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0INTERNATIONAL SCIENTIFIC AND PRACTICAL CONFERENCE "STATUS AND DEVELOPMENT PROSPECTS OF FUNDAMENTAL AND APPLIED MICROBIOLOGY: THE VIEWPOINT OF YOUNG SCIENTISTS" _25-26 SEPTEMBER, 2024_
MOLECULAR CHARACTERIZATION OF TOBAMOVIRUS ISOLATES NATURALLY INFECTING PEPPER IN
AZERBAIJAN
1Nargiz Fakhraddin Sultanova, 2Khadica Hasan Hasanli
Associated professor, Institute of Molecular Biology & Biotechnologies, Ministry of Science and Education of Azerbaijan Republic and Baku State University, SABAH Groups 2master student, Baku State University, SABAH Groups https://doi.org/10.5281/zenodo.13832618 Annotation. Pepper (Capsicum spp.) is an economically important crop worldwide, with significant production contributing to global food markets. However, viral diseases pose a major threat to pepper cultivation, leading to substantial yield losses. Pepper samples exhibiting tobamovirus-like symptoms were collected from the main vegetable-growing regions of South Azerbaijan, including Calilabad and Masalli, as well as from various gardens and greenhouses located on the Absheron peninsula. To identify the main causal agents of the observed symptoms, the collected samples were initially screened using lateral flow immunoassay techniques (AgriStrip kits targeting TMV, ToMV, PMMoV, TSWV) and enzyme-linked immunosorbent assays (DAS-ELISA and TAS-ELISA). These techniques allowedfor a rapid and preliminary detection of common viruses affecting pepper plants. To confirm and further characterize the presence of tobamoviruses in the samples, reverse-transcription polymerase chain reaction (RT-PCR) was performed using primer pair universal to partial the coat protein region of tobamovirus. The findings confirm the presence of single and mixed tobamoviruses in the symptomatic pepper plants and highlight the necessity for continued surveillance and characterization of viral pathogens in the region.
Keywords: Pepper, tobamovirus, ELISA, RT-PCR, molecular characterization
Pepper (Capsicum spp.), a member of the order Solanales and the family Solanaceae, is an annual dicotyledonous plant widely cultivated around the world. Native to the Americas, pepper has become an essential crop in various regions, including Asia, Europe, and Africa, due to its economic importance and versatility in culinary uses. The plant is grown for its fruits, which are consumed fresh, dried, or processed, making it a valuable staple in many global cuisines. Pepper is a globally significant crop, with an estimated production of over 36 million tons in 2021 (Food and Agriculture Organization of the United Nations, 2022). However, viral diseases represent a major threat to pepper cultivation, leading to substantial yield and quality losses [1]. A detailed review of current databases and literature has identified over 130 viruses, satellite viruses, and viroid species associated with pepper, spanning more than 15 families and 30 genera. These infections contribute to extensive economic losses in pepper production, with global yield losses estimated to range from 20% to 40%, translating into billions of US dollars annually. Mixed virus infections in pepper plants occur when two or more viruses simultaneously infect the same host, often leading to more severe symptoms and greater yield losses compared to single infections. These mixed infections can involve various combinations of viruses, including tobamoviruses, potyviruses, begomoviruses, and others. The interaction between these viruses can significantly
INTERNATIONAL SCIENTIFIC AND PRACTICAL CONFERENCE "STATUS AND DEVELOPMENT PROSPECTS OF FUNDAMENTAL AND APPLIED MICROBIOLOGY: THE VIEWPOINT OF YOUNG SCIENTISTS" _25-26 SEPTEMBER, 2024_
alter their individual effects, enhancing pathogenicity, and complicating disease management [2]. Among these, the tobamoviruses are particularly notorious, affecting a wide range of economically significant crops, including tomatoes and peppers. These viruses are conveyed via direct contact and seeds, rather than by invertebrate carriers. This study focuses on the molecular characterization of tobamovirus isolates naturally infecting peppers in Azerbaijan, aiming to enhance our understanding of their genetic diversity, transmission dynamics, and impact on crop health.
Multiple surveys were carried and plant samples with tobamovirus symptoms were collected from various gardens and greenhouses during the 2024 growing seasons. The collected pepper samples were tested on ToMV, TMV, CMV, TSWV, PMMoV viruses with a serological method such as diagnostic test-system using specific test-bands and immunoenzyme analysis (Bioreba AG, Sweden va Agdia Inc., USA) allowing the simultaneous analysis of a large number of samples. For this purpose, extracts from leaf samples were prepared using adequate buffers according to the protocol and specific agristrips were tested on each virus [3]. ELISA analysis was performed following the instruction of the manufacture. The enzymatic reaction occurring on ELISA plate was first assessed visually by color change. Absorbance at 405 nm was measured with an ELISA reader, Stat Fax Microplate, Awareness Technology, USA.
Total RNA was extracted from 100 mg of fresh leaf tissue using TriReagent (Life Technologies) following the manufacturer's instructions for isolating total RNA from plants. The leaf samples were homogenized, and RNA was extracted using a mixture of TriReagent and chloroform. Isopropanol was then used to precipitate the RNA from the aqueous layer. The resulting pellet was resuspended in RNase-free water. Subsequently, 5 of RNA was loaded onto a denaturing agarose gel to assess RNA integrity. The purity and concentration of the extracted RNA samples were measured spectrophotometrically. Following this, the RNA samples were analyzed using RT-PCR. The RT-PCR was performed with a reaction mix containing 4 |l RNA, 1.5 |il Tobamovirus universal primer (TobRT do2), 1.5 |l dNTP (25 mM), 4 |l RT (5x) buffer, 1 |l M-MLV (RT enzyme), and 8 |l ddH2O. The reaction was conducted for 1 hour at 42°C, and to terminate the reaction, the samples were heated at 65°C for 10 minutes. Following PCR, 5 |l of the product was electrophoresed in a 1.5% agarose gel, stained with ethidium bromide and recorded digitally using UV-Gel Doc system (UK).
The large crop survey to evaluate the incidence of virus diseases of pepper were realized in 2024. Symptoms such as leaf curling, extensive leaf yellowing, leaf deformation, and discoloration were observed in pepper plants naturally infected with tobamoviruses. During surveys 48 plants reminissent virus infections were collected (Figure 1). Symptomless plants were also sampled as negative control.
INTERNATIONAL SCIENTIFIC AND PRACTICAL CONFERENCE "STATUS AND DEVELOPMENT PROSPECTS OF FUNDAMENTAL AND APPLIED MICROBIOLOGY: THE VIEWPOINT OF YOUNG SCIENTISTS" 25-26 SEPTEMBER, 2024
Figure 1. Symptoms observed in pepper plants naturally infected with tobamoviruses, including leaf curling, extensive leaf yellowing, leaf deformation, and discoloration.
Results showed that Tomato mosaic virus (ToMV), Tobacco mosaic virus (TMV), and Pepper mild mottle virus (PMMoV), all related to tobamoviruses, were widely distributed in pepper plants. Immunostrip or AgriStrip-test bands and ELISA results were in complete agreement. These viruses were detected both as single infections and in various combinations, such as TMV + PMMoV, TMV + ToMV, and ToMV + PMMoV, across different pepper samples. To confirm the presence of tobamoviruses in pepper plants, RNAs were isolated from infected samples and analyzed using RT-PCR. TobRT do1 (5'-CGACATCAGCCGATGCAGC-3') and TobRT do2 (5'-ACCGTTTTCGAACCGAGACT-3') universal primers, targeting a part of the tobamovirus CP gene, were used for RT-PCR amplification. Symptomless plants showed no amplification, whereas the expected 320 bp amplicons, characteristic of tobamoviruses, were obtained from infected pepper samples. The ELISA and RT-PCR methods were used to detect tobamoviruses, revealing that the TMV and ToMV incidence rates were 2.9% (14/48) and 2.1% (10/48), respectively, whereas PMMoV was detected at rate of 2.3% (11/48). Based on field observations, the incidence of the surveyed viruses in all pepper gardens and greenhouses was less than 3%.
Tobamoviruses belong to the genus Tobamovirus within the family Virgaviridae. These viruses have a single-stranded, positive-sense RNA genome that is approximately 6.3 to 6.6 kb in length [4]. The genome is organized into four open reading frames (ORFs): the first ORF encodes the replication proteins, the second ORF encodes a movement protein, and the third ORF encodes the coat protein, which is critical for virus assembly and transmission. Tobamoviruses are known for their stability and resistance to environmental degradation, which makes them particularly challenging to manage. Transmission of tobamoviruses typically occurs through mechanical means, including contact with contaminated tools, hands, or other plant materials. The virus can also be transmitted through infected seeds, further facilitating its spread within and between crops. In addition to mechanical transmission, tobamoviruses can be dispersed by contaminated water or soil, leading to widespread infection in agricultural settings. In peppers, tobamoviruses can cause significant economic damage. Symptoms of infection may include mosaic patterns on leaves, mottling, leaf distortion, stunted growth, and reduced fruit yield and quality [5]. The severity of symptoms can vary depending on the specific tobamovirus strain, environmental conditions, and the susceptibility of the pepper variety. In some cases, infected plants may also display necrotic
INTERNATIONAL SCIENTIFIC AND PRACTICAL CONFERENCE "STATUS AND DEVELOPMENT PROSPECTS OF FUNDAMENTAL AND APPLIED MICROBIOLOGY: THE VIEWPOINT OF YOUNG SCIENTISTS" _25-26 SEPTEMBER, 2024_
lesions on leaves and stems, which can further compromise plant health and productivity. The widespread presence and impact of these viruses underscore the urgent need for improved detection, management, and control strategies to safeguard pepper crops against these significant threats.
This work was supported by the Azerbaijan Science Foundation-Grant AEF-MCG-2022-1(42)-12/07/3 -M-07.
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