Rapid Detection of Grapevine Leafroll-associated virus Type 3 using the reverse transcription loop-mediated amplification method

Dissertation (MSc)--University of Pretoria, 2013.

Grapevine Leafroll disease (GLD), one of the most destructive diseases of grapevines, has been found in every country where grapevines are grown. Grapevine Leafroll associated virus type 3 (GLRaV-3), one of several viruses associated with GLD globally, is the most prevalent virus in South African grapevines and therefore control of GLRaV-3 takes high priority in any strategy aimed at control of GLD. GLD can be controlled through the use of an integrated strategy which includes using certified plant material, controlling insect vectors through use of systemic insecticides and the removal of infected vines by roguing. Infected individuals are identified each autumn, using either symptom display (in red cultivars, where infected individuals display interveinal reddening and downward rolling of leaves) or ELISA (in symptomless white cultivars). ELISA is laborious, time consuming and relatively insensitivity compared to molecular techniques and a simpler, more rapid and more sensitive means of indentifying GLRaV-3 infected vines is required. A simple RNA extraction procedure combined with a single-tube reverse transcriptase loop-mediated amplification (RT-LAMP) has been developed which allows for the rapid, simple detection of GLRaV-3. Using RT-LAMP, a viral target can be amplified in 2 hours under isothermal conditions. This GLRaV-3 specific RTLAMP uses hydroxy napthol blue (HNB), a colourimetric indicator that changes from violet to sky blue only where a positive RT-LAMP reaction has occurred, making results quick and easy to interpret. The sensitivity of this technique was compared to ELISA and nested PCR by pooling samples at varying ratios of healthy to infected plants. Using nested PCR and RT-LAMP 1 infected sample could be detected amongst 50 healthy individuals while ELISA could only detect 1 amongst 30 infected making RT-LAMP more sensitive than ELISA. Further RT-LAMP could be performed in 2 hours compared to nested PCR and ELISA’s 8 and 48 hours respectively. Based on these results, RT-LAMP is viable alternative for ELISA for the detection of GLRaV-3 in the field. RT-LAMP was also tested for its ability to detect GLRaV-3 in grapevine rootstocks where, due to low viral titres and erratic distribution, it is notoriously difficult to detect. The rootstocks which were used for testing of GLRaV-3 had been tested in a previous study and it was found that only 28% of samples tested positive after 33 months (post inoculation). Using RT-LAMP, 78% of samples tested positive for GLRaV-3. Although further testing must be done, RT-LAMP may also be a viable alternative for testing grapevine rootstocks for GLRaV-3 infection.

gm2014

Microbiology and Plant Pathology

unrestricted