Ahmed Raza1,2,3*, Muhammad Taimoor Shakeel4, Sidra Iqbal5, Azhar Abbas Khan6, Tahira Abbas7, Ammarah Hasnain8, Mahmoud Ahmed Amer9, Abdul Majid2, Adnan Ahmad10, Ummad Ud Din Umar11, Muhammad Umer Iqbal12, Muhammad Naveed Aslam4, Xifeng Wang3, Mingzheng Duan1*
1Key Laboratory of Edible Fungi Resources Innovation Utilization and Cultivation, College of Agronomy and Life Sciences, Zhaotong,657000, China
2Plant Virology Lab, Crop Diseases Research Institute, National Agriculture Research Centre, Pakistan Agricultural Research Council, Islamabad, Pakistan
3State Key Laboratory for Biology of Plant Diseases and Insect Pests, Institute of Plant Protection, Chinese Academy of Agricultural Sciences, Beijing, China
4Department of Plant Pathology, Faculty of Agriculture & Environment, The Islamia University of Bahawalpur, Bahawalpur 63100, Pakistan
5Department of Plant Breeding and Genetics, University of Agriculture-Faisalabad, Constituent College Depalpur, Okara, 56300, Pakistan
6Department of Entomology, Faculty of Agricultural Sciences and Technology, University of Layyah, Pakistan
7Department of Horticulture, Faculty of Agricultural Sciences and Technology, University of Layyah, Pakistan
8Department of Biotechnology, Faculty of Biological Sciences, Lahore University of Biological and Applied Sciences, Pakistan
9Plant Protection Department, College of Food & Agriculture Sciences, King Saud University, P.O. Box 2460, Riyadh 11451, Saudi Arabia
10GenEd, Pakistan Council of Scientific and Industrial Research, Lahore, 54600, Pakistan
11Department of Plant Pathology, Faculty of Agriculture Science and Technology, Bahauddin Zakariya University, Multan 60800, Pakistan
12Department of Plant Pathology, Muhammad Nawaz Sharif University of Agriculture, Multan, Pakistan
Received: 13June 2024 / Accepted: 20 April 2025 / Published Online: 28 May 2025
Abstract
Tomato chlorosis virus (ToCV), causing yellowing disease in tomato, is a phloem-limited, whitefly-transmitted crinivirus that mainly affects plants by reducing chlorophyll content. During a survey conducted in 2019, cirinivirus-specific yellowing symptoms were observed in the tomato fields in Multan, Khanewal, and Lodhran districts of Pakistan. To confirm the etiology of the yellowing disease of tomato, a total of 76 symptomatic and 22 asymptomatic samples were collected out of which 72% of the tested plants were found to be positive for ToCV using RT-PCR. The full genomic nucleotide sequences of two selected ToCV-isolates were obtained by next-generation sequencing (NGS). RNA1 and RNA2 of each isolate comprised 8594 and 8242 nucleotides, respectively. The NGS results were verified by sequencing the amplified overlapping fragments of RNA1 and RNA2 using specific primers designed in this study. In BLASTn analysis for ToCV-Pak1 and ToCV-Pak2, RNA1 from both isolates had the highest similarities (99.41-99.46%) with a South Korean isolate; RNA2 had the highest similarities of 99.67-99.64% with a Greek isolate for Pak1 and Turkish isolate for Pak2, respectively. Phylogenetic analysis indicated that RNA1 of the Pakistani isolates clustered together, forming a subclade, and RNA2 from one of the Pakistani isolates (MN869006) clustered with a Brazilian isolate. At the same time, the other one (MN869007) fell close to a Turkish isolate. Statistical analysis indicated a low polymorphic frequency in RNA1 but a higher polymorphic frequency in RNA 2 quantifying the degree of variation in the analyzed isolates which may result in emergence of new strains with altered levels of virulence. One probable recombination event was detected in RNA1. Analysis revealed that RNA1 of Pak1 and Pak2 is recombinant between South Korean isolate (major parent) and Spanish isolates (minor parent) with recombinant breakpoints at 6172 and 6668 nucleotide positions from the 5′ end, respectively. This study provides the first full genomic analysis and the genetic diversity of ToCV-RNA1 infecting the tomato plant in Pakistan. Understanding the trends and rate of variation in the isolates may provide an insight into the development of resistance in tomato plants against tomato yellowing disease.
