Development of a RT-LAMP assay for real-time detection of criniviruses infecting tomato.

Yellowing symptoms caused by tomato chlorosis virus (ToCV) and tomato infectious chlorosis virus (TICV), both assigned to the genus Crinivirus, resemble nutrient deficiencies. Therefore, early diagnosis of infections will prevent crop damage and the spread of the viruses. In this study, we established a rapid detection method for ToCV and TICV by reverse transcription-loop-mediated isothermal amplification (RT-LAMP). We first designed primer sets for RT-LAMP specific for ToCV and TICV. Next, by selecting the optimum primer set and determining the optimum conditions for the RT-LAMP reaction, each virus was detected within 50 min by piercing the diseased area of a tomato leaf with a toothpick, immersing the toothpick in the reaction solution, and conducting the RT-LAMP reaction. To verify the accuracy of the procedure, 61 tomato leaf samples showing disease symptoms were collected from five regions of Indonesia, and the RT-LAMP results for the samples were identical to those obtained with the commonly used reverse transcription-polymerase chain reaction.

Complete Genome Sequence of a Pepper Yellow Leaf Curl Indonesia Virus Isolated from Tomato in Bali, Indonesia.

We report a complete genome sequence of a pepper yellow leaf curl Indonesia virus (PepYLCIV) isolated in Bali, Indonesia. This virus shares around 90% identity with other PepYLCIV DNA-As and 86% identity with DNA-Bs, suggesting that it is a novel isolate of PepYLCIV.

Complete Genome Sequence of Tomato Leaf Curl New Delhi Virus from Luffa in Indonesia.

This is the first report of a begomovirus infecting luffa in Indonesia. The genome of this virus shares a close identity with that of Tomato leaf curl New Delhi virus (ToLCNDV). There is a 36-nucleotide duplicated sequence in the DNA-B component, suggesting the occurrence of an intraviral recombination.

Development of a LAMP assay with a portable device for real-time detection of begomoviruses under field conditions.

The emergence of begomovirus infection is one of the most important problems affecting production of a variety of vegetable crops worldwide. Infection by begomoviruses has been detected and spread rapidly on Cucurbitaceae and Solanaceae plants in Indonesia. A rapid and simple detection assay for begomoviruses under field conditions for routine sampling of plants is needed. Primers for a loop-mediated isothermal amplification (LAMP) assay were designed based on the sequences of three Indonesian begomoviruses, namely Tomato leaf curl New Delhi virus (ToLCNDV), Pepper yellow leaf curl Indonesia virus (PepYLCIV), and Tomato yellow leaf curl Kanchanaburi virus (TYLCKaV), infecting Cucurbitaceae and Solanaceae plants. LAMP assays using a Genelyzer™ III portable fluorometer with a toothpick method successfully detected these begomoviruses in infected melon, pepper, and eggplant samples. LAMP assays conducted during a field survey for detection of the three begomoviruses on 104 fresh leaves indicated that most of the samples were positive; the findings were confirmed by PCR using universal primers of begomovirus as a common detection method. These results demonstrate that this simple and rapid LAMP assay using a fluorometer portable device may be used to achieve real-time detection of begomoviruses under field conditions.