Papaya virus e infecting tomato and uses thereof

Abstract

The application discloses a papaya virus E which invades tomatoes and an application thereof, and belongs to the technical field of plant virus disease prevention and treatment. The papaya virus E of the application has been preserved in the China General Microbiological Culture Collection Center on February 6, 2026, and the preservation number is CGMCC No. 47097. The papaya virus E isolated by the application solves the current technical blank related to PpVE prevention and control in tomatoes, has important application value in aspects of precise prevention and control of tomato virus diseases, disease-resistant breeding, green prevention and control technology research and development and epidemic monitoring and the like, and can significantly improve the comprehensive prevention and control capability of the tomato industry, and produce good economic benefits, social benefits and ecological benefits.

Description

Technical Field

[0001] This invention relates to the field of plant viral disease control technology, specifically to a papaya virus E that infects tomatoes and its application. Background Technology

[0002] The information disclosed in this background section is intended only to enhance understanding of the overall background of the invention and is not necessarily to be construed as an admission or in any way implying that such information constitutes prior art known to those skilled in the art.

[0003] tomato( Solanum lycopersicum As a widely cultivated and important vegetable crop globally, tomatoes possess both economic and nutritional value, occupying a crucial position in agricultural production. However, viral diseases are one of the main biological stresses restricting tomato yield and quality, posing a serious threat to tomato production and fruit quality.

[0004] Tomato viral diseases present with diverse symptoms, commonly including mosaic, yellowing, stunting, and deformities. Viral-infected tomato plants exhibit mottled, chlorotic, or necrotic leaf patterns; in severe cases, leaves turn yellow and fall off. Furthermore, viral infection can impair tomato growth and development, leading to stunted plants and deformed fruits. The emergence of new viruses, coupled with a lack of timely detection and early warning systems, will significantly impact the tomato industry.

[0005] In 2025, new tomato plants exhibiting virus-like symptoms were discovered in vegetable greenhouses in Dezhou City, Shandong Province. These plants showed obvious yellowing of the leaves, and on the surface of immature green fruits, dark green spots of varying sizes were visible; mature red fruits showed green and yellow spots, accompanied by discoloration. The incidence rate reached 10-60%, severely impacting tomato yield and quality. It is speculated to be a newly emerging plant virus infection, but currently, there is a lack of technology for isolating, identifying, detecting, monitoring, and controlling the pathogenic virus strain. Summary of the Invention

[0006] To address the shortcomings of the existing technologies, the purpose of this invention is to provide a papaya virus E strain that infects tomatoes and its applications. This invention is the first to isolate and identify a novel tomato-infecting virus strain, PpVE-Tomato-1, from diseased tomato plants. PpVE-Tomato-1 of this invention facilitates early detection, early warning, and early intervention of viral diseases caused by PpVE in tomatoes, providing fundamental support for precise field control.

[0007] Specifically, the present invention relates to the following technical solutions: In a first aspect, this invention provides a papaya virus E (PpVE) strain that infects tomatoes. The isolate from this tomato strain is named PpVE-Tomato-1. This virus strain was deposited on February 6, 2026, at the China General Microbiological Culture Collection Center (CGMCC, address: No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing), with accession number CGMCC No. 47097; and classified as papaya virus E. Betacytorhabdoviruspapaya virus E.

[0008] The papaya virus E (PpVE-Tomato-1) of this invention belongs to the Rhabdoviridae family ( Rhabdoviridae ), Beta-cytoplasmic rhabdovirus ( Betacytorhabdovirus A novel viral strain of this invention, whose complete genome sequence is shown in SEQ ID NO.1-SEQ ID NO.15, differs from the previously reported papaya virus E in that the PpVE-Tomato-1 strain of this invention can infect tomatoes and cause the following novel symptoms in tomatoes: The leaves turn yellow, dark green spots appear on the surface of immature green fruits, and green and yellow spots appear on the surface of mature red fruits, accompanied by discoloration.

[0009] In a second aspect, the present invention provides the application of the above-mentioned papaya virus E in the screening of tomato germplasm resources resistant to viral diseases caused by papaya virus E.

[0010] In the above applications, by inoculating papaya virus E, large-scale and standardized disease resistance screening of tomato germplasm resources can be carried out, and excellent germplasm resistant to PpVE-Tomato-1 can be quickly screened out, accelerating the breeding process of PpVE-resistant tomato varieties, improving breeding efficiency and targeting, and providing variety guarantee for the sustainable development of the tomato industry.

[0011] In a third aspect, the present invention provides the application of the above-mentioned papain virus E in drug screening against papain virus E.

[0012] Using PpVE-Tomato-1 as a target, drugs that are effective against papaya virus E can also be screened, providing new drug resources for the prevention and control of PpVE-Tomato-1.

[0013] In a fourth aspect, the present invention provides the use of the above-described papain virus E in the preparation of a vaccine against viral diseases caused by papain virus E.

[0014] In the above applications, the type of vaccine is an inactivated vaccine or a live attenuated vaccine.

[0015] In a fifth aspect, the present invention provides the application of the above-mentioned papain virus E as a target in the preparation of PpVE detection reagents.

[0016] In the above applications, the PpVE detection reagent is preferably an RT-PCR detection reagent, a real-time PCR detection reagent, an ELISA detection reagent, or a colloidal gold immunoassay strip.

[0017] In the above applications, the PpVE detection reagent uses the primers shown in SEQ ID NO.16 and SEQ ID NO.17 as active ingredients.

[0018] The beneficial effects of this invention are: This invention marks the first time a novel papaya virus E strain has been isolated from diseased tomato plants, filling a technological gap in the isolation, identification, and application of PpVE strains in tomatoes for the prevention and control of viral diseases. It offers the following beneficial effects: (1) The papaya virus E that infects tomatoes in this invention can be used as a positive control for various detection technologies (RT-PCR, quantitative PCR, ELISA, immunoassay strips, colloidal gold immunoassay strips, etc.), which significantly improves the accuracy, specificity and reliability of PpVE detection in tomatoes, enabling early detection, early warning and early blocking of PpVE virus disease in tomatoes, and providing basic support for precise field control.

[0019] (2) This invention provides key materials for the breeding of PpVE-resistant tomato varieties, overcoming the shortcomings of existing tomato breeding methods that lack a standard PpVE virus source, making it impossible to conduct disease-resistant germplasm screening, resistance gene identification, and variety disease resistance evaluation. Using the PpVE lines isolated by this invention, large-scale and standardized disease resistance screening of tomato germplasm resources can be carried out, quickly screening out superior PpVE-resistant germplasm, accelerating the breeding process of PpVE-resistant tomato varieties, improving breeding efficiency and targeting, and providing variety guarantees for the sustainable development of the tomato industry.

[0020] (3) This invention lays a solid material foundation for revealing the interaction mechanism between PpVE and tomato, clarifying its pathogenic characteristics and transmission patterns. The homozygous strains obtained in this invention can be used to systematically study the infection process, symptoms, replication and migration mechanisms, virulence characteristics, and combined infection effects of PpVE with other tomato viruses in tomatoes, clarifying its impact on tomato growth and yield, and providing important theoretical basis for the subsequent development of targeted control strategies. It also provides important support for the development of green and efficient control technologies for tomato PpVE virus disease, meeting the needs of green agricultural development.

[0021] (4) Based on the PpVE strains isolated in this invention, attenuated strains can be further screened for cross-protection and control, key pathogenic genes of the virus can be identified for RNAi antiviral variety breeding or antiviral biological agent development, and the vector of the virus can be identified to guide integrated management of the vector, reduce the use of chemical pesticides, reduce environmental pollution, and achieve green and sustainable control of tomato viral diseases. This provides a technical basis for tomato PpVE epidemic monitoring, risk assessment and quarantine control.

[0022] (5) This invention clarifies the biological characteristics and molecular features of PpVE isolates in tomatoes, which can be used to track the prevalent strains and variation trends of PpVE in the field, provide early warning of new strains with high pathogenicity and high transmissibility, and provide scientific support for the introduction and quarantine of tomatoes and the delineation of regional control, effectively prevent the spread of PpVE and ensure the safe production of the tomato industry. Attached Figure Description

[0023] Figure 1 Symptoms appearing on tomato leaves and fruits in the field; in the image, AD shows yellowing of leaves; EG shows dark green spots on the surface of green fruits accompanied by yellowing; HJ shows green and yellow patches on red fruits accompanied by yellowing.

[0024] Figure 2 Transmission electron microscopy image of the morphology of PpVE-Tomato-1 virus particles; the arrows in the image point to the virus particles, which are bullet-shaped.

[0025] Figure 3 Schematic diagram of the genome structure of PpVE-Tomato-1.

[0026] Figure 4 Phylogenetic tree based on the complete genome nucleotide sequence of PpVE-Tomato-1; the tree was constructed using the maximum likelihood (ML) method, and the information for each isolate is presented in the following format: virus name, isolate, accession number, country, host; at the nodes, a confidence value of more than 70% in 1000 replicates is displayed, and branches with a value of less than 50% have been merged.

[0027] Figure 5 : RT-PCR detection of field-collected samples; In the figure, A represents the detection of leaves and fruits of single plants, 1 represents upper leaves; 2 represents middle leaves; 3 represents fruits; CK represents PCR negative control; ubiquitin gene (UBI) is used as internal reference gene; B represents the detection of fruits of PpVE-infected tomatoes and healthy tomatoes, 1-5 represent the detection of random samples.

[0028] Figure 6Results of the investigation on the transmission mode of PpVE-Tomato-1; In the figure, A shows whiteflies and their eggs observed on the underside of leaves in the field; B shows whiteflies and their eggs collected in the field by RT-PCR detection, where 1 represents whiteflies reared in the laboratory, 2 represents whiteflies collected from diseased plants in the field, 3 represents whitefly eggs in the laboratory, and 4 represents whitefly eggs on diseased plants in the field. The whitefly ribosomal protein L29 gene (BtRPL29) is an internal reference gene; C shows the leaf symptoms of tomato plants after co-culturing with whiteflies for 15 days; D shows the diseased tomato leaves after co-culturing with whiteflies by RT-PCR detection, where 1 represents healthy tomatoes, 2 represents diseased tomatoes, and the tomato ubiquitin gene (UBI) is an internal reference gene. Detailed Implementation

[0029] It should be noted that the following detailed descriptions are exemplary and intended to provide further explanation of this application. Unless otherwise specified, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this application pertains.

[0030] To enable those skilled in the art to better understand the technical solutions of this application, the technical solutions of this application will be described in detail below with reference to specific embodiments. Unless otherwise specified, experimental conditions not detailed in the embodiments are generally based on conventional conditions or conditions recommended by the reagent company; reagents, consumables, etc., used in the following embodiments can be obtained commercially unless otherwise specified.

[0031] Example 1: Isolation and Identification of Viruses 1. Virus isolation: In January 2025, tomato plants exhibiting virus-like symptoms were discovered in greenhouses in Dezhou City, Shandong Province. These plants showed obvious yellowing of the leaves. Figure 1 (AD). On the surface of the unripe green fruit, dark green spots of varying sizes can be seen (AD). Figure 1 (Middle EG). Ripe red fruits develop green and yellow spots ( Figure 1 (HJ), and it is accompanied by discoloration.

[0032] Virus particles were extracted from diseased tomato fruits using the following method: Collect diseased tomato fruits, cut them into pieces, and weigh them. Add twice the volume of phosphate buffer and homogenize using a high-speed blender. Then filter the homogenized solution through sterile double-layered gauze to remove plant tissue. Pour the supernatant into an 80 ml centrifuge tube and centrifuge at 8000 rpm for 20 min at 4°C to remove the precipitate. Add 1% Triton-100, 6% PEG6000 (wt / vol), and 1% NaCl (wt / vol) to the supernatant and stir magnetically at 4°C for 3 h. Then centrifuge the sample at 10000 rpm for 30 min at 4°C. Resuspend the precipitate in phosphate buffer containing 1% Triton-100 (the buffer volume is 1 / 10 of the initial total volume). Incubate the resuspended sample magnetically at 4°C overnight. The next day, centrifuge the sample at 10000 g for 30 min at 4°C to remove insoluble matter and save the supernatant for subsequent experiments.

[0033] The extracted virus particles were observed using a transmission electron microscope. The extracted particles were bullet-shaped or rod-shaped. Figure 2 ), which is similar to the particles of a rhabdovirus.

[0034] 2. Virus identification: To determine the exact causative agent of the observed disease, metagenomic sequencing was performed on samples of diseased tomato fruit. Based on the sequencing data, a complete viral genome sequence of 13,474 nucleotides was constructed. BLASTn analysis with the GenBank database showed that the obtained sequence had the highest nucleotide homology (92.95%) with PpVE-IR-BA recently identified in Iranian bitter apples. However, significant differences were observed when the obtained sequence was compared with other typical PpVE strains from different hosts and geographical origins, with whole-genome nucleotide homology ranging from only 79.60% to 81.06%. These results indicate that the identified virus represents a novel PpVE strain for infecting tomatoes, which we named PpVE-Tomato-1.

[0035] (1) Genome structure analysis: The genome structure of PpVE-Tomato-1 is as follows: Figure 3As shown, it contains seven open reading frames (ORFs). The nucleotide sequence of the 5' untranslated region (UTR) is shown in SEQ ID NO.1, with a length of 152 nucleotides; the nucleotide sequence of ORF1 (positions 153-1508) is shown in SEQ ID NO.2, encoding a 51 kDa nucleoprotein (N); the spacer sequence between ORF1 and ORF2 is shown in SEQ ID NO.3; the nucleotide sequence of ORF2 (positions 1700-3019) is shown in SEQ ID NO.4, encoding a 48.4 kDa phosphatase protein (P); the spacer sequence between ORF2 and ORF3 is shown in SEQ ID NO.5; the nucleotide sequence of ORF3 (positions 3182-3751) is shown in SEQ ID NO.6, encoding a 21.1 kDa putative protein (P3); the spacer sequence between ORF3 and ORF4 is shown in SEQ ID NO.7; the nucleotide sequence of ORF4 (positions 3768-4007) is shown in SEQ ID NO.8, encoding a 9.3 kDa nucleoprotein. The sequence of the spacer between ORF4 and ORF5 is shown in SEQ ID NO. 9; the nucleotide sequence of ORF5 (positions 4295-4939) is shown in SEQ ID NO. 10, encoding a 24.5 kDalton matrix protein (M); the spacer sequence between ORF5 and ORF6 is shown in SEQ ID NO. 11; the nucleotide sequence of ORF6 (positions 5158-6717) is shown in SEQ ID NO. 12, encoding a 58 kDalton glycoprotein (G); the spacer sequence between ORF6 and ORF7 is shown in SEQ ID NO. 13; the nucleotide sequence of ORF7 (positions 6975-13316) is shown in SEQ ID NO. 14, encoding a 241.2 kDalton large RNA polymerase (L); the nucleotide sequence of the 3' untranslated region (UTR) is shown in SEQ ID NO. 15, with a length of 158 nucleotides.

[0036] The sequences shown in SEQ ID NO.1-SEQ ID NO.15 together constitute the whole genome sequence of PpVE-Tomato-1.

[0037] (2) Phylogenetic analysis: To determine the evolutionary relationships of the viruses, we retrieved the full-length genome sequences of PpVE isolates from different hosts and representative species of the same genus transmitted by different vectors, including LYMoV, ADV, NCMV, BYSMV, and RSMV, from the NCBI database. We then constructed a phylogenetic tree using the maximum likelihood (ML) method. The results showed that the PpVE-Tomato-1 isolate clustered tightly with the PpVE IR-BA isolate, forming a separate branch. Figure 4 ).

[0038] (3) Sequence consistency analysis: Among the reported sequences, PpVE-Tomato-1 is most closely related to the bitter apple strain IR-BA, with a genome-wide nucleotide identity of 92.7%. In contrast, PpVE-Tomato-1 shows only 76.4%–78.5% genome-wide identity with other PpVE strains (Table 1).

[0039] Table 1: PpEV Sequence Consistency Analysis At the open reading frame (OPF) level, PpVE-Tomato-1 was compared with representative PpVE isolates. Nucleotide and amino acid similarities ranged from 82.3% to 93.1% for N; 77.2% to 93.2% for P; 80.8% to 92.6% for P3; 78.6% to 93.0% for M; 76.6% to 93.6% for G; and 79.8% to 93.8% for L. P4 showed the lowest similarity, with nucleotide and amino acid similarities ranging from 19.6% to 89.1% and 3.7% to 81.0%, respectively. Notably, in isolates C1, C2, and C3, the genomic regions corresponding to P4 and G contained multiple small ORFs, rather than a single, continuous ORF encoding a single protein (Table 2).

[0040] Table 2: Identification analysis of nucleotide and amino acid sequences of each ORF in representative PpEV strains Note: The NL gene column presents the nucleotide identity rate (%) / amino acid identity rate (%) for each ORF.

[0041] In summary, this invention has isolated a new PpVE-Tomato-1 strain capable of infecting tomatoes, which belongs to the Rhabdoviridae family ( Rhabdoviridae ), Beta-cytoplasmic rhabdovirus ( BetacytorhabdovirusThis new strain exhibits less than 80% genome-wide similarity to existing PpVE strains and causes yellowing of tomato leaves, while its fruit displays novel symptoms such as small green spots and brown lesions. It is distinct from existing PpVE strains. This invention has been bio-preserved, and the preservation information is as follows: Reference biological material (strain): PpVE-Tomato-1 Classification and nomenclature: Papaya virus E Betacytorhabdoviruspapaya virus E Accession number: CGMCC No. 47097 Deposit date: February 6, 2026 Preservation Institution: China General Microbiological Culture Collection Center, China Microbiological Culture Collection Committee Example 2: Field Sample Detection After obtaining the viral genome sequence of PpVE-Tomato-1, we designed specific detection primers (Table 3) and used reverse transcription-polymerase chain reaction (RT-PCR) to detect the collected samples.

[0042] Table 3: Primer sequences for PCR detection The results are as follows Figure 5 As shown, the results revealed that: in the presence of Figure 1 PpVE-Tomato-1 was detected in the upper and middle leaves and fruits of tomato plants exhibiting the shown symptoms, but not in the corresponding tissues of asymptomatic healthy plants. Furthermore, symptomatic and asymptomatic fruits were randomly selected for individual testing. All symptomatic fruits tested positive for PpVE, while no positive results were detected in asymptomatic fruits.

[0043] Therefore, PpVE-Tomato-1 based on the present invention can be used as a target for various detection technologies (RT-PCR, ELISA, immunoassay strips, etc.) to achieve early detection, early warning and early blocking of PpVE disease in tomatoes.

[0044] Example 3: Investigation of PpVE-Tomato-1 propagation mode Adult whiteflies and their eggs were observed on the underside of the leaves of symptomatic tomato plants in the field. Figure 6 (A) Detection of whiteflies and eggs collected from symptomatic plants for the presence of PpVE-Tomato-1.

[0045] The expected PpVE-specific product was amplified from adult whiteflies and eggs collected from symptomatic plants by RT-PCR. Figure 6(B). Gene sequencing showed that the sequence of the amplified product matched the PpVE genome, indicating the presence of PpVE in whiteflies.

[0046] To further verify the virus transmission ability of whiteflies, a virus transmission experiment was conducted. First, axillary branches were taken from symptomatic tomato plants in the field and grafted onto healthy tomato seedlings. Then, 20 days after grafting, PpVE levels in the newly grown systemic leaves were measured. Subsequently, virus-free whiteflies were allowed to feed on the infected plants for five days. Next, the whiteflies were transferred to healthy tomato plants in an insect-protected cage for a five-day inoculation period. After the inoculation period, insecticides were used to kill the whiteflies. Twenty days later, four tomato plants showed mosaic symptoms (…). Figure 6 (C). RT-PCR testing confirmed the presence of PpVE (C) in these symptomatic plants. Figure 6 (D).

[0047] In summary, these results indicate that PpVE-Tomato-1 can be transmitted by both whiteflies and through grafting. Once its transmission mechanism is clarified, PpVE-Tomato-1 can be used for large-scale, standardized screening of tomato germplasm resources to rapidly identify superior PpVE-resistant germplasm and accelerate the breeding process of PpVE-resistant tomato varieties.

[0048] The above description is merely a preferred embodiment of this application and is not intended to limit the application. Various modifications and variations are possible for those skilled in the art. As research progresses, the virus name may change, but this will not affect the protection of the viral genome sequence and corresponding protein sequence provided by this application. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of this application should be included within the scope of protection of this application.

Claims

1. A papaya virus E strain that infects tomatoes, characterized by, Its accession number is: CGMCC No. 47097.

Images