SJA_41_4_1494-1506

Introduction

Tomato (Solanum lycopersicum) is an economical important vegetable crop that is consumed globally belongs to the family Solanaceae (Ahringer, 2006; Li et al., 2022; Wang et al., 2024). The cultivated tomato and its wild relative are diploid (2n=24) with similar chromosomes number of 12 (Dhaliwala et al., 2020). Tomato is a model plant for research on fruit developmental processes because of its short lifespan, succulent fruit and moderate genome size 900 Mb has been sequenced (Sato et al., 2012; Shahwar et al., 2023; Shirasawa and Ariizumi, 2024). It is an important commercial vegetable crop, grown in tropical, subtropical and temperate regions of the world, ranking second after potato in area and production worldwide (Lucier et al., 2000; Minfal, 2007; Gerhardt et al., 2009; Bakht and Khan, 2014; Iftikhar et al., 2021).

In Pakistan, it is grown both as daily based use vegetable and as commercial crop (Ayaz et al., 2015). Due to the natural variations in climatic conditions of different ecological zones of Pakistan, the cultivation and production of tomato spread over nearly the whole year in the country. Thus, its fresh fruit remains available in the market throughout the year. Moreover, it is a major source of health beneficial antioxidants such as vitamin C, vitamin E, carotenoids, lycopene, ascorbic acid and polyphenolic compounds (Zhu et al., 2018). These compounds play a vital role in human nutrition and contribute significantly to the prevention of diseases such as cancer, diabetics, cardiovascular and neurodegenerative diseases (Giovannucci, 1999; Shakoor et al., 2010).

In Pakistan, tomato was cultivated on an area of 68863 hectares with a production of 762736 tons and average yield of 11.076 tons per hectare (FAOSTAT, 2023). In Pakistan its average per hectare yield (11.076 ton) is very low as compare to other developed countries (Arooj et al., 2019). The major causes for this low yield include fluctuations in climatic conditions and diseases, as it is pruned to more than 200 diseases including viral, bacterial, fungal and nematode (Iquebal et al., 2013; Junaid et al., 2021; Khan et al., 2023; Lata et al., 2024). Tomato Yellow Leaf Curl Virus (TYLCV) is a member of the genus Begomovirus family Geminiviridae (Kumar et al., 2013; Xie et al., 2013). This TYLCV is one of the most severe pathogen causing overwhelming damage to tomato crop in tropical and subtropical regions of the world (Li et al., 2022). Due to fast spreading of this pathogen, the disease is now a serious problem in more than 30 countries of the world and the major affecting regions are Mediterranean basin, Southern Asia, Africa and South, Central and North America (Glick et al., 2009; Yan et al., 2021).

These begomoviruses are among the genera possessing both mono-partite (DNA-A) and dipartite (DNA-A and DNA-B) genome organization that infect most dicotyledonous plant species including tomato (Gronenbor, 2007; Briddon et al., 2008; Devendran et al., 2022). Both the mono-partite and dipartite are mainly composed of single-stranded (ss) DNA genome (Miozzi et al., 2014). Their genome size ranges from 2.5 to 3 kb and encodes viral factors essential for viral replication in the nuclei of the infected cells (Varma and Malathi, 2003; Baltes et al., 2014; Fiallo-Olivé et al. 2020). Its genome encodes 6-7 open reading frames and one of them codes for Coat Protein (CP) which indicates structure mass of viral particle (Glick et al., 2009). TYLCV is transmitted by a vector insects called as whitefly (Bemisia tabaci) and silver leaf whitefly (Bemisia argentifolii).

Early studies of diagnosing geminiviruses were mostly based on observations of symptoms which have been of limited use as symptoms vary greatly as function of soil, growth conditions and climate. However, the advent of DNA sequences has proved to be a tool of choice, for robust and accurate identification of viruses, their evaluation and relationship with other strains of TYLCV isolates (Kheyr-Pour et al., 1991; Noris et al., 1994; Rochester et al., 1994; Crespi et al., 1995; Hong et al., 1995). These high-throughput technologies allow studying the changes in gene expression upon virus infection at the genome level and further proved as powerful tools in evaluating the functions of these genes during infections (Sato et al., 2010).

The objective of this research was to identify the type and problem of virus occurring in local tomato and hybrid varieties in District Mansehra through high-throughput technology and find the solution. As result of this research, a new strain of virus was isolated and identified as TYLCV and named as Mansehra sample 1 (MS1) through analyzing the nucleotide sequences of TYLCV in tomato crop, grown in Mansehra Pakistan, and is reported for the first time.

Materials and Methods

Study area and samples collection

Leaf samples were collected from six tomato varieties and hybrids showing typical tomato yellow leaf curly virus (TYLCV) symptom, grown in five different areas of district Mansehra Khyber Pakhtunkhwa Pakistan which include Baffa, Bajna, Murghuzar, Shinkiari and Gandia. The tomato varieties and hybrids were named as Larica, Picasso, 1225-F1, Yaqui, 2202 and 1554 (Table 1).

Table 1: Names and distributors of tomato varieties used in this experiment.

Variety name	Distributors in Pakistan	Seed Source
Larica	Kisan agro, Saadat Zari	syngenta
Picasso	Mehar Mohd. Din and sons	Haris Morin
1225 F1	IcI Pakistan	Advanta
Yaqui	Mehar Mohd. Din and sons	Seminis
2202	Phool Zari Mansehra	unknown
1554	Akora seeds	Agritip Germany

The samples were brought to laboratory of the Department of Genetics, Hazara University, Mansehra and stored at −80 ℃ until further processing to be carried out in the same laboratory.

DNA extraction and amplification of TYLCV gene through PCR

DNA was extracted from the collected samples using CTAB method (Porebski et al., 1997). About 100 mg of leaf of each sample was taken. The leaf samples were crushed in 500 µl 10% CTAB extraction buffer. After crushing the mixture was centrifuged for 5 minutes at 14000 rpm. The pellet was discarded and the supernatant was taken with 50 µl of sodium acetate with final concentration of 0.3M and 500 µl isopropanol, the tubes were inverted several times. The mixture was then centrifuged at 14000 rpm for 10 minutes. The supernatant was discarded and 500 µl of ethanol (80% v/v) was added to the pellet and then centrifuged for 3 minutes at 14000 rpm. Then the pellet was air- dried over night and was dissolved in 50 µl of TE buffer. The DNA quality was checked on 1% agarose gel.

Four sets of specific primers including PAR (Bela-ong and Bajet, 2007), PAL (Bela-ong and Bajet, 2007), BEG (Wyatt and Brown, 1996) and WTG (Bhyan et al., 2007) were used to amplify the TYLCV. For simplicity purpose, the six samples studied in the present study were named as Mansehra sample 1 (MS1), Mansehra sample 2 (MS2), Mansehra sample 3 (MS3), Mansehra sample 4 (MS4), Mansehra sample 5 (MS5) and Mansehra sample 6 (MS6). Where MS1 is Larica, MS2 is Picasso, MS3 is 1225 F1, MS4 is Yaqui, MS5 is 2202 and MS6 is 1554 tomato genotype.

The PCR products were analyzed on 1% agarose gel. Briefly, 3µl loading dye was mixed with 5µl of PCR product for each sample and then the samples were loaded in the wells of gel. A DNA ladder was also run along with the samples. The gel was run for a period of 40 minutes at 70V and then visualized under UV using gel doc system.

Cleaning and sequencing of the PCR product

TAINGEL kit was used for cleaning of PCR product as per instructions given in the kit manual. Briefly, band from gel was cut and 200 µl PN buffer was added and kept 50 min. Once completely dissolved, it was centrifuged for two minutes at 8000 rpm. The supernatant was discarded and the spin column was washed two times with 500 µl PW buffer. The empty column was centrifuged for two minutes at 8000 rpm and then dried for 20 minutes at room temperature. The cleaned eppendorf tubes were properly labeled and the columns were placed in them along with 30 µl EB buffer and stored for 10 minutes at room temperature and then centrifuged for two minutes at 8000 rpm. The cleaned PCR products of these six samples were sent to the sequencing company, MACROGEN Inc. Korea. The first 30 nucleotides were excluded from analysis because of unclear peaks. The nucleotide sequence of clear peaks up to 430 nucleotides were considered for further analysis and the sequence after 430 nucleotides was removed by trimming.

Analysis of the gene sequences

To check the similarity and differences among the virus strains the results of TYLCV sequencing were analyzed using bioinformatics tools. The nucleotide sequences were identified with online available BLASTn program and online ClustalW2 tool was used for multiple sequence alignments.

Results

Symptoms of TYLCV in diseased tomato

Leaf samples of six tomatoes varieties and hybrids collected from five locations in district Mansehra of Pakistan showed typical TYLCV symptoms (Figure 1A-F). The samples of Larica and Picasso varieties (Figure 1 A-B) showed upward curling and uniform chlorosis of the leaves that eventually resulted in color change from green to yellow of the leaves. Other tomato varieties (Figure 1 C-F) showed symptoms of puckering, chlorotic leaf margins and also reduction in leaf size. The severely affected plants remained stunted with growth retardation and produced no fruit or very small sized fruits.

Cleaning of coat protein gene of TYLCV and amplification of PCR

Figures 2 and 3 show PCR amplification products resolved in agarose gel. Among the 4 specific primers pairs, WTG primers pair showed positive results in two tomato samples of tomato i.e. MS1 and MS2 (Table 2) showed clear bands of size 1.5 kb (Figure 2), while no band was observed for rest of the samples i.e., MS3, MS4, MS5 and MS6 (Figure 2).The PCR amplified DNA fragments of the respective samples were cut under UV light from the gel. The gel electrophoresis showed clear band for the respective sizes of 1.5 kb (Figure 3). The rest of the tomato samples (MS3, MS4, MS5 and MS6) did not produce any PCR product with any of the 4 sets of specific primers, revealing the absence of WTGs in these samples.

Table 2: Molecular detection of TYLCV using different PCR primers

Primer	Locus identified by PCR in the samples
Primer	Larica	Picasso	1225	Yaqui	2202	1554
PAR	-ve	-ve	-ve	-ve	-ve	-ve
PAL	-ve	-ve	-ve	-ve	-ve	-ve
BEG	-ve	-ve	-ve	-ve	-ve	-ve
WTG	+ve	+ve	-ve	-ve	-ve	-ve

equence analysis of TYLCV gene

MS1 sample: The sequencing results and subsequent alignment results of MS1 are shown in Figures 4, 5. Eztaxon (online search tool) provided the nucleotides with best peaks. The resulted new sequence was awarded accession number (BankIt2535530 sample OM100018) by the GenBank. The sequence of TYLCV gene showed high similarity index with a class of viruses which infect tomato and other plant species (Table 3). The highest similarity of 96% was observed with Tobacco curly shoot virus isolate TbSCVCN (KM383753.1) isolated from Bangladesh Chittagong. This was followed by tomato leaf curl Karnataka virus isolate (KM383757.1) isolated from Bangladesh, 94%, Tobacco curly shoot virus

isolate TbCSV (KM383754.1) 92% isolated from Bangladesh and Tomato leaf curl Karnataka virus isolate (JX987088.1) isolated from Lucknow India, Papaya leaf curl virus AV1, AV2, AC1, AC2, AC3, AC4, AC5 genes and IR region (Y15934.1) isolated from India, Tobacco curly shoot virus isolate FB- 01 segment DNA-A, (JQ733557.1) isolated from India with 91%. Tomato Leaf Curl Virus KM383758.1 and Radish Leaf Curl Virus EU19494.2 (90 %), Papaya LCV JN135233.1 (89%), Tomato leaf curl Oman virus complete sequence. clone Tom 80 Tomato Leaf Curl Virus HG969206.1 (88 %), Croton yellow vein mosaic virus isolate CrYVMV_Del-Cr from India JN817516.1 (87%), Croton yellow vein mosaic virus isolate CYVMV-Del-Turnip, complete genome (KF888655.1) 87%, tomato leaf curl Bangladesh virus ToLCBV-[BD:Cox:01:15:Tom:06], complete genome (KM383765.1) 87% and Euphorbia leaf curl isolates Shandong. (KC852148.1) 87% similarity. However, the least similarity was observed with Chili leaf curl Ahmedabad Virus-India (KM880103.1) isolated from India with 86% similarity.

Phylogenetic analysis of MS1 gene

To assess the genetic variability of MS1 gene, seventeen isolates of different types of plant viruses on the basis of similarity (16 selected from NCBI and MS1 from our samples) were used in the phylogenetic analysis. The analysis was conducted by using full length nucleotide sequence of each isolate of plant viruses. The dendrogram obtained from phylogenetic analysis using UGENE revealed 4 clusters (Figure 6).

Cluster 1 showed single isolate of TYLCV gene which was obtained during the present study. In cluster 2 a single isolate of Tobacco curly shoot virus isolate TbSCV- (KM383753.1) isolated from Bangladesh Chittagong showed 96% similarity with that of TYLCV isolated in this study. Cluster 3 comprised of seven isolates of viruses which showing different similarity with that of TYLCV. This cluster consists of Tomato leaf curl Oman virus complete sequence clone Tom 80 (HG969206.1) 88%. Croton yellow vein mosaic virus isolate CrYVMV_Del-Cr from India, (JN817516.1) 87%, Croton yellow vein mosaic virus isolate CYVMV-Del-Turnip, complete genome (KF888655.1) 87%, Tomato leaf curl Karnataka virus isolate Lucknow segment DNA-A, (JX987088.1) 91% isolated from Lucknow India. Tomato leaf curl Bangladesh virus isolate ToLCBV-[BD:Syl:01:21:Tom:06],(KM383758.1) 87% isolated from Bangladesh. Tomato leaf curl Bangladesh virus isolate ToLCBV-[BD:Cox:01:15:Tom:06], complete genome (KM383765.1) 87% and Tomatoleaf27curl Karnataka virus isolate begomovirus genome assembly, segment: I (KM383757.1) isolated from Bangladesh with 94% similarity with our sample. Cluster 4 is composed of eight isolates of virus which have different similarity level with our amplified sequence of TYLCV. This cluster include Papaya leaf curl virus, such as AV1, AV2, AC1, AC2, AC3, AC4, AC5 genes and IR region (Y15934.1) 91% isolated from India. Papaya leaf curl virus segment DNA-A, (HM143914.1) 87%. Tobacco curly shoot virus isolate TbCSV-CN [BD:Raj:01:24:Tom:10], (KM383754.1) isolated from Bangladesh. 92%, Chili leaf curl Ahmedabad virus–India [India/Ahmedabad/2014], (KM880103.1) 88% isolated from India. Radish leaf curl isolates Pusa Bihar, (EU194914.2) 90%. Euphorbia leaf curl isolates Shandong. (KC852148.1) 87%, Papaya leaf curl isolate Lucknow segment DNAA, (JN135233.1) 89%. Tobacco curly shoot virus isolate FB-01 segment DNA-A, and (JQ733557.1) isolated from India with 91% similarity (Table 3).

Discussion

Tomato (Solanum lycopersicum L.) is an important vegetable crop of Solanaceae family after potato that is grown throughout the world (Bakht and Khan, 2014; Asif et al., 2022; Wang et al., 2024). It is used in various forms such as fresh in cooking and salad as well as in processed form such as tomato ketch up (Khan et al., 2021). Use of tomato as a diet can also help in preventing several diseases such as diabetes, cancer and cardiovascular diseases as tomato is a main source of antioxidants such as carotenoids, lycopene, vitamins A, C, E, K, and phenolic compounds that play a vital role in human diet and also helps in prevention of these diseases (Shahidi et al., 2011; Bacanli et al., 2017; Coelho et al., 2023). Although, tomato is an important vegetable crop of Pakistan and is widely grown as a daily based used vegetable in most parts of the country, still its yield and fruit quality is greatly influenced by over 200 diseases caused by fungi, bacteria and viruses (Wani, 2011). Among them, viral diseases are the most important biotic constraints that affect fruit quality and reduce yield (Kumar and Kumar 2018). The most noticeable viral diseases that influence tomato crop include Tomato golden mosaic virus, Tomatochlorotic mottle virus, Tomato rugose mosaic virus (Abhary et al., 2007), Tomato spottedwilt virus (German et al., 1992), Tomato mottle virus (Simone et al., 1990), Pepino mosaicvirus (Vander-Vlugt et al., 2000). Most of the viral diseases that infect tomato crop belong to the family Geminiviridae. Tomato yellow leaf curl virus (TYLCV) also belongs to this family and is responsible for up to 100% yield loss in several tropical and subtropical regions (Pandey et al., 2010; Majid et al., 2023). Globally tomato yellow leaf curl virus disease is among the common devastating plant disease observed in several countries of the world (Kumar and Kumar 2018). Tomato yellow leaf curl virus is the most damaging virus for global tomato production and has become one of the most studied plant virus due to its vast economic importance (Li et al., 2022). This disease was first identified in Israel in 1960 and then became the most notorious disease in most regions of the world (Czosnek and Laterrot, 1997). Naturally, TYLCV is transmitted through whitefly (Bemisia tabaci Gennadius Hemiptera: Aleyrodidae) with monopartite ssDNA (Moriones and Navas-Castillo, 2000). Therefore, the use of molecular techniques is essential for proper detection and analysis of the viruses and effective management of the disease. Although, virus specific drug development is a costly and time consuming process, but it is the need of the day for effective management of the disease and the first step towards drug development is the in silico identification of reliable inhibitors (Prajapat et al., 2011; Prasad et al., 2012; Shikhi et al., 2013).

The present study was conducted with the objective to detect the presence of TYLCV in tomato fields of Mansehra by analyzing the tomato samples through high-throughput analysis. During this study, the samples were collected on the basis of symptoms like inward curling, uniform chlorosis and yellowing of the leaves, stunted growth and puckering from five locations (Baffa, Bajna, Murghuzar, Shinkiari and Gandia) in Mansehra District of Hazara Division Khyber Pukhtunkhwa Province and evaluated for the presence of TYLCV. The samples of Larica and Picasso (Figure 1A, B) showed the symptoms of upward curling and uniform chlorosis of the leaves and resulted in the yellowing of the leaves as reported by Bela-ong and Bajet. (2007); Zambrano et al. (2007); Ghimirey et al. (2024). While other tomato plants (Figure 1C-F) showed puckering, chlorotic leaf margins and reduced leaf size. Severely affected plants were stunted due to retardation of growth and produced no fruit, or in some cases produced small size fruits as reported by several other investigators (Ajlan et al., 2006; Zambrano et al., 2007; Petrov et al., 2024). Although all the tomato samples showed the typical symptoms of tomato TYLCV but among the four specific primers pairs, WTG primers pair showed positive results in only two samples of tomato i.e. MS1 and NS2 (Table 2) and showed clear bands of size 1.5 kb (Figure 2) while no band was observed for the other four samples i.e., MS3, MS4, MS5 and MS6 (Figure 2). The rest of the tomato samples (MS3, MS4, MS5 and MS6) did not produce any PCR product with any of the 4 sets of specific primers, revealing the absence of WTGs in these samples or strainal differences in the causal virus due to changes at nucleotide level (Verma et al., 2016). Yet the possibility of leaf curl symptoms typical of geminivirus infection cannot be ignored. The WTGs may be present in the leaves samples but they could not detect by PCR due to contaminants in the total nucleic acids used as templates (Bela-ong and Bajet, 2007).

From the pairwise comparison and correlation analysis, it was observed that tomato TYLCV MS1 has maximum 96% similarity with Tobacco curly shoot virus isolate TbCSV-CN [BD:Chi:02:14:Tom:06] (KM383753.1) isolated from Bangladesh Chittagong (https://www.ncbi.nlm.nih.gov/nuccore/KM383753.1/) followed by 94% similarity with Tobacco curly shoot virus isolate TbCSV-CN[BD:Cox:02:16:Tom:06] segment DNA-A, complete genome accession number (KM383757.1) isolated from Bangladesh submitted to NCBI by Akhond et al. (2014) (https://www.ncbi.nlm.nih.gov/nuccore/KM383757.1/) and the minimum similarity of 86% was observed with Chilli LCV(KM880103.1) Ahmedabad-India virus submitted by Chahwala et al. (2014) to NCBI (https://www.ncbi.nlm.nih.gov/nuccore/KM880103.1/) (Table 3 and Figure 5).

Our results confirmed the association of a begomovirus with TYLCV. Based on high nucleotide sequence identity (96% for DNA-B and 94% for DNA-A) with the strains of TbCSV-CN and the demarcation criteria in species demarcation (Fauquet et al., 2008), the MS1 sample of Begomovirus isolated from diseased tomato is considered as a variant of TbCSV-CN and we suggest the name Mansehra sample 1 (MS1).

The sequence comparison was also estimated through cluster analysis to observe the difference among different isolates of plant viruses. The same method was also used by Ghanim and Czosnek (2000) studying maize streak viruses coat protein gene in sugarcane. The Clustal W series of programs are mainly used in molecular biology for carrying out automatic multiple alignments of nucleotide or amino acid sequences and for preparing phylogenetic trees (Chenna et al., 2003). The MS1 sample TYLCV was compared with 16 other viruses isolates by sequence alignments. The resulted phylogenetic tree separated the 17 isolates into 4 clusters including our isolate (Figure 6). However, our sample was grouped in a separate cluster (Cluster I, Figure 6) than the other. The clustering in separate cluster of MS1 sample of TYLCV identified during the current study further strengthen the idea that MS1 is a new strain of viruses infecting tomato crop in Pakistan and has not been previously reported by other studies.

Conclusions and Recommendations

To our knowledge, this is the first report of a TYLCV variant infecting tomato crop in Mansehra. Based on the finding of the present study it is concluded that TYLCV genes are present in the tomatoes cultivated in Mansehra area of Khyber Pakhtunkhwa Province of Pakistan. The results obtained from sequencing of our samples and their pairwise comparison with different classes of plant viruses clearly indicates the presence of novel species of TYLCV viral gene different from the existing genes pool of Gemini viruses. From the pairwise sequence comparison and correlation analysis, it was observed that tomato TYLCV MS1 has maximum 96% similarity with Tobacco curly shoot virus isolate TbCSV-CN and is considered as a variant of TbCSV-CN and we suggest the name MS1 (Mansehra sample 1).

The phylogenetic analysis clustered the MS1 gene in a separate cluster in the dendrogram which further strengthen the theory of novelty of this gene. For future perspectives it is recommended on the basis of the results of the present research that more research must be performed to investigate all the species of viruses which cause the TYLCV in tomato and its alternative hosts in Mansehra. The research paper highlights the evolution of new strain of TYLCV through high throughput analysis and also emphasizes on new methods for development of antiviral strategies utilizing the genomic content of infecting virus. The farmers should also be informed about the precautionary measures of this disease and its alternate host before cultivating tomato.

Acknowledgements

The authors are grateful to the Department of Biotechnology and Genetic Engineering, Hazara University Mansehra, Khyber Pakhtunkhwa for providing the facilities for carrying out this research and molecular work at the molecular laboratory of the department.

Novelty Statement

To the best our knowledge it is the first time that our findings clearly show the presence of a new TYLCV gene with maximum 96% and minimum 86% similarity to the online gene bank and this is the first report of presence of TYLC viruses in local tomato crop of Mansehra areas of Khyber Pakhtunkhwa Pakistan that are not reported by other studies.

Author’s Contribution

Mohib Shah: Conceived and designed the experiment, carried out the experiment, recorded and analyzed the data and wrote the paper.

Noorullah Khan: Conceived the Idea, designed and supervised the research, helped in analysis, prepared the figures and Tables re-wrote and revised the manuscript and submitted and corresponded the manuscript.

Aziz ud Din: Supervised the research as internal supervise, provided technical support at each stage of the study and critically reviewed the manuscript.

Sajid Ul Ghafoor: Conceived the idea, helped in recoding and analysis of data, provided technical support at each and every stage of the research and critically review and revised the manuscript.

Sajjad Ullah Khan: Helped in carrying out the experiment, recording data, and critically reviewed and revised the manuscript.

Sabaz Ali Khan: Helped in molecular analysis and critically reviewed and re-wrote the manuscript.

Conflict of interest

The authors have declared no conflict of interest.

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