Molecular characterization of divergent isolates of Citrus bent leaf viroid (CBLVd) from citrus cultivars of Punjab, Pakistan

Amjad Ali; Ummad Ud Din Umar; Syed Atif Hasan Naqvi; Muhammad Taimoor Shakeel; Muhammad Nouman Tahir; Muhammad Fahad Khan; Muhammad Tanveer Altaf; Fatih Ölmez; Abdelfattah A Dababat; Zia Ul Haq; Muhammad Azhar Nadeem; Rüştü Hatipoğlu; Faheem Shehzad Baloch; Yong Suk Chung

doi:10.3389/fgene.2022.1104635

Molecular characterization of divergent isolates of Citrus bent leaf viroid (CBLVd) from citrus cultivars of Punjab, Pakistan

Front Genet. 2023 Jan 12:13:1104635. doi: 10.3389/fgene.2022.1104635. eCollection 2022.

Authors

Affiliations

¹ Department of Plant Pathology, Faculty of Agricultural Sciences and Technology, BahauddinZakariya University, Multan, Punjab, Pakistan.
² Faculty of Agricultural Sciences and Technologies, Department of Plant Protection, Sivas University of Science and Technology, Sivas, Turkey.
³ Department of Plant Pathology, The Islamia University Bahawalpur, Bahawalpur, Punjab, Pakistan.
⁴ Department of Plant Protection, Faculty of Agricultural Sciences, Ghazi University, Dera GhaziKhan, Punjab, Pakistan.
⁵ International Maize and Wheat Improvement Centre (CIMMYT), Emek, Ankara, Turkey.
⁶ Department of Field Crops, Faculty of Agriculture, Kirsehir Ahi Evran Universitesi, Kirsehir, Turkey.

Abstract

Citrus viroid infection is emerging as a serious threat because of its efficient systemic movement within the host plant and its quick spread due to contaminated pruning tools. A survey was conducted to investigate the primary distribution and molecular characterization of Citrus bent leaf viroid (CBLVd) and its variants in different citrus cultivars. A total of 154 symptomatic citrus samples were collected and detected by RT‒PCR with newly designed specific primers with the incidence of 36.33%. During biological indexing study on Etrog citron, expressions of reduced leaf size, yellowing with a light green pattern, and bending were observed. Amplified products were sequenced and analyzed using a nucleotide BLAST search, which showed 98% homology with other CBLVd isolates. The results of the phylogenetic tree analysis showed the presence of two main groups (A and B), with the predominant variants of CBLVd, i.e., CVd-I-LSS (Citrus viroid Low Sequence Similarity) sequences, clustering in subgroup A1 along with newly detected CVd-I-LSS from Palestinian sweet lime (Citrus limettioides), which has been identified as a new host of CVd-I-LSS in Pakistan. Further analysis of the sequences in subgroup A1 showed that the variant of CVd-I-LSS infecting citrus cultivars had a close relationship with isolates reported from China, Japan, and Iran, which may have resulted from the exchange of planting material. This study also unveiled the variability in nucleotide sequences of CBLVd, which made it unable to be detected by old primers. The results of this study indicate that the widespread presence of divergent variants of CBLVd is a major concern for the citrus industry in Pakistan and other countries where virulent isolates of CBLVd are prevalent. These findings suggest the need for future research on effective management and quarantine measures to stop the spread of CBLVd.

Keywords: RT-PCR; biological indexing; citrus orchards; phylogenetic analysis; sequencing analysis.

Grants and funding

Basic Science Research Program supported this research through the National Research Foundation of Korea (NRF), funded by the Ministry of Education (2019R1A6A1A11052070).