Srivastava Nishant, Prajapati Malyaj R, Kumar Rakesh, Bhardwaj Pooja, Gupta Nitika, Chandel Vanita, Sharma Susheel K, Baranwal Virendra K
Advanced Centre for Plant Virology, Division of Plant Pathology, ICAR-Indian Agricultural Research Institute, New Delhi 110012, India; Amity Institute of Virology & Immunology, Amity University Uttar Pradesh, Noida 201313, India.
Advanced Centre for Plant Virology, Division of Plant Pathology, ICAR-Indian Agricultural Research Institute, New Delhi 110012, India.
J Genet Eng Biotechnol. 2024 Dec;22(4):100442. doi: 10.1016/j.jgeb.2024.100442. Epub 2024 Nov 26.
Sugarcane is host of many viral pathogens that affects its growth and productivity. High-throughput sequencing (HTS) is comprehensive diagnostic platform that permit the precise detection of viral pathogens to resolve the disease epidemiology of the crop, thus providing the phytosanitary status of plants. The current work was designed to comprehend the virome profiling of sugarcane belonging to five varieties collected from the major crop producing states in India. Additionally, a duplex OneStep RT-PCR assay was optimized for simplified detection of prevalent viruses in single reaction run along with validation and confirmation of HTS results.
The complete genome sequences of sugarcane streak mosaic virus (SCSMV), sugarcane yellow leaf virus (SCYLV) and sugarcane mosaic virus (SCMV) consisted of 9790, 5849 and 9600 nucleotides (nt) respectively were obtained excluding 5' UTR and 3' poly (A) tail from sugarcane samples belonging to different varieties. SCSMV and SCMV had single ORF encoding 3130 and 3063 amino acids (aa) respectively, whereas SCYLV genome comprised of six ORFs. The proteolytic cleavage sites in polyprotein region of SCSMV and SCMV revealed the unique amino acid motifs. SCSMV generated the highest number of single nucleotide variants (SNVs) 876 suggesting that it is more susceptible to mutations than other elucidated viruses in HTS. Recombination events revealed the origin of SCSMV_UP isolate from Indian and Iranian isolates as major and minor parents respectively. Further, validation assay by simplified duplex OneStep RT-PCR revealed the prevalence of SCSMV and SCYLV as mixed infection in sugarcane samples with 28 % incidence. The assay could detect the viruses up to 100 pg/µL of RNA concentration.
The first comprehensive report of sugarcane virome and use of an optimized duplex OneStep RT-PCR assay revealed the prevalence of SCSMV and SCYLV in sugarcane from India. The study also provides an insight into genetic variations in the coding region of SCSMV and SCMV and emergence of diverse variants present in a viral population. A simplified duplex OneStep RT-PCR assay for simultaneous and expeditious detection of prevalent viruses in sugarcane would be useful in certification programme for production of virus-free planting materials.
甘蔗是多种病毒病原体的宿主，这些病原体影响其生长和生产力。高通量测序（HTS）是一个全面的诊断平台，能够精确检测病毒病原体，以解析作物的疾病流行病学，从而提供植物的植物检疫状况。当前的研究旨在了解从印度主要作物生产邦收集的五个甘蔗品种的病毒组概况。此外，还优化了一种双重一步法RT-PCR检测方法，用于在单次反应中简化对流行病毒的检测，并对HTS结果进行验证和确认。
从不同品种的甘蔗样本中获得了甘蔗线条花叶病毒（SCSMV）、甘蔗黄叶病毒（SCYLV）和甘蔗花叶病毒（SCMV）的完整基因组序列，分别由9790、5849和9600个核苷酸（nt）组成，不包括5'非翻译区和3' poly（A）尾。SCSMV和SCMV分别有一个编码3130和3063个氨基酸（aa）的开放阅读框（ORF），而SCYLV基因组由六个ORF组成。SCSMV和SCMV多蛋白区域的蛋白水解切割位点显示出独特的氨基酸基序。SCSMV产生的单核苷酸变异（SNV）数量最多，为876个，这表明它比HTS中其他已阐明的病毒更容易发生突变。重组事件表明，SCSMV_UP分离株分别以印度和伊朗分离株为主要和次要亲本。此外，通过简化的双重一步法RT-PCR进行的验证检测表明，SCSMV和SCYLV在甘蔗样本中以混合感染的形式流行，发病率为28%。该检测方法能够检测低至100 pg/µL RNA浓度的病毒。
关于甘蔗病毒组的首份全面报告以及优化的双重一步法RT-PCR检测方法的应用，揭示了印度甘蔗中SCSMV和SCYLV的流行情况。该研究还深入了解了SCSMV和SCMV编码区的遗传变异以及病毒群体中出现的各种变异。一种用于同时快速检测甘蔗中流行病毒的简化双重一步法RT-PCR检测方法，将有助于无病毒种植材料生产的认证计划。
