Tomato (Solanum lycopersicum L.) is an economically important vegetable grown worldwide in almost all climates and environments.

Tomato plants are affected by many pathogens (Blancard 2012; Jones et al. 2016), which cause serious economic losses to the quantity and quality of tomato production (Mrkvová et al. 2022; Panno et al. 2021; Tolman et al. 2004). 136 tomato virus species infect tomatoes naturally and they are the biggest threat to tomato production worldwide (Hanssen et al. 2010; Nadeem et al. 2022; Ullah et al. 2017; Ullah et al. 2019).

An important group of tomato pathogenic viruses is 37 virus species belonging to the Tobamovirus genus of the Virgaviridae family (ICTV 2021). Many tobamoviruses, such as Tomato mosaic virus (ToMV), Tobacco mild green mosaic virus (TMGMV), Tobacco mosaic virus (TMV), Tomato brown rugose fruit virus (ToBRFV), and Tomato mottle mosaic (ToMMV), infect tomatoes naturally (Hancinský et al. 2020; Luria et al. 2017; Mrkvová et al. 2022). Among them, the Tomato mosaic virus (ToMV) is one of the highly persistent, infectious, cosmopolitan tobamoviruses and is the main limiting factor in tomato production in open fields and greenhouses (Nadeem et al. 2022; Ullah et al. 2019). Disease caused by ToMV infection has been detected in all tomato-growing areas worldwide and causes significant economic losses (Lyu et al. 2023).

The ToMV virion, like other tobamoviruses, is rod-shaped with a length of about 300 nm and a diameter of 18 nm. The viral genome consists of positive-stranded ssRNA, about 6400 nucleotides long. It encodes four proteins: a 130-kDa protein, its read-through product of 180 kDa, a 30-kDa protein, and the CP (17.5 kDa) (Ishibashi and Ishikawa 2016). The 130-kDa and 180-kDa proteins are involved in viral RNA replication and are collectively called replication proteins. The 30-kDa protein is essential for cell-to-cell transmission of the virus and is therefore called the movement protein (MP). ORF4 encodes a coat protein (CP) of approximately 17.5 kDa (Meshi et al. 1992; Mrkvová et al. 2022). In addition to the function of virion production, the coat protein of ToMV also plays a role in long-distance transmission of the virus (Li et al. 2005).

Tomato mosaic virus is the most virulent tobamovirus and has a wide host plant range. Diseases caused by ToMV infection usually do not cause the death of plants, but a mosaic of plant leaves, deformation of stems and fruits, and development of growth buds and roots are negatively affected (Ishibashi et al. 2023). The development of disease symptoms varies significantly depending on virus isolates and plant genotype, time of infection, light intensity, and temperature (Broadbent 1976; Zitter 2014). Leaves are light to dark green, sometimes accompanied by yellowing, curling, or tapering (Zitter 2014). Due to the uneven distribution of pigment in the fruits of tomato plants with mosaic disease, abnormal signs such as uneven ripening and internal darkening of the fruit occur, resulting in a decrease in their quality and loss of marketability (Broadbent and Cooper 1964; Ishibashi et al. 2023; Zitter 2014).

There are two main strategies to overcome the problems, the most important of which is the accurate and sensitive diagnosis of ToMV, which is the most important tool in managing virus diseases in tomato production.

To eliminate the problem of ToMV in agriculture, early diagnosis of the virus is the most appropriate and practical approach. In the development of science-based ways of fighting viruses, it is necessary to plant virus-free seeds, timely disposal of infected equipment, and use modern diagnostic methods, as well as the development of rapid methods for timely detection and identification of phytopathogenic viruses requires (Jovlieva et al. 2024). Immunodiagnostic methods are examples of the most common methods used to test large numbers of samples for viruses (Boonham et al. 2014; Rubio et al. 2020).

Highly specific methods for the detection of viruses are based on the polymerase chain reaction (PCR) and together with the latest generation of sequencing technologies provide the possibility of accurate identification of viruses, detection of multiplex viruses, the amount of virus, as well as the discovery of newly emerging viruses (Mehetre et al. 2021; Rubio et al. 2020). The high specificity of PCR-based methods can sometimes lead to false-negative results as a result of mutations in the primer-binding genomic parts of the viral genome (Mrkvová et al. 2022). Immunological methods have several advantages over other methods due to their simplicity and the possibility of testing many samples at the same time (Fayziev et al. 2020; Jovlieva et al. 2024). To date, there are several phytoviruses in Uzbekistan: separation of phytopathogenic viruses such as Barley yellow dwarf virus (BYDV), Potato leafroll virus (PLRV), Potato virus X (PVX), Alfalfa mosaic virus (AMV), Plum pox virus (PPV), Maize dwarf mosaic virus (MDMV) and a specific serum was prepared for them (Fayziev et al. 2020; Jovlieva et al. 2024; Kholmatova et al. 2024; Khusanov et al. 2020; Makhmudov et al. 2023; Sattorov et al. 2020; Sobirova et al. 2023). To date, plants such as N. tabacum and N. glutinosa L. have been used to obtain the purified preparation of ToMV.

Serological methods using polyclonal antibodies provide a versatile tool for the broad screening of phytopathogenic viruses that infect different crops (Souiri et al. 2014). The researcher does not have detailed information about the antigen strain used to produce the available commercial polyclonal antibodies for ToMV detection. Such information is obtained in the process of antiserum development, which begins with the identification, isolation, characterization, and immunization of the virus (Mrkvová et al. 2022).

Like other viruses, ToMV stores antigenic determinants in its protein coat. If a homogenous virus preparation purified from other viruses and plant proteins is injected into an animal’s body, protective proteins, i.e., antibodies, are synthesized in the animal’s blood against the virus. Artificial antibodies are of great importance in the serological diagnosis of plant viruses. This is because a specific antiserum is required for many immunological studies. Such necessary antisera often appear as a special protection, an “immune system” against various antigens (AG) in the body of several animals such as rabbits, rats, and white mice (Kerstin et al. 2010).

High titer antiserum can be obtained by injecting purified virus antigen subcutaneously, intravenously, and intramuscularly in laboratory animals. Also, the intensity of the antiserum depends on the concentration of the antigen the frequency of immunization, and the process of re-immunization (Egorov et al. 1991; Jovlieva et al. 2024).

The complete process of preparation of polyclonal antiserum to ToMV is the aim of this experimental work, during the experiments, an effective polyclonal antiserum that can be used for the detection of ToMV was prepared, and we determined the sensitivity of the prepared antiserum for the serological detection of ToMV in different varieties of tomato plants.

ToMV was isolated from a tomato plant showing symptoms of virus infection from the tomato fields of Kybrai district, Tashkent region. The leaves of this plant were collected and stored at -80°C (BDF-86V588, Biobase, China). The nucleotide sequence of the ToMV isolate CP gene was determined by Senger sequencing and deposited in the GenBank nucleotide database (OR420713.1 available at https://www.ncbi.nlm.nih.gov/nuccore/OR420713.1?report=genbank&to=677).

Antigen preparation

To carry out the research, a 50 g sample of a tomato plant leaf, which was proven to contain ToMV by PCR, was ground in a porcelain mortar by adding phosphate buffer (10 mM, pH = 7.2) in a ratio of 1:1. The resulting homogenate is spun at 7,000 rpm. centrifuged (TG16.5, Bioridge, China) at high speed for 15 minutes. The supernatant was isolated and inoculated into the Chenopodium amaranticolor L. plant under laboratory conditions. One of the necroses formed on the leaf of the Ch. amaranticolor L. plant was isolated, re-homogenized, and mechanically infected with the Datura stramonium L. plant. The yellow mosaic symptom that appeared on the leaf of D. stramonium L. was removed from the existing leaf and homogenized. Biologically pure ToMV purified from the mixed infection was obtained by re-infection three times in the same manner. 1 kg of leaves of D. stramonium L. were collected and stored at -80°C. A purified preparation of ToMV was obtained from the collected leaves of D. stramonium L. using physicochemical and gel chromatography methods in laboratory conditions, the degree of purity and virus concentration was determined, and it was placed in separate test tubes at 1mg/ml and stored at -80°C.

Immunization of experimental rabbits. Healthy albino rabbits (weight 3 kg) were selected to receive polyclonal serum to ToMV. The virus preparation was injected into the rabbit body by ear vein immunization. This process was carried out as follows. When injecting into the rabbit’s ear vein, once a week, 1 mg/ml of the pure virus preparation was mixed with 1 ml of physiological solution (NaCl 0.9%) in a ratio of 1:1, for a total of 6 injections once a week. The amount of virus was increased at each injection period after the first injection. The immunization schedule used to obtain rabbit anti-ToMV sera is shown in Table 1 (Table 1).

Table 1 . Scheme of rabbit immunization to obtain antiserum against ToMV.

Injection number	The dose of purified virus injected is mg/ml
1 time	1
2 times	2
3 times	3
4 times	4
5 times	5
6 times	6

10 days after the last injection, 45 ml of blood was collected from the right ear vein of the rabbit. Blood was allowed to clot at room temperature for 1 day, then stored overnight at 4°C. The shaped elements of the blood were carefully separated from the serum part and spun for 5 minutes at 1500 rpm to remove the remaining shaped elements from the serum. It was centrifuged for 15 minutes (Egorov et al. 1991), and 25 ml of blood antiserum was separated from the obtained 45 ml blood sample.

30 days after blood collection, pure virus preparation was reimmunized into the rabbit ear vein. In this case, an amount of the virus preparation equal to the last dose of the virus was injected with a physiological solution in a 1:1 ratio. 10 days after reimmunization, 45 ml of blood was taken for the second time, and the serum part was purified from blood cells as mentioned above. 25 ml of antiserum was separated from 45 ml of blood taken the second time and placed in 1 ml test tubes and stored at -20°C.

To obtain hyperimmune antisera, laboratory rabbits with ToMV isolate and blood sampling procedures were carried out together with the employees of the vivarium department of the “Republican State Center for Diagnosis of Animal Diseases and Food Safety”. Experiments on rabbits from laboratory animals were carried out in compliance with all the normative rules contained in “Veterinary Legislation”. The animals were under daily clinical observation. No discomfort was observed in laboratory animals during the experiment and after the study.

Determination of antiserum titer using the double immunodiffusion method. For detection of ToMV by double immunodiffusion, 20 µl of blood serum was taken and mixed with the same amount of 0.9% NaCl solution. This serum was diluted 2 times (1:2) and was taken from this serum for further dilution. In this way, blood serum was diluted 1:2, 1:4, 1:8, 1:16, 1:32, 1:64, 1:128, 1:256, 1:512, 1:1024, 1:2048.

1% agarose gel was prepared to determine the titer of antiserum by the double immunodiffusion method. For this, 1 g of agarose gel was removed and 99 ml of 10 mM phosphate buffer (pH = 7.2) was added and the gel was completely dissolved in a microwave oven. 25 ml of gel was poured into a separate container and mixed well by adding 1 ml of antibiotic (streptomycin) to prevent bacteria from growing in liquid agar cooled to 50-55°C. It was poured onto a 9 × 12 cm glass plate placed horizontally on a flat surface. After the agarose gel solidified, wells were prepared in a row using special stamps with a hole spacing of 5 mm, and 20 µl of diluted antiserum was placed in the first row of these wells, and the same amount of viral sap, i.e., antigen (AG) was placed opposite it, the plates were kept in a desiccator with 500 ml of water and 100 µl of chloroform under them for 48 hours.

Obtaining a pure preparation of local isolates of a particular virus is important for solving practical problems such as studying its biochemical properties, preparing specific antiserum, and developing immunodiagnostic methods. ToMV was gel filtered using TSK HW-75 gel to obtain a pure virus preparation. When the optical absorption index of the obtained virus preparation was measured in a spectrophotometer (Cary-60, Agilent, USA), it was found that the peak of absorption of the preparation, that is, the maximum 260 nm, this indicator corresponds to a nucleoprotein, and the ratio of 260/280 1,2, that is, it was found to be characteristic of viruses formed based on helical symmetry. To determine the virulence of the purified virus preparation, the leaves of the N.glutinosa plant were infected mechanically, and 48 hours after inoculation, necrosis with a size of 3-4 mm typical of ToMV was observed (Fig. 1).

Figure 1. Virulence determination of purified ToMV preparation (a) N. glutinosa leaf inoculated with purified ToMV preparation; (b) control healthy leaf

The purified virus drug was injected into the rabbit body with the isotonic solution for 6 weeks, and polyclonal antiserum was obtained.

The obtained polyclonal antiserum was observed to be specific for ToMV. Tomatoes infected with ToMV reacted with the extracts of plants and formed precipitate lines, but it was observed that there was no reaction with extracts from healthy plants (Fig. 2).

Figure 2. Determination of the specificity of the antiserum (As) obtained to ToMV (a) plant sap obtained from a healthy tomato leaf; (b) sample from a tomato leaf infected with ToMV

The titer of antiserum isolated from the first blood (52 days after the first immunization) of an Albinos breed rabbit injected into the ear vein was 1:128 when determined by the double immunodiffusion method, and the titer of the antiserum isolated from the blood after reimmunization increased several times, that is, antiserum in the ratio 1:512 it was observed that precipitation lines were formed when diluted, and no such line appeared in antiserum diluted 1:1024 and 1:2048 (Fig. 3).

Figure 3. Determination of antiserum titer obtained after reimmunization by the double immunodiffusion method. As = antiserum; Ag = antigen

The titer of the antiserum obtained initially and after the reimmunization process was compared, the obtained results are presented in Table 2.

Table 2 . Comparison of titer of isolated antiserum before and after reimmunization.

	Dilution factor of antiserum
	C*	1/2	1/4	1/8	1/16	1/32	1/64	1/128	1/256	1/512	1/1024	1/2048
Antiserum obtained before reimmunization	++++	++++	+++	+++	++	++	++	+	-	-	-	-
Antiserum received after reimmunization	++++	++++	++++	++++	+++	+++	+++	++	++	+	-	-

Note: “++++” in the table indicates that the titer of antiserum (AS) is high, “+++” is moderately high, “++” is moderate, “+” is weak, “-” is the product of the precipitation line, and “*” indicates that undiluted antiserum was used as a control. means that it is not..

During the study, the titer of the ToMV antigen isolated from D.stramonium L. was also determined using the double immunodiffusion method and was evaluated based on the precipitation lines formed between AG-AS (Table 3).

Table 3 . Titer of ToMV isolated from D. stramonium L. plant.

		Dilution factor of antiserum
		C*	1:2	1:4	1:8	1:16	1:32	1:64	1:128	1:256	1:512	1:1024	1:2048
Dilution factor of antigen	C**	++++	++++	++++	++++	+++	+++	+++	++	++	+	-	-
	1:2	++++	++++	++++	++++	+++	+++	+++	++	++	+	-	-
	1:4	++++	++++	+++	+++	+++	++	++	+	+	-	-	-
	1:8	++++	+++	+++	+++	++	++	+	+	-	-	-	-
	1:16	++++	+++	+++	++	++	++	+	-	-	-	-	-
	1:32	++++	+++	+++	++	++	+	+	-	-	-	-	-
	1:64	++++	+++	+++	++	++	+	-	-	-	-	-	-
	1:128	++++	+++	++	+	-	-	-	-	-	-	-	-
	1:256	+++	++	+	+	-	-	-	-	-	-	-	-
	1:512	++	+	-	-	-	-	-	-	-	-	-	-
	1:1024	+	-	-	-	-	-	-	-	-	-	-	-

Note: “++++” in the table indicates that the antiserum—antigen (AS—AG) reaction is high, “+++” is moderately high, “++” is moderate, “+” is weak, “-” is a precipitation line, “*” is the undiluted antiserum, and “**” is the undiluted antigen. represents the non-formulation of..

It can be seen from the result that the titer of ToMV isolated from the D.stramonium L. plant was 1:1024 when tested based on the obtained antiserum.

The specificity of the obtained antiserum and the resistance of different varieties of tomato to ToMV were carried out in 22 varieties of tomatoes mechanically infected with the purified preparation of ToMV. In this case, 20 days after inoculation, the virus was taken from ToMV-infected tomato leaves and placed in separate porcelain mortars, and the plant juice was separated. The presence of ToMV in the isolated plant sap was determined based on the experiments conducted by the double immunodiffusion method (Fig. 4).

Figure 4. Analysis of ToMV infection rate of tomato cultivars
Note: AS = antiserum; AG = antigen; a tomato sample artificially infected with the virus was used.

Precipitation reactions between antigen and antiserum showed the presence of ToMV infection in 18 out of 22 tested samples. From the 22 samples examined based on the precipitation lines formed by antiserum obtained from ToMV, Darkon, Revansh, Blogodatniy, Perst were not infected with the virus, TMK, Sevara, Agro, Tvenid, Finish, Tanimi, Lojayin F1, N-2274, Chelnok varieties are relatively resistant and Zakovat, Fakhriy, Magnat, L-200, Ofarin, Yakut, Surkhan 142, Charodey, Ofarin-2 varieties were found to be resistant to ToMV.

Antiserum with virus-specific antibodies can be prepared by isolating the antigen from the infected plant, purifying virus particles, and immunizing animals (Garcia-Calvo et al. 2021). A common problem in obtaining specific antibodies to plant viruses is obtaining purified virus antigens to immunize laboratory animals (Lima et al. 2001). Obtaining polyclonal antiserum is considered important for quick and accurate diagnosis of viruses by ELISA method in phytopathology and agricultural practice. Polyclonal antibodies recognize several similar isolates of the antigen being detected, which increases the sensitivity of the assays (Ascoli and Aggeler 2018).

As a result of similar studies, polyclonal antisera to Potato virus X (PVX) and Maize dwarf mosaic virus (MDMV) virus were obtained and used in virus detection (Jovlieva et al. 2024; Sobirova et al. 2023). These researchers obtained antiserum by injecting a purified virus preparation subcutaneously or between the muscles of a Shinshila rabbit. Lima et al. obtained a specific polyclonal antiserum by oral immunization of rabbits with a purified preparation of Cowpea severe mosaic virus (CPSMV) and Papaya lethal yellowing virus (PLYV) (Lima et al. 2001). According to Strobel and Mowat, when administered orally to an animal, only a very small amount of antigen is absorbed into the blood as antigens without being enzymatically degraded in the animal’s intestine (Strobel and Mowat 1998). For this reason, studies on antigen transfer into the blood without degradation are of great importance (Mestecky et al. 1997).

However, the results of our research showed that intravenous immunization is effective in obtaining virus-specific serum, that is, antiserum accumulates more in the rabbit’s body.

In general, the use of polyclonal antibody sera in the diagnosis of viruses can potentially detect other similar viruses, although sometimes with lower specificity. From the point of view of phytopathology and agricultural practice, it is very important to identify viruses present in plants, even if there are small changes in the virus genome that prevent them from being identified by more precise methods (Rubio et al. 2020).

Immunodiagnostic tests such as ELISA are suitable for primary mass screening of virus presence in plants, and molecular genetic methods with minimal sensitivity and high specificity are desirable to use after initial screening (Boben et al. 2007).

Different mechanisms against phytopathogenic viruses have been formed in plants, and regardless of which of these mechanisms, they are considered useful for protection against viral diseases caused by pathogens (Khalid et al. 2017; Lindbo and Falk 2017). In the course of our research, it was found that the Darkon, Revansh, Blogodatni, and Perst varieties of tomatoes have useful resistance mechanisms.

The antiserum obtained during this study can also be used in the broad screening of ToMV in different tomato cultivars using immunological methods. In our experiments, the antiserum obtained against ToMV is also suitable for the detection of ToMV spread and plant-virus interactions in agro ecological conditions.

From the analysis of the results of the conducted experiment, it became clear that it was possible to obtain high titer antiserum as a result of intravenous isotonic solution in obtaining antiserum used for the detection of ToMV. From the experiences of several authors on obtaining antiserum to viruses and the results of our research, it was concluded that the use of an albino breed of rabbit in the preparation of antiserum facilitates the process of immunization to the ear vein, and it is appropriate to use it in obtaining antiserum with a high titer compared to the Shinshila breed.

The development of diagnostic tools for the development of ToMV-resistant tomato varieties highlights the importance of basic plant protection research. It is important to develop diagnostic tools for identifying endemic strains to understand and evaluate the interaction between viruses and host plants.

This work was carried out with the financial support of the “IZ-2020122512 Development of immunodiagnostics of tomato mosaic virus” grant of the Ministry of Higher Education, Science and Innovation.