Variability in indigenous Pakistani tomato lines and worldwide reference collection for Tomato Mosaic Virus (ToMV) and Tomato Yellow Leaf Curl Virus (TYLCV) infection

Local and exotic germplasm of tomato remains a major source for genetic improvement. Assessment of such lines for biotic stresses particularly viral diseases are the most important criteria for selection in Pakistan, where Tomato Yellow Leaf Curl Virus (TYLCV) and Tomato Mosaic Virus (ToMV) are the major diseases/viruses. A set of 40 accessions (including indigenous Pakistani lines and exotic germplasm from Europe, the United States, and Asia) were evaluated for their resistance/infection response to ToMV with artificial inoculation under greenhouse conditions. Infection response was quantified through disease scoring and DAS-ELISA test (for ToMV). A subset of 24 lines, was further screened for TYLCV using disease scoring and TAS-ELISA. The tested lines showed significant variability for resistance to ToMV. Only one accession (Acc-17878) was resistant to the ToMV whereas seven accessions i.e. Acc-17890, AVR-261, CLN-312, AVR-321, EUR-333, CLN-352, and CLN-362 expressed resistance to TYLCV. Correlation between phenotypic evaluation was confirmed by the ELISA results in both diseases, although both tools complemented to assess the viral infection status. In future, tomato breeding programs must consider breeding for ToMV and TYLCV resistance (using identified germplasm in our study) so as to deliver virus resistant tomato varieties. with artificial inoculation under greenhouse conditions. Our objectives were to assess: i). the status of ToMV and TYLCV resistance in tomato accessions, ii). the relationship of diversity in relative resistance status with the origin of these accessions, and iii). the correlation of phenotypic assessment with enzyme-linked immunosorbent assay (ELISA) based characterization. In this paper we describe the resistance status against ToMV and TYLCV in tomato accessions from various geographical origins.


Variability in indigenous Pakistani tomato lines and worldwide reference collection for Tomato Mosaic Virus (ToMV) and Tomato Yellow Leaf Curl Virus (TYLCV) infection
Variabilidade em linhagens indígenas de tomate do Paquistão e coleção de referência global para infecção por vírus do mosaico do tomateiro (ToMV) e vírus da folha amarela do tomate (TYLCV) (Mabvakure et al., 2016). The disease is quite frequent in the tomato growing countries of Asia, including Pakistan. Tomato crop faced severe infestation of TYLCV in Sindh province during 2012-13 in Pakistan. During the menace, tomato production in the province reduced up to approximately 19%, which significantly affected yield by devastating the planted crops on 22.5 thousand hectares alone in Sindh province. Thus, management of the two viral diseases are inevitable to attain a sustainable high yield of tomato in Pakistan.
Like many other horticultural crops, tomato diseases could be managed through various measures including cultural practices, phytosanitary chemicals, and genetic resistance (Hovmøller and Henriksen, 2008). However, considering the human health and environmental aspects of agrochemical use, coupled with the labor, time and resource investments in cultural management, development and deployment of genetic resistance is favorable option, particularly in developing countries like Pakistan (Ali et al., 2007;Hovmøller and Henriksen, 2008;Iqbal et al., 2020;Kutcher et al., 2018). In tomato, the use of genetic resistance to combat viral disease has been advocated (Segbefia et al., 2018), however, development and deployment of resistant varieties require regular assessment of germplasm with viral diseases to identify sources of durable resistance.
Numerous studies have been undertaken to assess diversity for morphological and yield potential in indigenous tomato germplasm (Iqbal et al., 2014;Ullah et al., 2016), the reaction of the indigenous Pakistani accessions to ToMV and TYLCV under artificial inoculation, in relation with the exotic lines introduced from various parts of the world is limited. Therefore, it is imperative to assess the reaction of the indigenous Pakistani accessions as well as exotic ones to ToMV and TYLCV under artificial inoculation and the controlled environmental conditions. These lines could represent variable sources of resistance and needs to be assessed under both field and greenhouse conditions. The present study was conducted to assess the resistance status in a set of indigenous and exotic tomato accessions to ToMV and TYLCV, with artificial inoculation under greenhouse conditions. Our objectives were to assess: i). the status of ToMV and TYLCV resistance in tomato accessions, ii). the relationship of diversity in relative resistance status with the origin of these accessions, and iii). the correlation of phenotypic assessment with enzyme-linked immunosorbent assay (ELISA) based characterization. In this paper we describe the resistance status against ToMV and TYLCV in tomato accessions from various geographical origins.

Germplasm selection
A set of 40 indigenous accessions were selected for the study, including accessions collected from Pakistan (obtained from Plant Genetic Resource Institute (PGRI), Islamabad, Pakistan) and exotic varieties from Federal Seed Certification & Registration Department (FSC & RD), Islamabad, Pakistan (Table 1). Three local types were

Introduction
Tomato (Lycopersicon esculentum L.) is one of the major vegetable crops of Pakistan and India, being part of many dishes those are widely prepared around the sub-continent. Tomato was first introduced into Indian sub-continent by British colonists at the commencement of 19 th century, with the first report about tomato farming in India in 1832, while in Punjab (Pakistan), it was first reported in 1916 in the book "Plants of the Punjab" (McCue, 1952). However, tomato production remains low in Pakistan, largely due to poor genetic material and lack of locally developed and adapted varieties. Genetic improvement of tomato has been the main priority of breeding programs in tomato growing areas (Agong et al., 2001). The improved varieties of any crop remain at risk to diseases due to the continuous adaptation and evolution of pathogens to the host resistance (Brown, 2015;Vallavieille-Pope et al., 2012). Tomato in Indian sub-continent is affected by many diseases, particularly those caused by viruses. Among viral diseases, the ones caused by Tomato Mosaic Virus (ToMV) and Tomato Yellow Leaf Curl Virus (TYLCV) are economically important and the most devastating (Arooj et al., 2017;Imran et al., 2013).
Tomato mosaic virus is a tobamovirus, a stable and RNA-virus with worldwide distribution (Hollings and Huttinga, 1976). The virion consists of capsid, which is un-enveloped and the nucleocapsid is elongated with helically symmetry. The typical symptoms of tomato mosaic include mosaic and curling of leaves and uneven fruit ripening. The virus is transmitted through mechanical methods, grafting and seed (Büchen-Osmond, 2003). Severe infection could lead to 25% loss, depending on the environment and host genotypes (Imran et al., 2013). This disease along with TYLCV represents a big risk to tomato production throughout the country (Imran et al., 2013).
Tomato Yellow Leaf Curl Virus is a Geminivirus, also associated with huge economic loss in tomato crops (Cohen and Harpaz, 1964;Czosnek et al., 1988). TYLCV species have been assigned to the Genus Begomovirus, based on the molecular diagnostic data. TYLCV is a monopartite virus with geminate, quasi-isometric particles of 20-30nm (A. et al., 1990). The disease caused by TYLCV has variable symptoms with severe stunted growth along with reduced leaf size and deformation (upward cupping) and discoloration. Intensity of the disease and thus expression of the symptoms could be variable as influenced by the time of infection, plant growth stage and prevailing environmental conditions. The disease could lead to 100% losses under the climatic conditions prevalent in the warm temperate and tropical zones.
Transmission of the virus occurs through whitefly (Bemisia tabaci), where the virus infects and circulate within the insect vector (Cohen and Nitzany, 1966). The worldwide spread of the insect vector is considered to be associated with the increased incidence of TYLCV into Europe (Bellows Junior et al., 1994). In Europe, the disease/virus has been reported in major tomato growing regions of France, Spain, Portugal and Italy. At the global scale the disease is prevalent in North African and Sub-Saharan countries, Australia, South America and Asia

Screening with ToMV
To assess the response of the tomato accessions to the viral infection under controlled environmental conditions, all 40 tomato accessions were grown under greenhouse conditions and were artificially inoculated. The seed coat was disinfected for the ToMV by treatment with 1% NaOCl for five minutes, transferred to 0.1N HCl for 10 min, and washed eight times with distilled water. All the plants were tested for viral infection before inoculation with Double Antibody Sandwich-Enzyme-linked Immune-Sorbent Assay (DAS-ELISA) test to further confirm their virus free status, using the LOEWE test kit. On the very next day, leaf inoculation was performed by rubbing infected plant leaf extract and carborundum powder on the leaves, which were properly tagged. Four plants per accession were seeded in the greenhouse under controlled environment in completely randomized design (CRD). Two pre-tested ToMV free plants were included in each experiment as negative control, while the known susceptible line ToMV-99-01 was included as positive control. Both visual scoring and DAS-ELISA test were used to assess the viral infection.
Infection was recorded 2-3 weeks after inoculation, based on symptoms as follows, local symptoms, systematic symptoms, leaf chlorosis, leaf necrosis, ring spots, leaf bubbling, vein clearing, and stunting. Leaf samples from each accession for DAS-ELISA test were collected separately to avoid contamination from other accessions. The ELISA procedures were performed in accordance with the protocols of the firms. Sample buffer was added to each sample separately and crushed to a uniform leaf extract for DAS-ELISA test. This color development was evaluated visually as well as measured in a spectrophotometer at 405 nm after 1-2 h. The same procedure for DAS-ELISA test was followed at 10 th , 12 th and 16 th leaf stages. Scoring was also made based on symptomatic expression of the disease on a 0-4 scale.

Screening for TYLCV
A subset of 24 accessions were screened to assess their level of resistance to TYLCV in controlled environment in greenhouse at the Department of Agroecology, Flakebjerg, Research Institute of Aarhus University, Denmark. The 24 accessions were selected as they showed resistance to ToMV. The experiment was conducted in Complete Randomized Design (CRD) with two replications (one pot with one plant ). During the experiment in December no outdoor populations of White Fly (Bemisia tabaci), the vector of the disease (Cohen and Harpaz, 1964), was present in Denmark, and mechanical inoculation is usually not successful (Avgelis et al., 2001); therefore, grafting healthy plants cuttings on infected rootstock was used for inoculation (Kashina et al., 2007b). Disinfected seed of 24 accessions were sown, where at the second permanent leaf stage pre-grafting samples from two plants of each accession were taken and tested, using Triple Antibody Sandwich-Enzyme-linked Immune-Sorbent Assay (TAS-ELISA), using the DSMZ test kit. After pre-grafting sampling, grafting was practiced on the TYLCV infected rootstock material of all 24 accessions as previously described (Kashina et al., 2007a). Longitudinal italic cuts from two sides were made on the root stock infected with TYLCV to form a V-shape. Same type of cut was made on scion from each accession, using sterile razor blade. The scions were then inserted in root stock V-shape cut in such a way that there was no space between the exposed tissues and have close contact with each other. Budding tape was used for the union to hold the scion and graft (Figure 1). The grafted plants were maintained in glasshouse at 20-30ºC and 72.4% relative humidity, for 5 weeks to ensure graft success, viral incidence and symptoms severity on each accession (Friedmann et al., 1998). After successful grafting and clearly visible symptoms, TAS-ELISA test was carried out to measure the resistance and susceptibility levels of all 24 accessions. Scoring was also made based on symptomatic expression of the disease on a 0-4 scale.

Interpretation of the ELISA test and data analyses
The ELISA test was recorded positive when the absorbance value of the sample was equal or greater than two times the absorbance of the healthy control. The phenotypic data was analyzed in MS Excel® and the R-statistical environment (Fox and Leanage, 2016).

Results
Significant variability was observed among the genotypes for their response to ToMV and TYLC infection. ToMV infection response was not correlated with TYLCV infection, under the tested conditions.

Progress of ToMV infection over time using DAS-ELISA test
Comparison of lines from different geographical origin revealed that the progress of ToMV over time varied significantly at different leaf stages i.e., Leaf 2, Leaf 6, Leaf 12 and Leaf 14, as assessed through DAS-ELISA test (Figure 2). Results indicated significant differences in overall mean values. Mean values for all 40 accessions ranged from 1.2 (Acc-17878) to 8.3 optical density (OD) (Acc-17870) with a total difference of 7.1 OD, followed by Acc-17883 (3.3 OD) at the minimum and Acc-10587 (7.7 OD) at the maximum (Figure 2).
European accessions EUR-303 and EUR-333 had almost similar level of ToMV infection (Figure 2). Pre-inoculation at two leaf stage by DAS-ELISA test, resulted values of 0.92 OD and 0.97 OD for both accessions EUR-303 and EUR-333, which was less than two (˂2), confirming absence for ToMV. Post-inoculation at sixth leaf stage, resulted high value of 8.0 OD for accession EUR-303 and then slightly reduced to 5.3 OD at 14 th leaf stage, but still it was in the range of positive ToMV infection. Similar post-inoculation results were shown by EUR-333 but with a little bit difference from EUR-303 at 12 th leaf stage; whereas, DAS-ELISA test result value 7.0 OD greater than the previous value of 6.4 OD at sixth leaf stage, again a slight decrease at 14 th leaf stage with value 5.7 OD. All the results in case of accession EUR-333 were positive for ToMV. Indigenous accessions showed significant differences at different leaf stages as well as in overall mean performance, during screening against ToMV, using DAS-ELISA test (Figure 2). Mean values of indigenous accessions ranged from 1.2 (Acc-17878) to 8.3 OD (Acc-17870) with a total difference of 7.1 OD, followed by Acc-17883 (3.3 OD) at the minimum and Acc-10587 (7.7 OD) at the maximum.
At two leaf stage, all the indigenous accessions showed absence for ToMV. Only accession Acc-17878 was found with low OD values at all leaf stages with a mean value of 1.2 OD, confirming a lack of the virus. The remaining indigenous genotypes were all positive, but two distinct patterns of the disease were observed. Accessions Acc-17867, Acc-17872, Acc-17874, Acc-17879, Acc-17882, Acc-19912, Sahel, STM-1, and STM-3 showed significant increase in disease severity from second permanent leaf stage after inoculation to 12 th leaf stage and a slight decrease at 14 th leaf stage (Figure 3). The remaining indigenous accessions i.e., Acc-06232, Acc-10572, Acc-10587, Acc17870, Acc-17877, Acc-17878, Acc-17883, Acc-17889, Acc-17890, Acc-19288, Acc-19289, Acc-19290, Acc-19893, and STM-2 disease severity was high at sixth leaf stage and then decreased significantly at 12 th and 14 th leaf stage. All the Taiwan accessions were positive for ToMV at different leaf stages except at two permanent leaf stage. A unique pattern of disease incidence was observed for AVR-341, where up to sixth leaf stage the OD value was 1.1, considered to lack any viral infection. The infection increased at 12 th leaf stage (7.3 OD), but significant decline was recorded at 14 th leaf stage. Accessions AVR-201, AVR-241, and AVR-251 showed relatively stable expression for disease incidence.
Accessions  States showed positive result for ToMV, with high infection at 6 th leaf stage, with the maximum and the minimum values of 9.8 and 6.2 OD for the accessions CLN-282 and CLN-362, respectively. All the US accessions showed significant decline in disease incidence at 12 th and 14 th leaf stage (Figure 2).

Symptomatic ToMV infection and its correlation with DAS-ELISA results
Considering the overall parameters i.e., ToMV symptoms, disease severity index, and percentage of infection, none of the accessions was found asymptomatic. Only accession Acc-17878 was resistant with no clear symptoms (Figure 2A), disease severity index of 1 and disease infection ranging 1-10%. None of the accessions showed sparse light-yellow spots, which comes under disease severity index two ranging infection from 11-20% with moderate resistance (MR) of the host. Acc-17883 expressed clear visible yellow patches on leaves, leaf bubbling, and a little bit chlorosis was observed which fulfils disease severity index three, falls in the range 20-30% of infection showing moderate susceptibility (MS) of host reaction ( Figure 3A).

Assessment of TYLCV infection in tomato accessions
Compared to ToMV, the infection of TYLCV was low on many tomato genotypes ( Figure 4A). No visible symptoms for TYLCV infection were observed for accessions Acc-17890, AVR-261, CLN-312, AVR-321, EUR-333, CLN-352, and CLN-362, and had normal growth and leaf size. These symptoms-based results were further confirmed by TAS-ELISA test, which showed lack of infection for these aforementioned accessions with values of 0.132, 0.123, 0.142, 0.127, 0.120, 0.121 and 0.132 OD respectively. These accessions were placed in the category of zero (asymptomatic) or resistant to TYLCV.

Discussion
Our study is the first one to report variability in Pakistan's indigenous tomato accessions for their response to ToMV and TYLC infections, in comparison with worldwide representative accessions. Although there was a relative variability in terms of infection for accessions from various geographical origins, only one accession was highly resistant to ToMV and seven to TYLCV. The work elucidated the variable response of the tested tomato accessions to ToMV and TYLC and that the infection to ToMV was not directly correlated with TYLCV infection, which could be expected as the two viruses are different and different loci controlling resistance could be involved. The progression of disease at various leaf stages revealed an increase in the disease on developing new leaves, suggesting that the disease was more sever on developing younger leaves. The variation in genetics could further be exploited for tomato genetic improvement (by using the resistant accessions for gene mapping, marker-assisted selection in breeding programs) in Pakistan, as suggested in previous studies (Osei et al., 2012;Qaryouti et al., 2003). The information is valuable for breeders as most of the previous studies emphasized on assessment of the diversity for morphological and yield potential in indigenous tomato germplasm (Iqbal et al., 2014;Ullah et al., 2016); the current work will be complementing previously published studies on these accessions. Utility and life span of tomato varieties is increased if the pre-approval testing consider information from assessment of the resistance to viral infections in tomato accessions.
Resistance and infection response to viral diseases have been assessed through both phenotypic scoring and ELISA based screening, where the former is subject to quantitative bias (subjective) by the scorer, while ELISA is a relatively sensitive technique (objective) and is widely used for preliminary identification and quantification of viruses in plants (Segbefia et al., 2018;Silva et al., 2010). An overall consensus was revealed between phenotypic and ELISA based scoring, which was in concordance with the previous results (Segbefia et al., 2018). Normal growth and leaf size in tomato accessions against TYLCV was observed when the values were less than the double of the value considered for absence of virus (0.226), which we also considered as a value for resistance against TYLCV as reported by Segbefia et al. (2018) for genotype Hyb-2 showing least value of 0.1860 (Zakay et al., 1991;Zhang and Klessig, 1998). Sample selection remains critical for serological detection of TYLCV, compared to ToMV, where fresh and young laves have more viral load, compared to older plant parts. The main advantage is the fact that once the test is established, it is relatively simple to perform and analyze the data resulting in rapid deployment and quarantine screening (Silva et al., 2010;Yan et al., 2018). It will enable to protect the crop against tomato viral diseases to protect yield and quality losses in a long run.
Disease management is essential to achieve higher yield and to protect yield loss through a sustainable disease management strategy including cultural practices, phytosanitary measures, and genetic resistance (Hovmøller and Henriksen, 2008;Kutcher et al., 2018). Genetic resistance remains, however, the most important considering the environmental impact and costliness of chemical control and labor demanding nature of cultural practices (Ali et al., 2007;Ullah et al., 2019). In tomato, the use of genetic resistance to control viral disease have been advocated in numerous studies (Segbefia et al., 2018;Ullah et al., 2019) and the assessment of infection status from the current study would reflect on the resistance of accessions, which is a prerequisite for the development and deployment of resistance in varieties.
Local and exotic germplasm of tomato remains a major source for genetic improvement. Assessment of such lines for biotic stresses particularly viral diseases are the most important criteria for selection in Pakistan, where TYLCV and ToMV are the major diseases. Future tomato breeding programs must consider breeding for ToMV and TYLCV resistance so as to deliver high yielding tomato varieties. This becomes even more important in the context of changing pathogen population due to recombination (Arooj et al., 2017) and invasions , not only at regional but at worldwide scale (Ali et al., 2014).

Conclusion
Our work reports on variability response to ToMV and TYLC infections in Pakistan's indigenous tomato accessions in comparison with worldwide representative accessions. Based on the variable response of the tested tomato accessions to ToMV and TYLC, we conclude that the infection to ToMV was not directly correlated with TYLCV infection, which could be expected as the two viruses are un-related and different loci could be involved in resistance against these diseases. The available variability should be useful for tomato genetic improvement to deliver high yielding resistant tomato varieties.