Abstract:
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.
