A multiplex PCR assay for rapid identification of major tospovirus vectors reported in India

Jangra, Sumit; Mittal, Anubha; Dhall, Heena; Jain, Rakesh; Ghosh, Amalendu

doi:10.1186/s12864-020-6560-x

Cited by 16 publications

(12 citation statements)

References 41 publications

(70 reference statements)

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“…An isofemale population of T. palmi maintained at Advanced Centre for Plant Virology, Indian Agricultural Research Institute (IARI), New Delhi, was used in the study. The identification of T. palmi was based on the morphological keys and mitochondrial cytochrome oxidase subunit I (mtCOI) sequences amplified in PCR using primer pairs, LCO 1490 and HCO 2198 ( Folmer et al, 1994 ; Jangra et al, 2020 ). The population was established from a single adult female of T. palmi on eggplant (var.…”

Section: Methodsmentioning

confidence: 99%

Groundnut Bud Necrosis Virus Modulates the Expression of Innate Immune, Endocytosis, and Cuticle Development-Associated Genes to Circulate and Propagate in Its Vector, Thrips palmi

et al. 2022

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Thrips palmi (Thysanoptera: Thripidae) is the predominant tospovirus vector in Asia-Pacific region. It transmits economically damaging groundnut bud necrosis virus (GBNV, family Tospoviridae) in a persistent propagative manner. Thrips serve as the alternate host, and virus reservoirs making tospovirus management very challenging. Insecticides and host plant resistance remain ineffective in managing thrips–tospoviruses. Recent genomic approaches have led to understanding the molecular interactions of thrips–tospoviruses and identifying novel genetic targets. However, most of the studies are limited to Frankliniella species and tomato spotted wilt virus (TSWV). Amidst the limited information available on T. palmi–tospovirus relationships, the present study is the first report of the transcriptome-wide response of T. palmi associated with GBNV infection. The differential expression analyses of the triplicate transcriptome of viruliferous vs. nonviruliferous adult T. palmi identified a total of 2,363 (1,383 upregulated and 980 downregulated) significant transcripts. The Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analyses showed the abundance of differentially expressed genes (DEGs) involved in innate immune response, endocytosis, cuticle development, and receptor binding and signaling that mediate the virus invasion and multiplication in the vector system. Also, the gene regulatory network (GRN) of most significant DEGs showed the genes like ABC transporter, cytochrome P450, endocuticle structural glycoprotein, gamma-aminobutyric acid (GABA) receptor, heat shock protein 70, larval and pupal cuticle proteins, nephrin, proline-rich protein, sperm-associated antigen, UHRF1-binding protein, serpin, tyrosine–protein kinase receptor, etc., were enriched with higher degrees of interactions. Further, the expression of the candidate genes in response to GBNV infection was validated in reverse transcriptase-quantitative real-time PCR (RT-qPCR). This study leads to an understanding of molecular interactions between T. palmi and GBNV and suggests potential genetic targets for generic pest control.

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Section: Methodsmentioning

confidence: 99%

Groundnut Bud Necrosis Virus Modulates the Expression of Innate Immune, Endocytosis, and Cuticle Development-Associated Genes to Circulate and Propagate in Its Vector, Thrips palmi

et al. 2022

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“…Rapid identification of T. palmi is crucial in pest and disease management and quarantine. Several methods have been reported to date for the molecular identification of T. palmi including PCR, RFLP, SCAR marker, multiplex PCR, real-time PCR, LAMP, and monoclonal antibodies (Paran and Michelmore 1993 ; Banks et al 1998 ; Walsh et al 2005 ; Yeh et al 2014 ; Nakahara and Minoura 2015 ; Przybylska et al 2015 , 2017 ; Jangra et al 2020 b). Most of these techniques cannot be done in a farmer’s field without access to a laboratory.…”

Section: Discussionmentioning

confidence: 99%

A rapid field-based assay using recombinase polymerase amplification for identification of Thrips palmi, a vector of tospoviruses

et al. 2020

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Thrips palmi (Thysanoptera: Thripidae) is an important pest of vegetables, ornamentals, and legumes worldwide. Besides damage caused by feeding, it transmits several tospoviruses. Identification of T. palmi at an early stage is crucial in implementing appropriate pest management strategies. Morpho-taxonomic identification of T. palmi based on the adult stage is time-consuming and needs taxonomic expertise. Here, we report a rapid, on-site, field-based assay for identification of T. palmi based on recombinase polymerase amplification (RPA), its first application in insects. RPA primers designed based on 3′ polymorphisms of the Internal Transcribed Spacer 2 region efficiently discriminated T. palmi without any cross-reactivity to other predominant thrips species. RPA was performed with crude DNA, extracted from single T. palmi simply by crushing in sterile distilled water and could be completed within 20 min by holding the reaction tubes in the hand. The assay was further simplified by using fluorescent as well as colorimetric dyes thus eliminating the gel-electrophoresis steps. The presence of T. palmi was visualized by a change in color from dark blue to sky blue. The assay was validated with known thrips specimens and found to be effective in diagnosing the presence of T. palmi in natural vegetation. This on-site, rapid assay for diagnosis of T. palmi can be used by non-expert personnel in the field of quarantine and pest management. Keywords RPA • Melon thrips • Insect vector • Tospovirus transmission • Thrips diagnostics Key message • Thrips palmi an important pest of vegetables and ornamentals transmitting several tospoviruses • Morpho-taxonomic identification stage-specific, timeconsuming, and needs taxonomic expertise • Rapid, on-site, field-based assay for identification of T. palmi using recombinase polymerase amplification • Assay uses crude extract of T. palmi, completed within 20 min by holding the reaction tubes in hand without use of sophisticated laboratory instruments • The presence of T. palmi visualized by a change in reaction color • Useful for non-expert personnel in field-based identification, quarantine, and adopting suitable pest management strategies

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“…A homogeneous population of T. palmi was developed from a single adult female collected from the stock culture maintained at Advanced Centre for Plant Virology, Indian Agricultural Research Institute (IARI), New Delhi. Identification of the thrips was based on established morphological keys [ 13 , 24 ] and on sequencing mitochondrial oxidase subunit I (mtCOI) [ 25 ]. The population was maintained on healthy eggplants ( Solanum melongena var.…”

Section: Methodsmentioning

confidence: 99%

Progression of Watermelon Bud Necrosis Virus Infection in Its Vector, Thrips palmi

Ghosh

Priti

Mandal

et al. 2021

Cells

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Thrips are important pests of agricultural, horticultural, and forest crops worldwide. In addition to direct damages caused by feeding, several thrips species can transmit diverse tospoviruses. The present understanding of thrips–tospovirus relationships is largely based on studies of tomato spotted wilt virus (TSWV) and Western flower thrips (Frankliniella occidentalis). Little is known about other predominant tospoviruses and their thrips vectors. In this study, we report the progression of watermelon bud necrosis virus (WBNV) infection in its vector, melon thrips (Thrips palmi). Virus infection was visualized in different life stages of thrips using WBNV-nucleocapsid protein antibodies detected with FITC-conjugated secondary antibodies. The anterior midgut was the first to be infected with WBNV in the first instar larvae. The midgut of T. palmi was connected to the principal salivary glands (PSG) via ligaments and the tubular salivary glands (TSG). The infection progressed to the PSG primarily through the connecting ligaments during early larval instars. The TSG may also have an ancillary role in disseminating WBNV from the midgut to PSG in older instars of T. palmi. Infection of WBNV was also spread to the Malpighian tubules, hindgut, and posterior portion of the foregut during the adult stage. Maximum virus-specific fluorescence in the anterior midgut and PSG indicated the primary sites for WBNV replication. These findings will help to better understand the thrips–tospovirus molecular relationships and identify novel potential targets for their management. To our knowledge, this is the first report of the WBNV dissemination path in its vector, T. palmi.

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A multiplex PCR assay for rapid identification of major tospovirus vectors reported in India

Cited by 16 publications

References 41 publications

Groundnut Bud Necrosis Virus Modulates the Expression of Innate Immune, Endocytosis, and Cuticle Development-Associated Genes to Circulate and Propagate in Its Vector, Thrips palmi

Groundnut Bud Necrosis Virus Modulates the Expression of Innate Immune, Endocytosis, and Cuticle Development-Associated Genes to Circulate and Propagate in Its Vector, Thrips palmi

A rapid field-based assay using recombinase polymerase amplification for identification of Thrips palmi, a vector of tospoviruses

Progression of Watermelon Bud Necrosis Virus Infection in Its Vector, Thrips palmi

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