Development and validation of a specific real-time PCR protocol for the detection of tomato leaf curl New Delhi virus

Luigi, M.; Manglli, Ariana; Bertin, Sabrina; Donati, Livia; Tomassoli, L.; Ferretti, L.; Faggioli, F.

doi:10.1007/s10658-020-02038-1

Cited by 7 publications

(8 citation statements)

References 11 publications

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“…Regarding the diagnosis, to date, serological and molecular methods are available for ToLCNDV detection. Serological methods include DAS-ELISA [ 28 ] and commercial lateral flow ImmunoStrip ® (LFA) (Agdia, Elkhart, IN, USA), while molecular methods include end-point PCR [ 2 , 22 ], rolling circle amplification PCR (RCA-PCR) [ 29 ], real-time PCR [ 30 ], nucleic acid spot hybridisation (NASH) using specific riboprobes [ 31 ], colorimetric loop-mediated isothermal amplification (LAMP) [ 32 ], LAMP-coupled CRISPR–Cas12a [ 33 ], and a commercial ready-to-use real-time LAMP kit combined with proprietary devices (i.e., an ICGENE ToLCNDV diagnostic kit).…”

Section: Introductionmentioning

confidence: 99%

Development of an In-Field Real-Time LAMP Assay for Rapid Detection of Tomato Leaf Curl New Delhi Virus

Caruso

Ragona

Bertacca

et al. 2023

Plants

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Tomato leaf curl New Delhi virus (ToLCNDV) represents a threat to economically important horticultural crops. A real-time loop-mediated isothermal amplification (LAMP) assay for in-field ToLCNDV detection was developed, coupled to a rapid sample preparation method, and tested both in field and laboratory conditions on zucchini squash, tomato, and pepper samples. A set of six LAMP primers was designed for specific ToCLNDV detection, targeting a 218-nucleotide sequence within the AV1 gene. The sensitivity, specificity and accuracy of the real-time LAMP assay and comparison with canonical PCR were evaluated. The real-time LAMP assay developed was about one-thousand times more sensitive than the conventional PCR method, detecting a total of 4.41 × 102 genome copies as minimum target; no cross-reactivity was detected with the other geminiviruses used as the outgroup. The rapid sample preparation method allows for a reliable detection with a low reaction delay (≈2–3 min) compared to canonical DNA extraction, providing results in less than 45 min. Lastly, an increase in ToLCNDV-positive sample detection was observed compared to PCR, in particular for asymptomatic plants (85% and 71.6%, respectively). The real-time LAMP assay developed is a rapid, simple, specific, and sensitive technique for ToLCNDV detection, and it can be adopted as a routine test, for both in-field and laboratory conditions.

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Section: Introductionmentioning

confidence: 99%

Development of an In-Field Real-Time LAMP Assay for Rapid Detection of Tomato Leaf Curl New Delhi Virus

Caruso

Ragona

Bertacca

et al. 2023

Plants

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“…Care was taken to ensure that the plant size was as uniform as possible. A pool of the seed batch used in the experiments was tested by qPCR [ 23 ] to exclude any ToLCNDV infection or contamination.…”

Section: Methodsmentioning

confidence: 99%

Effects of Organic Biostimulants Added with Zeolite on Zucchini Squash Plants Infected by Tomato Leaf Curl New Delhi Virus

Donati

Bertin

Gentili

et al. 2022

Viruses

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The use of organic substances in integrated pest management can contribute to human- and environment-safe crop production. In the present work, a combination of organic biostimulants (Fullcrhum Alert and BioVeg 500) and an inorganic corroborant (Clinogold, zeolite) was tested for the effects on the plant response to the quarantine pest tomato leaf curl New Delhi virus (ToLCNDV). Biostimulants were applied to healthy and infected greenhouse-grown zucchini plants, and the vegetative parameters and viral titer were evaluated. Although no antiviral effects were observed in terms of both virus replication and symptom expression, these biostimulants were shown to influence plant fitness. A significant increase in biomass and in leaf, flower, and fruit production was induced in both healthy and infected plants. Biostimulants also enhanced the production of metabolites commonly involved in plant response to virus infection, such as carbohydrates, phenylpropanoids and free amino acids. These results encourage new field trials to evaluate the actual productivity of infected plants after treatments and the possible application of organic biostimulants in agriculture.

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“…In Europe, all plant infections reported so far were caused by the ToLCNDV-ES strain. In the Mediterranean region, ToLCNDV was only sporadically reported in tomato, sweet pepper and eggplant (Fortes et al, 2016;Zaidi et al, 2017;Juárez et al 2019;Luigi et al, 2020, but it was mostly found in Cucurbitaceae: melon, zucchini, cucumber and pumpkin, but not in watermelon (Parrella et al, 2018, 2020, Orfanidou et al, 2019. Thus, the ToLCNDV-ES strain appears to be adapted to Cucurbitaceae plants as the preferred hosts.…”

Section: Conclusion On Life Cyclementioning

confidence: 99%

“…PCR assays by Figàs et al (2017) were carried out with primers To-1F and To-1R (López et al, 2015). Luigi et al (2020) designed a new primer and probe set, targeting the BC1 region of component B of the ToLCNDV genome, and developed a real-time PCR protocol (validated according to the guidelines reported in EPPO standard PM 7/98 (EPPO, 2019c)) able to detect ToLCNDV from both host plants and its vector Bemisia tabaci.…”

Section: Laboratory Testingmentioning

confidence: 99%

Pest survey card on tomato leaf curl New Delhi virus

Gemert¹,

Schenk²,

Candresse³

et al. 2020

EFSA Supporting Publications

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This pest survey card was prepared in the context of the EFSA mandate on plant pest surveillance (M-2017-0137) at the request of the European Commission. Its purpose is to guide the Member States in preparing data and information for tomato leaf curl New Delhi virus (ToLCNDV) surveys. These are required to design statistically sound and risk-based pest surveys, in line with current international standards. Tomato leaf curl New Delhi virus is a clearly defined taxonomic entity. Although the main potential pathway of entry and spread of this virus via plants for planting of host plants is subject to prohibitions and specific measures designed to prevent entry and movement, the widespread occurrence and polyphagous nature of its vector Bemisia tabaci constitutes a risk for introduction and further spread of ToLCNDV given its persistent transmission biology. The virus is currently present in parts of the EU, and surveys in Member States can have several objectives, e.g. substantiation of disease freedom for countries or pest-free areas, or disease monitoring. The major hosts of ToLCNDV belong to the Solanaceae and Cucurbitaceae families, and detection surveys should thus target cultivated species belonging to these families. ToLCNDV is expected to be able to become established in most or all areas of the EU where B. tabaci is able to become established-outdoors or under protected cultivation-and where Solanaceae and Cucurbitaceae crops are grown. Surveillance should target symptomatic host plants in a fully grown crop. Because of the severity of its symptoms, surveillance of ToLCNDV can rely on visual examination. However, because its symptoms are similar or identical to those of other begomoviruses, its identification requires the use of validated laboratory assays.

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Development and validation of a specific real-time PCR protocol for the detection of tomato leaf curl New Delhi virus

Cited by 7 publications

References 11 publications

Development of an In-Field Real-Time LAMP Assay for Rapid Detection of Tomato Leaf Curl New Delhi Virus

Development of an In-Field Real-Time LAMP Assay for Rapid Detection of Tomato Leaf Curl New Delhi Virus

Effects of Organic Biostimulants Added with Zeolite on Zucchini Squash Plants Infected by Tomato Leaf Curl New Delhi Virus

Pest survey card on tomato leaf curl New Delhi virus

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