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ARS Home » Northeast Area » Beltsville, Maryland (BARC) » Beltsville Agricultural Research Center » Molecular Plant Pathology Laboratory » Research » Publications at this Location » Publication #423575
Research Project: Discovery, Characterization, and Diagnostics of Endemic and Exotic Citrus Pathogens Using High Throughput Sequencing (HTS)

Location: Molecular Plant Pathology Laboratory

Title: Development of a multiplex TaqMan assay for rapid detection of groundnut bud necrosis virus: a quarantine pathogen in the USA

Author
item DERANIYAGALA, ANUSHI - University Of Georgia
item Roy, Avijit
item Tallury, Shyamalrau
item SUDHINI, HARI - International Crops Research Institute For Semi-Arid Tropics (ICRISAT) - India
item CULBREATH, ALBERT - University Of Georgia
item BAG, SUDEEP - University Of Georgia

Submitted to: Viruses
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 4/3/2025
Publication Date: 4/5/2025
Citation: Deraniyagala, A.S., Roy, A., Tallury, S.P., Sudhini, H.K., Culbreath, A., Bag, S. 2025. Development of a multiplex TaqMan assay for rapid detection of groundnut bud necrosis virus: a quarantine pathogen in the USA. Viruses. https://doi.org/10.3390/v17040532.
DOI: https://doi.org/10.3390/v17040532

Interpretive Summary: Groundnut bud necrosis orthotospovirus (GBNV), was first described in India and presently restricted to a few Asian countries only. GBNV is included in the list of pests of economic and environmental concerns in 2017 (Priority Pest List for 2017 Pests of Economic and Environmental Importance). Major hosts of GBNV include cowpea, mungbean, soybean, potato, and tomato with global crop losses of $89 million. The virus induces a variety of symptoms on peanuts such as chlorosis, necrosis, seedling wilt, stunting, altering size and discoloration of pods and seeds. The presence of major hosts (peanut, tomato, and potato) and 3 known thrips vectors within the USA make GBNV an important quarantine pathogen. If GBNV introduced, it could spread rapidly and cause unexpected consequences for food production. In this study, a sensitive, specific, multiplex real-time PCR assay was developed for rapid detection of GBNV in plant materials, for post-entry quarantine purposes. The developed assay represents a valuable tool for global peanut germplasm screening, followed by introduction or acquisition and exchange of improved cultivars. Overall, this assay could be incorporated into Standard Operating Procedures (SOPs) and applied at key facilities such as the U.S. National Plant Germplasm Inspection Station, USDA-APHIS, and ICRISAT, including at ports of entry. It can be used to prescreen commodities to detect GBNV infected materials before export, thereby minimizing the risk associated with the spread of GBNV.

Technical Abstract: Groundnut bud necrosis orthotospovirus (GBNV), a tripartite single-stranded RNA virus, poses a significant threat to the United States agriculture. GBNV is one of the quarantine pathogens, and its introduction could lead to severe damage to economically important crops such as groundnut, tomato, potato, pea, and soybean. For rapid and accurate detection of GBNV at points of entry, TaqMan real-time RT-qPCR assays were developed and validated the results using RT-PCR followed by Sanger sequencing. These assays target highly conserved regions of the nucleocapsid (NP) and movement (MP) proteins within the viral genome. Multiplex GBNV detection assays targeting the NP and MP genes, as well as an internal control gene ACT11, and showed efficiency rates between 90% and 100% and R² values of 0.98 to 0.99, indicating high accuracy and precision. Moreover, there was no significant difference in sensitivity between multiplex and singleplex assays, ensuring reliable detection across various plant tissues. This rapid, sensitive and specific diagnostic assay will provide a valuable tool at the port of entry to prevent the entry of GBNV into the United States.