Phylogenetic Analysis of Three Isolates of Grapevine leafroll-associated virus 1 (GLRaV-1) from Vineyards in Khorasan Razavi Province Based on The Nucleotide Sequence of a Portion of The Minor Capsid Protein 2 (Cpm2)

پژوهش های حفاظت گیاهان ایران

Article 5, Volume 38, Issue 4 - Serial Number 66, December 2025, Pages 361-371   

Document Type: Research Article

DOI: 10.22067/jpp.2024.89928.1204

Authors

Sara Gharouni^{* 1}; Mahmoud Reza Karimi Shahri¹; Fatemeh Azaddisfani¹; Samira Pakbaz²

¹Plant Protection Research Department, Khorasan Razavi Agricultural and Natural Resources Research and Education Center, AREEO, Mashhad, Iran

²Department of Plant Protection, Faculty of Agriculture, Lorestan University, Khorramabad, Iran

Abstract

Introduction
Grapevines, as a vital agricultural product, face significant challenges from pests and diseases that adversely affect both the quality and quantity of the crop. Among these, viruses are particularly detrimental, with 86 different species identified in grapevines. The viruses associated with grapevine leafroll disease (GLD) are considered the most prevalent in vineyards. Specifically, six species of grapevine leafroll-associated viruses (GLRaVs), belonging to the family Closteroviridae, are linked to this disease. These viruses typically enter vineyards through infected cuttings and are disseminated by vectors, notably mealybugs. The symptoms of GLD manifest as downward rolling of leaf margins, leaf interveinal reddening, and leaf interveinal chlorosis across various grapevine varieties. These symptoms negatively impact the quality of the berries, ultimately affecting yield and marketability. The GLRaV-1 genome is composed of positive-strand RNA and contains ten open reading frames (ORFs), which play crucial roles in its biology and pathogenicity. In Iran, GLRaV-1 has been identified as one of the most widespread grapevine viruses, exhibiting high genetic diversity that complicates management and control efforts. This study focuses on the identification of GLRaV-1 and the phylogenetic analysis of its isolates in the vineyards of Razavi Khorasan province. By understanding the genetic relationships and variability of GLRaV-1 isolates, this research aims to provide insights which could be useful for designing effective management strategies and improving the overall health of grapevine in the region.
 
Materials and Methods
In the spring of 2020, a study was conducted in the Kashmar region of Khorasan Razavi province, focusing on grapevine plants exhibiting distinct symptoms of viral infection. A total of 70 samples were collected from affected plants. The RNA was extracted from these samples to facilitate the detection of grapevine leafroll-associated virus 1 (GLRaV-1). The initial identification process involved reverse transcription-polymerase chain reaction (RT-PCR). This method confirmed the presence of GLRaV-1 in 22 samples, marking a crucial step in understanding the viral infection's prevalence. To ensure the accuracy of the RT-PCR results, Sanger sequencing was used on the amplified fragments from three selected samples. This technique provided definitive confirmation of the viral identity by analyzing the nucleotide sequences, thereby validating the initial findings from the RT-PCR. Data analysis included several critical components. Nucleotide sequence similarity analysis was performed to compare the identified GLRaV-1 isolates against known sequences in databases, assessing their similarity and identifying potential genetic variants. Following this, multiple sequence alignments were conducted using MAFFT software, arranging the sequences to highlight similarities and differences among the isolates. The phylogenetic tree was constructed using the Maximum-Likelihood method with MEGA7 software.
 
Results and Discussion
The samples collected from black grape vineyards in Kashmar exhibited mild yellowing and downward rolling of leaf margins, symptoms commonly associated with increased anthocyanin levels in red grape varieties. In samples infected with GLRaV-1, a fragment of the minor capsid protein 2 (Cpm2) gene was successfully amplified, while no such fragments were detected in healthy plants. This indicates the presence of GLRaV-1, a virus recognized as one of the common pathogens affecting grapes across various regions of Iran. The GLRaV-1 is primarily transmitted through vectors or contaminated cuttings, which emphasizes the importance of monitoring and controlling these pathways to prevent outbreaks. Sequence comparisons revealed that nucleotide identity among Iranian isolates ranged from 84.2% to 100%, while identity with GenBank isolates varied between 77.6% and 87.7%. This high level of genetic diversity among the isolates may enhance the virus's adaptability to different grape varieties, posing challenges for management and control. The observed diversity at the genomic level was consistent with previously reported data regarding the sequence divergence between the two GLRaV-1 groups (Alabi et al., 2011; Sabella et al., 2018; Fan et al., 2015). Phylogenetic analysis further demonstrated that Iranian isolates of GLRaV-1 clustered into two main groups, indicating that the phylogenetic relationships are not strictly related to geographical origin. In previous studies, global isolates of GLRaV-1 have also been distinguished into two different groups based on the gene encoding the minor coat protein 2 (Alabi et al., 2011; Elci, 2019). This finding suggests that the spread of the virus may occur independently of the location, highlighting the need for comprehensive management strategies. To mitigate the spread of GLRaV-1, the use of virus-free plant materials is essential (Alabi et al., 2011; Bruisson et al., 2017). The genetic diversity of GLRaV-1 significantly influences the epidemiology of grapevine leafroll disease, underscoring the importance of producing virus-free plants. Continued research is crucial to understand the underlying causes and effects of this virus. Additionally, developing more sensitive and specific diagnostic tests, along with new control strategies, is vital for effectively managing GLRaV-1 and safeguarding grapevine health.

Keywords

Ampelovirus univitis; Closteroviridae; Coat protein; Phylogenetic analysis; RT-PCR

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