Characterization of Tomato leaf curl New Delhi virus associated with leaf curl and yellowing disease of Watermelon and development of LAMP assay for its detection

Tomato leaf curl New Delhi virus (ToLCNDV) became an alerting virus in Europe from 2017 to 2020 because of its significant damage to Cucurbitaceae cultivation. Until now, just some cucurbit crops including sponge gourd, melon, pumpkin, and cucumber were reported to be resistant to ToLCNDV, but no commercial cultivars are available. In this study, a new isolate of ToLCNDV was identified in Pakistan and analyzed together with ToLCNDV-ES which was previously isolated in Italy. Furthermore, infectious clones of two ToLCNDV isolates were constructed and agroinoculated into different cucurbit crops to verify their infectivity. Results showed that both isolates exhibited severe infection on all tested cucurbit (>70%) except watermelon. Thus, those cultivars may be good candidates in the first step of screening genetic resources for resistance on both Southeast Asian and Mediterranean ToLCNDV isolates. Additional, comparison pathogenicity of different geographical ToLCNDV isolates will be aided to understand viral characterization as such knowledge could facilitate breeding resistance to this virus.

Section: Discussionmentioning

confidence: 99%

Different Infectivity of Mediterranean and Southern Asian Tomato Leaf Curl New Delhi Virus Isolates in Cucurbit Crops

Lal

et al. 2022

“…HNB dye can change from purple to sky blue during reactions. HNB detection for viruses [164][165][166][167][168], bacteria [169,170], and fungi [171][172][173][174] has also been developed. There is only a prototype of direct detection of plant pathogen via magnesium pyrophosphate precipitation [175].…”

Section: Colorimetric Detection Of Lamp Ampliconsmentioning

confidence: 99%

The Potential Use of Isothermal Amplification Assays for In-Field Diagnostics of Plant Pathogens

Иванов

Safenkova

Zherdev

et al. 2021

Rapid, sensitive, and timely diagnostics are essential for protecting plants from pathogens. Commonly, PCR techniques are used in laboratories for highly sensitive detection of DNA/RNA from viral, viroid, bacterial, and fungal pathogens of plants. However, using PCR-based methods for in-field diagnostics is a challenge and sometimes nearly impossible. With the advent of isothermal amplification methods, which provide amplification of nucleic acids at a certain temperature and do not require thermocyclic equipment, going beyond the laboratory has become a reality for molecular diagnostics. The amplification stage ceases to be limited by time and instruments. Challenges to solve involve finding suitable approaches for rapid and user-friendly plant preparation and detection of amplicons after amplification. Here, we summarize approaches for in-field diagnostics of phytopathogens based on different types of isothermal amplification and discuss their advantages and disadvantages. In this review, we consider a combination of isothermal amplification methods with extraction and detection methods compatible with in-field phytodiagnostics. Molecular diagnostics in out-of-lab conditions are of particular importance for protecting against viral, bacterial, and fungal phytopathogens in order to quickly prevent and control the spread of disease. We believe that the development of rapid, sensitive, and equipment-free nucleic acid detection methods is the future of phytodiagnostics, and its benefits are already visible.

“…Highly sensitive and efficient detection tools are required to identify resistant varieties and evaluate new germplasm in breeding programmes. Several tools (ELISA, PCR, NASH, IC-PCR, Real-time PCR, and LAMP) have been optimized for the detection and identification of ToLCNDV [ 9 , 10 , 11 , 12 ]. Commonly, ELISA has been used for routine virus detection owing to its simplicity, sensitivity, accuracy, and affordability.…”

Section: Introductionmentioning

confidence: 99%

“…LAMP is more sensitive than all these methods, and the reaction would be completed in less than one hour. Further, it does not require thermal cyclers, and the amplification can be carried out with a water bath or heating block [ 11 , 12 ]. Immunocapture, followed by the detection of viruses using nucleic acid-based tools (PCR, LAMP), is a versatile, sensitive, and robust diagnostic technique.…”

Section: Introductionmentioning

confidence: 99%

Detecting Tomato Leaf Curl New Delhi Virus Causing Ridge Gourd Yellow Mosaic Disease, and Other Begomoviruses by Antibody-Based Methods

Naganur

Shankarappa

Mesta

et al. 2023

The incidence and severity of begomovirus diseases have been increasing around the world recently, and the ridge gourd [Luffa acutangula (Roxb.) L.] is the latest example of a crop that has become highly susceptible to the outbreak of the tomato leaf curl New Delhi virus (ToLCNDV, genus Begomovirus) in India. Accurate diagnosis of causal agents is important in designing disease management strategies. In this study the coat protein (CP) gene from a ToLCNDV-Rg ridge gourd isolate was used to produce polyclonal antibodies (ToLCNDV-Rg-CP-PAb) in a rabbit. The antibodies successfully detected a 30.5 kDa ToLCNDV-Rg-CP in extracts of symptomatic ridge gourd leaf samples by several assays, such as Western Blotting (WB), Dot Immuno Binding Assay (DIBA), Direct Antigen Coating Enzyme Linked Immuno Sorbent Assay (DAC-ELISA), Immuno Capture Polymerase Chain Reaction (IC-PCR), and Immuno Capture Loop-Mediated Isothermal Amplification (IC-LAMP) assays. However, none of the negative samples tested positive in either of the detection methods. Among all the methods tested, the immunocapture assay, IC-LAMP, was the most sensitive in detecting ToLCNDV-Rg. Furthermore, antibodies generated in this study also detected other commonly occurring begomoviruses in South India, such as tomato leaf curl Palampur virus and squash leaf curl China virus in cucurbits. Together, ToLCNDV-Rg-CP-PAb can be used for detecting at least three species of begomoviruses infecting cucurbits. The obtained antibodies will contribute to monitoring disease outbreaks in multiple crops.