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The recent association of a DNA betasatellite with Tomato yellow leaf curl virus in Israel – A new threat to tomato production
Crop Protection 128 (2020) 104995
Contents lists available at ScienceDirect
Crop Protection journal homepage: www.elsevier.com/locate/cropro
Short communication
The recent association of a DNA betasatellite with Tomato yellow leaf curl virus in Israel – A new threat to tomato production Dana Gelbart a, Lea Chen a, Tamar Alon b, Svetlana Dobrinin b, Ilan Levin a, Moshe Lapidot a, * a b
Institute of Plant Sciences, Agricultural Research Organization, Volcani Center, Rishon LeZion, Israel Extension Service, Ministry of Agriculture and Rural Development, Rishon LeZion, Israel
A R T I C L E I N F O
A B S T R A C T
Keywords: TYLCV Betasatellite Tomato Resistance Ty-1
During November 2016, tomato yellow leaf curl virus (TYLCV)-resistant F1 hybrid indeterminate tomato plants cv. Diagrama, grown in a commercial greenhouse in Revaya, Israel, expressed severe symptoms of tomato yellow leaf curl disease (TYLCD). Samples were collected from symptomatic tomato plants, and using TYLCV-specific PCR primers, it was confirmed that these plants were indeed infected with TYLCV. Using the universal betasa tellite primer pair B01/B02, followed by cloning and sequencing, it was found that the plants were also infected with a betasatellite. The satellite sequence was deposited in NCBI (GeneBank Acc. No. MK456609). Phylogenetic analysis revealed that the closest similarity, with 96% nucleotide identity, was to cotton leaf curl Gezira beta satellite (CLCuGB). Sampling symptomatic tomato plants in major growing areas in Israel revealed that CLCuGB is disseminated throughout the country. To the best of our knowledge, this is the first identification of a beta satellite, associated with TYLCV, in tomato plants in Israel.
Viruses belonging to the genus Begomovirus, family Geminiviridae, are transmitted by whiteflies of the Bemisia tabaci complex in a persistent manner, and cause significant damages to many crop plants. During the late 1950’s, a new disease was detected in tomato plants in Israel. It was found that the disease was caused by a new virus, which was named tomato yellow leaf curl virus (TYLCV). TYLCV has a genome composed of a single circular ssDNA molecule, nearly 2.8 kb in size (Navot et al., 1991). TYLCV has a relatively small host range, and is mainly known due to its devastating effect on tomato (Solanum lycopersicum) produc tion world-wide (Navas-Castillo et al., 2011; Lapidot et al., 2014; Lap idot and Levin, 2017). TYLCV-induced disease symptoms include upward cupping and chlorosis of tomato leaves, combined with signif icant plant stunting and reduction of plant yield (Cohen and Harpaz, 1964; Lapidot et al., 1997). With time it became clear that tomato yel low leaf curl disease (TYLCD) is induced by more than ten different begomovirus species (Moriones and Navas-Castillo, 2001; Nav as-Castillo et al., 2011). Two different strains of TYLCV are prevalent in Israel, TYLCV-IL (the type strain of the species) and TYLCV-Mld (Anti gnus and Cohen, 1994; Anfoka et al., 2008). The populations of the whitefly vector of TYLCV tend to reach very high numbers, which makes TYLCV-management a challenge. Most so lutions are aimed at reduction of insect numbers, by using insecticides or
physical barriers such as 50-mesh nets. Still, breeding tomatoes for resistance to TYLCV, although slow and tedious, is highly desirable as genetic resistance is the best strategy to combat viral-induced damages. Most efforts have been concentrated on screening wild tomato species for resistance to the virus, since all genotypes of cultivated tomato tested were found susceptible to TYLCV. Hence, resistant loci discovered in wild Solanum species were introgressed into S. lycopersicum. Among these are: Ty-1, Ty-3, Ty-4 and Ty-6 that were introgressed from S. chilense accessions, Ty-2 from S. habrochaites, and ty-5 presumably from S. peruvianum (Lapidot and Levin, 2017). Commercial tomato cultivars resistant to TYLCV are readily avail able. Following TYLCV-infection, most show some degree of disease symptoms, as well as some yield loss (Lapidot et al., 1997, 2014; Vidavsky et al., 2008). As Ty-1 was the first TYLCV-resistant locus identified, and it is dominantly inherited (Zamir et al., 1994), many breeding programs have utilized it. As a result, many commercial tomato hybrids resistant to TYLCV carry Ty-1 either alone or in combination with other resistance genes. Three types of DNA satellites, alphasatellites betasatellites and del tasatellites, have been associated with monopartite begomoviruses (Briddon and Stanley, 2006; Zhou, 2013; Lozano et al., 2016). Betasa tellites are circular ssDNAs, approximately 1,350 nt in size, that utilize
* Corresponding author. E-mail address: [email protected] (M. Lapidot). https://doi.org/10.1016/j.cropro.2019.104995 Received 30 June 2019; Received in revised form 24 October 2019; Accepted 26 October 2019 Available online 31 October 2019 0261-2194/© 2019 The Authors. Published by Elsevier Ltd. This is an open (http://creativecommons.org/licenses/by-nc-nd/4.0/).
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Fig. 1. Cv. Diagrama tomato plants showing tomato yellow leaf curl disease symptoms, Revaya, November 2016. (A) General view of the greenhouse; (B) Close-up on a plant showing disease symptoms.
monopartite begomoviruses as helper viruses, contribute to the induc tion of disease symptoms and are prevalent mainly in eastern Asia. Betasatellites rely on the helper begomoviruses for replication, encap sidation and spread within the host. By definition, betasatellites share no sequence similarity with the helper virus other than the begomoviral conserved hairpin structure that serves as the origin of viral DNA replication (Laufs et al., 1995; Zhou, 2013). Betasatellites code for a single multifunctional βC1 protein, which is involved in enhancement of disease symptoms and suppression of both transcriptional and post transcriptional gene silencing (Saunders et al., 2004; Cui et al., 2005; Yang et al., 2011; Amin et al., 2011; Zhou, 2013). During November 2016, F1 hybrid TYLCV-resistant indeterminate tomato plants cv. Diagrama (Nunhems, The Netherlands), grown commercially in greenhouses in Revaya (approximately 30 Km south of the Sea of Galilee, Israel), unexpectedly displayed severe TYLCD symptoms (Fig. 1). Leaf samples were collected from 18 symptomatic tomato plants from two different greenhouses and total nucleic acid were extracted from each (Dellaporta et al., 1983). Using PCR primer pair specific to TYLCV, TYC1F and TYC1R (Lapidot, 2002), all the tested plants were confirmed to be infected with TYLCV. To discriminate be tween TYLCV-IL and TYLCV-Mld, PCR primer pairs TY78R and IL2629F for TYLCV-IL, and TY78R and Mld2277F for TYLCV-Mld were used ac cording to Belabess et al. (2015). It was determined that eleven plants were infected with TYLCV-IL while seven plants were infected with TYLCV-Mld (we have also cloned and fully sequenced two clones of each TYLCV-IL and Mld. The full sequences were as expected of both TYLCV strains). However, due to the unexpected severity of disease symptoms expressed by the TYLCV-resistant plants and to the earlier identification of a betasatellite associated with TYLCV in Jordan (a neighboring country whose border is about 10 Km east from Revaya) (Anfoka et al., 2014), the samples were also tested for the presence of betasatellites using the universal primer pair, B01 and B02 (Briddon et al., 2002). While all 18 plants tested positive for TYLCV, ten tested positive for a betasatellite DNA (six plants were determined to be co-infected with TYLCV-Mld, four with TYLCV-IL and one with both viral strains). The amplification products from four plants (two co-infected with TYLCV-Mld and two co-infected with TYLCV-IL) were cloned into the plasmid pGEM-T Easy (Promega, USA) and sequenced. All four se quences (one from each plant), 1329 nt in size, were found to be iden tical and deposited as a single accession in NCBI sequence database (https://www.ncbi.nlm.nih.gov/genbank/, accession number MK456609). Phylogenetic analysis revealed that the closest sequence similarity, with 95.8% nucleotide identity, was to cotton leaf curl Gezira betasatellite (CLCuGB) isolate IsSq-C112, followed by 94.3% identity to
Table 1 Satellites accessions with more than 90% nucleotide sequence identity to the satellite sequence identified in Revaya, November 2016. Satellite
Accession No.
Genome size (nt.)
Nucleotide identity (%)
Cotton leaf curl Gezira betasatellite isolate IsSqC112 Cotton leaf curl Gezira satDNA 10a Okra yellow vein disease associated sequencea Cotton leaf curl Gezira betasatellite isolate IsSq1 Cotton leaf curl Gezira satDNA 10 isolate Homraa Cotton leaf curl Gezira satDNA 10 isolate Ammana Cotton leaf curl Gezira satDNA 3a
KT099177
1324
95.8
AF397215
1305
94.3
AJ316039
1307
93.9
KU095847
1310
93.8
KJ396939
1320
93.4
JX649952
1339
92.6
AF397217
1350
90.4
a Orginally termed OkLC betasatellite, but later recognized as cotton leaf curl Gezira betasatellite (Briddon et al., 2008).
OkLCB identified in Egypt and by 93.9% to OkLCB from Jordan [Orig inally termed OkLCB but later recognized as CLCuGB (Briddon et al., 2008)] (Table 1). Interestingly, CLCuGB isolate IsSq-C112 was identified in whiteflies collected from squash plants in Israel in 2011. The collec tion was from a squash field just outside Kafr Manda, approximately 50 Km North-West of Revaya. This identification was part of a vector-enabled metagenomics study of begomovirus-associated satellite DNAs analyzing whiteflies collected in different locations around the world, Israel being one of them (Rosario et al., 2016). This shows that this betasatellite is present in Israel for at least eight years. Plants containing both TYLCV-Mld and satellite served as source plants for inoculation of F1 hybrid Diagrama test plants using whiteflies. Adult whiteflies were allowed a 48 h acquisition access period (AAP) on tomato source plants infected either with TYLCV-Mld or with TYLCVMld and CLCuGB isolate Revaya. Following the AAP, the whiteflies were allowed a 48 h inoculation access period on 12 TYLCV-resistant ‘Diagrama’ test plants. To remove the whiteflies the test plants were treated with imidacloprid (Confidor, Bayer, Leverkusen, Germany), kept in an insect-proof greenhouse at 26–32 � C and monitored for TYLCD symptoms. The inoculation experiment was repeated twice, at two different dates. Using a Ty-1 specific molecular marker (P19) (Verlaan et al., 2013), it was determined that all the Diagrama F1 hybrid plants contain the TYLCV-resistant locus Ty-1 in a heterozygous state. While 2
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Crop Protection 128 (2020) 104995
Fig. 2. CLCuGB isolate Revaya associated with TYLCV-Mld enhances disease symptoms. Fourteen days old TYLCV-resistant cv. Diagrama plants (A, B) were inoculated with either TYLCV-Mld (A) or with TYLCV-Mld and with CLCuGB (B) using whiteflies. Following inoculation the test plants were treated with imidacloprid (Confidor, Bayer, Leverkusen, Germany), kept in an insect-proof greenhouse at 26–32 � C and monitored for symptom development. Pictures were taken 21 days after inoculation.
Table 2 Identification of Cotton leaf curl Gezira betasatellite in tomato production greenhouses and open fields in Israel between October, 2016 to October 2018. Location
Sampling date (M-Y)
Genotype
Ty-1
No. of samples Total
Positive for TYLCV-IL
TYLCV-Mld
CLCuGB
A. Greenhouse production Revaya Elifaz Netiv HaAsara Beit Hanan Yesha Idan Mivtachim Ein Yahav Ranen Sde David Nachla
10–2016 2–2017 5–2017 9–2017 10–2017 10–2017 10–2017 10–2017 7–2018 10–2018 10–2018
Diagrama Eshkol Eshkol 3951 Eshkol Eshkol Olympicos Eshkol Bruno Toni Gila
þ/ þ/ þ/ þ/ þ/ þ/ þ/ þ/ þ/ þ/ þ/
18 4 2 7 6 4 2 4 6 4 4
11 0 1 7 5 2 1 2 1 1 1
7 3 1 1 1 2 1 2 5 3 4
10 3 2 7 6 4 2 2 6 3 4
B. Open field production Tomer Mop Darom Givat Yoav Afik
3–2017 10–2017 8–2018 8–2018
Unknown Unknown Shavit Shanty
/ / þ/ þ/
4 4 8 4
2 2 4 2
2 4 7 4
4 3 6 4
Varieties sampled: Diagrama (Nunhems, The Netherlands); Eshkol (Seminis, USA); Olympicos, Bruno, 3951, Shavit, Shanty (Hazera Seeds, Israel); Toni, Gila (Rimi, Israel). Presence or absence of Ty-1 was determined using a specific molecular marker (P19) according to Verlaan et al. (2013); þ/ denotes presence of Ty-1 in a heterozygous state; / denotes plants that do not carry Ty-1.
‘Diagrama’ plants inoculated only with TYLCV-Mld hardly showed any disease symptoms (Fig. 2), ‘Diagrama’ plants inoculated with TYLCV-Mld and CLCuGB isolate Revaya developed severe TYLCD symptoms within 14–21 days (Fig. 2). The inoculated test plants were tested by PCR for presence of TYLCV-Mld and CLCuGB 28 days after inoculation. Indeed, all the test plants inoculated only with TYLCV-Mld were tested positive for the virus and negative for the satellite, while all the plants inoculated with both TYLCV-Mld and CLCuGB were tested positive for both. Moreover, a few test plants inoculated with both TYLCV-Mld and CLCuGB were kept in a greenhouse for 6 months and monitored for disease development. No change in symptom severity was detected, and after 180 days the plants were tested again and found positive for TYLCV-Mld and CLCuGB. Following the identification of a betasatellite in Revaya, samples were collected from commercially grown tomato greenhouses and fields throughout the country (Table 2, Fig. 3). Samples were collected from plants that exhibited severe TYLCD symptoms. All the sampled symp tomatic plants were found positive for either TYLCV-IL or TYLCV-Mld, most were also coinfected with CLCuGB. As the majority of tomato ge notypes grown commercially in Israel are TYLCV-resistant, indeed most of the sampled plants were determined positive for Ty-1 (Table 2). Betasatellites from six samples (two from each site) from Idan, Elifaz and from Givat Yoav (Fig. 3) were cloned and sequenced. All six sequences
were found to be identical to the betasatellite detected earlier in Revaya (we had an odd nucleotide difference in one sequence, but it may be a result of the PCR amplification performed prior to cloning). The association of a betasatellite with TYLCV was reported for the first time in 2008 from Oman (Khan et al., 2008), but the satellite effect on disease severity was not clear since no comparison was made to infection with the virus without the satellite. Recently, Conflon et al. (2018) studied the effect that CLCuGB (accession No. FN554575) iso lated from Okra in Burkina-Faso associated with TYLCV may have upon disease severity and upon virus accumulation in the infected plant. The betasatellite was found to increase the disease severity induced by TYLCV. Moreover, a Ty-1 resistant tomato genotype (Pristyla, Gautier Semences) infected with both CLCuGB and TYLCV (either Mld or IL) exhibited mild disease symptoms at the early stages of infection while later on symptoms became milder and eventually disappeared (Conflon et al., 2018). However, in our case, following inoculation of Ty-1 plants with TYLCV-Mld and CLCuGB the plants expressed severe disease symptoms (see Fig. 2) and showed no recovery for at least 180 days. Moreover, the presence of the satellite was discovered in relatively mature (4 months old, see Fig. 1) TYLCV-resistant tomato plants showing severe symptoms of TYLCD. Thus our results suggest that the association of CLCuGB with TYLCV compromises Ty-1 resistance. However, although CLCuGB was associated with either TYLCV-IL or 3
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interests or personal relationships that could have appeared to influence the work reported in this paper. Acknowledgments This work was supported by the Chief Scientist of The Ministry of Agriculture, Israel (grant number 20-10-0045) within the ERANETARIMNet2 (ref. 302) program. References Anfoka, G., Haj Ahmad, F., Altaleb, M., Abadi, M., 2014. Detection of satellite DNA beta in tomato plants with tomato yellow leaf curl disease in Jordan. Plant Dis. 98, 1017. Anfoka, G., Abhary, M., Haj Ahmad, F., Hussein, A.F., Rezk, A., Akad, F., AbouJawdah, Y., Lapidot, M., Vidavski, F., Nakhla, M.K., Sobh, H., Atamian, H., Cohen, L., Sobol, I., Mazyad, H., Maxwell, D.P., Czosnek, H., 2008. Survey of tomato yellow leaf curl disease-associated viruses in the Eastern Mediterranean basin. J. Plant Pathol. 90, 311–320. Amin, I., Hussain, K., Akbergenov, R., Yadav, J.S., Qazi, J., Mansoor, S., Hohn, T., Fauquet, C.M., Briddon, R.W., 2011. Suppressors of RNA silencing encoded by the components of the Cotton leaf curl begomovirus-betasatellite complex. Mol. Plant Microbe Interact. 24, 973–983. Antignus, Y., Cohen, S., 1994. Complete nucleotide sequence of an infection clone of a mild isolate of tomato yellow lwaf curl virus (TYLCV). Phytopathology 84, 707–712. Belabess, Z., Dallot, S., El-Montaser, S., Granier, M., Majde, M., Tahiri, A., Blenzar, A., Urbino, C., Petrschmitt, M., 2015. Monitoring the dynamics of emergence of a noncanonical recombinant of Tomato yellow leaf curl virus and displacement of its parental viruses in tomato. Virology 486, 291–306. Briddon, R.W., Bull, S.E., Mansoor, S., Amin, I., Markham, P.G., 2002. Universal primers for the PCR-mediated amplification of DNA β. Mol. Biotechnol. 20, 315–318. Briddon, R.W., Stanley, J., 2006. Subviral agents associated with plant single-stranded DNA viruses. Virology 344, 198–210. Briddon, R.W., Brown, J.K., Moriones, E., Stanley, J., Zerbini, M., Zhou, X., Fauquet, C. M., 2008. Recommendations for the classification and nomenclature of the DNA- β satellites of begomoviruses. Arch. Virol. 153, 763–781. Cohen, S., Harpaz, I., 1964. Periodic rather than continual acquisition of a new tomato virus by its vector, the tobacco whitefly (Bemisia tabaci Gennadius). Entomol. Exp. Appl. 7, 155–166. Conflon, D., Granier, M., Tiendrebeogoc, F., Gentite, P., Peterschmitt, M., Urbino, C., 2018. Accumulation and transmission of alphasatellite, betasatellite and tomato yellow leaf curl virus in susceptible and Ty-1-resistant tomato plants. Virus Res. 253, 124–134. Cui, X.F., Li, G.X., Wang, D.W., Hu, D.W., Zhou, X.P., 2005. A begomovirus DNA betaencoded protein binds DNA, functions as a suppressor of RNA silencing, and targets the cell nucleus. J. Virol. 79, 10764–10775. Dellaporta, S.L., Wood, J., Hicks, J.B., 1983. A plant DNA minipreparation: version II. Plant Mol. Biol. Report. 1, 19–21. Khan, A.J., Idris, A.M., Al-Saady, N.A., Al-Mahruki, M.S., Al-Subhi, A.M., Brown, J.K., 2008. A divergent isolate of Tomato yellow leaf curl virus from Oman with an associated DNA β satellite: an evolutionary link between Asian and the middle eastern virus-satellite complexes. Virus Genes 36, 169–176. Lapidot, M., 2002. Screening common beans (Phaseoulas volgaris) for resistance to TYLCV. Plant Dis. 86, 429–432. Lapidot, M., Friedmann, M., Lachman, O., Yehezkel, A., Nahon, S., Cohen, S., Pilowsky, M., 1997. Comparison of resistance level to tomato yellow leaf curl virus among commercial cultivars and breeding lines. Plant Dis. 81, 1425–1428. Lapidot, M., Legg, J.P., Wintermantel, W.M., Polston, J.E., 2014. Management of Whitefly-transmitted viruses in open-field production systems. In: Loebenstein, G., Katis, I. (Eds.), Control of Plant Viruses in the Series “Advances in Virus Research”, vol. 90. Academic Press, Burlington, pp. 147–206. Lapidot, M., Levin, I., 2017. Genetic resistance to viruses in tomato. In: Achieving Sustainable Cultivation of Tomatoes (Autar Mattoo and Avtar Handa. Burleigh Dodds Science Publishing, pp. 381–400. Laufs, J., Traut, W., Heyraud, F., Matzeit, V., Rogers, S.G., Schell, J., Gronenborn, B., 1995. In vitro cleavage and joining at the viral origin of replication by the replication initiator protein of tomato yellow leaf curl virus. Proc. Natl. Acad. Sci. U.S.A. 92, 3879–3883. Lozano, G., Trenado, H.P., Fiallo-Oliv�e, E., Chirinos, D., Geraud-Pouey, F., Briddon, R.W., Navas-Castillo, J., 2016. Characterization of non-coding DNA satellites associated with sweepoviruses (Genus Begomovirus, Geminiviridae) – definition of a distinct class of begomovirus-associated satellites. Front. Microbiol. 7, 162. https://doi.org/ 10.3389/fmicb.2016.00162. Moriones, E., Navas-Castillo, J., 2001. Tomato yellow leaf curl virus, an emerging virus complex causing epidemics worldwide. Virus Res. 71, 123–134. 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Fig. 3. Distribution of CLCuGB associated with TYLCD in tomatoes grown commercially in greenhouses and open fields in Israel. Samples were collected from commercially grown tomato greenhouses and fields throughout the country, from October 2016 to October 2018 as described in Table 2.
TYLCV-Mld in most symptomatic plants sampled in the field, these plants were not infected by a single viral clone but rather with a mixture of isolates. Hence, we cannot rule out the possibility that one of these viral isolates, or a combination of isolates is overcoming the Ty-1 resistance, and not necessarily the betasatellite. To the best of our knowledge, this is the first identification of a betasatellite associated with TYLCD in tomato plants in Israel. The sat ellite enhances the effect of the disease, possibly compromises the TYLCV-resistance induced by Ty-1 to a significant extent, and may very well increase yield loss due to TYLCD. This is of great concern for tomato growers in the Mediterranean region. Declaration of competing interest The authors declare that they have no known competing financial 4
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Saunders, K., Norman, A., Gucciardo, S., Stanley, J., 2004. The DNA β satellite component associated with ageratum yellow vein disease encodes an essential pathogenicity protein (βC1). Virology 324, 37–47. Verlaan, M.G., Hutton, S.F., Ibrahem, R.M., Kormelink, R., Visser, R.G., Scott, J.W., Edwards, J.D., Bai, Y., 2013. The Tomato yellow leaf curl virus resistance genes Ty-1 and Ty-3 are allelic and code for DFDGD-class RNA-dependent RNA polymerases. PLoS Genet. 9, e1003399. Vidavski, F., Czosnek, H., Gazit, S., Levy, D., Lapidot, M., 2008. Pyramiding of Genes Conferring resistance to Tomato yellow leaf curl virus from different wild tomato species. Plant Breed. 127, 625–631.
Yang, X., Xie, Y., Raja, P., Li, S., Wolf, J.N., Shen, Q., Bisaro, D.M., Zhou, X., 2011. Suppression of methylation-mediated transcriptional gene silencing by betaC1SAHH protein interaction during geminivirus-betasatellite infection. PLoS Pathog. 7, e1002329. Zamir, D., Ekstein-Michelson, I., Zakay, Y., Navot, N., Zeidan, M., Sarfatti, M., Eshed, Y., Harel, E., Pleban, T., Van Oss, H., Kedar, N., Rabinowitch, H.D., Czosnek, H., 1994. Mapping and introgression of a tomato yellow leaf curl virus tolerance gene, TY-1. Theor. Appl. Genet. 88, 141–146. Zhou, X., 2013. Advances in understanding begomovirus satellites. Annu. Rev. Phytopathol. 51, 357–381.
5
Contents lists available at ScienceDirect
Crop Protection journal homepage: www.elsevier.com/locate/cropro
Short communication
The recent association of a DNA betasatellite with Tomato yellow leaf curl virus in Israel – A new threat to tomato production Dana Gelbart a, Lea Chen a, Tamar Alon b, Svetlana Dobrinin b, Ilan Levin a, Moshe Lapidot a, * a b
Institute of Plant Sciences, Agricultural Research Organization, Volcani Center, Rishon LeZion, Israel Extension Service, Ministry of Agriculture and Rural Development, Rishon LeZion, Israel
A R T I C L E I N F O
A B S T R A C T
Keywords: TYLCV Betasatellite Tomato Resistance Ty-1
During November 2016, tomato yellow leaf curl virus (TYLCV)-resistant F1 hybrid indeterminate tomato plants cv. Diagrama, grown in a commercial greenhouse in Revaya, Israel, expressed severe symptoms of tomato yellow leaf curl disease (TYLCD). Samples were collected from symptomatic tomato plants, and using TYLCV-specific PCR primers, it was confirmed that these plants were indeed infected with TYLCV. Using the universal betasa tellite primer pair B01/B02, followed by cloning and sequencing, it was found that the plants were also infected with a betasatellite. The satellite sequence was deposited in NCBI (GeneBank Acc. No. MK456609). Phylogenetic analysis revealed that the closest similarity, with 96% nucleotide identity, was to cotton leaf curl Gezira beta satellite (CLCuGB). Sampling symptomatic tomato plants in major growing areas in Israel revealed that CLCuGB is disseminated throughout the country. To the best of our knowledge, this is the first identification of a beta satellite, associated with TYLCV, in tomato plants in Israel.
Viruses belonging to the genus Begomovirus, family Geminiviridae, are transmitted by whiteflies of the Bemisia tabaci complex in a persistent manner, and cause significant damages to many crop plants. During the late 1950’s, a new disease was detected in tomato plants in Israel. It was found that the disease was caused by a new virus, which was named tomato yellow leaf curl virus (TYLCV). TYLCV has a genome composed of a single circular ssDNA molecule, nearly 2.8 kb in size (Navot et al., 1991). TYLCV has a relatively small host range, and is mainly known due to its devastating effect on tomato (Solanum lycopersicum) produc tion world-wide (Navas-Castillo et al., 2011; Lapidot et al., 2014; Lap idot and Levin, 2017). TYLCV-induced disease symptoms include upward cupping and chlorosis of tomato leaves, combined with signif icant plant stunting and reduction of plant yield (Cohen and Harpaz, 1964; Lapidot et al., 1997). With time it became clear that tomato yel low leaf curl disease (TYLCD) is induced by more than ten different begomovirus species (Moriones and Navas-Castillo, 2001; Nav as-Castillo et al., 2011). Two different strains of TYLCV are prevalent in Israel, TYLCV-IL (the type strain of the species) and TYLCV-Mld (Anti gnus and Cohen, 1994; Anfoka et al., 2008). The populations of the whitefly vector of TYLCV tend to reach very high numbers, which makes TYLCV-management a challenge. Most so lutions are aimed at reduction of insect numbers, by using insecticides or
physical barriers such as 50-mesh nets. Still, breeding tomatoes for resistance to TYLCV, although slow and tedious, is highly desirable as genetic resistance is the best strategy to combat viral-induced damages. Most efforts have been concentrated on screening wild tomato species for resistance to the virus, since all genotypes of cultivated tomato tested were found susceptible to TYLCV. Hence, resistant loci discovered in wild Solanum species were introgressed into S. lycopersicum. Among these are: Ty-1, Ty-3, Ty-4 and Ty-6 that were introgressed from S. chilense accessions, Ty-2 from S. habrochaites, and ty-5 presumably from S. peruvianum (Lapidot and Levin, 2017). Commercial tomato cultivars resistant to TYLCV are readily avail able. Following TYLCV-infection, most show some degree of disease symptoms, as well as some yield loss (Lapidot et al., 1997, 2014; Vidavsky et al., 2008). As Ty-1 was the first TYLCV-resistant locus identified, and it is dominantly inherited (Zamir et al., 1994), many breeding programs have utilized it. As a result, many commercial tomato hybrids resistant to TYLCV carry Ty-1 either alone or in combination with other resistance genes. Three types of DNA satellites, alphasatellites betasatellites and del tasatellites, have been associated with monopartite begomoviruses (Briddon and Stanley, 2006; Zhou, 2013; Lozano et al., 2016). Betasa tellites are circular ssDNAs, approximately 1,350 nt in size, that utilize
* Corresponding author. E-mail address: [email protected] (M. Lapidot). https://doi.org/10.1016/j.cropro.2019.104995 Received 30 June 2019; Received in revised form 24 October 2019; Accepted 26 October 2019 Available online 31 October 2019 0261-2194/© 2019 The Authors. Published by Elsevier Ltd. This is an open (http://creativecommons.org/licenses/by-nc-nd/4.0/).
access
article
under
the
CC
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license
D. Gelbart et al.
Crop Protection 128 (2020) 104995
Fig. 1. Cv. Diagrama tomato plants showing tomato yellow leaf curl disease symptoms, Revaya, November 2016. (A) General view of the greenhouse; (B) Close-up on a plant showing disease symptoms.
monopartite begomoviruses as helper viruses, contribute to the induc tion of disease symptoms and are prevalent mainly in eastern Asia. Betasatellites rely on the helper begomoviruses for replication, encap sidation and spread within the host. By definition, betasatellites share no sequence similarity with the helper virus other than the begomoviral conserved hairpin structure that serves as the origin of viral DNA replication (Laufs et al., 1995; Zhou, 2013). Betasatellites code for a single multifunctional βC1 protein, which is involved in enhancement of disease symptoms and suppression of both transcriptional and post transcriptional gene silencing (Saunders et al., 2004; Cui et al., 2005; Yang et al., 2011; Amin et al., 2011; Zhou, 2013). During November 2016, F1 hybrid TYLCV-resistant indeterminate tomato plants cv. Diagrama (Nunhems, The Netherlands), grown commercially in greenhouses in Revaya (approximately 30 Km south of the Sea of Galilee, Israel), unexpectedly displayed severe TYLCD symptoms (Fig. 1). Leaf samples were collected from 18 symptomatic tomato plants from two different greenhouses and total nucleic acid were extracted from each (Dellaporta et al., 1983). Using PCR primer pair specific to TYLCV, TYC1F and TYC1R (Lapidot, 2002), all the tested plants were confirmed to be infected with TYLCV. To discriminate be tween TYLCV-IL and TYLCV-Mld, PCR primer pairs TY78R and IL2629F for TYLCV-IL, and TY78R and Mld2277F for TYLCV-Mld were used ac cording to Belabess et al. (2015). It was determined that eleven plants were infected with TYLCV-IL while seven plants were infected with TYLCV-Mld (we have also cloned and fully sequenced two clones of each TYLCV-IL and Mld. The full sequences were as expected of both TYLCV strains). However, due to the unexpected severity of disease symptoms expressed by the TYLCV-resistant plants and to the earlier identification of a betasatellite associated with TYLCV in Jordan (a neighboring country whose border is about 10 Km east from Revaya) (Anfoka et al., 2014), the samples were also tested for the presence of betasatellites using the universal primer pair, B01 and B02 (Briddon et al., 2002). While all 18 plants tested positive for TYLCV, ten tested positive for a betasatellite DNA (six plants were determined to be co-infected with TYLCV-Mld, four with TYLCV-IL and one with both viral strains). The amplification products from four plants (two co-infected with TYLCV-Mld and two co-infected with TYLCV-IL) were cloned into the plasmid pGEM-T Easy (Promega, USA) and sequenced. All four se quences (one from each plant), 1329 nt in size, were found to be iden tical and deposited as a single accession in NCBI sequence database (https://www.ncbi.nlm.nih.gov/genbank/, accession number MK456609). Phylogenetic analysis revealed that the closest sequence similarity, with 95.8% nucleotide identity, was to cotton leaf curl Gezira betasatellite (CLCuGB) isolate IsSq-C112, followed by 94.3% identity to
Table 1 Satellites accessions with more than 90% nucleotide sequence identity to the satellite sequence identified in Revaya, November 2016. Satellite
Accession No.
Genome size (nt.)
Nucleotide identity (%)
Cotton leaf curl Gezira betasatellite isolate IsSqC112 Cotton leaf curl Gezira satDNA 10a Okra yellow vein disease associated sequencea Cotton leaf curl Gezira betasatellite isolate IsSq1 Cotton leaf curl Gezira satDNA 10 isolate Homraa Cotton leaf curl Gezira satDNA 10 isolate Ammana Cotton leaf curl Gezira satDNA 3a
KT099177
1324
95.8
AF397215
1305
94.3
AJ316039
1307
93.9
KU095847
1310
93.8
KJ396939
1320
93.4
JX649952
1339
92.6
AF397217
1350
90.4
a Orginally termed OkLC betasatellite, but later recognized as cotton leaf curl Gezira betasatellite (Briddon et al., 2008).
OkLCB identified in Egypt and by 93.9% to OkLCB from Jordan [Orig inally termed OkLCB but later recognized as CLCuGB (Briddon et al., 2008)] (Table 1). Interestingly, CLCuGB isolate IsSq-C112 was identified in whiteflies collected from squash plants in Israel in 2011. The collec tion was from a squash field just outside Kafr Manda, approximately 50 Km North-West of Revaya. This identification was part of a vector-enabled metagenomics study of begomovirus-associated satellite DNAs analyzing whiteflies collected in different locations around the world, Israel being one of them (Rosario et al., 2016). This shows that this betasatellite is present in Israel for at least eight years. Plants containing both TYLCV-Mld and satellite served as source plants for inoculation of F1 hybrid Diagrama test plants using whiteflies. Adult whiteflies were allowed a 48 h acquisition access period (AAP) on tomato source plants infected either with TYLCV-Mld or with TYLCVMld and CLCuGB isolate Revaya. Following the AAP, the whiteflies were allowed a 48 h inoculation access period on 12 TYLCV-resistant ‘Diagrama’ test plants. To remove the whiteflies the test plants were treated with imidacloprid (Confidor, Bayer, Leverkusen, Germany), kept in an insect-proof greenhouse at 26–32 � C and monitored for TYLCD symptoms. The inoculation experiment was repeated twice, at two different dates. Using a Ty-1 specific molecular marker (P19) (Verlaan et al., 2013), it was determined that all the Diagrama F1 hybrid plants contain the TYLCV-resistant locus Ty-1 in a heterozygous state. While 2
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Fig. 2. CLCuGB isolate Revaya associated with TYLCV-Mld enhances disease symptoms. Fourteen days old TYLCV-resistant cv. Diagrama plants (A, B) were inoculated with either TYLCV-Mld (A) or with TYLCV-Mld and with CLCuGB (B) using whiteflies. Following inoculation the test plants were treated with imidacloprid (Confidor, Bayer, Leverkusen, Germany), kept in an insect-proof greenhouse at 26–32 � C and monitored for symptom development. Pictures were taken 21 days after inoculation.
Table 2 Identification of Cotton leaf curl Gezira betasatellite in tomato production greenhouses and open fields in Israel between October, 2016 to October 2018. Location
Sampling date (M-Y)
Genotype
Ty-1
No. of samples Total
Positive for TYLCV-IL
TYLCV-Mld
CLCuGB
A. Greenhouse production Revaya Elifaz Netiv HaAsara Beit Hanan Yesha Idan Mivtachim Ein Yahav Ranen Sde David Nachla
10–2016 2–2017 5–2017 9–2017 10–2017 10–2017 10–2017 10–2017 7–2018 10–2018 10–2018
Diagrama Eshkol Eshkol 3951 Eshkol Eshkol Olympicos Eshkol Bruno Toni Gila
þ/ þ/ þ/ þ/ þ/ þ/ þ/ þ/ þ/ þ/ þ/
18 4 2 7 6 4 2 4 6 4 4
11 0 1 7 5 2 1 2 1 1 1
7 3 1 1 1 2 1 2 5 3 4
10 3 2 7 6 4 2 2 6 3 4
B. Open field production Tomer Mop Darom Givat Yoav Afik
3–2017 10–2017 8–2018 8–2018
Unknown Unknown Shavit Shanty
/ / þ/ þ/
4 4 8 4
2 2 4 2
2 4 7 4
4 3 6 4
Varieties sampled: Diagrama (Nunhems, The Netherlands); Eshkol (Seminis, USA); Olympicos, Bruno, 3951, Shavit, Shanty (Hazera Seeds, Israel); Toni, Gila (Rimi, Israel). Presence or absence of Ty-1 was determined using a specific molecular marker (P19) according to Verlaan et al. (2013); þ/ denotes presence of Ty-1 in a heterozygous state; / denotes plants that do not carry Ty-1.
‘Diagrama’ plants inoculated only with TYLCV-Mld hardly showed any disease symptoms (Fig. 2), ‘Diagrama’ plants inoculated with TYLCV-Mld and CLCuGB isolate Revaya developed severe TYLCD symptoms within 14–21 days (Fig. 2). The inoculated test plants were tested by PCR for presence of TYLCV-Mld and CLCuGB 28 days after inoculation. Indeed, all the test plants inoculated only with TYLCV-Mld were tested positive for the virus and negative for the satellite, while all the plants inoculated with both TYLCV-Mld and CLCuGB were tested positive for both. Moreover, a few test plants inoculated with both TYLCV-Mld and CLCuGB were kept in a greenhouse for 6 months and monitored for disease development. No change in symptom severity was detected, and after 180 days the plants were tested again and found positive for TYLCV-Mld and CLCuGB. Following the identification of a betasatellite in Revaya, samples were collected from commercially grown tomato greenhouses and fields throughout the country (Table 2, Fig. 3). Samples were collected from plants that exhibited severe TYLCD symptoms. All the sampled symp tomatic plants were found positive for either TYLCV-IL or TYLCV-Mld, most were also coinfected with CLCuGB. As the majority of tomato ge notypes grown commercially in Israel are TYLCV-resistant, indeed most of the sampled plants were determined positive for Ty-1 (Table 2). Betasatellites from six samples (two from each site) from Idan, Elifaz and from Givat Yoav (Fig. 3) were cloned and sequenced. All six sequences
were found to be identical to the betasatellite detected earlier in Revaya (we had an odd nucleotide difference in one sequence, but it may be a result of the PCR amplification performed prior to cloning). The association of a betasatellite with TYLCV was reported for the first time in 2008 from Oman (Khan et al., 2008), but the satellite effect on disease severity was not clear since no comparison was made to infection with the virus without the satellite. Recently, Conflon et al. (2018) studied the effect that CLCuGB (accession No. FN554575) iso lated from Okra in Burkina-Faso associated with TYLCV may have upon disease severity and upon virus accumulation in the infected plant. The betasatellite was found to increase the disease severity induced by TYLCV. Moreover, a Ty-1 resistant tomato genotype (Pristyla, Gautier Semences) infected with both CLCuGB and TYLCV (either Mld or IL) exhibited mild disease symptoms at the early stages of infection while later on symptoms became milder and eventually disappeared (Conflon et al., 2018). However, in our case, following inoculation of Ty-1 plants with TYLCV-Mld and CLCuGB the plants expressed severe disease symptoms (see Fig. 2) and showed no recovery for at least 180 days. Moreover, the presence of the satellite was discovered in relatively mature (4 months old, see Fig. 1) TYLCV-resistant tomato plants showing severe symptoms of TYLCD. Thus our results suggest that the association of CLCuGB with TYLCV compromises Ty-1 resistance. However, although CLCuGB was associated with either TYLCV-IL or 3
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interests or personal relationships that could have appeared to influence the work reported in this paper. Acknowledgments This work was supported by the Chief Scientist of The Ministry of Agriculture, Israel (grant number 20-10-0045) within the ERANETARIMNet2 (ref. 302) program. References Anfoka, G., Haj Ahmad, F., Altaleb, M., Abadi, M., 2014. Detection of satellite DNA beta in tomato plants with tomato yellow leaf curl disease in Jordan. Plant Dis. 98, 1017. Anfoka, G., Abhary, M., Haj Ahmad, F., Hussein, A.F., Rezk, A., Akad, F., AbouJawdah, Y., Lapidot, M., Vidavski, F., Nakhla, M.K., Sobh, H., Atamian, H., Cohen, L., Sobol, I., Mazyad, H., Maxwell, D.P., Czosnek, H., 2008. Survey of tomato yellow leaf curl disease-associated viruses in the Eastern Mediterranean basin. J. Plant Pathol. 90, 311–320. Amin, I., Hussain, K., Akbergenov, R., Yadav, J.S., Qazi, J., Mansoor, S., Hohn, T., Fauquet, C.M., Briddon, R.W., 2011. 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Fig. 3. Distribution of CLCuGB associated with TYLCD in tomatoes grown commercially in greenhouses and open fields in Israel. Samples were collected from commercially grown tomato greenhouses and fields throughout the country, from October 2016 to October 2018 as described in Table 2.
TYLCV-Mld in most symptomatic plants sampled in the field, these plants were not infected by a single viral clone but rather with a mixture of isolates. Hence, we cannot rule out the possibility that one of these viral isolates, or a combination of isolates is overcoming the Ty-1 resistance, and not necessarily the betasatellite. To the best of our knowledge, this is the first identification of a betasatellite associated with TYLCD in tomato plants in Israel. The sat ellite enhances the effect of the disease, possibly compromises the TYLCV-resistance induced by Ty-1 to a significant extent, and may very well increase yield loss due to TYLCD. This is of great concern for tomato growers in the Mediterranean region. Declaration of competing interest The authors declare that they have no known competing financial 4
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Yang, X., Xie, Y., Raja, P., Li, S., Wolf, J.N., Shen, Q., Bisaro, D.M., Zhou, X., 2011. Suppression of methylation-mediated transcriptional gene silencing by betaC1SAHH protein interaction during geminivirus-betasatellite infection. PLoS Pathog. 7, e1002329. Zamir, D., Ekstein-Michelson, I., Zakay, Y., Navot, N., Zeidan, M., Sarfatti, M., Eshed, Y., Harel, E., Pleban, T., Van Oss, H., Kedar, N., Rabinowitch, H.D., Czosnek, H., 1994. Mapping and introgression of a tomato yellow leaf curl virus tolerance gene, TY-1. Theor. Appl. Genet. 88, 141–146. Zhou, X., 2013. Advances in understanding begomovirus satellites. Annu. Rev. Phytopathol. 51, 357–381.
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