice that of the
healthy negative control (buffer inoculated) was considered positive for ToBRFV infection. To
determine whether a line is resistant or susceptible to ToBRFV, we evaluated a series of readings to
identify an absorbance reading as the threshold level for resistance or susceptibility. Although no
detectable readings were seen in systemic leaves from several selected lines, most of the test plants
had only some low levels of virus infection in selected resistant lines. An absorbance reading at
OD405nm in less than 0.31 was selected as the threshold for resistance.
4.4. Virus detection using reverse transcription quantitative polymerase chain reaction (RT-qPCR)
In addition to using the ELISA method to assess the relative virus titers, we also conducted
reverse transcription quantitative polymerase chain reaction (RT-qPCR) as described in detail [36].
By using the Ct values, we could achieve better understanding on the virus titers on each of the test
plants to assess their resistance or susceptibility to ToBRFV. Briefly, total plant RNA was extracted
from a systemic leaf tissue collected from tomato plants at four weeks post ToBRFV inoculation using
a TRIzol reagent following the manufacturer’s instructions (Thermo Fisher Scientific, Gaithersburg,
MD, USA). The RT-qPCR was conducted with the following primers and TaqMan probe (ToBRFV-
F1, 5’ GCCCATGGAACTATCAGAAGAA-3’; ToBRFV-R1, 5’ TTCCGGTCTTCGAACGAAAT-3’;
ToBRFV-P1, FAM-AGTCCCGATGTCTGTAAGGCTTGC-TAMRA) [36] using a One Step
PrimerScript RT-PCR kit following the manufacturer’s instructions (Takara Bio USA, Mountain
View, CA, USA). RT-qPCR was carried out on a AriaMX real-time PCR system (Agilent, Santa Clara,
CA, USA) using the following thermocycling program: reverse transcription at 50oC for 30 min,
followed by 1 cycle of denaturation at 95oC for 2 min, and 40 cycles of 95oC for 10 sec and 55oC for
30 sec.
4.5. Disease scoring and data analysis
To evaluate tomato plants with resistance/tolerance against ToBRFV, we conducted replicate
experiments through visual observation of symptom expression on each test plant weekly post
inoculation for 4-8 weeks. Symptom severities were scored in 1 to 5 scales, where (0): no visible
symptoms; (1): mild mosaic; (2): mosaic; (3): mosaic with leaf deformed; (4): severe mosaic with leaf
deformed and mottling, and (5): severe mosaic with deformed leaf, mottling, and shoestring-like
leaves (Figure 1).
Disease severity index was calculated by the formula:
DSI (%) = ∑
DSI (%) = [sum (class frequency × score of rating class)] / [(total number of plants) × (maximal
disease index)] × 100
Where i= class, Yi= number of plants in the class. A disease severity index (DSI) less than 20%
was considered tolerance and those with higher DSI in 20% to 100% were considered susceptible to
ToBRFV. To be considered as resistance, test plants in a germplasm would need to be asymptomatic
Preprints (www.preprints.org) | NOT PEER-REVIEWED | Posted: 10 January 2024 doi:10.20944/preprints202401.0785.v1
14
as well as in lower absorbence reading (OD405nm at 0.31) to undetectable absorbance readings as tested
by ELISA.
4.6. Advancing selected resistant lines through self-pollination or cross-pollination to generate F1 plants for
evaluation of their inheritability of resistance to ToBRFV
To verify the ToBRFV resistance or tolerance from those accessions identified in the preliminary
screening of the core tomato germplasms, selected lines were self-pollinated to generate seeds (S1).
The S1 plants from six S. pimpinellifolium lines were tested to confirm their resistant properties to
ToBRFV. These S. pimpinellifolium lines included four high resistant lines, line 328: PI 390713, line 329:
PI 390714, line 331: PI 390716 and line 332: PI390717 and one susceptible line, line 333: PI 390718 from
S. pimpinellifolium. The resistance properties in two other lines, 326 and 327, between the preliminary
screening and the retest using S1 plants were not consistent, due to genetic segregation in the
germplasm materials. In addition, cross-pollination was conducted to generate F1 seeds from selected
lines. The F1 seedlings generated from selected crosses were evaluated for their resistance to ToBRFV
through symptom observation followed by an ELISA test. Some of these crosses will be advanced for
more detailed genetic study to characterize the inheritance of resistance and molecular marker
development.
5. Conclusions
In the present study, we evaluated a total of 476 accessions from 12 Solanum species and
identified 44 accessions with resistance/tolerance to ToBRFV in five species, including S.
corneliomulleri, S. habrochaites, S. peruvianum, S. subsec. lycopersicon hybrid, and S. pimpinellifolium.
Upon closer examination and comparison with earlier reported studies, the 44 accessions identified
in the present study appeared to be new additions, which enrich the genetic pool for selection in
tomato breeding. To our knowledge, this is the first report to identify S. pimpinellifolium with true
resistance to ToBRFV. The resistant property was verified from at least four accessions of S.
pimpinellifolium that were originally collected from Peru (USDA GRIN
https://data.nal.usda.gov/dataset/germplasm-resources-information-network-grin). Although it is
necessary to follow up on genetic characterization on the inheritance of resistance, preliminary
analysis on the resistance in the F1 progenies derived from several S. pimpinellifolium crosses showed
to be controlled by a recessive gene(s), which appeared to be in a general agreement with the result
from Zinger et al. 2021 [42]. These ToBRFV-resistant S. pimpinellifolium could serve as foundation
materials for parents in tomato breeding programs to develop cultivars with ToBRFV resistance, to
study the genetic inheritance and for genomic analysis to develop molecular markers that could be
useful for marker-assisted selection.
Supplementary Materials: Supplementary Table S1. Screening the tomato core germplasm collections at TGRC
for their resistance to ToBRFV. Supplementary Table S2. Screening the USDA tomato core germplasm collections
for resistance to ToBRFV.
Author Contributions: Conceptualization, K.S.L.; methodology, B.C., A.G., N.J., and K.S.L.; validation, N.J. and
K.S.L.; formal analysis, K.S.L., B.C., N.J., A.S.; investigation, B.C., A.G., N.J., K.S.L., and A.S.; resources, K.S.L.;
A.S. data curation, B.C., N.J., A,G, and K.S.L; writing—original draft preparation, N.J., B.C. and K.S.L.; writing—
review and editing, B.C., A.G., N.J., A.S. and K.S.L.; visualization, N.J. and K.S.L.; supervision, K.S.L.; project
administration, K.S.L.; funding acquisition, K.S.L. All authors have read and agreed to the published version of
the manuscript.
Funding: This research was funded in part by USDA-ARS National Plant Disease Recovery System (NPDRS)
and Crop Germplasm Committee of tomato, grant number 6080-22000-032-000D.
Data Availability Statement: The datasets presented in this study are available in Tables, Figures,
Supplementary Tables, and Supplementary Figures.
Acknowledgments: Tomato germplasm materials were kindly supplied by the Tomato Genetics Resource
Center at the University of California, Davis and the USDA-ARS Plant Genetic Resources Unit in Geneva, NY.
We thank Bazgha Zia and Jing Zhou for their review of the manuscript. Mention of trade names or commercial
Preprints (www.preprints.org) | NOT PEER-REVIEWED | Posted: 10 January 2024 doi:10.20944/preprints202401.0785.v1
15
products in this article is solely for providing specific information and does not imply recommendation or
endorsement by the USDA. USDA is an equal opportunity provider and employer.
Conflicts of Interest: The authors declare no conflict of interest. The funders had no role in the design of the
study; in the collection, analyses, or interpretation of data; in the writing of the manuscript; or in the decision to
publish the results.
References
1. Salem, N.M.; Jewehan, A.; Aranda, M.A.; Fox, A. Tomato brown rugose fruit virus pandemic. Annual
Review of Phytopathology 2023, 61, 1 https://doi.org/10.1146/annurev-phyto-021622-120703 (last accessed
8/13/2023)
2. Zhang, S.; Griffiths, J.S.; Marchand, G.; Bernards, M.A.; Wang, A. Tomato brown rugose fruit virus: an
emerging and rapidly spreading plant RNA virus that threatens tomato production worldwide. Mol. Plant
Pathol. 2022, 23, 1262–1277.
3. Salem, N. M.; Mansour, A. N.; Abdeen, A. O.; Araj, S.; Khrfan, W. I. First report of tomato chlorosis virus
infecting tomato crops in Jordan. Plant Disease, 2015, 99, 1286. https://doi.org/10.1094/PDIS-01-15-0130-PDN
4. Luria, N.; Smith, E.; Reingold, V.; Bekelman, I.; Lapidot, M.; Levin, I.; Elad, N.; Tam, Y.; Sela, N.; Abu-Ras,
A.; Ezra, N.; Haberman, A.; Yitzhak, L.; Lachman, O.; Dombrovsky, A. A new israeli tobamovirus isolate
infects tomato plants harboring Tm-22 resistance genes. PLOS ONE 2017, 12, e0170429.
https://doi.org/10.1371/journal.pone.0170429
5. Abou Kubaa, R.; Choueiri, E.; Heinoun, K.; Cillo, F.; Saponari, M. First report of tomato brown rugose fruit
virus infecting sweet pepper in Syria and Lebanon. J. Plant Path. 2022, 104, 425.
https://link.springer.com/article/10.1007/s42161-021-00987-y
6. Alkowni, R.; Alabdallah, O.; Fadda, Z. Molecular identification of tomato brown rugose fruit virus in
tomato in Palestine. J. Plant Pathol., 2019, 101, 719–723. https://doi.org/10.1007/s42161-019-00240-7
7. Ghorbani, A.; Rostami, M.; Seifi, S.; Izadpanah, K. First report of tomato brown rugose fruit virus in
greenhouse tomato in Iran. New Disease Reports, 2021, 44, e12040. https://doi.org/10.1002/ndr2.12040
8. Hasan, Z.M.; Salem, N.M.; Ismail, I.D.; Akel, E.H.; Ahmad, A.Y. First report of Tomato brown rugose fruit
virus on greenhouse tomato in Syria. Plant Disease, 2022, 106, 772. https://doi.org/10.1094/PDIS-07-21-1356-
PDN
9. Sabra, A.; Saleh, M.A.A.; Alshahwan, I.M.; Amer, M.A. First report of Tomato brown rugose fruit virus
infecting tomato crop in Saudi Arabia. Plant Disease, 2021, 106, 1310. https://doi.org/10.1094/PDIS-05-21-
1065-PDN
10. Yan, Z.-Y.; Ma, H.-Y.; Han, S.-L.; Geng, C.; Tian, Y.-P.; Li, X.-D. First report of tomato brown rugose fruit
virus infecting tomato in China. Plant Disease, 2019, 103, 2973. https://doi.org/10.1094/PDIS-05-19-1045-PDN
11. Amer, M.A.; Mahmoud, S.Y. First report of tomato brown rugose fruit virus on tomato in Egypt. New
Disease Reports, 2020, 41, 24. https://doi.org/10.5197/j.2044-0588.2020.041.024
12. Alfaro-Fernández, A.; Castillo, P.; Sanahuja, E.; Rodríguez-Salido, M.C.; Font, M.I. First report of tomato
brown rugose fruit virus in tomato in Spain. Plant Disease, 2021, 105, 515. https://doi.org/10.1094/PDIS-06-
20-1251-PDN
13. Beris, D.; Malandraki, I.; Kektsidou, O.; Theologidis, I.; Vassilakos, N.; Varveri, C. First report of tomato
brown rugose fruit virus infecting tomato in Greece. Plant Disease, 2020. 104, 2035.
https://doi.org/10.1094/PDIS-01-20-0212-PDN
14. Fidan, H.; Sarikaya, P.; Calis, O. First report of tomato brown rugose fruit virus on tomato in Turkey. New
Disease Reports, 2019, 39, 18. https://doi.org/10.5197/j.2044-0588.2019.039.018
15. Hamborg, Z.; Blystad, D.-R. The first report of Tomato brown rugose fruit virus in tomato in Norway. Plant
Disease, 2022, 106, 2004 doi: 10.1094/PDIS-10-21-2208-PDN
16. Mahillon, M.; Kellenberger, I.; Dubuis, N.; Brodard, J.; Bunter, M.; Weibel, J.; Sandrini, F.; Schumpp, O.
First report of Tomato brown rugose fruit virus in tomato in Switzerland. New Disease Reports, 2022, 45,
e12065. https://bsppjournals.onlinelibrary.wiley.com/doi/full/10.1002/ndr2.12065
17. Menzel, W.; Knierim, D.; Winter, S.; Hamacher, J.; Heupel, M. First report of tomato brown rugose fruit
virus infecting tomato in Germany. New Disease Reports, 2019, 39, 1. https://doi.org/10.5197/j.2044-
0588.2019.039.001
18. Orfanidou, C.G.; Cara, M.; Merkuri, J.; Papadimitriou, K.; Katis, N.I.; Maliogka, V.I. First report of tomato
brown rugose fruit virus in tomato in Albania. J Plant Path., 2022, 104, 855. doi: 10.1007/s42161-022-01060-y
Preprints (www.preprints.org) | NOT PEER-REVIEWED | Posted: 10 January 2024 doi:10.20944/preprints202401.0785.v1
16
19. Panno, S.; Caruso, A. G.; Davino, S. First report of tomato brown rugose fruit virus on tomato crops in Italy.
Plant Disease, 2019, 103, 1443. https://doi.org/10.1094/PDIS-12-18-2254-PDN
20. Skelton, A.; Buxton-Kirk, A.; Ward, R.; Harju, V.; Frew, L.; Fowkes, A.; Long, M.; Negus, A.; Forde, S.;
Adams, I. P.; Pufal, H.; McGreig, S.; Weekes, R.; Fox, A. First report of Tomato brown rugose fruit virus in
tomato in the United Kingdom. New Disease Reports, 2019, 40, 12.
https://doi.org/https://doi.org/10.5197/j.2044-0588.2019.040.012
21. Skelton, A.; Gentit, P.; Porcher, L.; Visage, M.; Fowkes, A.; Adams, I.P.; Harju, V.; Webster, G.; Pufal, H.,
McGreig, S.; Ward, R.; Fox, A. First report of Tomato brown rugose fruit virus in tomato in France. New
Disease Reports, 2022, 45, e12061. https://bsppjournals.onlinelibrary.wiley.com/doi/10.1002/ndr2.12061
22. van de Vossenberg, B. T. L. H.; Visser, M.; Bruinsma, M.; Koenraadt, H.M.S., Westenberg, M.; Botermans,
M. Real-time tracking of Tomato brown rugose fruit virus (ToBRFV) outbreaks in the Netherlands using
Nextstrain. PLoS ONE, 2020, 15, e0234671. https://doi.org/10.1371/journal.pone.0234671
23. Cambrón-Crisantos, J.M.; Rodríguez-Mendoza, J.; Valencia-Luna, J.B.; Alcasio-Rangel, S.; García-Ávila,
C.J.; López-Buenfil, J.A.; Ochoa-Martínez, D.L. First report of tomato brown rugose fruit virus (ToBRFV) in
Michoacan, Mexico. Revista Mexicana de Fitopatología, 2018, 37, 185-192.
https://doi.org/10.18781/r.mex.fit.1810-5
24. Camacho-Beltrán, E.; Pérez-Villarreal, A.; Leyva-López, N.E.; Rodríguez-Negrete, E.A.; Ceniceros-Ojeda,
E.A.; Méndez-Lozano, J. Occurrence of tomato brown rugose fruit virus infecting tomato crops in Mexico.
Plant Disease, 2019, 103, 1440. https://doi.org/10.1094/PDIS-11-18-1974-PDN
25. Ling, K.-S.; Tian, T.; Gurung, S.; Salati, R.; Gilliard, A. First report of tomato brown rugose fruit virus
infecting greenhouse tomato in the United States. Plant Disease, 2019, 103, 1439.
https://doi.org/10.1094/PDIS-11-18-1959-PDN
26. Sarkes, A.; Fu, H.; Feindel, D.; Harding, M.; Feng, J. Development and evaluation of a loop-mediated
isothermal amplification (LAMP) assay for the detection of Tomato brown rugose fruit virus (ToBRFV).
PLoS ONE 2020, 15, e0230403. https://doi.org/10.1371/journal.pone.0230403
27. Dey, K.K.; Velez-Climent, M.; Soria, P.; Batuman, O.; Mavrodieva, V.; Wei, G.; Zhou, J.; Adkins, A.; McVay,
J. Frist report of tomato brown rugose fruit virus infecting tomato in Florida, USA. New Disease Report, 2021,
44, e12028 https://doi.org/10.1002/ndr2.12028
28. Obregón, V.G., Ibañez, J.M., Lattar, T.E., Juszczak, S., Groth-Helms, D. First report of tomato brown rugose
fruit virus in tomato in Argentina. New Disease Reports 2023, 48, e12203.
https://doi.org/10.1002/ndr2.12203
29. EPPO. 2022. Tomato brown rugose fruit virus (ToBRFV) datasheet. EPPO Global Database.
https://gd.eppo.int/taxon/TOBRFV/datasheet (last accessed 9/22/2023)
30. Fraser, R.S.S.; Loughlin, S.A.R. Resistance to tobacco mosaic virus in tomato: effects of the Tm-1 gene on
virus multiplication. J. General. Virol. 1980, 48, 87-96. https://doi.org/10.1099/0022-1317-48-1-87
31. Holmes, F.O. Inheritance of resistance to infection by tobacco-mosaic virus in tomato. Phytopathology 1954,
44, 640–642
32. Pelham, J. Strain-genotype interaction of tobacco mosaic virus in tomato. Ann. Appl. Biol. 1972, 71, 219–228.
33. de Ronde, D.; Butte rbac h, P. ; Kor meli nk, R . Dominant resistance against plant viruses. Front. Plant Sci. 2014,
5, 307
34. Pfitzner, A.J.P. Resistance to tobacco mosaic virus and tomato mosaic virus in tomato. In Natural Resistance
Mechanisms of Plants to Viruses, 2006. ed. G. Loebenstein, J.P. Carr, pp. 399–413. Dordrecht, Neth.:
Springer
35. Maayan, Y.; Pandaranayaka, E. P. J.; Srivastava, D. A.; Lapidot, M.; Levin, I.; Dombrovsky, A.; Harel, A.
Using genomic analysis to identify tomato Tm-2 resistance-breaking mutations and their underlying
evolutionary path in a new and emerging tobamovirus. Archives of Virology, 2018, 163, 1863–1875.
https://doi.org/10.1007/s00705-018-3819-5
36. Chanda, B.; Gilliard, A.; Jaiswal, N.; Ling, K.-S. Comparative analysis of host range, ability to infect tomato
cultivars with Tm-22 gene, and real-time reverse transcription PCR detection of tomato brown rugose fruit
virus. Plant Disease, 2021a, 105, 3643–3652. https://doi.org/10.1094/PDIS-05-20-1070-RE
37. Alexander, L.J. Transfer of a dominant type of resistance to the four known Ohio pathogenic strains of
tobacco mosaic virus (TMV) from Lycopersicon peruvianum to L. esculentum. Phytopathology 1963, 53, 869.
38. Paudel, D. B.; Sanfacon, H. Exploring the diversity of mechanisms associated with plant tolerance to virus
infection. Frontiers in Plant Science, 2018, 9, 1575.
Preprints (www.preprints.org) | NOT PEER-REVIEWED | Posted: 10 January 2024 doi:10.20944/preprints202401.0785.v1
17
39. Kang, B.-C.; Yeam, I.; Jahn, M. M. Genetics of plant virus resistance. Annu. Rev. Phytopathol., 2005, 43, 581-
621.
40. Ponz F.; Bruening G. Mechanisms of resistance to plant viruses. Ann. Rev. Phytopathol. 1986, 24, 355-381.
41. Calil, I. P.; Fontes, E. P. B. Plant immunity against viruses: antiviral immune receptors in focus. Ann. Bot.
2017, 119, 711-723.
42. Zinger, A.; Lapidot, M.; Harel, A.; Doron-Faigenboim, A.; Gelbart, D.; Levin, I. Identification and mapping
of tomato genome loci controlling tolerance and resistance to tomato brown rugose fruit virus. Plants, 2021,
10, 1–16. https://doi.org/10.3390/plants10010179
43. Jewehan, A.; Salem, N.; Tóth, Z.; Salamon, P.; Szabó, Z. Screening of Solanum (sections Lycopersicon and
Juglandifolia) germplasm for reactions to the tomato brown rugose fruit virus (ToBRFV). J. Plant Dis. &
Protect., 2022a, 129, 117–123. https://doi.org/10.1007/s41348-021-00535-x
44. Jewehan, A.; Salem, N.; Tóth, Z. et al. Evaluation of responses to tomato brown rugose fruit virus (ToBRFV)
and selection of resistant lines in Solanum habrochaites and Solanum peruvianum germplasm. J. Gen. Plant
Pathol. 2022b, 88, 187–196. https://doi.org/10.1007/s10327-022-01055-8
45. Kabas, A.; Fidan, H.; Kucukaydin, H.; Atan, H.N.; Kabas, A.; Fidan, H.; Kucukaydin, H.; Atan, H. N.
Screening of wild tomato species and interspecific hybrids for resistance/tolerance to Tomato brown rugose
fruit virus (ToBRFV). Chilean Journal of Agricultural Research, 2022, 82, 189–196.
https://doi.org/10.4067/S0718-58392022000100189
46. Dombrovsky, A.; Smith, E. Seed Transmission of Tobamoviruses: Aspects of Global Disease Distribution.
Interchopen 2017, 10.5772/intechopen.70244.
47. Chanda, B.; Shamimuzzaman, M.; Gilliard, A.; Ling, K.-S. Effectiveness of disinfectants against the spread
of tobamoviruses: Tomato brown rugose fruit virus and Cucumber green mottle mosaic virus. Virol. J.
2021b. 18, 7. https://doi.org/10.1186/s12985-020-01479-8
48. Davino, S.; Caruso, A.G.; Bertacca, S.; Barone, S.; Panno, S. Tomato brown rugose fruit virus: seed
transmission rate and efficacy of different seed disinfection treatments. Plants 2020, 9, 1615.
https://doi:10.3390/plants9111615
49. Dombrovsky, A.; Mor, N.; Gantz, S.; Lachman, O.; Smith, E. Disinfection Efficacy of Tobamovirus-
Contaminated Soil in Greenhouse-Grown Crops. Horticulturae 2022, 8, 563.
https://doi.org/10.3390/horticulturae8070563
50. Ehlers, J.; Zarghani, S.N.; Kroschewski, B.; Büttner, C.; Bandte, M. Cleaning of Tomato brown rugose fruit
virus (ToBRFV) from contaminated clothing of greenhouse employees. Horticulturae 2022a, 8, 751.
https://doi.org/10.3390/horticulturae8080751
51. Ehlers, J.; Zarghani, S. N.; Kroschewski, B.; Büttner, C.; Bandte, M. Decontamination of tomato brown
rugose fruit virus-contaminated shoe soles under practical conditions. Horticulturae 2022b, 8, 1210.
https://doi.org/10.3390/horticulturae8121210
52. Ling, K.-S.; Gilliard, A.C.; Zia, B. Disinfectants useful to manage the emerging tomato brown rugose fruit
virus in greenhouse tomato production. Horticulturae 2022, 8, 1193.
https://doi.org/10.3390/horticulturae8121193
53. Rodríguez-Díaz, C. I.; Zamora-Macorra, E. J.; Ochoa-Martínez, D. L.; González-Garza, R. Disinfectants
effectiveness in Tomato brown rugose fruit virus (ToBRFV) transmission in tobacco plants. Rev. Mex.
Fitopatol. (Mexican Journal of Phytopathology) 2022, 40, 240-253. https://doi.org/10.18781/r.mex.fit.2111-2
54. Samarah, N.; Sulaiman, A.; Salem, N.M. et al. Disinfection treatments eliminated tomato brown rugose fruit
virus in tomato seeds. Eur. J. Plant Pathol., 2021, 159, 153–162. https://doi.org/10.1007/s10658-020-02151-1
55. Kole, C. Wild crop relatives: genomic and breeding resources: vegetables. 2011, Springer, Berlin
56. Chanda, B.; Rivera, Y.; Nunziata, S.O.; Galvez, M.E.; Gilliard, A.C.; Ling, K.-S. Complete genome sequence
of a tomato brown rugose fruit virus isolated in the United States. Microbiol. Resour. Announc. 2020. 9,
e00630-20.
Disclaimer/Publisher’s Note: The statements, opinions and data contained in all publications are solely those
of the individual author(s) and contributor(s) and not of MDPI and/or the editor(s). MDPI and/or the editor(s)
disclaim responsibility for any injury to people or property resulting from any ideas, methods, instructions or
products referred to in the content.
Preprints (www.preprints.org) | NOT PEER-REVIEWED | Posted: 10 January 2024 doi:10.20944/preprints202401.0785.v1
