Genetic Diversity of CMV and ToMV Isolates in Tomato and Pepper Production Areas in Hakkari, Turkey

doi:10.21203/rs.3.rs-1451598/v1

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Genetic Diversity of CMV and ToMV Isolates in Tomato and Pepper Production Areas in Hakkari, Turkey

https://doi.org/10.21203/rs.3.rs-1451598/v1

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Background Tomato (Lycopersicum esculentum Mill.) and pepper (Capsicum anuum L.) are the host species for the Cucumber mosaic virus (CMV) and Tomato mosaic virus (ToMV). This article aims to report the detection of serolocigally and molecularly and identification of genetic diversity these viruses in Hakkari province, Turkey.

Methods and results A total of 184 leaf samples were collected from tomato and pepper plants that showed virus-like symptoms. Sample collection was carried out in Hakkari province in October, 2020. CMV and ToMV factors were investigated by Double Antibody Sandwich Enzyme-Linked Immunosorbent Assay (DAS-ELISA). DAS-ELISA test showed that 106 (57.60%) of the samples were infected with one or more viruses. For phylogenetic analyses, 10 samples which were positive for CMV and ToMV were tested using Reverse Transcription Polymerase Chain Reaction (RT-PCR) method. Coat protein (CP) and replicase genes of Hakkari CMV and ToMV isolates were amplified by RT-PCR using specific primers. The resulting DNA sequences were deposited in NCBI GenBank (Accession Nos: OM286731, OM286732, OM286733, OM286734, OM418629). When the replicase and coat protein gene sequences of four ToMV isolates and one CMV isolate were compared with the isolates in GenBank, a nucleotide homology of 98.1-99.6% and 91.7-99.4% were found, respectively. Phylogenetic analysis revealed a strong similarity between Hakkari ToMV and CMV isolates and other Turkish isolates. Consistent with the molecular studies conducted in Turkey on CMV and ToMV, the isolates detected in this study also clustered in subgroup Ia.

Conclusions With this study, CMV and ToMV factors were serologically and molecularly detected for the first time in Hakkari and the first nation-wide report for ToMV replicase sequences in infected tomato and peppers were submitted.

CMV

ToMV

DAS-ELISA

phylogenetic analysis

Hakkari

In tomato and pepper production areas, various fungal, bacterial and viral diseased lead to severe losses during the period between seedling and harvest. CMV and ToMV are the most important viruses encountered in tomato production areas [1, 2]. CMV, which was first reported in the United States of America in 1934 by Price, belongs to the Cucumovirus genus in the family Bromoviridae. It has an isometric structure and a diameter of 29 nm. It has a linear genome composed of single-stranded RNA [3, 4]. CMV can infect more than 1200 different plants belonging to 500 genera from 100 families and is non-persistently transmitted by more than 60 aphid species belonging to the family of Aphididae [3, 5, 6, 7]. It can also be transmitted through mechanical inoculation and in seeds. In infected plants, CMV leads to mosaics, leaf and fruit distortion, and even to plant death [8]. It is serologically categorized in subgroups I and II and in the phylogenetic analysis of the coat protein, it clusters in the subgroup Ia [9]. CMV subgroups Ia and II can be found worldwide [10].

Tobamovirus is the largest genus in the family Virgaviridae [11, 12]. Belonging to this genus, ToMV is a disease agent with a linear single-stranded RNA genome and has rod-shaped virions. It has a wide host range including members of the family Solanaceae such as tomato and pepper and it causes significant crop losses in tomato plants worldwide [2, 12, 13, 14, 15, 16]. It is the most persistent virus in terms of its ability to survive outside of plant cells or in dead tissues [17]. ToMV is known to decrease tomato production by up to 20% [1, 2]. ToMV causes mosaic, necrosis and senescence in pepper leaves, and light or dark green mosaic and distortion in tomato leaves. ToMV infection has also been detected in tomato and pepper seeds [14, 15]. There is a number of methods employed to manage tobamoviruses, including using resistant varieties, and resorting to biological or chemical control. It is difficult to control the spread of ToMV as it can be transmitted in seeds or through mechanical inoculation, contaminated tools or workers [2, 12, 13]. Within the three pathological groups of different ToMV populations, global ToMV isolates in Group I mostly infected solanaceous host species tested while the isolates in Group II did also infect other plants [18, 19].

Although CMV and ToMV are common in tomato and pepper production areas in our country, information about the diversity of these viruses is highly limited. The aim of this 2020 study is to demonstrate the genetic relationship and diversity of the coat protein (CP) and replicase genes of CMV and ToMV present in our country. To that end, viruses were detected by DAS-ELISA method and selected samples were used in molecular studies conducted by using RT-PCR test. In this study, researchers also tried to determine the phylogenetic diversity of CP and replicase genes of CMV and ToMV isolates from tomato and pepper. This is, to our knowledge, the second phylogenetic analysis conducted in our country on CMV and ToMV isolates from tomato and pepper production areas.

Field surveys

During the field surveys carried out in 10 villages of Hakkari province (Durankaya, Geçitli, Kırıkdağ, Otluca, Üzümcü, and Çimenli villages in the city center of Hakkari; and Geçimli, Taşbaşı, Doğanlı, and Gelinli villages in Çukurca district) in October 2020, 184 leaf samples were collected from tomato (94 samples) and pepper (90 samples) plants that showed symptoms like mosaic, deformation, warty formations, local necrotic lesions, growth reduction, vein banding, wilting, chlorosis and fruit distortion. The areas to be surveyed were selected according to the intensity of vegetable production. Leaf samples were taken from plants showing common symptoms caused by plant viruses, placed in sterile polyethylene bags together with the labels containing information on the land area, brought to the laboratory and stored at -20 ^oC for testing.

DAS-ELISA test

184 leaf samples were subjected to DAS-ELISA test. For serological tests, ELISA kits (Bioreba, Switzerland) that are specific to CMV and ToMV were used. The results were evaluated at 405 nm on the Versamax ELISA reader by using positive and negative controls. Samples with an absorbance value of at least twice the mean absorbance value of the negative control were considered positive.

Preparation of primers and total nucleic acid extraction

The primer pairs were synthesized to amplify a 678 bp fragment of CP and a 318 bp fragment of the replicase gene and these were used during PCR. Primers used in the study are shown in Table 1. RNA extraction from leaf samples of tomato and pepper plants were conducted by using the EURX GeneMATRIX Universal RNA Purification Kit (Poland) as specified in the manufacturer's protocol.

RT-PCR test

5 samples were taken from each group of CMV and ToMV positive samples, and CMV CP and ToMV replicase gene were amplified using RT-PCR method. For RT reaction, a mixture of 20 µl was prepared. The mixture consisted of 6 µl of RNA (10 ng-5 μg), 1 µl of primers, 1 µl of dNTP (10 mM), 4 µl of n5 x cDNA Buffer, 2 µl of DTT (0.1 M), 0.5 µl of RNase Inhibitor (50 U/μ), 1 µl of smART (200 U/μl), and PCR-grade water. It was incubated at 50 °C for 45 minutes and at 85°C for 5 minutes in the PCR device. 25 µl PCR reaction consisted of 3,54 µl of PCR buffer, 3,54 µl of MgCl2 (1,5 mM), 0,35 µl dNTP (0,2 mM), 1.77 µl of each primer (0,3 µM), 0,40 µl of Taq DNA Polymerase (2U), 3 µl of DNA template and 20,96 µl of PCR-grade water. PCR was conducted for 45 seconds at 94°C, for 45 seconds at 57°C (annealing) and for 1 minute at 72°C (elongation). 40 cycles were run followed by final extension for 5 minutes at 72°C and the temperature was decreased to 4°C. The resulting PCR products with 100 bp DNA markers were electrophoresed by 1.5% agarose gel and visualized under ultraviolet light using a Transilluminator (White/2UV) (UVP, USA).

Purification of PCR products

RT-PCR amplified DNA was purified with the MAGBIO "HighPrep™ PCR Clean-up System" (AC-60005) in accordance with the manufacturer's instructions.

Sequencing and sequence comparison

The nucleotide sequences of CMV CP and ToMV replicase gene were determined by using ABI 3730XL Sanger sequencer (Applied Biosystems, Foster City, CA) and BigDye Terminator v3.1 Cycle sequencing kit (Applied Biosystems, Foster City, CA). The obtained DNA sequences were transferred to the BioEdit program and aligned [22]. Hakkari CMV and ToMV isolates were compared with sequences in the NCBI GenBank database. The similarities of the nucleotide sequences in the replicase genes of Hakkari ToMV isolates were determined by making multiple sequence comparisons in the Clustal W program [23].

Phylogenetic analysis

Phylogenetic analysis was performed to determine the relationship between Hakkari CMV and ToMV isolates and related isolates in the NCBI GenBank database. The partial sequence of the CP and replicase genes of CMV and ToMV isolates was compared using Mega (version X) program [24]. With data obtained from the phylogenetic analysis, a phylogenetic tree was constructed using the neighbor-joining method which is applied to Kimura two-parameter algorithm. 1000 repetitive bootstrap analyses were performed in order to statistically determine the accuracy of the generated parentage [25].

Field observation

Field surveys were conducted in Çukurca district and city center of Hakkari province during the 2020 growing season. Suspicious plants thought to be contaminated with CMV and ToMV were photographed in the field (Fig. 1). Samples were collected in accordance with the common symptoms that are known to be caused by these viral agents on tomato and pepper plants.

DAS-ELISA test

DAS-ELISA test showed that out of 94 tomato samples, 34.04% was infected with CMV, 23.40% was infected with ToMV, and 5.31% had mixed infection of ToMV+CMV. In general, it was found that 62.77% of tomato samples were infected with one or more viruses. The rates of exposure to viral infections in tomato production areas were as follows: 100% in Doğanlı, 85.71% in Çimenli, 83.33% in Geçimli, 76.92% in Kırıkdağ, 50% in Üzümcü, 44% in Otluca and 25% in Gelinli. No infection was found in Durankaya. CMV+ToMV mixed infection was detected in 66.66% of the samples from Doğanlı and 4.76% of the samples from Çimenli.

DAS-ELISA test showed that out of 90 pepper samples, 11.11% was infected with CMV, 18.88% was infected with ToMV, and 1.11% had mixed infection of ToMV+CMV. In general, it was found that 31.11% of pepper samples were infected with one or more viruses. The rates of exposure to viral infections in pepper production areas were as follows: 66.67% in Doğanlı, 41.18% in Çimenli, 33.33% in Gelinli, 28.57% in Üzümcü, and 8.33% in Kırıkdağ. No infection was found in the pepper samples form Durankaya and Geçimli. In Otluca, single infection of CMV and ToMV was observed only and the infection rate was found as 45.83%. The rate of CMV infection was highest in Doğanlı and Gelinli with 33.33%, followed by Üzümcü with 21.42%, Çimenli with 11.76%, Kırıkdağ with 8.33%, and Otluca with 4.16%. No CMV infection was found in Durankaya and Geçimli. The rate of ToMV infection was highest in Otluca with 41.66% and Çimenli with 29.41%. No ToMV infection was found in Gelinli, Kırıkdağ, Durankaya and Geçimli. CMV+ToMV mixed infection was observed only in Doğanlı with a rate of 16.66% (Table 2).

Molecular detection

Total nucleic acids were isolated from 10 samples which were found positive for CMV and ToMV. Then these samples were tested for the presence of viral agents along with negative controls using RT-PCR. 678 bp and 318 bp fragments of CMV and ToMV CP and replicase genes were amplified (Fig. 2a, b). Results of these tests have definitely proven the existence of these agents in Hakkari.

Sequencing and phylogeny

DNA sequences of one tomato isolate for CMV and one pepper and three tomato isolates for ToMV were deposited in NCBI GenBank with accession numbers OM418629, OM286731, OM286732, OM286733 and OM286734. Hakkari CMV and ToMV isolates were named as NHP1, NHT1, NHT2, NHT3 and NHD1.

282 bp and 264 bp regions of the CP and replicase genes of Hakkari CMV and ToMV isolates were compared with the appropriate regions of the other CMV and ToMV isolates available in the NCBI GenBank database in order to determine the genetic diversity of Hakkari CMV and ToMV isolates. Partial replicase gene sequences of four Hakkari ToMV isolates were highly conserved with sequence identity of 99.6%-100% at the nucleotide level, respectively. A low variation (up to 0.4%) was found among the four Turkish isolates.

In order to determine the similarity in DNA sequence information, 21 CMV isolates deposited in NCBI GenBank from different countries including 2 whole genome, 5 partial genome and 14 coat protein gene sequences [China (Accession Nos: AJ575589, AJ575589), USA (Accession Nos: AJ271416, U20219), Hungary (Accession Nos: AJ511990, AJ517802), China (Accession No: AJ239098), Australia (Accession No: AJ585518), Germany (Accession Nos: AJ810262, AJ810263), Spain (Accession No: AJ829776), Vietnam (Accession No: AM048831), India (Accession No: DQ285569), Japan (Accession Nos: LC066467, LC066473), Korea (Accession Nos: MG251397, MG251398), Turkey (Accession Nos: MH426932, MZ711455, MZ711456, MZ711457), Italy (Accession No: Y10886)], were compared with Hakkari CMV (Accession No: OM418629) isolate (Table 3).

The sequences of the Hakkari CMV isolate and 21 international isolates were 91.7% to 99.4% identical in the nucleotide sequence of the partial coat protein gene. Thus, it was determined that the partial CP gene of the CMV isolate had a maximum sequence difference of 8.3%.

When the nucleotide sequence similarities were examined, it was seen that the maximum nucleotide sequence identities of Hakkari CMV strain was with isolates from Turkey (Accession Nos: MZ711455, MZ711456, MZ711456) with a rate of 99.2%-99.4%. The minimum nucleotide sequence similarity, on the other hand, was with isolates from Germany (Accession Nos: AJ810262, AJ810263) with a similarity rate of 91.7%-91.9%.

Phylogenetic analyses showed that CMV isolates are categorized in two major groups (I and II) according to their coat protein genes. These two groups are also categorized within themselves (Ia, Ib, IIa, IIb). When we examined the first major group according to the origins of the isolates, we saw that the Turkey strain in this study (Accession No: OM418629) clustered in the first group together with Korea (Accession Nos: MG251397, MG251398), Vietnam (Accession No: AM048831) and USA (Accession No: AJ271416) strains and in the subgroup Ia together with Turkey (Accession Nos: MZ711455, MZ711456, MZ711457), Iran (Accession Nos: LC066467, LC066473), India (Accession No: DQ285569) and USA (Accession No: U20219) strains. In the second major group, another strain from our country, Turkey (MH426932), was in the same subgroup (subgroup IIb) together with Spain (Accession No: AJ829776), Australia (Accession Nos: AJ517802, AJ585518), and Hungary (Accession No: AJ517802) strains.

The branches of the phylogenetic trees created were supported with bootstrap values ranging from 20-100, showing that the evaluations were not random and that most of the branches were statistically supported. The phylogenetic tree of Hakkari NHD1 isolate, which was constructed in accordance with the nucleic acid sequence of the coat protein genes of CMV isolates around the world, is shown in Fig. 3.

In NCBI GenBank, 29 different ToMV isolates were present including 21 whole genome, 3 partial genome and 5 replicase gene sequences. In order to determine the similarity in DNA sequence information, these 29 ToMV isolates from different countries [Japan (Accession Nos: AB083196, AB355139), Australia (Accession No: AF332868), Kazakhstan (Accession No: AJ243571), China (Accession No: FN985165, KY967219, KY967222, KY967225, KY967228, MF002479, MF002480, MF002490), USA (KR537870), Egypt (Accession No: KU321698), South Africa (Accession No: KX711903), Slovakia (Accession No: KY912162), Uganda (Accession No: MG456601), India (Accession No: MH038047), Korea (Accession Nos: MH063883, MH063884, MH063885, MH393621, MH393622, MH393623), South Korea (Accession Nos: MH507165, MH507166), Turkey (Accession No: MK652756), Slovenia (Accession No: MN267904), Russia (Accession No: Z92909)] were compared with the Hakkari ToMV isolates (Table 4).

Hakkari ToMV isolates and 29 international ToMV isolates were found to be 98.1% to 99.6% identical in the nucleotide sequence of the partial replicase gene. Thus, it was determined that the partial replicase gene of the Turkish CMV isolates had a sequence difference of 1.9%.

When the nucleotide sequence similarities were examined, it was seen that the maximum nucleotide sequence identities of Hakkari ToMV strains was with isolates from USA (Accession No: KR537870), Australia (Accession No: AF332868), South Africa (Accession No: KX711903), Kazakhstan (Accession No: AJ243571), Russia (Accession No: Z92909), Slovakia (Accession No: KY912162), Turkey (Accession No: MK652756) and Korea (Accession No: MH393621) with a rate of 99.2%-99.6%. The minimum nucleotide sequence similarity, on the other hand, was with isolates from Korea (Accession No: MH063884), China (Accession No: KY967225) and Japan (Accession No: AB083196) with a similarity rate of 98.1%-98.4%.

Phylogenetic analyses are highly effective in determining genetic relationships between different isolates. Using more and diverse isolates increases the reliability of phylogenetic trees constructed for identification and classification of isolates. Using maximum likelihood method, phylogenetic analyse showed that there are two major phylogenetic clusters (I and II) of the ToMV isolates (Fig. 4). Among these groups, group I is divided into subgroups Ia and Ib. Of these, Group I includes the majority of isolates. ToMV isolates from Turkey are in Group I. A phylogenetic tree based on partial replicase gene sequences revealed that the ToMV isolates used in this study belonged to Group Ia by clustering with USA (Accession No: KR537870) isolate. The second group, on the other hand, included strains from Vietnam (Accession Nos: MH063883, MH063884, MH063885, MH393622, MH393623). The branches of the phylogenetic trees created were supported with bootstrap values ranging from 20-86, showing that the evaluations were not random and that most of the branches were statistically supported.

CMV and ToMV are plant viruses that cause diseases in tomato and pepper plants all around the world, leading to economic losses. The presence of CMV and ToMV viral agents in these plants in our country has previously been detected. However, there is no study in the literature on the existence, prevalence and economic impacts of these agents in Hakkari province. In this study, we tried to fill this gap in the literature by conducting a survey study in different regions of Hakkari province and managed to detect for the first time the CMV and ToMV agents in the samples collected from these areas by DAS-ELISA method. We conducted PCR tests and phylogenetic analyses on the samples that were serologically found to be positive for these viruses and found which groups these samples belong to.

However, the number of molecular studies and phylogenetic analyses on CMV is limited. In a study conducted in Çanakkale province, 6 of 22 plants collected from cowpea and bean cultivation areas and subjected to DAS-ELISA test were found to be infected with CMV. CP gene of the CMV isolates was amplified, the RT-PCR products of the selected CMV isolates were purified and partial base sequencing was performed. Phylogenetic analyses determined that CWP17 isolate was in subgroup Ib. Similarly, a close phylogenetic relationship was found between CMV isolates from melon plants in our country and other isolates in subgroup Ib that were obtained from various plant sources in Turkey, Thailand, India and China [38, 39]. In another study, 66 spinach samples were collected from the spinach production areas in Çanakkale province, tested by DAS-ELISA method and CMV was detected in 4 of the samples. One of the infected isolates was randomly selected for molecular characterization and the phylogenetic analysis conducted determined that this isolate was in subgroup Ia [40]. Karanfil [31] conducted survey studies in tomato production areas in Marmara region and applied DAS-ELISA test on 113 tomato plants that showed virus and virus-like symptoms. Of 34 plants that were found to have CMV infection, 10 were selected for amplification of CP and movement protein (MP) genes by RT-PCR. Phylogenetic analyses showed that tomato CMV isolates were closely related for both gene regions. CMV isolates in Marmara region were found to be in subgroup Ia. Molecular studies of CMV showed that isolates in subgroup Ia are more common.

Borah et al. [41] detected the presence of CMV in Capsicum chinense Jacq. in India by RT-PCR and prepared the first report on molecular characterization of the virus. Phylogenetic analysis of the 593 bp coat protein gene of CMV demonstrated that the isolate studied was closely related to the African isolates in subgroup Ib and other global isolates. Mishchenko et al. [42] deposited the 443 bp CP gene sequence of the CMV isolate P-EP-Ukr-19, which infects Echinacea purpurea (coneflower) plants in Ukraine, in the NCBI GenBank with the accession no. MT978189. Phylogenetic analysis revealed that P-EP-Ukr-19 clustered in subgroup IB with a nucliotide sequence similarity of 93.7–99.7%. In a study conducted in Colombia, leaves of three chili pepper varieties (tabasco, cayenne and habanero) with viral symptoms were sampled, total RNA was purified and a fragment of CP of CMV was amplified by RT-PCR. It was then sequenced and analysed bioinformatically. 37 of 71 chili pepper samples (52.1%) were found positive for CMV. CMV sequence analysis showed the maximum identity (98.5%) of the isolates in the study was with a CMV isolated from bananas from Ecuador. CP analysis of CMV also demonstrated that it can be transmitted by the species Aphis gossypii [43]. CMV is one of the most harmful viruses causing disease outbreaks in agricultural products all around the world [21]. Studies to be conducted in other countries will be helpful in understanding the epidemiology of CMV and in designing an appropriate management program [41].

A study conducted in the Mediterranean region of Turkey showed that tomato CMV isolate clustered in subgroup Ia [44]. Hakkari CMV isolates in the present study also clustered in subgroup Ia. It is suspicious that the CMV isolates from our country cluster in the same group with the isolates from Iran in the phylogenetic classification. However, they also show similarity with the USA isolate, suggesting that geographical origins may not be very important in this case.

Studies conducted in our country on the genetic diversity of ToMV are limited. Yılmaz and Sipahioğlu [45] carried out a large-scale survey study to determine the presence and prevalence of ToMV, TYLCV, TSWV, ToRSV and PVY in tomato production areas in Diyarbakır province. In multiplex RT-PCR tests carried out to detect the presence of ToMV and PVY, DNA bands of 621 bp and 480 bp were obtained from infected plant samples, respectively. The nucleotide sequences of four isolates were deposited in the NCBI GenBank with the accession numbers MK992250, MK992251, MK992252 and MK992253. The ToMV isolates in the study were 99.2–99.8% identical with other ToMV isolates in GenBank. In the phylogenetic classification, DT1 and DT2 isolates clustered in the same group with the USA (KR537870) isolate. The fact that Hakkari ToMV isolates were also in the same group with the USA (KR537870) isolate suggests that there is not much difference according to geographical origin and hosts, as in CMV.

Duarte et al. [46] isolated ToMV from Hemerocallis sp. (tobamo-H) and Impatiens hawkeri (tobamo-I). They performed RNA extraction, RT-PCR amplification, CP gene sequencing and phylogenetic to compare and characterize homologous sequences. The isolates obtained were deposited in NCBI GenBank with accession numbers DQ230836 and AY063743 and they showed 98,5% nucleotide sequence identity with ToMV. Regarding Tobamo-H, 100% identity was found with ToMV from Australian and Peruvian tomatoes. It was also observed that variences in hosts did not lead to a difference in similarity rates. Fillmer et al. [35] deposited the first whole genome sequence of a ToMV isolate from jasmine in the NCBI GenBank with the accession number KR537870. In phylogenetic analysis, they found 99% identity with the reference ToMV sequence. Arinaitwe et al. [37] performed the first study on whole genome molecular characterization of ToMV isolates from Uganda, as well as from the East and Middle East. They showed that the isolate from Africa was 98–100% identical to the ToMV strains especially from China, Japan and Germany, and that this strain was a strain of the same virus species seen in Asia and Europe. Bae et al. [47] amplified the ToMV-specific RdRp region from tomato and pepper and deposited the ToMV strains isolated from tomato (Accession No: MH393621), pepper leaves (Accession No: MH393623), and chili seeds (Accession No: MH393622) in NCBI Genbank. They found that these isolates had 38–43 nucleotide differences and 99% similarity to the Queensland ToMV (Accession No: AF332868) isolate. Four Hakkari ToMV isolates in the present study were also found to be identical with Queensland ToMV isolats at rates ranging from 99.2–99.6%. In order to investigate the host range and genetic variability of ToMV in Iran, Aghamohammadi et al. [18] collected 849 symptomatic samples from different vegetables and weeds and they found ToMV infection in 45 of the samples. They also performed molecular characterization of the ToMV CP gene. An analysis of the biological characteristics of the selected ToMV isolates revealed three pathological groups, indicating the presence of different ToMV populations in Iran. It was found that Iranian isolates from group II (NL93 and NO1) did not infect solanecous host plants, while four Iranian ToMV isolates in group I, together with other global isolates, infected solanecous host species.

Hakkari ToMV strains from our country clustered in the same group with strains from other countries, except for those from Vietnam. This demonstrated that Hakkari ToMV isolates were independent of geographical origin, suggesting that strains from other countries were introduced into our country. This may have happened due to the exchange of plant material between countries. It has been suggested that plant-virus interaction may play an important role in the genetic differentiation of tobamoviruses [48]. The variation potential of ToMV may lead to outbreaks of virulent and resistance-breaking strains. Knowledge of genetic diversity and population structure is a prerequisite for the successful development of disease resistance against viruses [49]. The development of successful techniques to control ToMV infection is urgently needed for the protection of agriculture and food production [2, 12, 13].

The present study was carried out to detect CMV and ToMV in tomato and pepper production areas in Hakkari and to compare the CMV and ToMV isolates in Turkey with other isolates from different parts of the world by using CP and replicase genes of the viruses. The fact that, in the surveyed areas, the use of pesticides is not common and that local seeds are used was noted. Since these viral agents can be carried mechanically or by graft, vector, seed and pollen, it would be appropriate to comply with the internal quarantine rules. The number of isolates used in phylogenetic analyses in this study is sufficient. The fact that the compared isolates were from different geographical regions of the world increased the diversity of CMV and ToMV isolates. Whole or partial genome sequences are generally used to group isolates of a particular virus. The availability of whole genome sequences of CMV and ToMV isolates in NCBI GenBank allowed us to use the CP and replicase gene alone. The data presented here, along with other available sequences, provides useful information on CMV and ToMV isolates in Turkey. These findings are particularly relevant to the recent reports on the presence of CMV and ToMV in vegetables in Turkey. CMV CP gene and ToMV replicase gene are highly conserved in different hosts. For a more efficient grouping of CMV and ToMV isolates, studies on various isolates combined with their biological characterization and further analyses with different genomic regions or whole genome sequences of these isolates are needed.

Authors’ contributions All authors contributed to the study conception and design. Material preparation, data collection and analysis were performed by Nevin AKDURA and Handan ÇULAL KILIÇ. All authors read and approved the final manuscript.

Funding This work was supported by Scientific Research Projects Unit of Hakkari University, Project number: EF2021BAP1.

Data availability All data will be available on request.

Conflict of interest Author Nevin AKDURA and Handan ÇULAL KILIÇ have no relevant financial or non-financial interests to disclose. The authors declare that they have no conflict of interests.

Consent to participate All authors read and approved the manuscript.

Consent to publish All authors read and approved the manuscript.

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Table 1

Primer pairs used in the molecular characterization of CMV and ToMV isolates
Primer	Sequence	Specific Gene	Reference
CMV_Forward	5´-TTGAGTCGAGTCATGGACAAATC-3´	Coat protein gene	[20]
CMV_Reverse	5´-AACACGGAATCAGACTGGGAG − 3´	Coat protein gene	[20]
ToMV_Forward	5´-CGAGAGGGGCAACAAACAT − 3´	Replicase gene	[21]
ToMV_Reverse	5´-ACCTGTCTCCATCTCTTTGG − 3´	Replicase gene	[21]

Table 2

Places where the tomato and pepper samples were collected, number of samples taken and presence of the viruses
District	Collected Samples Tomato	Infected Samples			Infection rate (%)	Collected Samples Pepper	Infected Samples			Infection rate (%)
District	Collected Samples Tomato	CMV	ToMV	CMV + ToMV	Infection rate (%)	Collected Samples Pepper	CMV	ToMV	CMV + ToMV	Infection rate (%)
Çimenli	21	8	9	1	85.71	17	2	5	0	41.18
Otluca	25	0	11	0	44.00	24	1	10	0	45.83
Üzümcü	16	7	1	0	50.00	14	3	1	0	28.57
Doğanlı	6	2	0	4	100.00	6	2	1	1	66.67
Gelinli	4	0	1	0	25.00	3	1	0	0	33.33
Kırıkdağ	13	10	0	0	76.92	12	1	0	0	8.33
Durankaya	3	0	0	0	0.00	4	0	0	0	0.00
Geçimli	6	5	0	0	83.33	10	0	0	0	0.00
Total	94	32	22	5	62.77	90	10	17	1	31.11

Table 3

List of the CMV isolates used in this study
Accession Number	Country	CMV Gene Region	Host	Isolate	Origin	Reference
AJ239098	China	coat protein	tobacco	YN	China	[26]
AJ271416	USA	coat protein	-	-	USA	unpublished
AJ511990	Hungary	partial genome	Nicotiana glutinosa	Ns	Hungary	[27]
AJ517802	Hungary	partial genome	Raphanus sativus	Rs	Hungary	[27]
AJ575589	China	coat protein	Brassica juncea	ZJZC-CMV	China	unpublished
AJ585518	Australia	partial genome	rockmelon	237	Australia	unpublished
AJ810262	Germany	coat protein	bean	RT144	Germany	[28]
AJ810263	Germany	coat protein	cornflower	RT54	Germany	[28]
AJ829776	Spain	partial genome	melon	MAD99/1	Spain	[29]
AM048831	Vietnam	coat protein	Nicotiana tabaccum	VN-Bavi	Vietnam	unpublished
DQ285569	India	coat protein	Piper longum	-	India	unpublished
LC066467	Japan	complete sequence	Raphanus sativus	IRN-REY4	Iran	[30]
LC066473	Japan	complete sequence	Raphanus sativus	IRN-REY10	Iran	[30]
MG251397	Korea	coat protein	Gentiana scabra	CMV-YD2	South Korea	unpublished
MG251398	Korea	coat protein	Gentiana scabra	CMV-YD3	South Korea	unpublished
MH426932	Turkey	coat protein	canola	KNLc32	Turkey	Unpublished
MZ711455	Turkey	coat protein	tomato	TKR-81	Turkey	[31]
MZ711456	Turkey	coat protein	tomato	EDR-91	Turkey	[31]
MZ711457	Turkey	coat protein	tomato	EDR-97	Turkey	[31]
OM418629	Turkey	coat protein	tomato	NHD1	Turkey	this study
Y10886	Italy	coat protein	tomato	Tfn	Italy	unpublished
U20219	USA	3a protein and coat protein	tomato	Ixora	USA	[32]

Table 4. List of the ToMV isolates used in this study

Accession Number		Country	ToMV Gene Region		Host		Isolate		Origin		Reference
AB083196	Japan		complete genome	-		Japan		L11A-Fukushima		[33]
AB355139	Japan		complete genome	tobacco		Japan		L11Y		[34]
AF332868	Australia		complete genome	tomato		Australia		Queensland		unpublished
AJ243571	Kazakhstan		complete genome	-		Kazakhstan		K1		unpublished
FN985165	China		complete genome	-		China		XJT-1		unpublished
KR537870	USA		complete genome	jasmine		USA		99-1		[35]
KU321698	Egypt		complete genome	tomato		Egypt		AH4		unpublished
KX711903	South Africa		complete genome	tomato		South Africa		mutoko		[19]
KY912162	Slovakia		complete genome	tomato		Slovakia		SL-1		[36]
KY967219	China		complete genome	tomato		China		SX		direct submission
KY967222	China		complete genome	tomato		China		Tai'an		direct submission
KY967225	China		complete genome	tomato		China		LY		direct submission
KY967228	China		complete genome	tomato		China		HHHT		direct submission
MF002479	China		complete genome	tomato		China		HHHT4		direct submission
MF002480	China		complete genome	tomato		China		HHHT3		direct submission
MF002490	China		complete genome	tomato		China		JX		direct submission
MG456601	Uganda		complete genome	Solanum		Uganda		ToMV-Ug		[37]
MH038047	India		partial replicase gene	tomato		India		B10 Jodhe		direct submission
MH063883	Korea		partial replicase gene	tomato		Vietnam		BaoLoc		unpublished
MH063884	Korea		partial replicase gene	chili pepper		Vietnam		CPS		unpublished
MH063885	Korea		partial replicase gene	bell pepper		Vietnam		DonDuong		unpublished
MH393621	Korea		partial genome	tomato		Vietnam		ToMV_BaoLoc		unpublished
MH393622	Korea		partial genome	chili pepper seed		Vietnam		ToMV_chili		unpublished
MH393623	Korea		partial genome	bell pepper		Vietnam		ToMV_DonDoung		unpublished
MH507165	South Korea		complete genome	tomato		South Korea		GW1		unpublished
MH507166	South Korea		complete genome	tomato		South Korea		GW2		unpublished
MK652756	Turkey		partial replicase gene	eggplant		Turkey		TRAntToMVEgp		Unpublished
MN267904	Slovenia		complete genome	tobacco		Slovenia		ToMV_WW17-I-f16-18-Nocc-pos2		direct submission
OM286731	Turkey		partial replicase gene	pepper		Turkey		NHP1		this study
OM286732	Turkey		partial replicase gene	tomato		Turkey		NHT1		this study
OM286733	Turkey		partial replicase gene	tomato		Turkey		NHT2		this study
OM286734	Turkey		partial replicase gene	tomato		Turkey		NHT3		this study
Z92909	Russia		complete genome			Russia		K2 (Kazakhstan strain)		unpublished

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Genetic Diversity of CMV and ToMV Isolates in Tomato and Pepper Production Areas in Hakkari, Turkey

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